Fabrication And Performance Study Of The Fiber-based Bionic Periosteum With High Osteoinductive Activity

As a layer of highly vascularized connective tissue membrane covering the outer surface of cortical bone,the natural periosteum plays a vital role in the development and reconstruction of bone tissue.However,in the repair of critical size bone defects,more attention is paid to the bone itself,and various types of biomaterial-based bone grafts for bone tissue regeneration have been developed while ignoring the importance of periosteum.This results in 5-10% of patients with critical-size bone defects that will cause lifelong disability due to delayed or even non-union.This unsatisfactory repair effect is mainly due to the limited osteoinductive activity of the bone regeneration strategy lacking periosteum,which leads to poor osseointegration and subsequent vascular network regeneration.In view of the inherent defects such as difficulty in obtaining natural periosteum,insufficient donors,and rejection reaction,the use of artificial methods to prepare bionic periosteum with high osteoinductive activity to improve the osseointegration effect of artificial bone grafts has great research significance and clinical significance.Moreover,when encountering a critical size bone defect,the natural periosteum located on the surface of the cortical bone will inevitably be damaged.While repairing the bone tissue,the natural periosteum tissue is regenerated in situ,so as to achieve long-term maintenance of bone tissue development and functional reconstruction.Therefore,the development of a bionic periosteum with dual regeneration functions of bone and periosteum tissue is also of great clinical value.Based on this,this study mainly relied on the known advantage of nanofibers and extracellular matrix(ECM)in a structure similar to the development and preparation of high osteoinductive activity fiber-based bionic periosteum suitable for different ways of application.The main research contents are as follows:1.Optimized preparation of homogeneous polycaprolactone/tricalcium phosphate sol(PCL/TCP sol)bionic periosteum.The TCP sol was synthesized by the sol-gel method and uniformly loaded into PCL nanofibers by co-electrospinning to prepare a homogeneous PCL/TCP sol hybrid fibrous membrane.The effects of different TCP sol loadings on the apparent morphology,surface hydrophilicity,and mechanical properties of hybrid fibrous membranes were explored.The results of the study showed that as the TCP sol loading increased from 0 wt% to 16.7 wt%,the diameter of the nanofibers increased from 485.3 ± 85.4 nm to 673.5 ± 112.9 nm.Moreover,with the increase of TCP sol loading,the hydrophilicity of the hybrid fibrous membrane was significantly improved,and lower loading TCP sol(4.8 wt% and 9.1 wt%)had a positive effect on the mechanical properties of the hybrid fibrous membrane.Among them,the hybrid fibrous membrane(P10T1)loaded with 9.1 wt% TCP sol exhibited a moderately hydrophilic surface(48.9° ± 4.1°)suitable for cell growth and also had the best mechanical properties(tensile strength of 4.44 ± 0.12 MPa).2.Evaluation of osteogenic properties of homogeneous PCL/TCP sol bionic periosteum.In order to verify the advantages of P10T1 as a biomimetic periosteum,rod-shaped TCP nanoparticles were prepared by hydrothermal method,and a composite fibrous membrane of PCL/TCP nanoparticles with the same inorganic content as P10T1 was prepared by co-electrospinning.The system compared the effects of the incorporation of TCP sol or TCP nanoparticles on the in vitro degradation and biomineralization properties of the two types of fibrous membranes,as well as the cell proliferation and osteogenic differentiation properties.The results of the study showed that conventional composite fibrous membrane still exhibits a hydrophobic surface(129.4° ± 2.0°),and the mechanical property(tensile strength of 3.98 ± 0.18 MPa)was also weaker than the hybrid fibrous membrane.Moreover,the hybrid fibrous membrane had a higher degradation rate and better biomineralization properties,rat bone marrow mesenchymal stem cells(r BMSCs)incubated on it also showed better adhesion,spreading,proliferation,and osteogenic differentiation properties.3.The preparation and evaluation of the bionic periosteum with the function of simulating the periosteal microenvironment.The ideal bionic periosteum can simulate the microenvironment created by natural periosteum during bone regeneration,that is,it can continuously provide growth factors,recruit bone cells,and subsequently promote ECM mineralization.Based on this,based on the previous work,a calcium-binding peptide with excellent bioactivity was designed and prepared.It was uniformly loaded into PCL nanofibers by co-electrospinning,and combined with the solutioninduced crystallization method,a peptide-modified bionic periosteum with a shish-kebab structure was prepared.The results of the study showed that bone morphogenetic protein-2(BMP-2)conjugated with a heptaglutamate domain(E7)has an obvious negative potential(-10.67 ± 0.65 m V)and could form a strong electrostatic binding force with TCP sol with a positive potential(6.4 ± 0.32 m V)to synthesize calcium-binding peptides.The external structure(shish-kebab structure)could significantly improve the surface roughness of nanofibers(0.750 ± 0.021 ?m),and combined with the strong internal electrostatic effect,it greatly prolonged the release cycle of peptides(>28 days).Moreover,the presence of the rough surface was also conducive to the uniform mineralization of the bionic periosteum.In vitro cell experiments confirmed that the bionic periosteum could significantly promote the growth of r BMSCs(adhesion,spreading,proliferation,and osteogenic differentiation)under the synergistic effect of chemical composition and physical structure.In vivo repair experiments have also verified that the bionic periosteum could promote the regeneration of vascularized bone tissue.4.Preparation and evaluation of the bionic periosteum for dual regeneration of bone and periosteum.In view of the importance of in situ regeneration of the periosteum and the double-layer structure of the natural periosteum,an enhanced bionic periosteum that simultaneously mimics the structure and function of the natural periosteum was designed and prepared.It was mainly composed of a bioactive layer in contact with bone tissue,a guiding layer in contact with soft tissue,and a barrier layer between the two.The guiding layer is a macroporous aligned PCL fibrous membrane prepared by electrospinning combined with foaming technology,which was used to simulate the aligned fibrous layer of natural periosteum tissue.The bioactive layer is an inorganic membrane of TCP nanowires prepared by the hydrothermal method combined with vacuum filtration technology,which was used to simulate the cambium of natural periosteum tissue.The barrier layer is a PCL dense membrane prepared by solution casting combined with rapid volatilization,and the prepared guiding layer and bioactive layer were respectively conjugated on both sides of the PCL dense membrane by re-solidification to form a tri-layer enhanced bionic periosteum.The results of the study showed that the guiding layer exhibited a good aligned macroporous structure(average pore size of 24.886 ?m),which could guide the orientation and infiltration growth of the mouse fibroblasts(L-929),and the infiltration depth could reach 106 ?m in 7 days.The PCL cast membrane with a dense structure could significantly improve the mechanical properties and cell barrier properties of the bionic periosteum.The bioactive layer had excellent osteoinductive activity and could significantly promote the proliferation and osteogenic differentiation of r BMSCs incubated on it.The in vivo repair results also verify that the guiding layer could promote the regeneration of periosteum tissue with a certain thickness and aligned structure.The existence of the bioactive layer could greatly enhance the bone repair effect.At the same time,the macroporous structure of the guiding layer and the bioactive components of the bioactive layer could help the regeneration of the blood vessel network.It could be found that the enhanced bionic periosteum has an excellent repair function to promote double regeneration of bone and periosteum tissue and a barrier function to prevent soft tissue invasion.In summary,this research was based on the background of repairing critical size bone defects,and the starting point of improving the osseointegration effect of bone tissue engineering scaffolds,and the importance of periosteum tissue regeneration,a series of high osteoinductive activity fiberbased bionic periosteum suitable for different ways of application have been designed and prepared.The dual methods of chemical(optimization of bioactive components)and physical(microstructure design)are used to endow the fiber-based bionic periosteum excellent tissue repair characteristics,which enriches the related functional design and evaluation system of the bionic periosteum,and lays a solid foundation for the further optimization of the performance of the clinical bionic periosteum.