| Reconstruction of large bone defects formed as a result of trauma,resection,or congenital malformations remains problematic in orthopedic and craniofacial clinical practice.Bone tissue contains 65-70% wt of inorganic crystals,mainly hydroxyapatite(HA),and 30-35% wt of an organic matrix,mainly collagen.Thus,engineering bioactive scaffold that includes the organic-inorganic interphase of bone tissue with the regenerative capacity of the periosteum may be a potential solution of autograft substitutes.PCL is very attractive in tissue engineering because of its good biocompatibility and processability,but its high hydrophobicity and low degradability in vivo make it less suitable for long-term applications.However,PCL possesses excellent mechanical strength.The advantage of PLGA-formed scaffolds is their large specific surface area,which is beneficial for cell adhesion and nutrient diffusion and thus for the survival and growth of cells.n-HA is the most widely used not only because its composition and morphology are similar to the inorganic component of natural bone but also because it has excellent bioactivity and biocompatibility.However,n-HA also shows limitations,such as high brittleness,poor flexibility and processability,and difficulty in the adjustment of degradation time.Purified n-HA has a diameter of 20-40 nm and possesses an-OH functional group;to a certain extent,it can neutralize the acid products produced by the degradation of PLGA,thus reducing the inflammatory response from the surrounding tissues.Studies have shown that chitosan(CS)is a natural polysaccharide,which is similar to the main components of the extracellular matrix,which can promote the adhesion and expression of many kinds of cells.Polyamide(PA)is a kind of medical polymer material with excellent properties,which is very similar to the human collagen molecule in structure,so it has good biocompatibility.Bone marrow mesenchymal stem cells(BMSCs)is a kind of adult stem cells,with low immunogenicity,easy to obtain,less ethical issues and a lower risk of tumor development,BMSCs are an attractive candidate as the seed cells for bone tissue engineering.Based on the above background,this research prepared porous scaffold materials usingpolycaprolactone as matrix,using the melt-blending and particle-leaching processes,polymerization with different polymers,and introduction of nano hydroxyapatite to enhance biological activity of polymer,in which Na Cl and PVP were used as co-porogenic agents and the melting formation and porogenic agent leaching were integrated.The structure of the scaffold materials has been analyzed,and the performance of the scaffold has been evaluated.The adhesion,proliferation,and differentiation of BMSCs on scaffolds,as well as the expression of bone-related genes,have been explored.The main topics of this dissertation are as follows:1.Scaffold preparation: The novel functional porous scaffolds were prepared by the polymerization of PCL with different polymer(PLGA,CS,PA)and nano hydroxyapatite.2.Scaffold characterization: SEM showed that polymer fiber can be integrated into PCL matrix to connect the dispersed single pores and to maintain the necessary spatially interconnected porous networks.The porous rates of the scaffolds were 64.47±0.05% to 75.74±1.21%,and the average pore sizes of the scaffolds were 154.36±1.32 ?m to 179.07±0.75 ?m,which facilitates cell adhesion and migration,as well as nutrient transport.Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and thermogravimetric analysis confirm that the continuous nanostructure on the channel surface of the scaffold materials is composed of HA.The blending of PCL and polymer materials effectively improves the mechanical properties of the scaffold.The tensile strength of the scaffolds was 127±2 MPa to147±5 MPa,the compressive strength was 39±2MPa to 47±2 MPa,and 24-week degradation rate was 32.31±1.93% to 37.52±1.69%.The material can degrade between 3 and 6 months,which not only maintains the scaffold morphology during bone regeneration but also is degraded and adsorbed by the host after the scaffold is implanted into the organ lesion.3.In vitro assessment of the osteogenic differentiation potential of BMSCs on scaffold materials: Human mesenchymal stem cells were cultured in the scaffolds for 7-21 days in vitro.After 14 days,single cells were rare on the PCL matrix composite scaffolds,as all the cells grew inside the pores;the cell layer and ECM continued to thicken and almost completely covered the scaffold,and the surface of the ECM was very smooth.The cell proliferation results show that with increasing time in culture,the interconnecting nanometer network structure formed in the PCL matrix composite system provides more nutrients to the cells,thus promoting the proliferation of BMSCs.ALP,as a marker of the early stages of bone development,was used to evaluate the bone-inducing potential of the scaffold materials.A further test of ALP activity was also used to evaluate the potential for BMSCs to differentiate into osteoblasts.For the PCL matrix scaffolds,ALP showed high activity after 14 days in culture,reaching the highest activity level on day 7;ALP activity continued to decline from day 14 to day 21,suggesting that osteoblast markersdecreased and BMSCs continued to differentiate and gradually mature on the scaffolds.Alizarin red staining indicating the formation of large amounts of calcium and mineral deposits.The expression of the bone-related genes Runx2,OPN,OCN,BMP-2,collagen I,integrin a1,integrin b1,and SLP was markedly upregulated,suggesting that this scaffold can promote BMSCs differentiation,proliferation and maturation to osteoblasts.4.In vivo evaluation of the osteogenic bioactivity of the composite scaffolds: In vivo experiments,the scaffolds were implanted in a rabbit skull-defect model.Micro X-ray 3D imaging,HE,and immunohistochemistry revealed that the scaffold materials are degradable and also display excellent biocompatibility,along with the capacity to induce bone regeneration.In this work,we have developed a simple technique to prepare a porous composite PCL matrix bone scaffold material with a specific mechanical strength and a controllable degradation rate through the melt-blending/particle-leaching process.This scaffold material possesses composition and structural characteristics that closely mimic those of natural bone matrix,which supports the adhesion and spreading of cells,as well as their activity and proliferation.This preparation method avoids the effect of organic solvents on organs in the traditional synthesis processes and exhibits excellent biocompatibility.Mesenchymal stem cells were used in the in vitro bone-inducing activity experiments with the scaffold material.The capability of the scaffold to induce osteoblast differentiation in BMSCs and to repair craniofacial lesions in an animal model were also evaluated.The results show that the composite scaffold material exhibits clear advantages in the induction of bone regeneration and self-repair and thus can be used in bone tissue engineering,particularly for the reconstruction of craniofacial,non-weight-bearing bones.Therefore,this work will provide a good basis for future research. |
PDF Full Text Request