High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo.

Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

[1]  Marcel Karperien,et al.  Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair. , 2012, Biomaterials.

[2]  Damian Szklarczyk,et al.  The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored , 2010, Nucleic Acids Res..

[3]  D. D’Lima,et al.  Cartilage cell clusters. , 2010, Arthritis and rheumatism.

[4]  J. Bruun,et al.  Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology , 2010, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[5]  D. Samartzis,et al.  Effectiveness of autologous chondrocyte implantation in cartilage repair of the knee: a systematic review of controlled trials. , 2010, Osteoarthritis and cartilage.

[6]  Zhiyuan Zhong,et al.  Enzymatically crosslinked dextran-tyramine hydrogels as injectable scaffolds for cartilage tissue engineering. , 2010, Tissue engineering. Part A.

[7]  D. Wendt,et al.  Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages , 2010, Arthritis research & therapy.

[8]  D. Wendt,et al.  Cartilage tissue engineering using pre-aggregated human articular chondrocytes. , 2008, European cells & materials.

[9]  K. Lyons,et al.  CCN family 2/connective tissue growth factor modulates BMP signalling as a signal conductor, which action regulates the proliferation and differentiation of chondrocytes. , 2008, Journal of biochemistry.

[10]  C. Poole,et al.  Primary cilia in osteoarthritic chondrocytes: From chondrons to clusters , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[11]  M. Barbe,et al.  Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF‐β1 to induce mesenchymal cell condensation , 2007, Journal of cellular physiology.

[12]  L. Peterson,et al.  Autologous chondrocyte implantation. , 2006, Instructional course lectures.

[13]  Ali Khademhosseini,et al.  Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures. , 2006, Biomaterials.

[14]  Robert L Sah,et al.  Probing the role of multicellular organization in three-dimensional microenvironments , 2006, Nature Methods.

[15]  L. Griffith,et al.  Capturing complex 3D tissue physiology in vitro , 2006, Nature Reviews Molecular Cell Biology.

[16]  A. Curtis,et al.  Chondrocyte aggregation on micrometric surface topography: a time-lapse study. , 2006, Tissue engineering.

[17]  A. Khademhosseini,et al.  Microscale technologies for tissue engineering and biology. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Wan-Ju Li,et al.  Cartilage tissue engineering: its potential and uses , 2006, Current opinion in rheumatology.

[19]  Jean F Welter,et al.  High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells. , 2005, BioTechniques.

[20]  Martin Fussenegger,et al.  Microscale tissue engineering using gravity-enforced cell assembly. , 2004, Trends in biotechnology.

[21]  Junzo Tanaka,et al.  Rapid and Large-Scale Formation of Chondrocyte Aggregates by Rotational Culture , 2003, Cell transplantation.

[22]  A. Cole,et al.  Horizontally oriented clusters of multiple chondrons in the superficial zone of ankle, but not knee articular cartilage , 2002, The Anatomical record.

[23]  R. Tuan,et al.  Cellular interactions and signaling in cartilage development. , 2000, Osteoarthritis and cartilage.

[24]  A. Hinek,et al.  Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin. , 1999, Journal of biomedical materials research.

[25]  H J Mankin,et al.  Articular cartilage repair and transplantation. , 1998, Arthritis and rheumatism.

[26]  B. Cole,et al.  Autologous chondrocyte implantation: an overview of technique and outcomes , 2011, ANZ journal of surgery.

[27]  Zhiyuan Zhong,et al.  Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates. , 2007, Biomaterials.

[28]  A. Cole,et al.  Differential matrix degradation and turnover in early cartilage lesions of human knee and ankle joints. , 2005, Arthritis and rheumatism.

[29]  T B F Woodfield,et al.  Scaffolds for tissue engineering of cartilage. , 2002, Critical reviews in eukaryotic gene expression.

[30]  B. Swoboda,et al.  [Cell proliferation in human arthrotic joint cartilage]. , 2001, Zeitschrift fur Orthopadie und ihre Grenzgebiete.

[31]  G. Lee,et al.  The incidence of enlarged chondrons in normal and osteoarthritic human cartilage and their relative matrix density. , 2000, Osteoarthritis and cartilage.

[32]  J. Kourí,et al.  Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: An overview , 1998, Microscopy research and technique.

[33]  C. Rorabeck,et al.  Osteoarthritis in the human knee: a dynamic process of cartilage matrix degradation, synthesis and reorganization. , 1993, Agents and actions. Supplements.