Ionic versus Covalent Crosslinking of Xenograft Demineralized Bone Matrix Putty: Comparative in Vitro Stability and Osteoblast Compatibility for Spinal Fusion Applications

Abstract

Objective: To compare an ionic crosslinker, aluminium hydroxide [Al(OH)3] gel, and a covalent crosslinker, paraformaldehyde (PFA), in xenograft DBM putty with respect to in vitro stability and osteoblast compatibility.

Study Design: An in vitro experimental study
Place and Duration of Study: This study was conducted at the Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia, between November 2025 and April 2026.

Methods: An in vitro experimental study was conducted using bovine DBM putty composed of 70% DBM, 25% carboxymethylcellulose, and 5% crosslinker. Specimens were crosslinked with either Al(OH)3 gel or PFA and
sterilized by gamma irradiation. Stability was evaluated through swelling ratio, quantitative cohesiveness, and enzymatic degradation testing. Biocompatibility was assessed using 7F2 osteoblast viability and adhesion assays. Twenty-five specimens were analyzed per group.

Results: Swelling ratio was comparable between the Al(OH)3 gel and PFA groups (408.4% vs 444.4%, p = 0.068). PFA demonstrated lower mass loss during cohesiveness testing (26.0% vs 36.3%, p = 0.026) and lower enzymatic degradation (10% vs 25%, p = 0.025). Osteoblast adhesion was higher in the Al(OH)3 gel group than in the PFA group (44.7% vs 38.4%, p = 0.019). Cell viability did not differ significantly between groups (31.7% vs 30.0%, p = 0.210).

Conclusion: PFA was associated with greater cohesiveness and lower enzymatic degradation, whereas Al(OH)3 gel supported greater osteoblast adhesion. Both formulations demonstrated similarly low cell viability, highlighting the need for further formulation optimization.