Study Of Asymmetric Chitosan Guided Bone Regeneration Membrane

Objective: Both periodontitis and peri-implantitis can cause inflammation around the soft tissue as well as progressive bone loss. The resulting bone defect surrounding tooth or implant can lead to tooth loss and implant failure. The objective of this study was to prepare a novel asymmetric chitosan guided bone regeneration(GBR) membrane, which is composed of a dense layer isolating the bone defect from the invasion of surrounding connective fibrous tissue and a loose layer which can improve cell adhesion and stabilize blood clots, thus guiding bone regeneration.Guided bone regeneration techniques can achieve bone regeneration. However, infection caused by either pathogens colonization at the wound site or foreign body response of the implant materia is considered as one of the major reasons of GBR failure in clinical. The objective of this study was to prepare a novel asymmetric chitosan guided bone regeneration(GBR) membrane loaded with minocycline, which is composed of a dense layer isolating the bone defect from the invasion of surrounding connective fibrous tissue and a loose layer which can improve cell adhesion and stabilize blood clots, thus guiding bone regeneration.Methods:(1)The chitosan membrane was fabricated through liquid nitrogen quencher combined with lyophilization and cross-linked by sodium tripolyphosphate(TPP). The physical properties of asymmetric chitosan membrane were measured by scanning electron microscope(SEM), fourier-transform infrared(FTIR), x-ray diffraction(XRD) and tensile test machine. MTT assay and Live/Dead cell staining for MC3T3-E1 osteoblasts cultured on the membrane were used to characterize the biocompatibility of the membrane. In animal experiments, full-thickness and critical sized skull defects were made to evaluate the effect of the membrane on bone regeneration.(2)The chitosan nanoparticle loaded with minocyclin was fabricated through ionic gelation cross-linked by sodium tripolyphosphate(TPP). Then, the nanoparticles were added into chitosan solution(2 %(w/v)) to prepare the chitosan mixture solution, which perpared asymmetric chitosan membrane by phase separation method. After that, collagen solution cross-linked by EDC/NHS was pured on the dense layer of asymmetric membrane to form collagen-chitosan membrane. The morphological observation of membrane was measured by scanning electron microscope(SEM). Antibacterial activity of the membrane against Staphylococcus aureus(ATCC 6538) was examined and observed by a laser confocal microscopy. Live/Dead cell staining for MC3T3-E1 osteoblasts and L929 fibroblasts cultured on the membrane was used to characterize the biocompatibility of the membrane. In animal experiments, full-thickness and critical sized skull defects were made to evaluate the effect of the membrane on bone regeneration.Results:(1)The results of this study indicate that the asymmetric chitosan membrane can be built and cross-linked by TPP to enhance the tensile strength of the membrane. In vitro experiment showed that no significant numbers of dead cells were detected on the chitosan membrane, which indicating good biocompatibility. In animal experiments, the chitosan membrane had faster new bone formation, showing the capability to enhance bone regeneration.(2) The results of this study indicate that the asymmetric membrane can be built by phase separation method. TEM images also showed the morphological properties of nanoparticles which have smooth surface, nearly spherical shape, and size range of about 20 nm. Antibacterial experiment showed dead bacterials on the collagen chitosan membrane loaded with minocycline. In vitro experiment showed that no significant numbers of dead cells were detected, indicating that the membrane had good biocompatibility. In animal experiments, the membrane can be degradated and promote angiogenesis. The formation of new bone showed the capability of the membrane to enhance bone regeneration.Conclusions:(1)The chitosan membrane prepared in this study possess asymmetric structure; its tensile strength, biodegradation and biocompatibility fulfill the requirements of guided bone regeneration. Therefore, the asymmetric chitosan membrane is a promising GBR membrane for bone regeneration.(2) An asymmetric collagen/chitosan GBR membrane can be fabricated by loading minocycline encapsulated chitosan nanoparticles, and shows satisfactory biocompatibility and barrier function, which enhances bone regeneration. Therefore, the antibacterial GBR membrane is a promising therapeutic approach to prevent infection and guide bone regeneration.