Experimental Study Of Storage Methods Of Bio-Derived Bone Scaffold Materials

Objective To study the effect of various storage methods on physical and chemical property, cellular compatibility, ectopic osteogenesis and repairing bone defect of bio-derived bone. Methods Freeze-dried biomaterials had been stored in two different preservation solutions for three and six months, while the biomaterials stored for same period were observed as control group. (1 ) Effect of various storage methods on physical and chemical property of materials: The color, transparency and deposition of preservation solution were monitored.The size and volume of materials were observed.It was known preliminarily if degeneration and bacteria occurred in preservation solution and materials. The materials were observed under electronic scanning microscope in order to determine the aperture and porosity . The bacterial culture of materials was selectively examined. The mechanical strength of materials preserved for three and six months was detected.(2)Effect of various storage methods on cellular compatibility of materials: The experiment was divided into A, B, C group according to different storage methods.Group A: Osteoblasts were cocultured with materials stored in preservation solution 1. Group B: Osteoblasts were cocultured with materials stored in preservation solution 2. Group C:Osteoblasts were cocultured with freeze-dried materials. Osteoblasts in 2×10⁶/ml density had been cocultured with materials for 1,3,5,7days.The cell-material complex was observed under phase microscope and electronic scanning microscope in order to evaluate the adhesion and growth of osteoblasts, cell viability, ALP activity and BGP content were measured, cell cycle was analysed by flow cytometer.(3)Effect of various storage methods on ectopic osteogenesis of materials:The materials were implanted in bilateral muscle of spine in 60 New Zealand white rabbits whichwere divided into A, B, C group according to transplant materials preserved by various methods. A, B, C group were deeply divided into A₁ and A2, Bi and B2, C1 and C2 subgroup according to whether materials were cocuitured with osteoblasts. Tissue engineered bone was implanted in left muscle of spine in A1, B1, C1 subgroup,while simple material in right muscle of spine in A2, B2, C2 subgroup. When the samples were harvested at 4, 8 weeks postoperatively, a series of examination were carried out,including the Brdu labelled cell detection,histomorphological and computerized graphical analysis.?Effect of various storage methods on repairing bone defect of materials:The experimental model of 15mm radial segmental defect was produced in 120 New Zealand white rabbits which were divided into A,B,C group according to transplant materials preserved by various methods. A, B, C group were deeply divided into A1 and A2, B1 and B2, C1 and C2 subgroup according to whether materials were cocultured with osteoblasts. Tissue engineered bone was used to repair bone defect of left limbs in A1, B1, C1 subgroup,while simple material to repair defect of right limbs in A2, B2, C2 subgroup. When the samples were harvested at 4,8,16 weeks postoperatively, a series of examination were carried out,including the roentgenographical and histomorphological assay, the immunohistochemical detection of collagen I and BMP-2, the in situ hybridization of BMP-2 and computerized graphical analysis, the ratio of calcium to phosphorus in repairing area and mechanical strength of ulnar and radius.Results (1)Effect of various storage methods on physical and chemical property of materials: Preservation solution kept transparent and clear during storage procedure. Deposition was not seen in preservation solution. The size and volume of materials were not changed. There was no difference in aperture and porosity of materials preserved dy various methods. Bacteria was not detected during the selective examination of materials. The mechanical strength of materials stored in preservation solution was higher than freeze-dried materials preserved for same period(P<0.05),while there was no difference between the mechanical strength of materials stored in two different preservation solution.(2)Effect of various storage methods on cellular compatibility of materials: Osteoblasts adhered to materials preserved by different methods,differentiated and proliferated in the hole of materials. The difference of cell viability was not significantly in three groups at 1 and 3 days. The cell viability of osteoblasts adhered to three materials was A>OB group at 5 and 7 days(P<0.01,P<0.05). The ALP activity and BGP content of osteoblasts adhered to three materials were A>C>B group at 7 days(P<0.01).The cell cycle of different groups did not changed significantly.the abnormal cells were not seen.(3)Effect of various storage methods on ectopic osteogenesis of materials: The Brdu labelled cells were detected around the edge of materials in different groups. The comparision study showed that ability of new bone formation was ranged as follows: A1> C1> B1 group (P<0.01,P<0.05), according to histomorphological evaluation. The new bone formation increased with time.There was no ectopic osteogenesis in A2, B2, C2 group. (4) Effect of various storage methods on repairing bone defect of materials: All of the defects which had been treated with implants exhibited new bone formation at 4, 8, 16 weeks postoperatively, increasing with time.The comparision study showed that ability of new bone formation in 6 groups was ranged as follows: A1> C1 > A2 >C2> B1 > B2 group (P<0.001,P<0.05), according to the radiological and histomorphological evaluation, the detection of collagen I and BMP-2, the ratio of calcium to phosphorus and mechanical strength assay.The ability of new bone formation in A1 group was strongest. At 16 weeks the defect in A1 group was bridged with the appearance of marrow cavities, the biomechanical property in implants approached to those of normal bone. Conclusion The choice of proper preservation solution could optimize the physical and chemical property, cellular compatibility, ectopic osteogenesis and repairing bone defect of bio-derived bone.