Tissue Engineering Studies On Bone Formation Using New Extracellular Matrix Material With Bone Marrow Stromal Cells

[Objective] Three kinds of materials had been developed and studied heavily for using as extracellular matrix (ECM) materials in bone tissue engineering, nutural material, bioceramics and biodegradable polymers. But no one can meet the require of ideal ECM materials. The design and manufacture of an ideal compound extracellular matrix material is becoming a very important and urgent challenge for bone tissue engineering study and clinical applicaton. In this study , a compound new bone-matrix material (NBM) composed of both organic and inorganic materials was designed, which pocesses a morphological structure of three-dimensional foam. The osteogenic potential of NBM was studied both in vitro and in vivo. The important issue of how to promote the adhension characteristic of osteoblasts to biomaterials was addressed. A simple and reliable method was established to measure the quantity of osteoblasts attached to porous biomaterials. Basic fibroblast growth factor was used to promote the cell adhension to NBM. The main objective of this study includes: to investigate the biocompatibility of NBM with bone marrow stromal cells (BMSC5), to establish a method for measuring the characteristics of cell adhension to NBM, and to observe the osteogenic potential of NBM combined with BMSC in vivo. [Method] (1) BMSCs were cultured with NBM. The attaching and extending speed of the cells to the material , the proliferation and alkaline phosphatase activity were tested. (2) A simple and reliable method to measure the quantity of osteoblasts adhension to porous biomaterials was established. The influence of basic fibroblast growth factor (bFGF) on the characteristics of cell adhension to NBM was tested. (3) The composite of NBM with BMSCs was implanted into the skeletal muscles in rabbits experiment. All implants were examined by gross observation and histological examination in the 2nd 4th and 8th weeks post operatively. [Results] (1) No inhibition to cellular proliferation and ALP activity of BMSCs was observed when cultured with NBM in vitro. (2) BMSCs attach and proliferate well in the NBM, secrecting lots of extracellular matrix. (3) The improved fluorescence method is a simple and reliable quantitative assay for osteoblasts adhension to porous biomaterial. (4) After lOng/mi bFGF induction, cell adhension to the new material was increased significantly, while no effect of 1 000ngIml bFGF induction was observed. (5) In vivo experiment of the NBM with BMSCs group showed a large number of lymphacytes and phagocytes invasing into the inner of NBM in the rabbit skeletal muscle beds after 4 weeks implantation, no new bone formation was observed after 8 weeks. (6) But in the BGC with BMSCs group, there is no sign of significant immunoreaction around the impant, and new bone formation in the composite material was observed after 8 weeks. (7) NBM has a better biodegradability than BGC. [Conclusion] (1) NBM is a new compound bone-matrix material constituted of organic and inorganic materials, which pocesses a morphological structure of three-dimensional foam. The new material has a certain extent of mechanical strength in dry state. The structure of NBM meets mostly the require of an ideal bone-matrix material. (2) NBM showed a good biocompatibility with BMSCs in vitro culture. There was no signs of inhibition of NBM material to the attechment, proliferation and differantiation of BMSCs. (3) lOng/mi bFGF can promote cell adhension...