Biomimetic apatite functionalization of collagen-rich membranes: Mechanical behavior, cytocompatibility, and odontoblast-like cell differentiation.

Abstract

Objectives: To evaluate the effect of biomimetic apatite mineralization on the physicochemical properties, mechanical behavior, cytocompatibility, and odontogenic differentiation potential of collagen-rich membranes derived from eggshell membrane, equine tendon type I collagen, and bovine pericardium.

Methods: Membranes were mineralized for 7 days and characterized by X-ray diffraction, scanning electron microscopy with energy-dispersive X-ray spectroscopy, Fourier-transform infrared and Raman spectroscopy, and X-ray photoelectron spectroscopy. Mechanical performance was assessed by microtensile testing, and hDPSC viability by calcein-AM assays up to 21 days. Odontogenic differentiation was evaluated by RT-qPCR for RUNX2, COL-1A1, DSPP, and DMP-1, DMP-1 immunofluorescence, and Alizarin Red S staining and quantification at 21 days. Data were analyzed using one-way or two-way ANOVA followed by Tukey's post hoc test, as appropriate (α = 0.05).

Results: Biomimetic calcium phosphate coatings formed on all membrane types and enhanced biofunctional performance in a membrane-dependent manner. Mineralization significantly increased tensile strength and toughness in eggshell and pericardium membranes, whereas elastic modulus increased only in pericardium. Equine collagen membranes showed modest, non-significant mechanical changes. Cell viability increased significantly over time, with mineralized membranes promoting greater early viability. Apatite-functionalized membranes increased COL-1A1, DSPP, and DMP-1 expression, while RUNX2 remained comparable among formulations. DMP-1-associated staining was more evident in mineralized membranes, which also showed increased calcium-rich extracellular matrix deposition.

Significance: Biomimetic apatite mineralization enhanced the mechanical and biological performance of collagen-rich membranes in a substrate-dependent manner. All apatite-functionalized membranes showed favorable properties as bioactive scaffolds for dental pulp regeneration and regenerative endodontic applications.