Biomimetic Designation And Construction Of Artificial Periosteum And The Effects On Osteogenesis

The treatment of bone defects caused by trauma,degenerative diseases,tumor removal,and congenital malformations is a severe clinical challenge.Periosteum plays a key role in bone growth and development,normal function maintenance,and bone defect repair.As the "gold standard" of bone defect treatment,autologous bone transplantation,the loss of periosteum can reduce the formation of new bone by about 63% and increase the incidence of complications such as nonunion.In actual clinical practice,the injury of autograft or negligence in the design of stent materials often leads to the loss of periosteum,which seriously affects the effect of bone reconstruction.However,the source of autologous periosteum is limited,and there is a lack of mature artificial periosteum suitable for trauma orthopedics.Therefore,it is necessary to develop an artificial periosteum that can effectively improve bone regeneration and reconstruction.In this study,a series of artificial periosteum with good biocompatibility and osteogenesis ability were constructed through mimicking the structure,composition and function of natural periosteum,and the operability and the ability of bone defect repair were systematically evaluated.The main research content as follows:(1)The construction of tissue engineering mineralized collagen membrane and its osteogenesis and angiogenesis effect.Through the method of biomimetic mineralization in vitro,a porous mineralized collagen membrane with type Ⅰ collagen as a mineralization template was prepared.The effect of the material on human bone marrow mesenchymal stem cells(hBMSCs)was studied.The results showed that the porous mineralized collagen membrane can effectively promote cell adhesion,proliferation and osteogenic differentiation of hBMSCs by up-regulating the expression of related genes(ALP,COL-Ⅰ,OCN,OPN,RUNX2 and BSP)and promote angiogenesis by up-regulating the expression of VEGF protein.Through the rat subcutaneous implantation model,it is proved that at the early stage of 2 to 4 weeks,the implant exhibited good biocompatibility and weak inflammatory response.After implanted for 6 weeks,the implanted materials were greatly degraded and collagen fibers and blood vessels are formed,indicating that this porous structure promotes the growth and formation of tissues.In addition,this material was used as a carrier to carry human bone marrow mesenchymal stem cells to repair the in situ bone defect of the femur in nude mice,and a good repair effect was obtained.(2)Preparation of biomimetic three-layered artificial periosteum comprising(Poly)-ε-caprolactone,collagen and mineralized collagen by electrospinning and the effect of guiding bone regeneration.The multi-functional PCL/collagen/mineralized three-layered nanofiber composite membrane was prepared by electrospinning technology through biomimicking the multi-layered structure natural periosteum.The dense and hydrophobic PCL was selected as the outer layer to act as a barrier preventing the surrounding connective tissues from growing into the bone defect area.The PCL/collagen is designed as the middle layer to endow the construct with elastic properties.The PCL/collagen/mineralized collagen is designed as the inner layer,to offer a favorable microenvironment for recruited cells to differentiate into the osteogenic lineage and support new bone formation.The proportions of PCL,collagen and mineralized collagen in the inner layer were comparatively studied.The cell experiments showed that the fibrous membrane promotes the adhesion,proliferation,migration and osteogenic differentiation of hBMSCs cells.When the mineralized collagen content is 3.3 %,the expression of osteogenesis-related proteins(ALP,OPN,OCN and Col-Ⅰ)was the highest.The bilateral 5 mm critical defect on rat skull model was used to evaluate the ability to promote bone formation in vivo.After 4 and 12 weeks of implantation,the membrane with higher mineralized collagen content was more effective,significantly promoted the healing of skull defects.It is proved that the composite membrane material had the ability to recruit cells and induce osteogenic differentiation of cells and promote the new bone formation.(3)Construction and biological performance evaluation of double-layer collagen/hydroxyapatite ultra-long nanowires composite artificial periosteum.The calcium oleate was used as precursor in the hydrothermal reaction to synthesize a hydroxyapatite ultralong nanowire slurry with good dispersibility.The hydroxyapatite nanofiber membrane with highly flexible was prepare from the slurry,and is combined with the collagen membrane to obtain the collagen-hydroxyapatite ultra-long nanowire bilayered film.Through in vivo and in vitro experiments,the physical and chemical properties of the composite scaffold and the in vitro osteogenesis performance were investigated,which proved that the composite material is beneficial to promote the high expression of osteogenesis-related genes RUNX2,ALP,Col-Ⅰ and OPN.The 8 mm skull defect model in rats proved that the composite material had excellent ability to promote bone regeneration.In this research,mineralized collagen materials were prepared by biomimetic mineralization,and was proved its ability to promote blood vessels for the first time.Through biomimicking the structure and function of natural periosteum,a series of artificial periosteal materials with good biocompatibility,high activity and outstanding ability to guide bone regeneration were designed and prepared,which has important guiding significance for the design and development of artificial periosteum.