Fabrication And Research Of Chitosan-?-TCP Composite Lamellas Scaffold

Purposes: Repairing bone defects caused by trauma,infection,necrosis,tumor resection and other common causes is still a great challenge in clinical practice.Small bone defects can achieve self-healing,while large bone defects such as critical bone defects cannot achieve self-healing and therefore require external intervention.At present,common methods of bone defect repair,such as autologous bone graft and allograft have some limitations,such as functional malformation of donor site and poor adaptability to irregular defect.Bone tissue engineering as a research hotspot in recent years,has the potential to repair critical bone defects.As one of the important components of bone tissue engineering scaffold can replace and play the role of natural bone tissue.However,currently common scaffolds have certain limitations,such as high hardness,poor elastic performance,lack of porous structure or unreasonable porous structure,which may lead to fracture of scaffolds or lack of nutrition in the center of scaffolds resulting in poor repair effect or even absorption of surrounding natural bone tissue.In addition,shape memory ability and shape recovery ability can ensure that scaffolds can be implanted in a minimally invasive way and adapt to the shape of defects,which plays an important role in the field of tissue engineering.As a common natural polysaccharide,chitosan(CS)is widely used in bone tissue engineering duo to its biocompatibility,biodegrade and antibacterial and good shape-memory ability.?-tricalcium phosphate(?-TCP)is also widely used in bone defect regeneration resulting from its good compatibility and osteoconductive.Therefore,Chitosan-?-tricalcium phosphate(CS-?-TCP)elastic scaffold was prepared used bi-directional freeze-drying technology,and its structure and mechanical properties biocompatibility as well osteogenic differentiation ability were tested to explore its feasibility as a strategy for repairing bone defects,moreover its potential value in the field of minimally surgery,large long bone defect and biological carrier will be discussed.Methods: ?-TCP nanoparticles were mixed into CS solution with 1% CS solution and shook for 30 min with ultrasonic oscillator to make ?-TCP nanoparticles evenly dispersed in CS solution and CS-?-TCP scaffolds were prepared by bidirectional lyophilization.The microstructure of the scaffold was observed by Scanning electron microscope(SEM).Energy Dispersive Spectroscopy(EDS)and X-ray Diffractometer(XRD)were used to analyze the element distribution and composition of the scaffold,and the compression strength of the scaffold was tested by universal mechanical instrument.Bone marrow mesenchymal stem cells(BMSCSs)were loaded and co-cultured with CS-?-TCP scaffolds for 1,4 and 7 days.The biocompatibility of the scaffolds was detected by CCK-8 method,and the cell viability of the cells on CS-?-TCP scaffolds was detected by live/dead staining method.In order to explore the in vitro mineralization ability of the scaffold,the CS-?-tcp scaffold was immersed in SBF solution for 7d,and the surface mineralization was observed by SEM,and the phase transformation was analyzed by XRD.BMSCSs were co-cultured with CS-?-TCP scaffold for 24 h and the nuclei and cytoskeleton were stained with 4,6-diamino-2-phenylindole(DAPI)and Phalloidin,respectively,and detected by confocal laser microscopy.q-PCR was used to detect the expression of osteogenic genes: bone morphogenetic protein(BMP2),RUNX-associated transcription factor 2(RUNX2)and type I collagen(COL1).Alkaline phosphatase(ALP)staining was used to detect the osteogenic differentiation effect of BMSCs.Results: The CS-?-TCP scaffolds were comprising of parallel,aligned,and thin lamellas with porous structures.?-TCP particles were evenly distribution over CS framework layers and the CS-?-TCP scaffold possess excellent elastic property,shape memory ability and can restore to the initial shape in a state of compression.In vitro mineralization results showed that CS-?-TCP scaffolds had biological activity.SEM results showed that needle-like minerals appeared around ?-TCP particles,and XRD phase analysis showed that the mineralized substance was hydroxyapatite.CCK-8 showed that the absorbance value of CS-?-TCP scaffold was not statistically different from that of the control group,showing good biocompatibility,and the cells on CS-?-TCP scaffold showed high cell viability.q-PCR results showed that the BMP2,RUNX2 and COL1 genes were significantly up-regulated in CS-?-TCP group compared with the negative control group(pure CS group),and the positive ALP staining was more obvious in CS-?-TCP group,suggesting that CS-?-TCP scaffold has a certain bone induction ability.It can promote osteogenic differentiation of mesenchymal stem cells.Conclusion: CS-?-TCP scaffolds have excellent mechanical and biological properties and can induce the osteogenic differentiation of mesenchymal stem cells,which provides a feasible method for the repair of bone defects and a new idea for the repair of irregular defects and minimally invasive surgery.