Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro |
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Affiliation: | 1. Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States;2. Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People''s Hospital, Shanghai, China;3. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States;4. Department of Biomedical Engineering and Physiology, Mayo Clinic College of Medicine, Rochester, MN, United States;5. Department of Laboratory Medicine & Pathology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States;6. Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore;7. Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore;8. Department of Biochemistry & Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States |
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Abstract: | Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with l-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix. |
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Keywords: | Tendon Growth factors TGF (transforming growth factor) Microsphere |
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