Investigating the Osteoinductive Potential of a Decellularized Xenograft Bone Substitute

Abstract

1.AbstractBone grafting is the second most common tissue transplantation procedure worldwide. The gold standard for bone grafting is the autograft; however, due to morbidity and limited supply, new alternatives, including allograft and tissue-engineered bone substitutes, are needed to satisfy long-term demand. One of the most desired properties of tissue-engineered bone substitutes is osteoinductivity, defined as the ability to stimulate primitive cells to differentiate into a bone forming lineage. In the current study, we treated porcine bone with a decellularization protocol to produce a bone scaffold. We examined whether the scaffold possessed osteoinductive potential and could be used to create a tissue-engineered bone microenvironment. To test if the bone scaffold was a viable host, pre-osteoblasts were seeded, incubated in vitro, and analyzed for markers of osteogenic differentiation. To assess these properties in vivo, scaffolds with and without pre-osteoblasts pre-seeded were subcutaneously implanted in mice for four weeks. The scaffolds underwent micro-computed tomography (microCT) scanning before implantation. After retrieval, the scaffolds were analyzed for osteogenic differentiation or re-scanned by microCT to assess new bone formation with the subsequent histological assessment. The osteoinductive potential was observed in vitro with similar osteogenic markers being expressed as observed in demineralized bone matrix and significantly greater expression of these markers than controls. By microCT, paired t-tests demonstrated significantly increased bone volume:total volume (BV/TV) and trabecular thickness (Tb.Th) after explantation in all groups. Pentachrome staining demonstrated osteogenesis within the scaffold, and angiogenesis in the scaffold was confirmed by CD31 staining for blood vessels. These results demonstrate that porcine bone maintains its osteoinductive properties after the application of a novel decellularization and oxidation protocol. Future work must be performed to definitively prove osteogenesis of human mesenchymal stem cells, biocompatibility in large animal models, and osteoinduction/osseointegration in a relevant clinical model in vivo. The ability to create a functional bone microenvironment using decellularized xenografts will impact regenerative medicine, orthopaedic reconstruction, and could be used in the research of multiple diseases.