The enthesis is a complex anatomical and functional interface between tendon and bone. Once injured, this site does not readily heal and is repaired with limited success. To aid in repair of the enthesis a commercially available scaffold was chosen, from 3 candidate biomaterials, with fibroblast and osteoblast deposited extracellular matrix (ECM) to create a tendon and bone region, respectively, on the scaffold. To further enhance the ECM deposition, the seeded scaffold was mechanically stimulated in a custom built bioreactor for 35 days. The scaffolds were then evaluated by looking at tissue specific gene activation of mesenchymal stem cells (MSC)s due to the deposited ECM.Out of the three materials, non-degradable polyester fabric (PET), degradable polylactic acid (PLA) fabric, and biologic acellular dermal matrix (ACDM), the PLA fabric had the best combination of ECM deposition and mechanical strength for the project. After selecting a scaffold, we determined the parameters for co-culture medium, with respect to fibroblast and osteoblast mineralization. It was determined that standard growth medium, alpha-MEM + 10% fetal bovine serum + 1000 U/mL penicillin, 1000 μg/mL streptomycin, 2.5 μg/mL amphotericin-B + 3 mM beta-glycerophosphate + 25 μg/mL of ascorbic acid provided low fibroblast mineralization while still allowing for osteoblast mineralization. Fluorescence imaging demonstrated that a co-cultured scaffold could be seeded to produce two distinct tissue specific regions. The transition zone produced had values for collagen and glycosaminoglycan (GAG) deposition between that of the two tissue specific regions. Lastly after mechanical conditioning, stimulating the entire scaffold produced an increase in cell number, and the ratio of collagen to GAG in ECM compared to static culture. When the MSCs were exposed to the tissue specific regions, entirely stretched ECM caused an increase in collagen and tendon-specific GAG gene activation and a decrease in mineralization gene activation compared to tissue culture plastic. Cartilage specific markers were unchanged.In conclusion, a suitable commercially available scaffold was identified. The scaffold was seeded so a tendon specific and bone specific regions were distributed on the scaffold. Mechanically conditioning the scaffolds in a bioreactor increased the activation of tissue specific genes for tendon and bone compared to stem cells seeded on tissue culture plastic. Future work includes a functional scaffold testing in an in vivo tendon-to-bone animal model
