课题背景：课题为广西科技厅青年基金资助项目（桂科青0447055）。人们在临床实践中观察到骨折合并脑外伤患者骨痂过度生长，甚至出现异位骨化，骨折愈合也明显加快。过去认为通过神经体液因素可以对骨折的愈合发挥间接性调节作用。近来通过一些临床观察和实验证实，神经因素对骨代谢和生长具有直接性的调节作用，但具体机制尚无定论。

同行评价：脑外伤后骨折愈合加快的原因是人们一直想知道的问题，过去的报道已经知道神经系统对骨折愈合有正向调节作用，包括碱性成纤维细胞生长因子等，本文从神经肽水平入手分析，得出了结论，进一步加深了对上述问题的认识，对指导临床应用有一定参考价值。
偏倚或不足：文章结果虽然证实了脑外伤后骨折愈合加速，神经因素直接调控骨的生长，但具体的作用机制尚有待进一步揭示。神经因素是多因子组成，具体的每种神经因子对骨折作用的强弱尚没有揭示，不同脑损伤程度对骨折愈合影响的相关性也有待进一步观察。

摘要
目的：研究证明，含有P物质、降钙素基因相关肽、血管活性肠肽、神经肽Y和酪氨酸羟化酶等神经肽的肽能神经共同存在于骨组织中，主要分布于骨代谢活跃的区域，表明这些肽能神经与骨的生长、发育密切相关。观察脑损伤后大鼠胫骨骨痂中神经肽的表达。
方法：实验于2007-02/05在广西医科大学实验动物中心完成。①实验分组：雄性Wistar大鼠130只，体质量450~550 g，随机数字表法分为单纯骨折组（n =60），脑损伤合并骨折组（n =60），正常对照组（n =10）。②实验方法：麻醉后显露大鼠右颅顶骨，中线旁2 mm 处开直径5 mm 骨窗，液压打击致中度脑损伤，并制备大鼠胫骨骨折模型，其中单纯骨折组头部只做颅骨开窗，正常对照组不做任何处理。③实验评估：术后3，7，14，21，28，35 d 苏木精-伊红染色和神经肽免疫组织化学染色观察神经肽在大鼠胫骨中的分布及胫骨骨折骨痂的连续性及骨折愈合情况。计算机X射线摄像仪（CR）摄片测定术后14，21，28 d 脑损伤合并骨折组及单纯骨折组骨痂面积大小。
结果：纳入大鼠130只，均进入结果分析。①脑损伤合并骨折组早期形成大量纤维骨痂和软骨骨痂，骨痂中神经肽免疫阳性神经纤维较多，明显增厚的骨膜内层骨祖细胞、幼稚的软骨细胞胞质内降钙素基因相关肽、P物质、血管活性肠肽、酪氨酸羟化酶、神经肽Y强阳性表达。②脑损伤合并骨折组14 d 纤维骨痂中的软骨细胞团增大，骨膜下软骨细胞层增厚；21 d 小梁骨明显增厚，软骨岛增大；28 d 仍可见大量的纤维骨痂和软骨骨痂，软骨细胞团周边有少量结构稀疏的编织骨形成。单纯骨折组骨膜反应轻，纤维骨痂量少，骨内成骨和软骨内成骨并存，以前者为主，骨折愈合过程明显晚于脑损伤合并骨折组。③14，21 d 脑损伤合并骨折组骨痂面积较单纯骨折组大（P < 0.01）；21，28 d 脑损伤合并骨折组骨痂面积变化明显快于单纯骨折组，提示骨痂塑性快（P < 0.01）。CR摄片发现，各骨折组大鼠骨折端均未发现不愈合现象，单纯骨折组骨折线清晰，骨痂量较少；脑损伤合并骨折组骨性愈合较好，骨痂量较多，骨折线模糊。
结论：正常大鼠骨生长活跃区有丰富的肽能神经支配。脑损伤后骨痂中神经肽有显著改变，并引起骨痂量和质的改变，骨折愈合加速。
关键词：脑损伤；胫骨；骨折；神经肽

李兵，胡朝晖，罗同清.脑损伤并胫骨骨折大鼠骨痂中神经肽的分布及意义[J].中国组织工程研究与临床康复，2008，12(2):231-235 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-231(ps).pdf]

Expression and significance of neuropeptides in callus of rats after brain injury and tibial fracture

Abstract

AIM：It is suggested that the peptidergic nerves containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and tyrosine hydroxylase (TOH) co-exist in bone tissues and distribute in the active bone metabolism area, indicating these peptidergic nerves are closely related with bone growth and development. In this study, the expression of neuropeptides in tibial callus of rats after brain injury was observed.
METHODS: The experiment was conducted in the Experimental Animal Center of Guangxi Medical University from February to May 2007. ①130 male Wistar rats of 3-4 months old and 450-550 g were randomly divided into fracture group (n =60), brain injury plus fracture group (n =60) and control group (n =10). ②The right parietal bone of anesthetized rats was exposed, and bone window of 5 mm was opened 2 mm near median line, then moderate brain injury was made by sap pressure, and the rat models of tibial fracture were established. The fracture group was only subjected to cranial bone window, and the control group was not given any treatment. ③The distribution of neuropeptide in tibia and callus succession as well as fracture healing were observed by HE and immunohistochemical staining on postoperatively days 3, 7, 14, 21, 28, and 35 days. The callus area in brain injury plus fracture group and fracture group was detected by computed radiography (CR) at 14, 21 and 28 days after operation.
RESULTS: All 130 rats were involved in the result analysis. ①At early stage after brain injury, there were a quantity of fibrous callus and cartilaginous callus formation in brain injury and fracture group and many neuropeptides immunoreactive nerve fibers in callus were found. Strong immunoreactivites of CGRP, SP, VIP, NPY, TOH occurred to osteogenitor cells and chondroblast, which proliferated in thickened endothecium. ②In brain injury and fracture group on day 14, the chondrocyte group was enlarged and subperiosteum chondrocyte layer was thickened; on day 21, bone trabecula was significantly thickened, and cartilage islands were increased; on day 28, there were still quantity of fibrous callus and cartilaginous callus, and sparse woven bones surrounding chondrocyte groups. In fracture group, periosteal reaction was slight, and fibrous callus was few; osteogenesis in bone and cartilage occurred, but mainly in bone. Moreover, the fracture healing was remarkably later than brain injury and fracture group. ③Callus area in brain injury and fracture group was larger than that in fracture group on days 14 and 21 (P < 0.01); on days 21 and 28, the callus area in brain injury and fracture group changed distinctly compared with fracture group, indicating callus plasticity was faster (P < 0.01). CR photographs showed that no disunion was found in each group, and fracture line was clear in fracture groups with few callus; in brain injury and fracture group, the bone healing was good, with plenty callus but obscure fracture line.
CONCLUSION:Neuropeptide nerves innervate in the active area for bone growth. Neuropeptides in the callus change greatly after brain injury, which induce the rapid recovery of fracture and improve fracture healing.

Li B, Hu ZH, Luo TQ.Expression and significance of neuropeptides in callus of rats after brain injury and tibial fracture.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):231-235(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-231(ps).pdf]