Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system

Authors

1 Department of Anatomical Sciences and Molecular Biology, Medical Faculty, Isfahan University of Medical Sciences, Iran

2 Department of Biophysics, Medical Faculty, Isfahan University of Medical Sciences, Iran

Abstract

Background: The Autologous Chondrocytes Transplantation (ACT) method is being studied for repair of cartilage diseases. As the chondrocytes dedifferentiated during monolayer culture, three-dimensional cultures are suggested to redifferentiate them. The aim of this study was investigation of the effect of TGF-β3 growth factor on chondrocytes in pellet culture system.
Materials and Methods: The chondrocytes were isolated from three human articular cartilages by enzymatic digestion. The cells of the second passage were transferred to pellet culture system. We determined the chondrogenic medium with TGF-β3 as the experimental group and without it as the control group. After 2 weeks, the aggrecan production was investigated using histological and immunohistochemical (IHC) methods.
Results: The presence of glycosaminoglycans was proved through Toluiden blue staining. Comparison of IHC results using MATLAB software showed that aggrecan in the experimental group was significantly higher than in the control group (P ≤ 0.05).
Conclusion: The presence of TGF-β3 in the chondrogenic medium could lead to the production of more aggrecan in chondrocytes cultivated in pellet culture system.

Keywords

1. Stewart MC, Saunders KM, Wurster NB, Macleod JN. Phenotypic stability of articular chondrocytes in vitro: The effects of culture models, bone morphogenetic protein-2, and serum supplementation. JBMR 2000;15:166-74.  Back to cited text no. 1
    
2. Aigner T, Stove J. Collagens-major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair. Adv Drug Deliv Rev 2003;1569-93.  Back to cited text no. 2
    
3. Temenoff JS, Mikos AG. Tissue engineering for regeneration of articular cartilage. Biomaterials 2000;21:431-40.  Back to cited text no. 3
[PUBMED]    
4. Gomez-Camarillo MA, Almonte-Becerril M, Vasquez Tort M, Tapia-Ramirez J, Kouri Flores JB. Chondrocyte proliferation in a new culture system. Cell Prolif 2009;42;207-18.  Back to cited text no. 4
    
5. Hickey DG, Frenkel SR, DiCesare PE. Clinical applications of growth factors for articular cartilage repair. Am J Orthop (Belle Mead NJ) 2003;32:70-6.  Back to cited text no. 5
    
6. Buckwalter JA, Brown TD. Joint injury, repair, and remodeling: Roles in post-traumatic osteoarthritis. Clin Orthop Relat Res 2004;423:7-16.  Back to cited text no. 6
[PUBMED]    
7. Mithofer K, Williams EJ III, Warren RF, Potter HG, Spock CR, Jones EC, et al. The microfracture technique for the treatment of articular cartilage lesions in the knee: A prospective cohort study. J Bone Joint Surg Am 2005;87:1911-20.  Back to cited text no. 7
    
8. Steadman JR, Rodkey WG, Briggs KK. Microfracture to treat full-thickness chondral defects: Surgical technique, rehabilitation, and outcomes. J Knee Surg 2002;15:170-6.  Back to cited text no. 8
[PUBMED]    
9. Sterett WI, Steadman JR. Chondral resurfacing and high tibial osteotomy in the varus knee. AmJ Sports Med 2000;32:1243-9.  Back to cited text no. 9
    
10. Gobbi A, Nunag P, Malinowski K. Treatment of full thicknesschondral lesions of the knee with microfracture in a group of athletes. Knee Surg Sports Traumatol Arthrosc 2005;13:213-21.  Back to cited text no. 10
[PUBMED]    
11. Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. The reactions of articular cartilage to experimental wounding: Role of apoptosis. Arthraitis Rheum 2000;43:215-25.  Back to cited text no. 11
    
12. Hering T, Kollar J, Huynh TD, Varelas JB, Sandell LJ. Modulation of extracellular matrix gene expression in bovine high density chondrocyte cultures by ascorbic acid and enzymatic resuspension. Arch Biochem Biophys 1994;314:90-8.  Back to cited text no. 12
    
13. Lefebvre V, Garofalo S, Zhou G, Metsaranta M, Vuorio E, De Crombrugghe B. Characterization of primary cultures of chondrocyte from type 2 collagen/β-galactosidase transgenic mice. Matrix Biol 1994;14:329-35.  Back to cited text no. 13
    
14. Hiraki Y, Inoue H, Shigeno C, Sanma Y, Bentz H, Rosen DM, et al.Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of differentiated phenotype of rabbit chondrocyte and osteoblastic MC3T3-E1 cells invitro. J Bone Miner Res 1994;12:1373-85.  Back to cited text no. 14
    
15. Luyten FP, Chen P, Paralkar V, Reddi AH. Recombinant human bone morphogenetic protein -4, transforming growth factor beta-1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp Cell Res 1994;210:224-9.  Back to cited text no. 15
[PUBMED]    
16. Luyten FP, Yu YM, Yanagishita M, Vukicivic S, Hammonds RG, Reddi AH. Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explants cultures. J Biol Chem 1992;267:3691-95.  Back to cited text no. 16
    
17. Sailor LZ, Wang JH, Morris EA, Hewick RH. Bone morphogenetic protein-2 (BMP-2) maintains the phenotype of articular chondrocytes in long term monolayer culture. J Orthop Res 1996;14:937-45.  Back to cited text no. 17
    
18. Guo J, Jourdian GW, McCallum DK. Culture and growth characteristics of chondrocyes encapsulated in alginate beads. Connect Tissue Res 1989;19:277-97.  Back to cited text no. 18
    
19. Hauselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA, et al. Adult human chondrocyte cultured in alginate form a matrix similar to native human articular cartilage. Am J Physiol 1996;271:C742-52.  Back to cited text no. 19
    
20. Lemare F, Steimberg N, Le Greil C, Demignot S, Adolphe M. Differentiated chondrocytes cultured in alginate beads: Restoration of the phenotype and metabolic responses to interlukin-1β. J Cell Physiol 1998;176:303-13.  Back to cited text no. 20
    
21. Banu N, Tsuchiya T. Markedly different effects of hyaluronic acid and chondroitin sulfate-A on the differentiation of human articular chondrocytes in micromass and 3-D honeycomb rotation cultures. J Biomed Mater Res A 2007;80:257-67.  Back to cited text no. 21
[PUBMED]    
22. Rahman MS, Tsuchiya T. Enhancement of chondrogenic differentiation of human articular chondrocytes by biodegradable polymers. Tissue Eng 2001;7:781-90.  Back to cited text no. 22
[PUBMED]    
23. Bradham DM, Horton WE Jr. In vivo cartilage formation from growth factor modulated articular chondrocytes. Clin Orthop Relat Res 1998;352:239-49.  Back to cited text no. 23
[PUBMED]    
24. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. Invitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265-72.  Back to cited text no. 24
[PUBMED]    
25. Martin JA, Buckwalter JA. The role of chondrocyte-matrix interaction in maintaining and repairing articular cartilage. Biorheology 2003;37:129-40.  Back to cited text no. 25
    
26. HashemiBeni B, Razavi SH, Esfandiari E, Karbasi S, Mardani M, Sadeghi F, et al. Effect of transforming growth factor-β3 and bone morphogenetic protein-6 growth factors on chondrogenic differentiation of adipose-derived stem cells in alginate scaffold. J Isfahan Med Sch 2010;28;607-20.  Back to cited text no. 26
    
27. Hahemibeni B, Razavi SH, Esfandiary E, Karbasi S, Mardani M, Nasresfahani M. Induction of chondrogenic differentiation of human adipose-derived stem cells with TGF-β3 in pellet culture system. Iran J Basic Med Sci 2008;11:10-7.  Back to cited text no. 27
    
28. Ibold Y, Lubke C, Pelz S, Augst H, Kaps C, Ringe J, et al. Effect of different ascorbate supplementations on in vitro high-density pellet cultures. Tissue Cell 2009;41:249-56.  Back to cited text no. 28
    
29. Cheng C, Conte E, Pleshko-Camacho N, Hidaka C. Differences in matrix accumulation and hypertrophy in superficial and deep zone chondrocytes are controlled by bone morphogenetic protein. Matrix Biol 2007;26:541-53.  Back to cited text no. 29
[PUBMED]    
30. Grimaud E, Heymann D, Redini F. Recent advances in TGF-β effects on chondrocyte metabolism. Potetialtrapeutic roles of TGF-β in cartilage disorders. Cytokine Growth Factor Rev 2002;13:241-57.  Back to cited text no. 30
    
31. Fan H, Irrgang JJ, Anderson AF, Boland AL, Horner CD, Kurosako M, et al. Porus gelatin-chondroitin-hyaluronatetricopolymer scaffold containing microspheres loaded with TGF-β1 induces differentiation of mesenchymal stem cells invivo for enhancing cartilage repair. J Biomed Mater Res A 2006;77:785-94.  Back to cited text no. 31
    
32. Van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB. differential effects of local application of BMP-2 or TGF-β 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 1998;6:306-17.  Back to cited text no. 32
[PUBMED]    
33. Yang IH, Kim SH, Kim YH, Sun HJ, Kim SJ, Lee JW. Comparison of phenotypic characterization between alginate bead and pellet culture system as chondrogenic differentiation models for human mesenchymal stem cells. Yonsei Med J 2004;45:891-900.  Back to cited text no. 33
[PUBMED]    
34. Bernstein P, Dong M, Corbeil D, Gelinsky M, Gunther KP, Fickert S. Pellet culture elicits superior chondrogenic redifferentiation than alginate-based systems. Biotechnol Prog 2009;25:1146-52.  Back to cited text no. 34
    
35. Collier S, Ghosh P. Effects of transforming growth factor beta on proteoglycan synthesis by cell and explants cultures derived from the knee joint meniscus. Osteoarthritis Cartilage 1995;3:127-38.  Back to cited text no. 35
[PUBMED]    
36. Pangborn CA, Athanasiou KA. Growth factors and fibrochondrocytes in scaffolds. J Orthop Res 2005;23:1184-90.  Back to cited text no. 36
[PUBMED]    
37. De Hart WJ, Van Osch GJ, Verhaar JA. Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, b FGF and L- ascorbic acid on bovine articular chondrocytes. Acta Orthop Scand 1999;70:55-61.  Back to cited text no. 37
    
38. Gruber HE, Fisher EC Jr, Desaei B, Stasky AA, Hoelscher G, Hanley EN Jr. Human invertebral disc cells from the annulus: Three dimensional culture in agaroseor alginate and responsiveness to TGF-beta 1. Exp Cell Res 1997;235:13-21.  Back to cited text no. 38
    
39. Matsumura T, Whelan MC, Li XQ, Tripple SB. Regulation by IGF-1 of Swarm-rat chondrosarcoma chondrocytes. J Orthop Res 2000;18:351-5.  Back to cited text no. 39
    
40. Nixon AJ, Lillich JT, Burton-Wuster N, Lust G, Mohammed HO. Differential cellular function in fetal chondrocyte s cultured with insulin-like growth factor-1 and transforming growth factor-beta. J Orthop Res 1998;16:531-41.  Back to cited text no. 40
    
41. Boumediene K, Vivien D, Marco M, Bogdanovicz P, Lebrun E, Pujol JP.Modulation of rabbit articular chondrocyte (RAC) proliferation by TGF-beta isoforms. Cell Prolif 1995;28:221-34.  Back to cited text no. 41
    
42. Richmond RS, Carlson CS, Register TC, Shunker G, Loeser RF. Functional esterogen receptors in adult articular cartilage. Arthritis Rheum 2000;43:2081-90.  Back to cited text no. 42
    
43. Ushiyama T, Ueyama H, Inoue K, Ohkubo I, Hukuda S. Expression of genes for esterogen receptors alpha and beta in human articular chondrocytes. Osteoarthritis and Cartilage 1999;7:560-6.  Back to cited text no. 43
[PUBMED]    
44. Van Osch GJ, Van der Veen SW, Buma P, Verwoerd-Verhoef HL. Effects of transforming growth factor-β on proteoglycan synthesis by chondrocytes in relation to differentiation stage and presence of pericellular matrix. Matrix Biol 1998;17:413-24.  Back to cited text no. 44
[PUBMED]    
45. Wright M.TGF-β1 in bovine serum. Art Sci 2001;19:1-3.  Back to cited text no. 45
    
46. Gunja NJ, Uthamanthil RK, Athanasiou KA. Effects of TGF-β1 and hydrostatic pressure on meniscus cell-seeded scaffolds. J Biomater 2009;30:565-73.  Back to cited text no. 46
    
47. Lucas PA, Dziewiatkawski DD. Feedback control of selected biosynthetic activities of chondrocyte in culture. Connect Tissue Res 1987;16:323-41.  Back to cited text no. 47