非血管化游离骨移植及同期种植的实验研究
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摘要
非血管化游离骨移植(NVBG)的出现为众多颌骨缺损的患者带来了福音,直至目前这一技术仍是最常用的颌骨修复手段之一。对于NVBG的愈合过程,传统的“爬行替代”理论认为移植骨终将吸收,被新生骨组织所逐渐替代。在这一理论的指导下,NVBG同期种植过去一直被视为禁忌。本实验在课题组前期研究的基础上分别用犬和转基因小鼠构建NVBG植骨模型,以移植骨转归、新骨生成、血供重建、种植体骨结合为研究重点,试图全面反映移植骨愈合全貌;探讨新生骨的来源和促进种植体骨结合的因素,为NVBG及同期种植的临床应用提供理论和实验基础。
本实验分为3个部分。
第一部分构建犬下颌骨缺损游离髂骨移植的动物模型,并以此为基础,对移植骨的细胞活力、成骨方式、血供重建、VEGF和TGF-β1表达作一深入研究,探讨NVBG移植骨的愈合规律和促进因素。
实验方法:选用实验犬12只(术前1个月拔除双侧下颌前磨牙),选取一侧下颌骨造成2.5cm全层骨缺损,植入同样大小的全层自体髂骨,重建钛板固定。髂骨块离体时间控制在20min以内。术后1、2、4个月取材,行大体、X线、下齿槽动脉造影、组织学、墨汁灌注微血管计数、骨组织形态计量学、TGF-β1原位杂交、VEGF和TGF-β1免疫组织化学观察。
实验结果: X线观察表明移植骨区呈现渐进性的骨修复和改建的过程。术后1个月:移植骨块密度不均,近牙槽嵴顶处有少量吸收,交界区呈不规则透光影。术后2个月:移植骨较1个月前密度有所增加,但仍低于周边颌骨;交界区仍有部分透光影。术后4个月:植骨区形态不规则,密度明显高于周围颌骨;移植骨与宿主骨已形成完整连接。动脉造影发现下齿槽动脉中断,动脉末端可见细小分支发出。
组织学观察显示术后1个月:移植骨表层骨质大部分已被吸收,新骨生成以微血管为中心展开;骨膜下大量未分化间充质细胞聚集,可见软骨细胞团块。术后2个月:移植骨进一步吸收,仍可见到正常骨细胞形态。新生骨小梁粗大融合,连接成片,部分已矿化成熟,微血管数目较多。术后4个月:新生骨、移植骨与宿主骨已基本融为一体,新的骨髓腔开始形成,血管数量减少。
对移植骨陷窝的着色计数表明,术后1个月,移植骨有近30%骨细胞存活,2个月存活率接近20%。移植骨术后1~2个月边缘部位的骨矿化沉积率明显高于中间部位,也高于周围颌骨近2倍;术后4个月移植骨各部位矿化速率与正常骨质无明显差异。对矿化骨小梁相关参数(TV/BV、Tb.Th和Tb.Sp)、未矿化骨基质相关参数(OS/BS、O.Th)和骨吸收参数(N.Oc/BS、ES/BS)的形态计量学分析结果表明:术后1~2个月移植骨不同部位间上述3组参数均有明显差异。边缘部位在骨和骨基质的生成方面明显高于中间区域;而在骨吸收指标上低于中间区域。术后4个月不同部位间的成骨差异缩小,接近正常水平。从同一部位各个时间点的比较来看,无论是移植骨边缘还是中部,3组参数均有明显差异。微血管计数分析,移植骨近中、远中部位各时间点微血管数目并无明显差异,中部则处于明显乏血管区域。术后1~2个月,近、远中部位微血管数目均较周围颌骨显著增多,2个月时血管数量处于最高峰。而同期的中间部位血管数目则明显少于周边颌骨。术后4个月时各部位血管数目接近正常。结合血管和新生骨的计量分析结果,推测新生血管和新骨生成呈正相关关系,与骨吸收呈负相关关系。
VEGF免疫组化观察发现VEGF定位于血管内皮细胞,其表达与微血管数量呈明显量效关系。术后1~2个月,VEGF在血管内皮细胞中强阳性表达。术后4个月呈弱阳性表达。原位杂交和免疫组化观察发现TGF-β1在多种组织细胞内均有表达。术后1个月,TGF-β1在新生软骨细胞、成骨细胞、破骨细胞内呈强阳性表达;术后2个月,成骨细胞和破骨细胞内TGF-β1仍呈强阳性表达;移植骨只有少部分有弱阳性表达;术后4个月,新生骨细胞和基质内有阳性表达。
上述结果表明不同时期NVBG移植骨块均能保留部分活力,新骨在术后1个月开始大量长入,术后2个月进一步生长并持续矿化,术后4个月开始成熟改建。新骨生长以新生血管为中心展开,新骨生成与新生血管有正相关性。VEGF和TGF-β1在血管再生和新骨生成中发挥重要作用,二者之间有协同作用。
第二部分在前期游离植骨的基础上,同期种植体植入。以种植体骨结合为着眼点,探讨NVBG同期种植种植体骨界面和种植体周围骨质的生成和改建规律以及VEGF和TGF-β1在种植体骨结合中的作用。
实验方法:选用实验犬24只,分为2组,术前1个月拔除两侧下颌前磨牙。如上述方法形成下颌骨缺损的游离髂骨修复,A组在髂骨块的中部和侧方分别植入1枚种植体。B组将种植体与rhVEGF165和rhTGF-β1复合后再植入移植髂骨内。犬的另一侧下颌常规植入1枚种植体作为对照。术后1、2、4个月取材,行大体、X线、组织学、显微CT观察和种植体骨结合力测试。
实验结果: X线观察术后1个月,种植体周围有明显透光影;术后2个月,种植体周围骨质有轻度阻射,密度低于周围移植骨。术后4个月,种植体与周围骨质大部分形成直接结合,无透射间隙存在。从种植体骨结合形成时间来看,术后1个月,BIC不足5%,术后2个月为25%左右;而同时间对照组的BIC分别为10%、35%。种植体骨界面的力学测试也证明术后1~2个月骨移植组较对照组差异明显。术后4个月时,A、B两组边缘位置的BIC达到60%以上,种植体骨界面结合力接近220N,已形成较好的骨结合。M-CT骨形态计量分析表明术后各时间点A、B两组在TV/BV、Tb.Th、Tb.Sp、Tb.N四组参数测量上均与对照组有明显差异;边缘部位在术后1、2、4个月的骨质新生和改建速度明显高于中间部位;从同一部位不同时间分析,骨小梁呈现出从少到多的量和质的累计过程,而对照组的这一进程明显快于A、B组。从种植体周围骨质密度变化来看,各时间点A、B组骨小梁组织矿化密度和连接密度均小于对照组。边缘部位各时间点骨连接密度显著高于中间部位。VEGF和TGF-β1同时用于种植体局部,结果表明术后1、2个月A组BIC较B组显著增高;对种植体周围骨小梁的进一步分析表明,A组的成骨的作用较B组明显增强。
上述试验结果表明NVBG同期种植体植入早期(术后1~2个月),种植体骨界面以纤维连接为主,术后4个月,有效的种植体骨结合形成。边缘部位BIC和成骨效率高于中间部位。TGF-β1和VEGF对种植体骨结合早期(术后1~2个月)有明显的促进作用。
第三部分利用GFP-C57BL/6小鼠建立NVBG动物模型,以新生骨来源和移植骨的成骨能力为研究重点,通过观察在不同时间点移植骨和新生骨细胞GFP表达,判断移植骨活力和新生骨来源。
实验方法:1、取GFP-C57BL/6小鼠胫骨,作体外成骨细胞培养,观察成骨细胞的繁殖能力和连续传代对GFP表达的影响。2、取GFP-C57BL/6和C57BL/6小鼠各15只,切取GFP-C57BL/6 5mm胫骨,完整保留骨膜及部分肌肉附着,植入C57BL/6小鼠同法截骨后的骨缺损区。分别于术后3天、1、2、3、4周取材,行X线、组织学和荧光显微镜观察。
实验结果: X线观察术后3天~1周,移植骨与宿主骨间隙明显,未见明显移植骨吸收。术后2~3周,移植骨边缘隐约可见膨隆的骨痂形成,植骨间隙模糊。术后4周,移植骨与宿主形成明显的条索样骨性连接。组织学观察术后3天移植骨区出血炎症反应明显。术后1周移植骨表面进一步吸收,破骨细胞大量增生;未分化间充质细胞、成骨前体细胞聚集。术后2周移植区由外向内可分为6个层次,依次为肌肉、骨膜、新生软骨层、网状骨小梁层、移植骨、移植骨骨髓腔。术后3周新生骨小梁粗大,部分与移植骨相融合。有早期的骨髓腔形成。术后4周新生骨小梁进一步融合,外层已有早期板状骨形成,内层与余留移植骨融合不易区分。荧光显微镜观察术后各时间点移植骨周围的肌肉一直呈现亮绿色荧光;新生骨小梁周围的成骨细胞也有明显荧光表达。移植骨骨细胞内有部分荧光表达。
上述结果表明GFP-C57BL/6小鼠成骨细胞体外培养、传代后,仍对GFP蛋白有稳定表达。GFP-C57BL/6小鼠NVBG胫骨移植术后各时间点移植骨、肌肉、新生成骨细胞均有GFP荧光表达,充分说明在适宜的条件下NVBG移植骨并非完全经历“爬行替代”,移植骨自身有很强的成骨能力。移植骨膜在新骨生成中起重要作用。
本研究从多个角度、不同层面全景式的展现了NVBG的愈合全貌。结果发现NVBG移植骨并未完全经历“爬行替代”过程,NVBG同期种植在术后4月能形成较好的骨结合。这是对“爬行替代”理论的补充和修正,具有很强的临床指导意义。实验中首次将GFP转基因小鼠用于NVBG骨移植研究,结果表明该动物模型具有良好的直观性和特异性;首次将VEGF和TGF-β1联合用于NVBG同期种植,发现二者有明显促进种植体骨结合的作用。
NVBG, as an effective method for the repair of bone defects, is still widely used up to now. Conventional view of incorporation of non-vascularized bone graft to the recipient is called creeping substitution. The grafts will finally be totally replaced by the host and all bone-forming cells originate from the recipients. According to this theory, it’s impossible to perform NVBG with implants at the same time. Following our previous series studies, this research pays much attention to the turnover of the grafted bone, formation of the new bone, reconstruction of vascularization, and bone-implant osseointegration, using dogs and transgenic mice as NVBG animal model. The purpose of this study is 1. providing a whole picture to the healing process of NVBG with and without dental implants; 2. demonstrating the effect of VEGF and TGF-β1 in bone healing and bone-implant osseointegration; 3. analyzing the fate of the grafts and the origin of the new bone.
Part 1: In this part, we developed a NVBG experiment model in dogs, and then studied the cell viability of the grafted bone, new bone formation and angiogenesis, and discussed the rule of healing and roles of healing promoting factors.
Methods: A 2.5cm-long-defect was made in one side of 12 native dogs’mandible, and repaired with the same sized iliac bone taken from the pars iliaca of themselves. A rigid fixation was performed with reconstruction plate and steel-wires (Ex vivo time of the graft was less than 15 minutes). Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and examed by clinic, X-ray, inferior alveolar artery arteriography, histology, capillary vesscle count, bone histomorphometry, VEGF and TGF-β1 immunohistochemistry and TGF-β1 in situ hybridization.
Results: One dog suffered from infection and its grafted bone was totally necrosis. We supplemented a dog to continue the study. Others remained healthy during the healing period. X-ray image showed that 1 month after surgery there was a little absorption in the grafted bone and between the connected areas. Bone density of grafted bone was increased in 2 months after surgery, but still lower than that of the normal mandible. In 4 months, there was no distance between the grafted bone and the host, and the density of grafted area was even higher than the host. Arteriography showed the stop of inferior alveolar artery in the operation side.
Histologically, 1 month after surgery, parts of the superficial transplanted bone were absorbed and new bone formation was observed around the capillary vesscles. Large amounts of mesenchymal cells and chondrocytes were aggregated. Grafted bone continued to be absorbed and newly formed trabecular grew together and became mineralized in 2 months after surgery. In 4 months, newly formed bone incorporated to the grafted bone and it’s difficult to distinguish the host and the grafts.
The analysis of the grafted bone lacuna vacancy rate showed that nearly 30 percent of the grafted bone cells survived 1 month after transplantation and the data decrease to 20 percent another month later. The data of bone mineralization deposition rate showed that the marginal parts of the grafted bone was higher than the middle in 1-2 months after surgery, but showed no difference 4 months later. The results of bone histomorphometry showed that trabecular related parameters (TV/BV、Tb.Th and Tb.Sp),osteoid related parameters (OS/BS、O.Th) and bone absorption related parameters(N.Oc/BS、ES/BS)had statistic difference between the marginal position and middle position of the grafted bone in 1-2 months after surgery. For trabecular and osteoid related parameters, marginal parts are higher than middle parts, while it’s just opposite in the parameters of bone absorption. Four months later, the statistic difference between the positions of the grafted bone disappeared. In 1, 2 or 4 months after surgery, the marginal parts showed much higher level than the middle parts in all the parameters. From the results of capillary vesscle counts, it can be found that the marginal position, no matter the proximal or the distal, acquired much more blood supply than the middle part. In 1-2 months after surgery, the number of capillary vesscles was much larger in marginal parts, even more than the normal mandible area. Four months later, the data became to decrease to normal level. From above data, it could be presumed that angiogenesis have positive correlation with the new bone formation and negative correlation with bone resorption.
Immunohistochemistry staining showed that VEGF localized in the vascular endothelial cells. It showed a strong positive expression in ECs 1-2 months after surgery, while a weak positive expression 4 months later. Immunohistochemistry and in situ hybridization of TGF-β1 revealed that newly formed chondrocytes, osteoblasts and osteoclasts showed a stong positive expression 1 month after surgery and a weak positive expression 2 months after surgery. Four months later only newly formed osteocytes and matrix showed positive expression.
The results indicate that: 1. Parts of the NVBG grafted bone survived after transplantation. 2. New bone formation began 1 month after surgery and newly formed trabecular was gradually mineralized and became matured 4 months after surgery. 3. New bone formed arround the capillary vesscles and new bone formation have positive correlation with the angiogenesis. 4. VEGF and TGF-β1 played an important role in osteogenesis and angiogenesis.
PartⅡ: Based on the previous studies, in this part we put dental implants, attached with rhVEGF165 and rhTGF-β1, into the transplanted iliac bone and observed the interface between the bone and titanium implants.
Methods: Twenty-four dogs were divided into 2 groups and the NVBG was performed the same as before. In group A, two implants were put into the marginal part and the middle part of the transplanted iliac bone. In group B, the implants were attached with rhVEGF165 and rhTGF-β1 first and then were implanted into the grafted bone in the same position as group A. An implant was put into the other side of the mandible as control. Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and measured by clinic, X-ray, histology, M-CT and bone-implant bonding force test.
Results: One dog suffered from infection and the grafted bone with implants was exposed in the mouth. We supplemented a dog to continue the study. X-ray image showed that 1 month after surgery there was an obvious gap between implants and the surrounding bone and 4 months after surgery, no lucency was found in this area. BIC was no more than 5 percent 1 month after surgery and 20 percent 2 months after surgery in experiment group, while was 10 percent and 26 percent respectively in the control. Four months after surgery, 60 percent BIC acquired in experiment group and 70 percent in the control. The test of bone-implant bonding force showed that it’s nearly 220N in the position of marginal implants 4 months after surgery. Bone histomorphometry made by M-CT showed that the growth rate of new trabecular formation arounding implants in marginal position were higher than those in middle position. From the analysis of the same position in different time, trabecular around implants showed a gradually bone formation process. In this process control group showed much higher speed than group A or B. In group A, bone tissue mineral density and connectivity density around implant were much higher than that in group B. Connectivity density in marginal position was higher than that in middle position. For the application of the rhVEGF165 and rhTGF-β1, the effectiveness of bone and BIC formation was greater in group A than in group B.
The results indicate that: In early healing period of the NVBG with implants, the interface of bone and implant was full of connective tissues, and effective osseointegration was formed 4 months after surgery. VEGF and TGF-β1 can promote osteogenesis and bone implant osseointegration.
Part III: In this part, tibia grafts taken from GFP transgenic mice was put into its isogenous’tibia defect and the fate of the grafted bone and the origin of the new bone were analyzed by the GFP fluorescence expression.
Methods: 1.Cell culture. GFP-C57BL/6 osteoblasts were cultured and its’propagation ability and GFP fluorescence expression were observed in vitro. 2. Animal study. A 5mm-long-tibia with intact periosteum and parts of muscles was made in 15 GFP-C57BL/6 mice and was put into the same defect of 15 C57BL/6 mice. Animals were sacrificed at 3 days, 1, 2, 3 and 4weeks after surgery respectively and the grafts were harvested and observed by clinic, X-ray, histology and fluorescence microscopy.
Results: Two mice were suffered from infection, so we supplemented 2 GFP mice to continue the study. X-ray image showed that in 3 -7d after surgery, the lucency gap between the grafted bone and host bone was obvious. In 2-3 weeks after surgery, porosis around the grafted bone was formed and 4 weeks later, the connection of bone and host formed. Histologically, 3 days: There were lots of blood clots and inflammatory cells in the grafted areas. The transplanted bone was partly absorbed. 1 week: Blood clots were replaced by vascularized granulation tissues. The union between the grafted bone and recipient bone was full of mesenchymal cells undergoing chondrocytic differentiation. Extensive bone absorption was observed at the periphery of the bone graft indicated by the numbers of osteoclasts. 2 weeks: The grafted areas could be divided into five layers according to different tissues: grafted muscles, grafted periosteum, newly formed cartilage, newly formed trabeculae and grafted bone. 3 weeks: The remained grafted bone began to integrate with newly formed mineralized trabecular. 4 weeks: Newly formed bone incorporated to the grafted bone and it’s difficult to distinguish them. Bright green light was found in transplanted muscles all the time under fluorescent microscopy. The osteoblasts and newly formed trabeculae and parts of the grafted bone also showed GFP expression.
The results indicated that: 1. GFP-C57BL/6 osteoblasts showed normal propagation ability and a stable GFP fluorescence expression was found in the continuous passaged cells in vitro. 2. Parts of non-vascularized grafted bone can keep viability and had the potential of osteogenesis. The grafted periosteum played an important role in new bone formation.
本实验分为3个部分。
第一部分构建犬下颌骨缺损游离髂骨移植的动物模型,并以此为基础,对移植骨的细胞活力、成骨方式、血供重建、VEGF和TGF-β1表达作一深入研究,探讨NVBG移植骨的愈合规律和促进因素。
实验方法:选用实验犬12只(术前1个月拔除双侧下颌前磨牙),选取一侧下颌骨造成2.5cm全层骨缺损,植入同样大小的全层自体髂骨,重建钛板固定。髂骨块离体时间控制在20min以内。术后1、2、4个月取材,行大体、X线、下齿槽动脉造影、组织学、墨汁灌注微血管计数、骨组织形态计量学、TGF-β1原位杂交、VEGF和TGF-β1免疫组织化学观察。
实验结果: X线观察表明移植骨区呈现渐进性的骨修复和改建的过程。术后1个月:移植骨块密度不均,近牙槽嵴顶处有少量吸收,交界区呈不规则透光影。术后2个月:移植骨较1个月前密度有所增加,但仍低于周边颌骨;交界区仍有部分透光影。术后4个月:植骨区形态不规则,密度明显高于周围颌骨;移植骨与宿主骨已形成完整连接。动脉造影发现下齿槽动脉中断,动脉末端可见细小分支发出。
组织学观察显示术后1个月:移植骨表层骨质大部分已被吸收,新骨生成以微血管为中心展开;骨膜下大量未分化间充质细胞聚集,可见软骨细胞团块。术后2个月:移植骨进一步吸收,仍可见到正常骨细胞形态。新生骨小梁粗大融合,连接成片,部分已矿化成熟,微血管数目较多。术后4个月:新生骨、移植骨与宿主骨已基本融为一体,新的骨髓腔开始形成,血管数量减少。
对移植骨陷窝的着色计数表明,术后1个月,移植骨有近30%骨细胞存活,2个月存活率接近20%。移植骨术后1~2个月边缘部位的骨矿化沉积率明显高于中间部位,也高于周围颌骨近2倍;术后4个月移植骨各部位矿化速率与正常骨质无明显差异。对矿化骨小梁相关参数(TV/BV、Tb.Th和Tb.Sp)、未矿化骨基质相关参数(OS/BS、O.Th)和骨吸收参数(N.Oc/BS、ES/BS)的形态计量学分析结果表明:术后1~2个月移植骨不同部位间上述3组参数均有明显差异。边缘部位在骨和骨基质的生成方面明显高于中间区域;而在骨吸收指标上低于中间区域。术后4个月不同部位间的成骨差异缩小,接近正常水平。从同一部位各个时间点的比较来看,无论是移植骨边缘还是中部,3组参数均有明显差异。微血管计数分析,移植骨近中、远中部位各时间点微血管数目并无明显差异,中部则处于明显乏血管区域。术后1~2个月,近、远中部位微血管数目均较周围颌骨显著增多,2个月时血管数量处于最高峰。而同期的中间部位血管数目则明显少于周边颌骨。术后4个月时各部位血管数目接近正常。结合血管和新生骨的计量分析结果,推测新生血管和新骨生成呈正相关关系,与骨吸收呈负相关关系。
VEGF免疫组化观察发现VEGF定位于血管内皮细胞,其表达与微血管数量呈明显量效关系。术后1~2个月,VEGF在血管内皮细胞中强阳性表达。术后4个月呈弱阳性表达。原位杂交和免疫组化观察发现TGF-β1在多种组织细胞内均有表达。术后1个月,TGF-β1在新生软骨细胞、成骨细胞、破骨细胞内呈强阳性表达;术后2个月,成骨细胞和破骨细胞内TGF-β1仍呈强阳性表达;移植骨只有少部分有弱阳性表达;术后4个月,新生骨细胞和基质内有阳性表达。
上述结果表明不同时期NVBG移植骨块均能保留部分活力,新骨在术后1个月开始大量长入,术后2个月进一步生长并持续矿化,术后4个月开始成熟改建。新骨生长以新生血管为中心展开,新骨生成与新生血管有正相关性。VEGF和TGF-β1在血管再生和新骨生成中发挥重要作用,二者之间有协同作用。
第二部分在前期游离植骨的基础上,同期种植体植入。以种植体骨结合为着眼点,探讨NVBG同期种植种植体骨界面和种植体周围骨质的生成和改建规律以及VEGF和TGF-β1在种植体骨结合中的作用。
实验方法:选用实验犬24只,分为2组,术前1个月拔除两侧下颌前磨牙。如上述方法形成下颌骨缺损的游离髂骨修复,A组在髂骨块的中部和侧方分别植入1枚种植体。B组将种植体与rhVEGF165和rhTGF-β1复合后再植入移植髂骨内。犬的另一侧下颌常规植入1枚种植体作为对照。术后1、2、4个月取材,行大体、X线、组织学、显微CT观察和种植体骨结合力测试。
实验结果: X线观察术后1个月,种植体周围有明显透光影;术后2个月,种植体周围骨质有轻度阻射,密度低于周围移植骨。术后4个月,种植体与周围骨质大部分形成直接结合,无透射间隙存在。从种植体骨结合形成时间来看,术后1个月,BIC不足5%,术后2个月为25%左右;而同时间对照组的BIC分别为10%、35%。种植体骨界面的力学测试也证明术后1~2个月骨移植组较对照组差异明显。术后4个月时,A、B两组边缘位置的BIC达到60%以上,种植体骨界面结合力接近220N,已形成较好的骨结合。M-CT骨形态计量分析表明术后各时间点A、B两组在TV/BV、Tb.Th、Tb.Sp、Tb.N四组参数测量上均与对照组有明显差异;边缘部位在术后1、2、4个月的骨质新生和改建速度明显高于中间部位;从同一部位不同时间分析,骨小梁呈现出从少到多的量和质的累计过程,而对照组的这一进程明显快于A、B组。从种植体周围骨质密度变化来看,各时间点A、B组骨小梁组织矿化密度和连接密度均小于对照组。边缘部位各时间点骨连接密度显著高于中间部位。VEGF和TGF-β1同时用于种植体局部,结果表明术后1、2个月A组BIC较B组显著增高;对种植体周围骨小梁的进一步分析表明,A组的成骨的作用较B组明显增强。
上述试验结果表明NVBG同期种植体植入早期(术后1~2个月),种植体骨界面以纤维连接为主,术后4个月,有效的种植体骨结合形成。边缘部位BIC和成骨效率高于中间部位。TGF-β1和VEGF对种植体骨结合早期(术后1~2个月)有明显的促进作用。
第三部分利用GFP-C57BL/6小鼠建立NVBG动物模型,以新生骨来源和移植骨的成骨能力为研究重点,通过观察在不同时间点移植骨和新生骨细胞GFP表达,判断移植骨活力和新生骨来源。
实验方法:1、取GFP-C57BL/6小鼠胫骨,作体外成骨细胞培养,观察成骨细胞的繁殖能力和连续传代对GFP表达的影响。2、取GFP-C57BL/6和C57BL/6小鼠各15只,切取GFP-C57BL/6 5mm胫骨,完整保留骨膜及部分肌肉附着,植入C57BL/6小鼠同法截骨后的骨缺损区。分别于术后3天、1、2、3、4周取材,行X线、组织学和荧光显微镜观察。
实验结果: X线观察术后3天~1周,移植骨与宿主骨间隙明显,未见明显移植骨吸收。术后2~3周,移植骨边缘隐约可见膨隆的骨痂形成,植骨间隙模糊。术后4周,移植骨与宿主形成明显的条索样骨性连接。组织学观察术后3天移植骨区出血炎症反应明显。术后1周移植骨表面进一步吸收,破骨细胞大量增生;未分化间充质细胞、成骨前体细胞聚集。术后2周移植区由外向内可分为6个层次,依次为肌肉、骨膜、新生软骨层、网状骨小梁层、移植骨、移植骨骨髓腔。术后3周新生骨小梁粗大,部分与移植骨相融合。有早期的骨髓腔形成。术后4周新生骨小梁进一步融合,外层已有早期板状骨形成,内层与余留移植骨融合不易区分。荧光显微镜观察术后各时间点移植骨周围的肌肉一直呈现亮绿色荧光;新生骨小梁周围的成骨细胞也有明显荧光表达。移植骨骨细胞内有部分荧光表达。
上述结果表明GFP-C57BL/6小鼠成骨细胞体外培养、传代后,仍对GFP蛋白有稳定表达。GFP-C57BL/6小鼠NVBG胫骨移植术后各时间点移植骨、肌肉、新生成骨细胞均有GFP荧光表达,充分说明在适宜的条件下NVBG移植骨并非完全经历“爬行替代”,移植骨自身有很强的成骨能力。移植骨膜在新骨生成中起重要作用。
本研究从多个角度、不同层面全景式的展现了NVBG的愈合全貌。结果发现NVBG移植骨并未完全经历“爬行替代”过程,NVBG同期种植在术后4月能形成较好的骨结合。这是对“爬行替代”理论的补充和修正,具有很强的临床指导意义。实验中首次将GFP转基因小鼠用于NVBG骨移植研究,结果表明该动物模型具有良好的直观性和特异性;首次将VEGF和TGF-β1联合用于NVBG同期种植,发现二者有明显促进种植体骨结合的作用。
NVBG, as an effective method for the repair of bone defects, is still widely used up to now. Conventional view of incorporation of non-vascularized bone graft to the recipient is called creeping substitution. The grafts will finally be totally replaced by the host and all bone-forming cells originate from the recipients. According to this theory, it’s impossible to perform NVBG with implants at the same time. Following our previous series studies, this research pays much attention to the turnover of the grafted bone, formation of the new bone, reconstruction of vascularization, and bone-implant osseointegration, using dogs and transgenic mice as NVBG animal model. The purpose of this study is 1. providing a whole picture to the healing process of NVBG with and without dental implants; 2. demonstrating the effect of VEGF and TGF-β1 in bone healing and bone-implant osseointegration; 3. analyzing the fate of the grafts and the origin of the new bone.
Part 1: In this part, we developed a NVBG experiment model in dogs, and then studied the cell viability of the grafted bone, new bone formation and angiogenesis, and discussed the rule of healing and roles of healing promoting factors.
Methods: A 2.5cm-long-defect was made in one side of 12 native dogs’mandible, and repaired with the same sized iliac bone taken from the pars iliaca of themselves. A rigid fixation was performed with reconstruction plate and steel-wires (Ex vivo time of the graft was less than 15 minutes). Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and examed by clinic, X-ray, inferior alveolar artery arteriography, histology, capillary vesscle count, bone histomorphometry, VEGF and TGF-β1 immunohistochemistry and TGF-β1 in situ hybridization.
Results: One dog suffered from infection and its grafted bone was totally necrosis. We supplemented a dog to continue the study. Others remained healthy during the healing period. X-ray image showed that 1 month after surgery there was a little absorption in the grafted bone and between the connected areas. Bone density of grafted bone was increased in 2 months after surgery, but still lower than that of the normal mandible. In 4 months, there was no distance between the grafted bone and the host, and the density of grafted area was even higher than the host. Arteriography showed the stop of inferior alveolar artery in the operation side.
Histologically, 1 month after surgery, parts of the superficial transplanted bone were absorbed and new bone formation was observed around the capillary vesscles. Large amounts of mesenchymal cells and chondrocytes were aggregated. Grafted bone continued to be absorbed and newly formed trabecular grew together and became mineralized in 2 months after surgery. In 4 months, newly formed bone incorporated to the grafted bone and it’s difficult to distinguish the host and the grafts.
The analysis of the grafted bone lacuna vacancy rate showed that nearly 30 percent of the grafted bone cells survived 1 month after transplantation and the data decrease to 20 percent another month later. The data of bone mineralization deposition rate showed that the marginal parts of the grafted bone was higher than the middle in 1-2 months after surgery, but showed no difference 4 months later. The results of bone histomorphometry showed that trabecular related parameters (TV/BV、Tb.Th and Tb.Sp),osteoid related parameters (OS/BS、O.Th) and bone absorption related parameters(N.Oc/BS、ES/BS)had statistic difference between the marginal position and middle position of the grafted bone in 1-2 months after surgery. For trabecular and osteoid related parameters, marginal parts are higher than middle parts, while it’s just opposite in the parameters of bone absorption. Four months later, the statistic difference between the positions of the grafted bone disappeared. In 1, 2 or 4 months after surgery, the marginal parts showed much higher level than the middle parts in all the parameters. From the results of capillary vesscle counts, it can be found that the marginal position, no matter the proximal or the distal, acquired much more blood supply than the middle part. In 1-2 months after surgery, the number of capillary vesscles was much larger in marginal parts, even more than the normal mandible area. Four months later, the data became to decrease to normal level. From above data, it could be presumed that angiogenesis have positive correlation with the new bone formation and negative correlation with bone resorption.
Immunohistochemistry staining showed that VEGF localized in the vascular endothelial cells. It showed a strong positive expression in ECs 1-2 months after surgery, while a weak positive expression 4 months later. Immunohistochemistry and in situ hybridization of TGF-β1 revealed that newly formed chondrocytes, osteoblasts and osteoclasts showed a stong positive expression 1 month after surgery and a weak positive expression 2 months after surgery. Four months later only newly formed osteocytes and matrix showed positive expression.
The results indicate that: 1. Parts of the NVBG grafted bone survived after transplantation. 2. New bone formation began 1 month after surgery and newly formed trabecular was gradually mineralized and became matured 4 months after surgery. 3. New bone formed arround the capillary vesscles and new bone formation have positive correlation with the angiogenesis. 4. VEGF and TGF-β1 played an important role in osteogenesis and angiogenesis.
PartⅡ: Based on the previous studies, in this part we put dental implants, attached with rhVEGF165 and rhTGF-β1, into the transplanted iliac bone and observed the interface between the bone and titanium implants.
Methods: Twenty-four dogs were divided into 2 groups and the NVBG was performed the same as before. In group A, two implants were put into the marginal part and the middle part of the transplanted iliac bone. In group B, the implants were attached with rhVEGF165 and rhTGF-β1 first and then were implanted into the grafted bone in the same position as group A. An implant was put into the other side of the mandible as control. Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and measured by clinic, X-ray, histology, M-CT and bone-implant bonding force test.
Results: One dog suffered from infection and the grafted bone with implants was exposed in the mouth. We supplemented a dog to continue the study. X-ray image showed that 1 month after surgery there was an obvious gap between implants and the surrounding bone and 4 months after surgery, no lucency was found in this area. BIC was no more than 5 percent 1 month after surgery and 20 percent 2 months after surgery in experiment group, while was 10 percent and 26 percent respectively in the control. Four months after surgery, 60 percent BIC acquired in experiment group and 70 percent in the control. The test of bone-implant bonding force showed that it’s nearly 220N in the position of marginal implants 4 months after surgery. Bone histomorphometry made by M-CT showed that the growth rate of new trabecular formation arounding implants in marginal position were higher than those in middle position. From the analysis of the same position in different time, trabecular around implants showed a gradually bone formation process. In this process control group showed much higher speed than group A or B. In group A, bone tissue mineral density and connectivity density around implant were much higher than that in group B. Connectivity density in marginal position was higher than that in middle position. For the application of the rhVEGF165 and rhTGF-β1, the effectiveness of bone and BIC formation was greater in group A than in group B.
The results indicate that: In early healing period of the NVBG with implants, the interface of bone and implant was full of connective tissues, and effective osseointegration was formed 4 months after surgery. VEGF and TGF-β1 can promote osteogenesis and bone implant osseointegration.
Part III: In this part, tibia grafts taken from GFP transgenic mice was put into its isogenous’tibia defect and the fate of the grafted bone and the origin of the new bone were analyzed by the GFP fluorescence expression.
Methods: 1.Cell culture. GFP-C57BL/6 osteoblasts were cultured and its’propagation ability and GFP fluorescence expression were observed in vitro. 2. Animal study. A 5mm-long-tibia with intact periosteum and parts of muscles was made in 15 GFP-C57BL/6 mice and was put into the same defect of 15 C57BL/6 mice. Animals were sacrificed at 3 days, 1, 2, 3 and 4weeks after surgery respectively and the grafts were harvested and observed by clinic, X-ray, histology and fluorescence microscopy.
Results: Two mice were suffered from infection, so we supplemented 2 GFP mice to continue the study. X-ray image showed that in 3 -7d after surgery, the lucency gap between the grafted bone and host bone was obvious. In 2-3 weeks after surgery, porosis around the grafted bone was formed and 4 weeks later, the connection of bone and host formed. Histologically, 3 days: There were lots of blood clots and inflammatory cells in the grafted areas. The transplanted bone was partly absorbed. 1 week: Blood clots were replaced by vascularized granulation tissues. The union between the grafted bone and recipient bone was full of mesenchymal cells undergoing chondrocytic differentiation. Extensive bone absorption was observed at the periphery of the bone graft indicated by the numbers of osteoclasts. 2 weeks: The grafted areas could be divided into five layers according to different tissues: grafted muscles, grafted periosteum, newly formed cartilage, newly formed trabeculae and grafted bone. 3 weeks: The remained grafted bone began to integrate with newly formed mineralized trabecular. 4 weeks: Newly formed bone incorporated to the grafted bone and it’s difficult to distinguish them. Bright green light was found in transplanted muscles all the time under fluorescent microscopy. The osteoblasts and newly formed trabeculae and parts of the grafted bone also showed GFP expression.
The results indicated that: 1. GFP-C57BL/6 osteoblasts showed normal propagation ability and a stable GFP fluorescence expression was found in the continuous passaged cells in vitro. 2. Parts of non-vascularized grafted bone can keep viability and had the potential of osteogenesis. The grafted periosteum played an important role in new bone formation.
引文
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