U.S. patent application number 12/194771 was filed with the patent office on 2009-02-26 for method for use of a double-structured tissue implant for treatment of tissue defects.
This patent application is currently assigned to Histogenics Corporation. Invention is credited to HANS P. I. CLAESSON, ERIC J. ROOS, SONYA SHORTKROFF, ROBERT LANE SMITH, LAURENCE J. B. TARRANT.
Application Number | 20090054984 12/194771 |
Document ID | / |
Family ID | 40382913 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090054984 |
Kind Code |
A1 |
SHORTKROFF; SONYA ; et
al. |
February 26, 2009 |
Method For Use Of A Double-Structured Tissue Implant For Treatment
Of Tissue Defects
Abstract
A method for use of a double-structured tissue implant or a
secondary scaffold stand alone implant for treatment of tissue
defects. The double-structured tissue implant comprising a primary
scaffold and a secondary scaffold consisting of a soluble collagen
solution in combination with a non-ionic surfactant generated and
positioned within the primary scaffold. A method of use of a stand
alone secondary scaffold implant or unit for treatment of tissue
defects.
Inventors: |
SHORTKROFF; SONYA;
(Braintree, MA) ; TARRANT; LAURENCE J. B.;
(Northampton, MA) ; ROOS; ERIC J.; (Grafton,
MA) ; SMITH; ROBERT LANE; (Palo Alto, CA) ;
CLAESSON; HANS P. I.; (Wayland, MA) |
Correspondence
Address: |
PETERS VERNY , L.L.P.
425 SHERMAN AVENUE, SUITE 230
PALO ALTO
CA
94306
US
|
Assignee: |
Histogenics Corporation
Waltham
MA
|
Family ID: |
40382913 |
Appl. No.: |
12/194771 |
Filed: |
August 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11982268 |
Oct 31, 2007 |
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12194771 |
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11894124 |
Aug 20, 2007 |
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11982268 |
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60967886 |
Sep 6, 2007 |
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Current U.S.
Class: |
623/16.11 ;
128/898; 606/151 |
Current CPC
Class: |
A61F 2210/0076 20130101;
A61F 2310/00365 20130101; A61L 2430/06 20130101; A61L 27/44
20130101; A61L 27/56 20130101; A61L 27/24 20130101; A61L 2420/06
20130101; A61L 27/3604 20130101; C07K 14/78 20130101; A61F
2310/00389 20130101; A61L 27/48 20130101; A61F 2/02 20130101; A61F
2002/30766 20130101; A61F 2210/0061 20130101; A61L 27/54 20130101;
A61F 2002/3092 20130101; A61L 27/34 20130101; A61F 2/30756
20130101; A61F 2002/30075 20130101; A61L 27/48 20130101; C08L 89/06
20130101 |
Class at
Publication: |
623/16.11 ;
128/898; 606/151 |
International
Class: |
A61F 2/28 20060101
A61F002/28; A61B 19/00 20060101 A61B019/00; A61B 17/08 20060101
A61B017/08 |
Claims
1. A method for use of a double-structured tissue implant (DSTI)
for treatment of a tissue defect, said method comprising steps: a)
obtaining or preparing the DSTI comprising a primary scaffold and a
secondary scaffold integrated into said first scaffold wherein said
primary scaffold is a porous structure prepared from collagen or a
collagen-containing material, said porous structure comprising
randomly or non-randomly organized pores, said primary scaffold
providing a structural support for the secondary scaffold
incorporated therein; b) preparing a tissue defect for implantation
of said DSTI; c) cutting or trimming the DSTI into a size of the
tissue defect; d) rehydrating said DSTI with a physiologically
acceptable solution, collagen-containing solution, buffer or
saline; e) implanting said DSTI into said defect; and f) covering
said implanted DSTI with a tissue adhesive.
2. The method of claim 1 wherein said collagen or
collagen-containing material for preparation of the primary
scaffold is selected from the group consisting of Type I collagen,
Type II collagen, Type III collagen, Type IV collagen, Type V
collagen, gelatin, collagen-containing agarose, collagen-containing
hyaluronan, collagen-containing proteoglycan, collagen-containing
glycosaminoglycan, collagen-containing glucosamine,
collagen-containing galactosamine, collagen-containing
glycoprotein, collagen-containing fibronectin, collagen-containing
laminin, collagen-containing bioactive peptide, collagen-containing
growth factor, collagen-containing cytokine, collagen-containing
elastin, collagen-containing fibrin, collagen-containing polylactic
acid, collagen-containing polyglycolic acid, collagen-containing
polyamino acid, collagen-containing polycaprolactone,
collagen-containing polypeptide, a copolymer thereof, a precursor
thereof and a combination thereof, wherein said precursor is
selected from the group consisting of alpha 1 (Type I) peptide,
alpha 2 (Type I) peptide, 2 (alpha 1, Type I) peptide, 1 (alpha 2,
Type I) peptide, 3 (alpha 1, Type II), and a combination
thereof.
3. The method of claim 2 wherein in said DSTI, said secondary
scaffold is integrated into said primary scaffold by introducing a
composition comprising a soluble collagen or collagen-containing
compound in combination with a non-ionic surfactant into said pores
of said primary scaffold, stabilizing said composition within pores
of said primary scaffold by precipitation or gelling and subjecting
a resulting composite to at least lyophilization and dehydrothermal
treatment.
4. The method of claim 3 wherein said soluble collagen or
collagen-containing compound used for preparation of the
composition for the secondary scaffold is Type I collagen, Type II
collagen, methylated collagen, gelatin or methylated gelatin.
5. The method of claim 4 wherein said non-ionic surfactant used for
preparation of the composition for the secondary scaffold is a
PLURONIC.RTM.-type or a TRITON.RTM.-type surfactant comprising
polyethylene oxide with terminal oxide groups.
6. The method of claim 5 wherein said surfactant is a derivatized
polyethylene glycol or a block co-polymer of polyoxyethylene (PEO)
and polyoxypropylene (PPO) having the generic organization of
polymeric blocks PEG-PPO-PEG or PPO-PEG-PPO.
7. The method of claim 6 wherein said surfactant is TRITON.RTM.
X100, namely polyethylene glycol
p-(1,1,3,3-tetramethylbutyl)-phenyl ether, or PLURONIC.RTM. F127,
namely a polymer of polyoxyethylene (PEO) and polyoxypropylene
(PPO) with two 96-unit hydrophilic PEO chains surrounding one
69-unit hydrophobic PPO chain.
8. The method of claim 7 wherein said integration of the secondary
scaffold into the primary scaffold results in the double-structured
tissue implant comprising two structurally and functionally
distinct sections, wherein one or both sections may separately be
seeded with cells or incorporated with a pharmaceutical agent or
modulator.
9. The method of claim 8 wherein said DSTI is implanted as a dry or
rehydrated acellular DSTI or a dry or rehydrated DSTI seeded with
cells.
10. The method of claim 9 wherein said implant is resistant to
dissolution, resistant to change of size and shape, maintains
collagen retention, maintains pH and osmolality and has a
rehydration and wettability time within 30 seconds.
11. The method of claim 1 wherein in step b) preparation of a
tissue defect comprises debriding said defect or debriding and
subchondral microfracture.
12. The method of claim 1 additionally comprising step of coating
said defect with a tissue adhesive before said implantation of said
DSTI.
13. The method of claim 12 wherein said tissue adhesive is selected
from the group consisting of di-aldehyde starch, 4-armed
pentaerythritol tetra-thiol and polyethylene glycol diacrylate,
photo-polymerizable polyethylene glycol-co-poly(.alpha.-hydroxy
acid) diacrylate macromer, periodate-oxidized gelatin, serum
albumin and di-functional polyethylene glycol derivatized with
maleimidyl, serum albumin and di-functional polyethylene glycol
derivatized succinimidyl, serum albumin and di-functional
polyethylene glycol derivatized phthalimidyl, a copolymer of
polyethylene glycol and polylactide, a copolymer of polyethylene
glycol and polyglycolide, a copolymer of polyethylene glycol and
polyhydroxybutyrate, 4-armed polyethylene glycol derivatized with
succinimidyl ester and thiol and a cross-linked polyethylene glycol
with methylated collagen.
14. The method of claim 13 wherein said tissue adhesive is
polyethylene glycol cross-linked with methylated collagen.
15. The method of claim 1 wherein said rehydration step d) is
performed before implantation of said DSTI or in situ after
implantation of said DSTI in a dry form.
16. A method for treatment of a cartilage, bone, tendon, skin,
meniscus, ligament, skeletal, muscle, cardiac muscle or nervous
tissue defect using an implantable double-structured tissue implant
(DSTI), said method comprising steps: a) obtaining or preparing the
DSTI comprising a primary scaffold and a secondary scaffold
integrated into said first scaffold wherein said primary scaffold
is a porous structure prepared from collagen or a
collagen-containing material, said porous structure comprising
randomly or non-randomly organized pores, said primary scaffold
providing a structural support for the secondary scaffold
incorporated therein and wherein said secondary scaffold comprises
collagen or collagen-containing material in combination with a
non-ionic PLURONIC.RTM.-type surfactant; b) debriding or debriding
and microfracturing said tissue defect for implantation of said
DSTI; c) cutting or trimming the DSTI into a size of the tissue
defect; d) rehydrating said DSTI with a physiologically acceptable
solution, collagen-containing solution, buffer or saline; e)
implanting said DSTI into said defect; and f) covering said
implanted DSTI with a tissue adhesive.
17. The method of claim 16 wherein said physiologically acceptable
solution additionally contains differentiated or undifferentiated
cells, a pharmaceutical agent, drug, growth modulator, growth
hormone, mediator, enzyme promoting a cell growth, enzyme promoting
cell incorporation into said DSTI, enzyme promoting cell
proliferation, or enzyme promoting cell division, a
pharmaceutically acceptable excipient or additive.
18. The method of claim 17 wherein said differentiated cells are
chondrocytes, osteoblasts, tenocytes, fibroblasts,
fibrochondrocytes or ligament cells.
19. The method of claim 18 wherein said undifferentiated cells are
adult or immature mesenchymal cells derived from bone marrow
aspirates, iliac crest needle biopsies, immortalized cell lines,
hematopoietic stem cells, neural stem cells, embryonic stem cells
or stem cells obtained from other dissociable mesenchymal
tissues.
20. The method of claim 19 wherein said dissociable mesenchymal
tissue is a somite, muscle, or interstitial connective tissue.
21. The method of claim 19, wherein said cells are activated with
hydrostatic pressure regimen.
22. The method of claim 17 wherein said drug or modulator is
selected from the group consisting of a growth factor, morphogenic
factor, cytokine, membrane associated factor that promotes growth
or morphogenesis, cell attachment or adhesion protein, hormone,
pericellular matrix molecule, nutrient, nucleic acid,
anti-neoplastic agent, vitamin, anti-inflammatory agent, enzyme and
metabolic inhibitor and a combination thereof.
23. The method of claim 22 wherein said growth and morphogenic
factor is a transforming growth factor, insulin-like growth factor
1, platelet-derived growth factor or bone morphogenetic protein;
wherein said cytokine is interleukin, chemokine, macrophage
chemoattractant factor, cytokine-induced neutrophil
chemoattractant, integral membrane protein, integrin or growth
factor receptor; wherein said membrane associated factor that
promote growth and morphogenesis is a repulsive guidance molecule;
wherein said cell attachment or adhesion protein is fibronectin or
chondronectin; wherein said hormone is growth hormone, insulin or
thyroxine; wherein said pericellular matrix molecule is perlecan,
syndecan, small leucine-rich proteoglycan or fibromodulin; wherein
said nutrient is glucose or glucosamine; wherein said nucleic acid
is RNA or DNA; wherein said anti-neoplastic agent is methotrexate
or aminopterin; wherein said vitamin is ascorbate or retinoic acid;
wherein said anti-inflammatory agent is naproxen sodium, salicylic
acid, diclofenac or ibuprofen; wherein said enzyme is
phosphorylase, sulfatase or kinase; and wherein said metabolic
inhibitor is RNAi, cycloheximide and steroid.
24. A method for use of a secondary scaffold for treatment of a
tissue defect, said method comprising steps: a) preparing the
secondary scaffold comprising a soluble collagen or
collagen-containing compound in combination with a non-ionic
surfactant neutralized to pH above 7.4%, precipitated, lyophilized
and dehydrothermally treated; b) preparing a tissue defect for
implantation of said DSTI; c) cutting or trimming the DSTI into a
size of the tissue defect; d) rehydrating said DSTI with a
physiologically acceptable solution, collagen-containing solution,
buffer or saline; e) implanting said DSTI into said defect; and f)
covering said implanted DSTI with a tissue adhesive.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a Continuation-in-Part of the
application Ser. No. 11/982,268, filed on Oct. 31, 2007 which is
Continuation-in Part of the application Ser. No. 11/894,124, filed
on Aug. 20, 2007, and claims priority of the Provisional
application Ser. No. 60/967,886, filed Sep. 6, 2007 and 60/958,401,
filed Jul. 3, 2007, all incorporated herein by reference.
FIELD OF INVENTION
[0002] The current invention concerns a method for use of a
double-structured tissue implant for implantation into tissue
defects. In particular, the invention concerns use of a
double-structured tissue implant comprising a primary scaffold and
a secondary scaffold generated and positioned within the primary
scaffold. The primary scaffold is a porous collagen-comprising
material having randomly or non-randomly oriented pores of
substantially homogeneous defined diameter. Under the most
favorable conditions, the pores are through oriented, mostly
vertically, and represent a high percentage of the total volume of
the scaffold. The secondary scaffold is generated within the
primary scaffold by introducing a composition comprising a soluble
collagen solution in combination with a non-ionic surfactant into
the pores of the primary scaffold and solidifying said composition
within said pores using a novel process of the invention.
[0003] The method for use of the double-structured tissue implant
comprises implantation of the DSTI into the tissue defect either in
a rehydrated or dry form and sealing the implant within the defect
with a biodegradable tissue sealant.
[0004] The DSTI may be rehydrated and/or preloaded with cells,
drugs or growth modulators before or after implantation.
[0005] When formed, the double-structured tissue implant has
improved properties, such as stability, resistance to shrinkage,
swelling or dissolution, improved wetting, storageability and
longer shelf-life as compared to the properties of each scaffold or
the composite separately.
[0006] Furthermore, the double-structured tissue implant provides
an increased surface area for cell adhesion, growth and
differentiation without compromising the porosity of the
implant.
BACKGROUND AND RELATED DISCLOSURES
[0007] Collagen matrices for use as an implant for repair of
cartilage defects and injuries are known in the art. Of a
particular interest is a honeycomb structure developed by Koken
Company, Ltd., Tokyo, Japan, under the trade name Honeycomb Sponge,
described in the Japanese patent JP3170693, hereby incorporated by
reference. Other patents related to the current subject disclose
collagen-based substrates for tissue engineering (U.S. Pat. No.
6,790,454) collagen/polysaccharide bi-layer matrix (U.S. Pat. No.
6,773,723), collagen/polysaccharide bi-layer matrix (U.S. Pat. No.
6,896,904), matrix for tissue engineering formed of hyaluronic acid
and hydrolyzed collagen (U.S. Pat. No. 6,737,072), method for
making a porous matrix particle (U.S. Pat. No. 5,629,191) method
for making porous biodegradable polymers (U.S. Pat. No. 6,673,286),
process for growing tissue in a macroporous polymer scaffold (U.S.
Pat. No. 6,875,442), method for preserving porosity in porous
materials (U.S. Pat. No. 4,522,753), method for preparation of
collagen-glycosaminoglycan composite materials (U.S. Pat. No.
4,448,718), procedures for preparing composite materials from
collagen and glycosaminoglycan (U.S. Pat. No. 4,350,629) and a
crosslinked collagen-mucopolysaccharide composite materials (U.S.
Pat. No. 4,280,954).
[0008] However, many of the above disclosed structures have
uncontrolled parameters such as uneven and uncontrolled porosity,
uneven density of pores, uneven sizes of the pores and random
distribution of pores within the support matrix. Such uncontrolled
parameters lead to usable pore structures that represent only a
small percentage of the total implant. Additionally, when
introduced into tissue defects or cartilage lesions during the
surgery, these structures are difficult to handle as they are
unstable and do not have appropriate wetting properties in that
they can shrink or swell and are not easily manipulated by the
surgeon.
[0009] For a tissue implant to be suitable for implantation,
particularly for implantation into the cartilage lesion, the
implant needs to be stable, easily manipulated, easily stored in
sterile form and have a long shelf-life.
[0010] In order to provide a more uniform and sterically stable
support structure for implantation into a tissue defect or
cartilage lesion, inventors previously developed a collagen matrix
having narrowly defined size and density of pores wherein the pores
are uniformly distributed, vertically oriented and non-randomly
organized. This matrix is disclosed in the co-pending patent
application Ser. No. 11/523,833, filed on Sep. 19, 2006, hereby
incorporated by reference in its entirety. Additionally, the
acellular matrix suitable to be used as the primary scaffold is
described in the priority application Ser. No. 10/882,581, filed on
Jun. 30, 2004, issued as U.S. Pat. No. 7,217,294, on May 15, 2007,
hereby incorporated in its entirety. However, even with the
above-described improvements, a solution to problems faced by the
surgeon during surgery is still lacking. A practicality needed for
routine use of the tissue implants, such as, for example, the
articular cartilage implants by the orthopedic surgeons, where the
implant needs to be readily available, manipulatable, wettable,
stable, sterile and able to be rapidly prepared and used for
implantation, is still not achieved. All the previously described
and prepared matrices or scaffolds require multiple steps before
they are fully implantable.
[0011] Thus, it would be advantageous to have available an implant
that would be easily manufactured and packaged, would be stable for
extended shelf-life, would be easily manipulatable and rapidly
wettable upon introduction into the lesion, could provide a support
for cell migration or seeding and that could have, additionally,
pre-incorporated drug or modulator in at least one portion of the
implant. The implant should also allow the surgeon to introduce a
drug or modulator during the surgical procedure.
[0012] It would also be an advantage to provide a secondary
scaffold with an increased area of internal membranes which while
not interfering with cell migration and nutrient exchange,
nevertheless, would provide a substrate favorable to cell adhesion,
growth and migration.
[0013] It is, therefore, a primary object of this invention to
provide a method for treatment of tissue defects using a
double-structured tissue implant comprising a primary scaffold and
a secondary scaffold where each scaffold of the implant can assume
a different function, be incorporated with cells, different drugs
or modulators and/or be selectively chosen for performing a
different function following the implantation.
[0014] The current invention provides such double-structured
scaffold and a method for use for treatment of tissue defects by
providing a first scaffold comprising a sterically stable and
biocompatible support structure, preferably made of Type I
collagen, having defined pore sizes and density with said pores
organized vertically and a second scaffold wherein said second
scaffold is formed within said pores of said first scaffold. The
double-structured scaffold of the invention is stable, resistant to
shrinkage, swelling and dissolution, rapidly wettable, prepared in
the sterile storageable form having a long-shelf life that can be
easily surgically delivered and easily manipulated.
[0015] All patents, patent applications and publications cited
herein are hereby incorporated by reference.
SUMMARY
[0016] One aspect of the current invention is a method for use of a
double-structured tissue implant for treatment of tissue
defects.
[0017] Another aspect of the current invention is a collagen-based
double-structured tissue implant comprising a primary scaffold and
a secondary scaffold wherein said secondary scaffold is a
qualitatively different structure formed within a confine of the
primary scaffold wherein said implant is suitable for implantation
into tissue lesions or defects.
[0018] Another aspect of the current invention is a collagen-based
primary porous scaffold having randomly oriented open pores of
substantially homogeneous pore size, said primary scaffold suitable
for incorporation of a secondary scaffold wherein said secondary
scaffold is incorporated into said primary scaffold by introducing
a Basic Solution comprising collagen and a non-ionic surfactant
into said primary scaffold and subjecting said primary scaffold
incorporated with said Basic Solution for the secondary scaffold to
a process comprising precipitation, lyophilization and
dehydrothermal treatment.
[0019] Still yet another aspect of the current invention is a
method of use for a double-structured tissue implant having two
distinct qualitatively different structures wherein each of the
structures may be independently loaded with cells or prepared
already incorporated with drugs or growth modulators or wherein
both structures of the implant may comprise cells, pharmaceutical
agents or growth modulators.
[0020] Still another aspect of the current invention is a method of
use for a double-structured tissue implant empty or seeded with
cells or incorporated with drugs or growth modulators for
implantation into tissue lesions or defects wherein said implant is
placed into said lesion or defect and covered with an adhesive and
wherein when the implant is seeded with cells or in vitro cultured
cells, the sealant or adhesive is applied to both the bottom of the
lesion and at the top of the lesion.
[0021] Still another aspect of the current invention is a method of
use for a secondary scaffold as a stand alone implant or unit for
tissue implantation, wherein said secondary scaffold is prepared
from the Basic Solution comprising collagen and a surfactant
neutralized to pH of about 7.4 and subjected to lyophilization and
dehydrothermal treatment.
[0022] Yet another aspect of the current invention is a method for
use of a double-structured tissue implant or the stand alone
secondary scaffold implant for implantation into a tissue defect or
cartilage lesion wherein said implant, in dry or wet form,
optionally seeded with cells is implanted into said defect or
lesion during surgery and covered with at least a top adhesive.
[0023] Another aspect of the current invention is a process for
preparation of a double-structured implant by providing a primary
porous scaffold prepared from a biocompatible collagen material
wherein said scaffold has a substantially homogenous defined
porosity and uniformly distributed randomly and non-randomly
organized pores of substantially the same size of defined diameter
of about 200 to 300.+-.100 .mu.m, wherein said primary scaffold is
brought in contact with a soluble collagen based solution
comprising at least one non-ionic surfactant (Basic Solution),
wherein such solution is introduced into said pores of said primary
scaffold, stabilized therein by precipitation or gelling,
dehydrated, lyophilized and dehydrothermally processed to form a
distinctly structurally and functionally different second scaffold
within said pores of said primary scaffold.
BRIEF DESCRIPTION OF FIGURES
[0024] FIG. 1A is a photograph of the lyophilized double-structured
tissue implant (DSTI) that shows the porous nature and the
sturdiness of the implant prior to rehydration. FIG. 1B is a
photograph of the double-structured tissue implant (DSTI) packaged
in a sterile form ready for delivery wherein said implant may be
provided in a dry form optionally comprising covalently bonded
drugs or growth factors or in a wet form optionally comprising
cells, drugs or growth modulators.
[0025] FIG. 2 is a photomicrograph of a primary scaffold showing
pores having substantially the same size (4.times. magnification),
said scaffold used as the foundation and structural support for
preparation of the double-structured tissue implant. As shown, the
primary scaffold has a honeycomb structure of relative uniform pore
size and equal distribution.
[0026] FIG. 3 is a photomicrograph of a primary scaffold having
pores loaded with a soluble collagen/PLURONIC.RTM. surfactant
solution for secondary scaffold (Basic Solution) before
precipitation and processing with dehydrothermal treatment
(4.times. magnification) wherein the Basic Solution is prepared and
applied as an aqueous gel which evenly fills the pores.
[0027] FIG. 4A is a photomicrograph of a rehydrated
double-structured tissue implant (DSTI) showing a primary and
secondary scaffold (4.times. magnification). FIG. 4A demonstrates
the formation of the double-structured implant, where the secondary
scaffold is observed from the fibrous-like diffraction pattern
present within the pores of the primary scaffold. The diffraction
pattern is created from the polymerization of the collagen within
the pores. The collagen fibers interdigitate within the pores and
among the pores. FIG. 4B is a photomicrograph of the dehydrated
double-structured tissue implant prior to implantation showing a
primary and secondary scaffold (4.times. magnification). Similarly
to FIG. 4A, FIG. 4B shows the double-structured implant wherein the
secondary scaffold is seen as the fibrous-like diffraction pattern
present within the pores of the primary scaffold.
[0028] FIG. 5 is a photomicrograph of a double-structured tissue
implant seeded with chondrocytes after 14 days in culture showing a
primary scaffold, a secondary scaffold and chondrocytes attached to
or embedded within the secondary scaffold localized in the pores of
the primary scaffold (10.times. magnification). The DSTI shown in
FIG. 5 was dehydrothermally treated and subsequently has undergone
rehydration with a phosphate buffered saline and seeding with
chondrocytes that were maintained in culture over a period of 14
days. The cultured chondrocytes are shown to adhere to the fibrous
secondary scaffold, as well as aggregate within the pores, seen in
the upper left corner.
[0029] FIG. 6 is a graph demonstrating collagen retention in
phosphate buffered saline, and its resistance to dissolution from
three separate double-structured tissue implants (DSTIs) compared
to a composite consisting of a primary scaffold loaded with a
composition for a secondary scaffold but not lyophilized or
dehydrothermally processed (Composite). FIG. 6 demonstrates the
structural stability of the collagen network present in the
double-structured implant (DSTI) as a function of time (in days) in
an aqueous buffered saline solution. The data demonstrate that
during the first day following the rehydration, there is very
little dissolution of collagen from the DSTI and that the retention
of collagen is close to 100% during the initial first critical hour
in all three DSTIs (Lots 1-3). On the other hand, the dissolution
from the Composite in the initial hour is much higher and retention
decreases immediately to approximately 96% during that same
critical first hour. FIG. 6 thus clearly demonstrates a stability
of the DSTI.
[0030] FIG. 7 is a graph illustrating percent of surface area
change of double-structured tissue implant (DSTI) and a composite
(Composite) of a primary scaffold loaded with a composition for a
secondary scaffold before lyophilization and dehydrothermal
processing, from 1 to 24 hours. The implants were rehydrated with
an aqueous phosphate buffered saline and maintained in culture for
eight days. Results show that there is an insignificant small
change in the surface area during the first hour following the
rehydration in both DSTI and Composite. This figure confirms that
in the double-structured tissue implants (DSTI) subjected to the
dehydrothermal treatment there is a very small change in the
surface area and therefore no shrinkage or swelling following the
rehydration.
[0031] FIG. 8 is a graph showing production of S-GAG/DNA by
chondrocytes seeded in double-structured tissue implant (DSTI) and
in a composite (Composite) comprising a primary scaffold loaded
with a composition for a secondary scaffold before lyophilization
and dehydrothermal processing, after 14 days in culture. FIG. 8
demonstrates that secondary scaffold supports the growth of cells
and deposition of extracellular matrix measured here as sulfated
glycosaminoglycan. A comparison between the double-structured
tissue implant and the Composite showed comparable results with
little evidence of significant steric hindrance due to the added
structural components.
[0032] FIG. 9A is a schematic illustration of a basic method for
implantation of the double-structured tissue implants (DSTI) into
tissue defects or lesions. FIG. 9B shows the same method with
microfracture pretreatment. FIGS. 9A and 9B illustrate the
implantation method in a surgical operating room setting. The
defect site is prepared and an initially oversized
double-structured tissue implant (DSTI) is cut and trimmed to match
the size and shape of the defect. In FIG. 9B, subchondral plate of
the defect site is penetrated using the microfracture technique.
The precut DSTI is rehydrated with physiologically acceptable
solution and placed into the defect. Alternatively, the dehydrated
DSTI is implanted and rehydrated in situ. The DSTI is rehydrated
with a physiologically acceptable solution optionally containing
cells, cell progenitors or agents that stimulate healing. The
defect site is sealed with a tissue adhesive applied over the
implant. Optionally, the same or different adhesive is also applied
to the defect site to coat the defect site (FIG. 9C) before the
implant is placed. In case of a microfractured defect as seen in
FIG. 9D, the adhesive is applied between microfracture
penetrations. FIG. 9E illustrates a method for implantation of DSTI
seeded with cultured cells activated with a hydrostatic pressure
regimen.
DEFINITIONS
[0033] As used herein:
[0034] "Primary scaffold" means a porous honeycomb, sponge, lattice
or another structure made of collagen or collagen based material
having randomly or non-randomly oriented pores of substantially
homogenous defined diameter. Under the most favorable conditions,
the pores are vertically oriented and represent a high percentage
of the porosity of the scaffold
[0035] "Secondary scaffold" means a collagen based structured
prepared from a collagen or collagen based compound and a non-ionic
surfactant. The secondary scaffold is generated within the primary
scaffold by introducing a composition comprising a soluble collagen
solution in combination with a non-ionic surfactant (Basic
Solution) into the pores of the primary scaffold and solidifying
said composition within said pores using a process of the
invention.
[0036] "Basic Solution" means a solution comprising a collagen in
admixture with a surfactant, preferably PLURONIC.RTM.-type
surfactant, neutralized to the pH of about 7.4. Basic Solution is
used for preparation of the secondary scaffold.
[0037] "Composite" means a primary scaffold loaded with a
composition comprising a precipitated or gelled soluble collagen in
combination with a non-ionic surfactant (Basic Solution). The
composite is in a hydrated form because the Basic Solution is added
in a fluid form as a gel, suspension or solution.
[0038] "Lyophilized composite" means the hydrated "composite", as
defined above, that is subsequently subjected to a dehydration and
lyophilization step.
[0039] "Double-structured tissue implant" or "DSTI" means a tissue
implant prepared according to a process of the invention wherein
the primary scaffold is loaded with a Basic Solution thereby
forming a composite that is subsequently subjected to
precipitation, dehydration and lyophilization to obtain lyophilized
composite that is subsequently treated with dehydrothermal (DHT)
treatment to result in a stable double-structured tissue
implant.
[0040] "Surfactant" means a non-ionic or ionic surfactant polymer.
Suitable surfactants, such as PLURONIC.RTM.-type polymers or
TRITON.RTM.-type polymers, are non-ionic co-polymer surfactants
consisting of polyethylene and polypropylene oxide blocks.
TRITON.RTM.-type surfactants are commercially available derivatized
polyethylene oxides, such as for example, polyethylene oxide
p-(1,1,3,3-tetramethylbutyl)-phenyl ether, known under its trade
name as TRITON.RTM.-X100. Other TRITON.RTM.-type surfactants that
may be suitable for use in the instant invention are TRITON.RTM.
X-15, TRITON.RTM. X-35, TRITON.RTM. X-45, TRITON.RTM. X-114 and
TRITON.RTM. X-102. TRITON.RTM. surfactants are commercially
available from, for example, Union Carbide, Inc. PLURONIC.RTM.-type
surfactants are commercially available block co-polymers of
polyoxyethylene (PEO) and polyoxypropylene (PPO) having the
following generic organization of polymeric blocks: PEO-PPO-PEO
(Pluronic) or PPO-PEO-PPO (Pluronic R). Exemplary
PLURONIC.RTM.-type surfactants are PLURONIC.RTM. F68, PLURONIC.RTM.
F127, PLURONIC.RTM. F108, PLURONIC.RTM. F98, PLURONIC.RTM. F88,
PLURONIC.RTM. F87, PLURONIC.RTM. F77, PLURONIC.RTM. F68,
PLURONIC.RTM. 17R8 and PLURONIC.RTM. 10R8.
[0041] "The porosity" means a pore size defined by the diameter of
holes within the primary scaffold as well as density of the pore
distribution as a function of cross-sectional area in millimeters.
Porosity is defined as a total volume of pores relative to the
implant.
[0042] "Substantially homogeneous" means at least 85-99%
homogeneity. Preferable homogeneity is between 95% and 99%.
[0043] "Substantially homogeneous porosity" means that a pore size
and diameter is within pore size range of about 200 to 300.+-.100
cm, preferably 300+50 cm, in diameter.
[0044] "Wettability" means an ability to quickly absorb a fluid
into the DSTI without changes in the size and shape of the
implant.
[0045] "Shrinkage" means a volumetric reduction in surface area in
all dimensions of a double structured tissue implant.
[0046] "Swelling" means a volumetric increase of a surface area in
all dimensions of a double structured tissue implant.
[0047] "Dissolution" means the act of a solid matter being
solubilized by a solvent.
[0048] "Rehydration" means the act of hydrating, wetting or
rewetting a dehydrated composite, lyophilized composite, stand
alone secondary scaffold or double structured tissue implant.
Rehydration may be performed before the implantation or after the
implantation of the DSTI into the tissue defect. Rehydration
utilizes a medium or fluid consisting of physiological fluid alone
or mixed with cells, stem cells, bone marrow aspirate, bone marrow
stem cells, drugs, or growth modulators.
[0049] "Dehydrothermal treatment" means removing water at low
pressure and at high temperature for cross-linking of polymers.
[0050] "Top surface" means an apical or synovial side of the matrix
turned toward the joint.
[0051] "Bottom surface" means basal, closest to bone surface of the
matrix.
[0052] "Cell" or "cells" means chondrocytes, synovial cells, tendon
cells, ligament cells, bone cells, mesenchymal stem cells,
embryonic stem cells, satellite cells, progenitor cells, cell
lines, virally transfected cell lines and any other cells that have
capability to form a differentiated tissue, such as, for example,
connective muscle or endothelial tissue.
[0053] "Tissue" means cartilage, ligament, tendon, bone, connective
tissue, nervous tissue, muscle, heart tissue, endothelial or spinal
cord tissue.
[0054] "Chondrocytes" means the cells naturally residing in
articular cartilage.
[0055] "S-GAG" means sulfated glycosaminoglycan.
[0056] Substantially" means at least 70%.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The current invention is directed to a method of use of a
double-structured tissue implant suitable for implantation into
tissue defects and lesions in a clinical setting for repair of
tissue lesions. The double-structured implant has improved
properties compared to a single structured implant and provides for
variability in use. The double-structured tissue implant (DSTI)
comprises a primary collagen-based structure, hereinafter called a
primary scaffold, and a secondary collagen-based structure,
hereinafter called a secondary scaffold. The two scaffolds are
structurally and functionally different. Each scaffold is prepared
from a different collagen-based composition with the primary
scaffold prepared first and the secondary scaffold prepared by
introducing a different collagen-based composition within the
primary scaffold thereby forming a composite structure comprising
the primary scaffold incorporated with a solution comprising a
collagen in admixture with a surfactant (Basic Solution) for
preparation of said secondary scaffold. The composite structure
(Composite) is then subjected to a lyophilization and
dehydrothermal treatment process according to the invention.
[0058] The resulting double-structured tissue implant (DSTI) thus
contains a structurally different secondary scaffold incorporated
into and localized within pores of the primary scaffold.
[0059] The actual photograph of a double-structured tissue implant
provided to a surgeon is seen in FIG. 1 in two manifestations.
[0060] FIG. 1A shows a close-up view of a DSTI, as delivered to the
surgeon in ready form for implantation. Before implanting, the
surgeon cuts the piece of the implant and trims it to a size and
shape corresponding to the size and shape of the defect or lesion,
places the precut section into the defect and rehydrates the
implant with sterile phosphate buffered saline or another
physiologically acceptable solution optionally containing cells,
such as chondrocytes, fibroblasts, mesenchymal stem cells, bone
marrow aspirate, bone marrow stem cells or any other cells, cell
suspensions or solutions containing growth hormone, mediators,
drugs, etc., as appropriate. Alternatively, such rehydration may be
performed before implanting said implant into the defect. The
precut implant is suitable for placement within a full thickness
defect of a tissue, and particularly for placement into an
articular cartilage lesion. Once in place, the double-structured
tissue implant can be held in place by suture, biologically
acceptable adhesive or a combination of both.
[0061] FIG. 1B shows the double-structured tissue implant as a dry
sterile product enclosed in a shipping container within sterile
packaging. The packaged product is available as an off-the-shelf
DSTI for implantation in clinical settings. Alternatively, the DSTI
may be prepared in a sterile wet rehydrated form optionally
containing cells, drugs or modulators and delivered to a surgeon in
a ready-to-use form.
[0062] I. Double-Structured Tissue Implant
[0063] A double-structured tissue implant (DSTI) comprises two
separately prepared components, namely the primary scaffold that
provides a structural support for the secondary scaffold
incorporated within the primary scaffold.
[0064] A. The Primary Scaffold
[0065] The primary scaffold is a collagen-based matrix prepared as
a honeycomb, lattice, sponge or any other similar structure made of
a biocompatible and/or biodegradable collagen containing material
of defined density and porosity that is pliable, storageable and,
most importantly, highly porous.
[0066] Typically, the primary scaffold is prepared from collagen,
collagen-containing composition or collagen containing a polymer.
Representative compounds suitable for preparation of the primary
scaffold are a Type I collagen, Type II collagen, Type III
collagen, Type IV, Type VI collagen, gelatin, collagen containing
agarose, collagen containing hyaluronan, collagen containing
proteoglycan, collagen containing glycosaminoglycan, collagen
containing glycoprotein, collagen containing glucosamine, collagen
containing galactosamine, collagen containing fibronectin, collagen
containing laminin, collagen containing a bioactive peptide growth
factor, collagen containing cytokine, collagen containing elastin,
collagen containing fibrin, collagen containing polylactic,
polyglycolic or polyamino acid, collagen containing
polycaprolactone, collagen containing polypeptide, or a copolymer
thereof, each alone or in combination with other collagen, such as
Type IX and XI.
[0067] Additionally, the primary scaffold may be prepared from the
collagen precursors, such as, for example, peptide monomers, such
as alpha 1 (type I), and alpha 2 (type I) collagen peptide or alpha
1 (type I) alpha 2 (type I) peptides, alone or in combination, or
from a combination of precursors, such as 2 (alpha 1, type I)
peptide and 1 (alpha 2, type I) peptide.
[0068] The collagen containing material used for preparation of the
primary scaffold may further be supplemented with other compounds,
such as pharmaceutically acceptable excipients, surfactants,
buffers, additives and other biocompatible components.
[0069] Preferably, the primary scaffold of the invention is
prepared from collagen and most preferably from Type I collagen or
from a composition containing Type I collagen.
[0070] In one embodiment, the primary scaffold is a structure
containing a plurality of narrowly defined randomly or non-randomly
organized pores having a substantially homogeneous narrowly defined
size and diameter that are uniformly distributed through the
scaffold, dividing the scaffold space into columns or pore network.
Under the most favorable conditions, the pores are through and
mostly vertically oriented, and represent a high percentage of the
total volume of the scaffold. The exemplary primary scaffold is
described in the co-pending application Ser. No. 11/523,833, filed
on Sep. 19, 2006, herein incorporated by reference in its
entirety.
[0071] In another embodiment, the primary scaffold may be the Type
I collagen-based support matrix that is a collagen-based porous
honeycomb, sponge, lattice, sponge-like structure or honeycomb-like
lattice of defined porosity having randomly or non-randomly
organized pores of variable pore diameters such as described in,
for example, application Ser. No. 10/882,581, filed on Jun. 30,
2004, issued as patent 7,217,294 on May 15, 2007, herein
incorporated by reference in its entirety.
[0072] In yet another embodiment the primary scaffold is a
honeycomb collagen matrix developed by Koken Company, Ltd., Tokyo,
Japan, under the trade name Honeycomb Sponge, described in the
Japanese patent JP3170693, hereby incorporated by reference. The
primary scaffold according to the invention has, preferably, a
substantially defined pore size in diameter and pore density in
randomly or non-randomly organized manner that creates an apical
(top) or basal (bottom) surface to the implant where the sizes and
diameters of the pores on both the apical or basal surface are
substantially the same, that is, at least 70% of the pores have the
same size and diameter. When used as a primary scaffold only, the
scaffold provides conditions for a sterically-enhanced enablement
of cells. Chondrocytes, for example, produce an extracellular
matrix comprising glycosaminoglycan and Type II collagen within
said implant in ratios characteristic for a normal healthy
articular cartilage.
[0073] A microphotograph of the primary scaffold is shown in FIG.
2. FIG. 2 is a representation of one embodiment of the primary
scaffold that is obtained and used as the structural foundation for
preparation of the double-structured tissue implant. As seen in
FIG. 2, the primary scaffold has a porous honeycomb structure of
relatively uniform pore size and equal distribution.
[0074] A secondary scaffold structure is generated within the pores
of the primary scaffold. To that end, the primary scaffold is
loaded with a Basic Solution suitable for preparation of the
secondary scaffold (Basic Solution). Such Basic Solution comprises
a soluble collagen, collagen-containing or collagen-like mixture,
typically of Type I collagen, in combination with a non-ionic
surfactant. The primary scaffold loaded with the Basic Solution for
formation of the secondary scaffold is shown in FIG. 3.
[0075] FIG. 3 is a microphotographic representation of the primary
scaffold that has been loaded with the Basic Solution comprising
solubilized collagen/surfactant composition that forms a basis for
formation of the secondary scaffold. The Basic Solution is prepared
as a solution, suspension or as an aqueous gel at a dilute acidic
pH and is further neutralized to pH 7.4. The Basic Solution is then
applied to or is loaded into the primary scaffold such that it
evenly fills the porous structure of the primary scaffold.
[0076] B. The Secondary Scaffold
[0077] The secondary scaffold is created or generated within the
pores of the primary scaffold. The secondary scaffold is a
qualitatively different structure formed within the confines of the
first scaffold or as a stand alone unit (see below).
[0078] The secondary scaffold is generated by a process comprising
preparing a soluble collagen-based composition as described below,
further comprising a suitable non-ionic or ionic surfactant (Basic
Solution).
[0079] The secondary scaffold comprises a collagen, methylated
collagen, gelatin or methylated gelatin, collagen-containing and
collagen-like mixtures, said collagen being typically of Type I or
Type II, each alone, in admixture, or in combination and further in
combination with a surfactant, preferably a non-ionic surfactant.
The suitable surfactant is preferably a polymeric compound such as
a PLURONIC.RTM.-type polymer.
[0080] Additionally, the secondary scaffold may be used
independently of the primary scaffold as said secondary scaffold
stand alone implant or unit where the Basic Solution can be
introduced into a mold or container and subjected to precipitation,
lyophilization and dehydrothermal treatment.
[0081] In preparation of the DSTI, said composition suitable for
generation of the secondary scaffold within the primary scaffold is
brought into contact with a primary scaffold structure by
absorbing, wicking, soaking or by using a pressure, vacuum, pumping
or electrophoresis, etc., to introduce said composition for the
secondary scaffold into the pores of the primary scaffold. In
alternative, the primary scaffold may be immersed into the Basic
Solution for the secondary scaffold.
[0082] C. Double-Structured Tissue Implant
[0083] The double structured tissue implant (DSTI) is prepared by
treating the primary scaffold loaded with a Basic Solution
comprising combination of the soluble collagen and non-ionic
surfactant subjected to a process for preparation of the DSTI
described below in Scheme 1.
[0084] Briefly, the primary scaffold is loaded with the
collagen/surfactant combination, precipitated or gelled, washed,
dried, lyophilized and dehydrothermally treated to solidify and
stabilize the secondary scaffold within the pores of the primary
scaffold.
[0085] A dehydrated (dry) and rehydrated double-structured tissue
implant is seen in FIGS. 4A and 4B. In these figures, the primary
scaffold pores are seen as delineating black lines and the pores of
the primary scaffold are filled with the secondary scaffold, where
the secondary scaffold is observed from the fibrous-like
diffraction pattern present within the pores of the primary
scaffold. Such diffraction pattern occurs due to the polymerization
of the collagen within the pores. The collagen fibers interdigitate
within the pores and among the pores.
[0086] The double-structured tissue implant can be seeded with
cells, loaded with pharmaceutical agents, drugs or growth
modulators. Additionally and preferably, the two of its distinct
components, namely the primary scaffold and the secondary scaffold,
can each be independently loaded with living cells, cell
suspension, with a pharmaceutically effective agent or agents or
with growth modulators. These may be loaded into the implant
individually or in any possible combination, such as, for example,
where the cells may be introduced into one component, for example,
into the primary scaffold of the DSTI, and the drug is introduced
into the second component, for example, into the secondary scaffold
of the DSTI, or the drug is introduced into one component and the
modulator into the second component and/or any variation thereof.
Both components of the DSTI may be loaded with the same or
different agent or with a combination of agents.
[0087] The DSTI loaded with chondrocytes is shown in FIG. 5 wherein
the cells are attached to the secondary scaffold.
[0088] FIG. 5 is a microphotograph of the dehydrothermally treated
double-structured tissue implant that was subjected to rehydration
with a solution containing chondrocytes where the chondrocytes were
deposited within the DSTI after being maintained in culture over a
period of several days. FIG. 5 shows cells adherent to the fibrous
secondary scaffold, as well as being present as aggregates within
the pores.
[0089] D. The Drug Containing Double-Structured Tissue Implant
[0090] The double-structured implant of the invention provides for
a variability of uses. One embodiment of the use is the
double-structured tissue implant containing the pharmaceutical
agent, drug, growth modulator, growth hormone, mediator, enzyme
promoting cell incorporation, cell proliferation or cell division,
pharmaceutically acceptable excipient, additive, buffer etc.
[0091] The drug may be introduced separately into the primary
scaffold, into the secondary scaffold, or both, or be added at a
time of rehydration to a composition for the secondary scaffold
before its processing. Because of the collagen structure of the
DSTI, drugs or modulators may be bonded to the DSTI through a
covalent linkage, such as for example, amide or ester bonds, or
ionic charge or hydrogen bonding.
[0092] The pharmaceutical agents, drugs or modulators are selected
from the group consisting of:
[0093] growth and morphogenic factors, such as, for example,
transforming growth factor, insulin-like growth factor 1,
platelet-derived growth factor, bone morphogenetic proteins
(bmps);
[0094] cytokines, such as, for example, interleukins, chemokines,
macrophage chemoattractant factors, cytokine-induced neutrophil
chemoattractants (gro-1), integral membrane proteins such as
integrins and growth factor receptors;
[0095] membrane associated factors that promote growth and
morphogenesis, such as, for example, repulsive guidance
molecules;
[0096] cell attachment or adhesion proteins, such as, for example,
fibronectin and chondronectin;
[0097] hormones, such as, for example, growth hormone, insulin and
thyroxine;
[0098] pericellular matrix molecules, such as perlecan, syndecan,
small leucine-rich proteoglycan and fibromodulin;
[0099] nutrients, such as, for example, glucose and
glucosamine;
[0100] nucleic acids, such as, for example, RNA and DNA;
[0101] anti-neoplastic agents, such as, for example, methotrexate
and aminopterin;
[0102] vitamins, such as, for example, ascorbate and retinoic
acid;
[0103] anti-inflammatory agents, such as, for example, naproxen
sodium, salicylic acid, diclofenac and ibuprofen;
[0104] enzymes, such as, for example, phosphorylase, sulfatase and
kinase; and
[0105] metabolic inhibitors, such as, for example, RNAi,
cycloheximide and steroids.
[0106] These, and other similar compounds and/or compounds
belonging to the above-identified groups may be added individually
or in combination to a primary scaffold, to a secondary scaffold,
to a composition (Basic Solution) for formation of the secondary
scaffold or to the lyophilized composite or DSTI before, during or
after implantation.
[0107] Addition of agents such as growth factors, cytokines and
chemokines will increase cell migration, cell growth, will maintain
or promote appropriate cell phenotype and will stimulate
extracellular matrix synthesis. Loading the scaffold with
anti-inflammatory agents or other drugs can provide a local
site-specific delivery system.
[0108] The range of concentration of the added drug or compound
depends on the drug or compound and its function and it extends
from picograms to milligrams.
[0109] E. Cellular Deposition
[0110] The DSTI has the capacity for preloading of differentiated
or undifferentiated cells to augment tissue repair. In the case of
differentiated cells, chondrocytes, osteoblasts, tenocytes,
fibroblasts, fibrochondrocytes and ligament cells can be isolated
and applied by infusion, dropwise, wicking, pumping or injection,
for healing of cartilage, bone, tendon, skin, meniscus or ligament,
respectively, or for other tissue defects.
[0111] In the case of undifferentiated cells, adult or immature
mesenchymal cells derived from bone marrow aspirates, iliac crest
needle biopsies or other dissociable mesenchymal tissues, such as
somites, muscle, interstitial connective tissues, through enzymatic
dissociation and subsequent culture, can be applied in methods
similar or identical to differentiated cells. Other mature or
immature undifferentiated cells may be from immortalized cell
lines, hematopoietic stem cells, neural stem cells each having the
capacity for differentiation in situ or may include embryonic stem
cells which when isolated and placed within the localized defect
can undergo differentiation to the target tissue.
[0112] The number of cells to be applied may vary in accordance
with the differentiated state of the cells and with their inherent
proliferative capacity. The age of the cells may vary with origin
and with time in culture.
[0113] F. Secondary Scaffold as a Stand Alone Implant
[0114] In one embodiment, the secondary scaffold may be generated
as a stand alone structure. In this regard, a composition
comprising a soluble collagen, methylated collagen, gelatin or
methylated gelatin in an acidic solution further comprising a
non-ionic surfactant is subjected to neutralization, precipitation,
dehydration, lyophilization and dehydrothermal treatment under
conditions as described in the Scheme 1.
[0115] As described for the double structured tissue implant, the
secondary scaffold as a stand alone implant may be similarly loaded
with cells and may optionally contain a pharmaceutical agent,
growth modulator or another compound before or after implantation,
as described above.
[0116] The process for preparation of the stand alone secondary
scaffold is modified to the extent that the composition for
preparation of the secondary scaffold (Basic Solution) is placed
into a container suitable to permit gelling, precipitation,
dehydration, lyophilization and dehydrothermal treatment.
[0117] The stand alone secondary scaffold is used for implantation
in the same manner as described for double-structured tissue
implant. The stand alone secondary scaffold is useful in the
healing of tears in cartilage or skeletal tissues, such as, for
example, the meniscus where it can be charged and/or supplemented
with all of the tissue factors and cells, such as meniscal
fibroblasts.
[0118] The stand alone secondary scaffold can be used in a similar
fashion for bone, tendon and ligament repair.
[0119] Components and conditions suitable for preparation of the
secondary scaffold stand alone structure and evaluation of its
performance using cell viability are seen in Table 1.
TABLE-US-00001 TABLE 1 Pluronic Collagen F127 DHT, Dissolution Cell
conc. conc. 6 h at Rehydration stability in viability (mg/ml)
(mg/ml) 140.degree. C. time (s) PBS at 37.degree. C. (%)
Precipitated or gelled using ammonia vapor 3 0.25 Y 12 Stable -- 3
1 Y 13 Stable -- 3 1 Y <10 Stable 97 3 1 N <10 Dissolved -- 3
3 Y <10 Stable 98 3 3 N <10 Dissolved -- 2.9 0.29 Y 6 Stable
-- 2.9 0.29 N 3 Dissolved -- 2 0.165 Y 7 Stable -- 2 0.33 Y 3
Stable -- 2 0.67 Y 2 Stable -- 2 0.25 Y 15 Stable -- 2 0.5 Y 7
Stable -- 2 0.1 Y 4 Stable -- 2 2 Y <10 Stable 99 2 2 N <10
Dissolved -- 1.5 0.15 Y <10 Dissolved -- 1.5 0.15 N 16 Dissolved
-- Precipitated or gelled using NaOH 2.3 0.05 Y 23 Stable -- 2.3
0.1 Y 19 Stable -- 2.3 0.23 Y 15 Stable -- 2.4 0.24 Y 5 Stable
--
[0120] Table 1 summarizes experimental conditions used for
determination of optimization of conditions for preparation of a
secondary scaffold. The conditions tested and evaluated were a
collagen concentration, surfactant concentration, temperature and
time for dehydrothermal treatment (DHT), rehydration time,
stability of the DSTI determined by dissolution of the secondary
scaffold in phosphate buffer saline at 37.degree. C., and cell
viability.
[0121] The secondary scaffold was precipitated in the presence of
ammonia vapor or ammonia aqueous solution or in the presence of
0.1M sodium hydroxide (NaOH).
[0122] Results seen in Table 1 show the effectiveness of
dehydrothermal treatment for preparation of secondary scaffolds, in
terms of achieving the stability of the secondary scaffold, its
fast rehydration and assuring cell viability within the secondary
scaffold.
[0123] As seen from the results summarized in Table 1, following
ammonia precipitation of the collagen, dissolution stability was
not observed in the absence of dehydrothermal treatment in spite of
varying collagen and surfactant concentrations. In instances where
stability was achieved, there was excellent cell loading and
viability at collagen concentrations greater than 1-5 mg/ml and at
surfactant concentrations of 1, 2 and 3 mg/ml.
[0124] In an alternative approach, the precipitation of collagen by
neutralization with NaOH, in the presence of Pluronic surfactant
and subject to DHT, detected formation of a rapidly rehydrating and
stable secondary scaffold.
[0125] These results clearly show that the properties of the
secondary scaffold alone or the secondary scaffold incorporated
into the DSTI may be conveniently optimized to achieve fast
rehydration time, dissolution stability and excellent cell loading
and cell viability up to 99% within the secondary scaffold.
[0126] G. Surfactants
[0127] Improved properties of the DSTI, such as its rapid
wettability and resistance to shrinkage, swelling and dissolution,
are due to a presence of a secondary scaffold as a distinct
functional entity.
[0128] The secondary scaffold prepared according to the process of
the invention requires, as an essential part, a presence of a
surfactant, preferably a non-ionic or, in some instances, even an
ionic surfactant. The surfactant, preferably the non-ionic
surfactant of type such as TRITON.RTM. or PLURONIC.RTM., preferably
PLURONIC.RTM. F127, is an essential component of a composition used
for preparation of the secondary scaffold, or micellar substrate
bound to the implant. The presence of the surfactant improves
stability and particularly wettability and rehydration properties
of the implant without causing its shrinkage or swelling.
[0129] Suitable surfactants, such as PLURONIC.RTM.-type polymers or
TRITON.RTM.-type polymers, are non-ionic co-polymer surfactants
consisting of polyethylene and polypropylene oxide blocks.
[0130] TRITON.RTM.-type surfactants are commercially available
derivatized polyethylene oxides, such as for example, polyethylene
oxide p-(1,1,3,3-tetramethylbutyl)-phenyl ether, known under its
trade name as TRITON.RTM.-X100. Other TRITON.RTM.-type surfactants
that may be suitable for use in the instant invention are
TRITON.RTM. X-15, TRITON.RTM. X-35, TRITON.RTM. X-45, TRITON.RTM.
X-114 and TRITON.RTM. X-102. TRITON.RTM. surfactants are
commercially available from, for example, Union Carbide, Inc.
[0131] PLURONIC.RTM.-type surfactants are commercially available
block co-polymers of polyoxyethylene (PEO) and polyoxypropylene
(PPO) having the following generic organization of polymeric
blocks: PEO-PPO-PEO (Pluronic) or PPO-PEO-PPO (Pluronic R).
Exemplary PLURONIC.RTM.-type surfactants are PLURONIC.RTM. F68,
PLURONIC.RTM. F127, PLURONIC.RTM. F108, PLURONIC.RTM. F98,
PLURONIC.RTM. F88, PLURONIC.RTM. F87, PLURONIC.RTM. F77,
PLURONIC.RTM. F68, PLURONIC.RTM. 17R8 and PLURONIC.RTM. 10R8. The
most preferred non-ionic surfactant of PLURONIC.RTM.-type suitable
for use in the invention is a block co-polymer of polyoxyethylene
(PEO) and polyoxypropylene (PPO) with two 96-unit hydrophilic PEO
blocks surrounding one 69-unit hydrophobic PPO block, known under
its trade name as PLURONIC.RTM. F127. PLURONIC.RTM. surfactants are
commercially available from BASF Corp.
[0132] H. Properties of the Double-Structured Tissue Implant
[0133] The DSTI of the invention has distinctly improved properties
when compared to the primary scaffold alone, to the secondary
scaffold alone or to a composite loaded with a composition for
preparation of the secondary scaffold (Composite), unprocessed, or
to the Composite that has been dehydrated and lyophilized
(Lyophilized Composite).
[0134] Typically, a tissue implant is implanted into a tissue
defect during a surgery either already rehydrated (wet) or in a dry
form. Also typically, such surgery has a time-limit on implantation
that has about one hour window when the implant needs to be placed
into the defect. For these reasons, it is important that a
specification for an implantable double-structured tissue implant
provides stability, resistance to change in shape, size and
shrinkage or swelling, resistance to dissolution, consistency with
respect to pore size permitting an ingrowth of cells into the
implant and conditions for formation of extracellular matrix within
the implant. The DSTI appears to have all the above properties.
[0135] Furthermore, a presence of the secondary scaffold improves
the function of the DSTI by providing a multitude of small
membranous substrates which can provide cell anchorage and
phenotype stability while preserving the through porosity of the
overall implant, thereby allowing nutrients and growth factors and
migratory cells to permeate the implant.
[0136] In the case of cartilage lesion the migratory cells include
chondrocytes from the debrided lesion which have been freed from
damaged extra-cellular matrix through the upregulation of certain
matrix metalloproteinases.
[0137] I. Stability of the Double-Structured Tissue Implant
[0138] From the point of view of the implantability, stability of
the implant is one of the major requirements. The implant stability
depends on several factors. There must be minimally low or,
preferably, almost no initial dissolution of collagen from the
implant into the physiological fluids and there must be minimally
low or preferably no change in size and shape of the implant
following rehydration or wetting before, during or after surgery
prior to biodegradation in situ.
[0139] DSTI of the invention has a very minimal initial collagen
dissolution and a minimal change in size and shape during the
initial critical period.
[0140] 2. Collagen Retention and Resistance to Dissolution
[0141] One of the most important requirements for the implant is
its resistance to dissolution of its components upon wetting and
rehydration of said implant during implantation during preparation
of the implant for implantation and subsequently also after
implantation. A minimally low dissolution or, preferably, almost no
dissolution of the collagen component from the implant into the
physiologic solution after or before placement of the implant into
the tissue defect, and into an interstitial fluid, plasma or blood
following the surgery, under normal physiological conditions
ensures continued functionality of the implant following its
implantation into the tissue defect, such as, for example into the
cartilage lesion. Low or no dissolution of collagen from the
implant means the high retention of the collagen within the
implant.
[0142] FIG. 6 demonstrates the structural stability of the collagen
network present in the double-structured tissue implant as a
function of time following rehydration of the implant with an
aqueous phosphate buffered saline solution by comparing collagen
retention, in percent, within DSTI, to collagen retention within an
unprocessed composite (Composite) comprising a primary scaffold
loaded with a composition for a secondary scaffold (Basic Solution)
but not lyophilized or dehydrothermally processed.
[0143] Results seen in FIG. 6 demonstrate the structural stability
of the collagen network present in the double-structured tissue
implant as a function of time in an aqueous buffered saline
solution. The data demonstrate that during the first day following
the rehydration, there is very little dissolution of collagen from
the DSTIs (D#1-3) and that the retention of collagen is almost 100%
during the same initial first critical hour. On the other hand, the
dissolution from the Composite (-x-) in the initial hour is much
higher and the collagen retention drops immediately to
approximately 96% during that same critical first hour although it
stabilizes later on. FIG. 6 thus clearly demonstrates stability of
the DSTI.
[0144] In order to determine the stability of the implant subjected
to transport and handling, another study was performed with and
without agitation and the dissolution of collagen from of DSTI
under these conditions was compared to the dissolution of collagen
from the non-lyophilized composite (Composite). Results are not
shown. These studies confirmed that even with agitation, there is a
relatively small change in the accumulated release of protein into
the solution over a period of eight days but particularly during
the first hour following the rehydration.
[0145] 3. Resistance to Change in Size and Shape
[0146] Another important feature of the DSTI is its resistance to
change in size and shape. This feature is very important for
implant efficacy as any change in the size and shape by shrinking
or swelling can negatively affect the outcome of the implantation
surgery. An implant that would get smaller by shrinking will not
fill the defect, will not provide a structural support for
migration of cells from the surrounding tissue or cell integration
into the surrounding tissue and may also be dislodged from the
defect. Swelling of the implant could, on the other hand, cause the
implant to swell within the defect, decrease the structural support
for cells and be rejected or ejected from the defect because of its
larger size.
[0147] The resistance to change in shape and size means that for
implantation into a defect of discernable size, the functional
construct must not swell or shrink extensively upon rehydration
during time of preparation before surgery or after placement of the
implant into the defect.
[0148] FIG. 7 presents the percent change in surface area of DSTI,
rehydrated with and maintained in culture for more than 24 hours in
an aqueous phosphate buffered saline. The results seen in FIG. 7
show that there is very little change in size and shape during the
critical first hour in DSTI. In three lots of double-structured
tissue implants subjected to the dehydrothermal treatment there was
approximately a 2-5% change in surface area within the first hour
and such change was maintained within these parameters for more
than 24 hours following the rehydration.
[0149] 4. Cell Viability
[0150] Another important feature of the tissue implant is to
provide support and conditions for cell migration from surrounding
tissue or for the cell integration into surrounding tissue in the
case when the cells are seeded into the DSTI before implanting.
This feature is determined by cell viability within the DSTI and
provides another criteria for determining functionality and
usefulness of the DSTI.
[0151] In order for an implant to be functionally viable, the
implant must provide a structural support for cells as well as
provide or permit conditions to be provided for cell seeding into
the implant, cell growth within the implant and/or cell migration
into or from the surrounding tissue. Conditions for cell seeding,
their growth within the implant, their nutritional and metabolic
needs are designed based on the type of cells that the implant is
supposed to deliver and support. For example, if the implant is
designed for repair of a skin defect, the cells and their
requirement will be different than if the implant is designed for
repair of a chondral or bone lesion. Conditions for structural
support and conditions for promotion of cell growth, their
migration and/or integration into the surrounding tissue will be
adjusted based on the tissue where the DSTI will be implanted and
the function the implant will assume in repair of the tissue
defect.
[0152] While the DSTI of the invention is preferably suitable for
use in treatment and repair of chondral, subchondral or bone
lesions, the DSTI, as such, is suitable to be used for repair of
any other tissue or tissue defect.
[0153] To determine the cell survival within the DSTI, studies were
performed to determine the cell viability by determining their
survival and growth within the DSTI. Cell viability was determined
for three lots of DSTI that had been seeded with chondrocytes after
1 day and 21 days in culture. Results are seen in FIG. 8.
[0154] FIG. 8 shows production of sulfated glycosaminoglycan and
DNA (S-GAG/DNA) by chondrocytes seeded in double-structured tissue
implant (DSTI) and in a composite (Composite) comprising a primary
scaffold loaded with a composition for a secondary scaffold before
lyophilization and dehydrothermal processing, after 14 days in
culture.
[0155] Results seen in FIG. 8 demonstrate that inclusion of the
secondary scaffold within DSTI supports the growth of cells and
deposition of extracellular matrix measured here as production of
sulfated glycosaminoglycan. A comparison between the
double-structured tissue implant and the Composite showed
comparable results with little evidence of significant steric
hindrance due to the added structural components. All samples had
96-100% viability at both timepoints indicating no cell toxicity.
Furthermore, as with the DNA measurements, the total cell number
increased over time which shows that the cells were retained in the
DSTI, were viable and proliferated to fill the pores.
[0156] 5. Pore Size
[0157] The successful implant, such as, for example, wet or dry
DSTI implanted into the cartilage lesion, must provide conditions
allowing cells to form and generate a new extracellular matrix. In
this regard, the implant porous structure must allow cells to
migrate, be attached or aggregate into and within the pores and to
function similarly to their normal function in the healthy
tissue.
[0158] Consequently, the pore size of the implant and the
consistency with respect to pore size for the ingrowth of cells is
important both for cell adhesion, extracellular matrix production
and cell to cell contact and communication. Depending on the tissue
to be repaired, the pore size of the primary and/or secondary
scaffold will vary. For example, cartilage scaffolds would have an
optimal pore size of approximately 200 .mu.m and bone would have a
pore size in the range of 300 to 350 .mu.m.
[0159] A significant advantage of having a double-structured tissue
scaffold arises from the increase in mechanical integrity relative
to a primary porous collagenous material because the polymerization
creates fiber-like structure between the primary and secondary
scaffold that serves as a reinforcing network for cells.
[0160] 6. Surface Area
[0161] In addition, due to inclusion of the secondary scaffold
there is an increase in overall surface area within the DSTI that
permits cells spreading and migration throughout the interstices of
the DSTI. At the same time, the secondary scaffold must be designed
such that it is not of such high density that it becomes a blocking
agent that acts as a steric hindrance for cell ingrowth and tissue
repair.
[0162] The double-structured tissue implant of the invention
provides optimal conditions, such as implant stability, collagen
retention, resistance to change in size and shape of the implant,
pore size and surface area for viability and growth of cells within
the implant.
[0163] II. Process for Preparation and Use of the Double-Structured
Tissue Implant
[0164] The secondary scaffold is generated within confines of the
primary scaffold by a process comprising several stages and steps
as set forth in Scheme 1. The process stages comprise pre-loading,
loading, polymerization, treatment of composite double-structured
scaffold, dehydrothermal treatment, packaging and surgical
procedure.
SCHEME 1
Process for Production and Use of a Double-Structured Implant Stage
1-Pre-Loading
[0165] The pre-loading stage is a preparatory stage where the
primary scaffold is either obtained from commercial sources or is
prepared according to the procedure described in Example 1
Step 1
[0166] Step 1 comprises obtaining or preparing a primary scaffold,
typically a collagen containing honeycomb, sponge or lattice
providing a structural support for incorporation of the secondary
scaffold.
[0167] In one embodiment, a bovine Type I collagen matrix with
honeycomb (HC) like structure is obtained, for example, from Koken,
Inc. (Japan) or from other commercial sources and used as primary
scaffold. However, such commercially available honeycomb matrices
have typically randomly distributed pores of irregular shape and
size. The pores of these structures are not always vertically
positioned.
[0168] In another embodiment, and preferably, a primary honeycomb
scaffold is produced according to a process described in Example 1,
wherein said primary scaffold has randomly or non-randomly oriented
pores of substantially the same size and shape.
[0169] The shape and size of the primary scaffold determines a size
of the double-structured tissue implant (DSTI) ultimately delivered
to the surgeon for implantation into the tissue defect.
[0170] Typically the DSTI has a rectangular, circular or oval shape
with dimensions of about 50 mm and a vertical thickness of about 1
to 5 mm, preferable 1-2 mm. Preferred dimensions of the DSTI for
its preparation and, therefore, the dimensions of the primary
scaffold are 50.times.50 mm.times.1.5 mm, with pores oriented
substantially vertically, said pores having a pore size of from
about 100 to about 400 .mu.m, preferably about 200+100 .mu.m and
pore length of 1.5 mm. However, dimensions of the primary scaffold
may be any that are required by the tissue defect to be repaired
and that can be prepared by the process of the invention.
Step 2
[0171] Step 2 comprises preparing a composition for preparation of
a secondary scaffold (Basic Solution) and comprises neutralization
of a soluble collagen solution having an initial acidic pH of about
pH 1.5-4, preferably between about pH 1.9-2.2, a collagen
concentration from about 0.5 to about 10 mg/ml of collagen,
preferably about 2.9 to about 3.2 mg/ml, a surfactant concentration
from about 0.05 to about 10 mg/ml, preferably about 0.29 to about
0.32 mg/ml and osmolality from about 20 to about 400 mOsm/kg,
preferably about 28 to about 32 mOsm/kg. The soluble collagen
solution is then neutralized with any suitable base and/or buffer
to pH in a range from about pH 7.3 to about pH 7.7 to derive the
Basic Solution. Preferably, the solution is neutralized by
adjusting pH to neutrality 7.4 using a collagen/surfactant,
10.times. Dulbecco's phosphate buffered saline (DPBS) and 0.1 M
NaOH in 8:1:1 ratio or using an aqueous solution or ammonia vapor
in concentration sufficient to neutralize acid within the collagen
solution. The final osmolality and pH of the Basic Solution is
about 290 mOsm/kg and pH 7.4, respectively.
[0172] The suitable buffers for solubilization of the Type I
collagen are, for example, a formic acid containing buffer at pH
4.8, acetic acid containing buffer at pH 5.0 or a diluted
hydrochloric acid containing buffer at pH 3.0.
[0173] Neutralization is typically carried out using ammonia
aqueous solution or a vapor of ammonia, or in concentration
sufficient to neutralize the acidic pH over about 30 minutes to
about 24 hours, preferably for 12 to 24 hours. This factor has also
been found to affect the collagen polymerization and formation of
pores having homogeneous pore size. However, other means of
neutralization may also be conveniently used.
Stage 2--Loading and Precipitation
[0174] The primary scaffold is loaded with a Basic Solution for the
secondary scaffold comprising soluble collagen solution containing
a surfactant. This Basic Solution is subsequently precipitated
within pores of the primary scaffold.
[0175] Loading the primary scaffold with the Basic Solution for the
secondary scaffold is performed using any suitable method. Soaking,
wicking, submerging the primary scaffold in the solution,
electrophoresis and any other suitable means. Once the Basic
Solution for the secondary scaffold is introduced into the primary
scaffold, a composite of both is subjected to a process or
treatment that results in formation of the secondary scaffold
inside pores of the primary scaffold.
Step 3
[0176] The neutralized Basic Solution of step 2 is loaded into the
primary scaffold by placing from about 3.75 to about 7.5 ml
(approximately 1 to 2.times. volume), preferably a volume about 4.9
ml (approx. 1.3.times. volume of the primary scaffold) of the
secondary scaffold Basic Solution on the bottom of a dish and then
placing the primary scaffold in this solution and allowing it to be
soaked up.
Stage 3--Polymerization of a Soluble Collagen within a Primary
Scaffold
[0177] The primary scaffold loaded with the neutralized Basic
Solution comprising the soluble collagen and the surfactant is then
subjected to conditions resulting in precipitation of the
neutralized Basic Solution within the pores of the primary scaffold
thereby generating a structurally distinct secondary scaffold
(Composite).
[0178] Typically, and allowing for variability of the Basic
Solution or composition used for creating of the secondary
scaffold, the composition introduced into the pores of the primary
scaffold is gelled or precipitated within said primary scaffold and
may also be cross-linked using chemicals such as glutaraldehyde or
another multifunctional aldehyde, where the aldehyde reacts with
amino groups of the collagen yielding a Schiff base, which can be
stabilized by a reduction reaction; carbodiimide reagent, such as
carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)
with or without N-hydroxy-succinimide (NHS) where the HNS is used
to suppress side reactions. Additionally, EDC and NHS can be used
in combination with diamine or diacid compounds to introduce
extended cross-links; acyl azide where the acid are activated and
subsequently reacted with an advanced amine group; epoxy compounds
such as 1,4-butanediol diglycidyl ether, or cyanamide.
[0179] In addition, irradiation such as short wave length UV
irradiation (254 nm) can introduce cross-links in the collagen. The
primary scaffold loaded with the Basic Solution (neutralized
collagen/surfactant solution) in a range from about 1 to about 2
volumes of the primary scaffold is then placed in an incubator at a
temperature from about 25.degree. C. to about 38.degree. C.,
preferably to about 37.degree. C. temperature, typically for from
about 10 minutes to about 18 hours, more typically for about 20 to
about 100 minutes, preferably for about 40 to 60 minutes, and most
preferably for a time when the precipitation of the neutralized
collagen solution into a solid secondary scaffold occurs.
Step 5
[0180] In order to assure that the vast majority of the salt of the
precipitated collagen solution within the pores of the primary
scaffold is removed, a composite consisting of the primary scaffold
having the secondary scaffold precipitated within is subjected to a
washing step whereby the majority of the salts are removed.
[0181] The composite (Composite) comprising the primary scaffold
and the secondary scaffold precipitated within, is washed by
placing said composite in a volume of from about 20 ml to about 10
liters, preferably about 500 ml, of de-ionized water further
containing a non-ionic surfactant. The surfactant is typically
present in concentration from about 0.05 to about 1.0 mg/ml,
preferably about 0.23 mg/ml. Most preferred surfactant is
PLURONIC.RTM. F127.
[0182] Typically, the washing step takes approximately 30 minutes.
There may be one or several washing step repetitions. All excess
non-precipitated collagen is removed during the extraction from the
composite into the wash solution. Polymerizing of the collagen
present in the secondary scaffold solution loaded within the
primary scaffold pores results in formation of a stable
double-structured composite, as defined above, comprising the
primary scaffold and the secondary scaffold precipitated
therewithin having shape memory.
[0183] Following the precipitation or gelling and washing, the
composite is subjected to lyophilization and dehydrothermal
treatment.
Stage 4--Dehydration of Composite Double-Structured Scaffold
[0184] The solid double-structured composite is then dehydrated
using any method suitable for such dehydration. Typically, such
dehydration will be freeze-drying or lyophilization. Freezing is
typically carried out at temperature from about -10.degree. C. to
about -210.degree. C., preferably from about -80.degree. C., over a
period of about 2 to about 60 minutes. The frozen composite is then
lyophilized forming the Lyophilized Composite.
[0185] The gradual nature of the polymerization and slow process of
water removal typically maintains the architectural elements of the
secondary scaffold and achieves the proper orientation and diameter
of the pores.
Step 6
[0186] Dehydration is achieved with freezing the solid
double-structured composite by placing it on the metal shelf of a
freezer and adjusting the temperature to from about -10.degree. C.
to about -210.degree. C., preferably to about -60.degree. C. to
about -90.degree. C., and most preferably for about -80.degree. C.,
for about 2 to about 60 minutes, preferably for about 20-45 minutes
and most preferably for about 30 minutes.
Step 7
[0187] The frozen and dehydrated solid double-structured composite
is then subjected to lyophilization. The frozen dehydrated
composite is removed from the freezer and placed into a pre-cooled
lyophilization chamber. Lyophilization typically occurs in about
15-21 hours, depending on the size and shape of the composite but
is typically and preferably completed in about 18 hours.
Stage 5--Dehydrothermal Treatment
[0188] To further stabilize the composite and to achieve necessary
stability, resistance to dissolution and sterility of the final
product, the solid double-structured composite is subjected to
dehydrothermal (DHT) treatment. DHT treatment achieves
cross-linking of the collagen with the surfactant and at higher
temperatures also sterilizes the DSTI.
[0189] Cross-linking step prevents dissolution of the secondary
scaffold upon rehydration before or after implantation.
Step 8
[0190] This step is performed to sterilize and cross-link the
double structured tissue implant.
[0191] The lyophilized double-structured composite is placed into a
dry glass chamber or container and covered with the glass, aluminum
foil or another suitable material resistant to higher temperatures.
The container with the lyophilized double-structured composite is
placed into the pre-heated dehydrothermal oven and subjected to a
temperature in a range from about 70.degree. C. to about
200.degree. C., preferably from about 130.degree. C. to about
150.degree. C., and most preferably about 135.degree. C., under
vacuum, for about 30 minutes to about 7 days, preferably for about
5-7 hours and most preferably for about 6 hours.
[0192] Such treatment stabilizes the composite, makes it resistant
to collagen dissolution upon wetting, provides for rapid wetting
and assures none or minimal shrinkage or swelling upon wetting with
a physiological solution or buffer, and sterilizes the
double-structured tissue implant.
Stage 6--Packaging and Storage
[0193] The double-structured tissue implant fabricated by the
process described above is then ready for a sterile packaging and
storage. In this form, the DSTI has a long shelf-life.
Step 9
[0194] The double structured tissue implant is removed from the
dehydrothermal oven and transferred aseptically into sterile
environment, such as a Bio Safety Cabinet (BSC), where it is
packaged under conditions assuring sterility. The double-structured
tissue implant is then ready to be stored at room temperature until
its use.
Stage 7--Delivery by Implantation
[0195] Packaged double-structured tissue implant is delivered or
made available to a surgeon for implantation into a tissue
defect.
Step 10
[0196] During surgery, surgeon determines an extent of the defect
or lesion to be repaired, opens the packaged product, cuts the DSTI
to size of the defects and places the cut-to-size piece into said
defect. The implant may be wetted before the implantation and then
placed into the defect or alternatively, it may be placed into the
defect in a dry form and a suitable physiologically acceptable
solution may be then added to wet the implant in situ.
[0197] Since the implant is very stable, and does not change its
size or shape significantly by shrinking or swelling, the implant
fits tightly into the defect or lesion. To assure that the implant
stays within the defect or lesion, such defect or lesion is first
coated with a suitable tissue adhesive, sealant or glue that keeps
the implant in place. In alternative, the defect or lesion may be
pretreated with microfracture where the tissue underlying the
lesion or defect is microfractured with microchannels to permit the
blood and nutrient supply into the lesion or defect, lining the
defect or lesion but not the microfracture, with the adhesive, glue
or sealant and placing the implant as described above. In both
instances, the implant placed into the lesion or defect may
optionally be covered with another layer of the adhesive, sealant
or glue.
[0198] In some instances, cells, drugs or modulators may be loaded
into the DSTI or attached to the second scaffold before
implantation and wetting, during wetting following the
implantation, or independently provided after the implantation.
[0199] Results obtained for three separate lots containing three
rehydrated DSTIs per each lot, are seen in Table 2. The DSTI is
rehydrated by placing a droplet of phosphate buffer saline
(1.5.times. volume of PBS), on top of the DSTI and the rehydration
time is measured as the time it takes for the DSTI to be completely
hydrated.
TABLE-US-00002 TABLE 2 Number of Sample Results Attribute (n/lot)
Lot #1 Lot #2 Lot #3 Reydra- 3 <2 <2 <2 tion Time
(seconds) Re- 3 <2 <2 <2 hydrated pH Re- 3 317 .+-. 6 356
.+-. 4 319 .+-. 1 hydrated Osmo- lality (mOsm/ kg) Size 3 99.8%
.+-. 5.2.+-. 100.6% .+-. 10.0% 99.7% .+-. 2.3% Variation at
Hydration (%) Collagen 3 99.4% .+-. 0.2% 99.1% .+-. 0.1% 99.2% .+-.
0.2% Retention in PBS (%)
[0200] As seen in Table 2, results obtained in three different lots
in three different studies are closely similar confirming the
reproducibility of the process as well as consistency of the
parameters observed after rehydration.
[0201] The rehydration time for each lot is less then 2 second
evidencing a very fast wettability of the DSTI products.
[0202] The pH of the rehydrated DSTI products is between 7.7 and
7.8 in all lots.
[0203] Osmolality of the rehydrated DSTI products is between 317
and 356 mOsm/kg in all lots.
[0204] Variation in size of rehydrated DSTI products is negligible
evidencing that there is no shrinkage or swelling upon hydration of
DSTI
[0205] Collagen retention within the rehydrated DSTI is above 99%,
evidencing a great stability of the DSTI products.
[0206] III. Method of Use of Double-Structured Tissue Implant
[0207] Double-structured tissue implant of the invention is useful
for treatment and repair of tissue defects of various tissues. Such
treatment is achieved by implanting the DSTI into the defect in
surgical setting.
[0208] A. Implantation of DSTI
[0209] In this regard, the use of DSTI, as described herein in
FIGS. 9A-9E illustrates its implantation of DSTI into the articular
cartilage lesion. However, the same or similar process would be
used for implantation of the DSTI into defect of any other
tissue.
[0210] There are two basic methods suitable for implantation of
DSTI into the tissue defect or lesion. The first method comprises
implantation of the DSTI into the lesion without any special
pretreatment of the lesion other than debriding and removing any
undesirable debris from the defect or lesion before the DSTI
deposition. The first method is illustrated in FIG. 9A. The second
method comprises pretreatment of the defect or lesion with a
microfracture technique. In such a case, a subchondral plate of the
lesion is penetrated with microchannels connecting the bottom of
the lesion or defect with underlying bone to permit the migration
of cells, blood and nutrients into the deposited DSTI within the
lesion or defect. The second method is illustrated in FIG. 9B.
[0211] Additionally, the implantation method comprises two
variations in attaching and sealing the DSTI within the defect or
lesion. In the first and preferred mode, the DSTI is placed into
the defect site and the tissue adhesive, preferably methylated
collagen methylated collagen-polyethylene glycol, is placed over
the defect containing the DSTI. The second variation of the
implantation method comprises, additionally, a step of placing a
second adhesive at the bottom of the lesion or defect before the
placement of DSTI. This variation is seen in FIGS. 9C and 9D.
Although it is preferred to use only one adhesive over the
implanted DSTI, in some instances, the use of the second adhesive
placed at the bottom of the lesion is warranted. For example, when
the DSTI is prepared as an implant seeded with in vitro cultured
and/or activated cells when the implant itself is already filled
with cells and extracellular matrix, as seen in FIG. 9E, rather
than being rehydrated with solution of cells or bone marrow, the
deposition of the bottom adhesive may be required. Adhesives
suitable for sealing the defect and securing it may be the same or
different and are, typically, compounds polymerizable within short
time from about 30 seconds to about 4 minutes with or without use
of a curing means.
[0212] The cells that may be seeded into the DSTI in vitro may be
cultured and activated using an intermittent hydrostatic pressure,
as described by inventors in copending U.S. application Ser. Nos.
10/626,459, 10/104,677, 10/625,822, 10/625,245 and 10/882,581,
hereby incorporated by reference in their entirety. Intermittently
applied hydrostatic pressure has been shown to support development
of a new hyaline cartilage in articular joints.
[0213] FIG. 9A is a schematic illustration of a basic method for
implantation of the double-structured tissue implants (DSTI) into
tissue defects or lesions in an operating room setting. The first
step in the implantation method comprises preparing a defect site
for implantation of DSTI. Such preparation comprises debriding the
lesion or defect of tissue debris, blood, blood clots, etc. When
the defect is prepared for implantation, surgeon cuts and trims the
DSTI to a size and shape of the defect. DSTI is supplied to surgeon
in dry or wet form in a sterile packaging such as shown in FIG. 1B.
After trimming the DSTI into the appropriate size and shape, the
DSTI is rehydrated, typically with saline, collagen containing
solution or another physiologically acceptable solution, Such
solution may be added before the DSTI is placed into the defect or,
preferably, after it is placed into the defect. The added solution
may be without any additional components or it may contain cells,
pharmaceutical agents, growth hormones, modulators, blood thinners
or buffers. The DSTI placed into the defect or lesion is then
sealed using a biocompatible and biodegradable polymeric tissue
adhesive, (preferably methylated collagen polyethylene glycol).
[0214] FIG. 9B shows essentially the same method as described in
FIG. 9A with added step for microfracture pretreatment. The defect
site is prepared and an initially oversized double-structured
tissue implant (DSTI) is cut and trimmed to match the size and
shape of the defect. The subchondral plate of the defect site is
penetrated using the microfracture technique. The precut DSTI is
rehydrated with saline, collagen containing solution or other
physiologically acceptable solution and placed into the defect.
Alternatively, the dehydrated DSTI is implanted and rehydrated in
situ. The DSTI may be rehydrated with a physiologically acceptable
solution optionally containing cells, cell progenitors or agents
that stimulate healing. The defect site is sealed with a tissue
adhesive applied over the implant.
[0215] FIG. 9C is a schematic illustration of a variation of a
basic method for implantation of the double-structured tissue
implants (DSTI) into tissue defects or lesions during surgery. In
this method, the adhesive is added first to the bottom of the
defect and cured or left non-cured, depending on the DSTI.
Typically, the first adhesive is deposited and should be left
uncovered until it is completely or at least it is partially
polymerized. The first step in the implantation method comprises
preparing defect site for implantation of DSTI. Such preparation
comprises debriding the lesion and coating said defect with a
biocompatible biodegradable polymeric adhesive. When the defect is
prepared for implantation, surgeon cuts and trims the DSTI to a
size and shape of the defect. In this instance, the DSTI is
typically pre-seeded with cells that have been previously cultured
and activated using methods referred to above. The pre-seeded DSTI
is supplied to a surgeon in dry or wet form in a sterile packaging
similar to that shown in FIG. 1B. After trimming the DSTI into the
appropriate size and shape, the DSTI is rehydrated, typically with
saline or another physiologically acceptable solution, such
solution may be added before the DSTI is placed into the defect or,
preferably, after it is placed into the defect. The added solution
may contain pharmaceutical agents, growth hormones or modulators.
The DSTI placed into the defect or lesion is then sealed using a
biodegradable tissue adhesive, preferably methylated collagen
polyethylene glycol. The tissue adhesive used to coat the bottom of
the defect may be same or different adhesive used to seal the
implanted defect.
[0216] For a variation of the basic microfracture method seen in
FIG. 9B, the microfractured defect seen in FIG. 9D, is treated with
the bottom adhesive similarly to the method described in FIG. 9C,
except that the bottom adhesive is applied at the bottom of the
defect between microfracture penetrations.
[0217] FIG. 9E illustrates a method for implantation of DSTI seeded
with cultured cells activated with a hydrostatic pressure regimen.
The method is similar to that seen in FIG. 9C.
[0218] B. Tissue Adhesives and Sealants
[0219] The double-structured tissue implant is implanted into a
tissue defect or cartilage lesion covered with a biocompatible
adhesive, tissue sealant or glue. Typically, the sealant is
deposited at and covers the bottom of the defect or lesion and may
also be used to cover the implant after implantation.
[0220] Generally, the tissue sealant or adhesive useful for the
purposes of this application has adhesive, or peel strengths at
least 10 N/m and preferably 100 N/cm; has tensile strength in the
range of 0.2 MPa to 3 MPa, but preferably 0.8 to 1.0 MPa. In
so-called Alap shear" bonding tests, values of 0.5 up to 4-6 N/cm2
are characteristic of strong biological adhesives.
[0221] Such properties can be achieved by a variety of materials,
both natural and synthetic. Examples of suitable sealant include
gelatin and di-aldehyde starch described in PCT WO 97/29715,
4-armed pentaerythritol tetra-thiol and polyethylene glycol
diacrylate described in PCT WO 00/44808, photo-polymerizable
polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers
described in U.S. Pat. No. 5,410,016, periodate-oxidized gelatin
described in U.S. Pat. No. 5,618,551, serum albumin and
di-functional polyethylene glycol derivatized with maleimidyl,
succinimidyl, phthalimidyl and related active groups described in
PCT WO 96/03159.
[0222] Sealants and adhesives suitable for purposes of this
invention include sealants prepared from gelatin and dialdehyde
starch triggered by mixing aqueous solutions of gelatin and
dialdehyde starch which spontaneously react and/or those made from
a copolymer of polyethylene glycol and polylactide, polyglycolide,
polyhydroxybutyrates or polymers of aromatic organic amino acids
and sometimes further containing acrylate side chains, gelled by
light, in the presence of some activating molecules.
[0223] Another type of the suitable sealant is 4-armed polyethylene
glycol derivatized with succinimidyl ester and thiol plus
methylated collagen in two-part polymer compositions that rapidly
form a matrix where at least one of the compounds is polymeric,
such as polyamino acid, polysaccharide, polyalkylene oxide or
polyethylene glycol and two parts are linked through a covalent
bond, for example a cross-linked derivatized PEG with methylated
collagen, such as methylated collagen polyethylene glycol.
[0224] Preferable sealants are 4-armed tetra-succinimidyl ester
PEG, tetra-thiol derivatized PEG and PEG derivatized with
methylated collagen (known as CT3), commercially available from
Cohesion Inc., Palo Alto, Calif. and described in U.S. Pat. Nos.
6,312,725B1 and 6,624,245B2 and in J. Biomed. Mater. Res.,
58:545-555 (2001), J. Biomed. Mater. Res., 58:308-312 (2001) and
The American Surgeon, 68:553-562 (2002), all hereby incorporated by
reference.
[0225] Sealants and adhesives described in copending U.S.
application Ser. Nos.: 10/921,389 filed Aug. 18, 2004 and
11/525,782 filed Dec. 22, 2006, are hereby incorporated by
reference.
[0226] C. Activation of Cells Within DSTI
[0227] Activation of the cells prior to their seeding into a DSTI
comprises steps:
[0228] a. isolation and/or collection of cells, such as
chondrocytes or stem cells from a donor tissue; with or without
expansion of the number of cells.
[0229] b. seeding the cells in a DSTI;
[0230] c. subjecting the seeded DSTI to a static, constant or
cyclic hydrostatic pressure above atmospheric pressure (about
0.5-3.0 MPa at 0.5 Hz) with medium perfusion rate of 5 .mu.l/min
for several (5-10) days; and
[0231] d. subjecting the seeded DSTI to a resting period for ten to
fourteen days at constant (atmospheric) pressure.
[0232] Seeded DSTI obtained by the above-outlined conditions and
method show that a combination of hydrostatic pressure and static
pressure has advantage over conventional culture methods by
resulting in higher cell proliferation and extracellular matrix
accumulation within DSTI. Use of DSTI maintains uniform cell
distribution within the primary and secondary scaffolds that also
provides support for newly synthesized extracellular matrix.
Obtained seeded DSTI is easy to handle and manipulate and can be
easily stored and safely implanted in a surgical setting.
[0233] Combination of a period of cyclic hydrostatic pressure under
low medium perfusion rate followed up with a period of static
culture (resting period) results in increased cell proliferation,
increased production of Type II collagen, increased DNA content and
increased S-GAG accumulation.
[0234] Increased cell proliferation shows that the harvested
inactive non-dividing cells, particularly chondrocytes, have been
activated into active, dividing and multiplying chondrocytes.
Increased level of DNA shows genetic activation of inactive
chondrocytes. Increased production of Type II collagen and S-GAG
shows that production of the extracellular matrix has been
activated using the method for activation described above.
[0235] Although the optimized conditions described above is
preferred, it is to be understood that these conditions may be
advantageously changed using variations of ranges of cyclic
hydrostatic pressure, flow rate, duration of the pressure and
resting period, particularly when applied to different cells or
tissue. All variations of all conditions and combinations thereof
are intended to be within the scope of this invention.
[0236] D. Use of DSTI for Treatment of Chondral Defects
[0237] One example of utility of the DSTI is its use for treatment
of chondral defects.
[0238] To be successful for treatment of articular cartilage, the
DSTI must provide conditions allowing the chondrocytes or
mesenchymal stem cells seeded therein to be able to form, generate
or induce the generation of the new extracellular matrix. In this
regard, the DSTI pore structure must allow cells to migrate into
the pores and function similarly to their normal function in the
healthy tissue. The extracellular matrix formed by the cells seeded
within the DSTI then provides means for growing a new hyaline or
hyaline-like cartilage for treatment, replacement or regeneration
of the damaged or injured articular cartilage. Such treatment is
currently difficult because of the unique properties of the
articular cartilage that is not the same as and does not behave as
other soft tissues.
[0239] E. Use of DSTI For Treatment Of Other Conditions
[0240] In addition to cartilage repair, a number of other chronic
conditions represent instances where the implantation of the double
structured scaffold can provide a clinically important bridge for
tissue repair.
[0241] For example, genitourinary tissues have been fabricated from
a variety of materials. The DSTI once placed at the site of tissue
damage will provide a support for development of new tissues occurs
in accordance with predefined configuration. In these applications,
similar to cartilage, the DSTI must resist the dynamic forces
generated by the surrounding muscle and connective tissues and
maintaining its structure during a necessary period of cellular
infiltration and tissue formation.
[0242] The rapidity by which tissue differentiation and structural
integrity are established is subject to modulation through the use
specific signaling factors localized within the primary and
secondary collagenous composite. Although the limits by which, for
example, new muscle formation can be derived from progenitor cells,
suggests that localization of the mesenchymal cells to the site of
damage in response to homing molecules, such as chemokines and cell
receptor ligands, may be used to accelerate repair of muscle,
either cardiac or skeletal. DSTI may be used to deliver these cells
or modulators to the site of damage.
[0243] Finally, wound healing applications have remained a primary
goal in the use of tissue implants for cell-based tissue repair.
Treatment of acute and chronic wounds is dependent on a
multi-faceted transition by which progenitor cells encounter
soluble mediators, formed blood elements, extracellular matrix
macromolecules and parenchymal cells that then serve to reestablish
a body surface barrier through epithelialization. In this instance
either the double-structured scaffold or the stand alone secondary
scaffold implant may provide a novice stromal layer into which
blood vessels and progenitor cells can migrate. From this
migration, the progenitor cells may then undergo differentiation
into the fibroblast stromal cell and generate or recruit epithelial
cells to support reestablishment of dermal and epidermal layers at
the time of wound closure.
[0244] IV. Basic Requirements for DSTI
[0245] The collagen-based primary and secondary scaffolds of the
DSTI are essential components of the DSTI and are responsible for
capability of DSTI to initiate the repair and induction of repair
of tissue defects.
[0246] The first requirement is that the scaffolds are prepared
from the biocompatible and preferably biodegradable materials that
are the same or similar to those observed in the tissues to be
repaired, hence the instant DSTI are prepared from collagen or
collagen-like materials.
[0247] The second requirement is that the scaffolds have a spatial
organization and orientation similar to that of the tissue to be
repaired. The porous structure of both primary and secondary
scaffold provides such organization. The third requirement is that
the scaffold has a pore density permitting the seeding of the cells
into said scaffolds in numbers needed for initiation of a tissue
recovery or formation of new tissue in vivo. The substantially
homogenous pore size and distribution within the DSTIs allows the
cell seeding and assures cell viability.
[0248] The fourth requirement is that the scaffolds have sufficient
number of pores for the number of cells needed for initiation of
the tissue recovery and repair. The spatial organization of both
scaffolds has optimized number of pores.
[0249] The fifth requirement is that the pores have substantially
the same size and that such size is substantially the same from the
top apical to the bottom basal surface of the pores, said pores
being organized substantially vertically from the top to the
bottom. The primary scaffold has such organization.
[0250] The sixth requirement is stability of the DSTI. The
double-structured organization of the DSTI provides such stability
during wetting, reconstitution, and resistance to dissolution and
to shrinkage or swelling.
[0251] The seventh requirement is that DSTI provides support and
conditions for cell migration from the surrounding tissue, for
integration of seeded cells into the surrounding tissue and
generally that assures the cell viability. The DSTI provides such
conditions and the cells seeded within DSTI have almost 100%
viability.
EXAMPLE 1
Preparation of the Primary Scaffold
[0252] This example describes one exemplary method for preparation
of the collagen-based primary scaffold suitable as a structural
support for preparation of the DSTI. Type I collagen is dissolved
in a weak hydrochloric acid solution at pH 3.0 with the collagen
concentration and osmolality of the solution adjusted to about 3.5
mg/ml and 20 mOsm/kg, respectively. The solution (70 ml) is poured
into a 100 ml Petri dish and the Petri dish containing the collagen
solution is centrifuged at 400.times.g for 30 minutes to remove air
bubbles. Neutralization and subsequent precipitation or gelling is
carried out in a 7 liter chamber containing 10 ml of 15% ammonia
solution over a 45 minutes period. The precipitated collagen is
then washed in a large excess of de-ionized water. The water is
changed as many times as needed over next 3 days to remove formed
salts and excess ammonia.
[0253] The solution is then subjected to unidirectional freezing
over a period of about 4 hours. The Petri dish is placed on a
stainless steel disc which is partially submerged in a cooling
bath. The temperature of the cooling bath is increased from
0.degree. C. to -18.degree. C. at a rate of 0.1.degree. C./minute.
The frozen water is removed by lyophilization over a period of
about 3 days. The lyophilized primary scaffold is then
dehydrothermally (DHT) treated at 135.degree. C. for about 18 hours
before being precut into slices of an appropriate thickness.
[0254] The organization of the newly synthesized cartilage specific
matrix within the porous type I collagen is visualized and
quantified using histological and image analysis methods.
EXAMPLE 2
Preparation of a Basic Solution for a Secondary Scaffold
[0255] This example describes preparation of the Basic Solution
used for formation of the secondary scaffold. The Basic Solution
comprises a soluble collagen in admixture with a PLURONIC.RTM.
surfactant. The Basic Solution is incorporated into the primary
scaffold and processes into the double scaffold tissue implant or
processed as a stand alone implant.
[0256] Solution for the secondary scaffold is prepared by mixing
PLURONIC.RTM. F127 (2.32 mg, 0.29 mg/ml), obtained commercially
from BASF, Germany, with 8 ml of a solution containing 2.9 mg/ml
bovine type I collagen dissolved in hydrochloric acid (pH 2.0) at
room temperature. The resulting solution is neutralized with 1 ml
of 10.times. Dulbecco=s phosphate buffered saline (DPBS) and 1 ml
of 0.1M NaOH to the final pH of 7.4.
[0257] In the alternative, the neutralization is achieved by
ammonia aqueous solution or ammonia vapor in concentration
sufficient to neutralize acid within the collagen solution.
EXAMPLE 3
Preparation of the Secondary Scaffold as Stand Alone Unit
[0258] This example illustrates preparation of the secondary
scaffold as a stand alone implant or stand alone unit. For
preparation of the stand alone secondary scaffold, the Basic
Solution prepared in Example 3 is subjected to precipitation or
gelling followed by dehydrothermal treatment.
[0259] The Basic Solution (2 ml) comprising collagen and
PLURONIC.RTM. surfactant is placed in a small glass beaker and the
beaker is placed into a chamber (approximately 9 liters) charged
with 1% ammonia solution. The Basic Solution is allowed to
precipitate in the chamber over a period of 15 minutes. The gelled
or precipitated collagen is then washed in 500 ml of deionized
water over a period of 30 minutes. The washing step is repeated
three times. The washed gel or precipitate is placed on metal shelf
of a freezer at -80.degree. C. over a period of 30 minutes. The
frozen gel or precipitate is removed from the freezer and
lyophilized. Lyophilization is performed over a period of 10 hours.
The lyophilized construct is then dehydrothermally treated at
135.degree. C. under vacuum for a period of 6 hours to form the
secondary scaffold alone.
EXAMPLE 4
Preparation of the Double-Structured Tissue Implant
[0260] This example describes preparation of the double-structured
tissue implant (DSTI). The preparation of DSTI includes
incorporation of a Basic Solution for formation of a secondary
scaffold within the primary scaffold and its further processing
into DSTI.
[0261] 4.9 ml (1.3.times. volume of the primary scaffold) of the
neutralized basic collagen/PLURONIC.RTM. solution prepared in
Example 2, is placed in a dish and a primary scaffold, prepared in
Example 1, precut into a square having 50.times.50.times.1.5 mm
dimensions is then placed into the neutralized Basic Solution for
the secondary scaffold. The basic neutralized solution is absorbed
into the primary scaffold by wicking or soaking.
[0262] The primary scaffold containing the neutralized solution is
then placed in a 37.degree. C. incubator over a period of 50
minutes to precipitate or gel the neutralized collagen solution.
The composite consisting of the primary scaffold with the gelled or
precipitated neutralized solution within is then washed in 500 ml
of deionized water over a period of 30 minutes. The washed
composite is placed on metal shelf of a freezer at a temperature
-801C over a period of 30 minutes. The frozen composite is removed
from the freezer and lyophilized.
[0263] Lyophilization is performed over a period of 18 hours. The
lyophilized composite is then dehydrothermally treated at 1351C
under vacuum for a period of 6 hours to form the double-structured
tissue implant (DSTI).
[0264] The DSTI is removed from the dehydrothermal oven and
transferred aseptically into a Bio Safety Cabinet (BSC) where it is
packaged.
EXAMPLE 5
Determination of Retention of Collagen within DSTI
[0265] This example describes a procedure used for determination of
the stability of the double-structured tissue implant in vitro.
[0266] Three lots of DSTIs are prepared as described in Example 4
and cut to a size of 1.5.times.1.5.times.0.15 cm. Cut DSTIs are
placed in 35 mm Petri dishes, rehydrated with 450 Cl of phosphate
buffered saline and additional 2 ml of phosphate buffered saline
are added to each Petri dish containing the DSTI. The analysis for
each lot consists of three replicates for a total of 9 samples for
the three lots.
[0267] Dishes containing individual DSTIs are placed in the
incubator for the duration of testing. In the predetermined
intervals of zero hour, 1 hour, 3 days, 7 days and 14 days, 1 ml
aliquot of the phosphate buffered saline is removed from each
plate. Each removed 1 ml is replaced with 1 ml of a fresh phosphate
buffer saline. The removed aliquots are subjected to a calorimetric
protein assay for quantification of total collagen released into
the saline.
[0268] Cumulative collagen retention curves are generated by
subtraction of the amount of collagen released into the solution
from the theoretical collagen load estimated at 0.777 mg of
collagen/DSTI sample. Results are seen in FIG. 6.
EXAMPLE 6
Determination of Change of Size and Shape of DSTI Following
Rehydration
[0269] This example describes the retention of size and shape of
double-structured tissue implant in a phosphate buffered saline
solution over time.
[0270] The DSTI samples obtained in Example 5 are photographed at
the designated intervals and the images generated are measured by
ImageJ, publicly available Java-based image processing program
developed at the NIH. The photograph of each sample at each time
point is imported into Image and the region of interest (DSTI area)
is manually defined. The area was measured and the percent change
is determined by dividing the areas at each time point by the
rehydrated DSTI at time 0 and multiplying by 100. Results are seen
in FIG. 7.
EXAMPLE 7
Studies of Biocompatibility
[0271] This example describes procedures used to determine cell
biocompatibility with the primary scaffold and with the DSTI.
[0272] The primary scaffold and DSTIs are prepared in three lots as
described in Examples 1 and 4. The chondrocytes or other cells are
loaded into the three samples of primary scaffolds and into the
three lots of three samples each of DSTIs. Time intervals for
biochemical, image and cell viability determination is set to Day O
(24 hours) and Day 21 (21 days) of incubation in the culture
medium.
TABLE-US-00003 TABLE 3 Chondrocyte Compatibility Experimental
Groups Time Sample Numbers Group Description point Biochemistry
Images Viability A DSTI #1 Day 0 3 2 3 B DSTI #1 Day 21 3 2 3 C
DSTI #2 Day 0 3 2 3 D DSTI #2 Day 21 3 2 3 E DSTI #3 Day 0 3 2 3 F
DSTI #3 Day 21 3 2 3 G Primary Day 0 3 2 3 Scaffold H Primary Day
21 3 2 3 Scaffold DSTI-5 mm disks are cut from each lot of sheets.
Primary scaffold 5 mm disks are cut from primary scaffold.
[0273] The disks of the primary scaffold are loaded with the
chondrocytes dissolved in a collagen solution at a cell
concentration of 5.times.10.sup.6 cells/ml. The primary scaffold
does not contain the surfactant and is not subjected to
lyophilization or to a dehydrothermal treatment.
[0274] Both the primary scaffold and DSTI disks (total 24) are
placed in 24 well plates. Chondrocytes are seeded into the disks by
the addition of 20 .mu.l of 3D cell culture medium (DMEM/F-12+10%
FBS+1% ITS) at a cell concentration of 5.times.10.sup.6 cells/ml.
The disks loaded with cells are placed in the incubator for 1 hour
at 37.degree. C. and in 5% CO.sub.2. Then 400 .mu.l of medium is
added and the plates are placed in the low oxygen incubator
overnight. At 24 hours (Day 0), one set of samples from each lot is
removed from culture. The remaining disks loaded with chondrocytes
are transferred to 12 well plates with 2 ml of 3D culture medium
and are maintained in culture at 37.degree. C., 5% CO2, 2% O2 for
three weeks with medium changes once a week.
[0275] The primary scaffold disks are processed in the same way as
DSTI disks.
[0276] Evaluations include assessment of chondrocyte growth,
viability and phenotypic S-GAG expression in both the primary
scaffold and DSTIs. Results are seen in FIG. 4B, and FIG. 5 for
DSTI images, FIG. 8 for S-GAG and DNA production and in Table 1 for
cell viability.
EXAMPLE 8
Production of S-GAG/DNA
[0277] This example describes conditions used for evaluation of
production of S-GAG and DNA by seeded chondrocytes.
[0278] DSTI and primary scaffold disks are prepared as described in
Examples 1 and 4 and seeded with 200,000 chondrocytes in 2081 of 3D
culture medium by absorption at 37CC for 1 hour. 400 .mu.l of
medium is added and incubated overnight. Disks are removed for
analysis at predetermined intervals.
[0279] At termination, the disks are placed in papain digest
solution and incubated at 60.degree. C. overnight. An aliquot of
the digest from each disk is taken to measure S-GAG by the
dimethylmethylene blue assay. An aliquot from each disk is taken
for measurement of DNA by the Hoechst dye method. Results are shown
in FIG. 8.
EXAMPLE 9
Determination of Viability of Chondrocytes
[0280] This example describes procedure used for determination of
viability of chondrocytes seeded in the DSTIs or in the primary or
secondary scaffolds.
[0281] DSTI, primary scaffold or secondary scaffold disks are
seeded with approximately 200,000 chondrocytes dissolved in the 20
.mu.l of 3D culture medium by absorption and incubated at 37CC for
31 hours. 400 .mu.l of medium is added and incubation is continued
overnight.
[0282] Cell loaded disks are removed for analysis and 2 ml of
medium is added to remaining disks for continued incubation for 21
days.
[0283] At day 21, the chondrocyte-loaded DSTIs and the primary and
secondary scaffolds disks are placed in 1.5 ml microcentrifuge
tubes and further incubated overnight in 0.15% collagenase. The
digest is centrifuged at 2000 rpm for 5 minutes and the supernatant
aspirated. An aliquot of culture medium (0.1 ml) is added to the
cell pellets and an aliquot taken for counting. Cell viability and
total cell count is determined using trypan blue. Results are shown
in Table 1.
* * * * *