Ectopic vascularized bone formation by human mesenchymal stem cell microtissues in a biocomposite scaffold

Three-dimensional multicellular human bone marrow mesenchymal stem cells (hBM-MSCs) are showing a great promise in the repair of bone tissue due to its osteogenic differentiation potential, mimicking in vivo microenvironment and immunomodulatory property. In the present study, the potential of hBM-MSC microtissues (MTs) in combination with a biocomposite material to form vascularized bone-like tissue at an ectopic site in an immunocompromised mouse was evaluated. The scaffold was fabricated using gelatin, carboxymethyl cellulose, polyvinyl alcohol and nano-hydroxyapatite (GCnHP) by the freeze-drying method. The physico-chemico-biological characteristics were compared with control scaffold devoid of polyvinyl alcohol (GCnH). The scaffolds (GCnH and GCnHP) were highly porous and had interconnected pores. GCnHP showed higher mechanical strength, higher water adsorption and a lower rate of collagenase-mediated degradation in comparison to GCnH. The scaffolds also supported growth and proliferation of hBM-MSCs MTs and subsequent differentiation into osteoblast-like cells. The differentiated cells showed matrix mineralization and high expression of runX2, alkaline phosphatase, collagen type 1 and osteocalcin genes. A high expression of VEGF was also observed suggesting the potential of hBM-MSC MTs to induce angiogenesis. H&E and Masson's trichrome staining of the 4-weeks in vivo implanted scaffold revealed the presence of newly synthesized collagen and infiltration of host vasculature. IHC assessment showed expression of osteocalcin and osterix. These results demonstrate the efficacy of the combination of hBM-MSC MTs and biocomposite material as a promising approach for in vivo non-load bearing bone tissue repair for future clinical and various regenerative medicine applications.