U.S. patent application number 12/412692 was filed with the patent office on 2009-10-08 for methods and collagen products for tissue repair.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Martha M. Murray.
Application Number | 20090254104 12/412692 |
Document ID | / |
Family ID | 39283929 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090254104 |
Kind Code |
A1 |
Murray; Martha M. |
October 8, 2009 |
METHODS AND COLLAGEN PRODUCTS FOR TISSUE REPAIR
Abstract
Methods and devices for the repair of articular tissue using
collagen material are provided. Compositions of collagen material
and related kits are also provided.
Inventors: |
Murray; Martha M.;
(Sherborn, MA) |
Correspondence
Address: |
DAVID S. RESNICK
NIXON PEABODY LLP, 100 SUMMER STREET
BOSTON
MA
02110-2131
US
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
|
Family ID: |
39283929 |
Appl. No.: |
12/412692 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2007/021009 |
Sep 28, 2007 |
|
|
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12412692 |
|
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60847743 |
Sep 28, 2006 |
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Current U.S.
Class: |
606/151 ;
424/423 |
Current CPC
Class: |
A61L 2300/602 20130101;
A61L 27/56 20130101; A61L 2300/432 20130101; A61L 2300/64 20130101;
A61K 31/401 20130101; A61K 38/39 20130101; A61L 27/24 20130101;
A61L 27/50 20130101; A61L 2300/406 20130101; A61L 2300/252
20130101; A61L 2430/10 20130101; A61L 27/3616 20130101; A61K 35/17
20130101; A61L 27/58 20130101; A61K 45/06 20130101; A61K 35/19
20130101; A61L 27/54 20130101; A61P 19/02 20180101; A61L 2400/06
20130101; A61L 27/3662 20130101; A61K 38/39 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
606/151 ;
424/423 |
International
Class: |
A61B 17/08 20060101
A61B017/08; A61F 2/00 20060101 A61F002/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under NIH
K02 AR049346. Accordingly, the Government may have certain rights
in this invention.
Claims
1. A compressible expandable scaffold comprising a collagen sponge
in the form of or forming a tube or cylinder which can be inserted
into an intra-synovial environment and a means for attachment of
the sponge scaffold to bone, graft or ligament to aid in the
repair, augmentation or reconstruction of a ligament, tendon, or
labrum.
2. The scaffold of claim 1, further comprising a graft material for
reconstruction of a ligament.
3. The scaffold of claim 1, wherein the means of attachment are one
or more sutures, staples and anchors.
4. The scaffold of claim 3, wherein the sutures or anchors are
bioabsorbable.
5. The scaffold of claim 1, comprising type I soluble collagen and
platelet rich plasma.
6. The scaffold of claim 1, comprising a sheet which is rolled
around the repaired, augmented, or reconstructed ligament.
7. The scaffold of claim 1, further comprising one or more growth
factors, enzyme inhibitors, antibiotics, or extracellular matrix
proteins.
8. The scaffold of claim 1, further comprising an additional repair
material to reinforce the area of the ligament or graft around
which the sponge scaffold has been placed.
9. The scaffold of claim 1 in a sterile package in a kit further
comprising means for obtaining blood or platelet rich plasma.
10. A method for repairing, replacing, augmenting or reconstructing
a ligament, comprising arthroscopically inserting a collagen sponge
scaffold in the form of or forming a tube or cylinder, and a means
for attachment of the sponge scaffold to bone, graft or ligament,
into an intrasynovial environment, and attaching the means for
attachment to the bone or ligament, around or in place of a
repaired ligament, a reconstructed ligament, or to augment a
ligament, tendon, meniscus or labrum.
11. The method of claim 10, wherein the means for attachment are
one or more sutures, staples and anchors.
12. The method of claim 10 for repair of a ligament comprising
re-connecting torn pieces of ligament, then inserting the sponge
scaffold into the intrasynovial environment around the torn pieces
of ligament, then securing the sponge scaffold to the bone or
ligament.
13. The method of claim 10 for reconstructing a ligament comprising
removing torn ligament, inserting a ligament graft and securing it
to the attachment points of the torn ligament, then inserting the
sponge scaffold into the intrasynovial environment around the
graft, then securing the sponge scaffold to the bone or
ligament.
14. The method of claim 10, wherein the ligament is selected from
the group consisting of anterior cruciate ligament, lateral
collateral ligament, posterior cruciate ligament, medial collateral
ligament, volar radiocarpal ligament, dorsal radiocarpal ligament,
ulnar collateral ligament, and radial collateral ligament.
15. The method of claim 10, wherein the ligament is torn or
ruptured and the sponge scaffold is used to augment the torn or
ruptured area.
16. The method of claim 10, comprising removing the torn ligament
ends, attaching a graft in place thereof, then placing the sponge
scaffold around the graft.
17. The method of claim 16, wherein the graft is synthetic.
18. The method of claim 10, comprising reconnecting a torn
ligament, then placing the sponge scaffold around the reconnected
ligament.
19. The method of claim 10, comprising providing an additional
repair material to reinforce the area of the ligament or graft
around which the sponge scaffold has been placed.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Application of co-pending
International Application PCT/US2007/021009 filed Sep. 28, 2007,
which designated the U.S., and claims benefit under 35 USC 119(e)
of U.S. Ser. No. 60/847,743 filed on Sep. 28, 2006, the contents of
which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to methods and devices for
the repair of articular tissue using collagen materials.
BACKGROUND OF THE INVENTION
[0004] Intra-articular tissues, such as the anterior cruciate
ligament (ACL), do not heal after rupture. In addition, the
meniscus and the articular cartilage in human joints also often
fail to heal after an injury. Tissues found outside of joints heal
by forming a fibrin clot, which connects the ruptured tissue ends
and is subsequently remodeled to form scar, which heals the tissue.
Inside a synovial joint, a fibrin clot either fails to form or is
quickly lysed after injury to the knee, thus preventing joint
arthrosis and stiffness after minor injury. Joints contain synovial
fluid which, as part of normal joint activity, naturally prevent
clot formation in joints. This fibrinolytic process results in
premature loss of the fibrin clot scaffold and disruption of the
healing process for tissues within the joint or within
intra-articular tissues.
[0005] Enhancing healing of ligaments using growth factors has been
an area of great interest and research. While the majority of
studies have focused on the use of a single growth factor to
stimulate healing, the natural wound healing process is an
orchestration of multiple growth factors released by platelets and
other cells over time. To try to reproduce this in the in vitro and
in vivo environment, prior investigators have looked at sustained
release carriers and viral vectors for release of these cytokines
over days or weeks, as well as examining applications of multiple
growth factors. These studies have shown some additive effects of
applied combinations of growth factors on the wound healing of
ligaments; however, even with advanced application techniques, the
combinations of growth factors, timing of release and concentration
of release make optimization of these systems difficult.
[0006] An alternative method recently used to stimulate healing of
the anterior cruciate ligament is the application of activated
platelet-rich plasma (PRP). PRP is a combination of the
extracellular matrix proteins normally found in plasma (including
fibrinogen and fibronectin) and platelets. When platelets are
activated by the exposed collagen of a ligament injury, they begin
to aggregate and release multiple growth factors including
platelet-derived growth factor (PDGF.alpha..alpha., PDGF
.alpha..beta., PDGF .beta..beta.), transforming growth
factors-.beta. (TGF.beta.1, TGF .beta.2), vascular endothelial
growth factor, basic fibroblast growth factor (FGF2), IGF-1 and
epithelial growth factor. Growth factor release typically occurs
immediately upon platelet activation and is sustained at much lower
levels for the life-span of the platelet--up to 5-7 days.
[0007] PRP can be used to increase local concentrations of active
PDGF-.alpha..beta. and TGF-.beta.1 by over 300% when platelets are
concentrated in the plasma to a similar degree. This degree of
platelet concentration can be accomplished by several available
systems. As seen in vivo, these levels of cytokines released
locally by these platelet concentrates can result in increased
fibroblast DNA synthesis and up-regulation of type I collagen
production and changes in collagen organization, and indeed the use
of far lower concentrations (10 ng/ml TGF-.beta.1 and 20 ng/ml
PDGF-.alpha..beta. can influence fibroblast proliferation,
fibroblast chemotaxis, collagen production and collagen
organization. The use of PRP over purified growth factor
concentrates provides the added benefit of additional ECM proteins
which also stimulate cellular adhesion and collagen synthesis,
particularly in the presence of collagen fibrils.
SUMMARY OF THE INVENTION
[0008] The invention relates in some aspects to methods and
products that facilitate anterior cruciate ligament regeneration or
healing.
[0009] In some aspects the invention is a composition of a sterile
solution of solubilized collagen in a concentration of greater than
5 and less than or equal to 50 mg/ml and having a viscosity of
1,000-200,000 centipoise, hydroxyproline in a concentration of
0.1-5.0 .mu.g/ml, a neutralizing agent wherein the solution has an
osmolarity of 280-350 mOs/kg, wherein the composition is free of
thrombin.
[0010] In other aspects the invention is a composition of a sterile
solution of solubilized collagen in a concentration of greater than
1 and less than 5 mg/ml and having a viscosity of 1,000-200,000
centipoise, hydroxyproline in a concentration of 0.1-5.0 .mu.g/ml,
wherein the solution has an osmolarity of 280-350 mOs/kg, wherein
the composition is free of thrombin.
[0011] A dried powder composition of sterile solubilized collagen,
at least one of decorin and biglycan, and buffer salts, wherein the
composition is free of thrombin may be provided according to other
aspects of the invention.
[0012] In other aspects the invention is a quick set composition of
a sterile solution of solubilized collagen in a concentration of
greater than 5 and less than or equal to 50 mg/ml and having a
viscosity of 1,000-200,000 centipoises and a pH of 6.8-8.0, wherein
the solution has an osmolarity of 280-350 mOs/kg, wherein the
solution sets into a scaffold within 10 minutes of exposure to
temperatures of greater than 30.degree. C. The solution in some
embodiments may be a liquid or a gel.
[0013] In some embodiments the composition further comprises a
buffer. The composition may have a pH of 6.8-8.0. In some
embodiments the composition has a pH of 7.4. In some embodiments
the solution is maintained at a temperature of 4.degree. C.
[0014] In some embodiments the solubilized collagen is present in a
concentration of greater than 15 mg/ml. The collagen may be Type I,
II or III collagen in some embodiments. The collagen may be pepsin
solubilized collagen, enzyme solubilized collagen or it may be
atelocollagen in certain embodiments.
[0015] In some embodiments each of the compositions includes at
least one of decorin and biglycan. In other embodiments each of the
composition includes both decorin and biglycan.
[0016] The composition may include other components, such as, an
antibiotic, an anti-plasmin agent, a plasminogen activator
inhibitor, fibrinogen, a glycosaminoglycan, insoluble collagen, a
non-toxic cross-linking agent, or an accelerator. The composition
may also include platelets or white blood cells. In other
embodiments, the composition may include a neutralizing agent.
[0017] In other aspects, the invention is a method for preparing a
collagen scaffold, by preparing a sterile solution of solubilized
collagen in a concentration of greater than 5 and less than or
equal to 50 mg/ml and having a viscosity of 1,000-200,000
centipoises, and subjecting the sterile solution of solubilized
collagen to a temperature of at least 30.degree. C. wherein the
sterile solution of solubilized collagen forms a collagen
scaffold.
[0018] In some embodiments, the collagen scaffold includes any of
the optional components or has any of the properties described
above.
[0019] A method for preparing a collagen scaffold by preparing a
sterile solution of solubilized collagen in a concentration of
greater than 1 and less than 5 mg/ml and having a viscosity of
1,000-200,000 centipoises, and subjecting the sterile solution of
solubilized collagen to a temperature of at least 30.degree. C.
wherein the sterile solution of solubilized collagen forms a
collagen scaffold is provided according to other aspects of the
invention.
[0020] In some embodiments, the collagen scaffold includes any of
the optional components or has any of the properties described
above. In other embodiments the collagen scaffold includes an
accelerator.
[0021] In other aspects a kit, including a first container housing
a solubilized collagen solution in a concentration of greater than
5 and less than or equal to 50 mg/ml and having a viscosity of
1,000-200,000 centipoise, buffer salts housed in the first
container or in a second container, and instructions for preparing
a solution from the solubilized collagen solution and the buffer
salts is provided.
[0022] A kit, including a container housing a solubilized collagen
solution in a concentration of greater than 5 and less than or
equal to 50 mg/ml and having a viscosity of 1,000-200,000
centipoise, a device for housing blood, and instructions for
preparing a gel from the solubilized collagen solution and blood
components isolated from the blood housed in the device is provided
according to other aspects of the invention.
[0023] In another aspect, the invention is a kit, including a
container housing a powder comprising collagen, a device for
housing blood, and instructions for preparing a gel from the
solubilized collagen solution and blood components isolated from
the blood housed in the device. In one embodiment the powder
includes a neutralization agent.
[0024] In some embodiments, the collagen scaffold includes any of
the optional components or has any of the properties described
above. For instance, in some embodiments the solution is a liquid
or a gel.
[0025] In certain embodiments the buffer salts are housed in the
first container and are part of the solubilized collagen solution.
In other embodiments the buffer salts are housed in the second
container.
[0026] The kit may also include a container housing a
neutralization solution.
[0027] The kit may also include a device for housing blood. In some
embodiments the device for housing the blood is a syringe that is
capable of being used for collecting blood. In other embodiments
the device for housing the blood is a centrifuge tube. An
anticoagulant may optionally be included in the device for housing
the blood or in a separate container. In yet other embodiments the
kit includes a vortex tube.
[0028] The invention according to other aspects is a method
comprising contacting the ends of a ruptured articular tissue in a
subject with a sterile solution of solubilized collagen in a
concentration of greater than 5 and less than or equal to 50 mg/ml
and having a viscosity of 1,000-200,000 centipoises and a pH of
6.8-8.0, and hydroxyproline in a concentration of 0.1-5.0 .mu.g/ml,
wherein the solution has an osmolarity of 280-350 mOs/kg, wherein
the composition does not include thrombin, and allowing the
solution to set to treat the ruptured articular tissue.
[0029] In some embodiments the articular tissue is intra-articular
tissue. An intra-articular injury may be, for instance, a meniscal
tear, ligament tear or a cartilage lesion.
[0030] In other embodiments the articular tissue is extra-articular
tissue. An extra-articular injury may be, for instance, ligament,
tendon or muscle injury.
[0031] The method may involve mechanically joining the ends of the
ruptured tissue.
[0032] A method for replacing a ruptured articular tissue, by
mechanically securing a prosthetic device to tissue proximal to a
site of ruptured articular tissue, wherein the prosthetic device
has an inductive core and an adhesive zone disposed on at least a
portion of the inductive core and which is adapted to provide a
microenvironment between the tissue proximal to a site of ruptured
articular tissue and the inductive core to promote cell migration
from the tissue proximal to a site of ruptured articular tissue
into the inductive core; and allowing bonds to form between the
tissue proximal to a site of ruptured articular tissue and the
adhesive zone of the prosthetic device is provided according to
other aspects of the invention. in one embodiment the tissue
proximal to a site of ruptured articular tissue is bone. In another
embodiment the inductive core is a collagen sponge.
[0033] In another aspect, the invention is a method comprising
contacting the ends of a ruptured articular tissue in a subject
with a sterile solution of solubilized collagen and white blood
cells in a concentration of at least 4.times.10.sup.3 wbc/ml, and
allowing the solution to set to treat the ruptured articular
tissue.
[0034] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. Also, the
phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of
"including", "comprising", or "having", "containing", "involving",
and variations thereof herein, is meant to encompass the items
listed thereafter and equivalents thereof as well as additional
items.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The figures are illustrative only and are not required for
enablement of the invention disclosed herein.
[0036] FIG. 1 is a graph depicting release of PDGF-.alpha..beta.
over time from bovine thrombin-activated (BT) and
collagen-activated (CENTR (centrifuged PRP), PC (platelet
concentrate) and RBC Reduced platelet concentrate) PRP
hydrogels.
[0037] FIG. 2 is a graph depicting release of TGF-.beta.1 over time
from bovine thrombin-activated (BT) and collagen-activated (CENTR,
PC and RBC Reduced platelet concentrate) PRP hydrogels.
[0038] FIG. 3 is a graph depicting TGF-.beta.1 release as a
function of platelet concentration in the PRP at 12 hours after
platelet activation
[0039] FIG. 4 is a graph depicting PDGF-.alpha..beta. release from
the PRP gels as a function of platelet concentration in the PRP at
12 hours after platelet activation.
[0040] FIG. 5 is a graph depicting PDGF-.alpha..beta. elution over
time from the cell-seeded PRP hydrogels. The negative values over
time suggest cell-based consumption of the PDGF-.alpha..beta..
[0041] FIG. 6 is a graph depicting VEGF elution over time from the
cell-seeded PRP hydrogels. The positive trend over time suggests
continuing greater production than consumption of the VEGF by the
ACL cells.
[0042] FIG. 7 is a graph depicting cellular proliferation within
the gels.
[0043] FIG. 8 is a graph depicting results of ACL cell-mediated gel
contraction.
[0044] FIG. 9 is a graph depicting results of in vivo pig total ACL
transection treated with collagen slurry/buffer mixed with animal's
own PRP in the operating room, wherein the mixture is injected into
the gap between the cut ligament ends.
[0045] FIG. 10 is a graph depicting cell number as a function of
time in culture and collagen concentration.
[0046] FIG. 11 is a scan of an SDS-PAGE gel depicting the
components of a collagen solution of the invention.
[0047] FIG. 12 is a graph depicting VEGF release as a function of
granulocyte concentration in the PRP at 12 hours after platelet
activation.
[0048] FIG. 13A: Mean elastic modulus for the collagen-PRP
hydrogels as a function of mixing time. represents a statistically
significant difference between 30 seconds and 120 seconds.
represents a statistically significant difference between 60
seconds and 120 seconds. Error bars represent.+-.one standard
deviation.
[0049] FIG. 13B: Mean inelastic modulus for the collagen-PRP
hydrogels as a function of mixing time. represents a statistically
significant difference between 30 seconds and 120 seconds.
represents a statistically significant difference between 60
seconds and 120 seconds. Error bars represent.+-.one standard
deviation. good
[0050] FIG. 14A: Mean elastic modulus for the collagen-PRP
hydrogels as a function of mixing speed. The differences between
the three groups were not statistically significant. Error bars
represent.+-.one standard deviation.
[0051] FIG. 14B: Mean inelastic modulus for the collagen-PRP
hydrogels as a function of mixing speed. The differences between
the three groups were not statistically significant. Error bars
represent.+-.one standard deviation.
[0052] FIG. 15A: Mean elastic modulus for the collagen-PRP
hydrogels as a function of heating rate. The differences between
the groups were not statistically significant. Error bars
represent.+-.one standard deviation.
[0053] FIG. 15B: Mean inelastic modulus for the collagen-PRP
hydrogels as a function of mixing speed. The differences between
the groups were not statistically significant. Error bars
represent.+-.one standard deviation.
[0054] FIG. 16A: Mean elastic modulus for the collagen-PRP
hydrogels as a function of injection temperature. represents a
statistically significant difference between 24.degree.
C.-26.degree. C. and all other groups. represents a statistically
significant difference between 26.degree. C.-28.degree. C. and all
other groups. Error bars represent.+-.one standard deviation.
[0055] FIG. 16B: Mean inelastic modulus for the collagen-PRP
hydrogels as a function of injection temperature. represents a
statistically significant difference between 24.degree.
C.-26.degree. C. and all other groups. represents a statistically
significant difference between 26.degree. C.-28.degree. C. and all
other groups. Error bars represent.+-.one standard deviation.
[0056] FIG. 17: Mean time to 45.degree. for the collagen-PRP
hydrogels as a function of injection temperature. represents a
statistically significant difference between 24.degree.
C.-26.degree. C. and all other groups. represents a statistically
significant difference between 26.degree. C.-28.degree. C. and all
other groups. .diamond-solid. represents a statistically
significant difference between 28.degree. C.-30.degree. C. and all
other groups. Error bars represent.+-.one standard deviation.
[0057] FIG. 18 is a graph depicting in vivo results of failure
strength versus temperature at injection.
[0058] FIG. 19: Coronal (A) view of a caprine knee with an intact
ACL. The black arrows designate the ACL itself.
[0059] FIG. 20: Graft healing in the collagen group (sagittal
view). The graft appears similar to the appearance at implantation
(white arrow); however, there is scar mass present behind the graft
(black arrow).
[0060] FIG. 21: Graft healing in the collagen-platelet group. The
graft is larger than at implantation and appears grossly to be
synovialized and infiltrated with fibrovascular tissue. Good
integration was observed at the insertion sites. (21A=coronal view,
21B=sagittal view).
[0061] FIG. 22 is a graph depicting strength of the joint as a
function of platelet count.
[0062] FIGS. 23A and 23B are bivariate scattergrams with regression
95% confidence bands. FIG. 23A depicts fail load as a function of
platelet count. FIG. 23B depicts stiffness as a function of
platelet count.
[0063] FIG. 24: AP laxity jig assembled in Instron Machine. The
femoral shaft is secured in the upper fixture which can be rotated
to place the knee between 0 and 90 degrees of flexion for testing.
All testing in this experiment was performed with the knee at 60
degrees of flexion.
[0064] FIG. 25: Sample graph of the AP laxity testing load versus
displacement data. This test was for the sutures tied through both
the anterior and middle tunnels with the knee flexed 30 degrees.
The resulting AP laxity is 8.7 mm, measured as the distance on the
x axis between the two vertical regions of the curve.
[0065] FIG. 26: Anatomy of the ACL insertion in the porcine knee.
In the pig, there are two discrete tibial insertion sites of the
ACL--one is posterolateral, behind the anterior horn attachment of
the medial meniscus (white arrow), and the second is anteromedial,
located between the anterior horn attachment of the medial meniscus
and the anterior horn attachment of the lateral meniscus (forceps
are retracting the anterior horn attachment of the lateral
meniscus).
[0066] FIG. 27: Photographs of the Anterior, Middle and Posterior
tibial tunnel positions. White arrows emphasize the exit site of a
Hewson suture passer through the three sites respectively.
[0067] FIG. 28: Individual values for each of the six knees for the
various testing positions. The intact knee laxity (column 1) is
best restored in the groups where sutures passed through the
anterior or middle tunnels and tied with the knee in 60 degrees of
flexion (columns 6, 8 and 12).
[0068] FIG. 29: Differences from the intact AP laxity of the knee
for all repair conditions. Bars represent the mean, error bars
represent the standard error of the mean. N=6 for all groups.
Groups not significantly different from intact are marked by an
asterisk.
[0069] FIG. 30 is a graph depicting cell counts within the
sponge/PRP preparations measured at Day 2 and Day 10.
DETAILED DESCRIPTION OF THE INVENTION
[0070] Aspects of the invention relate to compositions and methods
for repairing damaged articular tissue. The invention involves
novel collagen based compositions and formulations for repairing
articular tissue, such as a ruptured or torn ligament for instance.
The compositions may be used alone or in combination with
three-dimensional (3-D) scaffolds or other traditional repair
devices. The material provides a connection between the ruptured
ends of the ligament and fibers, or provides a replacement, alone
or in combination with other devices, for a torn ligament, after
injury, and encourages the migration of appropriate healing cells
to form scar and new tissue, thus facilitating healing and
regeneration.
[0071] It is intended that the use of the compositions and methods
of the present invention involve the repair, replacement,
reconstruction or augmentation of specific tissue types. Articular
injuries include both intra-articular and extra-articular injuries.
Intra-articular injuries involve, for instance, injuries to
meniscus, ligament and cartilage. Extra-articular injuries include,
but are not limited to injuries to the ligament, tendon or muscle.
Thus, the methods of the invention may be used to treat injuries to
the Anterior cruciate ligament (ACL), Lateral collateral ligament
(LCL), Posterior cruciate ligament (PCL), Medial collateral
ligament (MCL), Volar radiocarpal ligament, Dorsal radiocarpal
ligament, Ulnar collateral ligament, Radial collateral ligament,
meniscus, labrum, for example glenoid labrum and acetabular labrum,
cartilage, for example, and other tissues exposed to synovial fluid
after injury.
[0072] The injury being treated may be, for instance, a torn or
ruptured ligament. A ligament is a short band of tough fibrous
connective tissue composed of collagen fibers. Ligaments connect
bones to other bones to form a joint. A torn ligament is one where
the ligament remains connected but has been damaged causing a tear
in the ligament. The tear may be of any length or shape. A ruptured
ligament is one where the ligament has been completely severed
providing two separate ends of the ligament. A ruptured ligament
may provide two ligament ends of similar or different lengths. The
rupture may be such that a ligament stump is formed at one end.
[0073] An example of a ruptured anterior cruciate ligament is
described for exemplary purposes only. The anterior cruciate
ligament (ACL) is one of four strong ligaments that connects the
bones of the knee joint. The function of the ACL is to provide
stability to the knee and minimize stress across the knee joint. It
restrains excessive forward movement of the lower leg bone, the
tibia, in relation to the thigh bone, the femur, and limits the
rotational movements of the knee. An anterior cruciate ligament is
ruptured such that it no longer forms a connection between the
femur bone and the tibia bone. The resulting ends of the ruptured
ACL may be of any length. The ends may be of a similar length, or
one end may be longer in length than the other.
[0074] The repair of the damaged tissue is achieved using collagen
based repair material alone or in combination with a tissue healing
device. A tissue healing device is a device other than the repair
material that aids in the repair of the damaged tissue and
includes, for instance, scaffolds, such as sponges and grafts and
mechanical devices, such as sutures and anchors.
[0075] The damaged or injured tissue is treated with a novel
composition which is a sterile solution of solubilized collagen.
Solubilized collagen, as used herein, is enzyme solubilized
collagen including one or more of Type I, II, III, IV, V, X
collagen. Preferably the enzyme solubilized collagen is
tropocollagen or Atelocollagen rather than fibrillar collagen in
order to reduce the antigenicity of the material. The collagen is
isolated from a source and mechanically minced and broken up in an
enzyme based acid media rather than aqueous or salt solution. For
instance, the collagen may be solubilized in pepsin. The step of
mechanically mincing the collagen is important for homogenization
to produce a material of uniform consistency that is free of
aggregates and lumps.
[0076] The pH of the solution during the solubilization is very
acidic, for instance, a pH 2.0 is normally obtained during
solubilization with pepsin. A preferred pH for storage of the
material is 2.0 to 6.5. Preferably the collagen is kept cold
(4.degree. C. or on ice) during storage and throughout the
preparation.
[0077] In one embodiment the solubilized collagen is Type I
collagen. As used herein the term, "Type I collagen" is
characterized by two .alpha.1(I) chains, and one .alpha.2(I) chains
(heterotrimeric collagen). The al (I) chains are approximately 300
nm long. Type I collagen is predominantly found in bone, skin (in
sheet-like structures), and tendon (in rope-like structures). Type
I collagen is further typified by its reaction with the protein
core of another connective tissue component known as a
proteoglycan. Type I collagen contains signaling regions that
facilitate cell migration.
[0078] The collagen is synthetic or naturally derived. Natural
sources of collagen may be obtained from animal or human sources.
For instance, it may be derived from rat, pig, cow, or human tissue
or tissue from any other species. Tendons, ligaments, muscle,
fascia, skin, cartilage, tail, or any source of collagenous tissue
are useful. The material is then implanted into a subject of the
same or different species. The terms "xenogeneic" and "xenograft"
refer to cells or tissue which originates with or is derived from a
species other than that of the recipient. Alternatively the
collagen may be obtained from autologous cells. For instance, the
collagen may be derived from a patient's fibroblasts which have
been cultured. The collagen may then be used in that patient or
other patients. The terms "autologous" and "autograft" refer to
tissue or cells which originate with or are derived from the
recipient, whereas the terms "allogeneic" and "allograft" refer to
cells and tissue which originate with or are derived from a donor
of the same species as the recipient. The collagen may be isolated
anytime before surgery.
[0079] The solubilized collagen may be in a concentration of 1-50
mg/ml in the solution. In some embodiments that concentration of
solubilized collagen is greater than 5 mg/ml and less than or equal
to 50 mg/ml. The concentration of collagen may be, for instance,
10, 15, 20, 25, 30, 35, or 40 mg/ml. Such high concentrations of
collagen are useful for producing viscosity levels that are
desirable for the methods of the invention. Most commercially
available collagen solutions are of lower concentrations. Higher
concentrations can be made, for instance, using the methods
described herein. In other embodiments the solubilized collagen
solution has a concentration of 1 mg/ml to less than 5 mg/ml. When
such lower concentrations of collagen are used, additional
components or steps are taken to increase the viscosity of the
material in order to be useful according to the methods of the
invention. Examples of viscosity inducing methods or components are
described herein.
[0080] The solution should be prepared, by varying the collagen
content and other components, to provide the desired flow
properties of the finished composition. In some embodiments the
solution has a collagen viscosity of 1,000 to 200,000
centipoise.
[0081] The collagen solution is sterile for in vivo use. The
solution may be sterilized and/or components of the solution may be
isolated under sterile conditions using sterile techniques to
produce a sterile composition. The final desired properties of the
composition may be determinative of how the solution is sterilized
because some sterilization techniques may affect properties such as
viscosity. If certain components of the solution are not to be
sterilized, i.e., the collagen isolated from natural sources, the
remaining components can be combined and sterilized before addition
of the collagen, or each component can be sterilized separately.
The solution can then be made by mixing each of the sterilized
components with the collagen that has been isolated using sterile
techniques under sterile conditions. Sterilization may be
accomplished, for instance, by autoclaving at temperatures on the
order of about 115.degree. C. to 130.degree. C., preferably about
120.degree. C. to 125.degree. C. for about 30 minutes to 1 hour.
Gamma radiation is another method for sterilizing components.
Filtration is also possible, as is sterilization with ethylene
oxide.
[0082] The solubilized collagen solution may contain additional
components, such as insoluble collagen, other extracellular matrix
proteins (ECM), such as proteoglycans and glycosaminoglycans,
fibronectin, laminin, entectin, decorin, lysyl oxidase,
crosslinking precursors (reducible and non-reducible), elastin,
elastin crosslink precursors, cell components such as, cell
membrane proteins, mitochondrial proteins, nuclear proteins,
cytosomal proteins, and cell surface receptors, growth Factors,
such as, PDGF, TGF, EGF, and VEGF, and hydroxyproline. In some
embodiments hydroxyproline may be present in the solution in a
concentration of 1 to 3.0 .mu.g/ml, which may be 8 to 9% of the
total protein in the collagen solution. In some embodiments, the
hydroxyproline is present in a concentration of 0.5 to 4.0 .mu.g/ml
in the collagen solution prior to the addition of any buffer. In
some embodiments the collagen solution is free of thrombin. "Free
of thrombin" as used herein refers to a composition which has less
than 1% thrombin. In some embodiments, free of thrombin refers to
undetectable levels. In other embodiments it refers to 0%
thrombin.
[0083] The collagen is mixed with one or more buffers to produce a
solution having a desirable pH range for subsequent mixing with
cells and application to the body. Ideally the buffer solution(s)
has no toxic components or residue, confers physiologic osmolarity
and has the capacity to keep the solution at physiologic pH. A
preferred buffer used in accordance with the invention is a HEPES
buffer. However, any buffer that is non-toxic and is capable of
regulating the pH and/or osmolarity to the levels described herein
is useful according to the invention. HEPES is
N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid, (molecular
weight and structure=238.31, C.sub.8H.sub.18N.sub.2O.sub.4S). For
instance, the inventors have found the following buffer solution
achieves the appropriate pH and osmolarity values: [0084] 0.1M
HEPES [0085] 10.times. Ham's F-10 medium [0086] 100.times.
antibiotic/antimycotic solution (10,000 I.U. Penicillin, 10,000
.mu.g/mL Streptomycin, 25 .mu.g/mL Amphotericin B from CellGro by
Mediatech) [0087] Ultra pure sterile water [0088] 7.5% sodium
bicarbonate [0089] NaHCO.sub.3
[0090] The components of 10.times. Ham's F-10 include the
following:
TABLE-US-00001 Formulation (as 10X): Mol. Component mg/lt Mol. Wt.
(mM) Amino Acids L-Alanine 89.10000 89.1 1.00 L-Arginine HCl
2107.00000 174.2 12.10 L-Asparagine H.sub.2O 150.10000 150.1 1.00
L-Aspartic Acid 133.10000 133.1 1.00 L-Cysteine HCl H.sub.2O
351.30000 175.6 2.00 L-Glutamic Acid 147.10000 147.1 1.00 Glycine
75.10000 75.07 1.00 L-Histidine HCl H.sub.2O 209.60000 209.6 1.00
L-Isoleucine 26.20000 131.2 0.20 L-Leucine 131.20000 131.2 1.00
L-Lysine HCl 293.00000 182.6 1.60 L-Methionine 44.80000 149.2 0.30
L-Phenylalanine 49.60000 165.2 0.30 L-Proline 115.10000 115.1 1.00
L-Serine 105.10000 105.1 1.00 L-Threonine 35.70000 119.1 0.30
L-Tryptophan 6.10000 204.2 0.03 L-Tyrosine 18.10000 181.2 0.10
L-Valine 35.10000 117.1 0.30 Vitamins Biotin 0.24000 244.3 0.0010
Choline Chloride 6.98000 139.6 0.05 D-Calcium Pantothenate 7.15000
238.3 0.03 Folic Acid 13.20000 441.4 0.03 myo-Inositol 5.41000
180.2 0.03 Nicotinamide 6.11000 122.13 0.05 Pyridoxine HCl 2.06000
205.6 0.01 Riboflavin 3.76000 376.4 0.01 Thiamine HCl 10.12000
337.3 0.03 Vitamin B12 13.60000 1355.4 0.01 Inorganic Salts Calcium
Chloride [CaCl.sub.2 2H.sub.2O] 441.00000 147 3.00 Dihydrate Cupric
Sulfate [CuSO.sub.4] 0.01600 159.68 0.0001 Ferrous Sulfate
[FeSO.sub.4 7H.sub.2O] 8.34 278 0.03 Heptahydrate Magnesium Sulfate
[MgSO.sub.4] 746.00000 120.4 6.20 Potassium Chloride [KCl]
2850.00000 74.55 38.23 Potassium Phosphate Monobasic 830.00000
136.09 6.10 [KH.sub.2PO.sub.4] Sodium Chloride [NaCl] 74000.00000
58.44 1266.26 Sodium Phosphate Dibasic 1562.00000 141.96 11.00
[Na.sub.2HPO.sub.4] Zinc Sulfate [ZnSO.sub.4 7H.sub.2O] 0.28800
287.5 0.0010 Heptahydrate Other Dextrose 11000.00000 180.2 61.04
Hypoxanthine 40.80000 136.1 0.30 Lipoic Acid 2.06000 206.3 0.01
Phenol Red Sodium Salt 12.40000 376.4 0.03 Sodium Pyruvate
1100.00000 110 10.00 Thymidine 7.27000 242.2 0.03
[0091] The above-described buffer is exemplary. Many of the
components are not essential For instance, it is not essential to
use sterile water, as long as the appropriate osmolarity is
maintained. The 10.times.F10 solution is also optional. The buffer
may be prepared without 10.times.F10 or equivalent solution.
Additionally glucose or other sugar may be used in place of the
10.times.F10.
[0092] The buffer may or may not include an antibiotic. For
instance, the antibiotic may be penicillin/streptomycin as
described above. Alternatively it may be a clinical antibiotic,
which is used in human patients for the treatment or prevention of
diseases, such as any of those described in Remington's
Pharmaceutical Sciences (Mack Publishing Co., Easton Pa.), which is
hereby incorporated by reference.
[0093] The buffer may be a single component or it may be multiple
components added at the same time or different times. If the buffer
is a single component it should have properties that enable it to
produce a solution having a desirable pH range and osmolarity. In
some instances it is desirable to have at least two buffer
components, a collagen buffer solution and a neutralizing buffer.
The collagen buffer solution may be used to prepare the collagen in
a solution. In some instances the prepared collagen solution may be
stored for extended periods of time.
[0094] A neutralizing buffer, also referred to as a neutralizing
agent, may be added as a solution or in the form of dried salts to
a collagen solution. Once the neutralizing buffer is added the
solution should be kept cold. If the materials are being processed
at room temperature for extended periods of time, it is preferred
that the neutralizing buffer be added to the collagen solution
after storage. Thus, a collagen solution without a neutralization
agent may be prepared ahead of time and stored or it may be
prepared during surgery and used immediately. The neutralization
agent may be added at surgery or ahead of time, but a neutralized
collagen solution preferably should be kept cold (4.degree. C. or
on ice).
[0095] After the neutralizing agent is added to the collagen
solution an osmolarity of 250 to 350 mOsm/kg is preferably
achieved. Osmolarity is a count of the total number of osmotically
active particles in a solution and is equal to the sum of the
molarities of all the solutes present in that solution. It is
defined as a measure of the osmoles of solute per litre of
solution. Osmolarity is a measure of the osmoles of solute per
kilogram of solvent. One of skill in the art can determine the
osmolarity of a solution by obtaining measurements using an
osmometer. An equation used to determine the osmolarity of a
solution is: Osm=.phi.nC
wherein [0096] .PHI. is the osmotic coefficient and accounts for
the degree of dissociation of the solute. .PHI. is between 0 and 1
where 1 indicates 100% dissociation. [0097] n is the number of
particles into which a molecule dissociates. [0098] C is the molar
concentration of the solution
[0099] Additionally, after the neutralizing agent is added,
preferably a pH between 6.8 and 9.0 is achieved. In some
embodiments a pH of 6.8-8.0 is preferred. In other embodiments a pH
of 7.2-7.6 or even 7.4 is preferred.
[0100] Preferably the buffer is sterile prior to addition to the
collagen solution. If it is unsterile then it should be sterilized
prior to addition to the collagen solution or the whole
collagen/buffer solution should be sterilized as described herein.
The components of the buffer may be unsterile and then filtered at
a point before it is mixed with the collagen.
[0101] In certain embodiments, the collagen solution is mixed with
cells such as platelets or white blood cells. In some embodiments,
the cells are derived from the subject to be treated. In other
embodiments, the cells are derived from a donor that is allogeneic
to the subject.
[0102] In certain embodiments, platelets may be obtained as
platelet rich plasma (PRP). This component contains fibrin and
platelets as well as other plasma proteins found in the blood.
There may also be some white blood cells (WBC) and red blood cells
(RBC) found in this preparation. Preferably the platelet
concentration of PRP is at least 100 K/ml, and preferably over 300
K/ml. For instance, the platelet concentration may be at least
1.times. what it is in the blood of the patient, and preferably
1.5.times. or greater In order to maintain the stability of the
cells a physiologic pH (i.e., 6.2 to 7.6) and a physiologic plasma
osmolarity (i.e., 280-360 osms/kg) is used. In order to enhance the
function of the PRP, preferably the PRP is used within 7 days of
being drawn from the patient or donor. Often the PRP is isolated
from the patient at time of surgery. Preferably it is stored at 20
to 24.degree. C. (room temp). However, isolation and storage of the
cells may be achieved by any methods and for any length of time
known in the art for maintaining the activity of the active
components.
[0103] In a non-limiting example, platelets may be isolated from a
subject's blood using techniques known to those of ordinary skill
in the art. As an example, a blood sample may be centrifuged at 700
rpm for 20 minutes and the platelet-rich plasma upper layer
removed. Platelet density may be determined using a cell count as
known to those of ordinary skill in the art. The platelet rich
plasma may be mixed with collagen and applied to the patient.
[0104] In a non-limiting example, white blood cells may also be
isolated from a subject's blood using techniques known to those of
ordinary skill in the art. As an example, a blood sample may be
centrifuged at 700 rpm for 20 minutes and the buffy coat containing
white blood cells removed. WBC density may be determined using a
cell count as known to those of ordinary skill in the art. The WBCs
can be mixed with collagen and applied to the patient.
[0105] The collagen solution may also include any one or more of an
anti-plasmin agent, an extracellular matrix (ECM) protein, other
protein or enzyme inhibitors, antibodies to plasmin, antibodies to
tissue plasminogen activator or urokinase plasminogen activator,
non-toxic crosslinkers, calcium, dextrose or other sugars and cell
nutrients in physiological concentrations. Anti-plasmin agents
include but are not limited to antifibrinolytic enzymes such as
plasminogen inactivator, plasminogen binding .alpha..sub.2
antiplasmin, non-plasminogen binding .alpha..sub.2 antiplasmin, U2
macroglobulin, .alpha..sub.2 plasmin inhibitor, .alpha..sub.2
antiplasmin, and thrombin activatable fibrinolysis inhibitor. Other
protein or enzyme inhibitors include but are not limited to
anti-enzymatic proteins including inhibitors of collagenase,
trypsin, matrix metalloproteinases, elastase and hyaluronidase. The
ECM is composed of fibrillar and non-fibrillar components. The
major fibrillar proteins are collagen and elastin. The ECM includes
for instance, diverse combinations of collagens, fibrinogen,
proteoglycans, elastin, hyaluronic acid, and various glycoproteins
including laminin, fibronectin, heparan sulfate proteoglycan, and
entactin. Non-toxic crosslinkers include but are not limited to
tissue transglutaminases, lysyl oxidase, fibrin, fibronectin, and
reducible and non-reducible crosslink precursor molecules.
[0106] The collagen solution, with or without any of the
above-described additional components, may be stored as a liquid or
gel material or may be dried and stored as a powder. For instance,
a collagen solution may be lyophilized to produce a powder. The
powder may then be reconstituted in a buffer solution. Neutralizing
agent may be present in the reconstitution buffer or may be added
as a separate buffer or as salts.
[0107] The final collagen solution includes collagen, buffer and
cells, such as PRP or WBCs. The components are mixed on a
microscopic level, rather than layered. Preferably it has a pH of
7.4 and a minimum viscosity of approximately 1,000 centipoise.
Preferably the viscosity is in the range of 1,000-200,000
centipoise.
[0108] While the degree of "solidness" may vary from application to
application, generally speaking collagen solutions of the present
invention will exhibit viscosities in the full range of from liquid
to gel-like to solid-like. A collagen solution having optimal
viscosity can be obtained directly from the source of collagen,
depending on the concentration of the collagen. However, a collagen
solution not having an optimal viscosity can be manipulated to
create the correct viscosity. The viscosity of a collagen solution
may be lowered by diluting the solution. The viscosity of a lower
viscosity collagen solution may be increased to increase gelation.
Gelation is the change in viscosity from a fluid-like composition
to a solid or gel-like composition. Gelation or viscosity of a
solution may be increased by adding one or more of the following:
other ECM molecules, including but not limited to, insoluble
collagen, fibrin, fibronectin, and cellulose; cell additions,
including but not limited to, platelets and fibroblasts; non-toxic
crosslinking agents, including but not limited to, tissue
transglutaminases, lysyl oxidase, fibrin, and fibronectin; and
other high viscosity materials with low osmolarity, including but
not limited to, alginate and synthetic filler materials.
[0109] One example of a method for preparing and using the collagen
solution of the invention is provided. The methods of the invention
are not so limited and the description is provided for exemplary
purposes only. Collagen is isolated from rat tails and processed 6
weeks to 6 months ahead of application time (surgery). A buffer
solution is mixed ahead of time as well. The buffer is designed so
collagen-buffer mixture will have pH of 7.4 and be iso-osmotic with
plasma. The PRP is obtained from blood taken from the patient
during anesthesia for surgery using a large bore needle and
anticoagulant. The blood is centrifuged to get a PRP with platelet
count of at least 1.times. normal. When the surgical site is ready,
the collagen and buffer (containing neutralizing agent) are mixed
first using vortex. The PRP is then added to the neutralized
collagen-buffer mixture. The PRP and collagen are combined using a
mixing process to produce a repair material. The resultant gel is
injected arthroscopically into the joint wound site to promote the
healing process.
[0110] The term "repair material" as used herein refers to the
final formulation of collagen solution with cells to be delivered
to the subject.
[0111] The collagen solution or repair material may include
additional materials, such as growth factors, antibiotics,
insoluble or soluble collagen, a cross-linking agent, thrombin,
stem cells, a genetically altered fibroblast, platelets, water,
plasma, extracellular proteins and a cell media supplement.
Alternatively the collagen solution or repair material may exclude
any of these components, and in particular thrombin. The additional
materials may be added to affect cell proliferation, extracellular
matrix production, consistency, inhibition of disease or infection,
tonicity, cell nutrients until nutritional pathways are formed, and
pH of the collagen solution or repair material. All or a portion of
these additional materials may be mixed with the collagen solution
or repair material before or during implantation, or alternatively,
the additional materials may be implanted proximate to the defect
area after the repair material is in place.
[0112] In general, the collagen solution is prepared in advance or
at the time of surgery. At a temperature of preferably 4.degree. C.
to around room temperature PRP or WBCs are added. The PRP/WBC
collagen mixture is kept on ice until use. Just prior to use the
mixture may be warmed to a temperature of 24-30.degree. C.
(preferably 28.degree. C.) and then immediately injected into the
subject. In the subject the material is subjected to body
temperatures in excess of 30 C to produce a gel.
[0113] The repair material of the invention, as discussed above,
may be applied directly to the tissue alone or it may be used in
combination with a tissue healing device such as a scaffold.
Scaffolds may be synthetic or naturally occurring, such as in a
graft. A device or scaffold may be any shape that is useful for
implantation into a subject. The scaffold, for instance, can be
tubular, semi-tubular, cylindrical, including either a solid
cylinder or a cylinder having hollow cavities, a tube, a flat sheet
rolled into a tube so as to define a hollow cavity, liquid, an
amorphous shape which conforms to that of the repair space, a
"Chinese finger trap" design, a trough shape, or square. Other
shapes suitable for the scaffold of the device as known to those of
ordinary skill in the art are also contemplated in the
invention.
[0114] The scaffold may be pretreated with the repair material
prior to implantation into a subject. For instance, the scaffold
may be soaked in a repair material prior to or during implantation
into a repair site. The repair material may be injected directly
into the scaffold prior to or during implantation. The repair
material may be injected within a scaffold at the time of
repair.
[0115] A scaffold is capable of insertion into a repair site and
either forming a connection between the ends of a ruptured tissue,
or forming around a torn tissue such that, in either case, the
integrity and structure of the tissue is maintained. A scaffold is
preferably made of a compressible, resilient material which has
some resistance to degradation by synovial fluid. Synovial fluid as
part of normal joint activity, naturally prevents clot formation.
This fibrinolytic process would result in the premature degradation
of the scaffold and disrupt the healing process of the tissue. The
material may be natural or synthetic and may be either permanent or
biodegradable material, such as polymers and copolymers. The
scaffold can be composed, for example, of collagen fibers, collagen
gel, foamed rubber, natural material, synthetic materials such as
rubber, silicone and plastic, ground and compacted material,
perforated material, or a compressible solid material.
[0116] A scaffold that is capable of compression and expansion is
particularly desirable. For example, a sponge scaffold may be
compressed prior to or during implantation into a repair site. A
compressed sponge scaffold allows for the sponge scaffold to expand
within the repair site. Examples of scaffolds useful according to
the invention are found in U.S. Pat. No. 6,964,685 and US Patent
Application Nos. 2004/0059416 and 2005/0261736, the entire contents
of each are herein incorporated by reference.
[0117] An important subset of natural matrices are those made
predominantly from collagen, the main structural component in
ligament. Collagen can be of the soluble or the insoluble type.
Preferably, the collagen is soluble, e.g., acidic or basic. For
example, the collagen can be type I, II, III, IV, V, IX or X.
Preferably the collagen is type I. More preferably the collagen is
soluble type I collagen. Type I collagen is the predominant
component of the extracellular matrix for the human anterior
cruciate ligament and provides an example of a choice for the basis
of a bioengineered scaffold. Collagen occurs predominantly in a
fibrous form, allowing design of materials with very different
mechanical properties by altering the volume fraction, fiber
orientation, and degree of cross-linking of the collagen. The
biologic properties of cell infiltration rate and scaffold
degradation may also be altered by varying the pore size, degree of
cross-linking, and the use of additional proteins, such as
glycosaminoglycans, growth factors, and cytokines. In addition,
collagen-based biomaterials can be manufactured from a patient's
own skin, thus minimizing the antigenicity of the implant (Ford et
al., 105 Laryngoscope 944-948 (1995)).
[0118] Numerous matrices made of either natural or synthetic
components have been investigated for use in tissue repair and
reconstruction. Natural matrices are made from processed or
reconstituted tissue components (such as collagens and GAGs).
Because natural matrices mimic the structures ordinarily
responsible for the reciprocal interaction between cells and their
environment, they act as cell regulators with minimal modification,
giving the cells the ability to remodel an implanted material,
which is a prerequisite for regeneration.
[0119] Synthetic matrices are made predominantly of polymeric
materials. Synthetic matrices offer the advantage of a range of
carefully defined chemical compositions and structural
arrangements. Some synthetic matrices are not degradable. While the
non-degradable matrices may aid in repair, non-degradable matrices
are not replaced by remodeling and therefore cannot be used to
fully regenerate ligament. It is also undesirable to leave foreign
materials permanently in a joint due to the problems associated
with the generation of wear particles, thus degradable materials
are preferred for work in regeneration. Degradable synthetic
scaffolds can be engineered to control the rate of degradation.
[0120] A scaffold may be a solid material such that its shape is
maintained, or a semi-solid material capable of altering its shape
and or size. A scaffold may be made of expandable material allowing
it to contract or expand as required. The material can be capable
of absorbing plasma, blood, other body fluids, liquid, hydrogel, or
other material the scaffold either comes into contact with or is
added to the scaffold.
[0121] A scaffold material can be protein, lyophilized material, or
any other suitable material. A protein can be synthetic,
bioabsorbable or a naturally occurring protein. A protein includes,
but is not limited to, fibrin, hyaluronic acid, or collagen. A
scaffold material may incorporate therapeutic proteins including,
but not limited to, hormones, cytokines, growth factors, clotting
factors, anti-protease proteins (e.g., alpha1-antitrypsin),
angiogenic proteins (e.g., vascular endothelial growth factor,
fibroblast growth factors), antiangiogenic proteins (e.g.,
endostatin, angiostatin), and other proteins that are present in
the blood, bone morphogenic proteins (BMPs), osteoinductive factor
(IFO), fibronectin (FN), endothelial cell growth factor (ECGF),
cementum attachment extracts (CAE), ketanserin, human growth
hormone (HGH), animal growth hormones, epidermal growth factor
(EGF), interleukin-1 (IL-1), human alpha thrombin, transforming
growth factor (TGF-beta), insulin-like growth factor (IGF-1),
platelet derived growth factors (PDGF), fibroblast growth factors
(FGF, bFGF, etc.), and periodontal ligament chemotactic factor
(PDLGF), for therapeutic purposes. A lyophilized material is one
that is capable of swelling when liquid, gel or other fluid is
added or comes into contact with it.
[0122] The repair material may also be used in combination with a
scaffold that is a graft, such as an ACL graft. Several types of
ACL grafts are available for use by the surgeon in ACL
reconstruction. The grafts may be autografts that are harvested
from the patient, for example patellar bone-tendon-bone grafts, or
hamstring grafts. Alternatively, the grafts can be xenografts,
allografts, or synthetic polymer grafts. Allografts include
ligamentous tissue harvested from cadavers and appropriately
treated and disinfected, and preferably sterilized. Xenografts
include harvested connective tissue from animal sources such as,
for example, porcine tissue. Typically, the xenografts must be
appropriately treated to eliminate or minimize an immune response.
Synthetic grafts include grafts made from synthetic polymers such
as polyurethane, polyethylene, polyester and other conventional
biocompatible bioabsorbable or nonabsorbable polymers and
composites, such as the scaffolds described herein.
[0123] Tissue healing devices also include mechanical devices such
as sutures and anchors. An anchor is a device capable of insertion
into a bone or tissue such that it forms a stable attachment to the
bone or tissue. In some instances the anchor is capable of being
removed from the bone if desired. An anchor may be conical shaped
having a sharpened tip at one end and a body having a longitudinal
axis. The body of an anchor may increase in diameter along its
longitudinal axis. The body of an anchor may include grooves
suitable for screwing the anchor into position. An anchor may
include an eyelet at the base of the anchor body through which one
or more sutures may be passed. The eyelet may be oval or round and
may be of any size suitable to allow one or more sutures to pass
through and be held within the eyelet.
[0124] An anchor may be attached to a bone or tissue by physical or
mechanical methods as known to those of ordinary skill in the art.
An anchor includes, but is not limited to, a screw, a barb, a
helical anchor, a staple, a clip, a snap, a rivet, or a crimp-type
anchor. The body of an anchor may be varied in length. Examples of
anchors, include but are not limited to, IN-FAST.TM. Bone Screw
System (Influence, Inc., San Francisco, Calif.), IN-TAC.TM. Bone
Anchor System (Influence, Inc., San Francisco, Calif.), Model 3000
AXYALOOP.TM. Titanium Bone Anchor (Axya Medical Inc., Beverly,
Mass.), OPUS MAGNUM.RTM. Anchor with Inserter (Opus Medical, Inc.,
San Juan Capistrano, Calif.), ANCHRON.TM., HEXALON.TM., TRINION.TM.
(all available from Inion Inc., Oklahoma City, Okla.) and
endobuttons and TwinFix AB absorbable suture anchor (Smith &
Nephew, Inc., Andover, Mass.). Anchors are available commercially
from manufacturers such as Influence, Inc., San Francisco, Calif.,
Axya Medical Inc., Beverly, Mass., Opus Medical, Inc., San Juan
Capistrano, Calif., Inion Inc., Oklahoma City, Okla., and Smith
& Nephew, Inc., Andover, Mass.
[0125] An anchor may be composed of a non-degradable material, such
as metal, for example titanium 316 LVM stainless steel, CoCrMo
alloy, or Nitinol alloy, or plastic. An anchor is preferably
bioabsorbable such that the subject is capable of breaking down the
anchor and absorbing it. Examples of bioabsorbable material
include, but are not limited to, MONOCRYL (poliglecaprone 25), PDS
II (polydioxanone), surgical gut suture (SGS), gut, coated VICRYL
(polyglactin 910, polyglactin 910 braided), human autograft tendon
material, collagen fiber, POLYSORB, poly-L-lactic acid (PLLA),
polylactic acid (PLA), polysulfone, polylactides (Pla), racemic
form of polylactide (D,L-Pla), poly(L-lactide-co-D,L-lactide),
70/30 poly(L-lactide-co-D,L-lactide), polyglycolides (PGa),
polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone
(PDS), polyhydroxyacids, and resorbable plate material (see e.g.
Orthopedics, October 2002, Vol. 25, No. 10/Supp.). The anchor may
be bioabsorbed over a period of time which includes, but is not
limited to, days, weeks, months or years.
[0126] A suture is preferably bioabsorbable, such that the subject
is capable of breaking down the suture and absorbing it, and
synthetic such that the suture may not be from a natural source.
Examples of sutures include, but are not limited to, VICRYL.TM.
polyglactin 910, PANACRYL.TM. absorbable suture, ETHIBOND.RTM.
EXCEL polyester suture, PDS.RTM. polydioxanone suture and
PROLENE.RTM. polypropylene suture. Sutures are available
commercially from manufacturers such as MITEK PRODUCTS division of
ETHICON, INC. of Westwood, Mass.
[0127] A staple is a type of anchor having two arms that are
capable of insertion into a bone or tissue. In some instances, the
arms of the staple fold in on themselves when attached to a bone or
in some instances when attached to other tissue. A staple may be
composed of metal, for example titanium or stainless steel,
plastic, or any biodegradable material. A staple includes but is
not limited to linear staples, circular staples, curved staples or
straight staples. Staples are available commercially from
manufacturers such as Johnson & Johnson Health Care Systems,
Inc. Piscataway, N.J., and Ethicon, Inc., Somerville, N.J. A staple
may be attached using any staple device known to those of ordinary
skill in the art, for example, a hammer and staple setter (staple
holder).
[0128] The device may be inserted into a repair site of the
ruptured or torn tissue. A repair site is the area around a
ruptured or torn tissue into which the material of the invention
may be inserted. A device may be placed into a repair site area
during surgery using techniques known to those of ordinary skill in
the art. If a scaffold is used in the methods, the scaffold can
either fill the repair site or partially fill the repair site. A
scaffold can partially fill the repair site when inserted and
expand to fill the repair site in the presence of blood, plasma or
other fluids either present within the repair site or added into
the repair site, such as the repair material.
[0129] The scaffold may be positioned in combination with a
surgical technique. For instance, a hole may be drilled into a bone
at or near a repair site of a ruptured or torn tissue and the
scaffold attached by a suture through the hole to the bone. A bone
at or near a repair site is one that is within close proximity to
the repair site and can be utilized using the methods and devices
of the invention. For example, a bone at or near a repair site of a
torn anterior cruciate ligament is a femur bone and/or a tibia
bone. A hole can be drilled into a bone using a device such as a
Kirschner wire (for example a small Kirschner wire) and drill, or
microfracture pics or awls.
[0130] A hole may be drilled into a bone on the opposite side to
the repair site. A suture may be passed through the hole in the
bone and attached to the bone. A scaffold is attached to the suture
to secure the scaffold between the bone and an end of a ruptured
tissue. A ruptured tissue provides two ends of the tissue that were
previously connected. A scaffold may be attached to one or both
ends of a ruptured tissue by one or more sutures. A suture may be
attached to a second bone site at or near the repair site. The
suture may be attached to the second bone using a second
anchor.
[0131] In a typical arthroscopic procedure, for instance of the
ACL, the surgeon prepares the patient for surgery by insufflating
the patient's knee with sterile saline solution. Several cannulas
are inserted into the knee and used as entry portals into the
interior of the knee. A conventional arthroscope is inserted
through one of the cannulas so that the knee may be viewed by the
surgeon remotely.
[0132] In surgical reconstruction of a tissue such as ACL the
surgeon may drill a tibial tunnel and a femoral tunnel in
accordance with conventional surgical techniques using conventional
surgical drills and drill guides. A replacement anterior cruciate
ligament graft is then prepared and mounted in the tibial and
femoral tunnels, and secured using conventional techniques and
known devices in order to complete the knee reconstruction.
[0133] The repair material is applied to a subject. The application
to the subject involves surgical procedures. The following is an
example of a surgical procedure which may be performed using the
methods of the invention. The affected extremity is prepared and
draped in the standard sterile fashion. A tourniquet may be used if
indicated. The intra-articular lesion is identified and defined,
the tissue ends are pretreated, either mechanically or chemically,
and if a scaffold is being used, the scaffold is introduced into
the tissue defect. If the scaffold has not been pre-soaked in the
repair material or if more repair material is desired, then the
repair material is added to the scaffold. The scaffold may be
reinforced by placement of sutures or clips. If no scaffold is used
the tissue defect is coated directly with repair material. The
post-operative rehabilitation is dependent on the joint affected,
the type and size of lesion treated, and the tissue involved.
[0134] The temperature of the repair material may be regulated to
optimize rapid gelatin in vivo. For instance, it is shown in the
examples that different temperatures at the time of injection of
the repair material into the body can influence the time required
for gelatin to occur. In some embodiments of the invention the
injection temperature is ideally between 24.degree. C. and
30.degree. C. 28.degree. C. may be an optimal temperature in some
settings to cause the quickest gelatin time. The injection
temperature can be achieved by warming the solution to the optimal
temperature immediately prior to injection.
[0135] The methods of the invention may be achieved using
arthroscopic procedures. Standard arthroscopy equipment may be
used. Initially, diagnostic arthroscopy may be performed to
identify the appropriate repair site. If a scaffold is used it
should be compressible to allow introduction through arthroscopic
portals, incisions and equipment. The repair material can be placed
in the repair site by direct injection. After the procedure the
arthroscopic portals can be closed and a sterile dressing
placed.
[0136] A subject includes, but is not limited to, any mammal, such
as human, non-human primate, mouse, rat, dog, cat, horse or cow. In
certain embodiments, a subject is a human.
[0137] The materials used in the invention are preferably
biocompatible, pharmaceutically acceptable and sterile. As used
herein, the term "biocompatible" refers to compositions (e.g.
cells, tissues, matrices, etc.) that do not substantially disrupt
the normal biological functions of other compositions to which they
contact. In selected embodiments, the present invention also
contemplates biocompatible materials that are both biodegradable
and non-biodegradable.
[0138] As described above, each of the components of the repair
material may be prepared sterilely. If however, one or more
components is not retrieved or processed in a sterile manner then
it can be sterilized prior to application to the subject. For
instance the material (preferably without the cells) may be
sterilized after production using gamma irradiation, ethanol,
autoclave sterilization or other known sterilization methods.
[0139] As used herein, the term "pharmaceutically acceptable" means
a non-toxic material that does not interfere with the effectiveness
of the biological activity of the scaffold material or repair
material. The term "physiologically acceptable" refers to a
non-toxic material that is compatible with a biological system such
as a cell, cell culture, tissue, or organism. The characteristics
of the carrier will depend on the route of administration.
Physiologically and pharmaceutically acceptable carriers include
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials which are well known in the art. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the scaffold material is combined to facilitate the
application. The components of the pharmaceutical compositions also
are capable of being co-mingled with the device of the present
invention, and with each other, in a manner such that there is no
interaction which would substantially impair the desired
pharmaceutical efficacy.
[0140] In some embodiments the repair material composition is
injectable. Injectable compositions may contain formulatory agents
such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for injection may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
materials may also contain suitable stabilizers.
[0141] The collagen solution may be in the form of a liquid, gel or
solid, prior to addition of the cells. Once the cells are added,
the repair material will begin to increase in gelation for
application to the body. If the collagen solution is a liquid or
gel the cells may be directly added to the solution.
[0142] Alternatively, the collagen solution may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. Neutralization agent may be added
before or after reconstitution. After the powder is reconstituted
it is mixed with cells to form the repair material.
[0143] As used herein, the term "gel" refers to the state of matter
between liquid and solid. As such, a "gel" has some of the
properties of a liquid (i.e., the shape is resilient and
deformable) and some of the properties of a solid (i.e., the shape
is discrete enough to maintain three dimensions on a two
dimensional surface). A gel may be provided in pharmaceutical
acceptable carriers known to those skilled in the art, such as
saline or phosphate buffered saline. Such carriers may routinely
contain pharmaceutically acceptable concentrations of salt,
buffering agents, preservatives, compatible carriers and optionally
other therapeutic agents.
[0144] An example of a gel is a hydrogel. A hydrogel is a substance
that is formed when an organic polymer (natural or synthetic) is
crosslinked via covalent, ionic, or hydrogen bonds to create a
three-dimensional open-lattice structure which entraps water
molecules to form a gel. A polymer may be crosslinked to form a
hydrogel either before or after implantation into a subject. For
instance, a hydrogel may be formed in situ, for example, at the
repair site. In certain embodiments, the repair material forms a
hydrogel within the repair site upon exposure to body
temperatures.
[0145] The repair material, including the collagen solution and the
cells will begin to set once it is created. The setting process can
be delayed by maintaining cold temperatures or it may be
accelerated by warming the mixture. In certain embodiments, a quick
set composition of the repair material is provided. The quick set
composition is capable of forming a set scaffold within 10 minutes
of mixture when the material is exposed to temperatures of greater
than 30.degree. C. In some embodiments formation of the scaffold
takes approximately 5 minutes at such temperatures. As discussed
above, setting times can be further accelerated by optimizing
injection temperatures. The quick set composition is achieved by
preparing the collagen solution at concentrations and viscosities
as described herein. The quick set nature can be further enhanced
by the addition of non-toxic cross linking agents. Such
compositions should be applied quickly to the tissue defect to
sufficiently set before closure of the defect and surgery area.
[0146] The invention also includes in some aspects kits for repair
of ruptured or torn articular tissue. A kit may include one or more
containers housing the components of the invention and/or for
collecting or storing blood or cells and instructions for use. The
kit may be designed to facilitate use of the methods described
herein by surgeons and can take many forms. Each of the
compositions of the kit, where applicable, may be provided in
liquid form (e.g., in solution), or in solid form, (e.g., a dry
powder). In certain cases, some of the compositions may be
constitutable or otherwise processable (e.g., to an active form),
for example, by the addition of a suitable solvent or other species
(for example, water or a cell culture medium), which may or may not
be provided with the kit. As used herein, "instructions" can define
a component of instruction and/or promotion, and typically involve
written instructions on or associated with packaging of the
invention. Instructions also can include any oral or electronic
instructions provided in any manner such that a user will clearly
recognize that the instructions are to be associated with the kit,
for example, audiovisual (e.g., videotape, DVD, etc.), Internet,
and/or web-based communications, etc. The written instructions may
be in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which instructions can also reflects approval by the agency of
manufacture, use or sale for human administration.
[0147] The kit may contain any one or more of the components
described herein in one or more containers. As an example, in one
embodiment, the kit may include instructions for mixing one or more
components of the kit and/or isolating and mixing a sample (e.g.,
blood taken from a subject) and applying to a subject. The kit may
include a container housing collagen. The collagen may be in the
form of a liquid, gel or solid (powder). The collagen may be
prepared sterilely, packaged in syringe and shipped refrigerated.
Alternatively it may be housed in a vial or other container for
storage. A second container may have buffer solution premixed
prepared sterilely or in the form of salts. Alternatively the kit
may include collagen and some buffer premixed and shipped in a
syringe, vial, tube, or other container. The mixture may or may not
include neutralization agent. The neutralization agent may be
included in a separate container or may not be included in the
kit.
[0148] The kit may have one or more or all of the components
required to draw blood from a patient, process the sample into
platelet concentrate or WBCs, and deliver the repair material to a
surgical site. For instance, a kit for withdrawing blood from a
patient may include one or more of the items required for such a
procedure. For example, typically when an injection is to be made,
the patient's skin is cleansed with a disinfecting agent, such as
an alcohol wipe; then a second disinfecting agent, such as iodine
or Betadine may be applied to the skin; an area is usually isolated
with a tourniquet to restrict the blood flow within the artery or
vein making the vessel more visible before the needle is inserted,
a needle attached to a collection device, such as a vacutainer tube
is injected through the patient's skin to withdraw the blood; the
needle is then removed and wiped clean; and the puncture site is
covered with an absorbent pad until after hemostasis.
[0149] The accessories included may be specifically designed to
allow the practitioner to withdraw blood from the patient. For
instance, the accessories may include one or more of the following
a tourniquet, a skin penetration instrument, a device for housing
blood, a collection tube, disinfecting agents or post-injection
bleeding patches.
[0150] The skin penetrating instrument for initiation of blood flow
may be a conventional device such as a needle. The needle may be
single or double ended and may be of any gauge, preferably 21 or 23
gauge. It optionally has a safety sleeve, may be attached to a
needle hub, and preferably is used with a conventional tube holder.
The needle may also be part of a conventional syringe assembly
including barrel and plunger. The needle may be part of a
conventional blood collection set in which a penetrating needle
having a grasping means, such as wings, is connected via a hub and
tubing to a delivery needle for puncture of a septum of an
evacuated tube.
[0151] The device for housing the blood may be any type of
container for receiving the blood sample, such as, for example, a
syringe barrel or it may be a device to which the blood sample is
transferred following collection, for example a tube. Preferred
devices for housing the blood are conventional tubes or vials
having a closed end and an open end. Such tubes may have an
internal volume of 100 .mu.l to 100 ml. Devices to house the blood
after it has been collected include for instance, vials, centrifuge
tubes, vortex tubes or any other type of container. The device for
receiving the blood may be an evacuated tube in which the open end
is covered by a puncturable septum or stopper, such as a vacutainer
tube. Evacuated tubes are generally used with a conventional tube
holder and blood collection set for collection of multiple larger
blood samples, and may contain any of a variety of conventional
blood analysis additives, such as anticoagulants. Preferred
anticoagulants are citrate and ethylenediaminetetra acetic acid
(EDTA).
[0152] The plasma, which contains the platelets, may be separated
from the whole blood. Any separation technique can be utilized, for
example, sedimentation, centrifugation or filtration.
Centrifugation can be carried out at about 500 g for about 20
minutes to obtain platelets. The supernatant, which contains the
plasma, can be removed by standard techniques. Filtration can be
carried out by passing the whole blood through a suitable filter
that separates blood cells from plasma.
[0153] Optionally the kits may include disinfecting agents and
post-injection bleeding patches. A means for sterilizing the
patient's skin in the area of intended puncture, such as a
disinfecting agent may be provided. A typical and conventional
disinfecting agent is a piece of fabric commonly referred to as a
gauze combined with a disinfectant. Some typical disinfecting
agents include rubbing alcohol, antibacterial agents, iodine, and
Betadine, which may or may not be provided with application pads in
individually sealed packets. The post-injection bleeding patch can
also vary from a relatively simple gauze pad plus adhesive strips,
to a bandage.
[0154] When a blood draw is to be made, the practitioner may open
the sealed kit; isolate a selected region of the patient's body,
such as the lower arm, with the tourniquet to restrict the blood
flow within the region and make the blood vessels more visible;
clean the injection site with one or more of the sterilizing
agents; attach the needle to the collection tube; inject the needle
into the patient's blood vessel and collect the blood sample in the
tube; withdraws the needle from the skin; and covering the puncture
site with an absorbent pad. The blood may then be processed to
produce a concentrate of platelets or white blood cells.
[0155] The kit may have a variety of forms, such as a blister
pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable
thermoformed tray, or a similar pouch or tray form, with the
accessories loosely packed within the pouch, one or more tubes,
containers, a box or a bag.
[0156] The kit may be sterilized after the accessories are added,
thereby allowing the individual accessories in the container to be
otherwise unwrapped. The kits can be sterilized using any
appropriate sterilization techniques, such as radiation
sterilization, heat sterilization, or other sterilization methods
known in the art.
[0157] The kit may also contain any other component needed for the
intended purpose of the kit. Thus, other components may be a
fabric, such as gauze, for removing the disinfecting agent after
the sterilizing step or for covering the puncture wound after the
sample is drawn. Other optional components of the kit are
disposable gloves, a support for the device for holding blood after
the sample is taken, adhesive or other device to maintain the
fabric in place over the puncture wound.
[0158] The kit may include disposable components supplied sterile
in disposable packaging. The kit may also include other components,
depending on the specific application, for example, containers,
cell media, salts, buffers, reagents, syringes, needles, etc.
[0159] The following examples are intended to illustrate certain
aspects of certain embodiments of the present invention, but do not
exemplify the full scope of the invention.
EXAMPLES
Example 1
Preparation of Collagen solution and testing of Properties
[0160] A. Minimal Gelation Achieved with Some Formulations
[0161] 1. Innocoll: Aliquots of Innocoll Collagen (Starting pH=4.1)
were Made.
[0162] Weights of aliquots were 0.380 mg collagen, pH=4.1. One half
of the samples had 5 microliters NaHCO.sub.3 added to bring pH to
between 7.0 and 8.0 and one half were not neutralized. 300
microliters fetal bovine serum was added to each aliquot. All
solutions were monitored for gelation at 37.degree. C. for 30
minutes. The solutions remained liquid, with viscosities similar to
that of water (approximately 1 centipoise). No increase in
viscosity with time was noted over the hour long period.
[0163] Identical experiments with Innocoll aliquots at starting
pH=2.5 were also performed. Additionally, experiments were
performed using ratios of collagen:FBS of 1:1, 2:1 and 3:1. None of
these materials produced a gelled product at 37.degree. C. The
solutions remained liquid, with viscosities similar to that of
water (approximately 1 centipoise). No increase in viscosity with
time was noted over the hour long period.
[0164] 2. EPC: Collagen Slurry was Obtained from Elastin Products
Company (Owensville, Mo.), Product Number C857, Lot Number 698,
Lyophilized Type I Acid Soluble Collagen from Calf Skin.
[0165] Prepared according to the method of Gallop and Seifter Meth.
Enzymol., 6, 635, (1963). In the method, fresh calf skin is
extracted with 0.5 M NaOAc to remove non-collagen proteins. The
soluble collagen is extracted with 0.075 M sodium citrate pH 3.7
and precipitated as fibrils by dialysis against 0.02 M
Na.sub.2HPO.sub.4. The product was soluble in 0.01 M to 0.5 M
acetic acid of maximum of 10 mg/ml and soluble in 0.075 M sodium
citrate pH 3.7 and in dilute acetic acid 0.01 M to 0.5 M.
[0166] Before combining with the platelet component of the
hydrogel, the collagen slurry was mixed with 0.1M HEPES Buffer 1M
solution (Cellgro, Mediatech, Inc, Herndon, Va.), 10.times. Ham's
F-10 medium (MP Biomedicals, LCC, Aurora, Ohio),
Antibiotic-Antimycotic solution (Cellgro, Mediatech, Inc., Herndon,
Va.) and sterile water. The collagen slurry was neutralized to a pH
of 7.4 using 7.5% sodium bicarbonate (Cambrex BioScience
Walkersville, Inc., Walkersville, Md.). Mixtures of collagen-PRP
were tested using ratios of collagen:FBS of 3:1. None of these
materials produced a gelled product at 37.degree. C. The solutions
remained liquid, with viscosities similar to that of water
(approximately 1 centipoise). No increase in viscosity with time
was noted over 1 hour or overnight.
[0167] 3. SERVA: Collagen Slurry was Obtained from Serva, Product
Number 47256, Lot Number 14902 Type I Rat Tail Collagen Solution at
4 mg/ml in 0.1% Acetic Acid (Heidelberg Germany.
[0168] The collagen slurry was mixed with 0.1M HEPES Buffer 1M
solution (Cellgro, Mediatech, Inc, Herndon, Va.), 10.times. Ham's
F-10 medium (MP Biomedicals, LCC, Aurora, Ohio),
Antibiotic-Antimycotic solution (Cellgro, Mediatech, Inc., Herndon,
Va.) and sterile water. The collagen slurry was neutralized to a pH
of 7.4 using 7.5% sodium bicarbonate (Cambrex BioScience
Walkersville, Inc., Walkersville, Md.).
[0169] Mixtures of collagen-PRP were tested using ratios of
collagen:FBS of 3:1. This material did not produce a gelled product
at 37.degree. C. The solutions remained liquid, with viscosities
similar to that of water (approximately 1 centipoise). No increase
in viscosity with time was noted over 1 hour or overnight.
[0170] 4. VITROGEN: Collagen Slurry was Obtained from Cohesion
Technologies (Palo Alto, Calif.). Vitrogen 100 Slurry, Lot Number
C101636 with a Collagen Concentration of 3.1 mg/ml was also
Tested.
[0171] Vitrogen Collagen In Solution is 99.9% pure collagen as
judged by SDS polyacrylamide gel electrophoresis in conjunction
with bacterial collagenase sensitivity and silver staining
techniques. The solution is 95-98% Type I collagen with the
remainder being comprised of Type III collagen. Vitrogen Collagen
In Solution is a native collagen as judged by polarimetry and
trypsin sensitivity, although it does contain a low percentage of
nicked or shortened helices.
[0172] The collagen slurry was mixed with 0.1M HEPES Buffer 1M
solution (Cellgro, Mediatech, Inc, Herndon, Va.), 10.times. Ham's
F-10 medium (MP Biomedicals, LCC, Aurora, Ohio),
Antibiotic-Antimycotic solution (Cellgro, Mediatech, Inc., Herndon,
Va.) and sterile water. The collagen slurry was neutralized to a pH
of 7.4 using 7.5% sodium bicarbonate (Cambrex BioScience
Walkersville, Inc., Walkersville, Md.). Mixtures of collagen-PRP
were tested using ratios of collagen:FBS of 3:1. This material did
not produce a gelled product at 37.degree. C. For the first thirty
minutes, the solutions remained liquid, with viscosities similar to
that of water (approximately 1 centipoise). No significant increase
in viscosity with time was noted until one hour had passed.
[0173] 5. Wake Forest Collagen Testing: Collagen Solutions were
Made from Pig Skin. the Skin was Washed with Water and the Hair and
Subcutaneous Fat Removed. the Skin was Minced and Further De-Fatted
with Acetone and Washed with Deionized Water.
[0174] The minced pieces were soaked in 10% NaCl at 4.degree. C.
for 24 hours then soaked in a citrate buffer (pH 4.3) for 48 hr and
homogenized in 0.5 M acetic acid at 4.degree. C. The homogenate was
then digested with pepsin at 4.degree. C. for 24 hr and
centrifuged. NaCl equivalent to 5% w/v was added to salt-out
atelo-collagen and the collagen washed with phosphate buffer and
dissolved in 0.5 M acetic acid and dialyzed.
[0175] The aliquots of collagen were held on ice until they were
mixed with FBS and Ham's F12. All solutions had a pH between 7.0
and 7.5. 150 microliters of FBS and 50 microliters Hams F12 were
added to each tube, mixed with vortexer, and placed in water bath
at 37.degree. C. One of five aliquots gelled at 7 minutes. None of
the other four gelled over the 30 minute time period of the test.
The tests were repeated for the 4 collagen samples that did not
gel. None of the repeated samples gelled during the 30 minute time
frame.
[0176] In order to confirm the accuracy of the gelation of the
single sample that produced a gelled product, a second trial was
conducted to repeat the study on the collagen type that did set in
first trial. In the second trial no gelation was observed, even
after 3 hours at 37.degree. C. The experiment was attempted again
with collagen and FBS only. The following was observed: ratio of
2:1 collagen:FBS produced no gelation and ratio of 3:1 collagen:FBS
did gel when placed in water bath at 37.degree. C. (gel softened
over the remaining hour). Repeat testing of this collagen type
showed inconsistent gelation which softened over time rather than
continuing to hold shape.
B. Rapid Gelation Observed with Formulations of the Invention.
[0177] Pig Patellar Tendons were minced, placed into 10% NaCl
solution to obtain salt-solubilized collagen. The collagen was
homogenized and centrifuged. The supernatant was aspirated and PRP
was added. 5 of the 6 samples tested resulted in rapid gelation
when exposed to temperatures of 37.degree. C. In one sample no
gelation was obtained.
[0178] Rat tail tendons were harvested. All steps were carried out
using sterile technique and solutions. Salt solubilized tendon
fascicles were centrifuged, the supernatant removed and replaced
with acetic acid and enzyme to solubilized the collagen further.
The resultant collagen slurry had a pH=3.0. Aliquots were made and
neutralized with NaHCO.sub.3 to pH=7.0. 500 microliters of PRP were
added to aliquots of 1 cc collagen slurry and vortexed (all on ice
prior to mixing). The mixture was placed in a water bath at
37.degree. C. Each of the samples formed a soft set gel within 5
minutes (partial gelation).
[0179] Additional aliquots with collagen slurry, and buffer
containing F10 culture media and antibiotics were tested. 150
microliters serum was added after neutralization with NaHCO.sub.3
and NaOH. The collagen gel set in 5 minutes at 37.degree. C. on
initial four tests. The fifth sample did not gel. Repeat testing
showed most slurries made with this protocol would set, but not all
(approx 60%).
[0180] The collagen solution was then prepared using different
buffer components. With the addition of HEPES buffer to help
maintain pH between 7.0 and 8.0 and adjusting other components of
buffer (antibiotics, F10 and sterile water) to bring osmolarity to
within 280 to 350 mOsm/kg, we were able to get reliable gelation
within 10 minutes for over 90% of aliquots tested in trials.
C. Cell Viability in Collagen/Buffer Mixture:
[0181] Drop testing: One million pig primary outgrowth ACL cells
were trypsinized and added to a neutralized collagen slurry to
produce a density of cells of 1.times.105 cells/cc. FBS was added
to produce a ratio of 3:1 and 4:1 collagen:FBS. Drops were placed
onto individual wells of a tissue culture plate. The next day, some
cell spreading was seen from 2 of 3 gels. At day 4, cells were
growing in 1 of 3 gels.
D. Evaluation of Whether the Proliferation of Pig ACL Cells Seeded
in Collagen Gels is Affected by the Final Collagen Concentration of
the Gels.
[0182] The following methods were performed:
[0183] Targeted cells were seeded at 5.times.10.sup.5 cells/ml. The
final collagen concentrations in the gels was 3.4, 1.7 and 0.8
mg/ml. These final concentrations were calculated based on slurry
#50's collagen concentration (10.5 mg/ml), the fact that the slurry
will be used at full, 1/2 and 1/4 strengths and how much the slurry
is diluted when making the gels. Time points were taken at 1, 5 and
10 days, resulting in a total of 9 data points (time/collagen
concentration). Each data point was run in quadruplicate with 2
cell-free controls at each data point.
[0184] The total amount of cell-seeded gels was 36 ml. Enough gel
for each data point was made using 1.5 ml slurry and 1.5 ml PRP.
The PRP needed for data points was 13.5 ml (1.5.times.9) and PRP
needed for cell-free gels was 2.2 ml. Total PRP needed was 15.7
ml.
[0185] The number of cells needed was
13.5.times.5.times.10.sup.5.times.3 (since PRP is diluted .about.3
times when making the gel) which results in 202.5.times.10.sup.5
(or 20.3.times.10.sup.6) cells.
Day 1--Make Cell-Seeded Gels:
[0186] The cells were trypsinized, centrifuged, resuspended in
complete media and counted to make sure there are enough cells for
the assay. The cell solution was centrifuged and the cells were
resuspended in PRP such that the cell concentration in (at least)
13.5 ml PRP was 15.times.10.sup.5 cells/ml (PRP was diluted
.about.3 times when making gels).
[0187] The gels were made using PRP with or without cells depending
on the case. 1 ml of gel was aliquoted on 42 wells of 12-well
plates and placed in an incubator. After 1 hour the complete media
was added on top of gels and the cells were equilibrated in gels
for .about.24 hours.
Days 2, 6 and 11--MTT Assay at Days 1, 5 and 10:
[0188] The plates were removed from the incubator and the media was
aspirated. With a sterile spatula all gels were transferred into
new 12-well plates. 1 ml of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye
was added to each well of time point 1-day. The gels were incubated
for 3 hours. The MTT solution was aspirated off and discarded. 1 ml
of sterile 1.times.DPBS was added to each well. Plates were placed
on a rotating platform and allowed gentle rinsing for 30 minutes.
200 .mu.l aliquot was removed from each well and the absorbance was
read for persistence of color removal. The remainder of DPBS was
removed and the DPBS steps were repeated twice. If absorbance
levels from DPBS after 3.sup.rd rinse are still above 0.1 a
4.sup.th rinse may be performed. Using a sterile spatula, the gels
were detached from sides/bottom of wells and transferred to new 3
ml tubes. 1 ml of detergent (20% SDS/Formamide) was added. The
mixture was incubated for 5 hours. The tubes were removed from the
incubator, briefly vortexed on high for .about.5 seconds and
samples were spun down at 1500 rpm for 5 minutes. 200 .mu.l of
supernatant was transferred to 96-well plate and the absorbance was
read.
Results
[0189] Cell proliferation in vitro was seen in final collagen
concentrations of 0.8 to 3.4 mg/ml (starting collagen
concentrations were 10.5, 5.3 and 2.6 mg/ml). Cell number increased
with time in culture for all groups between 1 and 10 days. The
results are shown in FIG. 10.
E. Viscosity of Collagen/Buffer Mixture:
[0190] At cold temperature viscosity was 70 cp and after heating to
37.degree. C., the viscosity was assessed at 3200 cp at shear rate
of 1/sec. At slower shear rate (0.3/sec), viscosity at 37.degree.
C. was determined to be 6,000-11,000 cp.
F. Ability of Collagen/Buffer Solution to Stimulate Platelets to
Release Growth Factors:
[0191] The collagen/buffer mixture (described in (B) above) was
added to a platelet mixture and the subsequent release of growth
factors from the platelets measured using an ELISA assay. The
thrombin-free preparations were compared to a preparation using
bovine thrombin to stimulate platelet release of growth factors.
Similar growth factor release was seen using the collagen slurry as
a platelet activator as with the bovine thrombin as an activator.
The results are described in Example 2.
G. Sterility
[0192] Multiple in vitro assays have been performed using the
Collagen/Buffer solution described above in (B) with no evidence of
bacterial or fungal contamination or infection in any of the
samples tested out to 10 days in vitro and up to 9 weeks in
vivo.
H. In Vivo Testing of Collagen/Buffer Solution
[0193] Model: Pig total ACL transection was treated with collagen
slurry/buffer mixed with animal's own PRP in the operating room.
The mixture was injected into the gap between the cut ligament
ends. Adding the Collagen/Buffer solution to the autologous PRP
resulted in more than doubling of the yield strength of the healing
ligament after four weeks in vivo compared with the use of sutures
alone for the repair. FIG. 9 is a graph depicting results of the in
vivo pig total ACL transection treated with collagen
slurry/buffer.
Example 2
[0194] In this Example, type I collagen was used to stimulate
activation of the fibrin clotting mechanism and platelet
activation. Initially, we tested collagen to determine if it would
result in more sustained release of two growth factors used as
markers of platelet function, namely TGF-.beta.1 and
PDGF-.alpha..beta., when compared with the use of exogenous
thrombin. Secondly, we tested whether the amount of this release
would be dependent on the platelet concentration in the PRP. The
release profiles of these growth factors from three types of
collagen-PRP gels were compared with the release profile from a PRP
clot created with exogenous bovine thrombin over 10 days.
Additionally, we determined whether the growth factor release from
collagen-activated PRP hydrogels would cause a physiologic changes
in ACL cells in terms of 1) cellular metabolism of growth factors,
2) cellular proliferation within the gels and 3) cell-mediated gel
contraction.
Materials and Methods
Preparation of Platelet-Rich Plasma: Centrifugation Method
[0195] Three hundred milliliters of whole blood was drawn from each
of five hematologically normal volunteers meeting all criterion of
the American Association of Blood Banks (Food and Drug
Administration, Center for Biologics Evaluation and Research).
Blood was collected in a bag to contain 10% acid-citrate dextrose
at the Center for Blood Research (Boston, Mass.). Forty five ml of
whole blood from each patient was centrifuged for 6 minutes at 200
g (Beckman GS-6 Centrifuge, Fullerton, Calif.). The supernatant was
aspirated and collected as PRP. Two additional groups of PRP
samples were made using the Harvest Smart PreP2 System (Harvest
Technologies, Plymouth, Mass.) as noted below.
PRP Preparation Using the Smart PreP2 System: Platelet Concentrate
Method
[0196] PRP was also produced using the Harvest Smart PreP2 System
(Harvest Technologies, Plymouth, Mass.). PRP was produced by the
method recommended by the manufacturer. Fifty four cc of whole
blood was anticoagulated using 10% acid-citrate dextrose and
transferred to the blood chamber of the device, and 2 ml ACD was
placed in the plasma chamber of the disposable blood processor
(DP). The blood is centrifuged in a container with a floating shelf
designed to rise to just below the buffy coat/red blood cell
interface. Following the separation of plasma from the red blood
cells, the centrifuge slows, and the platelets, plasma and white
blood cells are decanted into the plasma chamber. When the plasma
decant is complete, a second centrifugation step is used to form a
pellet of platelet concentrate in the bottom of the plasma chamber.
The plasma chamber contains the platelet concentrate (a button-like
precipitate) and platelet poor plasma (supernatant). The complete
process is entirely automatic and completed in approximately 14
minutes. Approximately 2/3 of the platelet poor plasma (PPP) is
removed. The platelet concentrate (PC) is then resuspended in the
remaining PPP.
PRP Preparation Using the Smart PreP2 System: RBC-Reduced
Method
[0197] PRP in this group was prepared using the platelet
concentrate as above with an additional step to remove the majority
of erythrocytes in the PRP. To accomplish this, 30 ml of platelet
concentrate from each patient was centrifuged in the Smart PreP2
system for an additional 2 minutes. The supernatant is then
aspirated and kept as the RBC-reduced (RBC-red) PRP.
[0198] Samples of whole blood and platelet rich plasma preparations
were analyzed for complete blood count with differential to
determine initial and final platelet and white blood cell
concentrations (Table 1 and Table 2)
TABLE-US-00002 TABLE 1 Platelet counts for each PRP preparations.
Baseline Centrifuged PC RBC Reduced Patient # Plt Count (% of
baseline) (% of baseline) (% of baseline) 1 316 434 (137%) 919
(291%) 1287 (407%) 2 204 286 (140%) 860 (422%) 1000 (490%) 3 194
249 (128%) 715 (368%) 794 (409%) 4 318 573 (180%) 1215 (382%) 1385
(436%) 5 246 479 (195%) 1057 (430%) 1131 (460%) Avg 256 404 (158%)
953 (373%) 1119 (438%)
TABLE-US-00003 TABLE 2 Differential in total cells/microliter.
Patient Baseline Centrifuged PC RBC Reduced # WBC GRN WBC GRN WBC
GRN WBC GRN 1 3,500 2,000 1,400 100 7,700 700 7,600 300 2 3,900
2,600 1,000 0 7,600 1,000 5,400 200 3 6,700 4,800 500 0 13,600
5,600 4,900 600 4 5,500 3,300 1,100 100 16,000 4,300 8,000 1,300 5
5,000 3,300 500 0 11,000 3,300 6,100 700 Avg 4,920 3,200 900 40
11,180 2,980 6,400 620
Manufacture of Acid-Soluble Collagen Used in the Hydrogels
[0199] Rat tails were obtained from control breeder rats undergoing
euthanasia for other Institutional Animal Care and Use Committee
approved studies. The rat-tail tendons were sterilely harvested,
minced, and solubilized in an acidified pepsin solution to obtain
the acid soluble collagen. Collagen content within the slurry was
adjusted to greater than 5 mg/ml using 0.01N hydrochloric acid.
Before combining with the platelet component of the hydrogel, the
collagen slurry was mixed with 0.1M HEPES Buffer 1M solution
(Cellgro, Mediatech, Inc, Herndon, Va.), 10.times. Ham's F-10
medium (MP Biomedicals, LCC, Aurora, Ohio), Antibiotic-Antimycotic
solution (Cellgro, Mediatech, Inc., Herndon, Va.) and sterile
water. The collagen slurry was neutralized to a pH of 7.4 using
7.5% sodium bicarbonate (Cambrex BioScience Walkersville, Inc.,
Walkersville, Md.).
Platelet Activation Exogenous Thrombin Group
[0200] Five milliliters of calcium chloride (100 mg/ml) were added
to 5,000 IU bovine thrombin (Bovine Thrombin--JMI, Jones Pharma
Inc, Bristol, Va.) to produce a 1,000 IU/ml solution. 80 ml of the
thrombin solution was then added to 720 ml of the Platelet
Concentrate group for each patient. Duplicate samples of the
mixture were injected into 2 ml centrifuge tubes and allowed to
form a clot. Clots were weighed and placed in a 37.degree. C.
incubator for 20 minutes prior to transfer to sterile 12-well
plates. One milliliter of Dulbecco's Modified Eagle's Medium (DMEM,
Cat# 10013CV, Cellgro, Mediatech, Inc., Herndon, Va.) with 2%
antibiotics (Cellgro, Mediatech, Inc., Herndon, Va.) was added to
each clot. Samples were cultured in a 37.degree. C. humidified
incubator.
Platelet Activation: Collagen Groups
[0201] For each sample, an equal volume of PRP and collagen
hydrogel were mixed and heated to 30.degree. C. over 1 minute.
Duplicate samples of each collagen-PRP mixture were injected into
two 2 ml centrifuge tubes. This was repeated for all test groups.
Gels were weighed and placed in a 37.degree. C. incubator for 20
minutes prior to transfer to sterile 12-well plates. One mL of DMEM
with 2% antibiotics (Cellgro, Mediatech, Inc., Herndon, Va.) was
added to each gel. Samples were cultured in a 37.degree. C.
humidified incubator.
[0202] Additionally, a collagen hydrogel-only (no PRP) was also
made and the release evaluated at 12 hours, 1 day, 3 days and 5
days.
Measurement of Growth Factor Levels:
[0203] At each time point (12 hours, 1, 3, 5, 7 and 10 days) media
was aspirated from around each sample and replaced with 1 mL of
fresh media (serum-free DMEM with 2% antibiotics added). Media
samples were stored in cryovials in a -80.degree. C. freezer until
all samples were collected. Concentrations of human PDGF
.alpha..beta., TGF .beta..sub.1 and VEGF were determined using the
commercially available Quantikine calorimetric sandwich ELISA kits
(R&D Systems, Minneapolis, Minn.). Assays were performed in
duplicate on media samples as described in the instructions of the
manufacturer. Dilutions of 1:20 (12 hour samples) and 1:10 (day 1,
day 3, day 5, day 7 and day 10 samples) were used for samples in
the PDGF.alpha..beta. assay; a dilution of 1:10 was used for all
samples in the TGF .beta..sub.1 assay; and no dilution was used for
the VEGF assay. These dilutions were accounted for in analysis.
[0204] The media concentration of each growth factor was determined
using the ELISA kit after performing the dilutions described above.
The plasma total TGF .beta..sub.1 was assayed after acid activation
of the plasma by adding 20 microliters of 1N HCl to 40 microliters
of media sample. The reaction solution was mixed and incubated at
room temperature for 10 minutes before it was neutralized by with
microliters of 1.2N of NaOH/0.5 M HEPES. It was further diluted to
1:10 in calibrator diluent before it was added to the ELISA
plate.
[0205] For each growth factor, the standard curve was produced by a
2-fold serial dilution of a known concentration of growth factor
provided in the kit to make final concentrations of 0, 31.2, 62.5,
125, 250, 500, 1000 and 2000 pg/ml. The color change of the final
reaction was measured at a wavelength of 450 nm for the optical
density and the standard curve concentrations vs absorbances was
linear using a four parameter logistic fit curve. The reported
minimal detection limit of TGF-.beta.1 was 4.61 pg/ml, 9.0 pg/ml
for VEGF and 1.7 pg/ml for PDGF-.alpha..beta..
[0206] Due to the media sampling technique described above, growth
factor concentrations reported in the results section reflect the
growth factor release in the time period since the prior media
change. For 12 hours and day 1, this is a 12 hour release and for
day 3, day 5 and day 7, it is a 48 hour release.
[0207] Analysis: The cumulative TGFb release was measured after 1,
5 and 10 days of culture of gels seeded with 3.times.105 ACL cells.
The effect of the seeded cells on TGFb release was calculated by
subtracting the cumulative TGFb release from cell-free gels at each
time point from the cumulative TGFb release from the cell seeded
gels at the same time point (both values calculated per ml gel to
account for possible differences in gel sizes during gel
manufacture).
[0208] The cumulative PDGF release was measured after 1, 5 and 10
days of culture of gels seeded with 3.times.105 ACL cells. The
effect of the seeded cells on PDGF release was calculated by
subtracting the cumulative PDGF release from cell-free gels at each
time point from the cumulative PDGF release from the cell seeded
gels at the same time point (both values calculated per ml gel to
account for possible differences in gel sizes during gel
manufacture).
[0209] The cumulative VEGF release was measured after 1, 5 and 10
days of culture of gels seeded with 3.times.105 ACL cells. The
effect of the seeded cells on VEGF release was calculated by
subtracting the cumulative VEGF release from cell-free gels at each
time point from the cumulative PDGF release from the cell seeded
gels at the same time point (both values calculated per ml gel to
account for possible differences in gel sizes during gel
manufacture).
Effect on Cell Proliferation: MTT Assay
[0210] Collagen-PRP hydrogels and thrombin-PRP clots containing
3.times.10.sup.5 cells/ml were prepared as follows. Primary
outgrowth human ACL cells were cultured from explants obtained from
2 women (ages 15 and 22) undergoing ACL reconstruction. Explants
were cultured in media containing 10% FBS (HyClone Inc., Cat. #
16777-006, South Logan, Utah), 1% AB/AM (Media Tech, Inc., Cat.
#30004067, Hemdon, Va.) and media changed 2 times per week until
confluent cultures established. The cells were trypsinized and
replated once onto T-75 flasks until use. First passage ACL cells
were trypsinized, resuspended in complete media, counted and four
pellets containing 6.times.10.sup.6 cells were prepared in 15 cc
centrifuge tubes. Each pellet was resuspended in 6.5 cc of one of
four solutions: PRP prepared by centrifugation, PC PRP, RBC-reduced
PRP or normal phosphate buffered saline (EMD Chemicals, Cat. #:
B10241-34, Gibbstown, N.J.).
[0211] Cell proliferation was measured using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay; this assay measures the ability of a cell's mitochondrial
dehydrogenase enzymes to convert yellow, soluble MTT salt into
purple formazan salt. The MTT was prepared at a concentration of 1
mg/ml in serum-free DMEM from the sterile stock MTT solution (5
mg/ml PBS). After the media was removed from each well, a sterile
spatula was used to transfer each gel to a sterile 12-well plate;
this allowed only those cells proliferating in the gel to be
labeled by the MTT. 1.2 ml of MTT solution (1 mg/ml) was added to
each well. Each gel was fully immersed in the MTT solution. After
the MTT was added, the plates were incubated for 3 hours (37 C, 5%
CO2). Subsequently, the excess MTT solution was removed and 1 ml of
sterile 1.times.PBS was added to each well, placed on a vertical
agitator (Fisher Scientific Clinical Rotator, 100 rpm) and left to
rinse at room temperature for 30 minutes. Afterwards, 150
microliters of PBS was removed from each well, transferred to a
sterile 96-well plate and the absorbencies read at 562 nm. This
rinse was repeated until all PBS aliquots read absorbencies under
0.100 nm. All PBS was then removed and each gel transferred with a
sterile spatula into a sterile 3.0 ml centrifuge tube. The gels
and? formazan crystals were then dissolved by adding 1 ml of a
detergent containing 20% aqueous SDS/formamide (1:1 volume ratio)
to each tube and incubating for 5 hours in a 37 C water bath.
Finally, the tubes were centrifuged for 5 minutes at 1500 rpm, and
aliquots of the supernatant from each tube (200 microliters) were
then transferred onto a sterile 96-well plate. The absorbencies
were measured at 562 nm, and the cell concentrations
determined.
[0212] We were unable to assess the effect of the PC on cell
proliferation as the control readings for the cell free gels were
higher than the absorbance reader tolerance, likely due to the high
number of red blood cells in this PRP preparation.
[0213] MTT controls were prepared identically to the method above
differing solely in their absence of cells. MTT protocol was again
performed at 1, 5, and 10 days allowing the controls to be
determined and when applied to the MTT results from the cell seeded
hydrogels, the effect of each gels' cells isolated and
compared.
Collagen Gel Contraction Assay
[0214] Both fibroblast and PRP-mediated collagen gel contraction
was assessed. The degree of contraction of collagen gels was
determined by measuring the area of each gel over time in culture.
Every two days (day 1, day 3, day 5 and day 7), the length and
width at the gel midpoint was measured using a millimeter ruler and
recorded. Comparisons between fibroblast-seeded gels with and
without PRP and cell-free gels with and without PRP were made.
Statistical Analysis
[0215] Two-factor ANOVA for group and time was used to compare the
growth factor release of the collagen-PRP hydrogels with those of
the thrombin-PRP clots, with values of p<0.05 considered
statistically significant. Bonferroni-Dunn post hoc testing was
used to determine the significance of observed differences between
groups in a pairwise analysis.
Results
[0216] Hypothesis One: That the Use of Collagen as a Platelet
Activator would Result in a More Sustained Release of Growth
Factors from a PRP Gel
[0217] In both the bovine thrombin-activated and collagen-activated
PRP gels, the highest release of PDGF-.alpha..beta. and TGF-.beta.1
occurred in the first twelve hours (FIGS. 1 and 2). For time points
greater than 3 days (delayed release), there was no difference in
release of PDGF-.alpha..beta. or TGF-.beta.1 between the bovine
thrombin-activated and collagen-activated groups. In both groups,
the release of PDGF-.alpha..beta. at 10 days, was higher than 1.9
ng/ml and for TGF-.beta.1, the release at 10 days was higher than
15 ng/ml for both bovine-activated and collagen-activated PRP
gels.
[0218] The results are shown in FIGS. 1 and 2. FIG. 1 is a graph
depicting the release of PDGF-.alpha..beta. over time from bovine
thrombin-activated (BT) and collagen-activated (Centr, PC and RBC
Reduced) PRP hydrogels. The release of TGF-.beta.1 over time from
bovine thrombin-activated (BT) and collagen-activated (Centr, PC
and RBC Reduced) PRP hydrogels is shown in FIG. 2.
Hypothesis Two: that Platelet Number in the PRP would Affect the
Release of Growth Factors from the PRP Gels.
[0219] There was a strong positive correlation between platelet
count in the PRP preparation and TGF-.beta.1 and PDGF-.alpha..beta.
release. For TGF-b, this was strongest at the 12 hour time point
(r2=0.608) and remained positive at the 10 and 12 day time points
(r2>0.35 for both correlations). A positive correlation was also
found between platelet concentration in the gel and PDGF release,
particularly at the 12 hour time point (r2>0.35). The results
are shown in FIGS. 3 and 4.
[0220] TGF-.beta.1 release as a function of platelet concentration
in the PRP at 12 hours after platelet activation is depicted in
FIG. 3. FIG. 4 shows the PDGF-.alpha..beta. release from the PRP
gels as a function of platelet concentration in the PRP at 12 hours
after platelet activation.
[0221] There was a strong positive correlation between platelet
concentration in the gels and gel contraction at all time points
(r2>0.64 at all time points), suggesting the contraction of the
gels was platelet-mediated. Much lower correlations were found
between granulocyte counts and gel contraction (r2<0.30 for all
correlations) and was more likely due to the correspondence between
platelet and granulocyte content in the PRP (r2=0.35).
[0222] Hypothesis Three That the Growth Factor Release from
Collagen-Activated PRP Hydrogels would Cause a Physiologic Changes
in ACL Cells in Terms of 1) Cellular Metabolism of Growth Factors,
2) Cellular Proliferation within the Gels and 3) Cell-Mediated Gel
Contraction.
[0223] 1) Cellular Metabolism of Growth Factors in Collagen-PRP
Gels
[0224] Less TGF-.beta.1 and PDGF-.alpha..beta. eluted from the
cell-seeded gels than from the cell-free gels, suggesting the cells
were metabolizing the TGF-.beta.1 and PDGF-.alpha..beta.. There was
no significant difference among groups (two-factor ANOVA with
p>0.2 for group and p>0.4 for time). In contrast, more VEGF
eluted from the cell-seeded gels, suggesting the cells were
producing additional VEGF. There was no significant difference
between the groups, however VEGF release did increase over time in
culture (two factor ANOVA with p>0.15 for group and p<0.02
for time, BFD post-hoc testing p<0.012 for comparison between 1
and 10 day values). On average, more than 4 times as much VEGF was
eluted from the cell-seeded gels than from the cell-free gels.
[0225] The PDGF-.alpha..beta. elution over time from the
cell-seeded PRP hydrogels is shown in FIG. 5. The negative values
over time suggest cell-based consumption of the PDGF-.alpha..beta..
VEGF elution over time from the cell-seeded PRP hydrogels is shown
in FIG. 6. The positive trend over time suggests continuing greater
production than consumption of the VEGF by the ACL cells.
[0226] 2) Cellular Proliferation within the Gels
[0227] The incorporation of centrifuged or RBC-depleted platelet
rich plasma in the collagen hydrogel resulted in a significant
increase in cell number within the gels over 10 days of culture in
vitro (one factor ANOVA, p<0.009) with an almost two-fold
increase in cell number between saline (0.165+/-0.03 mean+/-sd) and
centrifuged (0.316+/-0.04) groups and a more than 2-fold difference
between the saline and RBC-depleted (-0.359+/-0.11) groups. No
significant difference was seen between the centrifuged and RBC-D
groups (BFD, p>0.40). The results are shown in FIG. 7.
[0228] 3) ACL Cell-Mediated Gel Contraction
[0229] The addition of ACL cells to the gels resulted in a
stabilization of gel size during the days of culture (two factor
ANOVA, p<0.006 for time and p<0.001 for group, BFD p>0.003
for all comparisons between time points except between days 0 and 1
where p<0.001). The results are shown in FIG. 8.
Effect of Growth Factor Consumption on Gel Contraction
[0230] There was a positive correlation between gel contraction and
TGF-.beta.1 consumption, that is, the more TGF-.beta.1 consumed by
the cells, the greater the cell-mediated contraction of the gels.
This was most significant in the RBC reduced group (r2=0.38) and
less so in the PC (r2=0.24) and Centrifuged (r2=0.11) groups. There
was also a positive correlation between gel contraction and PDGF
consumption, that is, the more PDGF consumed by the cells, the
greater the cell-mediated contraction of the gels. This was most
significant in the Centrifuged (r2=0.46) and PC groups (r2=0.42)
than in the RBC red group (r2=0.27). In contrast, the more VEGF
produced by the cells, the greater the cell-mediated contraction of
the gels (r2=0.38). This was only significant in the Centrifuged
group (r2=0.38) and was not seen in the PC (r2=0.11) or the RBC
reduced group (r2=0.004).
Example 3
[0231] The components of a preferred collagen solution of the
invention was tested to identify components.
Methods
[0232] Collagen from various sources (Cellagen, MP Biomedicals,
Solon, Ohio (shown as lane 4 in FIG. 11); Elastin Products Company,
Inc., Owensville, Mo. (shown as lane 5 in FIG. 11); StemCell
Technologies (shown as lane 6 in FIG. 11), Becton Dickinson,
Franklin Lakes, N.J. (shown as lane 7 in FIG. 11);
fluorescein-labeled collagen (shown as lane 8 in FIG. 11)) were
prepared into aliquot samples. Total protein content of each
collagen sample was determined using a calorimetric assay (BCA
Protein Assay Kit, Rockford, Ill.) in order to aliquot samples
containing equal protein content. The aliquots were treated with
SDS and .beta.-mercaptoethanol and placed at 100.degree. C. for
five minutes for denaturation and then loaded onto a 4-12% SDS-PAGE
gel. The gels were stained with Coomassie Blue (Bio-Rad, Hercules,
Calif.) and washed with a 7.5% acetic acid and 5% methanol
destaining solution.
Results
[0233] The results of the staining of the SDS-PAGE gel are shown in
FIG. 11. The results demonstrated that the commercially available
products contained relatively pure Type I collagen (Lanes 4-8).
Lanes 1-2 serve as negative controls. The material shown in Lane 3
of the gel in FIG. 11 was prepared according to the methods
described herein, i.e. Example 1, "manufacture of acid soluble
collagen used in the hydrogels". The collagen preparation of the
invention produced additional bands migrating at about 39 KD,
signifying additional proteins present in this preparation. Two
additional bands were seen at approximately 39 KD, consistent with
decorin and biglycan, as well as several other bands. These bands
were only observed in the collagen preparations prepared according
to the methods of the invention, and not in any of the commercially
available collagen products tested.
Example 4
[0234] A challenge for stimulation of ACL healing has been to
create an activated delivery system that provides for the release
of growth factors found during the successful wound healing process
in other soft connective tissues The addition of white blood cells
(WBCs) to collagen solutions was tested. Surprisingly it was
discovered that detectable concentrations of WBCs in the platelet
rich plasma-collagen solutions described herein had a significant
effect on the immediate release of VEGF from the hydrogels.
Methods
[0235] Methods for Preparation of Platelet-rich Plasma, PRP
preparation using the Smart PreP2 system, Platelet and RBC-Reduced
Method, Manufacture of acid-soluble collagen used in the hydrogels,
and Platelet Activation: Collagen Groups were performed as
described in Example 2.
[0236] Samples of whole blood and platelet rich plasma preparations
were analyzed for complete blood count with differential to
determine initial and final platelet and white blood cell
concentrations. The data is shown in Table 2 above.
Measurement of Growth Factor Levels:
[0237] VEGF release from each gel was measured at 12 hours. Media
was aspirated from around each sample and replaced with 1 ml of
fresh media (serum-free DMEM with 2% antibiotics). Media samples
were stored in 1.3 ml cryovials in a -80.degree. C. freezer until
all samples were collected. Concentrations of human VEGF were
determined using the commercially available Quantikine calorimetric
sandwich ELISA kits (R&D Systems, Minneapolis, Minn.). Assays
were performed in duplicate on media samples as described in the
instructions of the manufacturer. No dilution was used for the VEGF
assay.
[0238] For each growth factor, the standard curve was produced by a
2-fold serial dilution of a known concentration of growth factor
provided in the kit to make final concentrations of 0, 31.2, 62.5,
125, 250, 500, 1000 and 2000 pg/ml. The color change of the final
reaction was measured at a wavelength of 450 nm for the optical
density and the standard curve concentrations vs absorbances was
linear using a four parameter logistic fit curve. The reported
minimal detection limit of TGF-.beta.1 was 4.61 pg/ml, 9.0 pg/ml
for VEGF and 1.7 pg/ml for PDGF-.alpha..beta..
Results
[0239] The results of the study are shown in FIG. 12. Linear
regression analysis demonstrated a positive correlation between
WBC, and in particular granulocyte, count and VEGF release at the
12 hour time point, with r.sup.2=0.35. The results demonstrate that
the inclusion of WBCs in the collagen-PRP materials of the
invention can result in improved conditions for healing and tissue
repair.
Example 5
Injection Temperature Significantly Effects In Vitro and In Vivo
Performance of Collagen-PRP Hydrogels
[0240] We have demonstrated the efficacy of the use of collagen-PRP
hydrogels to stimulate healing of the anterior cruciate ligament
(ACL) after partial and complete transection in animal models.
These hydrogels are thought to serve as a substitute provisional
scaffold in the ACL wound site. Important rheologic properties of
the provisional scaffold include its gelation characteristics
(including final modulus and time to gelation). As described above,
the modulus of the provisional scaffold must be sufficient to
maintain the provisional scaffold analog in the wound site and to
allow it to deform in a similar fashion to the surrounding wound
edges. A hydrogel with a modulus that is too low is more likely to
flow out of the wound site before wound healing can be stimulated.
The time to gelation is also important, as in a surgical procedure,
a provisional scaffold substitute that can achieve gelation in five
minutes is far more practical than a hydrogel that requires 60 or
more minutes to become firm enough to allow for closure of the
operative site.
[0241] In this experiment, we tested the mechanical perturbation
during the accumulation of collagen crosslinks as well as the
temperature at which this final perturbation occurred and
demonstrated that they had a significant effect on the mechanical
properties of the provisional scaffolds in vitro, and also in turn
significantly effected the function of these materials in an in
vivo model of ACL repair.
Materials and Methods
In Vitro Study: Experimental Design
[0242] Acid soluble collagen was neutralized and combined with
platelet rich plasma to form aliquots of provisional scaffold
matrix. Each aliquot was then mixed under specific heating and
mixing parameters using an automated device which could accurately
control the heating voltage, mixing speed and mixing time while
simultaneously recording temperature within the gel. After
processing, aliquots were injected onto the plate of a small
oscillation rheometer and modulus and time to gelation
recorded.
Preparation of Platelet-Rich Plasma (PRP)
[0243] A total of one thousand two milliliters of whole blood was
drawn from two hematologically normal pigs undergoing other
Institutional Animal Care and Use Committee approved studies. Blood
was collected in a bag containing 10% by volume acid-citrate
dextrose. The blood was transferred into fifteen milliliter
centrifuge tubes, ten milliliters per tube. The tubes were
centrifuged for six minutes at 150 g's (GH 3.8 rotor, Beckman GS-6
Centrifuge, Fullerton Calif.). The supernatant was collected as
PRP, and complete blood counts (CBC's) were taken.
Manufacturing of Acid-Soluble Collagen Used in Hydrogels
[0244] The collagen used in this study was derived from rat tails
which were obtained from control breeder rats undergoing euthanasia
for other Institutional Animal Care and Use Committee approved
studies. The rat-tail tendons were sterilely harvested, minced, and
solubilized. The collagen content in the resulting slurry was found
to be >5 mg/ml. The same collagen slurry was used in all
experiments.
[0245] The collagen slurry was neutralized using HEPES Buffer
(Cellgro, Mediatech, Inc, Herndon, Va.), Ham's F-10 medium (MP
Biomedicals, LCC, Aurora, Ohio), Antibiotic-Antimycotic solution
(Cellgro, Mediatech, Inc., Herndon, Va.) and sterile water. 7.5%
sodium bicarbonate (Cambrex BioScience Walkersville, Inc.,
Walkersville, Md.) was used to neutralize the acidic slurry to a pH
of 7.4. Aliquots of provisional scaffold analogs were created by
combining equal amounts of PRP and the neutralized collagen and
kept on ice until mixing as outlined below.
Apparatus for Mixing and Heating the Gels
[0246] Mixing speed, mixing time, and heating rate were controlled
using a cradle designed and built by TNCO Inc. (Whitman, Mass.). An
auger was designed to fit inside the 6 cc syringe held in the
cradle. This allowed for mixing of the collagen hydrogel
components. This device had a motor which was coupled to the auger
to allow for control of mixing speed and time, and a heating pad
under the syringe that allowed for control of heating rate. The
cradle was connected to a notebook computer running a custom
LabView (Austin, Tex.) application which allowed for control of the
variables, and logging of feedback data.
[0247] The experiments performed tested three different mixing
speeds (50 RPM, 100 RPM, and 150 RPM), three different mixing times
(30 seconds, 60 seconds, and 120 seconds), and three different
heating rates (9 mV, 11 mV, and 13 mV). All combinations of those
parameters were tested as seen in Table 3. The final temperature of
the gels were recorded for these mixing conditions. Additional
triplicate gels having an injection temperature of 24.degree.
C.-26.degree. C., 26.degree. C.-28.degree. C., 28.degree.
C.-30.degree. C., and 30.degree. C.-32.degree. C. were also tested.
The additional gels were prepared by mixing at 100 RPM and heating
at 11 mV for the time necessary for the gel to reach the required
final temperature.
TABLE-US-00004 TABLE 3 Gel Preparation Parameters Mixing Speed
Mixing Time Heating Rate RPMs (sec) (mV) 50 60 11 100 60 11 200 60
11 50 30 11 50 120 11 50 60 9 50 60 13
Preparation of Gels
[0248] One milliliter aliquots of the acid soluble collagen were
measured into 5 ml cryotubes. An appropriate quantity of buffer was
added to the tube and vortexed for five seconds. The auger was then
placed into the syringe, and used to aspirate the neutralized
collagen. An equal amount of PRP as acid soluble collagen was then
aspirated into the same syringe. The syringe was affixed in the
cradle and mixed accordingly.
Mechanical Testing
[0249] Mechanical properties of the gels were determined using Cone
on Plate Small Amplitude Oscillatory Shear Rheometry using a TA
Instruments AR 1000 Rheometer (New Castle, Del.). The rheometer was
fitted with a 60 mm 1.degree. acrylic cone, and the base plate was
heated to 25.degree. C. A gel was prepared as described above, and
one milliliter of the collagen-PRP gel was dispensed onto the
rheometer plate. The cone was lowered so that the gel was situated
in a 38 .mu.m layer between the cone and plate, and subjected to a
1% oscillatory strain. The viscoelastic complex modulus of the gel
was recorded as the gelation progressed. Elastic modulus (G'),
inelastic modulus (G''), and phase angle were measured for all of
the gels.
In Vivo Studies: Experimental Design
[0250] Five 30 kg female Yorkshire pigs were used in the study.
Four animals had bilateral ACL transections and for each of these,
one side was treated with a suture repair augmented with a
collagen-PRP hydrogel, while on the contralateral side, the
transection was treated with suture repair without hydrogel. In the
remaining animal, unilateral surgery was performed with the
augmented repair and the contralateral side left as a contemporary
intact control. One of the animals developed a post-operative
seroma which was treated with antibiotics on the collagen-PRP side.
This knee was excluded from the study. Therefore, there were a
total of four knees in the augmented repair group and four knees in
the non-augmented group. All animals were survived to 14 weeks and
then underwent MRI evaluation and euthanasia. Knees were
immediately harvested and frozen until biomechanical testing. Load
to yield, load to failure, maximum stiffness and displacement to
failure were measured.
Surgical Procedure
[0251] Institutional Animal Care and Use Committee approvals were
obtained for this study prior to any surgical procedures. Five 30
kg female Yorkshire pigs were used in this study. The pigs were
pre-medicated with telazol 4.4-6.6 mg/kg 1M, xylazine 1.1-2.2 mg/kg
1M, and atropine 0.04 mg/kg. They were intubated and placed on
isoflurane 1-3% for anesthesia maintenance. After anesthesia had
been obtained, the pigs were weighed and placed in the supine
position on the operating room table. Both hind limbs were shaved,
prepared with chlorhexidine followed by betadyne paint and
sterilely draped. No tourniquet was used. To expose the ACL, a
four-centimeter incision was made over the medial border of the
patellar tendon. The incision was carried down sharply through the
synovium using electrocautery. The fat pad was released from its
proximal attachment and partially resected to expose the
intermeniscal ligament. The intermeniscal ligament was released to
expose the tibial insertion of the ACL. A Lachman maneuver was
performed prior to releasing the ACL to verify knee stability. Two
#1 Vicryl sutures were secured in the distal ACL stump using a
modified Kessler stitch. The ACL was transected completely at the
junction of the middle and proximal thirds using a No 12 blade.
Complete transection was verified visually and with a repeat
Lachman maneuver that became positive in all knees with no
significant endpoint detected after complete transection. All knees
were irrigated with sterile saline to remove synovial fluid before
suture anchor placement. An absorbable suture anchor (TwinFix AB
5.0 Suture Anchor with DuraBraid Suture (USP#2); Smith and Nephew,
Inc, Andover Mass.) was placed at the back of the femoral notch.
The knee was irrigated with 500 cc of sterile normal saline to
remove all synovial fluid. Once hemostasis had been achieved, a
collagen sponge was soaked in cold collagen-PRP hydrogel and
threaded onto sutures and up into the region of the proximal ACL
stump in the notch. The sutures were tied using maximum manual
tension with the knees in resting flexion (approximately
70.degree.-40.degree. short of full extension in these animals). A
second batch of collagen-PRP hydrogel was mixed by sequentially
drawing up equal aliquots of neutralized collagen solution and
autologous PRP into the mixing and heating device (FIGURE) and
mixing for 1 minute at 50 rpm and 13 mV which resulted in injection
temperatures between 28.9 and 32.4.degree. C. This mixture was then
placed over the ACL repair to fill the intercondylar notch. The
knee was left in resting extension while the identical technique of
suture anchor repair was performed with an identical collagen
sponge, but without the addition of the collagen-PRP hydrogel. The
incisions were closed in multiple layers with absorbable
sutures.
[0252] The animals were not restrained post-operatively, and were
allowed ad lib activity. Once the animals recovered from
anesthesia, they were permitted to resume normal cage activity and
nutrition ad lib. Buprenex 0.01 mg/kg IM once and a Fentanyl patch
1-4 ug/kg transdermal were provided for post-operative analgesia.
All animals were weight bearing on their hind limbs by 24 hours
after surgery. After fourteen weeks in vivo, the animals were again
anesthetized and underwent in vivo MR imaging using the protocol
detailed below.
[0253] After the magnetic resonance images had been obtained, the
animals were euthanized using Fatal Plus at 1 cc/10 lbs. No animals
had any surgical complications of difficulty walking normally,
redness, warmth and swelling of the knee, fever or other signs of
infection that would have necessitated early euthanasia.
[0254] The six intact control knees were obtained from age-gender-
and weight-matched animals after euthanasia following surgical
procedures to the chest. The hind limbs were frozen at -20.degree.
C. for three months and thawed overnight at 4.degree. C. before
mechanical testing. All other testing conditions for these knees
were identical to those in the experimental groups.
Magnetic Resonance Imaging
[0255] In vivo magnetic resonance imaging was performed at 1.5
Tesla (GE Medical Systems, Milwaukee, Wis.) with an eight-channel
phased array coil at the specified time points. Scanning was
performed with the knees placed maximum extension (between 30 and
45 degrees of flexion). Conventional MR included multiplane T1, FSE
PD and T2 weighted images. Field of view (FOV): 16-18 cm, matrix:
256.times.256, (repetition time/echo time) TR/TE: 400/16, 2500/32,
3000/66 msec, echo train length (ETL): 8, bandwidth (BW): 15 kHz,
slice thickness: 3, interslice gap: 1 mm). Perfusion was evaluated
by using spoiled gradient echo sequence (TR/TE=200/2 ms, flip
angle=60, 3 mm slice thickness, and 0.625 mm in plane resolution)
with an intravenous contrast agent (Magnevist; Berlex, Wayne, N.J.)
0.2 ml/kg injected 10 s after the start of scan. Five images were
obtained per slice, 78 s apart. Post contrast T1-weighted images
were obtained (FOV: 16 cm, matrix: 256.times.256, TR/TE: 400/9
msec, slice thickness: 3 mm, interslice gap: 1 mm) in the coronal
and sagittal planes.
Biomechanical Testing
[0256] The bone-ligament-bone ACL complex from both knees for each
pig was tested in uniaxial tension. In brief, testing was performed
with the knee flexed at 30 degrees of flexion and at room
temperature. Immediately after preconditioning, each specimen was
tested to failure in uniaxial tension at 20 mm/min. Close-range
digital images were acquired at 3 Hz using a high resolution
digital camera with a macro lens (PixeLINK PLA662 Megapixel
Firewire camera, PixeLINK, Ottawa ON, Canada) to determine failure
mode. The yield load, displacement at yield, tangent modulus
(maximum slope of force-displacement curve), maximum load at
failure, displacement at failure and total work to failure (area
under force-displacement curve) were determined from the
force-displacement curve measured for each bone-ligament-bone ACL
complex. The yield load represented the point along the normalized
force-displacement curve where the mechanical behavior of the ACL
complex departed from "linear" behavior and for the purposes of
this analysis was defined as the point where the tangent modulus
declines by at least 2% from its maximum value. The displacement at
yield was the displacement recorded at this same point. The maximum
load is the maximal normalized load sustained by the ACL complex
prior to failure and the displacement at failure the displacement
recorded at the maximum load. The energy to failure was derived by
integrating the total area under the force-displacement curve.
Statistical Analysis
[0257] Mechanical testing measurements were compared at 4 weeks in
vivo between intact ACL and ACLs treated by suture anchor repair
alone and to those treated by suture anchor repair plus collagen
sponge using F-tests from multivariate analysis of variance
(MANOVA) with 95% confidence intervals (CI). A F-test exceeding the
critical value of 3.84 would be regarded as evidence for
statistical significance. Each of the six variables (load at yield,
maximum load, displacement at yield, displacement at failure,
tangent modulus, and energy to failure) followed a normal
(Gaussian-shaped) distribution and therefore data are presented in
terms of the mean and standard deviation (SD). Paired t-tests were
used to evaluate differences in ACLs treated with suture anchor
repair alone compared to the bilateral side receiving suture
anchors with PRP. Statistical analysis was performed using SPSS
version 14.0 (SPSS Inc., Chicago, Ill.). All values of p<0.05
were considered statistically significant.
Results
Hematology
[0258] A one milliliter sample of whole blood from each pig, and a
one milliliter sample of each of the PRPs were taken to the CBR
Institute for Biomedical Research (Boston, Mass.) and a complete
blood count was performed. The results are summarized in Table
4.
TABLE-US-00005 TABLE 4 Summary of Blood Results Whole Blood
Platelet Rich Plasma PRP #1 Platelet Count (Platelets/.mu.l) 3.71
.times. 10.sup.5 6.52 .times. 10.sup.5 Red Blood Cell Count
(RBC/.mu.l) 4.46 .times. 10.sup.6 3 .times. 10.sup.4 White Blood
Cell Count (WBC/.mu.l) 6.6 .times. 10.sup.3 2.2 .times. 10.sup.3
Hematocrit (%) 28.4 0.2 PRP #2 Platelet Count (Platelets/.mu.l)
3.30 .times. 10.sup.5 7.76 .times. 10.sup.5 Red Blood Cell Count
(RBC/.mu.l) 5.78 .times. 10.sup.6 4 .times. 10.sup.4 White Blood
Cell Count (WBC/.mu.l) 9.6 .times. 10.sup.3 2.7 .times. 10.sup.3
Hematocrit (%) 39.3 0.3
Mechanical Testing
[0259] 1.) The Effect of Mixing Time
[0260] There was no statistical difference between mixing the
components of the gels for 30 seconds or 60 seconds as measured by
the maximum elastic modulus (112.+-.34 Pa vs 142.+-.25 Pa). The
results are shown in FIG. 13A. However, mixing for 120 seconds
significantly decreased the maximum elastic modulus to only 5.+-.2
Pa (single variable ANOVA p<0.01). Similar findings were noted
for the inelastic modulus (shown in FIG. 13B). The inelastic
modulus for the samples mixed for 30 seconds was 27.+-.8 Pa, and
the inelastic modulus for the samples mixed for 60 seconds was
35.+-.7 Pa. This did not represent a statistically significant
difference. However, the inelastic modulus was significantly lower
for the 120 second mixing samples (2.+-.0.4 Pa) when compared to
the 30 second and 60 second mixing time samples (single variable
ANOVA p<0.001).
[0261] There was no statistically significant difference in the
rate of gelation as measured by time to 45.degree., the time to
G'max, and the time to G''max for the samples mixed for 30 or 60
seconds. For the samples mixed for 30 seconds, the time to
45.degree. was 3.1.+-.0.0 mins, the time to G'max was 16.+-.2.6
mins, and the time to G''max was 16.+-.2.8 mins. For the samples
mixed for 60 seconds, the time to 45.degree. was 2.7.+-.0.4 mins,
the time G'max was 14.2.+-.4.2 mins, and the time to G''max was
14.2.+-.4.3 mins. However, the samples mixed for 120 seconds had a
time to 45.degree. of 0.3.+-.0.03 mins, which represents a
statistically significant decrease (single variable ANOVA
p<0.001) when compared to both the 30 second and 60 second
mixing samples. Time to G'max (9.5.+-.5.2 mins) and G''max
(8.+-.6.8 mins) were not statistically significant when comparing
the 30 second and 60 seconds samples to the 120 second samples.
[0262] 2.) The Effect of Mixing Speed
[0263] Examining the affect that mixing speed had on the
rheological properties of the collagen-PRP hydrogels, there was no
statistically significant difference between the three mixing
speeds for both the elastic (FIG. 14A) and the inelastic modulus
(FIG. 14B) (single variable ANOVA). Mixing at 50 RPMs resulted in
an elastic modulus of 140.+-.42 Pa, and an inelastic modulus of
35.+-.11 Pa. Mixing at 100 RPMs resulted in an elastic modulus of
112.+-.41 Pa, and an inelastic modulus of 27.+-.10 Pa. Mixing at
200 RPMs resulted in an elastic modulus of 112.+-.30 Pa, and an
inelastic modulus of 27.+-.7 Pa.
[0264] Furthermore, the various mixing speeds did not have a
statistical affect on the speed at which gelation occurred as
measured by time to 45.degree., time to G'max, and time to G''max.
For the gels mixed at 50 RPMs, the time to 45.degree. was
2.3.+-.0.0 mins, the time to G'max 16.+-.2.4 mins, and the time to
G''max was 16.+-.2.5 mins. For the gels mixed at 100 RPMs, the time
to 45.degree. was 2.8.+-.0.5 mins, the time to G'max 16.+-.1.6
mins, and the time to G''max was 16.+-.1.7 mins. For the gels mixed
at 200 RPMs, the time to 45.degree. was 2.7.+-.0.6 mins, the time
to G'max 17.+-.4.1 mins, and the time to G''max was 16.7.+-.3.6
mins.
[0265] 3.) The Effect of Heating Rate
[0266] Increasing the heating rate of the collagen-PRP hydrogels
did not have a statistical effect on the viscoelastic modulus of
the gels. The gels heated at 9 mV recorded an elastic modulus of
106.+-.20 Pa (FIG. 15A), and an inelastic modulus of 25.+-.4 Pa
(FIG. 15B). The gels heated at 11 mV had an elastic modulus of
93.+-.13 Pa, and an inelastic modulus of 22.+-.3.1 Pa. The gels
heated at 13 mV had an elastic modulus of 89.+-.56 Pa, and an
inelastic modulus of 22.+-.16 Pa. None of these represent
statistically significant differences.
[0267] For the gels heated at 9 mV, the time to 45.degree. was
3.6.+-.0.4 mins, the time to G'max 17.4.+-.1.0 mins, and the time
to G''max was 16.4.+-.2.0 mins. The gels heated at 11 mV had a time
to 45.degree. of 2.4.+-.0.6 mins, a time to G'max of 16.+-.0.5
mins, and a time to G''max of 15.+-.0.8 mins. Finally, the gels
heated at 13 mV had a the time to 45.degree. of 2.0.+-.0.5 mins, a
time to G'max of 17.+-.1.2 mins, and a time to G''max of 16.+-.1.5
mins. Comparing these values, the only statistically significant
comparison was between the 9 mV and the 13 mV heating rates for
time to 45.degree. (single variable ANOVA p<0.01).
[0268] 4.) The Effect of Injection Temperature
[0269] An increase in injection temperature resulted in a decrease
in the mechanical properties of the Collagen-PRP hydrogels. The
gels injected onto the rheometer plate between 24.degree. C. and
26.degree. C. had an elastic modulus of 156.+-.26 Pa (FIG. 16A),
and an inelastic modulus of 39.4.+-.7.2 Pa (FIG. 16B). The gels
injected onto the rheometer plate between 26.degree. C. and
28.degree. C. had an elastic modulus of 128.7.+-.15.7 Pa, and an
inelastic modulus of 32.3.+-.2.7 Pa. The gels injected onto the
rheometer plate between 28.degree. C. and 30.degree. C. had an
elastic modulus of 90.3.+-.11.4 Pa, and an inelastic modulus of
22.3.+-.3.6 Pa. Finally, the gels injected onto the rheometer plate
between 30.degree. C. and 32.degree. C. had an elastic modulus of
54.6.+-.30.4 Pa, and an inelastic modulus of 14.2.+-.6.5 Pa. For
the elastic modulus, there was a statistically significant
difference between the gels injected between 24.degree. C. and
26.degree. C., and all other groups (single variable ANOVA
p<0.01), and a statistically significant difference between the
gels injected between 26.degree. C. and 28.degree. C., and the gels
injected between 30.degree. C. and 32.degree. C. For the inelastic
modulus, there was a statistically significant difference between
the gels injected at 24.degree. C.-26.degree. C., and the gels
injected at 28.degree. C.-30.degree. C., and the gels injected at
30.degree. C.-32.degree. C. (single variable ANOVA p<0.005).
There was also a statistically significant difference in inelastic
modulus between the gels injected at 26.degree. C.-28.degree. C.
and the gels injected at 30.degree. C.-32.degree. C. (single
variable ANOVA p<0.005).
[0270] The rate of gelation, as measured by time to 45.degree., the
time to G'max, and the time to G''max was affected by increasing
temperature of injection. For the gels injected between 24.degree.
C. and 26.degree. C., the time to 45.degree. was found to be
2.3.+-.0.1 mins, the time to G'max was 14.6.+-.4.5 mins, and the
time to G''max was 14.5.+-.4.7 mins. For the gels injected between
26.degree. C. and 28.degree. C., the time to 45.degree. was
1.6.+-.0.3 mins, the time to G'max was 10.5.+-.3.1 mins, and the
time to G''max was 9.4.+-.2.1 mins. For the gels injected between
28.degree. C. and 30.degree. C., the time to 45.degree. was found
to be 1.5.+-.0.0 mins, the time to G'max was 10.6.+-.3.3 mins, and
the time to G''max was 9.0.+-.2.1 mins. Finally, for the gels
injected between 30.degree. C. and 32.degree. C., the time to 450
was found to be 1.0.+-.0.2 mins, the time to G'max was 8.6.+-.0.8
mins, and the time to G''max was 8.5.+-.0.9 mins. Statistically,
there were no significant differences between the groups for time
to G'max and time to G''max; however, there were statistically
significant differences between the groups for time to 45.degree..
There was a significant decrease in time to 45.degree. when
comparing the gels injected between 24.degree. C. and 26.degree. C.
to all the other injection temperature groups (single variable
ANOVA p<0.003) (FIG. 17). There was also a significant
difference for the time to 45.degree. between the 26.degree.
C.-28.degree. C. group and the 30.degree. C.-32.degree. C. group,
and between the 28.degree. C.-30.degree. C. group and the
30.degree. C.-32.degree. C. group (single variable ANOVA
p>0.005).
[0271] In vivo results: Effect of injection temperature on repair
strength at 14 weeks is shown in FIG. 18. Average strength in the
sponge alone control group was 206N. The temperature of the gel at
injection significantly affects both the in vitro mechanical
properties of the hydrogel, as well as the in vivo properties of
tissue healing induced by the hydrogel. Temperatures of less than
26.degree. C. yielded the strongest gels in vitro and temperatures
of 28.degree. C. yielded the strongest in vivo healing
ligaments.
Example 6
Platelets Enhance ACL Graft Strength and Post Operative Knee
Laxicity in a Caprine Model
[0272] ACL injuries affect over 200,000 patients each year in the
US. While ACL reconstruction is a reliable procedure for grossly
restoring stability of the knee, normal biomechanics of the knee
are not restored and a clinically relevant percentage of patients
have excessive laxity post-operatively. The early healing of the
ACL reconstruction graft with decreased structural properties could
explain in part previous work in humans showing the majority of the
increase in knee laxity post-operatively occurs in the first
several months after surgery. Thus, strategies which could improve
the early structural properties (strength and stiffness) of the ACL
graft are desirable as a potential solution to reduce the risk of
abnormal knee laxity after ACL reconstructive surgery.
[0273] We have demonstrated by histological evaluation of healing
of a biomechanically stable partial ACL injury model the growth
factor profile. In normal extraarticular healing of medial
collateral ligament (MCL) and patellar tendon (PT) the expression
of and timing was qualitatively similar to placement of a
collagen-platelet hydrogel in the partial ACL. This is in contrast
to the lack of healing and severely limited growth factor
expression in the partial ACL injury without (control)
collagen-platelet hydrogel. Specific individual growth factors
(PDGF vs TGF and EGF vs VEGF) have been applied to ACL
reconstruction models in either sheep or canines. Both growth
factors that are abundant in platelets (PDGF and TGF) demonstrated
improved load and stiffness at 12 weeks postoperatively. However
the application of a growth factor to stimulate revascularization
(VEGF) weakened the graft at 12 weeks. The earlier time points (6
weeks), optimal dosage, combination or application method to
improve early structural properties when the ACL reconstruction
graft is near the weakest is unknown. Thus, the strategy to apply
the body's own growth factors to promote the return of structural
properties of the ACL reconstruction graft requires further
research.
[0274] This study was performed to show that placement of a
platelet gel around an ACL graft at the time of surgery would
improve the early mechanical properties of the graft (maximum load
and stiffness). It was also shown that the platelet concentration
around the graft would have a direct correlation with the early
load to failure of the ACL graft.
Materials and Methods:
Animal Model
[0275] Twelve 4-year old castrated male Nubian cross goats
underwent unilateral anterior cruciate reconstruction using a
bone-patellar tendon-bone autograft. In the experimental group six
goats had the graft augmented with a collagen-platelet hydrogel,
while the six control goats had augmentation with the
collagen-hydrogel only. The surgeries were alternatively performed
between the right and left knees within each treatment group. The
animals were allowed unrestricted cage activity while ACL
reconstruction grafts were healing. At six weeks postoperatively
they were euthanized with an overdose of pentabarbitol solution
(Euthasol; 1 cc/10 lbs). At the time of euthanasia, both the
reconstructed and contralateral control knees were harvested and
stored at -20.degree. C. prior to mechanical testing.
Surgical Procedure
[0276] The animals were tranquilized preoperatively using
acepromizine (10 mg IM). Anesthesia was then induced with sodium
pentothal (5-8 mg/Kg IV) and maintained during surgery using
isofluorane.
[0277] A 15 blade was used to make an incision from top of patella
to below the tibial tubercle just medial of midline. The
prepatellar bursa was cut in line with the skin incision to expose
the paratenon. A longitudinal cut was made centrally in the
paratenon to expose the patellar tendon. The medial and lateral
borders of the patellar tendon were palpated and a 6 mm wide graft
marked with the electrocautery. The patellar block was 15.times.6
mm, leaving 10 mm of patella intact superiorly. The tibial bone
block was 10.times.6 mm. The harvested graft was shaped to fit 6 mm
diameter bone blocks and 1.5 mm drill holes were placed in the bone
block on each side. Because the length of the patellar tendon is
greater than the ACL, the tibial bone block was folded over onto
the patellar tendon and sutured in place to make an 8 mm
bone-tendon block on this side to shorten it. Two #2 Ethibond
sutures were placed in each bone block. The intracondylar notch was
exposed through the central defect in the patellar tendon by
sectioning the fat pad. The intermeniscal ligament was not cut. A
Lachman test was checked for baseline stability of the knee. A #11
blade was used to release the ACL from the back of the notch, and
the ACL was removed by releasing the ligament from its tibial
insertion. A manual Lachman was performed to verify complete
functional loss of the ACL. The tibial tunnel was drilled using the
tibial aiming guide set at 65.degree.. The pin was over-drilled
with an 8 mm drill and all soft tissue removed. A notchplasty was
performed using a curette. The knee was hyperflexed and a 6 mm
offset femoral drill guide (Arthrex Inc, Naples Fla.) was placed
into the back of the notch at the 10:30 position. The passing pin
was drilled through the femur and then over-drilled with a 7 mm
drill to 20 to 25 mm. Integrity of the back wall of the femoral
tunnel was verified in all cases. The graft was placed into the
femoral tunnel first using the Ethibond sutures, and then secured
in the femur using a 5.times.20 mm interference screw (Arthex,
Inc.). The graft was then pulled retrograde into the tibial tunnel.
With the knee at 60 degrees of flexion, the graft was firmly
tensioned and secured in the tibial tunnel using a 6.times.20 mm
interference screw (Arthex, Inc.). Tibial fixation was augmented
with sutures to the periosteum if the tibial fixation was not
deemed stable enough.
[0278] For the experimental group the graft was augmented with a
collagen sponge placed between the ACL and LFC using a freer
elevator, with part of the sponge lying anteriorly to the graft.
Two cubic centimeters of a collagen-platelet hydrogel (n=6) was
placed over the sponge. The control group was identical except no
platelets were added to the collagen-hydrogel. After ten minutes,
the knee was closed in layers. The animals were kept under
anesthesia for 1 hour after gel placement to maintain the knees in
the resting position and allow complete gelation.
[0279] Post-operative analgesia was control using Buprenorphine
(0.01 mg/Kg 1M, twice daily) and Ketoprofen (1 mg/Kg 1M, once
daily) for five days. Ampicillin (10 mg/Kg SC, twice daily) was
administered for 10 days to reduce the risk of infection.
Collagen Gel and Collagen-Platelet Gel Manufacture
[0280] Rat tail collagen was acid-solubilized as described herein.
For the collagen group, the collagen was neutralized to a pH of 7.4
and added to the surgical site just after neutralization. To add
platelets to the gel, initially the production of platelet-rich
plasma was attempted; however, due to the similarity in size and
weight of the caprine platelet and red blood cell, centrifugation
protocols using 150 to 250 g between 20 and 30 minutes all resulted
in effective decrease of red blood cells in the PRP, but platelet
counts in the PRP fraction were less than 100% that of the whole
blood with all protocols. Using the most effective protocol
determined ex vivo, 250 g for 30 minutes, the platelet yield in the
caprine PRP averaged 102%+/-68% (mean+/-standard deviation) for the
12 goats in this study when the measured MPV was used to calculate
platelet number. In addition, there were large variations in
enrichment seen within the group. Therefore, we elected to use
whole caprine autologous blood for the collagen-platelet group.
This resulted in the peripheral blood platelet concentration
determining the concentration of platelets delivered in the
collagen-platelet hydrogel. In addition, we measured the platelet
concentration in the peripheral blood for all animals, both
experimental and control groups. Fifty-four cc of blood was drawn
from each animal into a syringe containing 6 cc of
acid-citrate-dextrose as an anticoagulant. At the time of gel
placement, the collagen was neutralized and mixed with the blood in
a 4:1 collagen:blood ratio and the collagen-platelet gel added to
the graft.
Mechanical Testing
[0281] After all specimens were collected, the knees were thawed
and prepared for laxity and failure testing. The soft tissues
surrounding the tibia and femur were dissected free leaving the
joint capsule intact. The distal tibia and proximal femur were then
potted in PVC pipes using a potting material (SmoothCast 200;
Smooth-On, Easton Pa.) so that they could be mounted for mechanical
testing.
[0282] The anteroposterior load-displacement responses of the
intact joints were measured using a custom designed fixture with
the knee locked at 30.degree. and 60.degree. of flexion (FIG. 19)
(Fleming et al JOR 19: 841, 2001). Anterior and posterior directed
shear loads of .+-.60 Newtons were applied to the femur with
respect to the tibia using a MTS 810 Materials Testing System (MTS,
Prairie Eden, Minn.) while the AP displacement was measured. Axial
rotation of the tibia was locked in the neutral position, and all
other motions were left unconstrained.
[0283] After completing the AP laxity tests, the tibia and femur
were positioned on the so that the mechanical axis of the ACL was
collinear with the load axis of the material test system (Woo et
al; AJSM 19: 217, 1991; Tohyama et al; AJSM 24: 608, 1996). The
knee flexion angle was set at 30.degree.. The tibia was mounted to
the base of the MTS via a sliding X-Y platform. The femur was
unconstrained to rotation. This enabled the specimen to seek its
own position so that the load was distributed over the cross
section of the healing graft when the tensile load was applied.
[0284] The joint capsule, menisci, collateral ligaments and the PCL
were dissected from the joint leaving the ACL graft and scar mass
intact. The femur-graft-tibia complexes were then loaded in tension
to failure at 20 mm/min while the failure load-displacement data
were recorded. Identical protocols were performed on the
contralateral ACL-intact knees. From the MTS load-displacement
tracing, the failure load, failure displacement, and the linear
stiffness were determined.
Exclusion
[0285] The first animal operated on was assigned to the
collagen-platelet group. There were technical difficulties with the
graft harvest in this animal and at the conclusion of surgery, it
was decided to exclude this animal from the analysis. In addition,
one of the animals in the collagen alone group had a graft with a
failure strength more than three times higher than any other animal
in either group, thus was excluded from the remaining analysis due
to the 3 sigma rule. Therefore, neither animal is represented in
any group statistic nor are they graphically depicted in any
figure.
Statistical Analyses
[0286] Comparisons of AP laxity values, failure strength, and
stiffness values between the collagen (carrier only) and platelet
collagen groups were made. The differences for each of these
parameters between the treated knee and the contralateral control
knee were calculated. Unpaired t-tests were performed to determine
if the differences were significant (p<0.05). Correlation
analyses were performed to determine the association between
systemic platelet count and the strength of the graft after 6 weeks
of healing.
Results
Surgical Outcome
[0287] Eleven out of the 12 animals recovered well from surgery.
However, one goat from the collagen only group died within one day
of surgery. Autopsy revealed extensive atherosclerosis and the
cause of death was thought to be cardiac-related. At the time of
euthanasia, all animals appeared to be walking normally.
Gross Appearance
[0288] Observers were blinded to group when harvesting and grading
the specimens. There was no difference between the collagen and
collagen-platelet groups on gross appearance in terms of rate of
reformation of the ligamentum mucosum, rate of scar or adhesion
formation from the notch scar mass to the harvest defect of the
patellar tendon or amount of joint adhesions observed. There was no
difference between the groups in whether the scar mass infiltrated
only the most cranial section of the graft or whether it bridged
from femur to tibia--both findings were seen in ligaments of both
groups.
Biomechanics
[0289] At a knee flexion angle of 60 degrees of flexion, the AP
laxity of the knees was 34% lower in the collagen-platelet group
than in the collagen group, a difference which was statistically
significant (17.2+/-3.3 mm vs 23.1+/-4.0 mm; mean+/-SD; p<0.05).
At 30 degrees of flexion, there was also a 40% decrease in AP
laxity of the knees in the collagen-platelet group; however, the
difference approached, but did not reach, statistical significance,
due in part due to the large standard deviations seen within each
group (collagen-platelet group 14.3+/-4.0 mm vs collagen group
20+/-4.5 mm; p<0.09).
[0290] The collagen-platelet group strength was 30% higher than the
collagen group (139+/-41N vs 108+/-47N; both mean+/-SD), a
difference that was not statistically significant (p>0.30). The
values in both groups were approximately 10% of the intact ACL
strength. Femoral tunnel size, collagen sponge size and collagen
gel amount were not found to be significant predictors of failure
strength.
[0291] There was no significant difference between the groups in
terms of failure displacement. The collagen group failed at
9.3+/-5.1 mm (mean+/-SD) and the collagen-platelet group failed at
8.4+/-4.4 mm (p>0.80). Interestingly, both values were far lower
than the failure displacement in the contralateral intact
ligaments, which averaged 20.8+/-1.8 mm. There was also no
significant difference in linear stiffness between groups, with the
collagen group having a stiffness of 22+/-15 N/mm and the
collagen-platelet group having a stiffness of 26+/-14 N/mm
(mean+/-SD; p>0.60). Both groups were less than one third of the
intact ligament average stiffness (90+/-34 N/mm; mean+/-SD).
[0292] Higher systemic platelet counts correlated significantly
with both higher failure load (FIG. 20: R 2>0.67) and higher
ligament linear stiffness (FIG. 21: R 2>0.80) using a linear
regression model. There was no significant correlation between
platelet concentration and failure displacement (R 2=0.40) or AP
laxity at 60 degrees (R 2=0.44) with the number of animals tested.
The data is depicted graphically in FIGS. 22 and 23. FIG. 22 is a
graph depicting strength of the joint as a function of platelet
count. FIGS. 23A and 23B are bivariate scattergrams with regression
95% confidence bands. FIG. 23A depicts fail load as a function of
platelet count. FIG. 23B depicts stiffness as a function of
platelet count.
[0293] Thus, AP laxity measured at 30 degrees of knee flexion was
significantly improved in the experimental platelet group when
compared to that treated with the carrier alone (154%+/-44% vs
355%+/-55%; p=0.03). There was no significant difference between
the maximum load of the ACL graft in the two groups; however, the
maximum load of the graft correlated directly with the systemic
platelet count in both the experimental (R2=0.95) and control
(R2=0.85) groups. The addition of platelet to the collagen-hydrogel
improved AP laxity when compared to previous reports of ACL
reconstructed knees at six weeks.
[0294] The addition of blood platelets to the collagen hydrogel
(experimental group) resulted in clinically significant reduction
in knee laxity at six weeks after autograft patellar tendon ACL
reconstruction. In addition, the systemic platelet count of the
animals correlated directly with the maximum load for both the
experimental group (collagen-platelet hydrogel) and the control
group (collagen hydrogel). Both of these findings highlight the
role blood platelets play in ACL reconstruction graft healing with
presumably clinically relevant reduction in early undesired
postoperative AP laxity.
[0295] The improvement in AP laxity was evident when the knees were
tested in 30 degrees of flexion (full extension in the caprine
knee). At this position, the knees treated with collagen-platelet
hydrogel had only 54% more AP laxity than the contralateral knees
with intact ACLs, while the knees treated with collagen hydrogel
alone were over 200% more lax than the intact knees. At 60 degrees
of flexion, the difference between groups was smaller and
insignificant. This finding suggests a potential role for reducing
early undesired clinical laxity. Whether the platelets' effect was
on the graft healing or capsular structures is unknown. Further
optimization of platelet concentration as well as timing of
administration is needed. The large increases in AP laxity seen in
the collagen hydrogel alone group are consistent with those of
previous studies of ACL reconstruction with autogenous patellar
tendon grafts using the goat model (Abramowitch JOR 21:707, 2003;
Cummings JOR 20:1003, 2002; Papageorgiou AJSM 29:620, 2001; Jackson
AJSM 21:176, 1993). Papageorgiou et al reported 238% and 285%
increases in AP laxity with the knee at 30 degrees and 60 degrees
of flexion, respectively, after 6 weeks of healing. The improvement
seen in the collagen-platelet group is also a marked improvement
from results published previously on ACLR in sheep where the AP
laxity of the knee increased from 2.0+/-0.7 mm in the intact knee,
to 8.3+/-2.3 mm at six weeks in a model using femoral and tibial
interference screw fixation as we did in this study (HUNT et al
Knee Surg Sports Traumatol Arthrosc. 2006 December;
14(12):1245-51).
[0296] The six week time point is well recognized as a nadir of
strength in ACL reconstruction for animal models. The strength
values in both groups were approximately 10% of the intact ACL,
which is slightly higher than previous reports of 3% in the sheep
model (Hunt et al Knee Surg Sports Traumatol Arthrosc. 2006
December; 14(12):1245-51), and typical for the goat model after
6-weeks of healing (Papgeorgiou AJSM 29:620, 2001; Abramowitch
21:708, 2003). By applying platelets in a stabilized
collagen-hydrogel we have simulated the "natural" environment found
with platelets being deposited in a fibrin clot for extraarticular
healing. Further, platelets contain a multitude of growth factors
in addition to TGF-.beta. and PDGF in the appropriate concentration
for extraarticular healing. Additionally, the cost of recombinant
TGF or PDGF or EGF far exceeds the cost to apply autograft
platelets from blood.
[0297] Limitations of the study include the inability to control
rehabilitation in the animals and the inability in the caprine
model to provide two- to four-fold increased platelet concentrates
in the collagen hydrogel. Ruminants must be upright standing in the
very early post-operative period, thus it is difficult in this
model to protect the ACL graft from weight bearing loads. Bandaging
and immobilization are not practical or effective in this animal
model. The inability to concentrate the blood platelets limits the
ability to discover whether increasing platelet count above that of
whole blood would continue to enhance ACL graft healing and reduce
postoperative laxity further. However, regardless of the
limitations of the model, the data clearly demonstrates that the
addition of platelets enhances graft healing and improves AP
stability of the knee after ACL reconstruction at an early time
point in the caprine model.
Example 7
Suture Techniques that Restore Normal AP Laxity of the Knee after
ACL Transection
[0298] The data described herein demonstrate suture techniques that
go from femur to tibia can restore the normal AP laxity of the knee
at time zero, particularly if they are tied in a small amount of
flexion and the tibial attachment point is within the normal ACL
footprint. As shown below, repair to the tibial stump of the ACL
(Marshall technique) resulted in knees with over 5 mm greater AP
laxity than knees with an intact ACL. Suture repair to bone using
fixation points within the normal ACL footprint resulted in knee
laxity within 0.5 mm of the knees with an intact ACL when the
sutures were tied with the knee flexed at 60 degrees. Laxity
increases of 1 to 3 mm were seen if the sutures were tied with the
knee in 30 degrees of flexion or in more posterior tunnels.
[0299] Primary repair of the ACL was pioneered by John Marshall in
the 1960's (Marshall J L, Warren R F, Wickiewicz T L, Clin Orthop.
1979; 143:97-106, Marshall J L, Warren R F, Wickiewicz T L. Am J
Sports Med. 1982; 10: 103-107). Favor for this technique was lost
due to the high re-rupture rate (Feagin J A, Jr., Curl W W. Am J
Sports Med. 1976; 4(3):95-100) and the limited improvement over
nonsurgical treatment seen in patients undergoing primary repair
(Engebretsen L, Benum P, Fasting 0, Molster A, Strand T. American
Journal of Sports Medicine. 1990; 18(6):585-590, Grontvedt T,
Engebretsen L, Benum P, Fasting O, Molster A, Strand T. Journal of
Bone & Joint Surgery--American Volume. 1996; 78(2):159-168).
However, recent discoveries by the inventor have suggested that
amplifying the repair response of the torn ACL using a
bioengineered scaffold to deliver growth factors into the wound
site may result in functional healing of the severed ACL (Murray M
M, Spindler K P, Devin C, et al. J Orthop Res. April 2006;
24(4):820-830, Murray M M, Spindler K P, Ballard P, Welch T P,
Zurakowski D, Nanney L B. J Orthop Res. Apr. 5 2007).
[0300] Since Marshall's work outlining his technique of primary
repair, there has not been much interest or published work on
comparison of suture techniques for primary repair of the ACL. This
is in stark contrast to the numbers of papers published each year
on ACL reconstruction technique, including papers on fixation
types, tunnel placement and number of bundles to reconstruct. With
the recent renewed interest in primary repair, additional work is
needed to define the AP laxity of the knee after suture repair of
the ACL using various techniques. Defining these surgical variables
will allow for more accurate testing of new tissue engineered
constructs in animal models, and also begin to define the most
appropriate techniques for eventual human use.
[0301] In this study two hypotheses were tested. A first hypothesis
was that suture repair could restore the normal AP laxity of the
knee at time zero, and a second hypothesis was that the angle of
flexion at which the sutures were tied would have a significant
effect on the resultant AP laxity of the knee.
Materials and Methods
[0302] Six hindlimbs were retrieved from 30 kg female Yorkshire
pigs at the time of euthanasia for other IACUC approved studies.
The limbs were frozen until the time of testing (approximately 3
weeks). The limbs were thawed in warm water on the morning of
testing. The knees were isolated by sectioning the femur just below
the lesser trochanter and sectioning the tibia 2 cm above the ankle
joint. The muscular attachments to tibia and femur were removed
with care taken not to violate the knee joint capsule. All
extra-capsular muscle was also removed. The femur and tibia had
drywall screws placed at intervals along the bone (four screws in
each bone) to assist with purchase in the potting material, then
the bones were sequentially potted in 2'' diameter polyvinyl
chloride piping using Smooth-On casting material. Knees were
wrapped in towels moistened with normal saline until testing.
[0303] The AP laxity testing was performed using a customized jig
mounted on an Instron testing machine (FIG. 24). The femur was
mounted in a movable fixture, which allowed for positioning the
knee in 60 degrees of flexion. The pig knee extends only to 30
degrees short of full extension, thus the 60 degree position was
thought to correspond to the 30 degree position in humans. A pilot
study demonstrated that the 30 degree position was less sensitive
to changes in AP laxity in the knee and therefore, only the 60
degree position was used in this study. Once the knee was
positioned in the fixture, a cyclic load of 30N was applied to the
femur. This resulted in an anterior femoral displacement at 30N
followed by a posterior femoral displacement relative to the tibia
to 30N. The magnitude of the displacements as load was applied was
measured for each cycle at 100 Hz and plotted using Excel to give a
load-displacement curve (FIG. 25).
[0304] Each of the six knees was tested in the intact state
(INTACT). After that testing, dissection was performed to remove
the patella and patellar tendon and expose the notch and the
testing repeated (PAT DEFICIENT). The specimen was brought back to
the dissection table and the ACL completely transected and testing
repeated (ACL DEFICIENT).
[0305] The knees were then prepared for various primary repair
techniques. First, a TwinFix 3.5 mm titanium anchor (Smith-Nephew,
etc) was placed in the posterolateral notch of the femur, at the
11:00 position for the right knees and the 1:00 position for the
left knees. This anchor had two Durabraid sutures passed through
the anchor eyelet, resulting in four strands available for repair.
These sutures were used for all tests. A four-stranded Marshall
repair technique was performed by passing two looped #1 Vicryl
sutures through the tibial stump at various depths and securing
these four ends to the four ends of the Durabraid for a
four-stranded Marshall repair. The sutures were first tied with the
knee first in 30 degrees of flexion (MARSHALL 30) and then in 60
degrees of flexion (MARSHALL 60). The sutures were unknotted and
the tibial stump of the ACL resected to reveal the tibial insertion
sites. In the pig, there are two discrete tibial insertion sites of
the ACL--one is posterolateral, behind the anterior horn attachment
of the medial meniscus, and the second is anteromedial, located
between the anterior horn attachment of the medial meniscus and the
anterior horn attachment of the lateral meniscus (FIG. 26). A
tibial aimer (ACUFEX) was used to place a 2.4 mm guide pin from the
anteromedial border of the tibia up to each of these insertion
sites. Care was taken to maintain a minimum of 5 mm between each of
the drill sites on the anteromedial tibia. A third drill hole was
made just medial to the apex of the lateral tibial spine. These
tibial drill sites were labeled ANTERIOR for the insertion of the
AM bundle, MIDDLE for the insertion of the PL bundle and POSTERIOR
for the lateral tibial spine site. In four of the knees, drilling
actually went through the potting material to get to the
appropriate site.
[0306] AP laxity testing was then performed with sutures passed
through the anterior, middle or posterior bone holes and tied over
an endobutton. Sutures through each tunnel were tied in first 30
degrees of flexion and then 60 degrees of flexion and then
underwent AP laxity testing at the 60 degree position. For example,
each knee had all four sutures placed through the anterior tunnel
and tied together over an endobutton with the knee at 30 degrees of
flexion. This test was labeled (ANTERIOR 30). The sutures were then
untied and re-tied with the knee in 60 degrees of flexion and the
AP laxity of the knee measured (ANTERIOR 60). Sutures were then
untied, passed through the middle tunnel, tied at 30 degrees and
tested (MIDDLE 30), and so on. In addition to all four sutures
going through the anterior, middle and posterior tunnels, a final
position with two sutures going through the anterior tunnel and two
sutures going through the middle tunnel was also tested (ANT-MID 30
and ANT-MID 60).
[0307] After each test, the knee was inspected to make sure the
suture anchor was still secure and this was verified. There was no
evidence of suture anchor pullout for any of the tests.
Statistical Analysis
[0308] Mixed model ANOVA with suture location and knee flexion
angle as repeated measures terms and subject factors included to
track individual specimens was used to determine the significance
of differences between groups. A compound symmetry covariance
structure (which demonstrated good fit based on Akaike's
Information Criterion (AIC)) was selected.
Results
[0309] The intact knees had an AP laxity of 4.9 mm+/-0.4 mm
(mean+/-SEM). Removal of the patella, patellar tendon, ligamentum
mucosum and fat pad had a negligible effect on the AP laxity of the
knee, with values for that group of 5.2+/-0.3 mm and t-testing
p>0.79 for comparison between the two groups). When the ACL was
sectioned, the laxity of the knee exceeded the maximum level set by
the testing device (32 mm), and even at those displacements, no
load was placed on the load cell. Therefore, this group was
assigned a displacement of 32 mm.
[0310] Primary repair using the Marshall technique resulted in
improved laxity in comparison with the ACL deficient knee, but
increased AP laxity when compared to the intact and patellar
deficient knee when the repair was done at both 30 degrees of
flexion and at 60 degrees of flexion (Table 5). These differences
between intact and Marshall technique ligament knee laxity were
statistically significant at both 30 and 60 degrees (p<0.002 for
both comparisons).
[0311] The AP laxity of the knee after suture repair was dependent
on the location of the tibial suture (F=35; p<0.001). Sutures
placed in the middle bone tunnel, located within the ACL tibial
insertion site, restored AP laxity of the knee to values similar to
that in the intact ACL knees with the patella removed (5.2+/-0.6 mm
vs 5.2+/-0.4 mm, p>0.99; mean+/-SEM). Sutures placed in the
anterior bone tunnel resulted in repairs with an average of 1.2 mm
greater laxity than sutures placed in the middle location
(6.4+/-0.4 mm), a difference which approached, but did not reach
statistical significance in this multiple comparison model
(p>0.05). Placement of the suture in a more posterior location
on the tibial spine or in the ACL tibial remnant resulted in knees
with significantly greater laxity than the knees with an intact ACL
or knees repaired to anterior or middle tunnels (p<0.05 for all
comparisons).
[0312] FIG. 27 is photographs of the anterior (27A), middle (27B),
and posterior (27C) tibial tunnel positions. FIG. 28 is a graph
depicting AP laxity values for all specimens. FIG. 29 is a graph
depicting differences from intact AP laxity values.
[0313] The AP laxity of the knee after suture repair was also
dependent on the knee flexion angle when the sutures were tied
(F=30, p<0.001). Laxity was greatest when the repairs were tied
at 30 degrees of flexion, with less laxity noted when the repairs
were tied at 60 degrees (p<0.001); however, the laxity in both
groups remained higher than that of the ACL intact knees
(p<0.02).
[0314] There was no interaction between tibial suture location and
knee flexion angle (p=0.67).
[0315] Suture techniques that go from femur to tibia can restore
the normal AP laxity of the knee at time zero, particularly if they
are tied in a small amount of flexion and the tibial attachment
point is within the normal ACL footprint. The data demonstrate that
suture repair to the tibial stump, as in the Marshall technique,
does not restore normal AP laxity of the knee, a finding which may
be one of the reasons this technique of ACL repair resulted in a
large percentage of patients having abnormal knee laxity
post-operatively.
[0316] However, in several of the groups, there were some knees
that had less AP laxity after suture repair than in the intact ACL
condition. This could potentially result in overconstraining of the
knee. Whether overconstraint or excess laxity are more likely to
proceed to early degenerative joint changes is as yet unclear, but
it is likely that repairs that result in large changes in knee
laxity may not be ideal.
TABLE-US-00006 TABLE 5 AP laxity as a Function of Repair Type Std.
95% Confidence Mean Error df Interval Lower Upper Lower Upper Lower
Location Bound Bound Bound Bound Bound Intact 4.933(a) 0.624 59.855
3.686 6.181 Patellar 5.200(a) 0.624 59.855 3.952 6.448 Deficient
Marshall 12.433(a).dagger. 0.444 52.094 11.542 13.325 Anterior
6.400(a) 0.444 52.094 5.509 7.291 Middle 5.192(a) 0.444 52.094 4.3
6.083 Posterior 7.658(a).dagger-dbl. 0.444 52.094 6.767 8.55
Ant-Mid 5.808(a) 0.444 52.094 4.917 6.7 (a)Based on modified
population marginal mean. .dagger.Statistically different from the
ACL intact knees (p < 0.002). .dagger-dbl.Statistically
different from ACL intact knees (p < 0.05).
Example 8
Use of 40 mg/ml High Density Sponges (HDBC Sponges)
[0317] A study comparing the effectiveness of standard density
collagen sponges (Gelfoam) and high density collagen sponges (HDBC)
was performed. 1 cm diameter sponges of both Gelfoam and HDBC
(3.times. increase in collagen concentration) were used as the
scaffold.
[0318] The HDBC sponge was prepared by lyophilizing a collagen
slurry and reconstituting it to a density of 40 mg/ml collagen. The
slurry was neutralized and allowed to gel at 38.degree. C. The
resulting gels were then lyophilized. Both GELFOAM and HDBC sponges
were seeded with cells suspended in platelet-rich plasma (PR or
cells suspended in collagen slurry+PRP (concentration
1.times.10.sup.6 cells/ml for both groups). Both cell solutions
were allowed to absorb into the sponges for 30 min in the
incubator, and then 2 drops of complete media was placed on top of
the sponges to keep them moist overnight. After 12 hours, 1 ml of
complete media was added to each well.
Results
[0319] The standard density sponges (GELFOAM) did not absorb the
PRP or collagen slurry very efficiently. Cell counts within the
sponges were measured at Day 2 and Day 10 (FIG. 30). The greatest
cell proliferation occurred in the HDBC+PRP group and the least
proliferation in the Gelfoam+PRP group.
[0320] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
[0321] All references disclosed herein are incorporated by
reference in their entirety.
* * * * *