骨损伤修复中H型血管的分布和作用及血管束神经束联合植入组织工程骨修复羊大段胫骨缺损的研究
发布时间：2018-12-17 03:00
【摘要】：骨组织是一个高度血管化和神经化的组织。无论在正常的骨损伤修复过程中还是在应用组织工程骨修复骨缺损的情况下,及时和丰富的血管再生是保证骨损伤正常修复和组织工程骨存活的基础。最近有文献报道在小鼠骨发育过程中存在两种血管内皮细胞亚型,即高表达CD31和Emcn的H型内皮细胞和低表达CD31和Emcn的L型内皮细胞。这两种内皮细胞亚型具有不同的形态、分布和功能特征。其中H型内皮细胞在介导小鼠骨发育成血管和成骨偶联的过程中起着重要作用。本研究第一部分对H型血管在正常成年大鼠长骨中的分布以及在正常和骨质疏松大鼠骨损伤修复中的分布和变化规律进行研究,目的是对H型血管在骨损伤修复中的地位和作用获得更深入的认识。通过研究我们发现,在正常成年大鼠长骨中也存在H型血管并特异的分布在靠近干骺端和骨内膜的骨小梁表面这些成骨活动活跃的部位;在正常大鼠骨损伤的修复过程中,H型血管在损伤后第3d即出现大量增生和分布;随着修复时间的进展,H型血管的量逐渐减少,但特异性的分布在靠近生长前沿的新生骨小梁的表面,在远离生长前沿的骨小梁表面分布显著减少;作为对照,在骨质疏松大鼠骨损伤修复的各个时期,H型血管增生的量均显著减少,并且未发现其特异性高分布,新生骨小梁数量和厚度显著减少且结构紊乱。结合以往的文献推测H型血管具有支持大鼠骨发育和骨损伤修复中新生骨小梁生长的作用,骨质疏松大鼠骨损伤修复中H型血管数量的显著减少是其成骨量少的重要原因。此外,我们还研究了在正常和骨质疏松大鼠骨损伤修复中成血管相关因子Hif-1α、VEGFA和成骨因子BMP2、Osterix转录水平的变化。研究发现在骨损伤后第3d,骨质疏松组Hif-1α和VEGFA的转录水平显著高于正常组;但在术后第7d和第14d,骨质疏松组Hif-1α和VEGFA的转录水平显著低于正常组;骨质疏松组成骨因子BMP2和Osterix的转录水平在术后3d、7d和14d均显著低于正常组;研究提示骨质疏松大鼠在损伤修复期成血管和成骨因子转录水平的显著降低也是其成血管和成骨量低下的原因。组织工程骨血管化和神经化程度不足是制约其向临床应用转化的重要原因。有学者发现将动静脉环或动静脉束移植入组织工程骨来修复骨缺损可以显著促进组织工程骨血管化和骨缺损的修复。另有学者发现将感觉神经束离断后移植入组织工程骨中也能起到与血管束植入相同的促成骨效果,其中的原因可能是移植入的神经束本身具有滋养血管,而且感觉神经束的植入可以通过CGRP、NPY和SP等外分泌递质的释放促进种子细胞的存活和成骨。据此,裴国献教授提出了组织工程骨血管化和神经化同步构建的理论。本研究第二部分通过分别将隐血管束与隐神经束同时植入组织工程骨、单纯隐血管束植入组织工程骨和单纯应用组织工程骨修复绵羊胫骨3cm大节段骨缺损,在术后3个月分别通过X线平片、微米X射线扫描后三维重建、生物力学检测以及VG染色评价各组骨缺损的修复效果和生物力学功能。结果显示,隐血管束和隐神经束联合植入组成骨量和材料的降解率均显著高于其他两组,并且获得了较其他两组更好的生物力学功能的恢复。本研究的结果提示组织工程骨血管化和神经化同步构建对于促进组织工程骨成骨和力学功能的恢复具有重要作用。
[Abstract]:Bone tissue is a highly vascularized and neurotic tissue. In the case of the normal bone injury repair or the application of the tissue engineering bone to repair the bone defect, the timely and abundant blood vessel regeneration is the basis for ensuring the normal repair of the bone injury and the survival of the tissue engineering bone. Recently, there are two types of vascular endothelial cell subtypes, namely, H-type endothelial cells with high expression of CD31 and Emcn, and L-type endothelial cells with low expression of CD31 and Emcn in the development of mouse bone. The two types of endothelial cells have different morphology, distribution and functional characteristics. in which H-type endothelial cells play an important role in mediating the development of a mouse bone into a blood vessel and a bone-forming coupling. The first part of this study is to study the distribution of H-type blood vessels in the long bones of normal adult rats and the distribution and the rule of change in the repair of bone injury in normal and osteoporotic rats. The purpose of this study is to get a more thorough understanding of the status and role of H-type blood vessels in the repair of bone injury. We have found that H-type blood vessels are also present in the long bones of the normal adult rats and are specifically distributed on the surface of the bone small beams close to the dry end and the inner surface of the bone, which are active parts of the bone; during the repair of the bone injury of the normal rats, In the third day after the injury, the H-type blood vessel has a large number of hyperplasia and distribution; with the development of the repair time, the amount of the H-type blood vessel is gradually reduced, but the specific distribution is close to the surface of the new-born bone small beam near the growth front, and the surface distribution of the small-beam surface which is far from the leading edge of the growth is significantly reduced; As a control, the amount of H-type vessel hyperplasia was significantly reduced in the various stages of bone injury repair in the osteoporosis rats, and no specific high profile was found, with a significant reduction in the number and thickness of the new bone and a structural disorder. Combined with the previous literature, the H-type blood vessel has the effect of supporting the growth of the new bone in the bone development and the bone injury repair of the rat, and the significant reduction of the number of H-type blood vessels in the bone injury repair of the osteoporosis rats is an important cause of the small amount of the bone. In addition, we have also studied the changes of vascular-related factors Hif-1, BFA and bone factor BMP2 and Osternix in the repair of bone injury in normal and osteoporotic rats. The results showed that the levels of Hif-1 and HFA in the osteoporosis group were significantly higher than those in the normal group after the bone injury, but the levels of Hif-1 and HFA in the osteoporosis group were significantly lower than that of the normal group at the 7th and 14th day after the operation, and the transcription level of the bone factors BMP2 and Osterx in the osteoporosis group was lower than that of the normal group. Both 7d and 14d were significantly lower than that in the normal group. The insufficient degree of vascularization and neurotization of the tissue engineering is an important factor in the transformation of the clinical application. It is found that the graft of the arteriovenous ring or the arteriovenous beam into the tissue engineering bone to repair the bone defect can significantly promote the tissue engineering bone vascularization and the repair of the bone defect. another scholar has found that it is also possible to implant the sensory nerve bundle into the tissue engineered bone after it is broken, which may be the same contributing bone effect as the implantation of the vascular bundle, where the cause may be that the implanted nerve bundle itself has a nourishing blood vessel, and the implantation of the sensory nerve bundle may be through the cgrp, The release of exogenous hormones, such as NPY and SP, promotes the survival of seed cells and the formation of bone. On the basis of this, the author of the founding of the Republic of China put forward the theory of the construction of the tissue engineering's vascularisation and the synchronization of the neurogenesis. in that second part of the study, the bone defect of the 3cm large section of the sheep's tibia was repaired with the bone of the tissue engineer and the saphenous nerve bundle at the same time as the saphenous bundle and the saphenous nerve bundle, and the bone defect of the 3cm large section of the sheep tibia was repaired by using the tissue engineering bone and the pure application tissue engineering bone, respectively, and the bone defect of the bone of the sheep tibia is repaired through the X-ray flat sheet at 3 months after the operation, Three-dimensional reconstruction, biomechanical testing and VG staining were used to evaluate the repair and biomechanical function of each group of bone defects. The results showed that the rate of bone mass and material degradation was significantly higher than that of other two groups, and the recovery of other two groups of better biomechanical function was obtained. The results of this study suggest that the construction of the tissue engineering bone and the synchronization of the neurogenesis play an important role in promoting the recovery of bone and mechanical functions of the tissue engineering.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68
，
本文编号：2383536
[Abstract]:Bone tissue is a highly vascularized and neurotic tissue. In the case of the normal bone injury repair or the application of the tissue engineering bone to repair the bone defect, the timely and abundant blood vessel regeneration is the basis for ensuring the normal repair of the bone injury and the survival of the tissue engineering bone. Recently, there are two types of vascular endothelial cell subtypes, namely, H-type endothelial cells with high expression of CD31 and Emcn, and L-type endothelial cells with low expression of CD31 and Emcn in the development of mouse bone. The two types of endothelial cells have different morphology, distribution and functional characteristics. in which H-type endothelial cells play an important role in mediating the development of a mouse bone into a blood vessel and a bone-forming coupling. The first part of this study is to study the distribution of H-type blood vessels in the long bones of normal adult rats and the distribution and the rule of change in the repair of bone injury in normal and osteoporotic rats. The purpose of this study is to get a more thorough understanding of the status and role of H-type blood vessels in the repair of bone injury. We have found that H-type blood vessels are also present in the long bones of the normal adult rats and are specifically distributed on the surface of the bone small beams close to the dry end and the inner surface of the bone, which are active parts of the bone; during the repair of the bone injury of the normal rats, In the third day after the injury, the H-type blood vessel has a large number of hyperplasia and distribution; with the development of the repair time, the amount of the H-type blood vessel is gradually reduced, but the specific distribution is close to the surface of the new-born bone small beam near the growth front, and the surface distribution of the small-beam surface which is far from the leading edge of the growth is significantly reduced; As a control, the amount of H-type vessel hyperplasia was significantly reduced in the various stages of bone injury repair in the osteoporosis rats, and no specific high profile was found, with a significant reduction in the number and thickness of the new bone and a structural disorder. Combined with the previous literature, the H-type blood vessel has the effect of supporting the growth of the new bone in the bone development and the bone injury repair of the rat, and the significant reduction of the number of H-type blood vessels in the bone injury repair of the osteoporosis rats is an important cause of the small amount of the bone. In addition, we have also studied the changes of vascular-related factors Hif-1, BFA and bone factor BMP2 and Osternix in the repair of bone injury in normal and osteoporotic rats. The results showed that the levels of Hif-1 and HFA in the osteoporosis group were significantly higher than those in the normal group after the bone injury, but the levels of Hif-1 and HFA in the osteoporosis group were significantly lower than that of the normal group at the 7th and 14th day after the operation, and the transcription level of the bone factors BMP2 and Osterx in the osteoporosis group was lower than that of the normal group. Both 7d and 14d were significantly lower than that in the normal group. The insufficient degree of vascularization and neurotization of the tissue engineering is an important factor in the transformation of the clinical application. It is found that the graft of the arteriovenous ring or the arteriovenous beam into the tissue engineering bone to repair the bone defect can significantly promote the tissue engineering bone vascularization and the repair of the bone defect. another scholar has found that it is also possible to implant the sensory nerve bundle into the tissue engineered bone after it is broken, which may be the same contributing bone effect as the implantation of the vascular bundle, where the cause may be that the implanted nerve bundle itself has a nourishing blood vessel, and the implantation of the sensory nerve bundle may be through the cgrp, The release of exogenous hormones, such as NPY and SP, promotes the survival of seed cells and the formation of bone. On the basis of this, the author of the founding of the Republic of China put forward the theory of the construction of the tissue engineering's vascularisation and the synchronization of the neurogenesis. in that second part of the study, the bone defect of the 3cm large section of the sheep's tibia was repaired with the bone of the tissue engineer and the saphenous nerve bundle at the same time as the saphenous bundle and the saphenous nerve bundle, and the bone defect of the 3cm large section of the sheep tibia was repaired by using the tissue engineering bone and the pure application tissue engineering bone, respectively, and the bone defect of the bone of the sheep tibia is repaired through the X-ray flat sheet at 3 months after the operation, Three-dimensional reconstruction, biomechanical testing and VG staining were used to evaluate the repair and biomechanical function of each group of bone defects. The results showed that the rate of bone mass and material degradation was significantly higher than that of other two groups, and the recovery of other two groups of better biomechanical function was obtained. The results of this study suggest that the construction of the tissue engineering bone and the synchronization of the neurogenesis play an important role in promoting the recovery of bone and mechanical functions of the tissue engineering.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68
，
本文编号：2383536
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