CA2192064A1 - Three-dimensional cartilage cultures - Google Patents

Abstract

The present invention relates to a method of stimulating the proliferation and appropriate cell maturation of a variety of different cells and tissues in three-dimensional cultures in vitro using TGF-.beta. in the culture medium. In accordance with the invention, stromal cells, including, but not limited to, chondrocytes, chondrocyte-progenitors, fibroblasts, fibroblast-like cells, umbilical cord cells or bone marrow cells from umbilical cord blood are inoculated and grown on a three-dimensional framework in the presence of TGF-.beta.. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope the framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. The living stromal tissue so formed provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo.

Description

V~0 9!~133821 2 l 9 ~ ~ ~ 4 .

T~REE - DI~bnoll ~T. CARTIL~GB CULTURES

The invention relates to growing stromal cells, such as uhondLuuyLes~ progenito~ -_hUI~dL ~Cy Les, fibroblasts and/or fibroblast-like cells on a three-dimensional scaffold or rL ~L~ in vitro under conditions which enhance the formation of cartilage in culture. A variety of biodegradable and nonbiodegradable matrices treated with sterilizing agents and/or ploaeduL~s can be used as the scaffold in accordance with the invention. A variety of culture conditions can be adjusted, ~nrll-~;ng the control of physical conditions such as yLas~uLe and/or the addition of growth factors. Additionally, the cultured cells can be genetically engineered to express gene products beneficial to growth, transplantation and/or amelioration of disease conditions.
The resulting three-ta~ ionAl cultures and biological rt~plAt --t cartilage tissue constructs have a variety of applications ranging from transplantztion or implantation in vivo, to screening the effectiveness of cytotoxic ,- a_ and pharmaceutical agents in vitro. The invention is " LLtlted by way of ~- ~loc describing the three-dimensional culture of ohund~u~yLes.

2 . ~r~'v~P. _ OF TXT;: 1L~
Articular cartilages are rt~cpt~ncihlr for providing moveable joints the ability for smooth gliding mction. The articular cartilages are firmly attached to the underlying bones and measure less than Smm in th1r~n~cc in human joints, with considerable variation at~pt~nt~;ng on joint and site within the joint. The articular cartilages are aneural, avascular, and alymphatic. In adult humans, they WO gC,3382l ~ 1 9 2 ~ ~ ~ PC~'flJg~S/0~96 derive their nutrition by a double diffusion system through the synovial membrane and through the dense matrix of the cartilage to reach the chul.dLv~yLe.
The bio~hem; QR ~ composition of articular cartilage includes up to 65-80% water (~r~; ng on the cartilage~, with collagen as the most prevalent organic constituent. Articular cartilage consists of highly speriRli7pd chondrocytes ~u,Lu~l.ded by a dense extracellular matri~ consisting mainly of type II
collagen, proteoglycan and water. Collagen (mainly type II) accounts for about 15-25% of the wet weight and about half the dry weight, except in the superficial zone where it accounts for most of the dry weight. Its concentration is usually ployLessively reduced with increasing depth from the articular surface. The proteoglycan content accounts for up to 10~ of the wet weight or about a quarter of the dry weight. Proteoglycans consist of a protein core to which linear sulfated polysaccharides are attached, mostly in the form of chondroitin sulfate and keratin sulfate. Tn addition to type II collagen, articular collR~n contains several other collagen types tIV, V, IX and X) with distinct structures. There are a variety of interactions between these individual macromolecules, which include both noncovalent associations between proteoglycans and collagens, and covalent bonds between different collagen species.
Resistance of the extracellular matrix to water flow gives cartilage its ability to ~iep~ee high joint loads. It absorbs shûck and ml~;m;7~e stress on s~ l bone (Now et al., 1984, J. Biomech.
17:377-394). Adult cartilage and bone have a limited ability of repair.
Damage of cartilage produced by disease, such as rheumatoid and/or osteoarthritis, or trauma can lead to serious physical deformity and debilitation. As human articular cartilage ages, its tensile properties WO9~1338ZI 2 1 9 2 0 ~ 4 PCTIUS9~107Z96 .

change. The superficial zone of the knee articular cartilage exhibits an increase in ten6ile strength up to the third decade of life, after which it decreases ~ markedly with age as detec~hle damage to type II
collagen occurs at the articular surface. The deep ~ zone cartilage also exhibits a ~Lv~Lessive decrease in tensile strength with increasing age, although coll~gPn content does not decrease. These oLseLvGtions indicate that there are changes in r- '-n;r~l and, hence, ~L-u~LuLGl organization of cartilage with aging that, if sufficiently developed, can prP~icpnce cartilage to traumatic damage. In osteoarthritic cartilage there is excessive damage to type II collagen, resulting in crimping of collagen fibrils. In rheumatoid arthritis, the combined actions of free radicals and pro~P;n~ces released from polymorpholeukocytes cause much of the damage seen at the articular surface. (Tiku et al., l99o, J.
Immunol. 145:690-696). Induction of cartilage matrix degradation and prote;n~CPc by chondrocytes is probably induced primarily by interleukin-1 ~IL-l) or tumor necrosis factor-~ (TNF-~) (Tyler, 1985, Biochem.
J. 225:493-507).
The current therapy for loss of cartilage is repl~r L with a prosthetic material, for example, cilicnnP for jr repairs, or metal alloys for ~oint r~l in L. Pl~ t of prosthetic devices i6 usually associated with loss of underlying tissue and bone without I~CV~LY of the full function allowed by the original cartilage. Serious long-term complications associated with the ~L ~~enCe of a peL~anel.L foreign body can include infection, erosion and instability.
Use of sterilized bone or bone powder or surgical steel seeded with bone cells which were eventually implanted have been largely lln uvv~''ful because of the nvn deyL~dable nature of the cell support.

WO95/33X2l 2 1 ~ 2 ~ 6 4 r c ~ 5 According to one procedure fibroblasts are exposed n vitro for a minimum of three days, to a soluble bone protein capable of stimulating in vitro and/or in vivo a ~L~-dLv~ ic ~espunse. The activated fibroblasts are then transferred in vivo by ~;nin~ them with a blodegradable matrix, or by intra-articular injection or atf~ ~ to allografts and prss~hetic devices.
The disadvantage of this method is that chondLocJ~ ic is not allowed to develop in the short-term cultures and there is an unduly heavy reliance for cartilage synthesis by the exposed fibroblasts at the implant site. Caplan, A., U.S. Pat. No. 4,609,551, issued September 2, 1986.
U.S. Pat. No. 5,041,138 to J.P. Vacanti et al., issued August 20, 1991, describes growth of cartilaginous ~LL~ULUL~R seeding ul.ondLouyLes on biodegradable matrices for ~JhC~ t implantation L~
vivo. Although this system offers the advantage of a greater surface area and exposure to nutrients, the conditions employed for culturing the chondrocytes are routine and no efforts have been made to optimize the conditions for the ~hul-dLuuyLes to produce collagen and other cartilage-type macromolecules.

2.1. Growtb Factors r-' H~ ~e~
Growth factors have paracrine or autocrine ef$ects on cell metabolism and can retard or enhance chondrocyte division, matrix synthesis, and degradation.

2.1.1. Transformin~ Growth Factor-~
TGF-~ refers to a growing family of related dimeric proteins which regulate the growth and differentiation of many cell types (Barnard et al., 1990, Biochem. Biophys. Acta. 1032:79-87; M~C5~
1990, Annu. Rev. Cell. Biol. 6:597-619; Roberts and Sporn, 1990, pp. 419-472 M.~. Sporn and A.B. Roberts W095133821 2 1 9 2 0 6 4 r~

(eds.), Peptide Growth Factors and Their Receptors I, Springer-Verlag, Berlin). Members of this family include TGF~-1 (Derynck et al., 1985, Nature 316: 701-705; Moses et al., 1981, Cancer Res. 41:2842-2848;
Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78, 5339-5343; Sharples et al., 1987, DNA 6:239-244), TGF-~2 (DeNartin et al., 1987, ENBO J. 6:3676-3677; Hanks et al., 1988, Proc. Natl. Acad. Sci. USA 85, 79-82;
Ikeda et al., 1987, Biorh~mi-stry 26, 2406-2410;
Madisen et al., 1988, DNA 7, 1-8; Marquardt et al., 1987, Biol. Chem. 262:12127-12131, Seyedin et al., 1987, J. Biol. Chem. 262:1946-1949), TGF-~3 (Derynck et al., 1988, EMBO J. 7:3737-3743; Jakowlew et al., 1988, Endocrinnol. 2, 747-755, TGF-~4 (Jakowlew et al., 1988, Mol. Endocrinnol. 2:1064-1069), TGF-~5 (~n~aiAh et al., 1990, J. Biol. Chem. 265:1089-1093), and the more distantly related Nullerian inhibitory substance (Cate et al., 1986, Cell. 45:685-698), the ;nh;hinc (Mason et al., 1985, Nature 318:659-663), the bone morphogenetic proteins (Wozney et al., 1988, Science 242:1528-1534) and OP-1 (Ozkaynak et al., 1990, EMBO J. 9:2085-2093). Newly discov~ed members include OP-2 (Ozkaynak et al., 1992, J. Biol. Chem.
267:25220-25227), GDF-l (Lee, 1990, Mol. Endocrinnol.
4:1034-1040); GDF-3 and GDF-9 (McPherron and Lee, 1993, J. Biol. Chem. 268:3444-3449) and Nodal (Zhou çt al., 1993, Nature 361:S43-546).
TGF-~ was first characterized for its effects on cell proliferation. It both stimulated the anchorage-in~ l~n~r~r~ growth of rat kidney fibroblasts (Roberts et al., 1981), and inhibited the growth of monkey kidney cells (Tucker et al., 1984, Proc. Natl.
Acad. Sci. USA 81:6757-6761). Since then, it has been shown to have many diverse biological effects: it stimulates bone formation (Noda and C~mi 11 ir~re, 1989, Endocrinnol. 124:2991-2995; Joyce et al., 1990, J.
Cell. Biol. 110:2195-2207; Marcelli et al., 1990, J.

Wo95133X21 2 ~ 9 2 0 6 ~ ~5 Bone Mineral Res. 5:1087-1096; Beck Ç~_B11, 1991, J.
Bone Mineral Res. 6:961; Mackie and Trechsel, 1990, J.
Cell. Biol. 110, 2195-2207), induces rat muscle cells to produce cartilag_ _~ecific macromolecules tSeyedin et al., 1984, J. Biol. Chem. 261:5693-5695; Seyedin et al., 1986, J. Biol. Chem. 261:56g3-5695; and Seyedin et al., 1987, J. Biol. Chem. 262:1946-1949~, inhibits the growth of early he~atopoietic progenitor cells (Goey et al., 1989, J. Immunol. 143:877-880), T cells (Kehrl et al., 1986, J. Exp. Med. 163:1037-1050), 8 cells (Kasid et al., 1988, J. Immunol. 141, 690-698), mouse keratinocytes (Pietenpol et al., 1990, Cell 61:777-785; Coffey et al., 1988, Cancer Res.
48:1596-1602) and several human cancer cell lines ~Roberts et al., 1985, Proc. Natl. Acad. Sci. USA
82:119-123; Ranrh~li~ et al., 1987, Biophys. Res.
Co~mun. 148:783-789). It increases the synthesis and secretion of c~ q~n and fibronectin (Ignotz and Massague, 1986, J. Biol. Chem. 261:4337-4345;
Centrella et ~1., 1987, J. Biol. Chem. 262:2869-2874;
Malemud et nl., 1991, J. Cell Physio. 149:152-159;
Galera et al., 1992, J. Cell Physio. 153:59~-606;
Phillips ç~_gl_, 1994, soc. Inv. Derm. 103-2:228-232), accelerates healing of incisional wounds t~ustoe et al., 1987, Science 237:1333-1335~, ~u~Less~s casein synthesis in mouse mammary eYrl~nt~ i n~on et al., 1993, J. Cell. Biol. 120:245-251), inhibits DNA
synthesis and pho~huLylation of pRb in rat liver epithelial aells (~hitson and Itakura, 1992, J. Cell.
Biochem. 48:305-315~, stimulates the production of BFGF binding proteoglycans (Nugent and Edelman, 1992, J. Biol. Chem. 267:21256-21264), modulates rh~srhorylation of the EGF receptor and proliferation of epidermoid carcinoma cells (Goldkorn and M~n~t~l5t~hn, 1992, Cell Growth and Differentiation) and can lead to apoptosis in uterine epithelial cells (Rotello et al., 1991, Proc. Natl. Acad. Sci. USA

WO9S/33821 2 1 9 2 0 6 ~ PCT/U595l07296 .

88:3412-3415), cultured hepatocytes and regressing liver (Oberhammer et al., 1992, Proc. Natl. Acad. Sci.
USA 89:5408-5412). It can mediate cardioprotection against reperfusion injury tLefer et al., 1990, Science 249, 61-64) by inhibiting neutrophil adherence to endothelium (Lefer et al., 1993, Proc. Natl. Acad.
Sci. USA 90:1018-1022), and it protects against experimental autoimmune ~i ~P~D~ in mice (Kuruvilla et al., 1991, Proc. Natl. Acad. Sci. USA 88:2918-2921).
In contrast to the foregoing reports of the ability of TGF-~ to induce the production of cartilage-speci~ic macromolecules in muscle cells and chondL~yLes~ TGF-~ was found to act synergistically with fibroblast growth factor to inhibit the synthesis of collagen type II by chicken sternal chondrocytes (Horton et al., 1989, J. Cell Physio. 141:8-15) and TGF-~ inhibited production of type II collagen in rat chondrocytes (Rosen et al., 1988, J. Cell Physio.
134:337-346). In fact, TGF-~ has emerged as the prototypical inhibitor of the proliferation of most normal cell types in culture as well as in vivo, exhibiting a remarkable diversity o~ biological activity (Alexandrow, M.G., and Moses, H.L., 1995, Cancer Res. 55:1452-1457).
TGF-~1 has been purified from human and porcine blood platelets, Assoian et al., 1983, from human pl~cPnta, Frolick et al., 1983, and rP~hin~nt TGF-~1 is currently available, Gentry et al., 1988, Mol.
- Cell. Biol. 7:3418-3427.

2.1.2. Inculin-like Growth Factors I ~n~ IGF-I ana IGF-II) - Insulin alone is much less potent than IGF-I in stimulating collagen matrix synthesis. Insulin, ~ however, Pnh~nCP~ proteoglycan synthesis in the pIes~n-e of a low concenLL~tion of serum (1%). IGF-I, previously designated somatomedin c, is a potent ~ sl3382l 21 9 ~ G ~
.

inducer of collagen and proteoglycan synthesis in vitro. tLindahl et al., 1987, J. Endocrinnol.
115:263-271; Markower et al., 1989, Cell. Biol. Int.
Rep. 13:259-270).
IGF-II stlmulates DNA and ~NA synthesis and is more potent than IGF-I in stimulating clonal growth in fetal cells, whereas IGF-I is more effective on adult chul.dLu~yLes. IGF-II can stimulate proteoglycan synthesis, but, like insulin, is much less effective than IGF-I (~cQuillan et al., 1986, Biochem. J.
240:423-430).

2.1.3. Growth Normone (GH~
Parenteral administration of GH can stimulate localized growth plate development in vivo.
Hypophysectomy leads to disappearance of IGF-I in growth plate chondrocytes, indicating a cessation of synthesis. On the other hand, treatment with GH, syst~ic~lly or locally, results in the appearance of IGF-I. Reports of direct stimulatory effects of GH on cell growth in vitro (Maro et al., 1989, Endocrinnology 125:1239-1445) conflict with reports that it has no effect (Burch et al., 1985, J. Clin.
Endccrinnol. Metab. 60:747-750).

2.1.~. Qther Growth Factors Epidermal growth factor (EGF) alone has no effect on ~I.ol.dLu~yLe proliferation. Together with insulin, EGF synergistically stimulates proteoglycan synthesis and induces prcliferation of chondLu~yLes. (Osborn et ~ , 1989, J. Orthop. Res. 7:35-42). 8asic fibroblast growth factor (bFGF) inhibits proteoglycan synthesis in fetal articular cartilage (~2 -- -n et al., 1986, J. Cell. Physiol. 127:317-322), but it appears to function additively with IGF-I in adult articular cartilage and stimulates proteoglycan synthesis (Osborn, K.D., et al., 1989, J. Orthop. Res. 7:35-42~.

W095/33821 2 ~ ~ ~ 0 ~ 4 ra~

Platelet-derived growth factor (PDGF) also r~nh Inr~r~c proteoglycan synthesis ~Prins et al., 1982, Arthritis Rheum. 25:1228-1238).

3. 8~MMaRY OF THE ~
The present invention relates to the growth and p~epaL~Lion of cartilage m vitro which can be used for a variety of ~u.~oses in vivo. In accordance with the invention, stromal cells which elaborate cartilage-specific macromolecules and extracellular matrix proteins, are inoculated and grown on three-dimensional f~ -~L~S or biodegradable scaffolds.
The stromal cells, which are inoculated onto the scaffold, may include ch~ndLu~yLes, chol.dLu~yLe-progenitors, fibroblasts, fibroblast-like cells and/or cells capable of producing collagen type II and other collagen types, and proteoglycans which are typically ~L~duaed in cartilaginous tissues tSee Table I, iB~E~). The stromal cells and connective tissue proteins secreted by the stromal cells attach to and substantially envelope the three-dimensional rL JL~
or construct, _ e' of a biocompatible non-living material formed into a three-dimensional ~LLU~LULe~
having interstitial spaces bridged by the stromal cells. The living stromal tissue so formed provides the support, growth factors, and regulatory factors nr~r~r~Sc~ry to sustain long-term active proliferation of stromal cells in culture and/or cultures implanted ln v vo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form e ts of adult tissue analogous to counterparts n vivo.
In another ~mho~i t of the invention, the stromal cells are inoculated and grown on a three-n~l rL JLk placed in any container that can be manipulated to allow intermittent p~essuLe changes or in a bioreactor system specially designed for the _ g _ W'O 95/33821 2 1 ~ 2 ~ 6 ~ PC 1'11~95/07296 .

n yitro production of cartilage tissue ~n~LLu~Ls, which allows for pressurization of the chamber during growth and an adequate supply of nutrients to stromal cells by convection.
In yet another c-~o~ of the invention, the stromal cells are stirulated to produce cartilage using ~ g~ cly added growth factors, e.q., TGF-~with or without ascorbate, in culture. Alternatively, the stromal cells can be genetically engineered to express the gsnes for specific types of TGF-~ se.q., TGF-~) for ,uccesDrul and/or i ~v~d LULII~V~L o~
cartilage production post-transplantation.
In yet another P~ho~i L of the invention, the stromal cells can be genetically engineered to express a gene product beneficial for successful and/or improved transplantation. For exanple, the stromal cells can be genetically engineered to express anti-inflammatory gene products to reduce the risk of degen~Lative ~ic~lc~c like rheumatoid arthritis resulting in failure of cartilage due to inflammatory reactions; ç~g., the stromal cells can be engineered to express peptides or polypeptides C~LL --L~ ;ng to the idiotype of neutralizing antibodies for granulocyte-m3~L~hag~ colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), interleukin-2 SIL-2), or other inflammatory cytokines and mediators.
Preferably, the cells are engineered to express such gene products transiently and/or under inducible control during the post-operative Lec~v~Ly period, or as a chimeric fusion protein anchored to the stromal cell, e.a., a chimeric 1ecllle -FZ~ of an intracellular andlor tr~ a~-e domain of a receptor or receptor-like molecule, fused to the gene product as the extracellular domain.
In another alternative embodiment, the stromal cells can be genetically engineered to "knock outH
expression of factors that promote re~ection or WO95/33821 2 ~ 9~0~ PCT~S95/07296 degenerative changes in articular cartilage due to aging, rheumatoid disease or inflammation. For example, expression of pro-inflammatory mediators such as GM-CSF, TNF, IL-l, IL-2 and cytokines can be knocked out in the stromal cells to reduce the risk of ~ inflammation. Likewise, the expression of MHC class II molecules can be knocked out in order to reduce the risk of rejection of the cartilage graft.
In yet another Pmho~ of the invention, the three-dimensional culture system of the invention may afford a vehicle for introducing genes and gene pLoduuLs i vivo to assist or improve the results of the transplantation and/or the use in gene therapies.
For example, genes that prevent or ameliorate symptoms of degenerative changes in cartilage such as rheumatoid disease or inflammatory reactions and bone resorption, may be undële~Lessed or overexpressed in disease conditions and/or due to aging. Thus, the level of gene activity in the patient may be increased or de~Leased, respectively, by gene repl~r t therapy by adjusting the level of the active gene product in gpnptic~lly Pngin~red stromal cells.
In a specific Pmho~i~~~t exemplified by the examples in Sections 6-8, infra, chondrocytes from articular cartilage of New Zealand rabbits or cows were grown in culture in monolayer or on three-dimensional biodegradable, bi- ltible fibrous fL ~Lk or scaffold formed of sterilized polymers such as polyglycolic acid, polylactic acid or other polymers. The ~L~ ~LkS were designed to allow adequate nutrient and gas PYrh~ng~c to the cells until engraftment at the site of engraftment takes place.
Particular benefits were achieved by maintaining the cultures under sterile conditions without inhibiting the growth of cartilage in biodegradable polymers sterilized by rhP~ir~l methods or radiation.
~ TGF-~ was added to the three-dimensional 2 1 9206~
WO95/33821 PCT~95~729~
.

cultures to achieve a greatly increased proliferation and differentiation of chondrocyte cells. The cultured cartilage was characterized by analyzing the cartilage constructs for glycn~min~glycan, collagen I
and II by histology and i ~ tonh~mi~try, bioch~m~ç~l quantitation, Northern-Blot analysis and i -blotting.

4. ~RIEF D~o~It~lON OF THE DRAWING
Figure l is a photograph of rabbit cartilage tissue grown Ln Yitro with or without TGF-~1.

Figure 2 is a photograph of Hematoxylin and Eosin stained cartilage tissue grown n yitro without TGF-~1.

Figure 3 is a photograph of Hematoxylin and Eosin stained cartilage tissue grown n vitro with TGF-~1.

Figure 4 is a photograph o~ Trichrome stained cartilage tissue grown in vitro without TGF-~l to show the ~Lesence o~ collagen.

Figure 5 is a photograph of Trichrome stained cartilage tissue grown n yitro with TGF-~1 to show the pL ~s~-.ce of collagen.

Figure 6 is a photograph of Alcan Blue stained cartilage tissue grown i yitro without TGF-~1 to show~
the presence of glycos~minnglycan.

Figure 7 is a photograph of Alcan Blue stained cartilage tissue grown L~ vitro with TGF-~1 to show the presence of GAG.

Figure 8 is a photograph of cartilage after growth in yitro for eight weeks with or without TGF-~1.

WO95/~3821 2 1 9 2 0 6 4 PCT~US9~1~7296 Figure 9 is a photograph of cartilage grown on radiation sterilized mesh in control, FGF-, and TGF-~l-treated cultures of cartilage.

Figure lO: TGF-~ and ascorbate increase the proliferation of bovine articular chondrocytes.

Figure ll: Detection of collagen type II and GAGs in bovine articular ~1.ondLu~yLe lysates.
A. Bovine articular chondrocytes were grown in complete media containing no additives (lanes l and 5), ascorbate t50 ug/ml, lanes 2 and 6~, TGF-~ (20 ngjm, lanes 3 and 7), or TGF-~ +
ascorbate (lanes 4 and 8). Cell lysates were prepared, fractionated by SDS-PAGE and analyzed by ;mmnn~hlotting using anti-chondroitin sulfate antibody (anti-CS; lanes 1-4) or normal rabbit serum (NRS; lanes 5-8).
B. Bovine articular ch~ndL~yLe lysates were fractionated as in panel A and i nnhlotted with antibody against collagen type II (lanes 1-4) or normal rabbit serum (lanes 5-8). Lanes l and 5, no additives; lanes 2 and 6 plus ascorbate; lanes 3 and 7, plus TGF-~; lanes 4 and 8, plus TGF-~and ascorbate.
C. RNA was prepared from ch~..dLo~yLes which were untreated (lane l), treated with ascorbate (50 ug/ml, lane 2), treated with TGF-~ (20 ng/ml, lane 3), or TGF-~ plus ascorbate (lane 4) and analyzed by Northern blotting using anti-collagen type II probe as described in Materials and Methods.

Figure 12: Immuno-histo~h~ic~l staining of cartilage constructs. Samples were stained with normal rabbit serum (panel A and B), anti-collagen type I (panel C
and D), or anti-collagen type II (panel E and F).

WO95/33821 ~ /9Z ~6 y PCT~S95/07296 Samples shown in panel A, C, and E were grown without TGF-~ while samples shown in panels B, D, and F were grown with TGF-~.

Figure 13: Cartilage-like tissue produced by bovine chu..d~o~yLes grown on PGA scaffolds. Bovine ~h~l~dL~yLes were seeded onto PGA scaffolds and grown for three weeks as described in Materials and Methods with (panel A) or without (panel 8) TGF-~ (20 ng/ml).

Figure 14: ~ematoxylin and eosin staining of 'n Yi~LQ
cartilage tissue. The samples were sectioned and stained with hematoxylin and eosin. Samples shown in panel A and B were grown without TGF-~ and samples shown in panel C and D were grown with TGF-~.

Figure 15 i5 a photograph of cartilage constructs A-grown under static conditions and B - grown in bioreactors.

Figure 16 describes cartilage produced in a bioreactor and stained with: A and B: Hematoxylin/eosin; C and D: Trichrome stain; E and F: Alcan Blue; and G and H: Safranin O. Panels I and J show hematoxylin/eosin and trichrome stain IeD~e~Lrully of a cartilage sample grown under static conditions.

Figure 17: Immunoblotting of bovine articular ~1.o1.d.~yLe lysate. Cell-lysates from bovine ..d,~yLes were PYAminPd by Western blotting using anti-type I collagen (lane l), anti-type II collagen (lane 2), normal goat serum (lane 3), anti-versican (lane 4), normal rabbit serum (lane 5), anti-chondroitin sulfate (lane 6), normal mouse serum (lane 7).

W0~5133821 PCT~SsS1072g6 ~ 21 92064 Figure 18 T -' i .cto~hpmi cal staining of cartilage constructs - cartilage uu.-sLL~uLs produced in a bioreactor was processed for ;- nh;ctochemiCal analysis and stained with: A - antitype II collagen;
B - anti-chondroitin; C - anti-type I collagen; and D
normal rabbit serum.

5, DT~TTT.!n DE~KI~-lON OF THE l~V
THE 8TTMHT~ OF CELL PROLIFERATION
AND APPROPRIATE CELL MATURATION
In accordance with the invention, stromal cells are inoculated onto a three-dimensional framework network or scaffold, and grown in culture to form a living cartilaginous material. The stromal cells may comprise chondrocytes, chondrocyte-progenitors, fibroblasts or fibroblast-like cells with or without additional cells and/or elements described more fully herein. The chondrocytes, fibroblast-like cells and other cells and/or elements that comprise the stroma may be fetal or adult in origin, and may be derived from convenient sources such as cartilage, skin, etc.
Such tissues and/or organs can be obtained by ~L V~L iate biopsy or upon autopsy; cadaver organs may be used to provide a generous supply of stromal cells and elements. Alternatively, umbilical cord and placenta tissue or umbilical cord blood may serve as an advantageous source of fetal-type stromal cells, e.q., chondLuuyLe-progenitors and/or fibroblast-like cells for use in the three-~ nc;nn~l system of the invention.~
Fetal fibroblasts and/or chundLuurLes can be inoculated onto the fL ..JLk to form a "generic"
living stromal tissue for culturing any of a variety of cells and tissues. However, in certain instances, it may be preferable to use a "specific" rather than "generic" stromal system, in which case stromal cells and elements can be obtained from a particular tissue, WO 95/33821 PCI'~US9~/07296 21 92~64 organ, or individual. For example, where the three-dimensional culture is to be used for ~u-yOSeb of transplantation or implantation in vivo, it may be preferable to obtain the stromal cells and elements from the individual who is to receive the transplant or implant. This approach might be ~cp~ri~lly advantageous where immunological rejection of the transplant and/or gra~t versus host disease is likely.
Once inoculated onto the three-~ ion~l matrix or LL . ~ k, the stromal cells will proliferate on the f- - JL~ and form the living stromal tissue which can be used n yivo. The three-dimensional living stromal tissue will sustain active proliferation of the culture for long periods of time. Because openings in the mesh permit the exit of stromal cells in culture, confluent stromal cultures do not exhibit contact inhibition, and the stromal cells continue to grow, divide, and remain functionally active.
The production of cartilage in the three-dimensional culture is i ov~d by the application of intermittent pressurization and adequate supply of nutrients to stromal cells by convection.
Growth factors are not n~r~Cc~ry since they are elaborated by the stromal support matrix. However, growth regulatory factors including, but not limited to, TGF-~ and ascorbate, may be added to the culture.
Because, according to the invention, it is i L~nt to recreate, in culture, the cellular microenvironment found i vivo for cartilage, the extent to which the stromal cells are grown prior to implantation L~ vivo or use n vitro may vary. In addition, the stromal cells grown in the system may be genetically engineered to produce gene products bene~icial to transplantation, e.a. anti-inflammatory factors, e,~., anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc.
Alternatively, the stromal cells may be genetically engineered to "knock out" expression o~ native gene W09~l33~21 P~
~ 2 1 ~0~4 products that promote inflammation, e.q., GM-CSF, TNF, IL-l, IL-2, or "knock out" expression of MHC in order to lower the risk of rejection. In addition, the stromal cells may be genetically ~ngin~red for use in gene therapy to adjust the level of gene activity in a patient to assist or improve the results of the cartilage transplantation.
The three-dimensional cultures may also be used Ln vitro for testing the effectiveness or cytotoxicity of pharmaceutical agents, and screening ~ uu--ds.
In yet another application, the three-dimensional culture system may be used in a "bioreactor" to produce cartilage tissue constructs which possess critical bio~h~mical~ physical and structural properties of native human cartilage tissue by culturing the tissue under environmental conditions which are typically experienced by native cartilage tissue. The three-dimensional culture system may be maintained under intermittent and periodic pressurization and chondrocytes are provided an adequate supply of nutrients by convection. Pressure facilitates flow of fluid through the mi~LupuI~Us three-dimensional cartilage construct, thereby improving the supply of nutrients and removal of waste from cells c ~ in the ~ol-aLLu~L.
Although the ApplicAnts are under no duty or obligation to explain the r- ' Ani~m by which the invention works, a number of factors inherent in the three-d;- -iqnAl culture system may contribute to its success:
la) The three-dimensional matrix provides a greater surface area for protein att~- L, and ccnsequently, for the adherence of stromal cells.
(b) Because of the three-dimensionality of the matrix, stromal cells continue to actively grow, in contrast to cells in monolayer cultures, which grow to confluence, exhibit contact inhibition, and cease to ~095/33821 2 1 9 2 064 PCT~SgS10729~

grow and divide. The elaboration of growth andregulatory factors by replicating stromal cells may be partially r~cp~nc1hle for stimulating proliferation and regulating differentiation of cells in culture.
(c) The three~ ;on~l matrix allows for a spatial distribution of c~ r elements which is more analogous to that found in the counterpart tissue ln vivo.
(d) The increase in potential volume for cell growth in the three-~;n~ n~l system may allow the establi6hment of localized microenviLv ~ conducive to c~ r maturation.
(e~ The three~ inn~l matrix r-Y;~iz~c cell-cell interactions by allowing greater potential for - ~ L of migratory cells, such as macrophages, monocytes and possibly lymphocytes in the adherent layer.
(f) It has been recogn;zed that maintenance of a differentiated cellular pheno-y~e requires not only growth/differentiation factors but also the appropriate c~ interactions. The present invention effectively recreates the tissue microenvironment.
The three-~;r-~cinn~l stromal support, the culture system itself, and its maintenance, as well as various uses of the three-dimensional cultures are described in greater detail in the subsections below.
The three-dimensional uLv~Lv~yLe cultures can be subjected to intermittent pL~s~uLlzation by creating elevated ~ essive forces through the plastic bag in which the cultures are housed by merely p;n~h;ng or clamping the outlet valve. The chvlldLv~yLes respond to the ambient ples~ule at the level of cell division.
There i5 an increase in the level of proteoglycans which a: ,~n;~c increases in D~A synthesis.
The three-~ n~l cartilage cultures of the invention are maintained in a bioreactor, a special WO95/33821 PCT~S95107296 2 ~ 9?06~
device for creating intermittent and periodic pressurization and chu.,dLouy-es are provided an adequate supply of nutrients by convection.
M~;ntAininq an adequate supply of nutrients to -undLo~yLe cells tl,Luu~LuuL a r~plAc L cartilage tissue constrUct of approximately 2-5mm ~h; r~n~5 iS
e~LL. -ly important as the apparent density of the construct increases. The bioreactors may include a number of designs inrln~;nq, but not limited to, the "piston-style," hard plastic bioreactor; bellows;
soft plastic bag with "pressure plate"; and soft plastic bag with "roller pins".

5.1. EstAhli~l t Of Three-dimensional Stromal Ti55ue The three-dimensional framework may be of any mater;al and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b~ allows cells to grow in more than one layer. A number of different materials may be used to form the matrix, including but not limited to: nylon (polyamides), dacron (polyestersj, poly~LyLene, polypropylene, polyacrylates, polyvinyl , ~c (ç.a., polyvinylchloride), poly~dlbo~ldte tPVC), polytetrafluorethylene (PTFE, teflon), thermanox ~TPX), nitrocellulose, cotton, polyglycolic acid (PGA), collagen (in the form of sponges, braids, or woven threads, etc.), cat gut sutures, cellulose, gelatin, or other naturally occurring biodegradable materials or synthetic materials, inrln~;ng, for example, a variety of polyl-yd-u~y~lkanoates. Any of these materials may be woven into a mesh, for example, to form the three-~ inn~l fL ~Lk or scaffold.
Certain materials, such as nylon, polystyrene, etc.
are poor substrates for cellular attachment. When these materials are used as the three-dimensional CL 'Olk~ it is advisable to pre-treat the matrix ~09~38~1 P~l~sssl~7~g6 2~9~0~ ~

prior to inoculation of stromal cells in order to enhance the att~l ~ of stromal cells to the matrix.
For example, prior to inoculation wlth stromal cells, nylon matrices could be treated with 0.1 M acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene could be similarly treated using sulfuric acid. Where the cultures are to be maintained for long periods of time or u~yupleserYed, non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc., may be preferred. A convenient nylon mesh which could be used in accordance with the invention is Nitex, a nylon filtration mesh having an average pore cize of 210 ~m and an average nylon riber diameter of 90 ~m (#3-210/36 Tetko, Inc., N.Y.).
Where the three-dimensional culture is itself to be implanted in vivo, it may be preferable to use biodegradable matrices such as polyglycolic acid, catgut suture material, collagen, or gelatin, for example. The polyglycolic acid is commonly sterilized in preparation for long-term in vitro, with ethylene oxide or by irradiating with an electron beam.
Unfortunately, both these p~uceduLes have deleteriou3 effects on the cells growing on the three-dimensional culture matrices. For exa~ple, ethylene oxide is toxic to the cells in culture and therefore, electron beam LL~al L is preferred. However, treatment with the electron beam results in cells falling off the rL ~ before depositing adequate extracellular matrix. The addition of TGF-~ to the cultured cells uve~s -- this problem.
Stromal cells comprising chondrocytes chondLu~yLe-progenitors~ fibroblasts or fibroblast-like cells, with or without other stromal cells and Pl~ c described below, are inoculated onto the framework. Growth factors, such as TGF-~ may be added to the culture prior to, during or 5llhc~qunnt to wo ss/33s2l r~ 5 2~ 9206~

inoculation of the stromal cells. The concenLL~tion of TGF-~ maintained in the cultures can be monitored and adjusted to optimize growth. Alternatively, host cells that are genetically engineered to express and produce TGF-~ may be included in the innC~llnm; such cells can include genetically engineered stromal cells. These cells would serve as a source of TGF-~
or other protein factor(s) in the culture.
Preferably, the gene or coding sequence for TGF-~
would be placed under the control of a regulated promoter, so that production of TGF-~ in culture can be controlled. The genetically engineered cells will be s~L~ened to select those cell types: 1) that bring about the amelioration of symptoms of rheumatoid disease or inflammatory reactions i~ v vo, and 2) escape immunoloyical surveillance and rejection.
Stromal cells such as ch~ndLo~yLes may be derived from articular cartilage, costal cartilage, etc. which can be obtained by biopsy (where appropriate) or upon autopsy. Fibroblasts can be obtained in quantity rather conveniently from foreskin or, alternatively, any a~L~pIiate cadaver organ. Fetal cells, ;n~ ;nq fibroblast-like cells, ~I.ol,d~o~yLe pL~y~llitors, may be obtained from umbilical cord or placenta tissue or nmhili~l cord blood. Such fetal stromal cells can be used to prepare a "generic" stromal or cartilaginous tissue. However, a "specific" stromal tissue may be prepaIed by inoculating the three-dimensional matrix with fibroblasts derived a particular individual who is later to receive the cells and/or tissues grown in culture in accordance with the three-dimensional system of the invention.
Fibroblasts may be readily isolated by di~ ey~ting an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art. For example, the tissue or _ . .. _ . . . . . .

wo g5133821 2 1 9 2 3 6 ~ r organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between nQ;ghh~ring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. ~nzy_atic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, ~ILY -tLy~sinl coll~gQnl~e, elastase, and/or hyaluronidase, ~nase, pronase, etc. MQ~h~nic~l disruption can also be accomplished by a number of methods including, but not limited to the use of grinders, blenders, sieves, h~ Qrs~ pressure cells, or sonicators to name but a few. For a review of tissue disaggregation techniques, see Freshney, Culture of Animal Cells. A
~anual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.
Fibroblast-like cells may also be isolated from human umbilical cords ~33-44 weeks~. Fresh tissues may be minced into pieces and washed with medium or snap-frozen in liquid nitrogen until further use. The t~mhiljr~l tissues may be disaggregated as described above.
Once the tissue has been reduced to a suspension of individual cells, the s~lcpon~io~ can be fractionated into sllhpop~ tions from which the fibroblasts and/or other stromal cells and/or elements can be ~ht~ i nQ~ . This also may be accomplished using standard te~hni~lQc for cell separation in~ ng but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adl.e~ence properties of the cells in the mixed WO95~33821 2 1 ~ 2 0 6 4 PCT~JS9~/~7296 population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation), unit gravity separation, counter current distribution, electrophoresis and fluu~escence activated cell sorting. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic T~rhniqll~c~ 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.
The isolation of chundIouyLes~ chondLuuyLe-progenitors, fibroblasts or fibroblast-like cells may, for example, be carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks bA1Anc~ salt solution (HBSS) in order to remove serum. The minced tissue is incubated from l-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation, the ~iCsociAted cells are snSp~n~d, pelleted by centrifugation and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown. The isolated stromal cells can then be grown to confluency, lifted from the confluent culture and inoculated onto the three-~;r -i~nAl support (see, Naughton et al., 1587, J. Med. 18(3&4):219-250).
Inoculation of the three-dimensional matrix with a high cu..c~n~5~tion of stromal cells, e.a., approximately 106 to 5 x 107 cells/ml, will result in the establishment of the three-dimensional stromal support in shorter periods of time.
In addition to chondrocytes, chondrocyte-progenitors, fibroblasts or fibroblast-like cells, other cells may be added to form the three-~ir -ionAl stromal tissue required to support long term growth in culture. For example, other cells found in loose c~nn~c1ive tissue may be inoculated onto the three-dimensional support along with chondrocytes or WO95/3~21 PCT~S9~0729C
21 92~6~ --fibroblasts. Such cells include but are not limited to endothelial cells, pericytes, ma~Lu~ha~s, monocytes, plasma cells, mast cells, adipocytes, etc.
These stromal cells may readily be derived from appropriate organs including umbilical cord or placenta or umbilical cord blood using methods known in the art such as those ~i~c~lcced above.
Again, where the cultured cells are to be used for transplantation or implantation n vivo it is preferable to obtain the stromal cells from the patient's own tissues. The growth of cells on the three-dimensional support may be further ~nh~nc~d by adding to the fL vlk or coating the fL s~Lk with proteins (e.q., collagens, elastic fibers, reticular fibers) glycoproteins, glycos~min~glycans te-q-~heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a celt~ r matrix, and~or other materials.
After inoculation of the stromal cells, the three-dimensional matrix should be incubated in an appropriate nutrient medium. Many commercially available mQdia such as DMEM, RPMI 1640. Fisher~s Iscove's, McCoy's, and the like may be suitable for use. It is important that the three-dimensional stromal matrix be Sn~p~n~ or floated in the mediu~
during the incubation period in order to ~ ize proliferative activity. In addition, the culture should be "fed" periodically to remove the spent media, ~pop~ te released cells, and add fresh media.
The ~ tion of TGF-~ may be adjusted during these steps. In ~1ulldLo~yL~ cultures, proline, a non-essential amino acid and ascorbate are also included in the cultures.
These procedures are greatly facilitated when carried out using a bioreactor, which i8 a closed system housing the three-dimensional framework inoculated with stromal cells. A bioreactor reduces W0~5/33821 PCT~S95/07296 ~ 2~92064 the possibility of contamination, maintains the cultures under intermittent and periodic pressurization to create environmental conditions that maintain an adequate supply of nutrients to ~h~..dLo~yLe cells thL~u~l.ou~ the cartilage tissue construct by convection.
During the incubation period, the stromal cells will grow linearly along and envelop and colonize the three~ -ci~n~l matrix before b~g;nn;ng to grow into the opr~n; ngc of the matrix. It is important to grow the cells to an appropriate degree which reflects the amount of stromal cells present in the in vivo tissue prior to inoculation of the stromal tissue with the tissue-specific cells.
The opPn;ngs of the fL ~Lk should be of an ~.u~Liate size to allow the stromal cells to stretch across the op~n;ngS. Maintaining actively growing stromal cells which stretch across the fL s~Lk onh~ncoc the production of growth factors which are elaborated by the stromal cells, and hence will support long term cultures. For example, if the opon;ngc are too small, the stromal cells may rapidly achieve confluence but be unable to easily exit from the mesh; trapped cells may exhibit contact inhibition and cease production of the appropriate factors n~cc~ry to support proliferation and maintain long term cultures. If the opon;ngc are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the ~Lu~.iate factors n~c~cq~ry to support proliferation and maintain long term cultures.
When using a mesh type of matrix, as exemplified herein we have found that op~n;nqC ranging from abut 150 ~m to about 220 ~m will work satisfactory.
However, dorr~n~; ng upon the three-dimensional structure and intricacy of the fL il~Lh, other sizes may work equally well. In fact, any shape or WO95/33821 2 1 9 2 0 6 4 PCT~lS95107296 structure that allow the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the invention.
Different proportions of the various types of collagen deposited on the LL ..~Lk can affect the growth of later inoculated tissue-specific pa~ 1.y."al cells. For three-dimensional skin culture systems, collAqDn types I and III are preferably deposited in the initial matrix. The proportions of collagen types deposited can be manipulated or ~nh~n~efl by selecting fibroblasts which elaborate the appropriate collagen type. This can be ~ h~d using monoclonal ant;bo~i~e of an appropriate isotypes or subclass that is capable of acti~ating complement, and which define particular collagen type. These antibodies and complement can be used to negatively select the fibrcblasts which express the desired collagen type.
Alternatively, the stroma used to inoculate the matrix can be a mixture of cells which synthesize the appropriate collagen types desired. The distribution and origins o~ the five types of collagen is shown ln Table I.

W09~/33821 P~
21 9~06~

TABLE I
DISTRIBUTIONS AND ORIGINS OF
THE FIVE TYPES oF CnT~T~A~.EN
Collagen Principal Tissue Distribution Cells of origin connective tissue; reticular cells;
collagen fibers smooth muscle cells Fibrocartilage 80ne Osteoblast Dentin Odontoblasts II Hyaline and elastic Chondrocytes cartilage Vitreous body of eye Retinal cells III Loose ccnnective tissue; Fibrcblasts and reticular fibers reticular cells Papillary layer of dermis Blood vessels Smooth muscle cells;
endothelial cells IV Basement membranes Epithelial and endothelial cells Lens capsule of eye Lens fibers V Fetal membranes; Fibroblast placenta Basement membranes Bone Smooth muscle Smooth muscle cells Thus, d~p~n~ i ng upon the tissue to be cultured and the collagen types desired, the appropriate stromal cell(s) may be selected to inoculate the three-~i- ion~l matrix. For example, for the growth and preparation of cartilage, ~LondL~yLes~ chondrocyte-W0~33821 2 ~ 9 2 0 ~ ~ r~l,u~

progenitors, fibroblasts or fibroblast-like cells should be used.
During incubation of the three-di~ensional stromal support, proliferating cells may be released from the matrix. These released cells may stick to the walls of the culture vessel where they may continue to proliferate and form a confluent monolayer. This should be prevented or ~;n;~;7~A~ for example, by removal of the released cells during feeding, or by transferring the three-dimensional stromal matrix to a new culture vessel. The ~L~Ecnce of a confluent monolayer in the vessel will "shut down" the growth of cells in the thrce-~; ~; on~ 1 matrix an~/or culture. Removal of the confluent monolayer or transfer of the matrix to fresh media in a new vessel will restore proliferative activity of the three-dimensional culture system. 5uch removal or transfers should be done in any culture vessel which has a stromal monolayer ~x~e~in9 25~ confluency.
Alternatively, the culture system could be agitated to prevent the released cells from sticking, or instead of perio~ically feeding the cultures, the culture system could be set up so that fresh media contimloncly flows through the system by convection.
The flow rate could be adjusted to both maximize proliferation within the three~ ion~l culture, and to wash out and remove cells released from the matrix, so that they will not stick to the walls of the vessel and grow to confluence. In any case, the released stromal cells can be collected and es~rv~d for future use.

5.2. Uses of the Three-Dimension~l Culture _vstem The three-dimensional culture system of the invention can be used in a variety of applications.
These include but are not limited to transplantation or implantation of either the cultured cells obtained W095/33821 2 1 9 2 ~ 6 4 PCT~S95/~7296 from the matrix, or the cultured matrix itself in vivo; screening the effectiveness and cytotoxicity of _ ~c, allergens, growth/regulatory factors, phAr~-~outical ~ , etc., n vitro; elucidating the - ~n;c~ of certain A;ce~c~c; studying the n; c~ by which drugs and/or growth factors operate; diagnosing and monitoring cancer in a patient; gene therapy; and the production of biologically active products, to name but a few.

5.2.l. Trans~lantation In Vivo The biological replacement cartilage tissue co1lDLLu~Ls p~uduced in the three-dimensional culture system of the invention can be used to replace or augment existing cartilage tissue, to introduce new or altered tissue, to modify artificial prostheses, or to join biological tissues or sLLuuLuLes. For example, and not by way of limitation, specific ~ -';r-nts of the invention would include i) hip prostheses coated with replA~ - t cartilage tissue oull~LLuuLs grown in three-dimensional cultures; ii~ knee leuu1l~LL~ction with cartilage tissue co1l~LLuuLs; and iii) prostheses of other joints requiring reconstruction and/or replacement of articular cartilage.
The evaluation of internal derangements of articular cartilage in several articulations, ;nr1uA;ng the knee, hip, elbow, ankle and the gl -~ ~l joint, has been made possible by arthroscopic techniques. Arthroscopic surgery has become increasingly popular as well as , ~c~c~rul, e.q., uus small cutting tools, 3 to 4mm in diameter can be used in the knee. Triangulation, in which the operating in~LL, Ls are brought into the visual field provided by the aLLhLoscu~e, requires multiple portals of entry; alternatively, the cutting tools can be passed through a channel in the aLLh~oscope itself in which case only one opening in wo gS133X21 2 1 9 2 0 6 4 .

the joint i5 n~u-SS~y (Jackson, R.W., 1983, J. Bone Joint Surg. [AM] 65:416. Selective removal of the injured or deteriorated portion with arthroscopic surgery, followed by cartilage grafting can be employed successfully. Cartilage tissue constructs can also be employed in major reconstructive surgQry for different types of joints. Detailed ~LuaeduL~5 have been described in Resnick, D., and Niwayama, G., eds., 1988, Dia~nosis of Bone and Joint Disorders, 2d ed.l W.B. Sanders Co.
Three-dimensional tissue culture implants may, according to the inventions, be used to replace or augment existing tissue, to il--Luduce new or altered tissue, or to join together biological tissues or ~LL U.;LUr~=S .

S.2.2. 6~ S~ ing Effectiveness and cvtotoxicitY o~ Comoouna~ In vitro The three-dimensional cultures may be used n vitro to screen a wide variety o~ compounds, for effectiveness and cytotoxicity of pharmaceutical agents, growth/regulatory factors, anti-inflammatory agents, etc. To this end, the cultures are maintainea n vitro and exposed to the ~ __ to be tested.
The activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture.
This may readily be AcR~Rs~A by vital staining techniques. The effect of growth/requlatory factors may be Acsa~ced by analyzing the cellular content of the matrix, e.a., by total cell counts, and differential cell counts. Thi8 may be accomplished using standard cytological and/or histological techniques ;n~lnAing the use of immunocytochemical techniques employing antibodies that define type-specific callulAr antiqens. The effect of various drugs on normal cells cultured in the three-di~~~~i~nAl gystem may be ARsP-se~A.

W09S/3382l 2 1 9 2 0 6 ~ PCT~S9S/~7~96 .

The three~ in~l cultures of the invention may be used as model systems for the study of physiologic or pathologic conditions. For example, joints that are immobilized suffer relatively quickly in a number of ~ea~evLL. The metabolic activity of chund~v~y~es appears affected, as loss of proteoglycans and an increase in water content are soon observed. The normal white, glistening appearance of the cartilage changes to a dull, bluish color, and the cartilage thickness is reduced.
However, how much of this process is due to nutritional deficiency and how much is due to upset in the stress-~pP~d~nt metabolic homeostasis is not yet clear. The three-~ ion~l chondLv~yLe culture system may be used to determine the nutritional requirements of cartilage under different physical conditions, e.q., intermittent pressurization and by pumping action of nutrient medium into and out of the cartilage c~naLLuu~. This may be especially useful in studying underlying causes for age-related, or injury-related decrease in tensile strength of articular cartilage, e.a., in the knee, that pr~ pnses the weakened cartilage to traumatic damage.
According to the present invention, the three-dimensional chondrocyte cultures may also be used to study the -echAnicp of action of cytokines and other pro-inflammatory mediators released in rheumatic disease in the synovial fluid, ç.q., IL-1, TNF and prostaglandins. The patient's own joint fluid eould be used Ln vitro to study the effect of these _ '- on chundlvvyLe growth and to screen cytotoxic andjor pharmaceutical agents that are most efficacious for a particular patient; i.e., those that prevent resorption of cartilage and enhance the b~lAnred growth of articular cartilage. Those agents could then be used to therapeutically treat the patient.

Wog~/33821 2 1 9 2 0 6 4 PCT~$~5/07296 .

5.2.3. GeneticallY En~ineered Cartil~~e The three~ n~;~nAl culture sy~tem of the invention may afford a vehicle for introducing genes and gene products n Yivo to assist or improve the results of the transplantation and/or for use in gene therapies. For example, the stromal cells can be genetically engineered to express anti-infla~at~Ly gene products to reduce the risk of failure or degenerative changes in the cartilage due to rheumatoid disease of inflammatory reactions. In thi~
regard, the stromal cells can be genetically ~nqin~ored to express anti-inflammatory gene products, for example, peptides or polypeptides corrPcpnn~ing to the idiotype of neutralizing an~i h9d; ~q for granulocyte-macrophage colony stimulating factor ~M-CSF), TNF, IL-1, IL-2, or other inflammatory cytokines. Il-1 has been shown to decrease the synthesis of proteoglycans and collagens type II, IX, and XI (Tyler et al., 1985, Biochem. J. 227:869-878;
Tyier et al., 1988, Coll. Relat. Res. 82: 393-405;
Goldring et al., 1988, J. Clin. Invest. 82:2026-203~;
and Lefebvre ct al., 1gso, siophys. Acta. 1052:366-372. TNF also inhibits synthesis of proteoglycans and type II collagen although it is much less potent than IL-l tYaron, I., et al., 1989, Arthritis Rheu~.
32:173-180; Ikebe, T., et al., 1988, J. Im~unol.
140:827-831; and Saklatvala, J., 1986, Nature 322:547-5~9.
Preferably, the cells are ~qin~red to exprsss such gene products transiently and/or under in~ ihl~
control during the post-operative Leau~Ly period, or as a chimeric fusion protein anul.uL~d to the stromal cells, for example, a chimeric molecule e -6?~ of an intracellular andlor tr~r ' ane domain of a receptor or receptor-like molecule, fused to the gene product as the extrRc~ r domain. In another n~, the stromal cells could be genetically W095l3 21 92064 3821 r~ Il-J~iY:lIU/~,_ PnginPered to express a gene for which a patient is deficient, or which would exert a therapeutic effect, e.~., TGF-~ to stimulate cartilage production, etc.
The genes of interest engineered into the stromal cells need to be related to rheumatoid or joint disease.
The stromal cells can be engineered using a ~ ~in~nt DNA cul,~Ll~L containing the gene used to transform or transfect a host cell which is cloned and then clonally PYp~nAPd in the three-dimensional culture system. The three-dimensional culture which expresses the active gene product, could be implanted into an individual who is deficient for that product.
For example, genes that prevent or ameliorate symptoms of various types of rheumatoid or joint diseases may be und~ .~ssed or down regulated under disease conditions. Specifically, expression of genes involved in preventing inflammatory reactions in rheumatoid or joint diseases may be down-regulated.
Alternatively, the activity of gene products may be fliminichPfl, leading to the manifestations of some or all of the above pathological conditions and eventual devPl~, L of symptoms of rheumatoid or joint ~ir~~ . Thus, the level of gene activity may be increased by either increasing the level of gene product present or by increasing the level of the active gene product which is present in the three-dimensional culture system. The three-dimensional culture which ex~l~s6es the active target gene product can then be implanted into the rheumatoid or joint disease patient who is deficient for that product.
"Target gene," as used herein, refers to a gene involved in rheumatoid or joint fl~cP~cPc in a manner by which modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of rheumatoid or joint fliCPICPq Wo9~133~1 2 1 9 2 ~ 6 4 PCT~9.~0729~) by preventing resorption of cartilage ~nd production of inflammatory mediators by ~hol.dLv~yLes.
Further, patients may be treated by gene rDpl~ t therapy during the post-recovery period after cartilage transplantation. Repl~~
cartilage tissue COI1~LU~LS or sheets may be ~iq specifically to meet the requirements of an individual patient, for example, the stromal cells may be genetically engineered to regulate one or more genes;
or the regulation of gene expression may be transient or long-term; or the gene activity may be non-inducible or inducible. For example, one or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein product with target gene function, may be inserted into human cells that populate the three-dimensional consLLu~ using either non-inducible vectors including, but are not limited to, adenovirus, adeno-associated virus, and retrovirus vectors, or inducible promoters, including metallothionien, or heat shock protein, in addition to other particles that il~LL~uc~ DNA into cells, such as li,-- -- or direct DNA injection or in gold particles. For example, the gene Dnco~ing the human complement regulatory protein, which prevents re~ection of the graft by the host, may be inserted into human fibroblasts. ~cCurry et al., 1995, Nature M~icin~
1:423-427.
The three-~i inn~l cultures containing such genetically DnginD~red stromal cells, e~a,, either mixtures of stromal cells each expressing a different desired gene product, or a stromal cell engineered to express several specific genes are then implanted into the patient to allow for the amelioration of the symptoms of rheumatoid or joint di5ease. The gene expression may be under the control of a non-inducible (i.e., constitutive) or i"~llcihl~ promoter. The level W0~5/338~1 2 1 q 2 ~ ~ 4 ~ ~ I/L~ JG
.

of gene expression and the type of gene regulated can be controlled ~PpPnrl;ng upon the treatment modality being followed for an individual patient.
The use of the three~ ncion~l culture in gene therapy has a number of advantages. Firstly, since the culture comprises eukaryotic cells, the gene product will be properly ~x~ssed and pLocessed in culture to form an active product. Secondly, gene therapy techniques are useful only if the number of transfected cells can be substantially ~nh~nrP~ to be of clinical value, relevance, and utility; the three-dimensional cultures of the invention allow for PYp~n~i~n of the number of transfected cells and amplification (via cell division) of transfected cells.
A variety of methods may be used to obtain the constitutive or transient expression of gene products engineered into the stromal cells. For example, the transkaryotic implantation technique described by Seldon et al., 1987, Science 236:714-718 can be used.
"Transkaryotic", as used herein, suggests that the nuclei of the implanted cells have been altered by the addition of DNA sequences by stable or transient transfection. The c811s can be engineered using any of the variety of vectors including, but not limited to, integrating viral vectors, e.q., retrovirus vector or adeno-associated viral vectors, or non-integrating replicating vectors, e.~., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors. Where transient expression is desired, non-integrating vectors and replication defective vectors may be preferred, since either j~r~l1r;hlP or constitutive promoters can be used in these systems to control expression of the gene of interest. Alternatively, integrating vectors can be used to obtain transient expression, provided the gene of interest is controlled by an ; n~nri hle promoter.

~V095~1 2 ~ ~ 2 ~ ~ 4 PCT~S9~10729C
.

Preferably, the expression control elements used should allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo. The promoter chosen would depend, in part upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e.q., those characterized by abundant rough Gn~t~F~ic reticulum, and golgi complex) are preferable. Hosts cells can be transformed with DNA controlled by appropriate expression control elements (e,g., promoter, Gnh~nrGr, s~q~1Gn~G~, transcription terminators, polyadenylation sites, etc.~ and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their ch. -- -- and grow to form foci which, in turn, can be cloned and PYp~n~G~ into cell lines. This method can advantageously be used to engineer cell lines which express the gene protein product.
Any promoter may be used to drive the expression of the inserted gene. For example, viral promoters include but are not limited to the CMV
promoter/Pnh~n~Pr~ SV 40, papillomavirus, ~pstein-Barr virus, elastin gene promoter and ~-globin. If transient expression is desired, such constitutive promoters are preferably used in a non-integrating and/or replication-defective vector. Alternatively, inducible promoters could be used to drive the expression of the inserted gene when necG~ry. For example, in~n~ihlP promoters include, but are not limited to, metallothionien and heat shock protein.
Examples of transcriptional control regions that exhibit tissue specificity which have been described WO95/~3821 2 ~ 6~ PCT~S9~/07296 .

and could be used, include but are nct limited to:
elastase I gene control region which is active in pancreatic acinar cells (Swit et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); immunoglobulin gene control region which is active in lymphoid cells (GL~JSS~
et al., 1984, Cell 3S:647-658: Adams et al., 1985, Nature 318:533-538; AlPY~n~r et al., 1987, Mol. Cell.
Biol. 7:1436-1444); myelin basic protein gene control region which is active in oliqod~rocyte cells in the brain (R~heAd et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314:283-286~; and gonadotropic releasing hormone gene control region which is active in the hypo~h~l (Mason et al., 1986, Science 234:1372-1378).
Once genetically engineered cells are implanted into an individual, the presence of the anti-inflammatory gene products, for example, peptides or polypeptides VV1L~LJOn~1;nq to the idiotype of neutralizing an~iho~i~c for GM-CSF, TNF, IL-1, IL-2, or other inflammatory cytokines, can bring about amelioration of the inflammatory reactions associated with rheumatoid or joint disease. IL-1 is a potent stimulator or cartilage resorption and of the production of inflammatory mediators by chu-.d~vvyLes (C -ll et al., 1991, J. Immun. 147:1238-1246).
The stromal cells used in the three-dimensional culture system of the invention may be genetically engineered to "knock cut" expression of factors that promote inflammation or rejection at the implant site.
Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are diccl~cs~d below. "Negative ~0~5l33x2t ~ PCTIllX~51~729~
2192064 ~

modulation", as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level andJor activity of the target gene product in the absence of the modulatory t~e , -nt. The expression of a gene native to stromal cell can be reduced or knocked out using a number of technigues, for example, expression may be inhiblted by inactivating the gene completely ~commonly termed "knockout") using the homologous I~ ~ ;n~tiO!I
technique. Usually, an exon ~nro~ i ng an important region of the protein tor an exon 5' to that region) is interrupted by a positive s~ler~hle marker (for example neo), preventing the production of normal mRNA
from the target gene and resulting in inactivation of the gene. A qene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene. By using a construct with two regions of homology to the target gene that are far apart in the genome, the s_~uen~es intervening the two regions can be deleted. Mombaerts et al., 1991, Proc. ~at. Acad.
Sci. U.S.A. 88:3084-3087.
Antisense and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of tar~et gene activity. For example, antisense RNA
molecules which inhibit the expression of major hi~- tibility gene complexes tHLA~ shown to be most versatile with respect to immune r~Cponc~.
Still further, triple helix ~lect~lPc can be utilized in reducing the level of target gene activity. These techniques are described in detail by L.G. Davis et al., eds, Basic Methods in Molecular Bioloqy, 2nd ed., Appleton & Lange, ~orwalk, Conn. 1994.
Using any of the foregoing techniques, the expression of IL-1 can be knocked out in the ~I.ol.dLu~y~es to reduce the risk of resorption of cartilage and production of inflammatory mediators by _ _ _ _ _ _ _ _ _ .

wo gsl33a2l 21 ~ 2 0 ~ ~ rcrluas~lo72sG

the chondrocytes. Likewise, the expression of MHC
class II molecules can be knocked out in order to reduce the risk of rejection of the graft.
In yet another ~ho~i L of the invention, the three-dimensional culture system could be used n vitro to produce biological products in high yield.
For example, a cell which naturally produces large quantities of a particular biological product (e.a., a growth factor, regulatory factor, peptide hormone, antibody, etc.), or a host cell genetically engineered to produce a foreign gene product, could be clonally ~YpAn~d using the three-dimensional culture system in vitro. If the transformed cell excretes the gene product into the nutrient medium, the product may be readily isolated from the spent or conditioned medium using standard separation techniques (e.c., HPLC, column chromatography, electrophoretic techniques, to name but a few). A "bioreactor" has been devised which takes advantage of the flow method for feeding the three-dimensional cultures in vitro. Essentially, as fresh media is passed through the three-dimensional culture, the gene product is washed out of the culture along with the cells released from the culture. The gene product is isolated (e.q., by HPLC column chromatography, electrophoresis, etc.) from the outflow of spent or conditioned media. The bioreactor system is specially designed to allow for YL ~S~UL ization of the chamber during growth of the cartilage tissue and supply nutrients to stromal cells by convection.

6. EXAMPLE: THREB-Dl~L~AI~ ~T~ O -_ L~
CULTURE SYSTBM
The three-dimensional culture of the present invention provides for the replication and colonization of cholldLo~yLes n vitro, in a system comparable to physiologic conditions. Importantly, WO 95133821 2 1 9 2 0 ~ 4 pCT/us9~)729~
.

the chondrocyte cells replicated in this system include all of the cells present in normal cartilage tissue, Acc~.~ing all cell types were present in the original chondrocyte inoculum used to initiate the cultures. Cartilage implants can be of one or more types of cartilage, ~p~n~ing primarily on the location of the implant and the type of cartilage cells seeded onto the polymeric matrix. The following ~ pl~s describe: (i) a method of gro~ing rabbit chondrocytes seeded on a biodegradable polyglycolic acid matrix sterilized by ethylene oxide or electron bea~ with or without TGF-~; (ii) a method of growing bovine chondrocytes on a polyglycolic acid matrix in a culture containing TGF-~ with or without ascorbate;
and iii) a method of growing rabbit chondrocytes on a polyglycolic acid matrix placed in a bioreactor.
Specific conditions used are described below.

6.1. Material an~ ~e~
G.1.1. Growth FActors Recombinant TGF-~ prepared according to Gentry ct al., 1987, Mol. Cell. Biol. 7:3418-3427, was u~ed at the conc~ L~tion of 20ng/ml. Recombinant human beta fibroblast growth factor ~FGF), Pepro Tech., Inc., Rocky Hill, N.J., was used at the concentration of lOmglml. Ascorbic acid or ascorbate was used at the con~ntration of 50~g/ml.

6.1.2. Cells -Cartilage was harvested from articular surfaces of healthy mature (2-3 years old) cows or New Zealand white rabbits (4-8 months old). The cartilage pieces were digested with collagenase (0.2~ weight/volume) in complete media DMEM containing 10% fetal bovine serum, 2mM L-glutamine, non-essential amino acids, 50mg~ml proline, l~M sodium pyruvate and 35 ~g/ml gentamicin for 20 hr. at 37~C. Liberated chondrocytes W0951338~1 2 1 9 2 0 6 ~ ~ r " .
.

were spun, rpcllcpDn~ in complete medium, counted and plated at 106 cells per T-150 flask. Cells were routinely passed at confluence (every 5-7 days).

6.1.3. Cell 8eedina Polyglycolic acid mesh t45mg/cc; non-heat plated;
2mm thick; lcm diameter) was sterilized by ethylene oxide or electron beam (E-beam, 3MR~ treatment and presoaked overnight in complete medium. The mesh was seeded in 6-well dishes with 3-4 x 10~ cells per mesh for 2 days at 37~C in complete medium and placed in an incubator or bioreactor ~p~n~; ng on the protocol used. Medium containing ascorbate was changed two-three times per week. Samples were then washed with PBS and processed for histology after a specific period of growth.

6.1.4. HistoloqY ana ~ ~~iqto~h~mictry Tissues were washed in PBS at harvesting and photographed. They were fixed in 10% buffered formalin, paraffin ~ e~ and stained with hematoxylin and eosin (HhE) and/or trichrome and/or Safranin 0. For some samples, i ~~ictorh~mir~
staining was also performed on paraffin . '~''ed tissue using Biotin-Streptavidin Amplified System tBiogenex Corp.) to unmask antigenic sites. They were then incubated with 3% hydrogen peroxide to block endo~enuus peroxidase activity, rinsed with tris-saline buffer and incubated with biotin block serum (Dako, Inc.) to reduce n~ncpecl~$c bauk~Luu..d.
Blo~ng serum was tapped off and primary ant~ho~i~c to collagen type I and collagen type II were added.
Sections were then incubated with biotinlated anti-lgG, and washed with tris-saline buffer. They were then incubated with horseradish peroxidase-conjugated ~LLe~ idin and washed with tris-saline. Sections WogSl3382l 2 ~ 9 2 0 6 4 PCT~59~/07296 were then incubated with 3,3~ ~;n~b~n~;~ine substrate and counterstained with hematoxylin.

6.1.5. ~ -hlottina Confluent monolayers of articular chondrocytes were scraped in PBS containing 1~ Tween 20. Lupis buffer (as described in Laemmli, U.R., 1970, Nature 227:680-685j was added and cell lysates were fractionated by SDS-PAGE and by ; ~hlotting as described in Burnette, W.N., 1981, Anal. Chem.
112:195-203. Antibodies to collagen type I and II
were obtained from Southern Biochemicals, Inc.
(Birmingham, AL) and anti-chondroitin sulfate antibody was from Sigma (St. Louis, MO).

6.1.6. Ouantitation of CollarJen ana R~a Constructs were frozen lyophilized and stored at -70~C until analysis. The CUI~LL~LS were digested with papain ~lmy/ml) in lOOmM phosphate buffer (pH
6.5) containing 5mM cysteine and 5mm EDTA at 65~C
overnight. Quantitztion of collagen and GAG were det~rmi n~d according to the methods described (Farndale et al., 1986, Biochem. Biophys. Acta 883:73-177; h~c-- er, J.F., 1961, Archiv. Biochem. Biophys.
93:440-447.

6.1.7. ~orthern Blot ~1Ysis RNA was isolated, fractionated on agarose-formaldehyde gels and probed with [37P}-labelled type II collagen cDNA as d,escribed (r~ _y~lski, P., and Sacchi, N., 1987, Anal. Bi-~' 162; Lehrach et al., 1977, Biochemistry 16:4743-4751; and Madisen et al., 1986, DNA 7:1-8).

WO95~33~21 ~ 92~4 PCT~S9~/07296 6.2. Effect of Ethylene Oxide or Electron Beam 8terilization on Rabblt Chond~v~yLe Cultures Polyglycolic acid mesh sterilized with: a~
ethylene oxide or b) electron beam was seeded in six well plates with 3-4 x 106 cells per mesh in a total volume of 10ml (50ml per side) and incubated for 3-4 hr. at 37~C in a tissue culture incubator. At this time, 1.5ml of media were added. The seeded mesh were incubated overnight. 5ml of media were added the next day. Media was changed three times per week for four weeks. Samples were then removed, washed with PBS, and processed for histology: hematoxylin/eosin, trichrome (for collagen); and Alcan blue (for glycosamineglycans).

6.3. Results 6.3.1. Effect of Ethylene Oxide or rl~ L v~ Bea~ 8terilization on Rabbit ~hol~aLv~yLe Cultures ~a) Ethylene Oxide Treated PolvqlYcolic Acid Mesh Cl.ol.dLv~yLes grown in the three-dLmensional matrix in the presence of TGF-~ produced cartilage tissue which was smoother, more glistening and had a more solid consistency than the tissue grown in cultures without the TGF-~ (Fig. 1).
Histological examination of the cartilage tissue using the hematoxylin-eosin stain showed an increase in c~ rity in the cultures without TGF-~ (Fig. 2) compared with those with TGF-~ (Fig. 3).
Staining with trichrome, an indicator of the plesellce of collagen, showed an increase in collagen deposition in cultures grown without TGF-~ (Fig. 4) compared with those grown in cultures with TGF-~(Figure 5).
Alcan blue staining indicated an even distribution and increase in GAG deposition throughout wo ~/33~21 r~

the cartilage tissue when samples were grown in the presence of TGF-~ ~Fig. 7) compared with those without TGF-~ (Fig. 6).
The gross appearance of the tis6ue after 8 weeks indicated that the cartilage grown in the presence of TGF-~ was substantially larger than that grown without TGF~ ig. 8~.
There was a two-fold increase in the dry weight of cartilage grown in cultures containing TGF-~, 20%
more collagen and 80% more GAG. See Table II below.

TABLE II - RABBIT CARTILAGE
Static: 8 Week Wet Dry Total Total% Collagen % GAG
Weight WeightCollagen GAG (Dry (Dry~
Sample(mg) (mg) (mg) (rng) Weight) Weight) TGF-~
64 (n=2) _ 4.8 1.4 273 (n=2) 8.Z 6.0 1.1 73.3 13 - TGF-,B
35(n=2) _ 1.9 0.3 2 50 (n=2) 4.2 2.6 0.3 61.9 7.1 Normal Rabbit Cartilage GAG 15-40%
Collagen 55-80%

(b) Bovine ~I.vdLv~y~es seeded on mesh sterilized by E-beam showed poor growth after four weeks. This is most likely due to the more rapid degradation of PGA after radiation treatment, causing the cells to fall off before being able to deposit adequate extrac~llu~ ~r matrix.

W095/338~l 2 1 9 2 0 6 4 ~CTIUS95/07296 Addition of fibroblast growth factor B (bFGF) had no effect. However, addition of TGF-~ resulted in growth of chondLo~yLes and formation of cartilage tissue by increasing the production of extracellular matrix by the uI.ondLu~yLes. Thus, addition of TGF-~~v~L~es the deleterious effects of sterilizing the mesh by the E-beam and ~nh~nrec the production of cartilage in the three-dimensional culture matrix.
(Fig. 9).

7. Effect of TGF-~ on Growth of Bovine Chondrocytes in ~onolayer Culture ~nd on Three-D;r---io--l FL ~ ..~r~ With or Without ascorb~te a) 80vine chondrocytes were seeded at 2.0 x 105 cells per T-25 flask. Twenty-four hours later they were treated with ascorbate, TGF-~ or both.
b) Polyglycolic acid mesh were seeded with 3 x 106 bovine ul~ulldlu~y~es and cultured in 6-well dishes for two days at 37~C in complete media containing TGF-and with 50mg/ml ascorbate or no ascorbate.

7.1. ESULTS
7.1.1. Effect of TGF-~ on Growth of Bovine Chondhv~es in ~onolayer Culture an~ on Three-D~- -ic--l FL .. ~L k~ With or Without ascorbate ~ a) TGP-~ increases Prolifer~tion A
Bovine Chondrocvtes in ~onolaYer Figure lOA shows that TGF-~ stimulated the growth of bovine chondrocytes in monolayer. Addition of ascorbate had a stimulating effect which was additive with TGF-~. Figure lOB shows a time course for the proliferative effect of TGF-~ in the ~ ence of ascorbate. Stimulation of chondrocyte growth was observed at O.lmg/ml TGF-~ (Figure lOC) and ~FGF had a slight inhibiting effect.
TGF-~ treat~ent also resulted in substantial increase in GAG synthesis (Figure llA) and had no W095~3~2t 2 1 9 2 0 6 4 PCTIU895~7~96 detectable ef~ect on collagen type II (Figure llB).
Northern blot analysis showed that TGF-~ had no effect on collagen type II mRNA levels (Figure llC).
T ~ tting with anti-type I collagen antibody was negative (Figure 12).
~b) Growth of Cartilage C~ns8Lu~Ls On ~kree-Dimension~l Fr~mewor~
Figure 13 shows the cartilage cu~laL~uL5 to be smooth and glistening and three-times larger in the presence of TGF-~ (Figure 13 and Table III). Cells were mostly cu..~enLL~ted on the outside edge while the centers were less dense and contained more undegraded polyglycolic acid fibers (Figure 14). Constructs stained positively for type II collagen (Figures 12E
and F) but not for collagen type I (Figures 12C and D); Table III shows that TGF-~ treatment resulted in about 2.5 fold increase in the dry ~eight of the constructs as well as in collagen and GAG.

TABLE III
BOVINE CARTILAGE
Total Tot~l G~Q
Dry Weight Collagen contont 8Ample TGF-~ (mg) ~g) ~g) 1 - 4.5 31.5 71.0 2 - ~.2 29.3 71.6 3 - 3.6 2S.2 69.3 ~ ~ 11.7 81.8 171.C
~ 11.0 76.9 180.5 6 ~ lt.2 78.3 189.~

These results indicate that TGF-~ is capable of increasing the proliferation of chondrocytes in monolayers as well as increasing cartilage production on three-~;r-n~lonAl fL JL~

Wos~3821 2 1 9 2 0 6 4 r 8. Cartilage Production by Rabbit ~ho~d~v~Le3 on Polyglycolic Ac$d FL - ..Or~ in a Closed Bioreactor 8vstem Polyglycolic acid mesh were seeded with 4 x 106 cells in 6-well dishes for two days and then placed into bioreactors where they were fed continuously using a 16-channel pump (Cole-Parmer: Masterflex with computerized drive, model no. 7550-90) with complete media (250ml for 5 bioreactors) at a flow rate of 50~g/ml for 28 days at 37~C with no media change.
Fresh ascorbate (50~g/ml final concentration) was added every three days. As a separate group of experiments, seeded mesh were cultured statically in 6 well dishes with 5ml of media which was changed twice weekly. The media used for growing cells on polyglycolic acid LL ~I~S was exactly the same as for cell growth in monolayer except for addition of ascorbate.

8.1. RESULTS
8.1.1. Cartilage Pro~v~t~n by Rabbit Chondrocytes on Polyglycolic Acid PrA~ ~L~S in a Closed Bioreactor 8YStem Figure 15A shows the gross appearance of seeded mesh grcwn statically. They were thinner than the c~nsLL~Ls grown in the bioreactor system, which were glistening and about 2mm in th i rkn~eS . Histologic examination with Hematoxylin/Eosin or Trichrome revealed cell growth and deposition of exacellular-matrix throughout the mesh (Figures 16A and 16C).
Alcan blue and Safranin 0 staining showed deposition of GAGs (Figures 16E-16H). Staining of COnSLLU~LS
grown statically showed far less matrix deposition (Figures 16I and J). T 7sLaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen (Figure 17). Cartilage produced in the bioreactors stained positively with anti-type II collagen and anti-WO ')5133g21 PCT1115!1~1072g6 2 1 920~4 chondroitin sulfate (Figures 18A and B) but not withanti-type I collagen lFigure 18C). Bior~
analyses showea collagen and GAG values to be 15~ and 25~ dry weiqht, respectively. These values in con~LLuuLs compare favorably with respective publLshed values in rabbit articular cartilage of 30-70~ and 10-30~ of dry weight, respectively.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations o~ individual aspects of the inventicn, and functionally equivalent methods and ~ t~ are within the scope of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and ~rc~~p~nying drawings. Such modifications are intended to ~all within the scope of the appended claims.

Claims (30)

What is claimed is:

1. A method for preparing a living stromal tissue in vitro, comprising culturing stromal cells inoculated onto a three-dimensional framework in a culture medium containing an effective amount of growth factors, so that the stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope the framework of a biocompatible, non-living material formed into a three dimensional structure having interstitial spaces bridged by the stromal cells to form into a three-dimensional construct.

2. The method of Claim 1 in which the stromal cells are chondrocytes.

3. The method of Claim 1 in which the stromal cells are fibroblasts or fibroblast-like cells.

4. The method of Claim 1 in which the stromal cells are umbilical cord cells or bone marrow cells from umbilical cord blood.

5. The method of claim 1 in which the stromal cells are a combination of chondrocytes, chondrocyte-progenitors, and fibroblasts, fibroblast-like cells, endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells, adipocytes, umbilical cord cells, and bone marrow cells from umbilical cord blood.

6. The method of Claim 1 in which the framework is composed of a biodegradable material.

7. The method of Claim 6 in which the biodegradable material is polyglycolic acid, cotton, cat gut sutures, cellulose, gelatin, collagen or polyhydroxyalkanoates.

8. The method of Claim 7 in which the polyglycolic acid is treated with ethylene oxide.

9. The method of Claim 7 in which the polyglycolic acid is treated with an electron beam.

10. The method of Claim 1 in which the framework is composed of a non-biodegradable material.

11. The method of Claim 8 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.

12. The method of Claim 1 in which the framework is a mesh.

13. The method of Claim 1 further comprising culturing parenchymal cells inoculated onto the living stromal tissue.

14. The method of Claim 13 in which the parenchymal cells comprise chondrocytes, chondrocyte-progenitors, fibroblasts, fibroblast-like cells umbilical cord cells, or bone marrow cells from umbilical cord blood.

15. The method of Claim 1 in which the culture medium further comprises of an effective amount of ascorbate.

16. The method of Claim 1 in which the culture medium is kept under static conditions.

17. The method of Claim 1 in which the culture medium is kept in dynamic state by convection and under periodic pressurization.

18. A method for transplantation or implantation of a living cartilage tissue construct comprising, (a) inoculating stromal cells on a three-dimensional framework;
(b) culturing the stromal cells so that they proliferate in vitro; and (c) implanting the cartilage tissue construct in vivo.

19. A living stromal cell-colonized three-dimensional framework prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells which are inoculated upon a three-dimensional framework such that the three-dimensional framework becomes populated with viable cells to form a three-dimensional structure having interstitial spaces bridged by the stromal cells.

20. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the stromal cells are chondrocytes.

21. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the stromal cells are fibroblasts or fibroblast-like cells.

22. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the stromal cells are umbilical cord cells or bone marrow cells from umbilical cord blood.

23. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the framework is treated with ethylene oxide.

24. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the framework is treated with an electron beam.

25. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the framework is composed of a biodegradable material.

26. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the biodegradable material is polyglycolic acid, cotton, cat gut sutures, cellulose, gelatin, collagen or polyhydroxyalkanoates.

27. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the framework is composed of a non-biodegradable material.

28. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.

29. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the framework is a mesh.

30. The living stromal cell-colonized three-dimensional framework of Claim 19 in which the stromal cells comprise chondrocyte-progenitors, fibroblasts, fibroblast-like cells, muscle cells, umbilical cord cells or bone marrow cells from umbilical cord blood.