An Experimental Study Of Prefabricating Vascularized Tissue Engineering Bone Using OB And VEC Co-culture Compounding Muscular Flap

In the oral maxillary surgery, it is a key how to reconstruct externality and function of bone and soft tissue and resume life quality of patients, which was due to oral tumor and trauma. In a long time many scholars have done many foundation research and clinical practice. At present autografting was the main method at repairing damage, but it difficult to reconstruct resume and made patient suffering from pain of operation again.In the near time with rapid development of tissue engineering, it is a new technique and means to repair bone damage using tissue engineered bone. Bone tissue engineering by means of cell amplification, which can be obtained from biopsy specimens, and seeded into biodegradable polymers scaffold as cell-polymer constructs to reconstruct the function of damaged tissue. A critical obstacle in tissue engineering is the ability to maintain large masses of cells alive following their transfer from in-vitro culture conditions into the host. In this study we research some topic about vascularized tissue-engineering bone: the interaction between osteoblasts and vascular endothelial cells co-culture in vitro; fabricating vascularization bone by co-culture of ROB and RVEC implanted into muscle flap; repairing mandibular defects with vascularized tissue engineering bone There are five parts in our study:Part one Culture and identification of rabbit osteoblasts andvascular endothelial cellsObjective: Culture and identification of rabbit osteoblasts and vascular endothelial cells in vitro. Methods: The tibiae and femora; the rental cortex of neonatal Japanese Rabbits born (within 24h) were isolated. Harvested cell were cultured and passaged. ROB was identified by alkaline phosphatase (ALP)staining and Von Kossa staining of calcified nodule. RVEC was identified by immunohistochemical SP methods. Results: The cultured cells were identified to be osteoblastic cells by the criteria of ALP, and calcifing nodules formation. RVEC was identified by immunohisto- chemical SP methods. 95% of the two kinds of cells were identified ROB or RVEC respectively. Conclusion: The generation of rabbit osteoblasts and vascular endothelial cell keep function and proliferation respectively.Part two Effects of PDLLA on the bioactivity of vascular endothelial cell Objective: To investigate effects of PDLLA on the bioactivity of vascular endothelial cell in order to confirm whether it can be a scaffold. Methods: Kidney cortex of neonatal Japanese Rabbits born (within 24h) were isolated, cut into Imm3 tissue piece and digested in mixture liquid of 0.25% trypsin and 0.1% collagenase at 37℃. RVEC were culture in an incubator containing 5% CO₂ at 37℃. PDLLA was immersed into the 75% alcohol 4h, distilled water 48h. lg PDLLA was dipped into 10%FBS DMEM for 7d in an incubator containing 5% CO₂ at 37℃. The extracts of PDLLA were divided into two groups: original extracts and 1/2 extracts. RVEC were implanted in PDLLA, at same time were cultured in the medium for 10d and observed using scan electron microscope (SEM). The RVEC were inoculated into the culture plate and added normal culture medium; original extracts and 1/2 extracts respectively. The samples were tested at 1, 2, 3, 5,7days using (MTT) assay and calculate its relative growth rate (RGR). RGR= light absorbance of experimental group/ light absorbance of negative group ×100%. Assess toxicity according to RGR, RGR ≥100% is 0, 75%-99% is I , 50%-74% is II, 25%-49%isIII, l%-24% is IV, 0 is V.Results: Scanning electron microscopic images showed polygonal cells attached to the surface of the scaffolds; there was no obvious change in morphology comparing with original cells. In some areas they stretched themselves and joined each other by their processes or there was even a subconfluent layer of cells visible. The MTT assay values in test groups and negative group were not significantlydifferent at all time points (P>0.05). The toxicity gradation of the material was 0-1, measured by A value of MTT assay. Conclusions: RVEC had the potentialof spread and proliferation in the scaffold. The PDLLA has no cell toxicity; it can be used as an ideal scaffold for tissue engineering.Part three Morphologic observation and interaction between osteoblastand vascular endothelial cells co-culture in vitroObjective: To study morphologic observation and interaction between rabbit osteoblasts(ROB) and rabbit vascular endothelial cells(RVEC) co-culture in vitro. Methods: Firstly Select rabbit osteoblast and rabbit vascular endothelial cell in ratios of 1:0, 1:1,2:1,4:1 co-culture, secondly Select rabbit osteoblast and rabbit vascular endothelial cell in ratios of 0:1, 1:1, 2:1, 4:1 co-culture. ALP activity was measured at 3d, 6d, 9d. 0.25% trypsin digest and collect cells, and which were sonicated for 4 min at 110 watts (60Hz) in ice and centrifugated at 4000/min for 10min. the supernatant fluid was measured by monitoping light absorbance by the solution at 560nm .the slope of the absorbance versus time plot was used to calculate the ALPase activity. Osteocalcin (OC) assay was measured at same time with ALP, the supernatant was collected, frozen, and the quantity of osteocalcin was determined using a radioimmunoassay. After RVEC was cultivated for Id, 3d, 5d, 7d, stained using immunochemical method and counted, then we protract the growth curve of RVEC. Results: There were good compatibility when co-cultured of osteoblasts and vascular endothelial cells. In the initial stages of coculture, the ROB and RVEC keep themselves morphology, with culture time prolonging to the 9 day; they lose their original morphology and presented long fusiform shape, but they had not been restrained each other.The activity of ALP and OC of group of in ratio of 2:1 were higher than the other two groups (P<0.05) , but no differences comparison group (P>0.05) .At same time the proliferation and division of RVEC were accelerated, which were higher in group of in ratio of 2:1 than the other two groups (P<0.05) . but no differences comparison group (P>0.05) .Conclusion: In vitro direct co-culture system, there were good cellular compatibility whencocultured of ROB and RVEC, the two kinds of cells had synergistic effect. Part four Primary study on fabricating vascularization bone byco-culture of OB and VEC implanted into muscle flapObjective: To observe the potential about prefabricating vascularized tissue engineering bone by compounding of cells and scaffold were embedded into latissimus dorsi muscle. Also to investigate relationship between vascularization and osteogenesis.Methods: A single 2.5 ×1 × 1cm scaffold (PDLLA) was made and coated by Collagen I. The tibiae and femora; the rental cortex of neonatal Japanese Rabbits born (within 24h) were isolated. Harvested cell were cultured and passaged. The two kinds of generation cells ROB and RVEC were processed by cryopreservation and resuscitation. Nine Japanese rabbits were devided into three groups, Group A: ROB and RVEC in ratio of 2:1 compounded PDLLA were implanted in the rabbit left latissimus dorsi muscle, ROB/PDLLA were implanted in the right latissimus dorsi muscle; Group B: left was ROB+RVEC/PDLLA, right was PDLLA; Group C: left was ROB/PDLLA, right was PDLLA. Specimens were harvested at 2w, 4w, 8w, postoperatively, and examined with histology for osteogenesis and vascularization. Results: At 2w, 4w, 8w, upon gross examination, the volume of scaffold of three groups were degraded. In the group of PDLLA, we can observe the facets of scaffold were filled with fibrous tissue, and there were capillary in part of area. The group of ROB/PDLLA and group of ROB+RVEC/PDLLA all can be found abundance of blood tissue. With implantation time prolonging, the rigidity of experimental group were higher than control group. Histologically, newly formed bone was odserved in all of the PDLLA seeded with ROB or ROB/RVEC, whereas it was not observed in the PDLLA alone. The control group showed only muscle cells and connective tissues even at 8 weeks. Bone tissue, on the other hand, was represented in the PDLLA loaded with ROB from 4 to 8 weeks, and it was always found to be deposited directly on the scaffold surface. Vascular tissues ingrowths were well demonstrated at the periphery of scaffold. In the group of ROB+RVEC/PDLLA,capillaries were seen running adjacent to the islands of bone tissue. The cross section of the experimental flaps at 4 and 8 weeks showed bone and vascular tissue arising in the central area, the osteogenesis ability and vascularization degree of latissimus dorsi muscle in addition of co-culture ROB with RVEC were better than ROB/PDLLA. Conclusion: This study demonstrates that it is an ideal method about prefabricating vascularized tissue engineered bone using ROB and RVEC in addition tolatissimus dorsi muscle.Part five Experiment study of repairing mandibular defects with vascularized tissue engineering boneObjective: The aim of this study was to fabricate a vascularized tissue engineering bone of ROB+RVEC/PDLLA and observe the bone regeneration after implanting it in the rabbit non-continuous defects. Methods: To culture ROB and RVEC in vitro, the two kinds of cells were reserved by cryopreservation, resuscitation and amplification. The PDLLA were executed for 15 × 10 ×7mm³ and coated by Collagen I , and then they were sterilized with ethylene oxide. A total of 12 adult Japanese White rabbits were used in this study. A rabbit mandibular body non-continuous defect modal of 1.5cm was excised; this study was divided into three groups: Group A was ROB+RVEC/PDLLA; Group B was ROB/PDLLA; Group C was PDLLA. The right defects of number 1-6 rabbits repaired using group A, The left defects of number 1⁶ rabbits repaired using group B; The right defects of number 7-9 rabbits repaired using group C, The left defects of number 7⁹ rabbits repaired using group A; The right defects of number 10-12 rabbits repaired using group B, The left defects of number 10-12 rabbits repaired using group C. After operation 4w and 8w, specimens were observed for formation of bone and angiogenesis through gross examination, histology and X-ray.Results: Up gross examination, defects site in group C was not still repaired until 8 weeks. There were no osteogenesis in the defects and the quality of material was soft, whereas the defects sites in group A were mostly reconstructed by bone tissue. The reconstructive range of group B smaller then group A. Histological examination of the group A showed that there were two kinds of bone formation: intramembranous ossification and cartilaginous ossification. Up to 8 weeks it also showed that there were vascularization in the central of scaffold and blood vessels were surrounded by newly formed...