Vergleichender Überblick über Verfahren zur Kultivierung artikulärer Chondrozyten

Zusammenfassung

Studienziel: In-vitro-Techniken für artikuläre Chondrozyten dienen nicht nur dem Studium pathophysiologischer Vorgänge sowie der Einflüsse von Mediatoren und Medikamenten zur Arthrose- bzw. Arthritisbehandlung, sondern auch der Herstellung von Knorpelersatzgewebe zur Defektauffüllung in vivo. Diese Arbeit soll einerseits einen Überblick über die Grundlagen verschiedener Techniken zur Kultivierung von Gelenkknorpelzellen geben, andererseits die spezifischen Anforderungen (phänotypische Stabilität mit Synthese von Aggrekan und Typ-II-Kollagen, fehlender direkter Zell-Zell-Kontakt, geringe Proliferationsneigung, geringer Umsatz der Matrixmoleküle) an solche Systeme aufzeigen. Methode: Anhand einer „Medline”-gestützten Recherche wurden die gegenwärtig verwendeten Techniken der Knorpelzellkultur und deren Entwicklung identifiziert. Die Bewertung erfolgte aufgrund eigener Erfahrungen und der in der Literatur angegebenen Daten. Ergebnisse: Es lassen sich zwei- und dreidimensionale Techniken zur Kultivierung von Gelenkknorpelzellen unterscheiden. Zweidimensionale (Monolayer-)Kulturen fördern insbesondere die Proliferation der Knorpelzellen, führen aber gleichzeitig innerhalb von Tagen zu einem Verlust des spezifischen Phänotyps artikulärer Chondrozyten. Dreidimensionale Anordnungen (z. B. Organ-, Alginat-, Agarosekulturen) gewährleisten nicht nur den Erhalt des Phänotyps über Monate, sondern erlauben (in Monolayer) dedifferenzierten artikulären Chondrozyten nach dem Transfer in eine dreidimensionale Umgebung die Wiederherstellung des ursprünglichen Phänotyps. Schlussfolgerung: Die Wahl der jeweiligen Methodik zur Kultivierung artikulärer Chondrozyten sollte an die zu beantwortende Fragestellung angepasst werden.

Abstract

Aim: In-vitro techniques for articular chondrocytes allow the analysis of their metabolism in the presence and absence of mediators or drugs against osteoarthritis or rheumatoid arthritis, as well as the synthesis of de-novo cartilage tissue for implantation into articular defects in vivo. This review aims to give an overview about the basics of different methods of cultivation of articular chondrocytes and about several specific demands (e.g., phenotypical stability with synthesis of aggrecan and type-II collagen, no cell-to-cell contact, low proliferation rates, low matrix molecule turn-over) to such methods. Method: Current techniques for the cultivation of articular chondrocytes and their development were identified via “medline”. Their evaluation was based on our own experience and on data from the literature. Results: Two- and three-dimensional culture systems are employed to maintain articular chondrocytes in vitro. Two-dimensional cultures (monolayer) support the proliferation of articular chondrocytes, but lead to a de-differentiation to fibroblast-like cells. Three-dimensional set-ups (e.g., organ, alginate, agarose cultures) not only maintain the articular cartilage phenotype, but they also support the re-differentiation of de-differentiated chondrocytes. Conclusion: The choice of a culture system for in-vitro studies with articular chondrocytes should be adapted to the question asked.

Artikuläre Chondrozyten - Gelenkknorpel - in vitro - Kultur - Phänotyp

Articular chondrocytes - articular cartilage - in-vitro - tissue culture - phenotype

Literatur

• 1 Adolphe M, Benoit B. Culture de chondrocytes articulaires humains. Interet en pharmacotoxicologie.  Ann Pharm Fr. 1994;  52 177-183
• 2 Adolphe M, Benya P. Different types of cultured chondrocytes - the in vitro approach to the study of biological regulation. In: M. Adolphe (Hrsg) Biological regulation of the chondrocytes. Ann Arbor, Michigan; CRC Press 1992: 105-139
• 3 Adolphe M, Thenet S. Le concept d’immortalite cellulaire, un mythe ou une realiter Exemple de chondrocytes articulaires „immortalises”.  Bull Acad Natl Med. 1990;  174 139-144 (discussion 144 - 146)
• 4 Aigner J, Tegeler J, Hutzler P, Campoccia D, Pavesio A, Hammer C, Kastenbauer E, Naumann A. Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester.  J Biomed Mater Res. 1998;  42 172-181
• 5 Archer C W, McDowell J, Bayliss M, Stephens M, Bentley G. Phenotypic modulation in sub-populations of human articular chondrocytes in vitro.  J Cell Sci. 1990;  97 361-371
• 6 Aulthouse A L, Beck M, Griffey E, Sanford J, Arden K, Machado M A, Horton W A. Expression of the chondrocyte phenotype in vitro.  In Vitro Cell Dev Biol. 1989;  25 659-668
• 7 Aydelotte M B, Greenhill R R, Kuettner K E. Differences between subpopulations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism.  Connect Tissue Res. 1988;  18 223-234
• 8 Aydelotte M B, Kuettner K E. Differences between subpopulations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production.  Connect Tissue Res. 1988;  18 205-222
• 9 Aydelotte M B, Schumacher B L, Kuettner K E. Heterogeneity of articular chondrocytes. In: K. E. Kuettner, R. Schleyerbach, J. G. Peyron, V. C. Hascall (Hrsg) Articular cartilage and osteoarthritis. New York; Raven Press 1992: 237-249
• 10 Aydelotte M S, Thonar E J-MA, Mollenhauer J, Flechtenmacher J. Culture of chondrocytes in alginate gel: Variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes.  In vitro Cell Dev Biol. 1998;  34 123-130
• 11 Baker T L, Goodwin T J. Three-dimensional culture of bovine chondrocytes in rotating-wall vessels.  In vitro Cell Dev Biol Anaimal. 1997;  33 358-365
• 12 Bassleer C, Gysen P, Foidart J M, Bassleer R, Frenchimont P. Human chondrocytes in tridimensional culture.  In Vitro Cell Dev Biol. 1986;  22 113-119
• 13 Bayliss M T, Venn M, Maroudas A, Ali S Y. Structure of proteoglycan from different layers of human articular cartilage.  Biochem J. 1983;  209 387-400
• 14 Benya P D, Nimni M E. The stability of the collagen phenotype during stimulated collagen, glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures.  Arch Biochem Biophys. 1979;  192 327-335
• 15 Benya P D, Padilla S R, Nimni M E. Independent regulation of collagen types of chondrocytes during the loss of differentiated function in culture.  Cell. 1978;  15 313-1321
• 16 Benya P D, Shaffer J D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.  Cell. 1982;  30 215-224
• 17 Binette F, McQuaid D P, Haudenschild D R, Yaeger P C, McPherson J M, Tubo R. Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro.  J Orthop Res. 1998;  16 207-216
• 18 Bonaventure J, Kadhom N, Cohen-Solal L, Ng K H, Bourguignon J, Lasselin C, Freisinger P. Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads.  Exp Cell Res. 1994;  212 97-104
• 19 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte Transplantation.  N Engl J Med. 1994;  331 889-895
• 20 Cao Y, Rodriguez A, Vacanti M, Ibarra C, Arevalo C, Vacanti C A. Comparative study of the use of poly(glycolic acid), calcium alginate and pluronics in the engineering of autologous porcine cartilage.  J Biomater Sci Polym Ed. 1998;  9 475-487
• 21 Carrel A. The preservation of tissues and its applications in surgery. (JAMA 1912; 59 : 523ff).  „The Classic” Clin Orthop. 1992;  278 2-8
• 22 Chandrasekhar S, Esterman M A, Hoffman H A. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride.  Anal Biochem. 1987;  161 103-108
• 23 Chang J, Poole C A. Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel.  Histochem J. 1997;  29 515-528
• 24 Chubinskaya S, Huch K, Mikecz K, Cs-Szabo G, Hasty K A, Kuettner K E, Cole A A. Chondrocyte MMP-8: Upregulation of neutrophil collagenase by interleukin-1β in human cartilage from knee and ankle joints.  Lab Invest. 1996;  74 232-240
• 25 Cima L G, Vacanti J P, Vacanti C, Ingber D, Mooney D, Langer R. Tissue engineering by cell transplantation using degradable polymer substrates.  J Biomech Eng. 1991;  113 143-151
• 26 D’Andrea P, Calabrese A, Grandolfo M. Intercellular calcium signaling between chondrocytes and synovial cells in co-culture.  Biochem J. 1998;  329 681-687
• 27 Delbrück A, Dresow B, Gurr E, Reale E, Schröder H. In-vitro culture of human chondrocytes from adult subjects.  Connect Tissue Res. 1986;  15 155-172
• 28 Deshmukh K, Kline W G. Characterization of collagen and its precursors synthesized by rabbit-articular-cartilage cells in various culture systems.  Eur J Biochem. 1976;  69 117-123
• 29 Dessau W, Vertel B M, von der Mark H, von der Mark K. Extracellular matrix formation by chondrocytes in monolayer.  J Cell Biol. 1981;  90 78-83
• 30 Fell H B, Jubb R W. The effect of synovial tissue on the breakdown of articular cartilage in organ culture.  Arthritis Rheum. 1977;  20 1359-1371
• 31 Fell H B, Robison R. The growth, development and phosphatase activity of embryonic avian femora and limb-buds cultivated in vitro.  Biochem J. 1929;  23 767-784
• 32 Fischer A. A pure strain of cartilage cells in vitro.  J Exp Med. 1922;  35 379-384
• 33 Flechtenmacher J, Huch K, Thonar E J-MA, Mollenhauer J, Davies S R, Schmid T M, Puhl W, Sampath T K, Aydelotte M B, Kuettner K E. Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes.  Arthritis Rheum. 1996;  39 1896-1904
• 34 Flint O P. A micromass culture method for rat embryonic neural cells.  J Cell Sci. 1983;  61 247-262
• 35 Freed L E, Martin I, Vunjak-Novakovic G. Frontiers in tissue engineering. In vitro modulation of chondrogenesis.  Clin Orthop. 1999;  367 Suppl 46-58
• 36 Galéra P, Rédini F, Vivien D, Bonaventure J, Penfornis H, Loyau G, Pujol J-P. Effect of transforming growth factor-β1 (TGF-β1) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the differentiation process.  Exp Cell Res. 1992;  200 379-392
• 37 Gionti E, Pontarelli G, Cancedda R. Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes.  Proc Natl Acad Sci USA. 1985;  82 2756-2760
• 38 Goldring M B, Birkhead J R, Suen L-F, Yamin R, Mizuno S, Glowacki J, Arbiser J L. lnterleukin-1β-modulated gene expression in immortalized human chondrocytes.  J Clin Invest. 1994;  94 2307-2316
• 39 Grande D A, Halberstadt C, Noughton G, Schwartz R, Manji R. Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts.  J Biomed Mater Res. 1997;  34 211-220
• 40 Gray M L, Pizzanelli A M, Grodzinsky A J, Lee R C. Mechanical and physicochemical determinants of the chondrocyte biosynthetic response.  J Orthop Res. 1988;  6 777-792
• 41 Green W T. Behavior of articular chondrocytes in cell culture.  Clin Orthop. 1971;  75 248-260
• 42 Gugala Z, Gogolewski S. In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold.  J Biomed Mater Res. 2000;  49 183-191
• 43 Guo J F, Jourdian G W, MacCallum D K. Culture and growth characteristics of chondrocytes encapsulated in alginate beads.  Connect Tissue Res. 1989;  19 277-297
• 44 Hamerman D, Janis R, Smith C. Cartilage matrix depletion by rheumatoid synovial cells in tissue culture.  J Exp Med. 1967;  126 1005-1012
• 45 Haudenschild D R, McPherson J M, Tubo R, Binette F. Differential expression of multipie genes during articular chondrocyte redifferentiation.  Anat Rec. 2001;  263 91-98
• 46 Häuselmann H J, Aydelotte M B, Schumacher B L, Kuettner K E, Gitelis S H, Thonar E J-MA. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads.  Matrix. 1992;  12 116-129
• 47 Häuselmann H J, Fernandes R J, Mok S S, Schmid T M, Block J A, Aydelotte M B, Kuettner K E, Thonar E J-MA. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads.  J Cell Sci. 1994;  107 17-27
• 48 Häuselmann H J, Masuda K, Hunziker E B, Neidhart M, Mok S S, Michel B A, Thonar E J. Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage.  Am J Physiol. 1996;  271 C742-752
• 49 Holtzer H, Abbott J, Lash J, Holtzer S. The loss of phenotypic traits by differentiated cells in vitro. I. Dedifferentiation of cartilage cells.  Proc Nat Acad Sci. 1960;  46 1533-1542
• 50 Horwitz A L, Dorfman A. The growth of cartilage cells in soft agar and liquid suspension.  J Cell Biol. 1970;  45 434-438
• 51 Huch K, Stöve J, Günther K P, Puhl, W. lnteractions between human osteoarthritic chondrocytes and synovial fibroblasts in co-culture.  Clin Exp Rheumatol. 2001;  19 27-33
• 52 Huch K, Wilbrink B, Flechtenmacher J, Koepp H E, Aydelotte M S, Sampath T K, Kuettner K E, Mollenhauer J, Thonar E. Effects of recombinant human osteogenic protein 1 on the production of proteoglycan, prostaglandin E2, and interleukin-1 receptor antagonist by human articular chondrocytes cultured in the presence of interleukin-1β.  Arthritis Rheum. 1997;  40 2157-2161
• 53 Jacoby R K. Effect of homologous synovial membrane on adult human articular cartilage in organ culture and failure to influence it with D-penicillamine.  Ann Rheum Dis. 1980;  39 53-58
• 54 Kawiak J, Moskalewski S, Darzynkiewicz Z. Isolation of chondrocytes from calf cartilage.  Exp Cell Res. 1965;  39 59-68
• 55 Keiser H D, Malemud C J. A comparison of the proteoglycans produced by rabbit articular chondrocytes in monolayer and spinner culture and those of bovine nasal cartilage.  Conn Tiss Res. 1983;  11 273-284
• 56 Kennedy R D, Plater-Zyberk C, Partridge T A, Woodrow D F, Muir R H. Morphometric comparison of synovium from patients with osteoarthritis and rheumatoid arthritis.  J Clin Pathol. 1988;  41 847-852
• 57 Kuettner K E, Memoli V A, Pauli B U, Wrobel N C, Thonar E J-MA, Daniel J C. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.  J Cell Biol. 1982;  93 751-757
• 58 Kuettner K E, Pauli B U, Gall G, Memoli V A, Schenk R K. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.  J Cell Biol. 1982;  93 743-750
• 59 Kupchik H Z, Langer R S, Haberern C, EI-Deriny S, O’Brien M. A new method for the three-dimensional in vitro growth of human cancer cells.  Exp Cell Res. 1983;  147 454-460
• 60 Kuroda Y. Studies on cartilage cells in vitro. II. Changes in aggregation and in cartilage-forming activity of cells maintained in monolayer-cultures.  Exp Cell Res. 1964;  35 337-348
• 61 Kurz B, Steinhagen J, Schünke M. Articular chondrocytes and synovialcytes in a co-culture system: influence on reactive oxygen species-induced cytotoxicity and lipid peroxidation.  Cell Tissue Res. 1999;  296 555- 563
• 62 Lemare F, Steimberg N, Le Griel C, Demignot S, Adolphe M. Dedifferentiated chondrocytes cultured in alginate beads: restoration of the dedifferentiated phenotype and of the metabolic responses to interleukin-1β.  J Cell Physiol. 1998;  176 303-313
• 63 Liu H, Lee Y-W, Dean M F. Re-expression of differentiated proteoglycan phenotype ba dedifferentiated human chondrocytes during culture in alginate beads.  Biochim Biophys Acta. 1998;  1425 505-515
• 64 Luyten F P, Chen P, Paralkar V, Reddi A H. Recombinant bone morphogenetic protein-4, transforming growth factor-β₁, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro.  Exp Cell Res. 1994;  210 224-229
• 65 Malemud C J, Norby D P, Soskoloff L. Explant culture of human and rabbit articular chondrocytes.  Connect Tissue Res. 1978;  6 171-179
• 66 Manning W K, Bonner W M. Isolation and culture of chondrocytes from human adult articular cartilage.  Arthritis Rheum. 1967;  10 235-239
• 67 Maroudas A, Bayliss M T, Uchitel-Kaushansky N, Schneiderman R, Gilav E. Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age.  Arch Biochem Biophys. 1998;  350 61-71
• 68 Maroudas A, Palla G, Gilav E. Racemization of aspartic acid in human articular cartilage.  Connect Tissue Res. 1992;  28 161-169
• 69 Maroudas A. Glycosaminoglycan turn-over in articular cartilage.  Philos Trans R Soc Lond B Biol Sci. 1975;  271 (912) 293-313
• 70 Melching L I, Roughley P J. Modulation of keratan sulfate synthesis on lumican by the action of cytokines on human articular chondrocytes.  Matrix Biol. 1999;  18 381-390
• 71 Mok S S, Masuda K, Häuselmann H J, Aydelotte M B, Thonar E J. Aggrecan synthesized by mature bovine chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools.  J Biol Chem. 1994;  269 33 021-33 027
• 72 Moscona A. The development in vitro of chimeric aggregates of dissociated embryonic chick and mouse cells.  Proc Nat Acad Sci. 1957;  43 184-194
• 73 Moscona H, Moscona A. The dissociation and aggregation of cells from organ rudiments of the early chick embryo.  J Anat. 1952;  86 287-301
• 74 Nevo Z, Silver J, Chorev Y, Riklis I, Robinson D, Yosipovitch Z. Adhesion characteristics of chondrocytes cultured separately and in co-cultures with synovial fibroblasts.  Cell Biol Intern. 1993;  17 255-273
• 75 Norby D P, Malemud C J, Sokoloff L. Differences in the collagen types synthesized by lapine articular chondrocytes in spinner and monolayer culture.  Arthritis Rheum. 1977;  20 709-716
• 76 Panagides J, Landes M J, Sloboda A E. Destruction of articular cartilage by arthritic synovium in vitro: mechanism of breakdown and effect of indomethacin and prednisolone.  Agents Actions. 1980;  10 22-30
• 77 Pazzano D, Mercier K A, Moran J M, Fong S S, DiBiasio D O, Rulfs J X, Kohles S S, Bonassar L J. Comparison of chondrogenesis in static and perfused bioreactor culture.  Biotechnol Prog. 2000;  16 893-896
• 78 Petit B, Masuda K, Dscuza A L, Otten L, Pietryla D, Hartmann D J, Morris N P, Übelhart D, Schmid T M, Thonar E J. Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct compartments.  Exp Cell Res. 1996;  225 151-161
• 79 Prudden T M. Experimental studies on the Transplantation of cartilage.  Am J Med Sci. 1881;  164 360-370
• 80 Pamachandrula A, Tiku K, Tiku M L. Tripeptide RGD-dependent adhesion of articular chondrocytes to synovial fibroblasts.  J Cell Sci. 1992;  101 859-871
• 81 Reddi A H. Role of morphogenetic proteins in skeletal tissue engineering and regeneration.  Nat Biotechnol. 1998;  16 247-252
• 82 Revell P A, Mayston V, Lalor P, Mapp P. The synovial membran in osteoarthritis: a histological study including the characterization of the cellular infiltrate present in inflammatory osteoarthritis using monoclonal antibodies.  Ann Rheum Dis. 1988;  47 300-307
• 83 Rinaldini L MJ. The isolation of living cells from animal tissues.  Int Review Cytology. 1959;  7 587-647
• 84 Robbins J R, Thomas B, Tan L, Choy B, Arbiser J L, Berenbaum F, Goldring M B. Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1beta.  Arthritis Rheum. 2000;  43 2189-2201
• 85 Sah R L-Y, Grodzinsky A J, Plaas A HK, Sandy J D. Effects of static and dynamic compression on matrix metabolism in cartilage implants. In: K. E. Kuettner, R. Schleyerbach, J. G. Peyron, V. C. Hascall (Hrsg) Articular cartilage and osteoarthritis. New York; Raven Press 1992: 373-391
• 86 Sailor L Z, Hewick R M, Morris E A. Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture.  J Orthop Res. 1996;  14 937-945
• 87 Schultz O, Keyszer G, Zacher J, Sittinger M, Burmester G R. Development of in vitro model systems for destructive joint diseases: novel strategies for establishing inflammatory pannus.  Arthritis Rheum. 1997;  40 1420-1428
• 88 Schwartz E R, Kirkpatrick P R, Thompson R C. Sulfate metabolism in human chondrocyte cultures.  J Clin Invest. 1974;  54 1056-1063
• 89 Sittinger M, Perka C, Schultz O, Häupl T, Burmester G R. Joint cartilage regeneration by tissue engineering.  Z Rheumatol. 1999;  58 130-135
• 90 Smith A U. Survival of frozen chondrocytes isolated from cartilage of adult mammals.  Nature. 1965;  205 782-784
• 91 Srivastava V M, Malemud C J, Sokoloff L. Chondroid expression by lapine articular chondrocytes in spinner culture following monolayer growth.  Connect Tissue Res. 1974;  2 127-136
• 92 Strangeways T SP. Observations on the nutrition of articular cartilage.  Br Med J. 1920;  661-663
• 93 Thonar E J, Buckwalter J A, Kuettner K E. Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage.  J Biol Chem. 1986;  261 2467-2474
• 94 Tyler J A. Chondrocyte mediated depletion of articular cartilage proteoglycans in vitro.  Biochem J. 1985;  260 493-507
• 95 Van Kampen G PJ, van de Stadt R J, van de Laar M AFJ, van der Korst J K. Two distinct matabolic pools of proteoglycans in articular cartilage. In: K. E. Kuettner, R. Schleyerbach, J. G. Peyron, V. C. Hascall (Hrsg) Articular cartilage and osteoarthritis. New York; Raven Press 1992: 281-290
• 96 Von der Mark K. Differentiation, modulation and dedifferentiation of chondrocytes. In: Rheumatology. Vol. 10 Basel; Karger 1986: 272-315
• 97 Willmer E N. Introduction. In: E. N. Willmer Cells and tissues in culture. Methods, biology and physiology. London, New York; Academic Press 1965: 1-17
• 98 Wolter J R, Meyer R F. Sessile macrophages forming clear endothelium-like membrane on inside of successful keratoprosthesis.  Trans Am Ophthalmol Soc. 1984;  82 187-82 202
• 99 Worster A A, Brower-Toland B D, Fortier L A, Bent S J, Williams J, Nixon A J. Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix.  J Orthop Res. 2001;  19 738-749
• 100 Zaucke F, Dinser R, Maurer P, Paulsson M. Cartilage oligomeric matrix protein (COMP) and collagen IX are sensitive markers for the differentiation state of articular primary chondrocytes.  Biochem J. 2001;  358 17-24