Preparation And Guided Bone Regeneration With Asymmetric Collagen-Chitosan Membranes Containing Aspirin-Loaded Chitosan Nanoparticles

Objective: Periodontal bone defects and patients with inadequate alveolar bone have always been the key and difficult problems in clinical treatment,and it is also the main cause of the failure of implants.Guided bone regeneration membrane is used as a barrier,placed on the surface of bone defects,preventing the connective tissues occupying the space of bone defects and provides the growth space for the bone regeneration and reconstruction.However,the current commercialized collagen membrane is difficult to load drugs or growth factors due to its complicated processing technic,and the ability to promote bone regeneration is not obvious.This research intend to fabricate a asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles,which is composed of a loose layer and a dense layer.The asymmetric membrane can sustained-release aspirin to promote bone regeneration.Methods: 1.The preparation and characterizations of asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles: The aspirin-loaded chitosan nanoparticles were fabricated through ironic gelation method.Transmission electron microscopy(TEM)was used to examine the micromorphology of aspirin-loaded chitosan nanoparticles.The drug release behavior of the nanoparticle was studied.The chitosan solution was poured into a polytetrafluoroethylene mold,and the mixed solution of collagen and nanoparticles was crosslinked by EDC-NHS and then spread on the chitosan membrane to form collagen-chitosan membrane.The obverse side,the reverse side,and the cross-section of the asymmetric membrane were observed using scanning electron microscopy(SEM).The degradation of pure chitosan membranes,pure collagen membranes,cross-linked collagen membranes,and the asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles was compared,and the mechanical properties of the material were evaluated.2.Biocompatibility and regeneration capacity of asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles: Cell proliferation was tested by CCK-8 assay by culturing bone mesenchymal stem cells(BMSCs)with the asymmetric membranes.Bone regeneration capacity was tested by ALP assay kit.AO/EB cell staining for BMSCs was used to characterize the biocompatibility of the asymmetric membrane.The critical-sized cranial defects on Sprague-Dawley rats were made to evaluate the effect of the asymmetric membrane on bone regenerationResults: 1.TEM showed that aspirin-loaded chitosan nanoparticles are were spherical with smooth surface and well dispersed,the diameter of the nanoparticles are uniform.The nanoparticles fabricated in this study could successfully sustain the release of aspirin until the 14 th day.SEM images indicated that the asymmetric membrane comprised a loose collagen layer and a dense chitosan layer.The crosslinking method can improve the mechanical properties of collagen and reduce its degradation rate.2.CCK-8 assay was used to evaluate the cell proliferation and experiment.The study showed that aspirin could promote the proliferation of BMSCs after 5th day and compared with the membrane without aspirin-loaded nanoparticles.ALP assay result showed that asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles have obvious advantages in bone function.Micro-computed tomography showed markedly more new bone formation in asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles group than that of the control group at the macro level.Conclusions: The asymmetric collagen-chitosan membranes containing aspirin-loaded chitosan nanoparticles fabricated in this research achieved a desirable controlled-release pattern,and the fabricated membranes showed good biocompatibility and osteogenic potential.The membranes also significantly increased new bone formation in a rat calvarial defect model.