A Collagen Membrane Pretreated with Citrate Promotes Collagen Mineralization and Bone Regeneration

Abstract

Purpose: Collagen membranes with biomimetic mineralization are emerging as promising materials for bone regeneration, owing to their high biocompatibility. In this study, we developed a biogenic collagen membrane by combining citrate (C) pretreatment and carboxymethyl chitosan (CMC)-mediated mineralization and further evaluated its bone healing potential. Methods: C-CMC collagen membranes were prepared by lyophilization. The mineral composition and content were tested through X-ray diffraction (XRD), Fourier transform infrared (FTIR), and thermogravimetric analysis (TGA). The micromorphology was observed using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning probe microscopy (SPM). Physical and mechanical properties, including the swelling rate, porosity, hydrophilicity, tensile strength, Young's modulus, degradation, and barrier function, were also evaluated. Bone mesenchymal stem cells (BMSCs) were cultured in vitro to observe their behavior. An in vivo critical-size rat calvarial defect model was used to validate the effects of the membrane on bone regeneration. Results: The C-CMC collagen membrane was successfully synthesized as a collagen-hydroxyapatite complex with intrafibrillar mineralization, exhibiting improved mechanical properties and an optimal swelling rate, porosity, hydrophilicity, and degradation rate. Additionally, the C-CMC collagen membrane promoted BMSC proliferation, adhesion, and osteogenesis while preventing epithelial cell infiltration. In vivo experiments indicated that C-CMC collagen membranes significantly stimulated bone regeneration without causing systemic toxicity. Conclusions: Our findings suggest that the C-CMC collagen membrane possesses satisfactory physical and mechanical properties, along with good biocompatibility and efficacy in bone defect regeneration, making it a potential candidate for a bioactive guided bone regeneration membrane in clinical applications.

Keywords: carboxymethyl chitosan; citrate; guided bone regeneration; intrafibrillar mineralization.

Figure 1

Schematic illustration of the remineralization procedure for collagen membranes via citrate pretreatment combined with CMC.

Figure 2

Mineral composition and content of COL, CMC, and C-CMC membranes. (A) FTIR spectra. (B) XRD spectra. (C) TGA patterns. (D) DTG patterns.

Figure 3

The morphology of collagen fibers and the membrane surface. (A) TEM images of collagen fibers; the red circle indicates the crystal deposits. Scale bar: 1 μm. (B) SEM image showing the surface of the collagen membrane. Scale bar: 10 μm. (C) SPM images of different membrane surfaces.

Figure 4

Physical and mechanical properties of pure, CMC, and C-CMC collagen membranes. (A) Swelling rate. (B) Porosity. (C) Water contact angle. (D) Tensile strength. (E) Young’s modulus. (F) Degradation in PBS. (G) Representative fluorescent images of HGECs, which were labeled with DAPI, cultured on the membrane surface, and images of the opposite side (** p < 0.005; *** p < 0.001; **** p < 0.0001).

Figure 5

Proliferation, adhesion, and osteogenesis of BMSCs on COL, CMC, and C-CMC membranes. (A) Cell proliferation evaluated by CCK8 assay. (B) Adhesion and morphology of BMSCs on different collagen membranes. (F-actin: red; vinculin: green; DAPI: blue) Magnification: 400×. (C) Cell areas and (D) mean fluorescence intensity of vinculin for BMSCs seeded on different collagen membranes. (E) ALP staining of BMSCs co-cultured with collagen membranes for 14 days. (a–c) represent the COL, CMC, and C-CMC groups, respectively (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 6

Systematic toxicity of blank control, COL, CMC, and C-CMC collagen membranes after 4 or 8 weeks. Magnification: 20×. Scale bar: 500 μm.

Figure 7

In vivo bone regeneration in calvarial defects. (A,B) The 3D reconstructions of rat craniums in 4 groups after being implanted for (A) 4 weeks and (B) 8 weeks. Dash circles indicate the region of interest. (C) The mean trabecular thickness (Tb.Th). (D) The bone volume fraction (BV/TV). (E,F) HE staining of rat calvarial defects at (E) 4 weeks and (F) 8 weeks. Black boxes indicate new bone formation. Scale bar: 300 μm. (G,H) OCN staining of calvarial defects at (G) 4 weeks and (H) 8 weeks. Scale bar: 50 μm. (I) The relative average optical density of OCN staining (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).