U.S. patent application number 11/100981 was filed with the patent office on 2005-08-18 for cosmetic repair using cartilage producing cells and medical implants coated therewith.
Invention is credited to Amir, Abraham, Amir, Reva.
Application Number | 20050181505 11/100981 |
Document ID | / |
Family ID | 26747125 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181505 |
Kind Code |
A1 |
Amir, Abraham ; et
al. |
August 18, 2005 |
Cosmetic repair using cartilage producing cells and medical
implants coated therewith
Abstract
A method of cosmetically repairing a skin contour irregularity
in a subject is provided. The method is effected by introducing
cartilage producing cells into the skin contour irregularity
thereby effecting cosmetic repair thereof. A medical implant coated
with cartilage producing cells is further disclosed.
Inventors: |
Amir, Abraham; (Tel Mond,
IL) ; Amir, Reva; (Tel Mond, IL) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Family ID: |
26747125 |
Appl. No.: |
11/100981 |
Filed: |
April 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11100981 |
Apr 7, 2005 |
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10066753 |
Feb 6, 2002 |
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6911202 |
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60266442 |
Feb 6, 2001 |
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Current U.S.
Class: |
435/396 ;
623/15.12; 623/23.72 |
Current CPC
Class: |
A61K 35/12 20130101;
A61L 27/3817 20130101; C12N 5/0655 20130101; C12N 2500/32
20130101 |
Class at
Publication: |
435/396 ;
623/015.12; 623/023.72 |
International
Class: |
C12N 005/08; A61F
002/10; A61F 002/02 |
Claims
1-8. (canceled)
9. A medical implant implantable in a subject comprising
non-biological implant material coated with cartilage producing
cells, said cartilage producing cells being for reducing a
physiological response to the implant in the subject.
10. The medical implant of claim 9, wherein said cartilage
producing cells are selected from the group consisting of
chondrocytes and chondrocyte progenitor cells.
11. The medical implant of claim 9, wherein said cartilage
producing cells are syngeneic with respect to the subject.
12. The medical implant of claim 9, wherein said physiological
response is selected from the group consisting of an immune
response, an inflammatory response, encapsulation, ossification,
calcification and infection.
13. The medical implant of claim 9, further comprising an
intermediate layer being for increasing adherence of said cartilage
producing cells to said non-biological material.
14. The device of claim 13, wherein said intermediate layer
includes at least one item selected from the group consisting of
fibronectin and silicone.
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention relates to methods of using cartilage
producing cells, such as chondrocytes, for repairing cosmetic
defects such as skin contour irregularities. The present invention
further relates to implantable medical devices coated with
chondrocytes and, more particularly to coating of implants (natural
and artificial) with chondrocytes and or and/or their progenitors
as a means of reducing unfavorable host response to the
implant.
[0002] Cosmetic Repair
[0003] Plastic surgeons, dermatologists and their patients
continually search for new and approved methods for treating
damaged or aging skin. Historically, the treatment of facial
wrinkles was primarily accomplished with the use of chemical peels
or dermabrasion. The use of chemical peels has fallen out of favor,
because it is difficult to accurately control and predict the depth
of tissue injury after such peels are applied. Deeper chemical
peels in particular have an increased risk of hypopigmentation and
scarring.
[0004] Soft tissue fillers, such as collagen, have also been used
for treatment of skin imperfections. These fillers are typically
injected subcutaneously in a manner which enables filling and thus
smoothing out skin contour irregularities such as wrinkles and
scars.
[0005] Use of collagen as a dermal filler has several inherent
disadvantages. These disadvantages typically include dissolution
within 3 months, localized hypersensitivity in 1-6% of subjects,
hypopigmentation, induration and multiple nodules as a result of
foreign body granulomas (Boss et al. (2000) Clinics in Plastic
Surgery 27(4): 613-626).
[0006] Synthetic dermal fillers offer an alternative to collagen.
However, commercially available fillers such as Artecoll.TM.,
silicone and hyaluronen.TM. have not been approved for use in the
United States. Further, some subcutaneous fillers, such as
AlloDerm.TM. and SoftForm.TM. require surgical insertion, and might
result in infection, reabsorption, malposition and rejection (Boss
et al. (2000) Clinics in Plastic Surgery 27(4): 613-626).
[0007] Medical Implants
[0008] Non-self implants are used in many fields of medicine, such
as: plastic surgery, orthopedics, maxillo-facial surgery, etc. The
main disadvantage of prior art implants is the physiologic response
they provoke following implantation. This response, which is
typically induced locally around such implants is the body's
attempt to reject the implant, or to isolate it from the body by
creating a capsule around it. While a capsule may isolate the
implant from a systemic immune response, it can also contract and
cause distortion and pain of the organ. For example, a breast
prosthesis induces capsule formation around it. In a study by
Melmed (Plast Reconstr Surgery (1998) 101(5): 1364-73) 66% of the
implanted women had severe capsular-contracture (III or IV Baker
Degree).
[0009] Several approaches for reducing the physiological response
to an implant have been suggested in the prior art. For example,
Nishibe et al. (J Cardiovasc Surg (Torino) 2001 42(5):667-73)
describe bonding of fibronectin to high porosity expanded
polytetrafluoroethylene grafts as a means of improving host
acceptance of implants. Rinsch et al. (Transplantation 2001 Feb
15;71(3):345-51) teach transient immunosuppression as a means of
increasing host tolerance for encapsulated xenogenic cells
implanted into a subject. Neither of these methods employs cells.
As such, the effect must be transient.
[0010] There is thus a widely recognized need for, and it would be
highly advantageous to have, methods of using chondrocytes to
effect cosmetic repair and chondrocyte coated implants devoid of
the above limitation.
SUMMARY OF THE INVENTION
[0011] According to one aspect of the present invention there is
provided a method of cosmetically repairing a skin contour
irregularity in a subject, the method including introducing
cartilage producing cells into the skin contour irregularity
thereby effecting cosmetic repair thereof.
[0012] According to another aspect of the present invention there
is provided a medical implant implantable in a subject including
non-biological implant material coated with cartilage producing
cells, the cartilage producing cells being for reducing a
physiological response to the implant in the subject.
[0013] According to further features in preferred embodiments of
the invention described below, the method further includes
harvesting and optionally culturing the cartilage producing cells
prior to the introducing.
[0014] According to still further features in the described
preferred embodiments the skin contour irregularity is selected
from the group consisting of a rhytid, a subcutaneous defect and a
depression. Depressions may be, for example, the result of scarring
or previous injury.
[0015] According to still further features in the described
preferred embodiments introducing is effected via subcutaneous
injection.
[0016] According to still further features in the described
preferred embodiments the physiological response is selected from
the group consisting of an immune response, an inflammatory
response, encapsulation, ossification, calcification and
infection.
[0017] According to still further features in the described
preferred embodiments the device includes an intermediate layer
being for increasing adherence of the cartilage producing cells to
the non-biological material.
[0018] According to still further features in the described
preferred embodiments the intermediate layer includes fibronectin,
silicone or a combination thereof.
[0019] According to still further features in the described
preferred embodiments the cartilage producing cells are selected
from the group consisting of chondrocytes and chondrocyte
progenitor cells.
[0020] According to still further features in the described
preferred embodiments the cartilage producing cells are harvested
from the subject.
[0021] According to still further features in the described
preferred embodiments cartilage producing cells are harvested from
a source syngeneic with respect to the subject.
[0022] According to still further features in the described
preferred embodiments the cartilage producing cells are harvested
from a source allogeneic with respect to the subject.
[0023] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
methods which utilize chondrocytes to effect cosmetic repair and
implantable medical devices coated with an immunoprotective layer
of chondrocytes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0025] In the drawings:
[0026] FIG. 1 schematically illustrates the steps employed by the
method of cosmetically repairing skin contour irregularities of the
present invention.
[0027] FIG. 2 is a schematic representation of a medical implant
coated with an immunoprotective layer of chondrocytes fabricated
according to the teachings of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention is of methods for using chondrocytes
to effect cosmetic repair of skin contour irregularities.
Specifically, the present invention can be used to repair cutaneous
contour irregularities in a minimally invasive fashion.
[0029] The present invention is further of implants coated with
chondrocytes or their progenitors as a means of reducing
unfavorable host response to the implant. Specifically implants
according to the present invention are resistant to encapsulation
and inflammatory response.
[0030] The principles and operation of methods and implants
according to the present invention may be better understood with
reference to the drawings and accompanying descriptions.
[0031] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description and pictured in the drawings. The invention is capable
of other embodiments or of being practiced or carried out in
various ways. Also, it is to be understood that the phraseology and
terminology employed herein is for the purpose of description and
should not be regarded as limiting.
[0032] While the use of chondrocytes in the art is well known, such
use has typically been limited to reconstructive surgery and tissue
repair.
[0033] Park and Ward (Facial Plast Surg (1995) 11(4): 278-83) teach
tissue-engineered cartilage for implantation and grafting. Their
teachings are directed to culture of chondrocytes on a
three-dimensional biodegradable template which is first briefly
incubated in vitro, then implanted into a recipient host. The
template then resorbs and is replaced with new cartilage produced
by the chondrocytes. Use of a template prevents injection of
cultured chondrocytes and requires invasive implantation techniques
that make these teachings ill suited to cosmetic repair of
cutaneous contour irregularities such as wrinkles and scars.
[0034] Kim et al (Plast Reconstr Surg (1994) 94(2): 233-40) teach
cartilage engineered in predetermined shapes employing cell
transplantation on synthetic biodegradable polymers. These
teachings are similar to those of Park and Ward and are ill suited
to cosmetic repair of cutaneous contour irregularities for the same
reasons.
[0035] Sims et al. (Plast Reconstr Surg 1996 98(5): 843-50) teach
injectable cartilage using polyethylene oxide polymer substrates.
Sims et al. specifically teach use of polyethylene oxide polymer
substrates and teaches against use of chondrocytes in cosmetic
applications. Specifically, Sims et al concluded that control
specimens from eight implantation sites consisting of chondrocytes
alone or polyethylene oxide substrates did not demonstrate any
gross or histologic evidence of neo-cartilage formation.
[0036] U.S. Pat. No. 5,944,754 also to Vacanti teaches tissue
resurfacing with hydrogel-cell compositions and methods for
generating new tissue on a surface, e.g., a surface of damaged or
lost tissue of a structure or organ in a mammal. The methods
involve applying a thin layer of a liquid hydrogel-cell composition
to the surface; and allowing the liquid hydrogel-cell composition
to solidify, thereby forming a matrix that enables the tissue
precursor cells to grow and generate new tissue. The surface can be
internal, e.g., the surface of an organ or the internal surface of
a blood vessel, or external, e.g., skin. These teachings require
matrix formation and dispersal of cells therein. Use of a matrix is
a disadvantage with respect to cosmetic repair of contour
irregularities.
[0037] U.S. Pat. No. 5,902,741 to Purchio et al. teaches
three-dimensional cartilage cultures. Specifically, Purchio teaches
a method of stimulating the proliferation and appropriate cell
maturation of cells and tissues in three-dimensional cultures in
vitro using TGF-.beta. Teachings of Purchio are limited to in-vitro
culture. Further, teachings of Purchio et al. require use of TGF
beta and the use of a three dimensional substrate which would
require an invasive procedure to implant. In summary, the teachings
of Purchio et al. can not be used for cosmetic repair of skin
contour irregularities.
[0038] U.S. Pat. No. 6,139,578 to Lee et al. teaches preparation of
cell seeded ceramic compositions, specifically a synthetic, poorly
crystalline apatitic (PCA) calcium phosphate material seeded with
cells. The compositions taught by Lee et al. are useful for a
variety of applications, including in vivo and in vitro tissue
growth (preferably bone or cartilage), osseous augmentation, and
methods of diagnosing disease states by assaying tissue-forming
potential of cells isolated from a host. The invention disclosed by
Lee et al. also provides in vitro cell culture systems and cell
encapsulation matrices. As with the patents cited hereinabove, Lee
teaches use of a matrix, rendering his teachings ill suited for
cosmetic repair of contour irregularities.
[0039] Brown et al. describe chondrocyte-smooth muscle cell
compositions suitable for reconstructive applications. Brown et al
note that the plastic properties of chondrocytes are sub-optimal
for reconstructive applications and as such suggest mixing in of
other cell types with the chondrocyte culture [e.g. chondrocytes
plus smooth muscle cells; Brown et al. in Tissue Eng (2000) 6(4):
297-305].
[0040] Although numerous prior art publications teach various uses
of chondrocytes in reconstructive surgery and tissue repair, none
describe or suggest cosmetic repair of skin contour irregularities
using chondrocytes or chondrocyte progenitors.
[0041] Thus, according to one aspect of the present invention there
is provided a method of cosmetically repairing a skin contour
irregularity in a subject. As used herein, the phrase "skin contour
irregularity" refers to wrinkles of the skin, dermal depressions
(rhytids), scars and the like.
[0042] The method according to this aspect of the present invention
is effected by introducing cartilage producing cells such as
chondrocytes or chondrocyte progenitors into the skin contour
irregularity of the subject.
[0043] The present invention requires the use of cartilage
producing cells, preferably chondrocytes, chondrocyte progenitor
cells or mixtures thereof. In contrast to the prior art, cultures
of these cartilage producing cells are preferably free of other
cell types. As is described by Brown et al. the addition of other
cell types (e.g., smooth muscle cells) can reduce the rigidity of a
resultant mixed cell population, a quality which is disadvantageous
in repair of skin contour irregularities which require a
substantially rigid filler material.
[0044] The cartilage producing cells utilized by this aspect of the
present invention can be obtained from a variety of tissue
sources.
[0045] Preferably, cartilage is harvested with minimal functional
harm and aesthetic damage. Preferably, harvest is from the auricle
of the ear, most preferably from the concha such that a virtually
invisible posterior scar remains. Alternately, harvest may be from,
for example, the septum of the nose. In a case where the subject is
undergoing surgery, and cartilage is being harvested for post
surgical scar repair, other sources of cartilage may present
themselves during surgery.
[0046] Typically, a piece of about 1.times.1 cm of the conchal
cartilage is harvested and chondrocytes released therefrom are
either used directly or cultured for a predetermined time period
prior to administration (for further detail see the Examples
section which follows).
[0047] Preferably, the cartilage is harvested from the subject to
be treated and thus constitute an autologous graft. Alternatively,
the cartilage producing cells are harvested from a source syngeneic
with respect to the subject.
[0048] Since chondrocytes and the cartilage produced therefrom
display weak immunogenicity, cells harvested from an allogeneic or
even xenogeneic tissue source can also used.
[0049] Allografts and xenografts offer tremendous advantage with
respect to autologous transplant. Use of allograft or xenograft
cultures, if properly engineered to be immunologically neutral,
allows maintenance of a small number of cell cultures for virtually
all recipient subjects. This reduces patient trauma from tissue
harvest, reduces the expense of generating primary cultures and
reduces the waiting time required for a procedure.
[0050] Introducing chondrocytes or chondrocyte progenitors into a
skin irregularity of the subject is carried out in a minimally
invasive fashion, most preferably by injection of the cartilage
producing cells subcutaneously. A small volume of cell suspension
injected into the site of the subcutaneous contour defect will
eventually fill the defect. Contact inhibition will prevent
over-filling of the defect so that depressions do not become bumps.
Chondrocytes harvested as described herein, and in particular
autologous chondrocytes, can also be used as an immunoprotective
layer for non-biological implants.
[0051] Thus, this aspect of the present invention provides novel
and simple methodology for repairing skin contour irregularities.
This methodology traverses the limitations of prior art methods by
providing a filler material (chondrocytes) which can be easily
produced and introduced into the subject's skin, does not elicit a
physiological response (e.g., immune response) and is not absorbed
over time. In addition, the cartilage formed from this filler
material is more capable of resisting facial muscles movements than
prior art fillers and as such, substantially reduce the chances of
the wrinkles or rhytids returning.
[0052] Use of chondrocytes as an immunoprotective layer for tissue
implants is known in the art (U.S. Pat. No. 5,741,685 to Vacanti).
However, Vacanti does not teach or suggest coating of
non-biological material with chondrocytes.
[0053] Thus, according to another aspect of the present invention
there is provided a medical implant implantable in a subject. The
medical implant is composed of non-biological implant material
which is coated with cartilage producing cells, such as
chondrocytes. For purposes of this specification and the
accompanying claims, the phrase "non-biological" refers to any
material which does not contain living cells.
[0054] The cartilage producing cells function to reduce a
physiological response to the implant in the subject. The
physiological response may be, for example, an immune response, an
inflammatory response, encapsulation, ossification, calcification
or infection.
[0055] To reduce such a physiological response, the cartilage
producing cells utilized by this aspect of the present invention
are preferably derived from the subject or a tissue source which is
syngeneic with respect to the subject. It will be appreciated
however, that other tissue sources (allogeneic, xenogeneic) may
also be used, provided measures are taken to substantially reduce
the immunogenicity of cartilage producing cells harvested from such
sources.
[0056] In order to facilitate increased adherence of the cartilage
producing cells to the device, an intermediate layer applied to the
device may be employed. While many materials may be useful in
construction of this intermediate layer will be known to those of
ordinary skill in the art, fibronectin, silicone and combinations
thereof have been found to be especially well suited for use in the
context of the claimed device.
[0057] Thus, this aspect of the present invention provides a
medical implant which does not elicit a physiological response
following implantation, thus traversing problems associated with
rejection of implants and as such prolonging the service life of
such an implant within the subject's body.
[0058] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0059] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0060] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include cellular and
biochemical techniques. Such techniques are thoroughly explained in
the literature. See, for example, "Current Protocols in Immunology"
Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds),
"Basic and Clinical Immunology" (8th Edition), Appleton &
Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), "Selected
Methods in Cellular Immunology", W. H. Freeman and Co., New York
(1980); "Animal Cell Culture" Freshney, R. I., ed. (1986); and
"Methods in Enzymology" Vol. 1-317, Academic Press; all of which
are incorporated by reference as if fully set forth herein. Other
general references are provided throughout this document. The
procedures therein are believed to be well known in the art and are
provided for the convenience of the reader. All the information
contained therein is incorporated herein by reference.
[0061] As mentioned hereinabove, the method of cosmetic repair and
the coated implants of the present invention both utilize cartilage
producing cells.
[0062] The following Examples describe methodology which can be
used for harvesting, culturing and utilizing such cells for
cosmetic repair and implant coating.
Example 1
Harvesting Cartilage From a Donor
[0063] A cartilage explant of about 1.times.1 cm. is removed from
an incision of approximately 1 cm in length along the posterior
side of the concha of the auricle. A number 15 scalpel blade is
well suited to this purpose, although other cutting implements may
be employed without significantly changing the outcome of the
harvest. The incision is then closed with resorbable suture
material. The resultant scar is small and hidden by virtue of its
location.
[0064] As is further described below, cartilage producing cells
enzymatically released from the explant (further described below)
can be used directly in cosmetic repair or coating of implants, or
alternatively, such cells can be cultured prior to use.
Example 2
Culture of Cartilage Producing Cells
[0065] Several methods can be used for harvesting chondrocytes from
the cartilage explant and for culturing harvested cells (see for
example, Robinson et. al. Autologous chondrocytes transplantation
for reconstruction of isolated joint defects: the Assaf Harofeh
Experience. Israel Medical Association Journal, Vol.
2:290-295).
[0066] The following describes a method suitable for chondrocyte
harvesting and culturing. The cartilage explant is digested with
collagenase (0.2% weight/volume) in complete media containing 10%
fetal bovine serum, 2 mM L-glutamine, non-essential amino acids, 50
mg/ml proline, 1 mM sodium pyruvate and 35 .mu.g/ml gentamicin for
20 hrs at 37.degree. C. Liberated cells (chondrocytes, chondrocyte
progenitors) are spun, resuspended in complete medium, counted and
plated at 10.sup.6 cells per T-150 flask. Cells can be passed at
confluence (every 5-7 days) until sufficient cell number are
achieved for purposes described in examples 3 and 4.
[0067] It is important that the stromal mass be suspended or
floated in the medium during the incubation period in order to
maximize proliferative activity. In addition, the culture should be
"fed" periodically to remove the spent media, depopulate released
cells, and add fresh media. Preferably, proline, a non-essential
amino acid and ascorbate are also included in chondrocyte
cultures.
Example 3
Cosmetic Repair By Introduction of Cartilage Producing Cells
[0068] Chondrocytes produced as describe above are dissociated by
brief trypsinization monitored by microscopy. When sufficient
disruption of the monolayer has been achieved, cells are washed 2
times in media without fetal calf serum and resuspended in saline
or any other physiologically acceptable buffer for injection.
[0069] In order to facilitate injection, a cell density of 10.sup.6
cells/ml is loaded into a syringe fitted with a needle suitable for
subcutaneous injection. Because it is desirable to leave no sign of
the injection, a narrow gauge needle is employed. Preferably a 25
gauge, more preferably a 30 gauge needle is used. In general,
approximately 1-2 ml. are required for each cosmetic repair site
(e.g. each wrinkle), although repair of large areas may require
larger volumes. However, it is generally best to prepare twice this
amount since the exact volume to be filled is difficult to
calculate. Injection of the chondrocytes is subcutaneous at the
site of the contour irregularity. Because the cells can grow and/or
proliferate, care is exercised not to overfill the contour
irregularity. It will be appreciated that since the metabolic
demand of chondrocytes and the cartilage produced therefrom is low,
the filler material is maintained in a viable state for long
periods of time by diffusion of nutrients and gasses from the
surrounding tissue.
[0070] FIG. 1 schematically exemplifies cosmetic repair according
to the teachings of the present invention.
[0071] Cosmetic repair is effected as follows, in a first step,
cartilage 28 is harvested from a human tissue such as an ear 24 as
described hereinabove. Cells 30 are dispersed and cultured 32 as
described hereinabove.
[0072] Cells 34 from culture 32 are then loaded into syringe 36 and
subcutaneously injected into the skin contour irregularity 26
(e.g., wrinkle scar, depression etc.) thereby effecting cosmetic
repair thereof.
Example 4
Coating of a Medical Implant with Cartilage Producing Cells
[0073] FIG. 2 illustrates a medical implant according to the
teachings of the present invention. The medical implant of the
present invention is fabricated by incubating non-biological
implant material 52 (e.g., breast implant) in a solution containing
5 .mu.g/ml of fibronectin overnight. Following incubation, the
fibronectin solution is removed and replaced with chondrocytes 34
predissociated by brief trypsinization monitored by microscopy. An
initial inoculum of 10.sup.6 cells/cm.sup.2 of surface area of the
implant material is employed. This allows confluent coverage of the
device in 14 days. An additional 21 days of incubation are carried
out in order to allow collagen 54 deposition by the newly formed
chondrocytes. Optionally, non-biological implant material 52 is
covered with an intermediate layer 56 which facilitates adherence
of chondrocytes 34.
[0074] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents, and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent, or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *