Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 μm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.
References
[1]
Harris JD, Siston RA, Brophy RH, Lattermann C, Carey JL, et al. (2011) Failures, re-operations, and complications after autologous chondrocyte implantation–a systematic review. Osteoarthritis Cartilage 19: 779–791.
[2]
Vavken P, Samartzis D (2010) Effectiveness of autologous chondrocyte implantation in cartilage repair of the knee: a systematic review of controlled trials. Osteoarthritis Cartilage 18: 857–863.
[3]
Lutzner J, Kasten P, Gunther KP, Kirschner S (2009) Surgical options for patients with osteoarthritis of the knee. Nat Rev Rheumatol 5: 309–316.
[4]
Perrot S, Menkes CJ (1996) Nonpharmacological approaches to pain in osteoarthritis. Available options. Drugs 52 Suppl 3: 21–26.
[5]
Cameron HU, Botsford DJ, Park YS (1997) Prognostic factors in the outcome of supracondylar femoral osteotomy for lateral compartment osteoarthritis of the knee. Can J Surg 40: 114–118.
[6]
Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, et al. (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331: 889–895.
[7]
Gillogly SD, Voight M, Blackburn T (1998) Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Ther 28: 241–251.
[8]
Kon E, Gobbi A, Filardo G, Delcogliano M, Zaffagnini S, et al. (2009) Arthroscopic second-generation autologous chondrocyte implantation compared with microfracture for chondral lesions of the knee: prospective nonrandomized study at 5 years. Am J Sports Med 37: 33–41.
[9]
Van Assche D, Staes F, Van Caspel D, Vanlauwe J, Bellemans J, et al. (2010) Autologous chondrocyte implantation versus microfracture for knee cartilage injury: a prospective randomized trial, with 2-year follow-up. Knee Surg Sports Traumatol Arthrosc 18: 486–495.
[10]
Boeuf S, Richter W (2010) Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors. Stem Cell Res Ther 1: 31.
[11]
Cherubino P, Grassi FA, Bulgheroni P, Ronga M (2003) Autologous chondrocyte implantation using a bilayer collagen membrane: a preliminary report. J Orthop Surg (Hong Kong) 11: 10–15.
[12]
Darling EM, Athanasiou KA (2005) Rapid phenotypic changes in passaged articular chondrocyte subpopulations. J Orthop Res 23: 425–432.
[13]
Holtzer H, Abbott J, Lash J, Holtzer S (1960) The Loss of Phenotypic Traits by Differentiated Cells in Vitro, I. Dedifferentiation of Cartilage Cells. Proc Natl Acad Sci U S A 46: 1533–1542.
[14]
Malda J, Kreijveld E, Temenoff JS, van Blitterswijk CA, Riesle J (2003) Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation. Biomaterials 24: 5153–5161.
[15]
Yen CN, Lin YR, Chang MDT, Tien CW, Wu YC, et al. (2008) Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: Effects of seeding density. Biotechnology Progress 24: 452–457.
[16]
Malda J, van Blitterswijk CA, Grojec M, Martens DE, Tramper J, et al. (2003) Expansion of bovine chondrocytes on microcarriers enhances redifferentiation. Tissue engineering 9: 939–948.
[17]
Banu N, Tsuchiya T (2007) 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 80: 257–267.
[18]
Goldberg AJ, Lee DA, Bader DL, Bentley G (2005) Autologous chondrocyte implantation. Culture in a TGF-beta-containing medium enhances the re-expression of a chondrocytic phenotype in passaged human chondrocytes in pellet culture. J Bone Joint Surg Br 87: 128–134.
[19]
Hsieh-Bonassera ND, Wu I, Lin JK, Schumacher BL, Chen AC, et al. (2009) Expansion and redifferentiation of chondrocytes from osteoarthritic cartilage: cells for human cartilage tissue engineering. Tissue Eng Part A 15: 3513–3523.
[20]
Imabayashi H, Mori T, Gojo S, Kiyono T, Sugiyama T, et al. (2003) Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis. Exp Cell Res 288: 35–50.
[21]
Tallheden T, Karlsson C, Brunner A, Van Der Lee J, Hagg R, et al. (2004) Gene expression during redifferentiation of human articular chondrocytes. Osteoarthritis Cartilage 12: 525–535.
[22]
Tare RS, Howard D, Pound JC, Roach HI, Oreffo RO (2005) Tissue engineering strategies for cartilage generation–micromass and three dimensional cultures using human chondrocytes and a continuous cell line. Biochem Biophys Res Commun 333: 609–621.
[23]
Giovannini S, Diaz-Romero J, Aigner T, Heini P, Mainil-Varlet P, et al. (2010) Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro. Eur Cell Mater 20: 245–259.
[24]
Croucher LJ, Crawford A, Hatton PV, Russell RG, Buttle DJ (2000) Extracellular ATP and UTP stimulate cartilage proteoglycan and collagen accumulation in bovine articular chondrocyte pellet cultures. Biochim Biophys Acta 1502: 297–306.
[25]
Anderer U, Libera J (2002) In vitro engineering of human autogenous cartilage. J Bone Miner Res 17: 1420–1429.
[26]
Siebold R, Sartory N, Yang Y, Feil S, Paessler HH (2011) Prone position for minimal invasive or all-arthroscopic autologous chondrocyte implantation at the patella. Knee Surg Sports Traumatol Arthrosc 19: 2036–2039.
[27]
Markway BD, Tan GK, Brooke G, Hudson JE, Cooper-White JJ, et al. (2010) Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant 19: 29–42.
[28]
Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE (2001) Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 3: 335–373.
[29]
Fu YQ, Colli A, Fasoli A, Luo JK, Flewitt AJ, et al. (2009) Deep reactive ion etching as a tool for nanostructure fabrication. Journal of Vacuum Science & Technology B 27: 1520–1526.
[30]
Cook MM, Futrega K, Osiecki M, Kabiri M, Kul B, et al. (2012) Micromarrows–three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells. Tissue Eng Part C Methods 18: 319–328.
[31]
Doran MR, Frith JE, Prowse AB, Fitzpatrick J, Wolvetang EJ, et al. (2010) Defined high protein content surfaces for stem cell culture. Biomaterials 31: 5137–5142.
[32]
Bhattacharya S, Datta A, Berg JM, Gangopadhyay S (2005) Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength. Journal of Microelectromechanical Systems 14: 590–597.
[33]
Bongaerts JH, Cooper-White JJ, Stokes JR (2009) Low biofouling chitosan-hyaluronic acid multilayers with ultra-low friction coefficients. Biomacromolecules 10: 1287–1294.
[34]
Tan GK, Dinnes DL, Butler LN, Cooper-White JJ (2010) Interactions between meniscal cells and a self assembled biomimetic surface composed of hyaluronic acid, chitosan and meniscal extracellular matrix molecules. Biomaterials 31: 6104–6118.
[35]
Kabiri M, Kul B, Lott WB, Futrega K, Ghanavi P, et al. (2012) 3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling. Biochem Biophys Res Commun 419: 142–147.
[36]
Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW (2008) Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS One 3: e1565.
[37]
Liebman J, Goldberg RL (2001) Chondrocyte Culture and Assay. Current Protocols in Pharmacology: John Wiley & Sons, Inc.
[38]
Bookout AL, Mangelsdorf DJ (2003) Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal 1: e012.
[39]
Lin RZ, Chang HY (2008) Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J 3: 1172–1184.
[40]
Moreira Teixeira LS, Leijten JC, Sobral J, Jin R, van Apeldoorn AA, et al. (2012) High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo. European cells & materials 23: 387–399.
[41]
Hirao M, Tamai N, Tsumaki N, Yoshikawa H, Myoui A (2006) Oxygen tension regulates chondrocyte differentiation and function during endochondral ossification. J Biol Chem 281: 31079–31092.
Doran MR, Markway BD, Clark A, Athanasas-Platsis S, Brooke G, et al. (2010) Membrane bioreactors enhance microenvironmental conditioning and tissue development. Tissue Eng Part C Methods 16: 407–415.
[44]
Shahin K, Doran PM (2011) Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors. PLoS One 6: e23119.
[45]
Cao Z, Hou S, Sun D, Wang X, Tang J (2012) Osteochondral regeneration by a bilayered construct in a cell-free or cell-based approach. Biotechnol Lett 34: 1151–1157.
[46]
Haasper C, Colditz M, Budde S, Hesse E, Tschernig T, et al. (2009) Perfusion and cyclic compression of mesenchymal cell-loaded and clinically applicable osteochondral grafts. Knee Surg Sports Traumatol Arthrosc 17: 1384–1392.
[47]
Grayson WL, Bhumiratana S, Grace Chao PH, Hung CT, Vunjak-Novakovic G (2010) Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion. Osteoarthritis Cartilage 18: 714–723.
[48]
Erisken C, Kalyon DM, Wang H, Ornek-Ballanco C, Xu J (2011) Osteochondral tissue formation through adipose-derived stromal cell differentiation on biomimetic polycaprolactone nanofibrous scaffolds with graded insulin and Beta-glycerophosphate concentrations. Tissue Eng Part A 17: 1239–1252.
[49]
Cheng HW, Luk KD, Cheung KM, Chan BP (2011) In vitro generation of an osteochondral interface from mesenchymal stem cell-collagen microspheres. Biomaterials 32: 1526–1535.
[50]
Klein TJ, Malda J, Sah RL, Hutmacher DW (2009) Tissue engineering of articular cartilage with biomimetic zones. Tissue engineering Part B, Reviews 15: 143–157.
