Tissue-engineered Bone Fabricated With Biphasic Seeding Technique In Vitro And Cultured In A Circulating-perfusion Device

Objective: To establish an efficient way to isolate collagen from porcine bone,and observe the effects on fabricating tissue engineering bone(TEB) with biphasic seeding technique,to trial-produce a new circulating-perfusion device for the culture of TEB to improve the efficiency of mass transfer,and evaluate the efficiency of the biphasic seeding technique combined with the circulating-perfusion culture of TEB.Methods: 1.Collagen was isolated from porcine bone with enhanced pepsin digestion. The gross observation,extraction rate calculating, amino-acid component analysis and molecular weight measurement of collagen were performed,and Mesenchymal Stem Cells(MSCs) growing in collagen gel were observed by inverted microscope and histology.2.Developing a circulating-perfusion device for the 3D culture of engineered tissue. Some capabilities including flow rate, homoisothermia effects, sterility and gas exchange efficiency were evaluated. 3. TEB was fabricated with biphasic seeding technique by the mediation of bone collagen. Then it was divided into two groups: experimental group and control group. TEB in the experimental group was cultured in the circulating-perfusion device while TEB in the control group was cultured in six-well plates. The adhersive rate was calculated and the cellular morphology and tissue structure were observed by means of gross ,inverted microscope ,scanning electron microscope(SEM).The structure of TEB and distribution of cells in TEB both in the two groups were compared by HE staining. The proliferation and differentiation of MSCs were also investigated by assay of alkaline phosphatase(ALP) activities, DNA and calcium contents and immunohistochemistry of osteocalcin.Results: 1. The extraction percentage of bone collagen which contained 35.6% glycine, 27.5% proline and oxyproline respectively was 94.04% by means of enhanced pepsin digestion. The peak wavelength of light absorption of bone collagen was 232nm. Protein electrophoresis revealed that collagen gel mainly contained monomer α1 and α 2,...