| BackgroundThe reconstruction of large bone segments remains a significant clinical problem in the case of extensive bone loss due to pathological events such as trauma, inflammation,and surgical treatment of tumors.Present therapeutic approaches include bone graft transplants(autologous,homologous or heterologous grafts), implants of different biomaterials or bone transport methods(Ilizarov technique),but none has proven to be fully satisfactory.Bone tissue engineering has the potential to become a real alternative to autologous bone grafts,but before this could happen it is necessary to solve the problem of homogenously supplying oxygen and nutrient to cells within a large scaffold.Sufficient blood-supply has the initial and decisive impact.Without efficient blood-supply,the tissue-engineered bone would be limited at repair the thin and small bone defects.The research of the distribution and function of nervous system in bone,has offered theoretical foundation and experience for the fact that the neural factor is incorporated to the construction of the tissue-engineered bone.In vivo studies,it maybe useful to test the feasibility and efficacy of using different approaches to facilitate the connection of the implant to the vascular network or innervation of the host.In this study,combining the tissue-engineering method and microsurgery skill, we implant the vascular bundle,sensory nerve bundle separately into the tissue-engineered bone to repair a great segmental femoral defect in rabbit,and observe the relationship between the osteogenesis effects and the reconstruction of vascular network or innervation in the tissue-engineered bone at early time.ObjectivesTo initially investigate the reconstruction of vascular network or innervation in the tissue-engineered bone with vascular bundle and nerve bundle implanted in vivo, and explore the mechanism of which the two methods affects osteogenesis.Materials and Methods1.42 New Zealand rabbits randomly assigned to 3 Groups were used for the experiment.Autologous bone marrow was harvested from the iliac crest of rabbits by needle aspiration and bone marrow mesenchymal stem cells(BMSCs) were proliferated and induction culture.These bone marrow-derived cells were seeded on beta-tricalcium phosphate(β-TCP).A stainless steel mini-plate was placed in front of the left femur.A reciprocating saw was then used to resect a 1.5-cm length segment of central diaphysis under saline irrigation.Tissue-engineered bone was imbedded into the defect at this location.A length of femoral vascular bundle was implanted to the side groove of tissue-engineered bone and fixed with thread(Group B,n=14). Similarly,an appropriate length of greater saphenous nerve was isolated,cut and then implanted to the side groove of tissue-engineered bone(Group C,n=14).Neither the vascular bundle graft nor the nerve graft was implanted into Group A(n=14).2.Four animals in each Group were sacrificed at 4,8 and 12 weeks after the implantations.Bone formation throughout the defects was assessed radiographically and evaluated by a radiologic score.Postmortem general and histology observation were also undertaken to examine bone formation.4%paraformaldehyde were used for the fixation of bone defects area and 10%EDTA were used for decalcification. The specimens was processed for routine paraffin imbedded to obtain serial paraffin sections(5μm)and the sections were then used to make HE and Masson sections to observe the osteogenesis of the tissue-engineered bone.Some of these sections were used for immunostaining or silver staining.3.The immunocytochemistry of vascular endothelial cell marker,CD34. Quantitative image analysis of the number of new vessels immunostained by CD34 was performed.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.4.Observed angiogenesis by ink perfusion.After 12 weeks we selected two animals in each group undertaken ink perfusion,and prepared frozen sections of tissue engineeried bone to observe the situation angiogenesis.5.The immunohistochemistry using antibodies directed against specific neuronal markers,neurofilament 200(NF200) and synaptophysin(SY).Quantitative image analysis of the integrated optical density(IOD) of immunostainings for these markers was performed.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.6.Observed the nerve reconstruction by silver staining.Gave each group samples conventional silver staining at different time points to observe nerve reconstruction of tissue engineering bone.Results1.Radiologic scoreThe radiologic scores of all the defects were increased in all groups at three time points post-implantation(P＜0.05).No significant difference between Group B and Group C in each time points(P＞0.05),but the scores in these two Groups were significantly higher than in Group A at 4,8 and 12 weeks.2.Histological observationThe histology observation of specimens in 3 Groups at 3 time points after operation.Calcification and degradation ofβ-TCP was found in all Groups at 4,8 and 12 week after operation.In Group B and Group C,material was divide into mini-particle by new bone formation,the degree of ossification around the material was higher;massive material was not degradation in Group C,and the degree of ossification around the material was lower.In all 3 Groups,new bone formation occurred around the vessel,the major way of bone formation is enchondral ossification.The extent of neovascularization and enchondral ossification in Group B and Group C were higher than Group A observed under Microscope.3.Immunohistochemistry stain results of CD34We observed the new vessels immunostained by CD34 in each group,and the total number of these new vessels was quantified.With the time prolong,the number of new vessels in each group were gradually decreased,the difference has statistical significance(F=222.578,P=0.000).The effects of sensory nerve bundle or vascular bundle implantation in the number of new vessels have statistical significance (F=1189.181,P=0.000).Factorial design ANOVA showed time and group have interaction(F=2.964,P=0.038).At 4,8 and 12 weeks,the number of new vessels in all 3 group have statistical significance(P＜0.05);In each group,the number of new vessels at all time point have statistical significance(P＜0.05).4.Ink perfusion observationsAfter 12 weeks,blood vessels structure can be seen clearly in vascular bundle implantation group material and there are a number of mature vessels with thick morphology,which are near adjacent osteoblasts.The internal blood vessels in nerve bundles implanted group with mild morphology are relatively mature,whose number is less than that of the vascular bundles implanted group.There are a small number of less mature internal blood vessels with general morphology in bone tissue engineering materials group.5.Immunohistochemistry stain results of SY and NF200With the time prolong,the expression of SY in each Group were gradually decreased,the difference has statistical signifcance(F=130.722,P=0.000).The effects of sensory nerve bundle or vascular bundle implantation in the expression of SY have statistical significance(F=103.523,P=0.000).Factorial design ANOVA showed time and group have interaction(F=4.867,P=0.004).At 4 and 8 weeks,the IOD of SY in all 3 group have statistical significance(P＜0.05);At 12 weeks,no significant difference was seen between Group B and Group C(P＞0.05),but they were obviously higher than in Group A(P＜0.05).In Group A,the IOD of SY at each time point have statistical significance(P＜0.05);In Group B and Group C,no significant difference was seen between 8 and 12 weeks(P＞0.05),but they were obviously lower than 4 weeks(P＜0.05).Small amounts of expression initially occurred at the edge of newly generated bone and periosteum and then decreased over time.With the time prolong,the expression of NF200 in each group were gradually decreased,the difference has statistical significance(F=152.848,P=0.000).The effects of sensory nerve bundle or vascular bundle implantation in the expression of NF200 have statistical significance(F=146.814,P=0.000).Factorial design ANOVA showed time and group have interaction(F=5.575,P=0.002).At 4 weeks,the IOD of SY in all 3 group have statistical significance(P＜0.05);At 8 and 12 weeks,no significant difference was seen between Group B and Group C(P＞0.05),but they were obviously higher than in Group A(P＜0.05).In each group,the IOD of SY at all time point have statistical significance(P＜0.05).The distribution of NF200 was similar with SY.Small amounts of expression initially occurred at the edge of newly generated bone and periosteum and then decreased over time.6.The results of silver stainingSilver-staining results showed that in four weeks the nerve fibers appear outside in each group of tissue-engineered bone and nerve fiber-like structures with poor form is only visible in the tissue gap inside the material.At 8 weeks,a large number of silver staining nerve fibers can be seen in the gap of new bone tissue and there are more in sensory nerve bundle implantation group and the vascular bundle implantation group than other groups.The staining nerve fibers with clear structures are staggering with each other.At 12 weeks,there are nerve fibers with good structure inside bone tissues and clear silver staining fibers are around the blood vessels.Conclusions1.In the early stage,both tissue-engineering bone implanted with vascular bundle and that implanted with sensory nerve bundle can enhance the repairing of large segmental defects in vivo,but there is no significant difference between two groups.2.In the early stage,both tissue-engineering bone implanted with vascular bundle and that implanted with sensory nerve bundle can obviously promote the reconstruction of blood supply in tissue-engineered bone,and the former group is better than the latter.3.In the early stage,both tissue-engineering bone implanted with vascular bundle and that implanted with sensory nerve bundle can obviously improve the reconstruction of innervation in tissue-engineered bone,but there is no significant difference between two groups.4.Angiogenesis and innervation in tissue-engineered bone decreased at different content in all groups during the early stage,suggesting that the early phase(4～8 weeks) maybe essential to the reconstruction of blood supply and innervation.5.The reconstruction of blood supply and innervation play an important role in the osteogenesis of tissue-engineered bone and the reconstruction of large bony defects.
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