Different effects of implanting sensory nerve or blood vessel on the vascularization, neurotization, and osteogenesis of tissue-engineered bone in vivo.

To compare the different effects of implanting sensory nerve tracts or blood vessel on the osteogenesis, vascularization, and neurotization of the tissue-engineered bone in vivo, we constructed the tissue engineered bone and implanted the sensory nerve tracts (group SN), blood vessel (group VB), or nothing (group Blank) to the side channel of the bone graft to repair the femur defect in the rabbit. Better osteogenesis was observed in groups SN and VB than in group Blank, and no significant difference was found between groups SN and VB at 4, 8, and 12 weeks postoperatively. The neuropeptides expression and the number of new blood vessels in the bone tissues were increased at 8 weeks and then decreased at 12 weeks in all groups and were highest in group VB and lowest in group Blank at all three time points. We conclude that implanting either blood vessel or sensory nerve tract into the tissue-engineered bone can significantly enhance both the vascularization and neurotization simultaneously to get a better osteogenesis effect than TEB alone, and the method of implanting blood vessel has a little better effect of vascularization and neurotization but almost the same osteogenesis effect as implanting sensory nerve.

[Tissue engineering vascularized bone repairing segmental femoral bone defects in rabbits].

OBJECTIVE: To investigate the effectiveness and mechanism of tissue engineering vascularized bone in repairing segmental femoral bone defects in rabbits. METHODS: Thirty-two rabbits were randomized into two groups (n = 16 each). A segmental and critical bone defect of 15 mm in length was made at left femur. In experimental group, the tissue engineering bone constructed from autologous bone marrow mesenchymal stem cells plus beta-tricalcium phosphate (beta-TCP) and vascular bundle was implanted into bony defect. In control group, there was no implantation of vascular bundle. Animals were sacrificed at 2, 4, 8 and 12 weeks post-implantation respectively. Histological observation was conducted to determine the process of new bone formation and remodeling. The expression of vascular endothelial growth factor (VEGF) in new bone was measured by immunohistochemistry, real-time PCR and Western blot. RESULTS: As indicated by histological observations over time, new bone formation increased in both groups. It was better in the experimental group than the control group at the beginning of 4 weeks. The expression level of VEGF gradually decreased in each group after an initial rise. And the expression of VEGF was significantly higher than the control group after implantation at all time points and peaked at 4 weeks. CONCLUSION: Tissue engineering vascularized bone accelerates bone repair in critical size defect model of femur in rabbit. Implantation of vascular bundle can promote the secretion of VEGF. And VEGF is an essential mediator of both angiogenesis and ossification.

Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized ß-tricalcium phosphate scaffold and mesenchymal stem cells.

Although vascularized tissue-engineered bone grafts (TEBG) have been generated ectopically in several studies, the use of prevascularized TEBG for segmental bone defect repair are rarely reported. In current study, we investigated the efficacy of prevascularized TEBG for segmental defect repair. The segmental defects of 15 mm in length were created in the femurs of rabbits bilaterally. In treatment group, the osteotomy site of femur was implanted with prevascularized TEBG, which is generated by seeding mesenchymal stem cells (MSCs) into ß-TCP scaffold, and prevascularization with the insertion of femoral vascular bundle into the side groove of scaffold; whereas in the control group, only MSC mediated scaffolds (TEBG) were implanted. The new bone formation and vascularization were investigated and furthermore, the expression of endogenous vascular endothelial growth factor (VEGF) which might express during defect healing was evaluated, as well. At 4, 8, and 12 weeks postoperatively, the treatment of prevascularized TEBG led to significantly higher volume of regenerated bone and larger amount of capillary infiltration compared to non-vascularized TEBG. The expression of VEGF in mRNA and protein levels increased with implantation time and peaked at 4 weeks postoperatively, followed by a slow decrease, however, treatment group expressed a significant higher level of VEGF than control group throughout the whole study. In conclusion, this study demonstrated that prevascularized TEBG by insertion of vascular bundle could significantly promote the new bone regeneration and vascularization compared to non-vascularized TEBG, which could be partially explained by the up-regulated expression of VEGF.

Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in vivo.

We investigated whether implantation of vascular bundles or sensory nerves affected the expression of calcitonin gene-related peptide type I receptor (CGRP1R) and neuropeptide Y1 receptor (NPY1R) in tissue-engineered bone. We implanted osteogenically induced bone marrow mesenchymal stem cells (BMSCs) with ß-tricalcium phosphate (ß-TCP) as the scaffold material either with sensory nerve tracts (group I, n = 18), vascular bundles (group II, n = 18) or alone (group III, n = 18) to repair a 1.2 cm femur defect in the rabbit. Better osteogenesis was observed by x-ray and histology in groups I and II than in group III at 4, 8 and 12 weeks. Within the new bone, the mRNA levels of the two neuropeptide receptors determined by real-time PCR increased through week 8, and then gradually decreased (P < 0.05). Expression of the neuropeptide receptors determined by immunohistochemistry was lowest at 4 weeks (P < 0.05) and was higher in group II than in group I (P < 0.05). Expression was significantly higher in groups I and II than in group III at all time points. We conclude that implanting vascular bundles into tissue-engineered bone can significantly improve the early expression of CGRP1R and NPY1R. In contrast, implantation of sensory nerves did not show the same dramatic effect as implantation of vascular bundles.

[Effect of tissue engineered bone implantation with vascular bundle and sensory nerve bundle on expression of neurokinin 1 receptor and vasoactive intestinal peptide type 1 receptor in vivo].

OBJECTIVE: Vascular bundle and sensory nerve bundle implantation can promote the osteogenesis of tissue engineered bone. To investigate whether vascular bundle and sensory nerve bundle implantation will affect the expressions of neurokinin 1 receptor (NK1R) and vasoactive intestinal peptide type 1 receptor (VIPR1). METHODS: Fifty-four 5-month-old New Zealand rabbits were selected. Autologous bone marrow was aspirated from the posterior iliac spine of rabbits, and the bone marrow mesenchymal stem cells (BMSCs) were proliferated in vitro. At the 3rd passage, the BMSCs were cultured in the osteogenic culture medium for 7 days. The tissue engineered bone was prepared by the combined culture of these osteoblastic induced BMSCs and beta tricalcium phosphate scaffold material. A 1.5 cm segmental bone defect was created at the right femur of rabbits. After the plate fixation, defects were repaired with sensory nerve bundle plus tissue engineered bone (group A, n = 18), with vascular bundle plus tissue engineered bone (group B, n = 18), and tissue engineered bone only (group C, n = 18). X-ray examination was used to evaluate the degree of the ossification. The expression levels of NK1R and VIPR1 were measured by the immunohistochemistry analysis and the mRNA expression of NK1R and VIPR1 by real-time PCR at 4, 8, and 12 weeks after operation. RESULTS: The better osteogenesis could be observed in group A and group B than in group C at all time points. X-ray scores were significantly higher in group B than in groups A and C (P < 0.05) at 4 weeks, and in groups A and B than in group C (P < 0.05) at 8 and 12 weeks. The mRNA expressions of NK1R and VIPR1 were highest at 8 weeks in groups A and B and gradually decreased at 12 weeks (P < 0.05); the expressions were higher in groups A and B than that in group C (P < 0.05), and in group B than group A (P < 0.05). Immunohistochemistry analysis showed that the expressions of NK1R and VIPR1 were highest at 8 weeks in 3 groups, and the expressions were higher in groups A and B than in group C. CONCLUSION: Implanting vascular bundles into the tissue engineered bone can significantly improve the expression levels of NK1R and VIPR1. It is an ideal method to reconstruct composite tissue engineered bone.

[Experimental study on construction of neurotization tissue engineered bone for repairing large bone defects in rabbit].

OBJECTIVE: Construction of viable tissue engineered bone is one of the most important research fields in the clinical application of bone tissue engineering, to investigate the function of nerve factors in bone tissue engineering by cell detection in vitro and construction of neurotization tissue engineered bone in vivo. METHODS: Fifty-four healthy New Zealand white rabbits, male or female, weighing 2-3 kg, were involved in this study. Bone marrow mesenchymal stem cells (BMSCs) from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates, using the LG-DMEM complete medium as control. The proliferation and osteogenic differentiation of the cells were observed and tested by the MTT assay, alkaline phosphatase (ALP) stain, and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in beta tricalcium phosphate (beta-TCP) biomaterial scaffold and cultured for 72 hours, then the beta-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups (n = 18): sensory nerve bundle (group A) or motor nerve bundle (group B) were transplanted into the side groove of beta-TCP scaffold, group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th, 8th, and 12th weeks after operation. Bone mineral density (BMD) detection and S-100, calcitonin gene-related peptide (CGRP) immunohistochemistry stain were used at the 12th week to evaluate the effects of bone formation and discuss the mechanism. RESULTS: MTT assay indicated that the absorbance (A) value of each group increased with culture time. From the 6th day, the A values of both the sensory nerve and motor nerve homogenate groups were lower than that of the control group, showing significant difference (P < 0.01). On the 8th and 10th days, the A value of the sensory nerve homogenate group was lower than that of the motor nerve homogenate group, showing significant difference (P < 0.05). ALP stain and collagen type I immunocytochemistry identification indicated that the positive cells in both the sensory nerve and motor nerve homogenate groups were less than that of control group after culturing 7 days. And the positive expression of collagen type I was just visible in the cells of control group. The Yang's scores increased gradually in three groups, the score of group A was significantly higher than those of group B and group C (P < 0.01) at the 8th week. The BMD value of group A was significantly higher than those of group B and group C (P < 0.01) at the 12th week. The S-100 and CGRP expressions were high in group A, and low in group B and group C. CONCLUSION: Homogenates of sensory nerve and motor nerve have inhibitory effects on the proliferation and osteogenic differentiation of BMSCs. The osteogenesis and remodeling of the neurotization tissue engineered bone are more closely related with sensory nerves.