Autologous bone grafts are the gold standard treatment for large bone defects but the available quantify of these grafts is limited, highlighting the need for alternative graft sources. Endochondral bone formation can be achieved by chondrogenically priming mesenchymal stem cell (MSC) pellets in vitro and implanting in vivo. In this study we investigated the effects of shorter in vitro priming on chondrogenesis and subsequent in vivo bone formation. We also characteriaed a novel micropellet (μpellet) construct to possibly enhance in vivo bone formation. Chondrogenically differentiated MSC pellets and μpellets were encapsulated using alginate or fibrin and cultured for 2, 5, 7, 10, 14, or 28 days. Gene expression was analysed and safranin O staining was performed. Due to consistent ColII and ColX upregulation and positive GAG staining after 7 days of culture, this time point was chosen for further in vivo study. μpellet-fibrin constructs cultured in vitro for 7 and 28 days retain a similar chondrogenic potential and showed upregulation of hypertrophic genes including ColX and BMP2. Fibrin constructs were subcutaneously implanted in athymic nude mice for 8 weeks. Similar volumes of calcified tissue between μpellet and pellet conditions were found in ex vivo μCT scans. Bone and bone marrow were found in both pellet and μpellet-fibrin constructs after either 7 or 28 days of in vitro priming. Here we have shown that bone formation can be achieved after only 7 days of in vitro chondrogenic priming. Interestingly, μpellet constructs formed comparable quantities of bone like tissue to pellets and show upregulation of hypertrophic genes in vitro when encapsulated in fibrin, indicating μpellet-fibrin constructs could possibly be cultured for an even shorter period in vitro and form bone in vivo. With the proper optimisation these μpellets could also be utilised as an injectable bone graft for future regenerative medicine applications.
Disclosure: The authors declared no competing interests.