Generation of double-layered equine mesenchymal stromal cell-derived osteochondral constructs

Background: In vitro tissue engineering of osteochondral implants is an attractive solution to circumvent the limitations of these therapies by providing readily available grafts for patients. Objective: Generate and evaluate the mechanical properties of osteochondral constructs through double indentation and stress-relaxation tests. Methods: A stepwise approach was used to generate and improve osteochondral constructs. Un- confined compression and double indentation tests were used to obtain mechanical properties. Appearance of neocartilage was examined through histochemistry, immunohistochemistry, ter- minal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and live/dead staining. Results: We developed a method of cartilage generation from mesenchymal stromal cells (MSCs) on a porous calcium polyphosphate substrate, improving upon previous iterations to avoid the digestion of chondrocytes and circumvent cell contraction through the transient supplementation of a Rho-associated kinase (ROCK) inhibitor. We further improved neocartilage by producing them with an even surface and increasing their thickness through a double layering approach. Mechanical properties of osteochondral constructs were lower than native equine joint cartilage, however, stress-strain values at equilibrium was comparable to the medial trochlea and patellar groove regions from the native joint. Conclusions: MSC-derived osteochondral constructs generated through double-layering permit the formation of flat and thicker cartilage, with load-bearing capacity comparable to non-weight bearing regions from the native joint.