Study Of Different Vascularization Strategies For Tissue Engineered Bone

Objective:The rising and development of bone tissue engineering provides a promising way to repair large size bone defects. However, as for reconstruction of large bone defects or the poor recipient bed vascularity in large mammals, engineered bone formation are often imcompleted due to insufficient blood supply in the initial phase after implantation. Thus, the vascularization strategy became the research focus of tissue engineered bone (TEB) study. Currently, the main vascularization strategy in used included designing novel biomaterials and innovation scaffold fabrication techniques, the application of cytokines, combining endothelial progenitor cells in vitro and surgery prevascularization techniques in vivo etc. For combined endothelial progenitor cells(EPC), there were only few study was reported in large animal models; Although surgical revascularization in vivo techniques have been widely used in vary kind of animal models, there was still a lack of cross compare on the effects between different surgical techniques of it. To clarify above issues, we constructed TEB by using different vascularization strategy in beagle dogs to identify the osteogenic promoting effects of combined endothelial progenitor cells. At the same time, we also made the comparisons of angiogenic and osteogenic effects of two kind of surgical prevascularization in vivo techniques on TEB. The aim of our study was try to provide a reference for the construction of vascularized TEB in future clinical application.Methods1. BMSCs were isolated from bone marrow of Beagle dogs by using density gradient centrifugation combined with adherent screening method. The BMSCs were cultured, expanded and induced into osteoblast, chondroblast or adipocyte in vitro. Endothelial progenitor cells (EPCs) derived from beagle dog bone marrow were isolated by using density gradient centrifugation combined with differential velocity adherent method and identificated by surface markers and cell function assay;2. EPCs/BMSCs/TCP group, EPCs/TCP group, BMSCs/TCP group were constructed in vitro respectively and implanted in nude mouse subcutaenously;The TEB grafts were extracted6and12weeks post operation and the effects of osteogenesis were compared by micro-CT and histological assay;3. EPCs/BMSCs/TCP group, EPCs/TCP group, BMSCs/TCP group were constructed in vitro respectively and implanted intramuscularly in the back of beagle dogs; The effects of vascularization and osteogenesis of each groups were compared by micro-CT, histological and immunohistochemistry assay12weeks post operation;4. An AV Loop was made by microsurgery anastomosis of saphenous artery and vein in lower limbs of beagle dogs to establish an AV-loop model of a vascularized TEB. VB vascularized TEB models were established by using above vascular bundle with the ligation of vessels distal site. The feasibility of the model was confirmed by CTA and real time contrast-enhanced B model ultrasonic inspection;5. The vascularized TEBs were extracted6months post implantation. The difference of vascularization between both TEB groups was examined by gross observation, Micro-CT (after Microfil perfusion), histological and immunohistochemistry assay;6. The CT value of vascularized TEBs was measured at different time point post implantation. The vascularized TEBs were extracted6months post implantation. The difference of vascularization between AV Loop and VB group was examined by Micro-CT and histological assay.Results1. The isolation, culture and identification of BMSCs:The BMSCs which was isolated from bone marrow of beagle dogs can be induced into osteoblast, chondroblast or adipocyte in vitro. EPCs derived from beagle dogs bone marrow can be isolated by using density gradient centrifugation combined with differential velocity adherent method and was intake DiI-ac-LDL, adsorb with FITC-UEA-1, own the ability of forming tube structure in matrigel and positive on VWF immunohistochemistry assay.2. Construction of vascularized TEB combined with EPC in nude mouse:The bone mineral density and bone formation aria of EPCs/BMSCs/TCP group was higher than BMSCs/TCP group (p<0.05);3. Construction of vascularized TEB combined with EPC in beagle dogs: Micro-CT and histological analysis demonstrated that there was no significant difference on bone mineral density and bone formation Area between combined EPC group and BMSC group (p>0.05). The VWF immunohistochemistry assay analysis demonstrated that the micro vascular density of combined EPC group was higher than BMSCs group (p<0.05). CT value assay showed that that the relative CT value of combined EPC group was higher than BMSCs group (p<0.05). Histological analysis showed that the osteogenic Area of combined EPC group was higher than BMSCs group (p<0.05)4. The construction of in vivo prevascularized TEB model:CTA assay demonstrated that AV Loops were successfully made and the patency of the loops were confirmed by CTA2weeks,4weeks and8weeks post operation. The real time contrast-enhanced B model ultrasonic inspection indicated that the patency of the loops were maintained6month post implantation. The VB group vascular bundle was not displayed in CTA after operation;5. The comparisons of angiogenesis of different prevascularized in vivo strategy of TEB:The Microfil perfusion and Micro-CT reconstruction analysis displayed that there was no significant difference on the total blood vessel volume and surface between AV Loop and VB group (p>0.05). The VWF immunohistochemistry assay analysis demonstrated that there was no significant difference on the micro vascular density of AV Loop and VB group was detected (p>0.05);6. The comparison of osteogenesis of different prevascularized in vivo strategy of TEB:There was no significant difference on the osteogenesis between AV Loop and VB group was found by CT, Micro-CT and histological analysis (p>0.05).Conclusions1. Combined EPCs can promote the osteogenesis of BMSCs in nude mouse. Combined EPCs owned the ability to promote the angiogenesis and osteogenesis of TEB grafts in beagle dog.2. The saphenous artery and vein of lower limbs of Beagle dog can be used successfully to establish AV-loop and VB vascularized TEB model, which provided a reference method for construction of large size vascularized TEB graft.3. Both of AV-Loop and VB in vivo vascularized strategy can improve the vascularization and osteogenesis of TEB and there was no significant difference on it between these two groups. However, VB vascularization strategy owned the advantage of easy manipulation and high success rate.