Tissue Engineering and Regenerative Medicine

, Volume 15, Issue 2, pp 163–172 | Cite as

Chondrogenic Potential of Dedifferentiated Rat Chondrocytes Reevaluated in Two- and Three-Dimensional Culture Conditions

  • Guang-Zhen Jin
  • Hae-Won Kim
Original Article


For the cartilage repair, the cell sources currently adopted are primarily chondrocytes or mesenchymal stem cells (MSCs). Due to the fact that chondrocytes dedifferentiate during 2-dimensional (2D) expansion, MSCs are generally more studied and considered to have higher potential for cartilage repair purposes. Here we question if the dedifferentiated chondrocytes can regain the chondrogenic potential, to find potential applications in cartilage repair. For this we chose chondrocytes at passage 12 (considered to have sufficiently dedifferentiated) and the expression of chondrogenic phenotypes and matrix syntheses were examined over 14 days. In particular, the chondrogenic potential of MSCs was also compared. Results showed that the dedifferentiated chondrocytes proliferated actively over 14 days with almost 2.5-fold increase relative to MSCs. Moreover, the chondrogenic ability of chondrocytes was significantly higher than that of MSCs, as confirmed by the expression of a series of mRNA levels and the production of cartilage extracellular matrix molecules in 2D-monolayer and 3-dimensional (3D)-spheroid cultures. Of note, the significance was higher in 3D-culture than in 2D-culture. Although more studies are needed such as the use of different cell passages and human cell source, and the chondrogenic confirmation under in vivo conditions, this study showing that the dedifferentiated chondrocytes can also be a suitable cell source for the cell-based cartilage repair, as a counterpart of MSCs, will encourage further studies regarding this issue.


Dedifferentiated chondrocytes Mesenchymal stem cells Chondrogenesis Three-dimension 



The present research was conducted by the research fund of Dankook University in 2016.

Compliance with ethical standards

Conflict of interest

Authors (GZJ and HWK) declare no conflict of interests exists.

Ethical statement

The cell isolation from Sprague–Dawley rats was according to the consent from Dankook University Institutional Animal Care and Use Committee (DKU-IRB-2014-039).


  1. 1.
    Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage. 2002;10:432–63.CrossRefPubMedGoogle Scholar
  2. 2.
    Karuppal R. Current concepts in the articular cartilage repair and regeneration. J Orthop. 2017;14:A1–3.CrossRefPubMedGoogle Scholar
  3. 3.
    Park DY, Min BH, Lee HJ, Kim YJ, Choi BH. Repair of partial thickness cartilage defects using cartilage extracellular matrix membrane-based chondrocyte delivery system in human Ex Vivo model. Tissue Eng Regen Med. 2016;13:182–90.CrossRefGoogle Scholar
  4. 4.
    Jang JH, Lee JS, Lee EY, Lee EA, Son YS. Disc-type hyaline cartilage reconstruction using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes and maintenance of its shape and phenotype after transplantation. Tissue Eng Regen Med. 2016;13:352–63.CrossRefGoogle Scholar
  5. 5.
    Yamashita A, Nishikawa S, Rancourt DE. Identification of five developmental processes during chondrogenic differentiation of embryonic stem cells. PLoS One. 2010;5:e10998.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wei Y, Zeng W, Wan R, Wang J, Zhou Q, Qiu S, et al. Chondrogenic differentiation of induced pluripotent stem cells from osteoarthritic chondrocytes in alginate matrix. Eur Cell Mater. 2012;23:1–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Mousavinejad M, Andrews PW, Shoraki EK. Current biosafety considerations in stem cell therapy. Cell J. 2016;18:281–7.PubMedPubMedCentralGoogle Scholar
  8. 8.
    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;331:889–95.CrossRefPubMedGoogle Scholar
  9. 9.
    Kim JM, Han JR, Shetty AA, Kim SJ, Choi NY, Park JS. Comparison between total knee arthroplasty and MCIC (autologous bone marrow mesenchymal-cell-induced-chondrogenesis) for the treatment of osteoarthritis of the knee. Tissue Eng Regen Med. 2014;11:405–13.CrossRefGoogle Scholar
  10. 10.
    do Amaral RJ, Matsiko A, Tomazette MR, Rocha WK, Cordeiro-Spinetti E, Levingstone TJ, et al. Platelet-rich plasma releasate differently stimulates cellular commitment toward the chondrogenic lineage according to concentration. J Tissue Eng. 2015;6:2041731415594127.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Im GI. Tissue engineering for osteochondral defects. Tissue Eng Regen Med. 2008;5:552–8.Google Scholar
  12. 12.
    Sancho-Tello M, Martorell S, Mata Roig M, Milián L, Gámiz-González MA, Gómez Ribelles JL, et al. Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds. J Tissue Eng. 2017;8:2041731417697545.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Browne JE, Anderson AF, Arciero R, Mandelbaum B, Moseley JB Jr, Micheli LJ, et al. Clinical outcome of autologous chondrocyte implantation at 5 years in US subjects. Clin Orthop Relat Res. 2005;436:237–45.CrossRefGoogle Scholar
  14. 14.
    Benya PD, Padilla SR, Nimni ME. Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell. 1978;15:1313–21.CrossRefPubMedGoogle Scholar
  15. 15.
    Lin Z, Fitzgerald JB, Xu J, Willers C, Wood D, Grodzinsky AJ, et al. Gene expression profiles of human chondrocytes during passaged monolayer cultivation. J Orthop Res. 2008;26:1230–7.CrossRefPubMedGoogle Scholar
  16. 16.
    Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C, et al. Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res. 1994;212:97–104.CrossRefPubMedGoogle Scholar
  17. 17.
    Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. 1992;10:745–58.CrossRefPubMedGoogle Scholar
  18. 18.
    Guha Thakurta S, Budhiraja G, Subramanian A. Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis. J Tissue Eng. 2015;6:2041731414566529.PubMedGoogle Scholar
  19. 19.
    Blashki D, Murphy MB, Ferrari M, Simmons PJ, Tasciotti E. Mesenchymal stem cells from cortical bone demonstrate increased clonal incidence, potency, and developmental capacity compared to their bone marrow-derived counterparts. J Tissue Eng. 2016;7:2041731416661196.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lee KB, Hui JH, Song IC, Ardany L, Lee EH. Injectable mesenchymal stem cell therapy for large cartilage defects—a porcine model. Stem Cells. 2007;25:2964–71.CrossRefPubMedGoogle Scholar
  21. 21.
    Emadedin M, Aghdami N, Taghiyar L, Fazeli R, Moghadasali R, Jahangir S, et al. Intra-articular injection of autologous mesenchymal stem cells in six patients with knee osteoarthritis. Arch Iran Med. 2012;15:422–8.PubMedGoogle Scholar
  22. 22.
    Uematsu K, Hattori K, Ishimoto Y, Yamauchi J, Habata T, Takakura Y, et al. Cartilage regeneration using mesenchymal stem cells and a three-dimensional poly-lactic-glycolic acid (PLGA) scaffold. Biomaterials. 2005;26:4273–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Zheng L, Fan HS, Sun J, Chen XN, Wang G, Zhang L, et al. Chondrogenic differentiation of mesenchymal stem cells induced by collagen-based hydrogel: an in vivo study. J Biomed Mater Res A. 2010;93:783–92.PubMedGoogle Scholar
  24. 24.
    Zhou XZ, Leung VY, Dong QR, Cheung KM, Chan D, Lu WW. Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold. Int J Artif Organs. 2008;31:480–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Seo SJ, Mahapatra C, Singh RK, Knowles JC, Kim HW. Strategies for osteochondral repair: focus on scaffolds. J Tissue Eng. 2014;5:2041731414541850.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Freyria AM, Mallein-Gerin F. Chondrocytes or adult stem cells for cartilage repair: the indisputable role of growth factors. Injury. 2012;43:259–65.CrossRefPubMedGoogle Scholar
  27. 27.
    Leijten JC, Emons J, Sticht C, van Gool S, Decker E, Uitterlinden A, et al. Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis. Arthritis Rheum. 2012;64:3302–12.CrossRefPubMedGoogle Scholar
  28. 28.
    Mueller MB, Tuan RS. Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum. 2008;58:1377–88.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Jikko A, Kato Y, Hiranuma H, Fuchihata H. Inhibition of chondrocyte terminal differentiation and matrix calcification by soluble factors released by articular chondrocytes. Calcif Tissue Int. 1999;65:276–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Kang SW, Yoo SP, Kim BS. Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage. Biomed Mater Eng. 2007;17:269–76.PubMedGoogle Scholar
  31. 31.
    Caron MM, Emans PJ, Coolsen MM, Voss L, Surtel DA, Cremers A, et al. Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage. 2012;20:1170–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Li X, Zhang Y, Qi G. Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells. Cytotechnology. 2013;65:323–34.CrossRefPubMedGoogle Scholar
  33. 33.
    Osipova EY, Shamanskaya TV, Kurakina OA, Nikitina VA, Purbueva BB, Ustugov AY, et al. Biological characteristics of mesenchymal stem cells during ex vivo expansion. Br J Med Med Res. 2011;1:85–95.CrossRefGoogle Scholar
  34. 34.
    Jin GZ, Kim JJ, Park JH, Seo SJ, Kim JH, Lee EJ, et al. Biphasic nanofibrous constructs with seeded cell layers for osteochondral repair. Tissue Eng Part C Methods. 2014;20:895–904.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Jin GZ, Kim JH, Park JH, Choi SJ, Kim HW, Wall I. Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect. J Mater Sci Mater Med. 2012;23:1739–48.CrossRefPubMedGoogle Scholar
  36. 36.
    Jin GZ, Park JH, Lee EJ, Wall IB, Kim HW. Utilizing PCL microcarriers for high-purity isolation of primary endothelial cells for tissue engineering. Tissue Eng Part C Methods. 2014;20:761–8.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lee SI, Yeo SI, Kim BB, Ko Y, Park JB. Formation of size-controllable spheroids using gingiva-derived stem cells and concave microwells: Morphology and viability tests. Biomed Rep. 2016;4:97–101.CrossRefPubMedGoogle Scholar
  38. 38.
    Jin GZ, Kim HW. Porous microcarrier-enabled three-dimensional culture of chondrocytes for cartilage engineering: a feasibility study. Tissue Eng Regen Med. 2016;13:235–41.CrossRefGoogle Scholar
  39. 39.
    Olmos Buitrago J, Perez RA, El-Fiqi A, Singh RK, Kim JH, Kim HW. Core–shell fibrous stem cell carriers incorporating osteogenic nanoparticulate cues for bone tissue engineering. Acta Biomater. 2015;28:183–92.CrossRefPubMedGoogle Scholar
  40. 40.
    Meinhart J, Fussenegger M, Höbling W. Stabilization of fibrin-chondrocyte constructs for cartilage reconstruction. Ann Plast Surg. 1999;42:673–8.CrossRefPubMedGoogle Scholar
  41. 41.
    Häuselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Aydelotte MB, et al. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci. 1994;107:17–27.PubMedGoogle Scholar
  42. 42.
    Park KD, Hwang JY, Kim C, Kang JY, Chun HJ, Han DK. Dedifferentiated chondrocyte culture using alginate microbead prepared from microfluidic technique. Tissue Eng Regen Med. 2009;6:353–9.Google Scholar
  43. 43.
    Bryant SJ, Bender RJ, Durand KL, Anseth KS. Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: engineering gel structural changes to facilitate cartilaginous tissue production. Biotechnol Bioeng. 2004;86:747–55.CrossRefPubMedGoogle Scholar
  44. 44.
    Greco KV, Iqbal AJ, Rattazzi L, Nalesso G, Moradi-Bidhendi N, Moore AR, et al. High density micromass cultures of a human chondrocyte cell line: a reliable assay system to reveal the modulatory functions of pharmacological agents. Biochem Pharmacol. 2011;82:1919–29.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Zhang Z, McCaffery JM, Spencer RG, Francomano CA. Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants. J Anat. 2004;205:229–37.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Lewis NS, Lewis EE, Mullin M, Wheadon H, Dalby MJ, Berry CC. Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence. J Tissue Eng. 2017;8:2041731417704428.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Sekiya I, Vuoristo JT, Larson BL, Prockop DJ. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A. 2002;99:4397–402.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Mwale F, Girard-Lauriault PL, Wang HT, Lerouge S, Antoniou J, Wertheimer MR. Suppression of genes related to hypertrophy and osteogenesis in committed human mesenchymal stem cells cultured on novel nitrogen-rich plasma polymer coatings. Tissue Eng. 2006;12:2639–47.CrossRefPubMedGoogle Scholar
  49. 49.
    Mwale F, Stachura D, Roughley P, Antoniou J. Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation. J Orthop Res. 2006;24:1791–8.CrossRefPubMedGoogle Scholar
  50. 50.
    Pelttari K, Winter A, Steck E, Goetzke K, Hennig T, Ochs BG, et al. Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis Rheum. 2006;54:3254–66.CrossRefPubMedGoogle Scholar
  51. 51.
    Mandl EW, van der Veen SW, Verhaar JA, van Osch GJ. Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity. Tissue Eng. 2004;10:109–18.CrossRefPubMedGoogle Scholar
  52. 52.
    Neri S, Mariani E, Cattini L, Facchini A. Long-term in vitro expansion of osteoarthritic human articular chondrocytes do not alter genetic stability: a microsatellite instability analysis. J Cell Physiol. 2011;226:2579–85.CrossRefPubMedGoogle Scholar
  53. 53.
    Xia Z, Duan X, Murray D, Triffitt JT, Price AJ. A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage. Cell Tissue Bank. 2013;14:267–76.CrossRefPubMedGoogle Scholar
  54. 54.
    Jakob M, Démarteau O, Schäfer D, Hintermann B, Dick W, Heberer M, et al. Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J Cell Biochem. 2001;81:368–77.CrossRefPubMedGoogle Scholar
  55. 55.
    Grigolo B, Lisignoli G, Piacentini A, Fiorini M, Gobbi P, Mazzotti G, et al. Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYAff 11): molecular, immunohistochemical and ultrastructural analysis. Biomaterials. 2002;23:1187–95.CrossRefPubMedGoogle Scholar
  56. 56.
    Quintana L, zur Nieden NI, Semino CE. Morphogenetic and regulatory mechanisms during developmental chondrogenesis: new paradigms for cartilage tissue engineering. Tissue Eng Part B Rev. 2009;15:29–41.CrossRefPubMedGoogle Scholar
  57. 57.
    Nejadnik H, Hui JH, Feng Choong EP, Tai BC, Lee EH. Autologous bone marrow-derived mesenchymal stem cells versus autologous chondrocyte implantation: an observational cohort study. Am J Sports Med. 2010;38:1110–6.CrossRefPubMedGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Institute of Tissue Regeneration Engineering (ITREN)Dankook UniversityCheonanKorea
  2. 2.Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative MedicineDankook UniversityCheonanKorea
  3. 3.Department of Biomaterials Science, School of DentistryDankook UniversityCheonanKorea

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