Experimental Study Of Cryopreservaton Of Tissue Engineered Bone

Objective To study the effect of various methods of cryopreservation on physical and chemical property, capability of osteogenesis and repairing bone defect of tissue engineered bone, and to explore feasibility and effect of cryopreservation for tissue engineered bone. Methods and result Tissue engineered bone had been store in 4℃ and -196℃ for 3 and 6 months respectively,while the tissue engineered bone without cryopreservation and bio-derived bone were observed as control group.The experiment was divided to two parts:â‘ the experimental part in vitro, â‘¡the experimental part in vivo. â‘ The experimental part in vitro(Effect of various methods of cryopreservation on property of tissue engineered bone):The experiment was divided into 7 groups. Group A3: Tissue engineered bone were preserved at 4 ℃ for 3 months. Group A6: Tissue engineered bone were preserved at 4℃ for 6 months. Group B3: Tissue engineered bone were preserved at -196℃ for 3 months .Group B6: Tissue engineered bone were preserved at -196℃ for 6 months . Group C: Tissue engineered bone without cryopreservation. Group D: Bio-derived bone.Group S:Fresh human bone(only for biomechanics).The cell-material complex was observed under phase microscope and electronic scanning microscope in order to evaluate the adhesion and growth of osteoblasts ,and biomechanics,ALP activity, α₂ β₁ Fluorescence intensity, collagen I, Area of nodules and Cell survival rate of tissue engineered bone were measured.Result show:Osteoblasts adhered tomaterials preserved by difference methods,differentiated and proliferated in the hole of materials. THE biomechanics showed no significant differences among A3, A6, B3, B6, D,P>0.05.but the biomechanics between groups of A3, A6, B3, B6, D and group S showed significant differences,P<0.05.The comparision study showed that ALP activity of tissue engineered bone was ranged as follows: C>B3>B6>A3>A6. B3 vs B6,P>0.05.A3 vs A6,P< 0.05.A3 vs B3 and A6 vs B6,P<0.01. Group of A3,A6,B3,B6 vs group C respectively,P<0.01. a 2 g l Fluorescence intensity was ranged as follows: C >B3>B6>A3>A6. A3 vs A6,P<0.05.B3 vs B6 ,A3 vs B3 and A6 vs B6,P<0.01. Group of A3,A6,B3,B6 vs group C respectively,? < 0.01. Collagen I was ranged as follows: OB3>B6>A3>A6.A3 vs A6 and B3 vs B6,P>0.05.A3 vs B3 and A6 vs B6,P<0.01.Group of A3,A6,B3,B6 vs group C respectively,P<0.01.Area of nodules and Cell survival rate was ranged as follows:C>B3>B6>A3>A6. A3 vs A6,B3 vs B6,A3 vs B3 and A6 vs B6,P<0.01.Group of A3,A6,B3,B6 vs group C respectively,P<0.01. (2) The experimental part in vivo (Effect of various methods of cryopreservation on repairing bone defect of tissue engineered bone):The experimental model of 15mm radial segmental defect was produced in 112 Japan white rabbits which left limb was regarded as experimental limb and right limb was regarded as control limb. The experiment was divided into groups A3,A6,B3,B6,C and D.Group A3: Tissue engineered bone were preserved at 4°C for 3 months. Group A6: Tissue engineered bone were preserved at 4°C for 6 months.Group B3: Tissue engineered bone were preserved at -196°C for 3 months. Group B6: Tissue engineered bone were preserved at -196°C for 6 months. Group C: Tissue engineered bone without cryopreservation. Group D: bio-derived bone.When the samples wereharvested at 2,4,6,12 weeks postoperatively,a series of examination were carried out,including macroscopical, histologial and roentgenographical assay, computerized graphical analysis,the immunohistochemical detection of collagen I and mechanical strength of unlar and radius. Results show:Effect of various methods of cryopreservation on repairing bone defect of tissue engineered bone:All of the defects which had been treated with implants exhibited new bone formation at 2,4,6,12 weeks postoperatively.The result of macroscopical, histologial and roentgenographical assay, computerized graphical analysis,the immunohistochemical detection of collagen I and mechanical strength of unlar and radius show no significant differences among A3, A6, B3, B6, C,P>0.05.but the result above between groups of A3, A6, B3, B6, C and group D showed significant differences,?< 0.05. No obvious immunological rejection reaction can be seen in all groups. Conclusions (DCryopreservation of 4°C and -196°C produced unapparent influence on biomechanics of bio-derived bone. ?Cryopreservation of 4°C and -196°C produced obvious influence on survival rate and viability of osteoblasts,but both methods were able to preserve survival rate and viability of osteoblasts to some extent. ?Effect of repairing bone defect was ranged as follows: C> B3 > B6 > A3 > A6 > D,without significant differences among A3, A6, B3, B6,C,P>0.05,but with significant difference between groups of A3, A6, B3, B6, C and groupD respectively, P<0.05. ? Cryopreservation (4 °C and -196 °C) were capable of preserve tissue engineered bone for long time. Cryopreservation of -196°C is better than cryopreservation of 4 °C ,but cryopreservation of 4 °C was simple, convenient and inexpensive,it was still a applied method.Both methods still need to improved and perfected especially cryopreservation of 4°C.