The Fabrication Of Bone Tissue Engineering Scaffolds With PHB And NHAP/PHB Composite Fibrous Materials By Electrospun Technology And The Studies On Their Structures And Properties

Bone tissue engineering scaffold material refers to the biodegradable materials that can integrate with osteoblasts and be implanted into the body, the source and property of which has limited, to some certain extent, the studies and applications of the bone tissue engineering. Comparing to the traditional bone tissue engineering scaffolds, electrospinning material exhibits high specific surface area, high porosity and the ability of interacting with osteoblasts, and can be used as the basic structure of the tissue-engineered bone.Objectives:Based on the electrospinning technology, this study intends to use PHB and nHAP/PHB as raw material to fabricate the fiber scaffold materials with a certain shape, thickness and mechanical strength. In details, the objectives include the following aspects:1. to contrast and test the surface morphology and mechanical strength of these two kinds of fiber scaffolds:2. to conduct comparative study on the material compatibility of the osteogenic cells;3. in vivo, to explore the histocompatibility difference of these two kinds of materials, namely, to observe the differences of the osteogenesis of the BMSCs(bone marrow stromal cells)-fiber scaffold implanted subcutaneously into nude mice.Methods and Results:1. This study adopted the electrospinning technology to fabricate slide fiber membrane scaffolds and cascading massive scaffolds of PHB and nHAP/PHB polymer materials. SEM (Scanning Electron Microscope) was applied to observe the microscopic morphology of these two scaffolds materials. In addition, the mechanical strength of the membrane was obtained by electronic universal testing machine. The results showed that certain differences existed between the two types of materials in the aspects of the microscopic morphology and mechanical strength.2. In vitro experiment, MG-63(human osteosarcoma cell line) cells were seeded on the surfaces of these two types of fiber membranes. By adopting the FIM, SEM observation, MTT assay, ALP(Alkaline phosphatase) activity assay and RT-PCR technology, we detected the cell surface adhesion, proliferation and differentiation status of MG-63on the surfaces of the scaffold materials. The results indicated that MG-63showed different adhesion morphology and cell proliferation on the surfaces of these two types of materials; and the difference was significant in terms of cell proliferation rate and osteogenic differentiation. The fiber scaffold materials promoted the osteogenic differentiation of MG-63at a certain degree, indicating by the levels of the three kinds of osteogenic markers mRNA RT-PCR results (bone osteocalcin (OCN), bone alkaline phosphatase (BALP) and type Ⅰ collagen (COL Ⅰ)).3. In vivo histocompatibility experiment, this study used the femur of the2-3weeks old neonatal rabbits to fabricate the primary BMSCs. The one-week continuous cultured primary BMSCs were inoculated, separately, onto the surfaces of the layered scaffolds from two types of material, and were implanted subcutaneously into nude mice. The tissue samples were collected separately at one month or two months after transplantation, and were explored by H&E. Masson’s tri-color and IHC staining technologies to observe their osteogenic differentiation statuses. The results indicated that these two kinds of implants had osteogenic differentiation at a certain degree, and a difference existed in term of the differentiation level of these two tissue blocks.Conclusion:The results showed that the diameters of eletrospun scaffold materials fabricated in the study were of the micron level and their rough surfaces benefited cell adhesion. The mechanical strength of these two types of materials prevented the excessive deformation of the implants. These two types of materials promoted the adhesion, proliferation and differentiation of MG-63cells. As implants. these two kinds of BMSCs-material complex showed osteogenic differentiation. Comparing to the pure PHB scaffold, the nHAP/PHB composite scaffold performed a better biocompatibility. However, the subcutaneous tissue blood vessels of the nude mice were not further ingrowth due to the excessive dense implant materials and therefore the incomplete degradation of the fiber scaffold. This reduced the ossification of the tissue blocks. Even though, the scaffold material fabricated by electrospinning technology have a bright future to be applied to the treatment of bone defect and bone implantation.