Preliminary Study on the Antigen-Removal from Extracellular Matrix via Different Decellularization

Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological functionality of the tissue engineering scaffolds. However, decellularization is necessary to remove antigenic components in the ECM that may trigger adverse immune response. Freeze-thaw (FT) cycles and treatment with Triton X-100/ammonium hydroxide (TN) are two commonly used decellularization methods for ECM, but their effects on both growth factor retention and antigen removal are still controversial. The objectives of this study are to compare the preservation of ECM texture and beneficial ingredients and the removal of cellular antigens by these two methods. First, the constructs combined bone marrow mesenchymal stem cell-derived ECM and poly(lactic-co-glycolic acid) (PLGA) membrane are prepared and decellularized using FT and TN treatments. Moreover, the effects of decellularization on the ultrastructure and the composition of ECM-decorated PLGA membrane are compared by scanning electron microscope observation and protein quantification. Furthermore, the ECM deposited on PLGA is stripped off and then implanted subcutaneously in rats, and the host macrophage and local lymphocyte responses were investigated. Finally, ECM-decorated porous PLGA scaffolds are implanted into rat calvarial defects, and the new bone formation is evaluated. Our results showed that both methods effectively removed DNA. TN treatment partially retained collagen, glycosaminoglycan, bone morphogenetic protein-2, and vascular endothelial growth factor, and better preserved structural integrity than FT treatment. ECM implants decellularized by both methods induced a mild host response after subcutaneous implantation. Although the total content of residual DNA in the two ECMs digested by the DNA enzyme seemed to be similar and very low, the interfaces between implanted materials and natural tissues in the TN group recruited lower numbers of CD68⁺ macrophages, CD68⁺CD86⁺ (M1) macrophages, and CD4⁺ T lymphocytes than that in FT group, implying that there exist other ECM antigens to influence immune response besides DNA. Furthermore, ECM-decorated scaffolds decellularized by TN treatment induced greater bone formation than that of bare scaffolds in vivo, demonstrating the effective retention of ECM bioactive components after decellularization. This study showed that TN treatment was a more effective and safer decellularization method than FT cycles. Impact statement Decellularization is a prerequisite for extracellular matrix (ECM) application, but there is still no standard for its selection. This study demonstrated that detergent treatment was more effective than freeze-thaw (FT) cycles in removing ECM antigens besides DNA, and the prepared ECM elicited a milder allogenic immune response, which ensured the safety of ECM. Moreover, detergent better preserved the ECM integrity than FT cycles, and effectively retained growth factors, and the decellularized ECM-decorated scaffolds significantly promoted bone repair, which ensured the effectiveness of ECM. This study provides the theoretical and experimental bases for the decellularization strategy of ECM-modified tissue engineering scaffolds.