Different Effects Of Implanting Vascular Bundles And Sensory Nerve Tracts On The Expression Of Neuropeptide Receptors In Tissue-Engineered Bone In Rabbits

ObjectivesBone defect malunion, delayed union or nonunion caused by trauma, infection and tumor has always been a threat to human health, and how to repair the large segmental bone defect is still a clinical problem.Now we can use the method like transplantation of the bone graft, transplantation of the biological materials and Ilizarov technique to solve this problem,but they remain some limitations. Bone tissue engineering applications of new technologies invented for human healing nonunion brought us hope,but how to build a similar physiological condition of bone tissue engineering is our hope diligently pursuing. Use of vascularized and neurotized tissue-engineered bone for reconstructive surgery is in harmony with the bionics perspective and may be an ideal solution to large segmental bone defects.We suggested that impanting the vascular bundles and sensory nerve into the tissue-engineering bone can promote the bone formation. On one hand, the vascular bundles and the sensory nerve bundle can supply oxygen and nutrients to the cellular constructs engineered in vitro. On the other hand, several neuropeptides released from the vascular bundles and sensory nerves, such as calcitonin gene-related peptide (CGRP),substance P(SP),neuropeptide Y (NPY) and Vasoactive intestinal peptide(VIP) may also play importantroles intheseexperimental results.Consequently, in this study, autogenous sensory nerves or femoral vascular bundles were implanted into cellular constructs engineered in vitro and used to repair segmental bone defects in an animal model. Our aims were to evaluate the osteogenicity of the vascularized and neurotized bone grafts and to analyze the levels of the neuropeptide receptors(CGRP1R,NK1R, NPY1R and VIPR1) expressed in the tissue-engineered bone.Materials and MethodsPart I The expression of the neuropeptide receptors in BMSCs and osteogenetically induced BMSCs1. The New Zealand rabbits were purchased from the Experimental Center of the Southern Hospital. Autologous bone marrow was isolated from the rabbits and bone marrow mesenchymal stem cells were proliferated in vitro. At the third passage,part of the BMSCs were induced osteogenetically and were cucluring for 21 days.And then the ALP staining and nodules of calcium staining was used to identify the differentiation ablity.2. Using the immunofluorescence staining to detect the expression of the neuropeptide receptors(CGRP1R,NK1R, NPY1R and VIPR1) in BMSCs and osteogenetically induced BMSCs 3. Using the QPCR to detect the expression of mRNA of the neuropeptide receptors(CGRP1R,NK1R, NPY1R and VIPR1) in BMSCs and osteogenetically induced BMSCsPart II Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in rabbits in short-term 1. Fifty-four New Zealand rabbits weighing between 2 and 3 kg were purchased from the Experimental Center of the Southern Hospital and randomly assigned to 3 groups of 18 rabbits each. Autologous bone marrow was isolated from the rabbits and bone marrow mesenchymal stem cells were proliferated in vitro. At the fourth passage, 4×106—6×106/ml cells were seeded onto 15mm diameter porousβ-tricalcium phosphate (P-TCP) (Bio-lu Biomaterials Company Limited) scaffolds and cultured in vitro for seven days prior to implantation.2. A 6-hole reconstruction common steel plate was placed in front of the right femur and a 1.5-cm length of femur was intercepted between the second and the third hole of the plate. Tissue-engineered bone was imbedded into the defect at this location. An appropriate length of greater saphenous nerve was isolated, cut and then implanted to the side groove of tissue-engineered bone (Group I, n=18). Similarly, A length of deep branch of the femoral vascular bundle was implanted to the side groove of tissue-engineered bone and fixed with thread (Group II, n=18). Neither the vascular bundle graft nor the nerve graft was implanted into GroupⅢ(n=18).3. Rabbits(6 animals per 4 months in each group) underwent radiography of the osteotomized femurs immediately after surgery and at 4,8 and 12 weeks after surgery.All radiographs were scored by five experienced radiologists to evaluate the degree of the bone defect repairing.4. At 4 w,8 w,12w postoperatively, two animals from each group were killed for the observation of new bone formation, reacts with host’s bone interface and interior bridging callus. The bone defect side femur was intercepted completely, fixed and demineralized, then embedded for immunostaining. Paraformaldehyde were used for the fixation of bone defects area, EDTA were used for decalcification. Paraffin imbedding, using HE、MAS SON stain and immunohistochemistry staining (CGRP1R,NK1R, NPY1R and VIPR1).5.4 animals in each group were sacrificed at 4,8 and 12 weeks after the implantations.And total RNA was extracted from tissue samples according to the manufacturer’s instructions. Reverse transcription (RT) was performed. cDNA was used as a template, and quantitative real-time RT-PCR was performed. Values were expressed as relative percentages compared toβ-actin mRNA levels. Relativeexpression levels of each gene were calculated using the 2-ΔΔC T method.6. Statistical software was used for statistical analysis.3×3 factorial design ANOVA was used to analyze the mean of each group. When differences were statistically significant, the LSD method was applied for multiple comparisons.Part III Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in rabbits in middle and long term1.Thirty-six New Zealand rabbits were purchased from the Experimental Center of the Southern Hospital and randomly assigned to 3 groups of 12 rabbits each. Autologous bone marrow was isolated from the rabbits and bone marrow mesenchymal stem cells were proliferated in vitro. At the fourth passage,4×106—6×106/ml cells were seeded onto 15mm diameter porousβ-tricalcium phosphate ((3-TCP) (Bio-lu Biomaterials Company Limited) scaffolds and cultured in vitro for seven days prior to implantation.2. About the preparation of the animal model, the Part II can be taken for reference.3. Rabbits(6 animals per 4 months in each group) underwent radiography of the osteotomized femurs immediately after surgery and at 24 and 48 weeks after surgery.All radiographs were scored by five experienced radiologists to evaluate the degree of the bone defect repairing.The continuous trend can be observe connected with the result in part I.4. At 24w,48w postoperatively, two animals from each group were killed for the observation of new bone formation, reacts with host’s bone interface and interior bridging callus. The bone defect side femur was intercepted completely, fixed and demineralized, then embedded for immunostaining. Paraformaldehyde were used for the fixation of bone defects area, EDTA were used for decalcification. Paraffin imbedding, using HE、MAS SON stain and immunohistochemistry staining(CGRP1R,NK1R, NPY1R and VIPR1).5.4 animals in each group were sacrificed at 24 and 48 weeks after the implantations.And total RNA was extracted from tissue samples according to the manufacturer’s instructions. Reverse transcription (RT) was performed. cDNA was used as a template, and quantitative real-time RT-PCR was performed. Values were expressed as relative percentages compared toβ-actin mRNA levels. Relative expression levels of each gene were calculated using the 2-ΔΔC T method.6. Statistical software was used for statistical analysis.3×2 factorial design ANOVA was used to analyze the mean of each group. When differences were statistically significant, the LSD method was applied for multiple comparisons.ResultsPart I The expression of the neuropeptide receptors in BMSCs and osteogenetically induced BMSCs1. During primary culture of BMSCs in vitro, specific colony-forming unit-fibroblastoids (CFU-Fs) were observed on the sixth day after seeding. CFU-Fs, present at approximately five or six per dish, displayed the morphology of fibroblasts, with aspindle-shaped structure existing in a monolayer rculture. When the primary cultured cells reached9 0% confluence, the BMSCs were subcultured and exhibited rapid proliferation in the subsequent passages. In addition, BMSCs cultured in osteogenic medium demonstrated lower growth speed and the polygonal cells can be observed.2. BMSCs cultured in osteogenic medium for 21 days demonstrated osteogenic phenotypes such as deposition of calcium and positive staining for ALP. 3.Positive neuropeptide receptors staining,characterized by positive green stains in the membrane of cells can be observed using the immunofluorescence staining for the neuropeptide receptors in BMSCs and osteogenetically induced BMSCs. 4.The mRNA of the CGRP1R,NK1R, NPY1R and VIPR1 can be detected in the BMSCs and osteogenetically induced BMSCs using QPCR method.Part II Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in rabbits in short-term1. Observation on the samples:new bone formation were gradually increased in every group after operations. Callus in group I and II were superior than that in group III in the first two time points. The roentgenographic score in group II was higher than in the other two groups(p<0.05),which were not significantly different from each other at 4 weeks after surgery (p> 0.05).At 8 and 12 weeks after surgery, the roentgenographic scores in groups I and II were each higher than in group III (p< 0.05), but they were not different from each other (p> 0.05). 2. The histological observation:With the time prolong, new bone formation were gradually increased. The formation of new bone in group I and group II were superior than in group III at every time point.3. Immunohistochemistry stain results of CGRP1R,NK1R, NPY1R and VIPR1:With the time prolong, the expression of CGRP1R,NK1R, NPY1R and VIPR1 in each group were gradually increased. The CGRP1R,NK1R, NPY1R and VIPR1 expression of group I and group II was superior than group III. They were mainly distributed in the edge of new bone, periosteum, and around the blood vessels.4. The expression of mRNA of CGRP1R:Over time, experimental group within the new bone tissue CGRP1R mRNA was gradually reduced after the first increase in the trend. The expression of CGRP1R mRNA at 8th week was superior than that of at 4th week and 12th week. The expression of CGRPIR mRNA at 4th week was the lowest in three time point. The difference had statistical significance. (F=75.265,58.831, 60.946,p<0.05). There was significant difference between the three groups at the same time point. (F=393.510,290.439,157.263, p< 0.05). The expression of CGRPIR mRNA in group II was superior than that of group I (p<0.05), furthermore, the expression of CGRPIR mRNA in group I and group II were superior than that of group III(p<0.05).5. The expression of mRNA of NKIR:Over time, experimental group within the new bone tissue NKIR mRNA was gradually increasing. The expression of NKIR mRNA at 4th week was the lowest in three time point. The difference had statistical significance. (F=60.993,61.634,30.602, p< 0.05). There was significant difference between the three groups at the same time point. (F=578.477, 632.202,164.934,p<0.05). The expression of NKIR mRNA in groupⅡwas superior than that of group I (p<0.05), furthermore, the expression of NKIR mRNA in group I and group II were superior than that of groupⅢ(p<0.05).6. The expression of mRNA of NPY1R:Over time, experimental group within the new bone tissue NPY1R mRNA was gradually reduced after the first increase in the trend. The expression of NPY1R mRNA at 8th week was superior than that of at 4th week and 12th week. The expression of NPY1R mRNA at 4th week was the lowest in three time point. The difference had statistical significance. (F=125.793,74.177, 99.460, p<0.05). There was significant difference between the three groups at the same time point. (F=395.125,849.587,138.529, p<0.05). The expression of NPYIR mRNA in group II was superior than that of group I (p<0.05), furthermore, the expression of NPYIR mRNA in group I and group II were superior than that of group III(p<0.05).7. The expression of mRNA of VIPR1:Over time, experimental group within the new bone tissue VIPR1 mRNA was gradually increasing. The expression of VIPR1 mRNA at 4th week was the lowest in three time point. The difference had statistical significance. (F=20.774,89.201,17.486, p<0.05). There was significant difference between the three groups at the same time point. (F=19.909,49.558,28.377, p<0.05). The expression of VIPR1 mRNA in group II was superior than that of group I (p< 0.05), furthermore, the expression of VIPR1 mRNA in group I and group II were superior than that of group III(p<0.05).Part III Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in rabbits in middle and long term1.X-ray:24 weeks after operation,theβ-TCP have been absorbed and degraded, the amont of the callus was decreasing compared with that in 12 weeks,and it began the bone moulding stage.The marrow cavity recanalization can be observed in the zone into which the bone grafts were implanted,and the degree of the bone moulding in Group I and II was higher than than in Group III. In the 48 weeks after operation the appearance of the bone defect in Group I and II is similar with the normal bone,and in Group III the bone cortex arranged in disorder. At both 24 and 48weeks after surgery, the roentgenographic scores in Groups I and II were higher than in Group III (P<0.05); and there were no significant differenced between Groups I and II (P>0.05).2. H&E and masson staining of repaired bone tissues:. Increased formation of new bone could be observed in all groups over time,whose appearance under the microscope looked like the normal bone. The thickness of cortical bone in GroupsⅠandⅡexceeded that in GroupⅢat all time points.3. Immunohistochemical staining for CGRP1R,NK1R, NPY1R and VIPR1:GroupsⅠGroupⅡboth showed a greater abundance of CGRP1R,NK1R, NPY1R and VIPR1 than the control group throughout the 48-week period. At weeks 24, all groups showed no more intense staining for CGRP1R than at week 48.The positive staining can be observed around the lacuna,the bone cell and the vascular among the bone.4. The expression of mRNA of CGRP1R:Over time, experimental group within the new bone tissue CGRP1R mRNA was expressed stably. The expression level of CGRP1R mRNA at 24th week was similar with that of at 48 week. The difference had no statistical significance. (F=4.809,0.354,0.220, p>0.05). There was significant difference between the three groups at the same time point. (F=52.604, 26.081, p<0.05). The expression of CGRP1R mRNA in groupⅡwas superior than that of groupⅠ(p<0.05), furthermore, the expression of CGRP1R mRNA in groupⅠwere not superior than that of groupⅢ(p>0.05).5. The expression of mRNA of NK1R:Over time, experimental group within the new bone tissue NK1R mRNA was expressed stably. The expression level of NKIR mRNA at 24th week was similar with that of at 48 week. The difference had no statistical significance. (F=0.404,0.121,0.247, p>0.05). There was significant difference between the three groups at the same time point. (F=186.233,124.068, p<0.05). The expression of NK1R mRNA in groupⅡwas superior than that of groupⅠ(p<0.05), furthermore, the expression of NK1R mRNA in groupⅠand groupⅡwere superior than that of groupⅢ(p<0.05).6. The expression of mRNA of NPY1R:Over time, experimental group within the new bone tissue NPY1R mRNA was expressed stably. The expression level of NPY1R mRNA at 24th week was similar with that of at 48 week. The difference had no statistical significance. (F=0.026,0.065,0.671, p>0.05). There was significant difference between the three groups at the same time point. (F=83.247,36.481, p<0.05). The expression of NPY1R mRNA in groupⅡwas superior than that of groupⅠ(p<0.05), furthermore, the expression of NPY1R mRNA in groupⅠand group Ⅱwere superior than that of groupⅢ(p<0.05).7. The expression of mRNA of VIPR1:Over time, experimental group within the new bone tissue VIPR1 mRNA was expressed stably. The expression level of VIPR1 mRNA at 24th week was similar with that of at 48 week. The difference had no statistical significance. (F=0.150,0.293,2.400, p>0.05). There was significant difference between the three groups at the same time point. (F=16.543,10.174, p<0.05). The expression of VIPR1 mRNA in groupⅡwas superior than that of groupⅠ(p<0.05), furthermore, the expression of VIPR1 mRNA in groupⅠwere superior than that of groupⅢat 24th week(p<0.05) and. the expression of VIPR1 mRNA in groupⅠwere not superior than that of groupⅢat 48th week(p>0.05)Conclusions1. CGRP1R, NK1R, NPY1R, VIPR1 can be expressed on the cell membrane of the BMSCs and osteogenetically induced BMSCs.2.The expression of the mRNA of the factor can be detected using QPCR,so this method can be used to detecte the hard tissue in the molecular level.3.Implanted separately with pure sensory nerve bundle or vascular bundle can promote osteogenesis.The expression of neuropeptide receptors can be detected in all groups using the immunohistochemistry staining,and the CGRP1R,NK1R, NPY1R and VIPR1 expression of groupⅠand groupⅡwas superior than groupⅢ. They were mainly distributed in the edge of new bone, periosteum, and around the blood vessels. 4.The expression of mRNA of the neuropeptide receptors in groupⅠandⅡwas superior than that in groupⅡin the short term, and the level of the expression were highest in 8th weeks, and then declined.5.The roentgenographic scores were increasing in all groups as time went by in the middle and lone term. The appearance of the bone defect in GroupⅠandⅡin grossly and under the microscope is similar with the normal bone,and the scores in the GroupⅢwas lowest.6.GroupsⅠandⅡboth showed a greater abundance of CGRP1R,NK1R, NPY1R and VIPR1 than the control group throughout the 48-week period.and can be observed around the lacuna,the bone cell and the vascular among the bone. 7.Over time, experimental group within the new bone tissue mRNA of the neuropeptide receptors was expressed stably in the middle and long term.The level of the expreesion in GroupⅡwas highest.8.Implanting vascular bundles can accelerate not only the process ofv ascularization, but also neurotization of the tissue-engineered bone. Our study provides a theoretical basis for constructing vascularized and neurotized tissue-engineered bone using implanted vascular bundles alone without need to use nerve tissue.