Construction of antibacterial functional material for hard tissue regeneration

Abstract

Introduction: Apatite plays important roles in hard tissue regeneration including the regeneration of enamel-like structure for dental caries treatment and the regeneration of bone for bone defect treatment. Since bacterial infection during or after the regeneration of hard tissues, may hinder the regeneration period or destroy the regenerated structure, an antibacterial material binding with apatite may terminate or prevent the happening of infection. Regenerated enamel-like structures or boneinductive materials should have antibacterial activities to ensure that they can perform their functions stably during service. Aims: In order to assemble fluorapatite into an enamel-like structure with antibacterial properties, acid-resistant properties, and enamel-comparable mechanical properties and to induce the intrafibrillar mineralization of potential guided bone regeneration (GBR) membrane with antibacterial properties and osteogenesis activities. Methods: Low-molecular-weight polyacrylic acid (LPAA) was applied to modulate crystal growth and to assemble fluorapatite into an enamel-like structure via an evaporation strategy. Microstructure characterization on the enamel-like structure was processed by scanning electron microscope (SEM) and transmission electron microscope (TEM); composition of the material was evaluated by fourier transform infrared (FTIR), X-ray diffraction (XRD), and high-resolution transmission electron microscopy (HRTEM); physicochemical properties including zeta potential, contact angle, organic content ratio, and mechanical properties of the enamel-like structure was evaluated. The antibacterial properties of the enamel-like structure and LPAA were evaluated by co-culture with streptococcus mutans (S. mutans) or staphylococcus aureus (S. aureus). Intrafibrillar mineralized eggshell membrane (ESM) was generated by LPAA stabilized amorphous calcium phosphate (LPAA-ACP) and LPAA contained calcification solution (LPAA-CaP). The mechanical properties of the mineralized ESM as a potential GBR membrane were evaluated by microtensile test, osteogenesis of the mineralized ESM was evaluated by alkaline phosphatase staining, Alizarin Red S staining, and quantitative real-time PCR (qRT-PCR). Its antibacterial properties were evaluated by co-culturing with S. aureus. Finally, the osteogenesis activities of the mineralized ESM were verified on rabbit bilateral femur. A circular critical size defect (CSD) was made at the near-end of the femur and the membrane was covered with membranes as GBR membrane. The regenerated new bone was evaluated by micro- CT at the 2nd, 4th, and 12th weeks. Results: The enamel-like structure was composed of parallel and densely arranged fluorapatite in a 97 wt.%, similar to the dental enamel. The structure had comparable mechanical properties to dental enamel and a stronger acid-resistant ability. Also, The LPAA at a ratio of 3 wt.% in the enamel-like structure was demonstrated to have antibacterial properties to S. mutans and S. aureus. LPAA-ACP mineralized ESM was demonstrated to have osteogenesis activities when compared with PAA-CaP mineralized ESM. LPAA-ACP had antibacterial properties to S. aureus and the antibacterial mechanism of PAA was demonstrated to be related directly to carboxy groups and the molecular weight of the polymer. Conclusions: LPAA was proven to control fluorapatite crystal growth and play important roles in assembling these crystals into an enamel-like structure. LPAA-ACP was also proven to induce intrafibrillar mineralization of ESM with antibacterial properties. The functionalized membrane may be applied as a GBR membrane.