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

Abstract

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. Unconfined compression and double indentation tests were used to obtain mechanical properties. Appearance of neocartilage was examined through histochemistry, immunohistochemistry, terminal 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.

Keywords

• Mesenchymal stromal cell
• Osteochondral construct
• Scaffold-free tissue-engineering