Viable tendon neotissue from adult adipose-derived multipotent stromal cells

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21 days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21 days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21 days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.