Comparative Evaluation of Three Different Demineralisation Protocols on the Physicochemical Properties and Biocompatibility of Decellularised Extracellular Matrix for Bone Tissue Engineering Applications.

Background With three-dimensional (3D) bioprinting emerging as the ultimate pinnacle of personalised treatment for achieving predictable regenerative outcomes, the search for tissue-specific bioinks is on. Decellularised extracellular matrix (DECM), which provides the inherent biomimetic cues, has gained considerable attention. The objective of the present study was to compare the efficacy of three different demineralisation protocols to obtain DECM for bone tissue engineering applications.  Methodology Goat femurs were treated using three demineralisation protocols to obtain DECM. Group A was treated with demineralisation solution at 40 rpm for 14 days, Group B with freeze-thaw cycles and 0.05M hydrochloric acid (HCl) and 2.4 mM ethylenediamine tetra-acetic acid (EDTA) at 40 rpm for 60 days, and Group C with 0.1M HCl at 40 rpm for three days. After washing, neutralization, 0.05% trypsin-EDTA treatment for 24 hours, and lyophilisation, DECM was obtained. Assessments included scanning electron microscope (SEM) analysis, energy dispersive X-ray (EDX) analysis, hematoxylin and eosin (H&E) staining, and biocompatibility analysis.  Results On comparative analysis, the protocol followed by Group C revealed good surface properties with patent and well interconnected pores with an average pore size of 218.87µm. Group C also revealed carbon and oxygen as predominant components with trace amounts of calcium, proving adequate demineralisation. Group C further revealed optimal demineralisation and decellularisation under histological analysis while maintaining biocompatibility. DECM obtained in Group C should be further processed for bioprinting applications.  Conclusion The three protocols explored in this study hold potential, with Group C's protocol demonstrating the most promise for DECM-based bioink applications. Further research is needed to evaluate the suitability of the obtained DECM for preparing tissue-specific bioinks for 3D bioprinting.