U.S. patent application number 09/996474 was filed with the patent office on 2002-05-09 for methods and compositions for bone graft implants.
Invention is credited to Gainey, Glenn Morris, Landesberg, Regina.
Application Number | 20020054901 09/996474 |
Document ID | / |
Family ID | 22992665 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020054901 |
Kind Code |
A1 |
Gainey, Glenn Morris ; et
al. |
May 9, 2002 |
Methods and compositions for bone graft implants
Abstract
An autologous platelet gel for bone grafts is comprised of a
mixture of platelet-rich-plasma activated by calcium chloride
solution, and an aqueous suspension of partially frayed Type I
collagen.
Inventors: |
Gainey, Glenn Morris; (New
York, NY) ; Landesberg, Regina; (Stamford,
CT) |
Correspondence
Address: |
Law Offices of Susanne M. Hopkins
7101 Bloomsbury Lane
Spotsylvania
VA
22553
US
|
Family ID: |
22992665 |
Appl. No.: |
09/996474 |
Filed: |
November 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09996474 |
Nov 26, 2001 |
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09261291 |
Mar 2, 1999 |
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6322785 |
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Current U.S.
Class: |
424/426 ;
435/366 |
Current CPC
Class: |
A61L 2430/02 20130101;
A61K 35/16 20130101; A61K 35/16 20130101; A61K 2300/00 20130101;
A61L 27/38 20130101; A61L 27/3608 20130101; A61L 27/3691 20130101;
A61L 27/3616 20130101; A61L 27/365 20130101 |
Class at
Publication: |
424/426 ;
435/366 |
International
Class: |
C12N 005/08; A61K
045/00 |
Claims
What is claimed:
1. An autologous gel, comprising; an aqueous suspension of Type I
fibrillar collagen alkaline treated to created partially frayed
termini; calcium chloride; and platelet-rich-plasma processed to
form an in vitro formable matrix.
2. A method of making an implant gel, comprising the steps of:
making (( an admixture of Type I collagen in physiological
suspension, an aqueous ( solution of calcium chloride and bone
particulate; and adding to said admixture platelet-rich-plasma in
an amount sufficient to establish a viscous formable gel.
3. The method as recited in claim 2 including the step of heating
said gel at around body temperature to accelerate formation of the
gel.
4. In a platelet-based sealant characterized by a mixture of
calcium chloride, thrombin and a source of platelets, the
improvement comprising: substituting an effective amount of
terminally frayed Type I microfibrillar collagen for said
thrombin.
5. A cellular matrix for cellular moieties, comprising by volume 10
to 60 parts of platelet-rich-plasma; 1 to 5 parts calcium chloride;
5 to 60 parts of partially defibrillated Type I collagen; and 5 to
40 parts bone particulate.
6. A method of making an in-vitro thrombic agent for use in
platelet-rich-plasma compositions comprising the steps of: forming
a mixture of Type I fibrillar collagen and water; adjusting the
mixture to a pH in the range of about 8 to 12; stirring said
mixture at said range for a sufficient time to establish a stable
suspension; and readjusting the suspension to a pH in the
physiological range.
7. The method as recited in claim 7 wherein said mixture has a pH
in the range of 9.5 to 110 and said physiological range is between
6.8 and 7.2.
8. A method of separating from whole blood a predominantly
platelet-rich-plasma fraction comprising the steps of: centrifuging
a volume of whole blood at a first separating condition at
centrifugal forces in the range of about 150 g to 400 g and a time
sufficient to differentiate a top layer, a middle layer and a
bottom layer; separating said top layer and said middle layer from
said bottom layer; recentrifuging the top layer and the middle
layer at a second separating condition at centrifugal forces in the
range of about 225 g to 300 g for a time to differentiate further
said top layer from said middle layer; and separating said middle
layer from said top layer.
9. The method as recited in claim 9 wherein said centrifugal forces
in at said first separating condition are in the range of about 175
g to 250 g.
10. The method as recited in claim 9 wherein said centrifugal
forces at said second separating condition are in the range of
about 225 g to 275 g.
11. A cellular matrix comprising: a cellular moiety in admixture
with an autologous platelet-based gel including autologous
platelet-rich-plasma, terminally frayed microfibrillar Type I
collagen, and a gelling initiator.
12. The matrix as recited in claim 11 wherein said cellular moiety
is pancreatic islets.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for bone repair and, in particular, an implantable, thrombin-free,
autologous platelet gel and matrix that promotes bone augmentation
and regeneration.
BACKGROUND OF THE INVENTION
[0002] Fibrin glue, also known as fibrin sealant or fibrin gel, is
one of many materials developed in response to a recognized need
for improved hemostatic agents and sealant (Ref. 1). Platelet-based
implantable gels have been used extensively as wound healing and
bone regeneration agents in preferred substitution for fibrin
glues. Fibrin glues are a two component system comprising, as a
first component, concentrated fibrogen, a fibrin stabilizing factor
and fibroconectin. The second component includes thrombin, calcium
chloride, and an inhibitor of fibrinolysis. The constituents when
combined form a fibrin gel or clot. Thrombin and the calcium cleave
the fibrogen to fibrin in the coagulation cascade and activate
factor XIlI which crosslinks fibrin into an organized clot. The gel
may be topically applied or in-vitro molded prior to
implantation.
[0003] The fibrogen content is customarily plasma based. Such
plasma may be derived from a variety of sources including random
donor or single-donor cryoprecipitate or from autologous plasma.
Homologous sourcing presents numerous quality control problems.
Procured from donor blood, certain patient risks may be encountered
including compatibility problems, disease transmission dangers,
clerical and storage errors. While an autologous cryoprecipitate
overcomes some of the above, the donor blood must be procured
substantially in advance of surgery, 3 to 5 days or more.
[0004] Autologous platelet gel was developed in further response to
the above. Therein, whole blood is obtained from the patient in the
preoperative period and processed in the operating area. Through
differential centrifugation, a fraction representing the platelet
strata is separated. This strata or platelet-rich-plasma (PRP) is
combined with thrombin and calcium chloride representatively using
the technique set forth in Whitman et.al, Ref above. It has also
been proposed that such resultant clots be supplemented with
calcium phosphate minerals and other osseoparticulates including
autologous bone and marrow material for use in oral and
maxillofacial surgery.
[0005] Autologous platelet gel differs from fibrin glue in the
presence of a high concentration of platelets and a high
concentration of native fibrogen. The platelets, activated by the
thrombin, release factors and form scaffolding for the development
of a clot. Two of the growth factors, platelet derived growth
factor (PDGF) and transforming growth factor-beta (TGF-B) are known
to promote wound healing. PDGF is an activator of collangenase
during wound healing allowing reshaping of collagen for wound
strength. It also is known to be chemotactic for monocytes and
macrophages. TGF-B is known to activate fibroblasts to form
procollagen resulting in collagen deposition within the wound.
[0006] Autologous platelets gels have gained acceptance in the area
of reconstructive oral surgery in connection with ablative surgery
of the maxillofacial region, mandibular reconstruction, surgical
repair of alveolar clefts and associated oral-antral/oral-nasal
fistulas, and adjunctive procedures related to the placement of
osteointegrated implants. Such platelet gels have also been used in
combination with particulate cancellous bone and marrow grafts
(PCBM) (Ref. 1, Ref. 2). It is reported that such platelet gels
with the graft material evidenced substantially greater maturation
rates and bone density than such implants without the platelet-
rich-plasma. The PDGF and TGF growth factors were amplified in this
approach and the probable primary initiators of the results.
[0007] The action of the thrombin in such autologous platelet gels
has been recognized as the primary biological release mechanism of
these growth factors as discussed in U.S. Pat. No. 5,165,938 to
Knighton. While other biological release agents such as collagen,
ADP, and srotonin have been suggested for activating, the
performance of thrombin was preferred and appears to have been
adopted in the art as the agent of choice. The thrombin customarily
used in both platelet gels and fibrin systems has been a bovine
derivative. To reduce potential xenographic effects, the bovine
thrombin has been used in highly purified form.
[0008] Notwithstanding the improved results reported with the
thrombin- based fibrin and platelet systems, there are numerous
reports detailing adverse clinical effects that have been linked
potentially to the bovine thrombin. Sosolik et. al. reported a
prolongation of thrombin time was associated with the presence of
anti-bovine thrombin antibodies following surgical procedures when
fibrin glues or bovine thrombin preparations were applied topically
and it was suggested that such exposure could lead to serious
bleeding complications during surgery or the postoperative period
(Ref. 3). Spero concluded that bovine-induced coagulopathy may
occur following surgical exposure to topical bovine thrombin and
may result in both postoperative morbidity and mortality in a
subset of patients resulting from topical-induced antibodies to
clotting factor V following neurolosurgical procedures (Ref. 4).
Cmolik et. al. reports coagulopathy occasioned by bovine
thrombin-induced factor V deficiency after exposure to bovine
thrombin in topical hemostatic agents during cardiovascular or
vascular operation (Ref. 5). Muntean et. al. reported inhibitors to
factor V following exposure to fibrin sealant during cardiac
surgery and concluded that exposure to topical thrombin
preparations may lead to the development of inhibitors in the
postoperative period that may cause bleeding complications (Ref.
6). Based on the foregoing and other reports, Landsberg et, al.
cautioned against use of bovine topical thrombin-based platelet
gels in oral and maxilofacial procedures and expressed the need for
alternative methods of activating PRP in the oral surgery area (Ref
7).
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides an autologous platelet gel
and bone graft matrix that is thrombin-free and promotes
osteoconduction, osteoinduction and osteogenesis at a bone
augmentation site, and initiates and augments the bone replacement
process. The implant compositions of the present invention comprise
an admixture of platelet-rich-plasma, osseoparticulates, a gelling
initiator, and a osseoinductive carrier comprising a suspension of
terminally defribillated fibrillar collagen. The admixture is
in-vitro cured to form a gelled composition that is formable for
implantation into bone deficient sites. Osseoparticluates in a
variety of forms can be utilized as the osseocondutive medium
including autologous bone and marrow, allographic bone particulate,
xenographic particulate bone substitutes and other calcium
phosphate minerals. The collagen suspension is a microfibrillar
Type I collagen processed to undergo partial unwinding of the
collagen stands at the ends thereof sufficient to maintain
suspension in a physiological medium and effective to act as a
carrier for the scaffolding and as an osseogenetic promoter.
[0010] REFERENCES. The publications set forth above are hereby
incorporated by reference.
[0011] 1. "Platelet Gel: An autologous Alternative to Fibrin Glue
with Applications in Oral and Maxilofacial Surgery", Whitman, D H
et al., J. Oral Maxilofacial Surgery, 1294-1299 (1997).
[0012] 2. "Platelet-rich plasma: Growth factor enhancement for bone
grafts", Mars, RE et al., Oral Surg Oral Med Oral Pathol Oral
Radiol Endod, Vol. 85, 638-646, (1998).
[0013] 3. "Anti-Bovine Thrombin Antibody", Sosolik, R C et al.,
Laboratory Medicine, Vol. 27, No. 10, 651-653 (1998).
[0014] 4. "Bovine thrombin-induced inhibitor of factor V and
bleeding risk in postoperative neurosurgical patients", Spero J A,
J Neurosurg, Vol 78, 817-820 (1993).
[0015] 5. "Redo cardiac surgery. Late bleeding complications tom
topical thrombin-induced factor V deficiency", Cmolik, B L, J
Thorac Cardiovasc Surg, Vol 105, 222-228 (1993).
[0016] 6. "Inhibitor to factor V after exposure to fibrin sealant
during cardiac surgery in a two-year-old child", Muntean, W, Acta
Paediar, Vol. 83, 84-87 (1994).
[0017] 7. "Risks of using platelet-rich-plasma gel", Landesberg, R,
J Oral Maxilofac Surg, Vol. 56, 1116-1117 (1998).
Description of the Preferred Embodiments
[0018] The autologous platelet gel and matrix of the present
invention is produced using patient derived platelet-rich-plasma
fraction derived from preoperative donated blood. For use as an
autologous platelet sealant/bone graft matrix, the composition
comprises by volume about 10 to 60 parts of platelet-rich-plasma; 1
to 5 parts calcium chloride; 5 to 60 parts of partially
defibrillated Type I collagen; and 5 to 40 parts osseoparticulate.
Such composition is made by: forming initially a mixture of
alkaline induced terminally frayed Type I fibrillar collagen and
water; adjusting the mixture to a pH in the range of about 8 to 12;
stirring said mixture at said range for a time sufficient to
establish a stable suspension; readjusting the suspension to a pH
in the physiological range; combining the suspension with calcium
chloride solution as an activator system; and combining the
activator system with autologous platelet-rich-plasma under time
and temperature conditions yielding a clotted, formable gel,
supplemented in accordance with the application by
osseoparticulate.
[0019] The procedure for harvesting the platelet-rich-plasma may be
practiced in many variations, however, the procedure as described
in Marx (Ref. 2) may be beneficially used herein. Such procedure
involves obtaining from the prospective implant patient,
immediately preoperative, whole blood, which is transferred into a
collection reservoir containing a citrate- phosphate-dextose
anticoagulant. The anticoagulated whole blood is transferred to a
centrifulge for separation at about 5600 rpm into plural layers
comprising: platelet-poor-plasma, upper layer: erythrocytes, lower
layer; and the "buffy coat", middle layer, containing the
platelet-rich-plasma fraction. The platelet-poor-plasma, upper
layer fraction, is removed by aspiration and the remainder
recentrifuiged at 2400 rpm to further define the remaining
fractions. The but coat middle layer containing the
platelet-rich-plasma is removed and stored at room temperature for
future use. Such technique is reported to yield 500,000 to
1,000,000 platelets in the PRP from a unit of whole blood.
[0020] While the foregoing separation procedure produces generally
acceptable results, the present invention has determined that
greater yield and improved morphology can be obtained by more
gently sequestering the platelets. Herein, a sample of whole blood
is transferred to a. Centrifuge tube containing a suitable
coagulant such as citrate solution. The tube is centrifuged in the
range of 175 g to 300 g for a period of time, 5 to 15 minutes,
sufficient to delineate the sample into three distinct layers; a
top layer containing platelet poor plasma, a buffy coat middle
layer containing platelet-rich-plasma, and a lower layer containing
the red blood cells. Thereafter, the top and middle layers are
transferred to a second Centrifuge tube. The tube is centrifuged
again at a gentle speed in the range of 200 g to 300 g for a period
of time, generally 5 to 15 minutes, sufficient to delineate clearly
the upper layer of platelet-poor-plasma and the now lower layer
containing the platelet-rich-plasma. The layers are thereafter
separated, and the platelet-rich-plasma reserved for use. Such
gentle sequestration of the platelet-rich-plasma has been found to
produce increased yields of intact platelets exhibiting a
morphology substantially unaffected by the separation process.
[0021] The gelling initiator or clotting activator is an inorganic
solution compatible with the graft constituents for effecting
gelling or clotting of the autologous platelet gel and matrix. For
the collagen-based system of the present invention a calcium
chloride aqueous solution is preferred.
[0022] The carrier and osseoinductive constituent and second
constituent of the activator is preferably a Type I fibrillar
collagen derived from allographic or xenographic sources. Type I
Bovine collagen is preferred, however, other suitable animal
sources such as mammalian or avian may be used. The collagen is
alkaline treated to promote strand uncurling at the ends of the
fibers. Such processing permits the collagen to form a stable
aqueous suspension, Thereafter, the suspension is neutralized to
physiological conditions for use in the gel and platelet
compositions as hereinafter described.
EXAMPLE 1
Preparation of Conventional Thrombin-Based Platelet Gel
[0023] Platelet-rich-plasma was obtained as set forth above, In a
mixing bowl, 1 ml of platelet-rich-plasma, 60 nl of 91M calcium
chloride, and 10,000 U of Type 1 bovine thrombin were mixed to form
a moldable viscous clot with the consistency of a gel.
EXAMPLE 2
Preparation of Collagen-Based Platelet Gel
[0024] In a mixing bowl, 1 ml of platelet-rich-plasma, 60 nl of
0.91M calcium chloride and .5 ml of collagen suspension (1 mg/ml)
made in accordance with example 3 below were thoroughly mixed and
transferred to a heating surface maintained at 37.degree. C. for
about 10 minutes until a viscous gel was formed.
EXAMPLE 3
Preparation of Collagen Suspension
[0025] A collagen suspension was prepared by admixing .5 grams of
fibrillar Type I Bovine Collagen (J&J Medical Systems, Product
No 1984) in 5 ml of distilled water. The pH of the mixture was
increased to 10.2 with the addition of 10M Sodium Hydroxide. The
adjusted mixture was gently stirred for a period of 8 hours at
which time the collagen remained in stable suspension. The adjusted
suspension was neutralized to physiologic conditions at a pH of 7.2
by the addition or 10M hydrochloric acid. Microscopic examination
of the collagen fibers indicated frayed termini on the fiber ends
resultant from above processing.
EXAMPLE 4
Determination of PDGF and TGF-B Content
[0026] In order to determine the effects of the thrombin-based gel
as prepared in accordance with Example 1 and the collagen-based gel
of the present invention as prepared in accordance with Example 2
in the release of Platelet Derived Growth Factor (PDGF) and
Transforming Growth Factor-Beta (TGF-B), 10 ml samples based on
different blood sources were analyzed in accordance with
established protocols
[0027] The results are set forth below in Table A
1 TABLE A Sample A B C PDGF (Units) Thrombin 157.3 113.7 107.4
Collagen 155.4 127.7 130.3 TGF-B (Units) Thrombin 118.0 123.9 154.7
Collagen 111.9 130.9 169.21
[0028] The foregoing demonstrates that both gel preparations are
substantially equally effective in releasing the noted growth
factors associated with bone and tissue augmentation.
EXAMPLE 5
Gel Conditions
[0029] To illustrate the effect of time, temperature and calcium
chloride concentration on clotting time and consistency three
samples of gels in accordance with Example 2 above were prepared
with the exceptions that two samples used the 10% calcium chloride
and one sample an equal volume of 2M solution. The 10% samples
clotted at room temperature in 11.5 minutes and at 37.degree. C. in
4 and 4.5 minutes. The 2M solution did not clot at either
temperature.
EXAMPLE 6
Clot Consistency
[0030] To illustrate the effect of calcium chloride concentration
on the collagen-based platelet gel, three samples of the gel were
prepared in accordance with Example 2 in the following
proportions;
2 Sample A B C PRP 100 ml 100 ml 100 ml Calcium Chloride 14 ml 50
ml 6 ml Collagen 20 ml 50 ml 100 ml Clotting Time no clot no clot
7.6 min
[0031] Such conditions indicated that increased amounts of the
calcium chloride adversely affected the desired clotting
characteristics, even in the presence of temperature
activation.
EXAMPLE 7
Preparation of Platelet Gel for Implantation
[0032] The collagen suspension prepared in Example 5, 10 ml was
mixed in a sterile glass bowl with 10 ml of 0.91M calcium chloride.
The collagen mixture was then mixed with 50 ml. of plasma rich
platelets contained in a second glass container and mixed until
evenly distributed. Preliminary gelling was noted. The glass
container was placed on a hot plate maintained at 37.degree. C. for
clotting. After 30 minutes of heating, the resultant platelet gel
was removed and found to be uniformly clotted and readily moldable
into retentive shapes conformal to all implant site.
EXAMPLE 8
Preparation Platelet Augmentation Osteograph Composition
[0033] Prior to heating, 25 mg. of cadavillar particulate was added
to the ungelled mixture and mixed until well incorporated. The
resultant mixture in the glass container was placed on a hot plate
maintained at 37.degree. C. for gellation. After 30 minutes of
heating, platelet gel with particulate was removed and bound to be
readily moldable into typical dental implant shapes.
EXAMPLE 9
Implantation of Osteogrph Composition.
[0034] A quantity of composition prepared in accordance with
Example 8 was preliminary molded and inserted into a mandibular
void of a subject. The composition was further defined to desired
shape. The surrounding tissue was then closed by sutures. Visual
inspection during the postoperative period did not indicate any
inflammation or swelling attributable to the implant. Subsequent
visual and radiological observation indicated progressive increase
in both load-bearing and complete osteointegration in accordance
with conventional analysis. No allergic or antibody reaction was
noted for a period of up to 4 months. At the end of 5 months, the
implant was fully integrated and load supporting.
[0035] The bone graft material of the present invention has
application in craniofacial reconstruction, periodontal defects,
joint reconstruction, fracture repair, orthopedic surgical
procedures, spinal fusion, bone defects, odontolological defects in
osteoconductive/osteoinductive grafting applications.
[0036] The gel matrices of the present invention also have
applications in any surgical or invasive technique in which
manipulative or promotion of wound tissue deficit healing is
intended.
[0037] The gels and matrices of the present invention also have
application as matrices for the storage and encapsulation of
cellular moieties such as pancreatic islets, xenographic or
allographic, hepatocytic cells and the like.
[0038] Various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are intended to
be within the scope of the appended claims.
* * * * *