In Vitro Experimental Study Of Construction And Vitrification Preservation Of Tissue Engineered Bone

ObjectiveTissue-engineered bone(TEB) is one of ideal bone graft material for repairing bone defects.It will take some time to construct TEB by traditional method,which is unable to meet the instantly clinical requirements of repairing bone defects.This research has conducted two projects of in vitro experimental study in view of this question.1 Experiments have reported that the instantly constructed TEB with bone marrow mononuclear cells(BMNCs) and materials of scaffold had the certain effect of immediately repairing bone defects in vivo.However,there was not in vitro experiment to prove the osteogenic effect of this project,which was further verified by in vitro tests under the osteogenically inducted environment in our study.2 If the ideal cryopreservation effects of TEB constructed by traditional method can be obtained,it is possible to be used as another method to meet the immediately clinical requirements of repairing bone defects.At present,vitrification preservation is the most promising methods of cryopreservation.In this study,we examined the feasibility of cryopreservation of TEB by vitrification in vitro.Methods1 BMNCs of Beagle were separated by 1.063g/mL Percoll gradient solution. Prepared from porcine femoral head,Partially demineralized bone matrix (pDBM) scaffolds were molded into discs(5 mm in diameter and 1 mm in thickness).The ideal seeding density was obtained by detecting the seeding efficacy of canine bone marrow mononuclear cells(cBMNCs) on pDBM.After the cBMNCs of this density were cultured in osteogenic culture medium(OCM)or basic culture medium(BCM) respectively,conventionally constructed TEB with osteo-induced or non-induced cBMSCs at P2 and pDBM was compared with immediately constructed TEB composed of cBMNCs and pDBM under OCM together for analyzing respectively the cell activity and osteogenic capacity in vitro.2 Using the different composition and concentration of vitrification fluid,the freezing experiments on traditionally constructed TEB with osteo-induced cBMSCs at P2 and pDBM were performed under the different preconditioning so that the proper vitrification media and optimal pretreatments were chosen.Afterwards,TEB in VS442 was subjected to vitrification preservation for 7 days and 3 months,respectively.Cell viability,proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons made with those cryopreserved in VS55 vitreous cryoprotectant.Results1 With the density gradient centrifugation method,cBMNCs were successfully isolated,and 1.063g/mL Percoll medium could obtain more purified monocytes.The optimal seeding density of cBMNCs on pDBM was 30×10~6/mL.After analyzing the cell activity and osteogenic capacity of conventionally constructed TEB,which were composed of osteo-induced and non-induced cBMSCs at P2 and pDBM,and immediately constructed TEB with cBMNCs and pDBM under the same culture of osteogenic induction in vitro, it was found that despite that of immediately constructed TEB were the lowest, the potentiality of enhancement would be bigger following the culture time.2 In the experiments of vitrification preservation for TEB conventionally constructed with osteo-induced cBMSCs at P2 and pDBM,the effect of vitreous cryopreservation with VS442 under 0℃/15min of preconditioning was better.After cell viability,proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons made with those cryopreserved in VS55 vitreous cryoprotectant,post-rewarm cell viability and osteogenic capability of TEB in VS442 were significantly higher.Furthermore,we observed that extending the cryopreservation of TEB from 7 days to 3 months did not have a significant impact on its survival and osteogenic potential.Conclusions1 cBMNCs of Beagle were isolated through the 1.063g/mL Percoll density gradient centrifugation.The optimal seeding density of cBMNCs was 30×10~6/mL.In vitro examination under the osteogenically inducted environment confirmed that the directly replantation of instantly constructed TEB might satisfy the immediately clinical requirements of repairing bone defects.2 VS442 was an optimal vitreous cryoprotectant for vitrification preservation of TEB.As compared with VS55,VS442 was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB.Consequently,TEB cryopreserved in VS442 by vitrification could satisfy the instantly clinical requirements of repairing bone defects.