U.S. patent application number 09/878641 was filed with the patent office on 2002-09-19 for ligament and tendon replacement constructs and methods for production and use thereof.
Invention is credited to Attawia, Mohammed A., Cooper, James A., Ko, Frank K., Laurencin, Cato T., Lu, Helen H..
Application Number | 20020133229 09/878641 |
Document ID | / |
Family ID | 26887624 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020133229 |
Kind Code |
A1 |
Laurencin, Cato T. ; et
al. |
September 19, 2002 |
Ligament and tendon replacement constructs and methods for
production and use thereof
Abstract
Degradable, polymeric fiber-based, three-dimensional braided
scaffolds for use as graft materials in ligament and tendon repair,
reconstruction and replacement are provided. Also provided are
methods for preparing these scaffolds.
Inventors: |
Laurencin, Cato T.; (Elkins
Park, PA) ; Ko, Frank K.; (Philadelphia, PA) ;
Cooper, James A.; (Philadelphia, PA) ; Lu, Helen
H.; (Philadelphia, PA) ; Attawia, Mohammed A.;
(Wayne, PA) |
Correspondence
Address: |
LICATLA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
26887624 |
Appl. No.: |
09/878641 |
Filed: |
June 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09878641 |
Jun 11, 2001 |
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09814427 |
Mar 22, 2001 |
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60191999 |
Mar 24, 2000 |
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Current U.S.
Class: |
623/13.17 ;
623/13.19 |
Current CPC
Class: |
A61F 2/08 20130101; A61L
27/56 20130101; A61F 2210/0004 20130101; A61F 2/38 20130101; A61F
2230/0019 20130101; A61L 27/18 20130101; A61L 27/58 20130101; A61F
2/40 20130101; A61F 2230/0006 20130101; A61L 2430/10 20130101 |
Class at
Publication: |
623/13.17 ;
623/13.19 |
International
Class: |
A61F 002/08 |
Goverment Interests
[0002] This invention was supported in part by funds from the U.S.
government (NIH Grant Nos. 5 F31 GM18905-02 and AR46117 and NSF
Presidential Grant BES9553162/BES981782) and the U.S. government
may therefore have certain rights in the invention.
Claims
What is claimed is:
1. A replacement construct comprising a degradable, polymeric
fiber-based, three-dimensional braided scaffold.
2. A replacement construct comprising a degradable polymeric
fiber-based, three-dimensional braided scaffold seeded with cells,
ingrowth of which is support by the scaffold.
3. The replacement construct of claim 2 where the cells are
mesenchymal in origin.
4. The replacement construct of claim 2 wherein the cells generate
mesenchymal cells.
5. The replacement construct of claim 4 wherein the cells are
pluripotent stem cells.
6. A method for repairing, replacing or reconstructing a damaged
tendon or ligament in a patient comprising implanting at a damaged
tendon or ligament the replacement construct of claim 1.
7. A method for repairing, replacing or reconstructing a damaged
tendon or ligament in a patient comprising implanting at a damaged
tendon or ligament the replacement construct of claim 2.
8. A method for producing a graft material composed of living cells
in a degradable matrix comprising: (a) harvesting, growing and
passaging cells in tissue culture; and (b) seeding the cultured
cells onto the degradable, polymeric fiber-based, three-dimensional
braided scaffold of claim 1.
9. The method of claim 8 wherein the cells are mesenchymal in
origin.
10. The method of claim 8 wherein the cells generate mesenchymal
cells.
11. The method of claim 10 wherein the cells are pluripotent stem
cells.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/814,427, filed Mar. 22, 2001, which claims
the benefit of priority from U.S. Provisional Application Serial
No. 60/191,999, filed Mar. 24, 2000.
FIELD OF THE INVENTION
[0003] The present invention relates to use of fiber technologies
to design useful matrices for tissue engineering. In particular, a
viable replacement construct of human ligaments and tendon is
provided. This replacement construct comprises a degradable,
polymeric fiber-based, three-dimensional braided scaffold. In one
embodiment, the replacement construct is seeded with cells,
preferably cells of mesenchymal origin or stem cells capable of
generating mesenchymal cells. The biocompatability of this
replacement construct coupled with the tissue engineering based
design is expected to promote healing and repair of the damaged
ligament or tendon.
BACKGROUND OF THE INVENTION
[0004] In orthopaedic reconstruction, surgeons often replace
damaged tissue resulting from trauma, pathological degeneration, or
congenital deformity with autogenous grafts (Langer, R. and
Vacanti, J. P. Science. 1993 260:920). Reconstructive surgery is
based upon the principle of replacing these types of defective
tissues with viable, functioning alternatives. The grafting of bone
in skeletal reconstruction has become a common task of the
orthopaedic surgeon with over 863,200 grafting procedures performed
each year in the U.S. For cartilage replacement, there are over
1,000,000 procedures of various types performed each year and for
ligament repairs, there are approximately 90,000 procedures
performed per year (Langer, R. and Vacanti, J. P. Science. 1993
260:920). Currently, autografts (Friedman et al. Clin. Ortho. 1985
196:9; Jackson et al. Amer. J. Sports Med. 1990 18:1) (tissue taken
from the patient) and allografts (Gadzag et al. J. Amer. Acad
Ortho. Surg. 1995 3:1; Shinoet al. J. Bone and Joint Surg. 1988
7011:556; Jackson et al. Arthroscopy 1994 10:442)(tissue taken from
a cadaver) are the most common replacement sources for the
treatment of musculoskeletal problems. In repair of ligament
injuries, such as injury of the anterior cruciate ligament (ACL), a
segment of the patellar tendon has been frequently used (Jackson et
al. Amer. J. Sports Med. 1990 18:1). For cartilage and bone repair,
transplantation of autogenous grafts has been the current treatment
of choice.
[0005] However, there are various problems associated with these
treatments. For example, for autogenous tissue, key limitations are
donor site morbidity where the remaining tissue at the harvest site
is damaged by removal of the graft, and the limited amount of
tissue available for harvesting. The use of allografts attempts to
alleviate these problems. However, this type of graft is often
rejected by the host body due to an immune response to the tissue.
Allografts are also capable of transmitting disease. Although a
thorough screening process eliminates most of the disease carrying
tissue, this method is not 100% effective.
[0006] As a result of the limitations with conventional
reconstructive graft materials, surgeons have looked to synthetic
alternatives.
[0007] Synthetic ligament grafts or graft supports include carbon
fibers, Leeds-Keio ligament (polyethylene terephthalate), the Gore
Tex prosthesis (polytetrafluoroethylene), the Stryker-Dacron
ligament prosthesis made of Dacron tapes wrapped in a Dacron sleeve
and the Gore-Tex ligament augmentation device (LAD) made from
polypropylene. These grafts have exhibited good short term results
but have encountered clinical difficulties in long term studies.
Limitations of these synthetic ligament grafts include stretching
of the replacement material, weakened mechanical strength compared
to the original structure and fragmentation of the replacement
material due to wear.
[0008] The ideal ligament or tendon replacement is biodegradable,
porous, biocompatible, exhibits sufficient mechanical strength and
promotes formation of ligament or tendon tissue.
[0009] Various researchers have disclosed potential ligament
constructs comprising collagen fibers, biodegradable polymers and
composites thereof. For example, collagen scaffolds for ACL
reconstruction seeded with fibroblasts from ACL and skin have been
described (Dunn et al. The Tissue Engineering Approach to Ligament
Reconstruction. Material Research Society Symposium Proceedings
331, 13-18, 1994, Boston, Materials Research Society; Bellincampi
et al. J. Orthop. Res. 1998 16:414-420). WO 95/2550 also discloses
a prosthetic device for ligament repair comprising an arrangement
of collagen threads.
[0010] A bioengineered ligament model, which differs from other
ligament models in the addition of ACL fibroblasts to the
structure, the absence of cross-linking agents and the use of bone
plugs to anchor the bioengineered tissue, has also been described
(Goulet et al. Tendons and Ligaments. In R. P. Lanza, R. Langer,
and W. L. Chick (eds), Principles of Tissue Engineering, pp.
639-645, R. G. Landes Company and Academic Press, Inc. 1997).
[0011] U.S. Pat. No. 4,792,336 discloses a device with an
absorbable component comprising a glycolic or lactic acid ester
linkage. The device comprises a plurality of fibers comprising the
absorbable component which can be used as a flat braid in the
repair of a ligament or tendon.
[0012] The present invention relates to a graft material for use in
ligament and tendon repair and reconstruction composed of a
degradable, polymeric, fiber-based, three dimensional braided
scaffold.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide a
replacement construct comprising a degradable, polymeric,
fiber-based, three-dimensional braided scaffold.
[0014] Another object of the present invention is to provide a
replacement construct comprising a degradable, polymeric,
fiber-based, three-dimensional braided scaffold which has been
seeded with cells, the ingrowth of which is supported by the
scaffold.
[0015] Another object of the present invention is to provide a
method for repairing a damaged ligament or tendon in a human which
comprises implanting at the damaged area a degradable, polymeric,
fiber-based, three-dimensional braided scaffold.
[0016] Another object of the present invention is to provide a
method for repairing a damaged ligament or tendon in a human which
comprises implanting at the damaged area a degradable, polymeric,
fiber-based, three-dimensional braided scaffold which has been
seeded with cells, the ingrowth of which is supported by the
scaffold.
[0017] Yet another object of the present invention is to provide a
method for producing a graft material composed of living cells in a
degradable matrix for use in ligament repair and reconstruction
which comprises harvesting, growing and passaging cells in tissue
culture and seeding the cultured cells onto a degradable,
polymeric, fiber-based, three-dimensional braided scaffold.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to an approach to tissue
repair based upon the principle of using bioresorbable scaffolds to
serve as templates for tissue regeneration. In particular, the
present invention relates to degradable scaffolds, preferably
polymeric, fiber-based three-dimensional (3-D) braided scaffolds
based on hierarchical design methodology.
[0019] Fiber-based braided scaffolds of the present invention were
compared with microfiber nonwoven matrices for tissue replacement
applications.
[0020] An electrospinning technique was used to fabricate
microfiber nonwoven matrices. The basis of this technique is the
generation of an electric field between an oppositely charged
polymer fluid and a collection screen. A polymer solution is added
to a glass syringe with a capillary tip. An electrode is placed in
the solution with another connection made to a copper screen. As
the power is increased, the polymer solution becomes charged and is
attracted to the screen. Once the voltage reaches a critical value,
the charge overcomes the surface tension of the droplet and a jet
of microfibers is produced. As the charged fibers are splayed, the
solvent quickly evaporates and the fibers randomly accumulate on
the surface of the collection screen. This results in a nonwoven
mesh of micron scaled fibers. Fiber diameter and mesh thickness can
be controlled by a number of different parameters including
solution viscosity, voltage, distance between screen and tip, and
duration of electrospinning.
[0021] The 3-D braided scaffolds of the present invention were
formed using a 3-D textile braiding technique. An exemplary 3-D
textile braiding technique is the 4-step process which uses a track
and column method to create the fiber matrix. However, as will be
understood by those of skill in the art upon reading this
disclosure, other techniques for preparing 3-D braided scaffolds
can also be used.
[0022] The 4-step braiding equipment consists of slotted tracks
where bobbins and yarn carriers are located. Movement of the
bobbins and carriers within the tracks is used to create vertical
columns in the 3-D structure. Alternating rows and columns of the
carriers in the braiding lattice are shifted to create the 3-D
braid. The geometric parameters which determine the shape and fiber
architecture of 3-D braids includes braiding angle distribution,
yarn volume fraction, number of carriers, and braiding yarn width.
This highly versatile system allows for the formation of a variety
of 3-D braided structures with different architecture and
mechanical properties.
[0023] Based on these fiber and textile technologies, a microfiber
nonwoven mesh and two rectangular 3-D braids were fabricated for
cell culture experiments.
[0024] In these experiments, the response of cells to the
hierarchical structure of the two fiber based matrices was
compared. In particular, the ability of these matrices to serve as
cellular scaffolds was evaluated using osteoblasts and fibroblasts
in an in vitro environment.
[0025] Electron microscopy of the three matrix structures was first
performed. Low magnification images showed basic matrix structure
and organization. SEM analysis of the microfiber matrix showed a
highly porous, fibrous structure resulting from the random
arrangement of the fibers. PLAGA [50:50] fibers ranged in diameter
from approximately 2-7 .mu.m. Images of the 3-D braided matrices
showed a highly organized fibrous structure resulting from the 3-D
braiding process. The difference in the number of fibers/yarn was
clearly evident in these two structures. Braid #1 which was
fabricated from 30 yarn having 30 fibers/yarn had more individual
braids throughout the structure than the Braid #2 matrix fabricated
from 60 yarn with 60 fibers/yarn. These structures can be
attributed to the packing density of the fibers. With half as many
fibers per yarn, the 30 yarn of Braid #1 was able to pack into a
tighter structure with a braid unit cell smaller than the 60 yarn
matrix. SEM evaluation of these structures indicated that all
matrices possessed the structural characteristics needed to
function as a cellular scaffold.
[0026] However, the results of the in vitro study revealed that the
cellular response was dependent on matrix structure. Both
fibroblasts and osteoblasts had the same morphology on the
microfiber nonwoven matrix. After one day of culture on the
microfiber matrix, cells appeared spindle shaped and exhibited
spreading over the surface. Slight cytoplasmic projections were
seen extending from the body of the cells to the surface of the
matrices. However, SEM did not reveal a microfiber structure in any
of the samples, regardless of time point. Since only 50,000 cells
were plated on a 1 cm.sup.2 matrix, it is believed that the cells
had completely spread over the surface obscuring the microfiber
structure. The spindle shaped morphology observed at day 1 is
indicative of initial attachment and not the formation of a
cellular monolayer.
[0027] A degradation study was also performed to evaluate any
changes to matrix structure due to degradation in the tissue
culture media. This study revealed that the matrix quickly degraded
while in the cell culture media. It is believed that exposure to
DMEM caused the swelling and aggregation of the microfibers.
Swelling was so significant in some samples that the structure lost
almost all of its porosity. Thus, this degradation changed the
matrix from a porous microfiber matrix to a non-porous mass of
polymer during the course of the cell culture study.
[0028] Unlike the microfiber matrix, cell morphology on the 3-D
braid differed between osteoblasts and fibroblasts. Over the course
of the 2 week experiment, both cell types followed the
characteristic sequence of events describing cell attachment,
spreading and proliferation. However, the rate at which these
events occurred differed for osteoblasts and fibroblasts. Further,
cellular attachment appeared to be more pronounced with osteoblasts
than fibroblasts. For example, at one day of cell culture on 3-D
Braid #1, the osteoblasts showed significant spreading over the
surface and the formation of a cellular layer. In comparison, the
day 1 fibroblasts still retained a spindle shaped morphology
characteristic of initial attachment. In addition, the fibroblasts
had organized along the length of the fibers. The cells appeared to
have grouped together along the groove created by two adjacent
fibers. Slight cytoplasmic extensions were seen between the aligned
cells.
[0029] Thus, as demonstrated by the cellular response observed in
these experiments, hierarchical structure plays an important role
in cellular morphology and organization. Cells responded
dynamically to the changing structure of the quickly degrading
matrix comprising the nonwoven microfiber. The cells did not
organize on such a structure and morphology of the specific cell
types was similar. In contrast, in the slowly degrading fiber
structure of the 3-D braid, fibroblasts organized along the length
of the fibers, and osteoblasts showed a distinctly different
morphology than fibroblasts.
[0030] Accordingly, use of fiber technology in tissue engineering
holds several advantages over a number of non-fibrous 3-D
structures. Importantly, the ability to impart high levels of
structural organization to the matrix allows for precise control of
matrix structure. The 3-D braided and nonwoven matrices are
exemplary of the range of 3-D fiber architectures that can be
designed and produced. The braided matrix consisted of highly
organized PLAGA yarns woven into a 3-D structure. Although the
nonwoven matrix was the result of randomly oriented microfibers,
the structure was highly uniform. Thus, both the 4-step 3-D
braiding technique and the electrospinning process are useful
fabrication methods showing high levels of versatility for various
tissue engineering application. The ability to manufacture a
variety of different matrices and to maintain precise control over
matrix fabrication are extremely important factors in the design of
a tissue engineered scaffold.
[0031] For example, the human knee contains large ligaments such as
the ACL which connects the femur to the tibia and participates in
motion control, acting as a stabilizer of joint movement. ACL is
the most commonly replaced ligament of the knee, with over 250,000
patients each year diagnosed with ACL injury. This type of injury
often occurs during sports and physical exercise, and frequently
results in disabilities that can be permanent and disabling to the
patient. Other exemplary ligaments which are oftentimes injured and
require repair and/or replacement include, but are not limited to,
the medical collateral ligament, the anterior talo-fibular ligament
of the ankle and the glenohumeral ligaments. Exemplary tendons
which are oftentimes injured and require repair and/or replacement
include, but are not limited to, the patellar, the Achilles tendon
and the rotator cuff.
[0032] It is believed that the 3-D braided scaffolds will be
particularly useful as replacement constructs for the
above-described exemplary ligaments and tendons, as well as any
other ligaments or tendons which have been damaged, as these
scaffolds are degradable, porous, biocompatible, exhibit sufficient
strength and promote formation of ligament and tendon tissue. The
fiber based design of the scaffold emulates the natural ligament or
tendon and the braided structure offers mechanical strength as well
as needed porosity for cell attachment and ingrowth.
[0033] While PLAGA fibers were used in the braided scaffold in the
experiments described herein, as will be understood by those of
skill in the art upon reading this disclosure, any and all
biodegradable polymers can be used. Preferred biodegradable
polymers are those degraded by hydrolysis. Examples of polymeric
fibers useful in the present invention include, but are not limited
to, fibers comprised of poly(hydroxy)esters, such as polylactic
acid, polyglycolic acid and co-polymers thereof. Preferred
biodegradable polymers are lactic acid polymers such as
poly(L-lactic acid (PLLA), poly(DL-lactic acid (PLA), and
poly(DL-lactic-co-glycolic acid) (PLGA). The co-monomer
(lactide-glycolide) ratios of the poly(DL-lactic-co-glycolic acid)
are preferably between 100:0 and 50:50. Most preferably, the
co-monomer ratios are between 85:15 (PLGA 85:15) and 50:50 (PLGA
50:50). Blends of PLLA with PLGA, preferably PLGA 85:15 and PLGA
50:50 can also be used for these scaffolds. Other exemplary
biodegradable polymers useful in the scaffolds of the present
invention include, but are not limited to, polyorthoesters,
polyanhydrides, polyphosphazenes, polycaprolactones,
polyhydroxybutyrates, degradable polyurethanes,
polyanhydrideco-imides, polypropylene fumarates, and
polydiaxonane.
[0034] To aid in selection of polymer fibers to be used for the
braiding of 3-D constructs for ligament and/or tendon replacement,
the degradation characteristics of three types of polymer fiber
bundles and the effect of degradation on long-term mechanical
properties of these polymers was examined. The three polymers
examined were multifilament fibers of L-poly-lactide (PLA, 70
denier), poly-glycolide (PGA, 60 denier) and their 82:18 co-polymer
(PLAGA, 70 denier) laced into 10 multi-fiber bundles. The mass
retention and mechanical properties of all the polymers decreased
with increasing immersion time in both phosphate buffered saline
(PBS) and cell culture medium (.alpha.MEM). However, PGA bundles
exhibited the most rapid loss of strength, mass and yarn integrity,
and this polymer had largely degraded after 2 weeks and broken up
into small fibers. PLA and PLAGA bundles degraded more slowly as
reflected in decreases in their mechanical strength, mass retention
and molecular weight. After 4 weeks, PLA sustained higher maximum
tensile load than PLAGA. It was found that polymer mass retention
was independent of changes in mechanical strength and molecular
weight.
[0035] PLAGA molecular weight decreased to half of its original
value after 2 weeks of immersion in .alpha.MEM, which may be too
fast for ligament healing to take place. As the polymers degraded,
the pH of PBS decreased as acidic degradation products were
released. While an initial decrease in pH was measured in
.alpha.MEM, the solution later returned to control values. This is
likely due to protein adsorption and the higher buffering potential
of .alpha.MEM, rendering it a more realistic solution in which to
model polymer degradation in vivo.
[0036] Thus, based on examination of changes in molecular weight,
mechanical strength and mass retention as the polymer degraded, PLA
(in comparison to PLAGA 82:18 or PGA) has specific advantages for
use in the braided, tissue-engineered 3-D ACL replacement
constructs of the present invention. Due to its accelerated
degradation and loss of mechanical properties, PGA may be less
preferred for ACL replacement.
[0037] Mechanical testing can be used to characterize the 3-D
fibrous construct's stress-strain relationship. It is believed that
similar stress-strain relationships to the rabbit ACL can be
engineered with a hierarchical design using 3-D braiding of a fiber
based absorbable scaffold. Accordingly, a structure to model a
rabbit ligament can be created. This synthetic ligament should have
a total gauge length of 1 cm. Mechanical tests are preferably
performed with a sample number of 6 for each particular test.
[0038] Tensile tests are preferably performed at strain rates
0.01%/s, 2.2%/s, and 50%/s as this helps to determine whether the
material is strain rate dependent. It is preferred that a sample
size of 18 be tested as suggested by the Food and Drug
Administration (Guidance Document for the Preparation of
Investigational Device Exemptions and Premarket Approval
Applications for Intra-Articular Prosthetic Knee Ligament Devices,
1987).
[0039] In a preferred embodiment of the present invention, the
braided construct is composed of three regions, with two end
sections designated for attachment of the construct, and the middle
region which serves as the replacement ligament or tendon. In this
embodiment, the middle region differs from the two end-regions in
size, braiding angle, porosity and mechanical strength. The length
and width of the replacement construct can be customized as
needed.
[0040] For ligament or tendon repair and reconstruction, the 3-D
braided scaffolds are preferably seeded with cells, preferably
mammalian cells, more preferably human cells. Various cell types
can be used for seeding. In a preferred embodiment, for ligament
and tendon replacement, the cells are either mesenchymal in origin
or capable of generating mesenchymal cells. Accordingly, preferred
cell types are those of the connective tissue, as well as stems
cells, more preferably pluripotent stem cells. For repair or
reconstruction of the ACL ligament, it may be preferable to seed
the scaffold with ACL host cells. As will be understood by those of
skill in the art upon reading this disclosure, however, the
scaffolds of the present invention can actually be seeded with any
cell type which exhibits attachment and ingrowth and is suitable
for the intended purpose of the braided scaffold. Some exemplary
cell types which can be seeded into these scaffolds include, but
are not limited to, osteoblast and osteoblast-like cells, endocrine
cells, fibroblasts, endothelial cells, genitourinary cells,
lymphatic vessel cells, pancreatic islet cells, hepatocytes, muscle
cells, intestinal cells, kidney cells, blood vessel cells, thyroid
cells, parathyroid cells, cells of the adrenal-hypothalamic
pituitary axis, bile duct cells, ovarian or testicular cells,
salivary secretory cells, renal cells, chondrocytes, epithelial
cells, nerve cells and progenitor cells such as myoblast or stem
cells, particularly pluripotent stem cells.
[0041] Cells used in the present invention are first harvested,
grown and passaged in tissue cultures. The cultured cells are then
seeded onto the 3-D braided scaffold to produce a graft material
composed of living cells and degradable matrix. This graft material
can then be surgically implanted into a patient at the site of
ligament or tendon injury to promote healing and repair of the
damaged ligament or tendon. Additional advantages of the braided
structure include its increased ease in implantation compared to
prior art constructs prepared from fiber bundles.
[0042] Design parameters such as polymer composition and the
response of primary ACL cells to 3-D braided constructs were
examined. Fibronectin (FN), one of the most abundant extracellular
adhesion proteins found in the body, is believed to be up-regulated
during ligament formation. Consequently, for these experiments
constructs were pre-coated with FN to enhance initial cell
adhesion. The attachment and growth of ACL cells on three types of
degradable polymers with various porosities were examined Scaffold
porosity ranged from 54% to 63%, with PLA constructs having a
porosity of 53.5.+-.6.9%, PGA having a porosity of 63.3.+-.7.3%,
and PLAGA constructs having an average porosity of 62.9.+-.3.6%.
Average pore diameter was similar between PLAGA and PLA (235-250
.mu.m) constructs, but smallest for PGA (177 .mu.m).
[0043] Primary ACL ligament-like cells exhibited semi-ovoid,
fibroblast-like morphology and when confluent, formed
multinucleated cultures with specific growth orientations. Cell
growth and morphology was dependent on polymer composition and
porosity. Extensive sheets of cells were observed on all three
types of polymers, but the morphology and cell spreading were
different from PLAGA to PLA scaffolds. Cell spreading was found to
be less on PLAGA, while the surface on both PGA and PLA were
smoother and had fewer cellular bundles. Quantitative cellular
growth (n=4) also revealed higher cells numbers on PLAGA and PLA,
when compared to PGA. Pre-coating the construct with fibronectin
resulted in an increase in proliferation, as reflected in a more
rapid decrease in solution pH when compared to uncoated constructs,
and controls without cells or fibronectin. It is likely that
fibronectin increased the initial number of cells attached to the
construct and consequently increased cellular growth and metabolism
in the long-term cultures. Thus, the ACL cellular response was
dependent on polymer composition and porosity. Further, pre-coating
of constructs with fibronectin increased cell attachment and growth
on these scaffolds.
[0044] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Microfiber Matrices
[0045] An electrospinning technique was used to produce
biodegradable non-woven fiber scaffold with an approximate
thickness of 0.5 mm. In this procedure, PLAGA (50:50) was dissolved
in methylene chloride to produce a 1:4 weight:volume solution. In
the electrospinning process, a 20 kV electric potential was applied
to the polymer solution and a collection screen to create an
electric field. The polymer solution was then sprayed onto the
collection screen for 30 minutes. This resulted in a uniform
non-woven microfiber matrix attached on the screen. The matrix was
removed, and cut into 1 cm.sup.2 pieces.
Example 2
3-Dimensional Fiber Braid
[0046] Three-dimensional fibrous matrices were fabricated using a
3-D braiding process as described by Ko, F. K. in Textile
Structural Composites, eds. Chou, T. W. and Ko., F. K. (Elsevier,
Amsterdam, 1989). In this procedure, PLAGA fiber (5:95 PLAGA) was
laced to produce yarns with a fiber density of 30 and 60 fibers per
yarn. Yarns were then placed in a custom built braiding loom with a
6 by 12 carrier arrangement. Sequential motion of the carriers
[alternating rows and columns] resulted in the formation of two
rectangular 3-D braids: a 30 yarn braid [braid #1] and a 60 yarn
braid [braid #2].
Example 3
In Vitro Cell Culture
[0047] Matrices were evaluated in a 2-week cell culture study using
fibroblasts and primary culture osteoblasts. All matrices were UV
sterilized for 24 hours per side prior to cell culture. Primary
culture osteoblasts isolated from neonatal rat calvaria were grown
to confluence in Ham's F-12 medium (GIBCO), supplemented with 12%
fetal bovine serum [FBS] (Sigma), as described by Jarcho, M. Clin.
Ortho. 1981 157:259. Mouse fibroblast cells (BALB/C C7 purchased
from ATCC: Arlington Va.) were grown to confluence in DMEM
supplemented with 10% FBS. Cells were seeded onto UV sterilized
matrices at a density of 5.times.10.sup.5 cells/matrix. Cells were
cultured on the matrices for 1, 3, 7, 10, and 14 days, and were
maintained with DMEM (10% FBS). At the various time points, cells
were fixed in glutaraldehyde, and dehydrated through a series of
ethanol dilutions. Samples for scanning electron microscopy [SEM]
were sputter coated with gold (Denton Desk-1 Sputter Coater).
Matrix and cellular structure was visualized by SEM (Amray 3000) at
an accelerating voltage of 20 kV.
Example 4
Degradation Properties of Various Polymers
[0048] Multifilament fibers of L-poly-lactide (PLA, 70 denier),
poly-glycolide (PGA, 60 denier) and their 82:18 co-polymer (PLAGA,
70 denier) were laced into 10 multi-fiber bundles for use in
degradation studies. The bundles were cut to a length of 6 cm and
sterilized with 70% alcohol followed by UV irradiation. The polymer
bundles were soaked in 10 ml of phosphate buffered saline (PBS,
pH=7.3), and in 10 ml of cell culture medium (.alpha.MEM, pH=7.3)
supplemented with 10% Fetal Bovine Serum, L-glutamine and 1%
antibiotics. The samples were shaken and maintained at 37.degree.
C. in a water bath for up to 3 weeks. The immersion ratios for both
solutions were as follows, PLA at 0.6 mg/ml, PLAGA at 0.8 mg/ml and
PGA at 0.7 mg/ml. The solutions were changed weekly, and at 1, 2, 3
and 4 weeks, pH (n=8) was measured and the amount of monomer in
solution were quantified by high performance liquid chromatography
(HPLC).
[0049] At 2 and 4 weeks after immersion, molecular weight, mass
retention and mechanical properties of the bundles (n=5) were
determined. Degradation-related morphological changes were examined
using scanning electron microscopy. For mass retention
measurements, the bundles were rinsed and lyophilized for 24 hours.
The dry weight was recorded (n=4) and the same samples were used
for molecular weight (MW) determination. Molecular weights (n=3)
for PLA and PLAGA (82:18) were measured by gel permeation
chromatography in tetrahydrofuran, using polystyrene standards. The
mechanical properties of the yarn under tension were tested on a
Instron machine (Model 4442, Instron Inc., MA), using a 500 N load
cell (gauge length =3 cm), at a strain rate of 2% per second.
Example 5
Effect of Polymer Construct on Morphology and Growth of Anterior
Cruciate Ligament Cells
[0050] Fibrous scaffolds were fabricated using the 3-D braiding
process described in Example 2. Fibers of L-polylactide (PLA, 70
deniers), polyglycolide (PGA, 60 deniers) and
poly-lactideco-glycolide 82:18 (PLAGA, 70 denier) were laced into
10 fiber/yarn bundles and these yarns were then braided using a 3-D
circular braiding machine. Circular 3-D braids of 24 yarns were
formed and cut into 1.5 cm lengths for these experiments. Dacron
constructs were similarly formed and used as controls.
[0051] The porosity, pore diameter and total pore area of the
construct were determined using the Autopore III porosimeter
(Micromimetics). Scanning Electron Microscopy (SEM) was used to
confirm pore distribution and examine pore geometry. The samples
were UV sterilized prior to culture. The constructs were each
coated with reconstituted human fibronectin (10 .mu.g/ml) for 30
minutes.
[0052] Primary ACL cells were isolated from 1 kg New Zealand white
rabbits. The excised ACL was digested using a 0.1% collagenase
solution, and only cells collected from fourth digestion were
selected for the study. Cells were cultured in .alpha.MEM+10% fetal
bovine serum, L-glutamine and 1% antibiotics at 37.degree. C. and
5% CO.sub.2. ACL cells were seeded on the scaffolds at a density of
80,000 cells/scaffold and grown for up to 28 days. Tissue culture
plastic and Dacron served as control groups. Media were exchanged
every two days and for each time point, the pH was measured. Cell
growth was measured using the cell-titer 96 assay. Cell morphology
and growth on the scaffolds were imaged using SEM.
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