CA2316413A1 - Use of adipose tissue-derived stromal cells for chondrocyte differentiation and cartilage repair - Google Patents
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Abstract
Methods and compositions for directing adipose-derived stromal cells cultivated in vireo to differentiate into cells of the chondrocyte lineage are disclosed.
The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.
The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.
Description
n~u. au uum n~~ A~ ~. CA 02316413 2000-08-18 ATTO1ZNEY DOCKET NO: 5750-12 USE OF ~D1POS~ TISSUE-DERIVED STROMAL CELLS FOR CIiONDROCYTE
DIFFERENTIATION AND CARTILAGE. RCPAIR
FIELD OF INVENTION' The present invention relates to methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage and particularly to such directed lineage indueiion prior to, or at the time of, their implantation into a recipient or host for the therapeutic treatment of pathologic conditions in humans and ocher species.
DACKGROU'N1~ OF THE TNVFNTION
Mesenchymal stem cells (MSCs) are the formative pluripotent blast or ~o embryonic-like cells found in bone marrow, blood, dermis, and periosteum that are capable oFdifferentiating into specific types ofmesenchymal or connective tissues including Adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues. The specific differentiation pathway which these cells enter depends upon various influences from mechanical influences andlor endogenous bioactivc factors, such l5 as growth factors, cytokines, andlor local microenvironmental conditions established by host tissues.
In prenatal organisms, the differentiation of MSCs into specialized connective tissue cells is well established; for example embryonic chick, mouse or human limb bud mesenchymal cells differentiate into cartilage, bone and other connective tissues (Caplan 2o A I (1981) In: 39th Annual Symposium of the Society for Developmental Biology, ed by S. Subtelney and U Abbott, pp 3'768_ New Yorbc, Alan R Liss Inc; Elmer et al.(1981) Teratology, 24:215-223; Hauschka S.D. (1974) Developmental Biology (1974) 37:345 3G8; Solursh er al.(l9$1) Developmental Biology, B3v9-19; Swalla et. al.
(1986) RTAaI/Z076~fi6v1 Attorney Docket No.: X750-IZ
" v ~' V ~~ ~~ ~ ~ y ' CA 02316413 2000-08-18 Developmentallliolagy, lIG:31-38. In addition, a clonal rat fetus calvarial cell line has also been shown to differentiate into muscle, fat, cartilage, and bone (Goshlma et al.(1991) Chn Orrhop Rel Res. 269:274-283. The existence of MSCs in post-natal organisms has not been widely studied with the objective of showing the differentiation of post-embryonic cells into several mesodermal phenotypes. The few studies which have been done involve the formation ofbonc and cartilage by bone marrow cells following their encasement in diffusion chambers and in vivo transplantation (Ashton et al.(1980) Clln Orthop Rel Res, lS1:294-307; Hruder et al.(1990) Bane Mineral, 11;141-151, 1990). Recently, cells from chick periosteum have beers isolated, expanded in ~o culture, and, under high density conditions in vtrro, shown to differentiate into cttrtilase and bone [Nakahata et al. (199I) Exp Cell Res, J95:492-503). Ral bone marrow-derived mcscnchymal cells have been shown to have the capacity to differentiate into osteobtasts and chondroeytes when implanted in vivo (Dennis ct al.(1991) Call Transpl, 1:2332:
Goshima et al.(1991) Clfn Orthop Rel Res. 269:274-283). Work by Johnstone et al, 1J.S, 1 s Pat. No. 5,908,784 has shown the ability of mesenchymal cells derived from skin to differentiate into cells biochemically and phenotypically similar to chondrocytes.
The adult bone marrow microenvironment is a potential source fot these hypothetical mesodermal stem cells. Cells isolated from adult mawow are referred to by a variety of names, including stromal cells, stromal stem cells, mesenchymal stem cells zo (MSCs), mesenehymal fbroblasts, reticular-endothelial cells, and Westen-Bainton cells (Gimble et al, (Nov. 1996) Bone I9(S): A~21-8). In vlrro studies have determined that these cells can differentiate along multiple mesodennal or mesenchymal lineage pathways. These include, but arc not limited to. adipocytcs (Gimble, er vl_ (1992) J. Cell Biochem. SU:73-82, chondrocytes; Caplan, el al. ( I 998) J Bone Joint Surg.
Am.
25 80(12):1745-57; hematopoietic supporting cells, Gimble, et al. (1992) J.
Cell Biochem.
50:73-82; myocyles, Prockop, el ah (1999) J. Cell Biochem.72(4);570-85;
myocytes, Charbord, et al.(1999) Exp. Hematol. 27(12):1762-95; and osteobtasts, Beresford et al.
(1993) 1. Cell Phy~siol.75.4:317-328), The bone marrow has been proposed as a source of stromal stem cells for the regeneration of bone, cartilage, muscle, adipose tissue, and 30 other mesenchymai derived organs. The major limitations to the use of these cells are the RTA01/207G I 6G -2- Anornay Docket No. 5750-12 ,"". ,." "" ;...., ~." "_ CA 02316413 2000-08-18 difficulty and risk attendant upon bone marrow biopsy procedures and the low yield of stem cells from this souroe.
Adipose tissue offers a potential altentative to the bone marrow as a source of multipotential stromal stem cells. Adipose tissue is readily accessible and abundant in many individuals_ Obesity is a condition of epidemic proportions in the United Stales, where over 50% of adults exceed the recommended BMI based on their height.
Adipocytes can be harvested by liposuction on an outpatient basis, This is a relatively non-invasive procedure with cosmetic effects that are acceptable to the vast majority of patients. It is well documented that adipocytes are a replenishable call population. Cven I 0 after surgiegl removal by liposuction or other procedures, it is common to see a recurrence of adipocytes in an individual over time. This suggosts that adipose tissue contains stroma) stctn cells which arc capable of self renewal.
Pathologic evidence suggests that adipose-derived stromal cells are capable of differentiation along multiple mesenchymal llneages. The most common soft tissue I 5 tumor, liposareomas, develop from adipocyte-like cells. Soft tissue tumors of mixed origin are rLlatively common. These may include elements of adipose tissue, muscle (smooth or skeletal), cartilage, and/or bone. Just as bane fonoing cells within the bone marrow can diffcrentiace into adipocytes or fat cells, the extramedullary adipocytes are capable of forming osteoblasts (T-lalvorsen WO 99128444).
20 Cartilage is a ltyperhydrated structure with water comprising 70°1° to BO% of its weight. The remaining ~0% to 3Q% comprises type I1 collagen and proteoglycan.
The collagen usually accounts for 70% of the dry weight of cartilage (in "Pathology" ( 1988) Eds. LRubin 8c Farber, J. B. Lippincott Company, PA. pp. 1369-1371).
Proteoglycans are composed of n central protein care from which long chains of polysaccharides extend.
25 These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate. Cartilage has a characteristic structural orgt~nization consisting of chondrogenic cells dispersed within an endogenously prodtteed and secreted extracellular matrix. The caviiies in the matrix which contain the chondroeytes are called cartilage lacunae. Unlike bone, cartilage is neither innervated 30 not' penetrated by either the vascular or lymphatic systems (Ctemente (~
984) in "Gray's Anatomy, 30th Edit," Lca ~. Febiger).
RTA01/2076166 -3- Attorney Docket No. S7S0-12 .~uv. au uw..v.r ..v~v.
. CA 02316413 2000-08-18 'w-a Three types of cartilage are present in mammals and include: hyaline cartilage;
fibrocartilage and elastic cartilage (Rubin and Farber, supra). Hyaline cartilage consists of a gristly mass having a firm, classic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found an the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal earlilage, bronchial cartilage and nasal cartilage. Fibrocartilagc is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage. The collagenous fibers arc arranged in bundles, with the cartilage cells located between the bundles. Fibrocartitage is found commonly in the annulus fibrosis of to the invcrtebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules. Elastic cartilage also is sitnilar to hyaline cartilage except that ii conh3ins fibers of elastin. 1t is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fbers embedded in the cartilage matrix. Typically, classic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.
The surfaces of articulating bones in mammalian joints are covered with articular cadilage. The articular cartilage prevents direct contact of the opposing bone surfaces and pcrtnits the near l~riccionless movement of the articulating bones relative to one another (Clemenle, supra). Two types of articular cartilage defects are commonly 2d observed in mammals and include full-thickness and partial-thickness defects. The two-types of defects differ not only in the extent of physical damage but also in the nalurc of repair response each type of lesion elicits:
Full-thickness artieular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified Layer of earlilage located between the articular cartilage and the subchondral hone. Full-thickness defects typically arise during severe trauma of the joint or during the late sTages of degenerative joint diseases, for example, during osleoarthritis. Since the subchondral bone tissue is both innervaled and vascularirxd, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of 3o tibrocartilage at the site of the fall-thickness defect. Fibrocartilage, however, lacks the RTAOtI2U761GG -4- Attorney hockct No. 5750-12 "",. ", ~~ " ~., , . ~ ~" CA 02316413 2000-08-18 v biomechanical properties of articular cartilage and fails to persist in the joint on a lotlg term basis.
Isartia!~thickness articular cartilage defects are resCrieted to the cartilage tissue itself these defects usually include fiissures or clefts in the articulating surface ofthc cartilage. Partial-thickness defects are caused by mechanical arrangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.
tn In accordance with the present invention ii has barn observed by the inventors that when human adipose tissue-derived stromal cells are associated in a lhree-dimensional format they can be induced to commit and differentiate along the chondrogenic pathway when contacted in vitro with certain chondroinductive agents or factors. The three dimensional format is critical to the in vitro chondrogenesis of the invention and the cells are - preferably condensed together, for example, as a packed or pelleted cell mass or in an alginate matrix. This invention presents examples of methods and composition Ior the isolation, differentiation, and characterization of adult human cxtramedullary adipose tissue stromal cells along the chondroeyte lineage and outli~tes their use for the treatinent of a number of human conditions and diseases.
This in vitro 2o process is believed to recapitulate that which occurs in vivo and can be used to facilitate repair of cartilage in vivo in mammals.
SUMMARY OF INVENTION
The present invention provides methods and composition for consistent and z5 quantitative induction of stromal cells derived from subcutaneous, mammary, gonadal, or omcntal adipose tissues into fully ritnctional chondroeytes. the methods comprise incubation of isolated adipose tissue-derived stromal cells, plated at densities of 500 to 20,000 cells/cntz in a chemically defined culture medium having or supplemented with (1) a chondroinductive agent that can activate any cellular lransduction pathway 30 leading to the mature chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement RTA01/2076166 -S- Attorney Doclcet No. 5750-12 __. .. __ , .. , -- -- CA 02316413 2000-08-18 L
such as fetal bovine serum or horse scrum; (4) ascorbate or related vitamin C
analogue;
and (5) a glucoeortieoid or another chemical agent capable of activating the cellular glucocorticoid receptor.
The present invention also provides a method for differentiating adipose tissue derived stromal cells into chondrocytice cells by pellcting stromal cells in medium such as DMEM or alpha-MEM or RPMI 1640 and supplementing the medium with (I) a ehondroinductive agent that can activate any cellular transduetion pathway leading to the mature chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement such as fetal bovine serum or horse serum; (4) sscorbate or related vitamin C
1o analogue; and (5) a glueocorlicoid or another chemical agent capable of activating the cellular glucocorticoid receptor.
The present invention also provides a method for differentiating adipose tissue derived stromal cells into chondrocytic cells by suspending the cells in a ca1ciurn alginate or other bioeompatible lattice or matrix capable of supporting chondrogenesis in a three ~ s dimensional configuration.
The present invention provides methods for determining the ability of these culture conditions and agents to direct the differentiation and function of the adipose tissue-derived stromal cells, for the transduction of viral vectors carrying regulatory genes info the stromal cells, for the transfection ofplQSmid vectors carrying regulatory 2o genes into the stxomal cells, for the tracking end detention of functional proteins encoded by these genes, and for developing biomechanical carriers for the ro~introduotion of these cells into a living organism.
This invention further provides methods for the introduction of these chondrocytes into cartilage defect areas far repair.
25 The methods and composition have use in drug discovery for compounds and proteins with relevance to the differentiated cell-related disease states and traumatic injuries including but not limited to: anterior cnicia ligament fears, full-thickness articular cartilage defects, partial-thickness articular cartilage defects.
RTAO11ZU761b6 -G- Altomey Docket No. 5750-12 i ."". ," "" .,., " ""
BRIEF DESCIZ11'1'lON OF THE DRAWINGS
s Figurc 1 shows the immunodetection of collagen type I1 in human adipose stromal cells from monolayer cultures. Phase contrast microscopy is used in the upper panel;
lmmunoiluorescence is used in the lower panel.
Figure 2 shows immunodetection of collagen type II in human adipose stromal to cells from pellet cultures. Phase contrast microscopy is used in the upper panel; lmmuno-fluorescence is used in the lower panel.
Figure 3 shows immunodetection of collagen type II in human adipose stromal cells from alginate cultures. Phase contrast microscopy is used in the upper panel;
15 Immunofluorescence is used in the lower panel.
Figure 4 shows Collagen type V1 expression when cells were cultured in an alginate matrix at 2 weeks without TGF-beta (control) and with TGF-beta-2o Figure 5 shows a Western blot of results when cells were grown as tnonolayors or in nn alginate suspension for the expression of different proteins including: collagen type VI, link, aggrecan. collagen type 1, and actin.
The present invention provides methods and a composition for the differentiation and culture of adipose tissue-derived stromal eGlls into chondroeytes. The cells produced by the methods of invention ace useful in providing a source of fully dificrentialed and 30 functional cells for r~se~erch, transplantation, and development of tissue engineering products for the treatment of human disease and traumatic injury repair. Thus, in one RTA01/2076166 -7- Attorney Dockol No, 5750-12 a aspect, the invention provides a method for diffcrcntiating adipose tissue-derived stromal cells into chondrocytes comprising culturing etromal cells in a composition which comprises a medium capable of supporting the growth and differentiation of stromal cells into functional ehondrocytes. This invention further providos methods for the introduction of these chondrocytes into cartilage defect arCaS for repair.
"Adipose stromal cells" refers to stromal cells that originate from adipose tissue, ay "adipose" is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omentallvisceral, mammary, gonadal, or other adipose tissue site, Preferably, the adipose is subcutaneous white adipose tissue.
Sueh cells may 1o comprise a primary cell culture or sn immortqlized cell line. The adipose tissue may be from any organism having fat tissue. Preferably, the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue or the method of isolation of adipose tissue is not critical to the invention. If stromal cells are desired for autologous transplantatiozt itato a subject, the adipose tissue will be isolated from that subject.
"Chondrocytes (cartilage cells)" refers to cells that are capable of expressing characteristic biochemical markers of chondrocyles, including but not limited to collagen type II, ehondt~oitin sulfate, keratin sulfate and characteristic morphologic markers of smooch muscle, including but not limited to the rounded morphology obsorved in culture, and able to secrete collascn type II, including but not limited to the generation of tissue or matrices with hemodynamic properties of cartilage in vitro.
Any medium capable of supporting stromal cells in tissue cultztre may be used.
Media formulations that will support the growth of fibrobiasts include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential zs Medium (a.MEM), and Roswel) Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, 0 to 20°/a retal Bovine Serum (FBS) or 1-20% horse serum will be added to the above media in order to support the growrth of slromal cells and/or chondrocytes. rlowever, a def ned medium could be used if the necessary growth factors, eytolcines, and hormones in FBS for stromal cells and chondrocyles arc idcntitied and provided ai appropriate concentrations in the growth medium- Media useful in tl~e methods of tho invention may oontain one or more compounds of interest, including, but R'I'AD1/2U76166 -B- Attorney Docket No. 5750-l2 i nuv. Au uuww .u~v, a not limited to antibiotics mitogcnic or diffcrcntiativc compounds for stromal cells, The cells will be grown at temperatures between 3l°C to 37°C in a humidi .Tied incubator. The carbon dioxide content will be maintained between 2% to 10% and the oxygen content between 1 ~o and 22%. Cells will remain in this environment for periods of up to 4 weeks, Antibiotics which can supplemented into the medium include, but are not limited to penicillin and streptomycin. The concentration of penicillin in the chemically dcfncd culture medium is about 10 to about 200 units per m1. The concentration of streptomycin in the chemically defined culture medium is about 10 to about 200 ug/ml.
Glucocorticoids that can be used in the invention include but are not limited to Io hydrocortisone and dexamethasone. The concentration of dexamethasonc in the medium is about 1 to about 100 nM. The concentration of hydroeorl7sone in the medium is about 1 to about 100 nM.
As used herein the terms "chondroinductive agent" or "chondroinductive factor"
refers to any natural or synthetic, organic or inorganic chemical or biochemical I S compound or combination or mixture of compounds, or any mechanical or other physical device, container, influence or farce that can be applied to human adipose tissue-derived slromal cells so as to effect their in vitro chondrogenic induction or the production of chondrocytes. The chondroinductive agent is preferably selected, individually or in combination, froth the group consisting of (i) a glucocorticoid such as dexamethasone;
zo (ii) a m~:mber of the transforming growth factor-(3 superfatnily such as a bone morphogenic protein (preFerably BMP-2 or BMP-4), TOF- ail, TQr-(52, TGF-(33, insulin-like growth factor (1GI=), platelet derived growth factor (PDGF), epidennal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFHF), hepatocytic growth factor (HGF), keratocyte growth factor (1CGF), 25 osteogenic proteins (OP-l, OP-2, and OP-3), inhibin A or claondrogenic stimulating activity factor (CSA); (iii) a component of the collagenous extracellular matrix such as collagen I {particularly in !he form of a gel); and (iv) a vitamin A analogue such as retinoic acid and; (v) ttscorbaCe or other related vitamin C analogue.
The concentration of transfotTning growth factor-beta is about 1 to about 100 30 ng/ml. The concentration of retinoic acid is about 0, l to about I ug/ml.
lt'fAUII207ti161 -9- Attorney Docket No. 5750-12 i nuu. xo uuww xu~uu Examples of compounds that arc stromal cell milogens include but are trot limited to transforming growth factor (i; fibroblast growth factor, bone morphogenetic protein and stroma! cell differentiating factors include but are not limited to dexamethasone, hydrocortisone, transforming growth factor p, fibroblast growth factor, and bone morphogenetic protein and the like.
Preferably. the adipose tissue derived stromal cells are isolated from the adipose tissue of the subject into which the final differentiated cells are to be introduced.
However, the stromal cells may also be isolated from any organism of the same or different species as the subject. Any organism with adipose tissue can be a pnlcntial ~n candidate. Preferably, the organism is mammalian, most preferably the organism is human.
The present invention also provides a method for differentiating adipose derived stromal cells into chondrocytic cells by suspending the cells in a calcium alginate or another biocompatible lattice or matrix capable of supporting chondrogenesis in a three dimensional conrguralion. Examples of lattice materials include (1) calcium alginate, a Polysaccharide of cross linked 1 -giucuronic and D-mannuronic acid., at concentrations of between 1% l0 4%; (2) fibrin; (3) collagen type II; or (4) agarose gel. The lattices or matrixes containing the cells are transferred to culture dishes containing: ( I ) a chondroinductive agent that can activate any cellular trtxnsduction pathway leading io the mat>are chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement such as fecal bovine scrum or horsy scrum: (4) ascorbatc or related vitamin C analogue: and (5) a glucocorticoid or another chemical agent capable of activating the oellular glucocorlicoid receptor.
The adipose tissue derived stromal cells may be stably or transiently transfected or transduced with a nucleic acid of interest using a plasmid, viral or alternative vector strategy. Nucleic acids of interest include, but are not limited to, those encoding gene products which enhance the production of exiracellular matoix components found in cartilage; examples include transforming growth factor (i, bone morphogcntic protein, acti.vin and insulin-like grourth factor.
The transduelion of viral vectors canying regulatory genes into the stron~al cells can be performed with viral vectors (adenovirus. retrovirus. adeno-associated virus, or RTAOI/z076tGG -10- Attorney Docket Nn. 5750-12 ~u~. xu uuwm au.uu CA 02316413 2000-08-18 w other vector) purified by cesium chloride banding or other method at a multiplicity of infection (viral units:cell) of between 10:1 to 2D00:1. Cells will bo exposed to the virus in serum free or serum-containing medium in the absence or presence of a cationic detergent such as polyethyleneimine or Lipofectsmine~~M for a period of 1 hour to 24 hours (Hyk T. ef al. (1998) Human Gene Therapy 9:2493-2502; Sommer B_eI al.
(7999) Calcif. Tissue Int. 6x:45-.49), The transfeetion of plastnid vectors carrying regulatory genes into the stromal cells can be introduced into the cells in monolayer cultures by use of calcium phosphate DNA precipitation or cationic detergent methods (LipofeetaminerM, DOTAP) or in threi:
1o dimensional eultur~s by incorporation of the plasmid DN~1 vectors directly into the biocompatible polymer (Donadio J. et al. ( 1999) Nar. Med. 5:753-759).
rot the tracking and detection of functional proteins encoded by these genes, the viral or plasmid DNA vectors will contain a readily detectable marker gene, such as the green fluorescent protein or beta-galactosidase enayme, both of which can be tracked by histochemical means.
f or the development of. biomechanical carriers for Lhe re-introduction of the stromal cells into a living organism, the carriers include but arc not limited to calcium r~lginale, agarose, types 1, iT, IV or other collagen isofocm, fibrin, poly-lactic/poly-gtycolic acid, hyaluronate derivatives or other materials (Perka C, et al.
(2000) J. Biomed.
Mater. Res. 49:305-31 l: Sechricst Vr. et al. (2000) J. Biomed. Morer_ Res.
49:534-541;
Chu CR et al. (1995) J. l3iomed Mater. Res. 29;1147-1 154; I~iendrickson DA et al.
( 1994) Orthop.Res.12:485-497).
Another object of the invention is to provide for the identification and study of compounds that enhance the differenlietion of adipose tissue derived sCromal cells into chon4rocyles. Compounds which enhance differentiation may be of value in the treatment of partial or full cartilage defects, osteoarthtitis, traumatized eattilage, cosmetic surgery of inborn defects including cleft palate or deviated septum. Methods include but are not limited to the development of three-dimensional in vitro cultures maintaining adipose tissue-derived siroma~l cells as chondrocytes that can be subsequently exposed to 3o novel compounds of interest.
RTA01/2076166 -I 1- A~lorncy Docket No. 5750-12 Any compound may be tested for its ability to affect the differentiation of adipose tissue derived stromal cells into chondrocytes. Appropriate vehicles compatible with the compound to be tested arc known to those skilled in the art and may be found in the current edition of Ramington's Pharmaceutical Sciences (1995, Mack Publishing Co., Easton, PA) the contents of which arc incorporated herein by reference.
The features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention.
1 o EXPERIMENTAL
Differentiation of Adipose Tissue-Derived Stromal Cells into Chandrocytcs E~CAMPLC 1: In vitro Chondrogenesis using Dexamethasonc Stromal cells are isolated from human subcutaneous adipose tissue according to methods described in ''Methods and Composition oflhe D~erentialion ojHumnn Preadipocyles inter Adipocyrcs" Serial Number 09/240,029 Filed Januaty 29, 1999.
These cells are plated at a density of 500 to 20,000 cells per em'. The ptasent invention contemplates that the creation of a prccartilagc condensation in vitro promotes chondrogcnesis in mesenchymal progenitor cells derived from human adipose tissue, This is accomplished by methods including, but not limited to:
( 1 ) The pellet culture system, which was developed for use with isolated growth plate cells (ICato et al, (1988) PNAS 85:9552-9556 : Ballock & Reddi, J. Cell 9iol. (1994) 126(5):1311-1318) and has been used to maintain expression of the cartilage phenotype of chondrocytcs placed in culture (Solursh ( 1991) J.
Cell ~inchem. 45:258-260).
DIFFERENTIATION AND CARTILAGE. RCPAIR
FIELD OF INVENTION' The present invention relates to methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage and particularly to such directed lineage indueiion prior to, or at the time of, their implantation into a recipient or host for the therapeutic treatment of pathologic conditions in humans and ocher species.
DACKGROU'N1~ OF THE TNVFNTION
Mesenchymal stem cells (MSCs) are the formative pluripotent blast or ~o embryonic-like cells found in bone marrow, blood, dermis, and periosteum that are capable oFdifferentiating into specific types ofmesenchymal or connective tissues including Adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues. The specific differentiation pathway which these cells enter depends upon various influences from mechanical influences andlor endogenous bioactivc factors, such l5 as growth factors, cytokines, andlor local microenvironmental conditions established by host tissues.
In prenatal organisms, the differentiation of MSCs into specialized connective tissue cells is well established; for example embryonic chick, mouse or human limb bud mesenchymal cells differentiate into cartilage, bone and other connective tissues (Caplan 2o A I (1981) In: 39th Annual Symposium of the Society for Developmental Biology, ed by S. Subtelney and U Abbott, pp 3'768_ New Yorbc, Alan R Liss Inc; Elmer et al.(1981) Teratology, 24:215-223; Hauschka S.D. (1974) Developmental Biology (1974) 37:345 3G8; Solursh er al.(l9$1) Developmental Biology, B3v9-19; Swalla et. al.
(1986) RTAaI/Z076~fi6v1 Attorney Docket No.: X750-IZ
" v ~' V ~~ ~~ ~ ~ y ' CA 02316413 2000-08-18 Developmentallliolagy, lIG:31-38. In addition, a clonal rat fetus calvarial cell line has also been shown to differentiate into muscle, fat, cartilage, and bone (Goshlma et al.(1991) Chn Orrhop Rel Res. 269:274-283. The existence of MSCs in post-natal organisms has not been widely studied with the objective of showing the differentiation of post-embryonic cells into several mesodermal phenotypes. The few studies which have been done involve the formation ofbonc and cartilage by bone marrow cells following their encasement in diffusion chambers and in vivo transplantation (Ashton et al.(1980) Clln Orthop Rel Res, lS1:294-307; Hruder et al.(1990) Bane Mineral, 11;141-151, 1990). Recently, cells from chick periosteum have beers isolated, expanded in ~o culture, and, under high density conditions in vtrro, shown to differentiate into cttrtilase and bone [Nakahata et al. (199I) Exp Cell Res, J95:492-503). Ral bone marrow-derived mcscnchymal cells have been shown to have the capacity to differentiate into osteobtasts and chondroeytes when implanted in vivo (Dennis ct al.(1991) Call Transpl, 1:2332:
Goshima et al.(1991) Clfn Orthop Rel Res. 269:274-283). Work by Johnstone et al, 1J.S, 1 s Pat. No. 5,908,784 has shown the ability of mesenchymal cells derived from skin to differentiate into cells biochemically and phenotypically similar to chondrocytes.
The adult bone marrow microenvironment is a potential source fot these hypothetical mesodermal stem cells. Cells isolated from adult mawow are referred to by a variety of names, including stromal cells, stromal stem cells, mesenchymal stem cells zo (MSCs), mesenehymal fbroblasts, reticular-endothelial cells, and Westen-Bainton cells (Gimble et al, (Nov. 1996) Bone I9(S): A~21-8). In vlrro studies have determined that these cells can differentiate along multiple mesodennal or mesenchymal lineage pathways. These include, but arc not limited to. adipocytcs (Gimble, er vl_ (1992) J. Cell Biochem. SU:73-82, chondrocytes; Caplan, el al. ( I 998) J Bone Joint Surg.
Am.
25 80(12):1745-57; hematopoietic supporting cells, Gimble, et al. (1992) J.
Cell Biochem.
50:73-82; myocyles, Prockop, el ah (1999) J. Cell Biochem.72(4);570-85;
myocytes, Charbord, et al.(1999) Exp. Hematol. 27(12):1762-95; and osteobtasts, Beresford et al.
(1993) 1. Cell Phy~siol.75.4:317-328), The bone marrow has been proposed as a source of stromal stem cells for the regeneration of bone, cartilage, muscle, adipose tissue, and 30 other mesenchymai derived organs. The major limitations to the use of these cells are the RTA01/207G I 6G -2- Anornay Docket No. 5750-12 ,"". ,." "" ;...., ~." "_ CA 02316413 2000-08-18 difficulty and risk attendant upon bone marrow biopsy procedures and the low yield of stem cells from this souroe.
Adipose tissue offers a potential altentative to the bone marrow as a source of multipotential stromal stem cells. Adipose tissue is readily accessible and abundant in many individuals_ Obesity is a condition of epidemic proportions in the United Stales, where over 50% of adults exceed the recommended BMI based on their height.
Adipocytes can be harvested by liposuction on an outpatient basis, This is a relatively non-invasive procedure with cosmetic effects that are acceptable to the vast majority of patients. It is well documented that adipocytes are a replenishable call population. Cven I 0 after surgiegl removal by liposuction or other procedures, it is common to see a recurrence of adipocytes in an individual over time. This suggosts that adipose tissue contains stroma) stctn cells which arc capable of self renewal.
Pathologic evidence suggests that adipose-derived stromal cells are capable of differentiation along multiple mesenchymal llneages. The most common soft tissue I 5 tumor, liposareomas, develop from adipocyte-like cells. Soft tissue tumors of mixed origin are rLlatively common. These may include elements of adipose tissue, muscle (smooth or skeletal), cartilage, and/or bone. Just as bane fonoing cells within the bone marrow can diffcrentiace into adipocytes or fat cells, the extramedullary adipocytes are capable of forming osteoblasts (T-lalvorsen WO 99128444).
20 Cartilage is a ltyperhydrated structure with water comprising 70°1° to BO% of its weight. The remaining ~0% to 3Q% comprises type I1 collagen and proteoglycan.
The collagen usually accounts for 70% of the dry weight of cartilage (in "Pathology" ( 1988) Eds. LRubin 8c Farber, J. B. Lippincott Company, PA. pp. 1369-1371).
Proteoglycans are composed of n central protein care from which long chains of polysaccharides extend.
25 These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate. Cartilage has a characteristic structural orgt~nization consisting of chondrogenic cells dispersed within an endogenously prodtteed and secreted extracellular matrix. The caviiies in the matrix which contain the chondroeytes are called cartilage lacunae. Unlike bone, cartilage is neither innervated 30 not' penetrated by either the vascular or lymphatic systems (Ctemente (~
984) in "Gray's Anatomy, 30th Edit," Lca ~. Febiger).
RTA01/2076166 -3- Attorney Docket No. S7S0-12 .~uv. au uw..v.r ..v~v.
. CA 02316413 2000-08-18 'w-a Three types of cartilage are present in mammals and include: hyaline cartilage;
fibrocartilage and elastic cartilage (Rubin and Farber, supra). Hyaline cartilage consists of a gristly mass having a firm, classic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found an the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal earlilage, bronchial cartilage and nasal cartilage. Fibrocartilagc is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage. The collagenous fibers arc arranged in bundles, with the cartilage cells located between the bundles. Fibrocartitage is found commonly in the annulus fibrosis of to the invcrtebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules. Elastic cartilage also is sitnilar to hyaline cartilage except that ii conh3ins fibers of elastin. 1t is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fbers embedded in the cartilage matrix. Typically, classic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.
The surfaces of articulating bones in mammalian joints are covered with articular cadilage. The articular cartilage prevents direct contact of the opposing bone surfaces and pcrtnits the near l~riccionless movement of the articulating bones relative to one another (Clemenle, supra). Two types of articular cartilage defects are commonly 2d observed in mammals and include full-thickness and partial-thickness defects. The two-types of defects differ not only in the extent of physical damage but also in the nalurc of repair response each type of lesion elicits:
Full-thickness artieular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified Layer of earlilage located between the articular cartilage and the subchondral hone. Full-thickness defects typically arise during severe trauma of the joint or during the late sTages of degenerative joint diseases, for example, during osleoarthritis. Since the subchondral bone tissue is both innervaled and vascularirxd, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of 3o tibrocartilage at the site of the fall-thickness defect. Fibrocartilage, however, lacks the RTAOtI2U761GG -4- Attorney hockct No. 5750-12 "",. ", ~~ " ~., , . ~ ~" CA 02316413 2000-08-18 v biomechanical properties of articular cartilage and fails to persist in the joint on a lotlg term basis.
Isartia!~thickness articular cartilage defects are resCrieted to the cartilage tissue itself these defects usually include fiissures or clefts in the articulating surface ofthc cartilage. Partial-thickness defects are caused by mechanical arrangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.
tn In accordance with the present invention ii has barn observed by the inventors that when human adipose tissue-derived stromal cells are associated in a lhree-dimensional format they can be induced to commit and differentiate along the chondrogenic pathway when contacted in vitro with certain chondroinductive agents or factors. The three dimensional format is critical to the in vitro chondrogenesis of the invention and the cells are - preferably condensed together, for example, as a packed or pelleted cell mass or in an alginate matrix. This invention presents examples of methods and composition Ior the isolation, differentiation, and characterization of adult human cxtramedullary adipose tissue stromal cells along the chondroeyte lineage and outli~tes their use for the treatinent of a number of human conditions and diseases.
This in vitro 2o process is believed to recapitulate that which occurs in vivo and can be used to facilitate repair of cartilage in vivo in mammals.
SUMMARY OF INVENTION
The present invention provides methods and composition for consistent and z5 quantitative induction of stromal cells derived from subcutaneous, mammary, gonadal, or omcntal adipose tissues into fully ritnctional chondroeytes. the methods comprise incubation of isolated adipose tissue-derived stromal cells, plated at densities of 500 to 20,000 cells/cntz in a chemically defined culture medium having or supplemented with (1) a chondroinductive agent that can activate any cellular lransduction pathway 30 leading to the mature chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement RTA01/2076166 -S- Attorney Doclcet No. 5750-12 __. .. __ , .. , -- -- CA 02316413 2000-08-18 L
such as fetal bovine serum or horse scrum; (4) ascorbate or related vitamin C
analogue;
and (5) a glucoeortieoid or another chemical agent capable of activating the cellular glucocorticoid receptor.
The present invention also provides a method for differentiating adipose tissue derived stromal cells into chondrocytice cells by pellcting stromal cells in medium such as DMEM or alpha-MEM or RPMI 1640 and supplementing the medium with (I) a ehondroinductive agent that can activate any cellular transduetion pathway leading to the mature chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement such as fetal bovine serum or horse serum; (4) sscorbate or related vitamin C
1o analogue; and (5) a glueocorlicoid or another chemical agent capable of activating the cellular glucocorticoid receptor.
The present invention also provides a method for differentiating adipose tissue derived stromal cells into chondrocytic cells by suspending the cells in a ca1ciurn alginate or other bioeompatible lattice or matrix capable of supporting chondrogenesis in a three ~ s dimensional configuration.
The present invention provides methods for determining the ability of these culture conditions and agents to direct the differentiation and function of the adipose tissue-derived stromal cells, for the transduction of viral vectors carrying regulatory genes info the stromal cells, for the transfection ofplQSmid vectors carrying regulatory 2o genes into the stxomal cells, for the tracking end detention of functional proteins encoded by these genes, and for developing biomechanical carriers for the ro~introduotion of these cells into a living organism.
This invention further provides methods for the introduction of these chondrocytes into cartilage defect areas far repair.
25 The methods and composition have use in drug discovery for compounds and proteins with relevance to the differentiated cell-related disease states and traumatic injuries including but not limited to: anterior cnicia ligament fears, full-thickness articular cartilage defects, partial-thickness articular cartilage defects.
RTAO11ZU761b6 -G- Altomey Docket No. 5750-12 i ."". ," "" .,., " ""
BRIEF DESCIZ11'1'lON OF THE DRAWINGS
s Figurc 1 shows the immunodetection of collagen type I1 in human adipose stromal cells from monolayer cultures. Phase contrast microscopy is used in the upper panel;
lmmunoiluorescence is used in the lower panel.
Figure 2 shows immunodetection of collagen type II in human adipose stromal to cells from pellet cultures. Phase contrast microscopy is used in the upper panel; lmmuno-fluorescence is used in the lower panel.
Figure 3 shows immunodetection of collagen type II in human adipose stromal cells from alginate cultures. Phase contrast microscopy is used in the upper panel;
15 Immunofluorescence is used in the lower panel.
Figure 4 shows Collagen type V1 expression when cells were cultured in an alginate matrix at 2 weeks without TGF-beta (control) and with TGF-beta-2o Figure 5 shows a Western blot of results when cells were grown as tnonolayors or in nn alginate suspension for the expression of different proteins including: collagen type VI, link, aggrecan. collagen type 1, and actin.
The present invention provides methods and a composition for the differentiation and culture of adipose tissue-derived stromal eGlls into chondroeytes. The cells produced by the methods of invention ace useful in providing a source of fully dificrentialed and 30 functional cells for r~se~erch, transplantation, and development of tissue engineering products for the treatment of human disease and traumatic injury repair. Thus, in one RTA01/2076166 -7- Attorney Dockol No, 5750-12 a aspect, the invention provides a method for diffcrcntiating adipose tissue-derived stromal cells into chondrocytes comprising culturing etromal cells in a composition which comprises a medium capable of supporting the growth and differentiation of stromal cells into functional ehondrocytes. This invention further providos methods for the introduction of these chondrocytes into cartilage defect arCaS for repair.
"Adipose stromal cells" refers to stromal cells that originate from adipose tissue, ay "adipose" is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omentallvisceral, mammary, gonadal, or other adipose tissue site, Preferably, the adipose is subcutaneous white adipose tissue.
Sueh cells may 1o comprise a primary cell culture or sn immortqlized cell line. The adipose tissue may be from any organism having fat tissue. Preferably, the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue or the method of isolation of adipose tissue is not critical to the invention. If stromal cells are desired for autologous transplantatiozt itato a subject, the adipose tissue will be isolated from that subject.
"Chondrocytes (cartilage cells)" refers to cells that are capable of expressing characteristic biochemical markers of chondrocyles, including but not limited to collagen type II, ehondt~oitin sulfate, keratin sulfate and characteristic morphologic markers of smooch muscle, including but not limited to the rounded morphology obsorved in culture, and able to secrete collascn type II, including but not limited to the generation of tissue or matrices with hemodynamic properties of cartilage in vitro.
Any medium capable of supporting stromal cells in tissue cultztre may be used.
Media formulations that will support the growth of fibrobiasts include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential zs Medium (a.MEM), and Roswel) Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, 0 to 20°/a retal Bovine Serum (FBS) or 1-20% horse serum will be added to the above media in order to support the growrth of slromal cells and/or chondrocytes. rlowever, a def ned medium could be used if the necessary growth factors, eytolcines, and hormones in FBS for stromal cells and chondrocyles arc idcntitied and provided ai appropriate concentrations in the growth medium- Media useful in tl~e methods of tho invention may oontain one or more compounds of interest, including, but R'I'AD1/2U76166 -B- Attorney Docket No. 5750-l2 i nuv. Au uuww .u~v, a not limited to antibiotics mitogcnic or diffcrcntiativc compounds for stromal cells, The cells will be grown at temperatures between 3l°C to 37°C in a humidi .Tied incubator. The carbon dioxide content will be maintained between 2% to 10% and the oxygen content between 1 ~o and 22%. Cells will remain in this environment for periods of up to 4 weeks, Antibiotics which can supplemented into the medium include, but are not limited to penicillin and streptomycin. The concentration of penicillin in the chemically dcfncd culture medium is about 10 to about 200 units per m1. The concentration of streptomycin in the chemically defined culture medium is about 10 to about 200 ug/ml.
Glucocorticoids that can be used in the invention include but are not limited to Io hydrocortisone and dexamethasone. The concentration of dexamethasonc in the medium is about 1 to about 100 nM. The concentration of hydroeorl7sone in the medium is about 1 to about 100 nM.
As used herein the terms "chondroinductive agent" or "chondroinductive factor"
refers to any natural or synthetic, organic or inorganic chemical or biochemical I S compound or combination or mixture of compounds, or any mechanical or other physical device, container, influence or farce that can be applied to human adipose tissue-derived slromal cells so as to effect their in vitro chondrogenic induction or the production of chondrocytes. The chondroinductive agent is preferably selected, individually or in combination, froth the group consisting of (i) a glucocorticoid such as dexamethasone;
zo (ii) a m~:mber of the transforming growth factor-(3 superfatnily such as a bone morphogenic protein (preFerably BMP-2 or BMP-4), TOF- ail, TQr-(52, TGF-(33, insulin-like growth factor (1GI=), platelet derived growth factor (PDGF), epidennal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFHF), hepatocytic growth factor (HGF), keratocyte growth factor (1CGF), 25 osteogenic proteins (OP-l, OP-2, and OP-3), inhibin A or claondrogenic stimulating activity factor (CSA); (iii) a component of the collagenous extracellular matrix such as collagen I {particularly in !he form of a gel); and (iv) a vitamin A analogue such as retinoic acid and; (v) ttscorbaCe or other related vitamin C analogue.
The concentration of transfotTning growth factor-beta is about 1 to about 100 30 ng/ml. The concentration of retinoic acid is about 0, l to about I ug/ml.
lt'fAUII207ti161 -9- Attorney Docket No. 5750-12 i nuu. xo uuww xu~uu Examples of compounds that arc stromal cell milogens include but are trot limited to transforming growth factor (i; fibroblast growth factor, bone morphogenetic protein and stroma! cell differentiating factors include but are not limited to dexamethasone, hydrocortisone, transforming growth factor p, fibroblast growth factor, and bone morphogenetic protein and the like.
Preferably. the adipose tissue derived stromal cells are isolated from the adipose tissue of the subject into which the final differentiated cells are to be introduced.
However, the stromal cells may also be isolated from any organism of the same or different species as the subject. Any organism with adipose tissue can be a pnlcntial ~n candidate. Preferably, the organism is mammalian, most preferably the organism is human.
The present invention also provides a method for differentiating adipose derived stromal cells into chondrocytic cells by suspending the cells in a calcium alginate or another biocompatible lattice or matrix capable of supporting chondrogenesis in a three dimensional conrguralion. Examples of lattice materials include (1) calcium alginate, a Polysaccharide of cross linked 1 -giucuronic and D-mannuronic acid., at concentrations of between 1% l0 4%; (2) fibrin; (3) collagen type II; or (4) agarose gel. The lattices or matrixes containing the cells are transferred to culture dishes containing: ( I ) a chondroinductive agent that can activate any cellular trtxnsduction pathway leading io the mat>are chondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement such as fecal bovine scrum or horsy scrum: (4) ascorbatc or related vitamin C analogue: and (5) a glucocorticoid or another chemical agent capable of activating the oellular glucocorlicoid receptor.
The adipose tissue derived stromal cells may be stably or transiently transfected or transduced with a nucleic acid of interest using a plasmid, viral or alternative vector strategy. Nucleic acids of interest include, but are not limited to, those encoding gene products which enhance the production of exiracellular matoix components found in cartilage; examples include transforming growth factor (i, bone morphogcntic protein, acti.vin and insulin-like grourth factor.
The transduelion of viral vectors canying regulatory genes into the stron~al cells can be performed with viral vectors (adenovirus. retrovirus. adeno-associated virus, or RTAOI/z076tGG -10- Attorney Docket Nn. 5750-12 ~u~. xu uuwm au.uu CA 02316413 2000-08-18 w other vector) purified by cesium chloride banding or other method at a multiplicity of infection (viral units:cell) of between 10:1 to 2D00:1. Cells will bo exposed to the virus in serum free or serum-containing medium in the absence or presence of a cationic detergent such as polyethyleneimine or Lipofectsmine~~M for a period of 1 hour to 24 hours (Hyk T. ef al. (1998) Human Gene Therapy 9:2493-2502; Sommer B_eI al.
(7999) Calcif. Tissue Int. 6x:45-.49), The transfeetion of plastnid vectors carrying regulatory genes into the stromal cells can be introduced into the cells in monolayer cultures by use of calcium phosphate DNA precipitation or cationic detergent methods (LipofeetaminerM, DOTAP) or in threi:
1o dimensional eultur~s by incorporation of the plasmid DN~1 vectors directly into the biocompatible polymer (Donadio J. et al. ( 1999) Nar. Med. 5:753-759).
rot the tracking and detection of functional proteins encoded by these genes, the viral or plasmid DNA vectors will contain a readily detectable marker gene, such as the green fluorescent protein or beta-galactosidase enayme, both of which can be tracked by histochemical means.
f or the development of. biomechanical carriers for Lhe re-introduction of the stromal cells into a living organism, the carriers include but arc not limited to calcium r~lginale, agarose, types 1, iT, IV or other collagen isofocm, fibrin, poly-lactic/poly-gtycolic acid, hyaluronate derivatives or other materials (Perka C, et al.
(2000) J. Biomed.
Mater. Res. 49:305-31 l: Sechricst Vr. et al. (2000) J. Biomed. Morer_ Res.
49:534-541;
Chu CR et al. (1995) J. l3iomed Mater. Res. 29;1147-1 154; I~iendrickson DA et al.
( 1994) Orthop.Res.12:485-497).
Another object of the invention is to provide for the identification and study of compounds that enhance the differenlietion of adipose tissue derived sCromal cells into chon4rocyles. Compounds which enhance differentiation may be of value in the treatment of partial or full cartilage defects, osteoarthtitis, traumatized eattilage, cosmetic surgery of inborn defects including cleft palate or deviated septum. Methods include but are not limited to the development of three-dimensional in vitro cultures maintaining adipose tissue-derived siroma~l cells as chondrocytes that can be subsequently exposed to 3o novel compounds of interest.
RTA01/2076166 -I 1- A~lorncy Docket No. 5750-12 Any compound may be tested for its ability to affect the differentiation of adipose tissue derived stromal cells into chondrocytes. Appropriate vehicles compatible with the compound to be tested arc known to those skilled in the art and may be found in the current edition of Ramington's Pharmaceutical Sciences (1995, Mack Publishing Co., Easton, PA) the contents of which arc incorporated herein by reference.
The features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention.
1 o EXPERIMENTAL
Differentiation of Adipose Tissue-Derived Stromal Cells into Chandrocytcs E~CAMPLC 1: In vitro Chondrogenesis using Dexamethasonc Stromal cells are isolated from human subcutaneous adipose tissue according to methods described in ''Methods and Composition oflhe D~erentialion ojHumnn Preadipocyles inter Adipocyrcs" Serial Number 09/240,029 Filed Januaty 29, 1999.
These cells are plated at a density of 500 to 20,000 cells per em'. The ptasent invention contemplates that the creation of a prccartilagc condensation in vitro promotes chondrogcnesis in mesenchymal progenitor cells derived from human adipose tissue, This is accomplished by methods including, but not limited to:
( 1 ) The pellet culture system, which was developed for use with isolated growth plate cells (ICato et al, (1988) PNAS 85:9552-9556 : Ballock & Reddi, J. Cell 9iol. (1994) 126(5):1311-1318) and has been used to maintain expression of the cartilage phenotype of chondrocytcs placed in culture (Solursh ( 1991) J.
Cell ~inchem. 45:258-260).
(2) The alginate suspension method, where cells are maintained in a caleit~m alginzce suspension to prevent cell-cell contact and maintain a oharacteristic rounded morphology promoting the maintenance or acquisition of the cliondracyte phenotype.
RTA01/2076 I GG ~ I Z- Altamey pocket No. 5750-1 Z
nun. au uumw Av~uu ' CA 02316413 2000-08-18 human adipose tissue-derived cells are isolated as described above, For pellet cultures, aliquots of 200,000 cells were centrifuged at SOOg for 10 minutes in sterile 15 ml conical polypropylene tubes in DMEM with 10% fetal bovine serum, 50 ng/tnl ascorbate-2-phosphate, 100 nM dexamethasone (DEX) and then incubated ac 37° C.in a Sq/° COZ incubator for up to 3 weeks. For alginate cultures, cells were suspended at a density of 1 million cells per ml in 1.2% calcium alginate and maintained in DMEM
with 10% fetal bovine serum, 50 ng/ml ascorbate-2-phosphate, 100 nM
dexamethasone (DEX) and then incubated at 37° C in a 5% CO~ incubator for up to 3 weeks. After 2 or 4 1o weeks, the cells were isolated, fixed and analyzed for chondrocyte lineage markers by immunohistochcmistry with appropriate antibody reagents or by staining with tolui4ine blue to detect the presence of sulfated proteoglycans in the extracellular matrix.
Results obtained with an antibody detecting a representative chondrocyte marker protein, collagen l1, rue shown in Figures 1-3. The cells maintained in pellet culture I5 (Figure 2) or calcium alginate (Figure 3) stained positive by immunofluorescence far the intracellular presence of the collagen II protein. These results are to he contrasted with identical analysis of adipose tissue-derived cells maintained for 3 weeks in monolaycr culture as shown in Figure 1; here, no staining whatsoever is observed.
lmmunohistochemical results with an antibody reagent detecting the chondrocyte markor z0 protein, collagen VI, arc shown in rigure 4. Adipose tissue-derived siromal cells were maintained in 1.2% calcium alginate and maintained in DMEM with 10% fetal bovine scrum, 50 ng/ml ascorbate-2-phosphate, 100 nM dexamethasane (l~l;X) in the absence or presence of transforming growth factor (i (10 ng/ml) and then incubated at 37° C in a 5%
CO? incubator for up to 2 weeks. Immunohistochemistry revealed a dense deposition of 25 the collagen Vl protein surrounding those cells maintained in the presence, but not the absence, of transforming growth factor p, Polymerase chain reaction results deteoling representative gene markers associated with chondrogenesis are shown in Figure 5. Adipose tissue-derived stromal cells were maintained in l.2% calcium alginate (AIg) or in monolayer (Mono) cultures 3o and maintained in DMEM with 10% fetal bovine serum, 50 ng/m) sscorbatc-2-phosphate, 100 nM dexamethasone (DEC) in the absence (TGF(i -) or presence (TGl~(3 (ZTA01 /3076 t GG -13- Attorney Docket No. 5750-12 1VV~ AU UV ll mn ~-V VV
~-) of transforming growth factor (i (10 ng/ml) far a period of 4 weeks. Total RNA was isolated tfom the individual cultures and used in polymerase chain reactions with primers specific for eollagens types I or VI, the proteoglycan link (Link) protein, aggrecan, or actin. The collagen markers and actin were detected under all growth conditions.
s However, the link mR.NAs were most abundant under alginate suspension conditions and aggrecan was only present under alginate conditions in the presence of TGF(i.
These results demonstrate that, through a combination of creating an in vitro cell condensation and adding the appropriate permissive factors, we are able to produce the expression of chondrocyte markers consistent with chondrogenesis in cells frotr to subcutaneous adipose tissue.
Example 2: Preparation of Synthetic Cartilage Patch 15 Following proliferation, the chondrogenic cells still having chondrogenic potential may be cultured in an anchorage-independent manner, i.e., in a well having a cell contacting, cell adhesive surface, in order to stimulate the secretion of cairtiltlge-speci Iic extracel lular tnatt'ix components.
Heretofore, it has been observed that chondrogenic cells proliferatively expanded 20 in an anchorage-dependent manner usually dedifferentiate and lose their ability to secret cartilage-specific type II collagen and sulfated proteoglycan . (Mayne et al.
(1984) Exp.
Cell. Res. 151(1): 171-82; Mayne et al. (I97b) PNAS73(S): 1674-8; Okayama et al, (1976) PN~tS 73(9):3224-8; Pacifci et al. (1981) J.Btol Chem. 256(2); 1029-37;
Pacifici et al. (1980) Cancer Res_ 40(7): 2461-4; Pacif:ici et al. (1977) Cell 4: 891-9; von der 25 Mark et al. (1977) Nature 267(561 l):531-2; Wesl et al. (1979) Cell 17(3):491-501;
Ocgama er al. ( 1981 ) J. Biol. Chem. 256(2):1015-22; Bcnya et al ( 1982) Cell 30( 1 ):215-24).
It has been discovered that undifferentiated chondrogenic cells, when seeded info, and cultured in a well having a cell contacting surface that discourages adhesion of 3o cells to the cell contacting surface, the cells rcdiffcrcntiatc and start to secrete cartilage-IZTAO 112076 i 66 -14- Attorney Docket No. 575U-13 .'1UU. AO Uut~W ~~ Au~u a specific collagen and sulfated proleoglyeans thereby to form a patch of synfihetic cartilage in vitro (US Patent Nos. 5,902,741 and 5,723,331.)_ In addition, it has been found that culturing the cells in a pre-shaped well, enables tine to manufacture synthetic canilage patches of pre-determined thickness and volume.
It is appreciated, however, chat the volume of the resulting patch of cartilage is dependent not only upon the volume of the well but also upon the number of chondrogenic cells seeded into the well. Cartilage of optimal pre-determined volume may be prepared by routine experimentation by altering either, or both of the aforcmentianed parameters, to A. Preparation of Pre-shaped Well.
Several approaches are available far preparing pre-shaped wells with cell contacting, cell adhesive surfaces.
The cell contacting surface of the well may be coated with a molecule that discourages adhesion of ehondrogenic cells to the cell contacting surface.
Preferred 15 cowling reagents include silicon based reagents i.e..
dichlorodimethylsilane or polytetrafluoroethylcnc based reagents, i.e., '1 eflon.k'fM.. Procedures for coating materials with silicon based reagents, specifically dichlorodimethylsilane, arc well known in the art. See for example, 6ambrook el al. (1989) "Molecular Cloning A
Laboratory lvl;anual", Cold Spring Harbor Laboratory Press, the disclosure of which is incorporated 2D by reference herein. It is appreciated that other biocompatiblc rcascnts that prevent the attachment of cells to the surface oJ: the well nrtay b~ useYul in the practice of the instant invention.
Alternatively, the well may be cast froth a pliable or moldable biocompatible material that does not permit attachment of cells per se. Preferred materials that prevent zs such cell attachment include, but are not limited to, agarose, glass, untreated cell culture plastic and polytctrafluorocthylcnc, i.c., Tcflon® Untreated cell culture plastics, 1. e., plastics that have not been treated with or made from materials that have an electrostatic charge arc conuncrcially available, and may be purchased, for example, from Falcon Labware, Becton-Dickinson, Lincoln Park, N.J. 'fhe aforementioned materials, however, 3U are not meant to be limiting. It is appreciated that any other pliable or moldable tt'l'AU L /207I~ 166 - I 5- AClomey Uockct No. 5 750-12 1v u. a a a a p W . i a a ~ a bioeompatible material that inherently discourages the attachment of chondrogenic cells may be useful in the practice of the instant invention.
T he size and shape of the wet) may be determined by the size rind shape of the articular cartilage defect to be repaired. For example, it is contemplated that the well may have a cross-sectional surface area of 25 cm2. This is the average cross-sectional surface area of an adult, human femoral ehondyle. Accordingly, il is anticipated that a single piece of synthetic cartilage may be prepared in accordance with the invention in order to resurface the entire femoral chondyle. The depth of the well is preferably greater than about 0.3 cm and preferably about 0.6 cm in depth. The thickness of natural I 0 arlicular cartilage in an adult articulating joint is usually about 0.3 cm. Accordingly, the depth of the well should be large enough to permit a cartilage patch of about 0.3 cm to form. However, the well should also be deep enough to contain growth medium overlaying lhc cartilage patch.
>'t is contemplated also that a large piece of cartilage prepared in accordance with 15 the invention may be "trimmed" to a pre-selected size and shape by a surgeon performing surgical repair of the damaged earlilr~ge. Trimming may be performed with the use of a sharp cutting implement, i.e., a scalpel, a pair of scissors or an arthroscopic device fatted with a cutting edge, using procedures well known in the art.
The pre-shaped well preferably is cast its a block of agarose gel utt.der aseptic 2n conditions. Agaroae is an economical, biocompatible, pliable and moldable material that can be used co cast pre-shaped wells, duickly and easily. As rrtentioned above, the dimensions of the well may dependent upon the size of the resulting cartilage plug that is desired.
A pre-shaped well may be prepared by pouring a hat solution of molten LT
zs agarose (l3ioRad, Richmond, CA) into a tissue culture dish containing a cylinder. The cylinder having dimensions that minor the shape of the well to be formed. The size and shape of the well may be chosen by the artisan and may be dependent upon the shape of the articular cartilage defect to be repaired. Once the agarose has cooled and solidified around the cylinder, the cylinder is carefully removed with forceps. The surface or the tissue culture dish that is cxpo5cd by vhe removal of the cylinder is covered with molten agarose. This seals the bottom of the well and provides a cell. adhesive surface at the base RTAOlIZ07614G -16- Attorney Docket No. 5750-12 1VV~ AV VVt~~~~~ ~u~yy oTthe well. When the newly added molten LT agarose cools and solidifies, the resulting pre-shaped well is suitable for culturing, and stimulating the redifferentiation of proliferated cJtondrogonic colts. It is appreciated, however, t113t alternative methods may be used to prepare a pre-shaped well useful in the practice of the invention.
B. Growth of Cartilage Patch.
Proliferated chondrogenic cells in suspension may be seeded into and cultured in the pre-shaped well. The cells may be diluted by the addition of cell culture medium >;o a cell density of about 1 x 105 to 1 x 109 ohondrogenio oells per ml. A
preferred cell culture to medium comprises DMEM supplemented with 10% fetal bovine serum.
Within about four hours of seeding the chondrogenic cells into the well, the cells may coalesce to form a cohesive plug of cells. After about 4-10 days, the cel)s will start to secrete cartilage-specific sulfated proteoglycaos and type lI collagen.
After prolonged periods of time in culture the collagen expressed by the chondrogenic cells in the well 15 will be predominantly type II collagen. It is contemplated however, chef the cohesive plug of cells formed within four hours mtty be removed from the well and surgically iraplanted into the cartilage defect. It is anlicipattJ that the undiffcrenliatcd chondrogenic cells subsequently tray redifferentiate in situ thereby to form synthetic cartilage within the joint.
2o It is contemplated that chondrocytic differentiation or stimulatory factors may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the produotion of artioular oarlilage specific proteoglycans and/or collagen (Luyten & Iteddi (1992) in "Biological Regulation of the Chondrocytes". CRC Press. Boca Raton, Ann Arbor, London, and Tokyo, p.p. 227-236). Preferred growth factors include, hut are not zs limited to transforming growth factor-p (TGr-(i), insulin-like growth factor (IGF), platelet derived growth factor (PDGr), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor, (IiGF) keralinocyle growth factor (KGF), the bone morphogenic factors (BIvIPy) i.c., DMP-1, BMP-2, 13MP-3, EiMP-4, EMP-5 and BMP-6 and the ostengenic proteins [OPs), 3o i.e. OP-1, OP-2 and OP-3. 1'referrcd concentrations of ~'fGl=-~, lCi'F, fllGf, EGh, al~I3F, RTA01 /30761 G<, -t7~ Attorney Docket No, 5750-I2 1VV. AV VV lllv./ n.~ Vv ' CA 02316413 2000-08-18 bFHF, HGF, and KGF, range from about 1 to 100 ng/ml. Preferred eonoentrations of the BMP's and OP's range from about 1 to about 500 ng/ml.
however, these particular growth factors are not limiting. Any polypeptide growth factor capable of'stimulating or inducing the production of cartilage specific proteoglycans and collagen may be useful in the practice ofthe instant invention.
rn addition, it is contemplated that ascorbate may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and collagen. Preferred concentrations of ascorbate range from afoul 1 to about 1000 pg/ml.
Example 3: Surgical Repair of Articular cartilage Dcfcct Cartilage defectc in mammals are readily identl~.able visually during anhroscopic examination or during open surgery of the joint. Cartilage defects may also be identified 1 s inferentially by using computer aided tomography (CAT scanning), X-ray examination, magnetic resonance imaging (MRI), analysis of synovial fluid or serum markers or by any other procedures known in the art. 'treatment of the defects can be effected during an arthroscopic or open surgical procedure using the methods and compositions disclosed herein.
2o Accordingly, once the defect has been idcntif'icd, the defect may be treated by the following steps of (1) surgically implanting at the pre-determined site, a piece of synthetic articular cartilage prepared by the methodologies described herein, and (2) permitting the synthetic a~ticular cartilage to integrate into pre-determined site.
The synthetic cartilage patch optimally has a size and shape such that when the 25 patch is implanted into the defect, the edges of the implanted tissue contact directly the edges of the defect. In addition, the synthetic cartilage patch may be fixed in placed during the surgical procedure. This can be effected by surgically faing the patch info the defect with biodegradable sutures, i.e., (Ethicon, Johnson & Johnson) andlor by applying iriclu~e~~ui'are riottiirit~e8 Ior°r~rin-lhu°rt~'in ~A~'~
St~'ni~a~rP~O LnOrs"e~"~s~'~0~f~c. t'it1 i~~"f~~_ No. 2 4~4~8 900; Pr. Pat. No. 2 448 90l and LP.S.N. 88401961.3 and synthetic RTAOt~207GLGG -18- Attorney Docket No. 5750-t~
1 V V ~ A V V V 1 . m ~ n n. V V a bioadhesives similar to those disclosed in U.S. Pat. No. 5,197,97. It is contemplated, however, that alternative types of sutures and biocompatible glues may be useful in the practice of the invention In some instances, damaged articuiar cartilage maybe surgically excised prior the s to implantation of the patch of synthetic cartilage. Additionally, the adhesion of the smthetic cartilage patch to the articular cartilage defect may be enhanced by treating the defect with transglutaminase (Ichinose et al. (1990) J. Biol. Chem.
265(3);13411-13414;
Najjar e~ al. (1984) in "Transglutaminascs", Boston, Martinuse-Nijhoffj.
Initially, the cartilage defect is dried, for example by using cottonoi:d, and filled with a solution of to transglutaminasc. The solution is subsequently removed, for example, by aspiration, leaving a film containing transglutaminase upon the cartilage, The synthetic cartilage patch is implanted subsequently into the defect by the methods described above.
In addition the synthetic cartilage may be useful in the repair of human articular cartilage defecis_ Accordingly, chondrogenic cells may be differentiated from human 1 s adipose tissue-derived stromal cells, i.e, human subcutaneous adipose tissue.
Surgical procedures for effecting the repair of articular cartilage defects are well lcnown in the art. See for example: Luyten & Reddi (1992) in "Biological Regulation of the Chondrocytes", CRC Press, Hoca Raton, Ann Arbor, London, & Tokyo, p.p. 227-236, the disclosure of which is incorporated by reference herein.
20 'Che above demonstrates a culture system in which human adipose tissue-derived slromal cells differentiate into hypcrtrophic chondrocytes. Since all components are defined, the system can be used far studies of the affects of growth factors etc, on the progression of chondrogcncsis. Irr vt~ro systems have been used by us and others to show that these cell populations have osteogenic and adipocytic potential. We demonstrate 25 here that this population has chondrogenic potential. This has clinical applicability for cartilage repair, The invention also provides a process for inducing chondrogencsis in human adipose tissue-derived stromal cells by contacting such cells with a chondroinductive agent en vitro where die atromal cells rue associated in a three dimensionrsl format.
RTA01/2076166 -19- Attorney Docket No. 5'750-12 nuu. ,.~ u~~~,~..~ ..~.~~ CA 02316413 2000-08-18 a The invention also provides a prooess for using in vitro differentiated chondrocytes from adipose-derived stromal cells in the zepair of cartilage tissue in mammals, including humans.
In the above methods, the siromal cells are preferably isolated, culture expanded s human adipose tissue-derived stromal cells in a chemically defined environment and are condensed into close proximity, such as in the form of a three dimensional cell mass, e.g.
packed cells or a centrifugal cell pellet. Further, the contacting preferably comprises culturing a pellet of human adipose tissue-derived strornal cells in a chemically defined medium which comprises Dlvi~M with 10% serum, 50 nglml ascorbatc-2-phosphate, 10'' M dexantethasone. The differentiated cells are then introduced into the surgery site to repair cartilage. Since all components of the system are defined, the system can be used as a product for cartilage repair in mammals, including man and horses.
RTA01/2o761 GG -20- Accomcy Docket No. 5750-12
RTA01/2076 I GG ~ I Z- Altamey pocket No. 5750-1 Z
nun. au uumw Av~uu ' CA 02316413 2000-08-18 human adipose tissue-derived cells are isolated as described above, For pellet cultures, aliquots of 200,000 cells were centrifuged at SOOg for 10 minutes in sterile 15 ml conical polypropylene tubes in DMEM with 10% fetal bovine serum, 50 ng/tnl ascorbate-2-phosphate, 100 nM dexamethasone (DEX) and then incubated ac 37° C.in a Sq/° COZ incubator for up to 3 weeks. For alginate cultures, cells were suspended at a density of 1 million cells per ml in 1.2% calcium alginate and maintained in DMEM
with 10% fetal bovine serum, 50 ng/ml ascorbate-2-phosphate, 100 nM
dexamethasone (DEX) and then incubated at 37° C in a 5% CO~ incubator for up to 3 weeks. After 2 or 4 1o weeks, the cells were isolated, fixed and analyzed for chondrocyte lineage markers by immunohistochcmistry with appropriate antibody reagents or by staining with tolui4ine blue to detect the presence of sulfated proteoglycans in the extracellular matrix.
Results obtained with an antibody detecting a representative chondrocyte marker protein, collagen l1, rue shown in Figures 1-3. The cells maintained in pellet culture I5 (Figure 2) or calcium alginate (Figure 3) stained positive by immunofluorescence far the intracellular presence of the collagen II protein. These results are to he contrasted with identical analysis of adipose tissue-derived cells maintained for 3 weeks in monolaycr culture as shown in Figure 1; here, no staining whatsoever is observed.
lmmunohistochemical results with an antibody reagent detecting the chondrocyte markor z0 protein, collagen VI, arc shown in rigure 4. Adipose tissue-derived siromal cells were maintained in 1.2% calcium alginate and maintained in DMEM with 10% fetal bovine scrum, 50 ng/ml ascorbate-2-phosphate, 100 nM dexamethasane (l~l;X) in the absence or presence of transforming growth factor (i (10 ng/ml) and then incubated at 37° C in a 5%
CO? incubator for up to 2 weeks. Immunohistochemistry revealed a dense deposition of 25 the collagen Vl protein surrounding those cells maintained in the presence, but not the absence, of transforming growth factor p, Polymerase chain reaction results deteoling representative gene markers associated with chondrogenesis are shown in Figure 5. Adipose tissue-derived stromal cells were maintained in l.2% calcium alginate (AIg) or in monolayer (Mono) cultures 3o and maintained in DMEM with 10% fetal bovine serum, 50 ng/m) sscorbatc-2-phosphate, 100 nM dexamethasone (DEC) in the absence (TGF(i -) or presence (TGl~(3 (ZTA01 /3076 t GG -13- Attorney Docket No. 5750-12 1VV~ AU UV ll mn ~-V VV
~-) of transforming growth factor (i (10 ng/ml) far a period of 4 weeks. Total RNA was isolated tfom the individual cultures and used in polymerase chain reactions with primers specific for eollagens types I or VI, the proteoglycan link (Link) protein, aggrecan, or actin. The collagen markers and actin were detected under all growth conditions.
s However, the link mR.NAs were most abundant under alginate suspension conditions and aggrecan was only present under alginate conditions in the presence of TGF(i.
These results demonstrate that, through a combination of creating an in vitro cell condensation and adding the appropriate permissive factors, we are able to produce the expression of chondrocyte markers consistent with chondrogenesis in cells frotr to subcutaneous adipose tissue.
Example 2: Preparation of Synthetic Cartilage Patch 15 Following proliferation, the chondrogenic cells still having chondrogenic potential may be cultured in an anchorage-independent manner, i.e., in a well having a cell contacting, cell adhesive surface, in order to stimulate the secretion of cairtiltlge-speci Iic extracel lular tnatt'ix components.
Heretofore, it has been observed that chondrogenic cells proliferatively expanded 20 in an anchorage-dependent manner usually dedifferentiate and lose their ability to secret cartilage-specific type II collagen and sulfated proteoglycan . (Mayne et al.
(1984) Exp.
Cell. Res. 151(1): 171-82; Mayne et al. (I97b) PNAS73(S): 1674-8; Okayama et al, (1976) PN~tS 73(9):3224-8; Pacifci et al. (1981) J.Btol Chem. 256(2); 1029-37;
Pacifici et al. (1980) Cancer Res_ 40(7): 2461-4; Pacif:ici et al. (1977) Cell 4: 891-9; von der 25 Mark et al. (1977) Nature 267(561 l):531-2; Wesl et al. (1979) Cell 17(3):491-501;
Ocgama er al. ( 1981 ) J. Biol. Chem. 256(2):1015-22; Bcnya et al ( 1982) Cell 30( 1 ):215-24).
It has been discovered that undifferentiated chondrogenic cells, when seeded info, and cultured in a well having a cell contacting surface that discourages adhesion of 3o cells to the cell contacting surface, the cells rcdiffcrcntiatc and start to secrete cartilage-IZTAO 112076 i 66 -14- Attorney Docket No. 575U-13 .'1UU. AO Uut~W ~~ Au~u a specific collagen and sulfated proleoglyeans thereby to form a patch of synfihetic cartilage in vitro (US Patent Nos. 5,902,741 and 5,723,331.)_ In addition, it has been found that culturing the cells in a pre-shaped well, enables tine to manufacture synthetic canilage patches of pre-determined thickness and volume.
It is appreciated, however, chat the volume of the resulting patch of cartilage is dependent not only upon the volume of the well but also upon the number of chondrogenic cells seeded into the well. Cartilage of optimal pre-determined volume may be prepared by routine experimentation by altering either, or both of the aforcmentianed parameters, to A. Preparation of Pre-shaped Well.
Several approaches are available far preparing pre-shaped wells with cell contacting, cell adhesive surfaces.
The cell contacting surface of the well may be coated with a molecule that discourages adhesion of ehondrogenic cells to the cell contacting surface.
Preferred 15 cowling reagents include silicon based reagents i.e..
dichlorodimethylsilane or polytetrafluoroethylcnc based reagents, i.e., '1 eflon.k'fM.. Procedures for coating materials with silicon based reagents, specifically dichlorodimethylsilane, arc well known in the art. See for example, 6ambrook el al. (1989) "Molecular Cloning A
Laboratory lvl;anual", Cold Spring Harbor Laboratory Press, the disclosure of which is incorporated 2D by reference herein. It is appreciated that other biocompatiblc rcascnts that prevent the attachment of cells to the surface oJ: the well nrtay b~ useYul in the practice of the instant invention.
Alternatively, the well may be cast froth a pliable or moldable biocompatible material that does not permit attachment of cells per se. Preferred materials that prevent zs such cell attachment include, but are not limited to, agarose, glass, untreated cell culture plastic and polytctrafluorocthylcnc, i.c., Tcflon® Untreated cell culture plastics, 1. e., plastics that have not been treated with or made from materials that have an electrostatic charge arc conuncrcially available, and may be purchased, for example, from Falcon Labware, Becton-Dickinson, Lincoln Park, N.J. 'fhe aforementioned materials, however, 3U are not meant to be limiting. It is appreciated that any other pliable or moldable tt'l'AU L /207I~ 166 - I 5- AClomey Uockct No. 5 750-12 1v u. a a a a p W . i a a ~ a bioeompatible material that inherently discourages the attachment of chondrogenic cells may be useful in the practice of the instant invention.
T he size and shape of the wet) may be determined by the size rind shape of the articular cartilage defect to be repaired. For example, it is contemplated that the well may have a cross-sectional surface area of 25 cm2. This is the average cross-sectional surface area of an adult, human femoral ehondyle. Accordingly, il is anticipated that a single piece of synthetic cartilage may be prepared in accordance with the invention in order to resurface the entire femoral chondyle. The depth of the well is preferably greater than about 0.3 cm and preferably about 0.6 cm in depth. The thickness of natural I 0 arlicular cartilage in an adult articulating joint is usually about 0.3 cm. Accordingly, the depth of the well should be large enough to permit a cartilage patch of about 0.3 cm to form. However, the well should also be deep enough to contain growth medium overlaying lhc cartilage patch.
>'t is contemplated also that a large piece of cartilage prepared in accordance with 15 the invention may be "trimmed" to a pre-selected size and shape by a surgeon performing surgical repair of the damaged earlilr~ge. Trimming may be performed with the use of a sharp cutting implement, i.e., a scalpel, a pair of scissors or an arthroscopic device fatted with a cutting edge, using procedures well known in the art.
The pre-shaped well preferably is cast its a block of agarose gel utt.der aseptic 2n conditions. Agaroae is an economical, biocompatible, pliable and moldable material that can be used co cast pre-shaped wells, duickly and easily. As rrtentioned above, the dimensions of the well may dependent upon the size of the resulting cartilage plug that is desired.
A pre-shaped well may be prepared by pouring a hat solution of molten LT
zs agarose (l3ioRad, Richmond, CA) into a tissue culture dish containing a cylinder. The cylinder having dimensions that minor the shape of the well to be formed. The size and shape of the well may be chosen by the artisan and may be dependent upon the shape of the articular cartilage defect to be repaired. Once the agarose has cooled and solidified around the cylinder, the cylinder is carefully removed with forceps. The surface or the tissue culture dish that is cxpo5cd by vhe removal of the cylinder is covered with molten agarose. This seals the bottom of the well and provides a cell. adhesive surface at the base RTAOlIZ07614G -16- Attorney Docket No. 5750-12 1VV~ AV VVt~~~~~ ~u~yy oTthe well. When the newly added molten LT agarose cools and solidifies, the resulting pre-shaped well is suitable for culturing, and stimulating the redifferentiation of proliferated cJtondrogonic colts. It is appreciated, however, t113t alternative methods may be used to prepare a pre-shaped well useful in the practice of the invention.
B. Growth of Cartilage Patch.
Proliferated chondrogenic cells in suspension may be seeded into and cultured in the pre-shaped well. The cells may be diluted by the addition of cell culture medium >;o a cell density of about 1 x 105 to 1 x 109 ohondrogenio oells per ml. A
preferred cell culture to medium comprises DMEM supplemented with 10% fetal bovine serum.
Within about four hours of seeding the chondrogenic cells into the well, the cells may coalesce to form a cohesive plug of cells. After about 4-10 days, the cel)s will start to secrete cartilage-specific sulfated proteoglycaos and type lI collagen.
After prolonged periods of time in culture the collagen expressed by the chondrogenic cells in the well 15 will be predominantly type II collagen. It is contemplated however, chef the cohesive plug of cells formed within four hours mtty be removed from the well and surgically iraplanted into the cartilage defect. It is anlicipattJ that the undiffcrenliatcd chondrogenic cells subsequently tray redifferentiate in situ thereby to form synthetic cartilage within the joint.
2o It is contemplated that chondrocytic differentiation or stimulatory factors may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the produotion of artioular oarlilage specific proteoglycans and/or collagen (Luyten & Iteddi (1992) in "Biological Regulation of the Chondrocytes". CRC Press. Boca Raton, Ann Arbor, London, and Tokyo, p.p. 227-236). Preferred growth factors include, hut are not zs limited to transforming growth factor-p (TGr-(i), insulin-like growth factor (IGF), platelet derived growth factor (PDGr), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor, (IiGF) keralinocyle growth factor (KGF), the bone morphogenic factors (BIvIPy) i.c., DMP-1, BMP-2, 13MP-3, EiMP-4, EMP-5 and BMP-6 and the ostengenic proteins [OPs), 3o i.e. OP-1, OP-2 and OP-3. 1'referrcd concentrations of ~'fGl=-~, lCi'F, fllGf, EGh, al~I3F, RTA01 /30761 G<, -t7~ Attorney Docket No, 5750-I2 1VV. AV VV lllv./ n.~ Vv ' CA 02316413 2000-08-18 bFHF, HGF, and KGF, range from about 1 to 100 ng/ml. Preferred eonoentrations of the BMP's and OP's range from about 1 to about 500 ng/ml.
however, these particular growth factors are not limiting. Any polypeptide growth factor capable of'stimulating or inducing the production of cartilage specific proteoglycans and collagen may be useful in the practice ofthe instant invention.
rn addition, it is contemplated that ascorbate may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and collagen. Preferred concentrations of ascorbate range from afoul 1 to about 1000 pg/ml.
Example 3: Surgical Repair of Articular cartilage Dcfcct Cartilage defectc in mammals are readily identl~.able visually during anhroscopic examination or during open surgery of the joint. Cartilage defects may also be identified 1 s inferentially by using computer aided tomography (CAT scanning), X-ray examination, magnetic resonance imaging (MRI), analysis of synovial fluid or serum markers or by any other procedures known in the art. 'treatment of the defects can be effected during an arthroscopic or open surgical procedure using the methods and compositions disclosed herein.
2o Accordingly, once the defect has been idcntif'icd, the defect may be treated by the following steps of (1) surgically implanting at the pre-determined site, a piece of synthetic articular cartilage prepared by the methodologies described herein, and (2) permitting the synthetic a~ticular cartilage to integrate into pre-determined site.
The synthetic cartilage patch optimally has a size and shape such that when the 25 patch is implanted into the defect, the edges of the implanted tissue contact directly the edges of the defect. In addition, the synthetic cartilage patch may be fixed in placed during the surgical procedure. This can be effected by surgically faing the patch info the defect with biodegradable sutures, i.e., (Ethicon, Johnson & Johnson) andlor by applying iriclu~e~~ui'are riottiirit~e8 Ior°r~rin-lhu°rt~'in ~A~'~
St~'ni~a~rP~O LnOrs"e~"~s~'~0~f~c. t'it1 i~~"f~~_ No. 2 4~4~8 900; Pr. Pat. No. 2 448 90l and LP.S.N. 88401961.3 and synthetic RTAOt~207GLGG -18- Attorney Docket No. 5750-t~
1 V V ~ A V V V 1 . m ~ n n. V V a bioadhesives similar to those disclosed in U.S. Pat. No. 5,197,97. It is contemplated, however, that alternative types of sutures and biocompatible glues may be useful in the practice of the invention In some instances, damaged articuiar cartilage maybe surgically excised prior the s to implantation of the patch of synthetic cartilage. Additionally, the adhesion of the smthetic cartilage patch to the articular cartilage defect may be enhanced by treating the defect with transglutaminase (Ichinose et al. (1990) J. Biol. Chem.
265(3);13411-13414;
Najjar e~ al. (1984) in "Transglutaminascs", Boston, Martinuse-Nijhoffj.
Initially, the cartilage defect is dried, for example by using cottonoi:d, and filled with a solution of to transglutaminasc. The solution is subsequently removed, for example, by aspiration, leaving a film containing transglutaminase upon the cartilage, The synthetic cartilage patch is implanted subsequently into the defect by the methods described above.
In addition the synthetic cartilage may be useful in the repair of human articular cartilage defecis_ Accordingly, chondrogenic cells may be differentiated from human 1 s adipose tissue-derived stromal cells, i.e, human subcutaneous adipose tissue.
Surgical procedures for effecting the repair of articular cartilage defects are well lcnown in the art. See for example: Luyten & Reddi (1992) in "Biological Regulation of the Chondrocytes", CRC Press, Hoca Raton, Ann Arbor, London, & Tokyo, p.p. 227-236, the disclosure of which is incorporated by reference herein.
20 'Che above demonstrates a culture system in which human adipose tissue-derived slromal cells differentiate into hypcrtrophic chondrocytes. Since all components are defined, the system can be used far studies of the affects of growth factors etc, on the progression of chondrogcncsis. Irr vt~ro systems have been used by us and others to show that these cell populations have osteogenic and adipocytic potential. We demonstrate 25 here that this population has chondrogenic potential. This has clinical applicability for cartilage repair, The invention also provides a process for inducing chondrogencsis in human adipose tissue-derived stromal cells by contacting such cells with a chondroinductive agent en vitro where die atromal cells rue associated in a three dimensionrsl format.
RTA01/2076166 -19- Attorney Docket No. 5'750-12 nuu. ,.~ u~~~,~..~ ..~.~~ CA 02316413 2000-08-18 a The invention also provides a prooess for using in vitro differentiated chondrocytes from adipose-derived stromal cells in the zepair of cartilage tissue in mammals, including humans.
In the above methods, the siromal cells are preferably isolated, culture expanded s human adipose tissue-derived stromal cells in a chemically defined environment and are condensed into close proximity, such as in the form of a three dimensional cell mass, e.g.
packed cells or a centrifugal cell pellet. Further, the contacting preferably comprises culturing a pellet of human adipose tissue-derived strornal cells in a chemically defined medium which comprises Dlvi~M with 10% serum, 50 nglml ascorbatc-2-phosphate, 10'' M dexantethasone. The differentiated cells are then introduced into the surgery site to repair cartilage. Since all components of the system are defined, the system can be used as a product for cartilage repair in mammals, including man and horses.
RTA01/2o761 GG -20- Accomcy Docket No. 5750-12
Claims (18)
1, A medium for differentiating adipose tissue derived stromal cells into chondrocyte cells, comprising: a chemically defined culture medium having or supplemented with (i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype (ii) an antibiotic (iii) a nutrient supplement such as 1-20% fetal bovine serum or 1-20% horse scrum or any other biological or synthetic equivalent combination of proteins (iv) ascorbate or related vitamin C analogue and (v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorlicoid receptor.
2. The medium of claim 1, wherein the chondroinductive agent or stromal cell mitogen is selected individually or in combination from the group consisting of: a glucocortieoid; a member of the transforming growth factor-beta superfamily; a collagenous extracellular matrix molecule; and a vitamin A analog.
3. The medium of claim 1, wherein said antibiotic is penicillin.
4. The medium of claim 1, wherein said antibiotic is streptomycin.
5. The medium of claim 3, wherein the concentration of penicillin is about to about 200 units per ml.
6. The medium of claim 4, wherein the concentration of streptomycin is about 10 to about 200µg per ml.
7. The medium of claim 2, wherein the glucocoritcoid is hydrocortisone.
8. The medium of claim 2, wherein said glucocorticoid is dexamethasone.
9. The medium of claim 8, wherein the concentration of dexamethasone is about 1 to about 100 nM.
10. The medium of claim 7, wherein the concentration of hydrocortisone is about 1 to about 100 nM.
11. The medium of claim 2, wherein said transforming growth factor-beta is selected from the group consisting of: bone morphogenic protein-2, bone morphogenic protein-4, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, IGF, PDGF, EGF, aFBF, bFBF, HGF, KGF, inhibin A, and chondrogenic stimulating factor.
12. The medium of claim 11, wherein the concentration of transforming growth factor-beta is about 1 to about 100 ng per ml.
13. The method according to claim 11, further comprising TGF.beta.-1 at concentrations from about 1 ng/ml to about 10 ng/ml.
14. The medium of claim 2, wherein the collagenous extracellular matrix molecule is collagen 1.
15. The medium of claim 2, wherein the vitamin A analog is retinoic acid.
16. The medium of claim 15, wherein the concentration of retinoic acid is about 0.1 ng per ml to about 1 µg per ml.
17. A method for differentiating adipose tissue derived stromal cells into chandrocytic cells, comprising:
a) pelleting said stromal cells by centrifuging between 50,000 to 5 million cells at 500 X g for 2 to 20 minutes in sterile tubes containing a medium such as Dulbecco's Modified Eagle's Medium (DMEM) or alpha modified Minimal Essential Medium (.alpha.MEM) or Roswell Park Memorial Institute media 1640 (RPMI Media 1640;
b) plating isolated stromal cells at a density of 500 to 20,000 cells/cm2 in a differentiating medium;
c) supplementing said medium with.
(i) a chondroinductive agent capable of activating any cellular signal transduction pathway leading to the mature chondrocyte phenotype (ii) an antibiotic (iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins (iv) ascorbate or related vitamin C analog (v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and d) incubating said cells at about 31°C to 37°C for about 3-4 weeks in with 5% CO2 and between 1% and 20% oxygen.
a) pelleting said stromal cells by centrifuging between 50,000 to 5 million cells at 500 X g for 2 to 20 minutes in sterile tubes containing a medium such as Dulbecco's Modified Eagle's Medium (DMEM) or alpha modified Minimal Essential Medium (.alpha.MEM) or Roswell Park Memorial Institute media 1640 (RPMI Media 1640;
b) plating isolated stromal cells at a density of 500 to 20,000 cells/cm2 in a differentiating medium;
c) supplementing said medium with.
(i) a chondroinductive agent capable of activating any cellular signal transduction pathway leading to the mature chondrocyte phenotype (ii) an antibiotic (iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins (iv) ascorbate or related vitamin C analog (v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and d) incubating said cells at about 31°C to 37°C for about 3-4 weeks in with 5% CO2 and between 1% and 20% oxygen.
18. A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising;
a) suspending stromal cells at a concentration of 0.5 to 10 million cells per ml in calcium alginate or any other biocompatible lattice or matrix of supporting chondrogenesis in a three-dimensional configuration;
b) transferring cells to 35 mm culture dishes and plating cells at a density of 500 to 20,000 cells/cm2 in a differentiating medium comprising a chemically defined culture medium having or supplemented with:
(i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype;
(ii) an antibiotic;
(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;
(iv) ascorbate or related vitamin C analog;
(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and c) incubating said cells at about 31 to 37°C for about 3-4 weeks in an incubator with 5% CO2 and between 1% and 20% oxygen.
a) suspending stromal cells at a concentration of 0.5 to 10 million cells per ml in calcium alginate or any other biocompatible lattice or matrix of supporting chondrogenesis in a three-dimensional configuration;
b) transferring cells to 35 mm culture dishes and plating cells at a density of 500 to 20,000 cells/cm2 in a differentiating medium comprising a chemically defined culture medium having or supplemented with:
(i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype;
(ii) an antibiotic;
(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;
(iv) ascorbate or related vitamin C analog;
(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and c) incubating said cells at about 31 to 37°C for about 3-4 weeks in an incubator with 5% CO2 and between 1% and 20% oxygen.
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US20050112761A1 (en) | 2005-05-26 |
ATE330000T1 (en) | 2006-07-15 |
TW200516149A (en) | 2005-05-16 |
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US6841150B2 (en) | 2005-01-11 |
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TWI283707B (en) | 2007-07-11 |
US7033587B2 (en) | 2006-04-25 |
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US8911994B2 (en) | 2014-12-16 |
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