Comparative analysis of the effectiveness of bone matrix purification protocols

Background: This article describes the protocol for the purification of xenogenic bone matrix tested by a team of authors in the article «Determining the effectiveness of a xenogeneic bone matrix decellularization protocol in in vitro and in vivo studies» (the test results were described in the journal N.N. Priorov Journal of Traumatology and Orthopedics. 2023;30(4):431–443, doi: https://doi.org/10.17816/vto622849). AIM: To conduct a comparative analysis of methods of physical and chemical purification of xenogeneic spongy bone tissue by tomographic and morphological studies. MATERIALS AND METHODS: Xenogenic bovine femoral spongiosa tissue was fragmented to the size of 10×10×10 mm and treated with water, hypertonic, hypotonic, hypotonic solutions, and 3% or 6% hydrogen peroxide solution in various combinations. Deep secondary purification with organic solvents or supercritical fluid extraction was then applied, followed by 1H NMR to determine traces of reagents. The efficiency of the optimal protocol was determined by histologic and tomographic studies with calculation of the purification factor by densitometric indices. RESULTS: In accordance with the purification coefficient calculated by densitometric indicators, the intertrabecular space of bone tissue after exposure to flowing water and hypo- and hypertonic solutions followed by cleaning with a 3% H2O2 solution is not sufficiently purified; histological analysis showed the presence of 0 to 60% osteocytes for different cleaning protocols. When replaced with a 6% H2O2 solution, the purification coefficient was higher, but bone destruction was observed. Additional deep purification allows a high purification rate while preserving the structure, but when organic solvents are used, their traces are detected in the matrix; therefore, the use of supercritical fluid extraction is more effective. CONCLUSION: The sequential use of flowing water, 0.5% NaCl solution, 3% H2O2 solution followed by sc-CO2 treatment is an effective protocol for the purification of xenogeneic spongy bone tissue.