| ObjectivesAs the bone tissue engineering is studied deeply and gradually aimed to theclinical application, a main problem is how to construct large tissue-engineered boneto alive and give play to the role of function in the human body. Sufficientblood-supply has the initial and decisive impact .Without efficient blood-supply, thetissue-engineered bone would be limited at repair the thin and small bone defects. Theresearch of the distribution and function of nervous system in bone, has offeredtheoretical foundation and experience for the fact that the neural factor is incorporatedto the construction of the tissue-engineered bone. Utilizing the tissue-engineeringmethods and microsurgery skill, jointly plant the blood vessles, sense nerves, motornerves and the two into the big section of tissue engineering bone separately, toinvestigate the osteogenesis effects of nerves-tract and blood vessles implant intolarge tissue engineered bone in vivo at early time.Using immunohistochemistry stainof Calcitonin gene related peptide(CGRP), protein S-100 and Neuropeptide Y(NPY)in order to observes it's mechanism of the osteogenesis effects. Methods1. osteogenesis effects of nerves implantation in the large tissue engineered bone1.1 48 New Zealand rabbits were used for the experiment. Autologous bonemarrow stromal cells(BMSCs)was obtained from the iliac crest of rabbits by needleaspiration and implanted into beta-tricalcium phosphate bring side trough. Theanimal used for experiment is randomly divided into 4 groups: Group A,tissue-engineering group; Group B, tissue engineered bone+motor nerve tract group;Group C, tissue engineered bone+sensory nerve tract group; Group D, tissueengineered bone+sensory and motor nerve tract group. 1.5cm long bone defects wasmade in the rabbit's femur and fixed with four-hole plate. Then, the defects wereimplanted with tissue-engineered bone accompanied with nerves.1.2 At 4w, 8w, 12w postoperatively ,four animals from each group were killedfor the observation of new bone formation, reacts with host's bone interface andinterior bridging callus .Paraformaldehyde of 40g/L were used for the fixation ofbone defects area, EDTA of 150g/L were used for decalcification .Paraffin imbedding,using HE and MASSON stain. Using the image analysis system to detect thepercentage of new bone in defects area. The data were treated with 4×3 factorialdesign ANOVA, any significant deviation were detected with LSD multiplecomparison, in order to compare the reparation of bone defects area.1.3 Immunohistochemistry stain of CGRP and protein S-100 were used. Theimage analyses determined the grey scale of each group, calculate out the averagegrey scale. The data were treated with 4×3 factorial design ANOVA, any significantdeviation were detected with LSD multiple comparison, in order to approach themechanism of osteogenesis.2.The osteogenesis effects of vessel tract or sensory nerve tract implantationin large tissue-engineered bone2.1 36 New Zealand rabbits were used for the experiment. Tissue-engineeredbone was made as described in part one. Vessel tract and sensory nerve tract wereimplanted into it separately.2.2 At 4w, 8w, 12w postoperatively ,four animals from each group were killed for the observation of new bone formation, reacts with host's-bone interfaceand interior bridging callus. Paraformaldehydeof 40g/L were used for the fixationof bone defects area, EDTA of 150g/L decalcification and paraffin imbedding,using HE and MASSON stain. Using the image analysis system to detect thepercentage of new bone in defects area. The data were treated with 3×3 factorialdesign ANOVA, any significant deviation were detected with LSD multiplecomparison, in order to compare the reparation of bone defects area.2.3 Immunohistochemistry stain of CGRP and NPY were used. The image analysesdetermined the grey scale of each group, calculate out the average grey scale. Thedata were treated with 3×3 factorial design ANOVA, any significant deviation weredetected with LSD multiple comparison, in order to approach the mechanism ofosteogenesis.Results1.osteogenesis effects of nerves implantation in the large tissue engineered bone1.1 Observation on the specimens: new bone formation were gradually increasedin every group after operations. Callus in group C and D were superior than in groupA and D.1.2 Histological observation and results analysis: With the time prolong, newbone formation were gradually increased, the difference has statistical significance(F=456.427,P=0.000). The effects of sensory nerve tract implantation inosteogenesis has statistical significance (F=112.359,P=0.000). Factorial designANOVA showed time and sensory nerve tract implantation have interaction(F=3.111,P=0.015). The formation of new bone in group C and group D weresuperior than in group A and group B at every time point by multiple comparison ofLSD. No obvious difference were seen between group A and group B. No obviousdifference were seen between group C and group D.1.3 Immunohistochemistry stain results of CGRP and S-100: The expression ofCGRP in new bone were grown with time, the difference has statistical significance(F=258.046,P=0.000 ) The effects of sensory nerve tract implantation inpromoting the expression of CGRP has statistical significance (F=689.552,P= 0.000). Factorial design ANOVA showed time and sensory nerve tract implantationhave interaction (F=25.915,P=0.020). The gray scale of CGRP in group C andgroup D were superior than in group A and group B at every time point by multiplecomparison of LSD. No obvious difference were seen between group A and group B.No obvious difference were seen between group C and group D.The expression of S-100 in new bone were grown with time, the difference hasstatistical significance (F=346.524,P=0.000). The effects of sensory nerve tractimplantation in promoting the expression of S-100 has statistical significance(F=713.807,P=0.000). Factorial design ANOVA showed time and sensory nervetract implantation have interaction (F=2.909,P=0.020). The gray scale of S-100 ingroup C and group D were superior than in group A and group B at every time pointby multiple comparison of LSD. No obvious difference were seen between group Aand group B. No obvious difference were seen between group C and group D.2.1 Observation on the specimens: new bone formation were gradually increasedin every group after operations. Callus in group C and B were superior than in groupA.2.2 Histological observation and results analysis: With the time prolong, newbone formation were gradually increased, the difference has statistical significance(F=105.735,P=0.000). The effects of sensory nerve tract or vessel tractimplantation in osteogenesis have statistical significance (F=290.008,P=0.000).Factorial design ANOVA showed time, sensory nerve tract and vessels tractimplantation have interaction (F=3.177,P=0.029). The formation of new bone ingroup B were superior than in group A and group C at the four weeks, group C weresuperior than group A. Group B and group C were superior than group A at the eightand twelve weeks. No obvious difference were seen between group C and group B atthe eight and twelve weeks.2.3 Immunohistochemistry stain results of CGRP and NPY: With the timeprolong, the expression of CGRP in each group were gradually increased, thedifference has statistical significance (F=95.392,P=0.000). The effects of sensory nerve tract or vessel tract implantation in increasing the expression of CGRP havestatistical significance (F=415.324,P=0.000). Factorial design ANOVA showedtime, sensory nerve tract and vessels tract implantation have interaction (F=16.475,P=0.000). The gray scale of CGRP in group B were superior than in group A andgroup C at the four weeks, group C were superior than group A. Group B and group Cwere superior than group A at the eight and twelve weeks. No obvious differencewere seen between group C and group B at the eight and twelve weeks.With the time prolong, the expression of NPY in each group were graduallyincreased, the difference has statistical significance (F=892.240,P=0.000). Theeffects of sensory nerve tract or vessel tract implantation in increasing the expressionof NPY have statistical significance (F=118.373,P=0.000). Factorial designANOVA showed time, sensory nerve tract and vessels tract implantation haveinteraction (F=30.5.9,P=0.000). The gray scale ofs-I00 in group B were superiorthan in group A and group C at the four weeks, group C were superior than group A.Group B and group C were superior than group A at the eight and twelve weeks. Noobvious difference were seen between group C and group B at the eight and twelveweeks.Conclution1. Implantation of sensory nerve tract or sensory nerve tract associated withmotor nerve tract into tissue-engineered bone can improve osteogenesis, butimplantation of motor nerve tract can not achieve the similar efffect according tothis experiment.2. Implantation of sensory nerve tract or sensory nerve tract associated withmotor nerve tract into tissue-engineered bone can increase the expression ofCGRP and S-100 in newly formed bone, but implantation of motor nerve tract cannot achieve the similar efffect according to this experiment.3. In the early time(four weeks), vessel tract implantation was better thansensory nerve tract implantation. At eight and twelve weeks, there were no obviousdifference between the two groups. 4. Implantation of vessel tract into tissue-engineered bone can increase theexpression of CGRP and NPY in newly formed bone.5. Implantation of vessel tract into tissue-engineered bone can increase therebuilding surface to improve the rebuilding speed, then raise the speed andquality of osteogenesis.
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