Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro

Abstract

Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18–24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.

Response of human peripheral blood monocyte-derived macrophages (PBMM) to demineralized and decellularized bovine bone graft substitutes

“…By conducting comprehensive immuno-compatibility assessments, valuable insights could be gained into the graft’s potential immunogenicity, possible delayed adverse reactions, and develop strategies to mitigate any risks. This study has paved the way for safer and more successful clinical applications of DCC grafts in bone tissue regeneration when its processing techniques was able to produce a bone substitute that meets the recommended acceptable level of residual nucleic acid content in biological tissue scaffolds for safe transplantation [ 16 , 21 ]. While the DMB demonstrated partial elimination of cellular nuclear material, DCC achieved a more comprehensive removal of cells and genetic material [ 21 ], which render it both bio and immuno-compatible.…”

“…The DMB and DCC bovine bone samples were prepared according to a previously reported standard procedures [ 21 ]. Cancellous bone grafts were prepared from bovine femoral heads that has been stored at -80°C until the time of bone substitutes preparation.…”

“…Following washing in running tap water and water jet spray washing (high-pressure water-jet spray with pressure not exceeding 160 psi), the bone cubes were suspended in sterile distilled water and centrifuged for 24h in mild rotation (300 g) to remove debris and red blood cells. The cleaned bone cubes were divided into two groups for demineralization and decellularization treatments [ 21 ]. At the end of the treatment procedures, both the DMB and DCC scaffolds were crushed into granules using an IKA A10 S9 bone grinder (Staufen, Germany).…”

“…PBMMs enable researchers to evaluate the response of innate immune cells, assess cytokine production, and gain insights into the potential inflammatory reactions induced by the biomaterials. In a previous study, we reported the development of a novel decellularization technique for the production of safe decellularized bovine bone (DCC) substitute, as well as a demineralization technique for creating demineralized bovine bone (DMB) substitutes specifically designed to support bone regeneration [ 21 ]. On comparison, DCC demonstrated superior biocompatibility and exhibited promising potential for osteogenesis and bone regeneration both in vitro [ 21 ] and in vivo [ 22 ], than DMB substitutes.…”

“…In a previous study, we reported the development of a novel decellularization technique for the production of safe decellularized bovine bone (DCC) substitute, as well as a demineralization technique for creating demineralized bovine bone (DMB) substitutes specifically designed to support bone regeneration [ 21 ]. On comparison, DCC demonstrated superior biocompatibility and exhibited promising potential for osteogenesis and bone regeneration both in vitro [ 21 ] and in vivo [ 22 ], than DMB substitutes. However, understanding the immune response to these bone substitutes is crucial for assessing their safety and optimizing their long-term performance in bone regeneration applications.…”