Construction And Osteogenesis Capacity Study Of Biomimetic Poly(L-lactic Acid) Nanofiber Bone Tissue Engineering Scaffolds

In tissue engineering,scaffolds play a crucial role because they represent an alternative to the conventional implantation of organs and tissues.Structural parameters of scaffold such as pore size（usually ranging from 50μm to 1000μm）,pore shape and porosity have to be controlled so that living cells can grow throughout the entire scaffold,and nutrients and metabolic wastes can be readily transported into or out of the scaffold.For bone tissue engineering,it is generally considered that the suitable pore size is from 200μm to 400μm.Porous scaffolds composed of nanofibre mimicing the architecture of natural ECM component such as the fibrillar structure of collagen（50 nm to 500 nm in diameter）,have shown to promote the cell attachment,proliferation and differentiation.In this study,the cloud point thermally induced phase separation（CP-TIPS）method was developed on the basis of traditional thermally induced phase separation technology.In the PLLA/dioxane/H₂O ternary system,the porous PLLA scaffolds with 300μm⁴00μm pore size and nanofiber structure were prepared in one step.The effects of traditional thermally induced phase separation and cloud point thermally induced phase separation on the surface morphology,phase transformation,crystallization behavior,crystal structure,molecular chain conformation and arrangement of PLLA porous scaffolds were investigated.It was found that under the high-water content（14%）conditions,the PLLA in the porous scaffolds prepared by traditional thermally induced phase separation method was mainly platelet-likeα′-form crystal.Cloud point thermally induced phase separation method could change the crystallization behavior of PLLA in the phase separation process.The crystallization was mainlyα′-form crystal,and the pore wall of the scaffolds maintained nanofiber structure.The PLLA scaffold prepeared by CP-TIPS showed well elasticity and flexibility after swelling.This property further enabled a facile“mechanical squeeze and intake”of high concentration viscous chitosan solution（3%w/v）into the porous structure to modify the composite scaffolds.The effect of chitosan concentration on the morphology,mechanics,and water uptake ability of the scaffolds was investigated.The hybrid scaffolds were cultured with mouse bone marrow stromal cells（mBMSCs）to examine the cell adhesion,viability and osteogenic differentiation on the scaffolds.Finally,the bone formation in vivo was examined by a critical-sized calvarial bone defects rat model.The introducing of chitosan enhanced the mechanical property,hydrophilicity and alleviating pH decreases during the degradation of the scaffold.The PLLA/CS scaffolds showed better cell affinity and promoted osteogenic differentiation of mBMSCs.In vivo,the PLLA/CS scaffolds promoted new bone regeneration in the calvarial bone defect.In addition,PLLA/lecithin scaffolds with open pore structure were prepared by thermally induced phase separation.The effects of lecithin concentration on the morphology,crystallization,hydrophobicity,cytocompatibility and osteogenic properties of PLLA scaffolds were studied.The addition of lecithin greatly enhanced the hydrophilicity of PLLA scaffolds.The PLLA/lecithin scaffolds were cultured with mBMSCs to examine the cell adhesion,viability and osteogenic differentiation on the scaffolds.Finally,the bone formation in vivo was examined by a critical-sized calvarial bone defects rat model.The ideal bone tissue engineering scaffold requires high porosity to ensure the growth of the cells in the scaffold,the development of the new tissue and the realization of vascularization.At the same time,it was hoped that the scaffold can have certain mechanical strength,which can maintain its shape,and partially restoring the function of bone tissue.However,in the scaffold preparation and design process,the requirements for macroporous with high porosity structure and good mechanical properties are contradictory.How to solve the contradiction between high porosity and high mechanical properties of the scaffolds has always been a difficulty in tissue engineering.In this study,by imitating the structure of cancellous bone and dense bone in human body,a imitating bone tissue gradient scaffold was prepared by thermal phase separation method to solve the contradiction between high porosity and high mechanical properties.The imitating bone tissue gradient scaffold was cylindrical.It was consists by two different structures and components.The outer layer was made of high concentration（More than 10%w/v）PLLA solution,which provides good mechanical properties for the scaffold.And the inner layer was prepared with low concentration（less than 4%w/v）of PLLA solution,forming a scaffold with large pore structure to providing space for the cells and new tissues.The factors affecting the preparation of the gradient scaddolfs were studied.Several thermally induced phase separation methods were tried to combine with eath other,and several gradient scaffolds with complex structure and chemical composition were successfully prepared.The cell compatibility of gradient scaffold was preliminarily studied by mBMSCs cells.