【作者】 李瑛；

【导师】 邹季；

【作者基本信息】 湖北中医学院 ， 中医骨伤科学， 2008， 博士

【摘要】 一、课题研究的背景和意义骨折固定是治疗骨折的重要环节和基本手段之一，也是骨折按期愈合的基本条件。目前全世界临床应用的骨折固定技术主要流派有：AO(Arbeitsgeme inschaft for Osbeosynthese fragen)内固定学派；BO(BiologicalOsteosynthesis)生物学接骨术学派，ilizarov外固定学派，即DO(distractionOsteosynthesis)学派；samiento外固定支架学派；CO(中国接骨术ChineseOsteosynthesis)学派。其中AO为“刚性固定”法，其他属“弹性固定法”，以CO法为弹性固定理念的经典性代表。以瑞士学者Muller为代表所创立的AO内固定技术，建立在结构解剖学和功能解剖学及生物力学基础上，融固定器材设计规范化，固定技术专业化，固定操作设备配套化为一体，在全世界范围内风行近40年，有效解决了多发性及复杂性骨折及其某些特殊类型和特殊部位的骨折，如关节内骨折，但同时，随着临床上广泛推广，经验积累，在当代国际骨科界占统治地位的AO学派，其骨折固定的理论与技术体系，在近年来作出了重大的改变。AO派学着们逐渐认识到他们原来所持的“刚性固定”理念的缺陷，如“应力遮挡”产生的负面效应等。于是近年来，他们逐渐扬弃AO学派们对骨折实行”安全休息，绝对固定”的治疗观点，积极改进了骨折的内外固定方法，发展和完善了AO内固定技术，穿针外固定技术和局部外固定技术。在AO基础上提出BO的新理念，BO的骨折治疗的基本内容是微创操作，其核心为对长骨骨折不再强求解剖复位，而着重恢复力线和长度，并更加重视对骨折部位血供的保护和手术以后的早期功能锻炼，BO是对AO理论的完善和进步，但BO还只是一种治疗手段，BO的内容并不是新的理论，也没有反映二十世纪骨折愈合生物学研究的丰硕成果，同样无法解释应力遮挡，骨延期愈合机制等重大理论问题，但BO观点的提出，却使骨折愈合研究重新回到重视骨痂作用的正确轨道上来，从AO到BO是历史在更高层次上的回归，BO的提出，正预示骨折治疗的新趋势，AO学派的变化由刚性固定转变为弹性固定，顺应了人类对健康的更高要求，就医疗理念与技术内涵而言，无疑是受了小夹板固定治疗骨折所蕴藏的“弹性固定”原理的启迪和影响，即受到“动静结合，筋骨并重”原则的影响。更值得关注的是，在脊柱外科方面，近年来也出现了类似的变化，国际骨科界至少已经在骨折固定和脊柱固定两个领域，正在与刚性固定渐行渐远，而与弹性固定日益靠拢。弹性固定中的DO技术是也是受CO理念启发的结果，DO是指用牵张装置将揭开的骨块以一定的速率和频率牵开，在牵开间隙内刺激骨痂的形成，进而成骨的一种修复方法，它强调的是，把握骨折修复过程中动与静的关系。CO技术源于中国的小夹板技术，是应用现代结构解剖和功能解剖及生物力学对传统小夹板技术进行整理和提高的结果，它以“弹性固定”理念为精髓。C0汲取传统中医之精，并紧密结合现代科学技术的发展，其指导思想“动静结合，筋骨并重，内外兼治，医患合作”，CO治疗骨折以非手术治疗为主，辅以有限手术，与BO思想十分贴近，也就是说CO的观点是先进的，甚至是超前的，符合骨折治疗的最新趋势。至此，国际骨伤界在经历了大量临床和实验研究的正反两方面的经验教训后，终于基本上达成了共识：弹性固定是最合理、最有利于骨折愈合的治疗理念和技术。所谓“弹性固定”，就是骨折愈合过程中，允许骨折端之间有“微动”现象，对骨折愈合最有利，但骨折端之间活动过大显然更不利于骨折愈合，甚至造成骨折不愈合或延迟愈合，这更是多年临床实践证实和学术界早已公认的事实。骨折断端微动和应力是骨折愈合的发生发展最重要的原动力之一。“微动”是国内外承认的促进骨折愈合的治疗手段，但一些实验大都停留在内固定和外固定支架对骨折愈合的促进作用的证实方面，以及对其机理的探讨上，而对小夹板固定对骨折愈合的影响还很模糊。夹板局部外固定治疗骨折可以控制不利于骨折愈合的活动。它通过布带的约束力，夹板的杠杆力及纸压垫的效应力来平衡引起骨折再移位的肢体重力及肌肉张力。这些力学的平衡是通过肢体软组织的两个主要机制来实现的：一个是不可压缩性，充满肌肉的间隔由夹板限制时形成了一个不变体系，由肌肉包绕的骨折端也达到了相对稳定的状态。小夹板局部外固定不是企图将骨折断端绝对固定，只是保持骨折断端的相应解剖关系，肢体活动使骨折断端始终承受一定的压力及应力，这是一种生理性刺激，有利于骨折愈合。骨折愈合进程对局部力学环境变化很敏感，骨折部位的应力改变依据加于骨折的载荷和骨折部组织的特性所决定。小夹板由于具有合理的构型和力学优势，具有轴向持骨的稳定性，贯彻了功能活动的原则，能促进骨痂的形成，并使骨痂能在功能状态下得到良好的塑形，因此在骨的功能适应性上有明显的优点。这种优势最根本的体现在与“刚性固定”方法不同，小夹板所实现的是一种“动静结合”的“弹性固定”，这是它的精髓与灵魂。小夹板弹性固定提供给骨折端相对固定的力学环境，在保证骨折部稳定可靠的前提下，允许骨折端有纵轴上的活动。这种活动对骨折愈合是有利的，可促进血肿吸收骨膜反应性肥厚增生，骨细胞分化提前，血管再生丰富，骨痂生长及钙化迅速，愈合时间提前。小夹板外固定对骨折端无应力遮挡效应，对皮质骨血运亦无破坏，对骨折自然愈合的过程无干扰。另外，这种固定方法，不需剥离骨膜，软组织损伤较轻，对局部血液供应破坏轻，对骨折愈合有利；同时，小夹板固定骨折不固定关节，不影响关节活动，使肢体功能恢复与骨折愈合过程基本达到同步。这一点尤其符合目前国际医学界追求的共同医疗理念——以医疗的“终点目标”为原则。由此可见，小夹板治疗骨折的技术包含的科学内涵十分丰富，具有符合生物学基本规律的科学性、合理性、先进性，是中医学闪光部分，是中华民族极为宝贵的文化技术遗产。然而非常令人遗憾的，由于历史条件的限制，它一直停留在“经验”状态，其力学本质以及定量关系至今尚未被认识，缺乏系统而科学的发掘、整理，其特色和优势也没有得到系统的诠释和阐明，因此，影响与国际医学界的交流致使在医疗实践中，这些年来正日益受到冷漠，在不知不觉中有被淹没以及失传的趋势。以历史的责任感，采用现代的技术方法，整理、挖掘、提高这一传统的技术，深刻地阐明其机理，予以继承和创新，并推广应用于临床，这是开展本课题研究的背景和意义。本课题在国内外首次将传统的中国骨伤接骨术及小夹板固定技术用于实验性骨折中；采用先进检验指标评估骨折愈合质量，监测骨折愈合过程，及其有利于或促进骨折愈合的机理；将应变式压力传感器用于骨折外固定器中，较为准确地测出骨折端的微动幅度，为设计新的骨折外固定器提供了思路；得出的研究结果，将为传统的小夹板固定技术由经验型转变为科学型提供实验和理论基础。该项研究不仅能指导临床工作具有较高的实用价值，而且可以帮助我们挖掘、整理、提高祖国医学骨伤技术精华，并使之与现代科学相融合，使传统的技术精华发扬光大，造福人民，具有重要的学术价值和理论意义。这项研究，将为小夹板固定治疗四肢骨干骨折提供科学依据，将为小夹板外固定普及、推广奠定新的指标，提示探索骨折固定新方法应遵循的规律，对中西医结合治疗骨折的发展前景具有前瞻性指导意义，对中医的医疗、教学、科研均有指导意义。二、课题研究的目的本研究以实验性兔胫骨骨折动物模型为研究对象，分别采用小夹板固定和钢板固定，小夹板固定中，通过调节小夹板扎带的松紧度使骨折断端承受不同的压力，观察骨折断端在二种不同的压力作用下对骨折愈合的影响，并与钢板固定组相比较，主要观察下列指标：用免疫组化方法检测兔骨折愈合的不同阶段血管内皮生长因子(VEGF)、生长转化因子β1(TGF-β1)的表达；采取电镜技术对骨折愈合的不同阶段骨痂组织的超微结构进行观察；采用光镜技术对骨折愈合的不同阶段作组织学观察；观察小夹板固定对兔实验性骨折愈合骨痂生物力学的影响，从而了解骨折愈合的质量；观察小夹板固定对兔实验性骨折愈合骨痂骨密度的影响；观察小夹板固定对兔实验性骨折愈合不同阶段血清碱性磷酸酶含量，以及对骨折局部的放射性观察。从不同的角度来评估小夹板弹性固定对骨折愈合的影响，监测骨折愈合过程，同时探讨小夹板固定骨折促进骨折愈合的机理，并进一步证实微动可刺激骨痂形成，加速骨折愈合。通过这项研究，为小夹板固定治疗四肢骨干骨折提供了科学依据，使小夹板得到更广泛的应用，为设计弹性固定新产品提供理论数据和实验基础。三、课题研究的方法：实验一1实验材料1．1动物兔45只，体重2-2．5kg。由湖北省科学研究院动物实验中心提供。1．2试剂抗VEGF抗TGF-β1均由北京中杉试剂公司提供1．3仪器光学显微镜免疫组化仪器数件夹板：自制小夹板，由较好弹性杉树皮制作。分前、后、内、外侧四块夹板，夹板上宽下窄。前侧夹板长11cm，上端宽约2．4cm；后侧夹板长10cm，上端宽约2．4cm；内、外侧夹板长均为10cm，上端宽均为1．9cm。在前侧、后侧夹板靠近胫骨结节部各刺一1．5mm小孔。钢板：江苏金鹿集团医疗有线公司提供压力传感器：由武汉超宇测控技术公司提供。构造如下图所示。压力传感器主要有压力盒、导气管、三通、注气管、敏感元件以及半导体应变片等组成。其原理为：在压力测量之前，利用注气管将气体注入，使得气管内部充满气体，即使压力传感器的敏感元件两侧压力平衡，敏感元件不受应力作用。当压力盒受压力作用时，导气管内的气体受压使得敏感元件变形而产生应变，粘贴在敏感元件上的半导体应变片将压力变化产生的应变转换为电阻的变化，即建立了电阻变化与压力之间的定量关系。使用全差动测量电桥检测电阻变化，即可得出压力的变化关系。使用时，将压力传感器包入夹板内，通过传感器数值，确定夹板系带松紧程度。2．实验方法2．1动物造模与分组选用45只健康家兔，在左胫骨中下三分之一处，复制3mm标准横形骨折模型，随机分成A组、B组和C组，A组和B组用石膏固定，C组用四孔钢板内固定，五天后小心撤除A组和B组石膏，改用小夹板局部外固定。在兔胫骨结节处横穿一枚直经为1．5mm克民针，再穿入前后侧夹板的眼内，以防夹板滑脱，压力传感器置于前侧夹板内面骨折的断端，再用四条扎带捆住四块夹板，夹板上扎带的松紧度不同，A、B组扎带上下移动分别约为3mm和7mm。此时读出A组压力传感器读数是18kPa，B组压力传感器读数是12kPa。2．2标本的采集与处理分别于术后14天，24天，34天处死A、B、C组每组各五只兔子，取胫骨骨折部上下方各1cm的骨干，10％福尔马林固定三天后，经5％硝酸脱钙，流水冲洗，酒精梯度脱水，浸腊包埋，纵向切片厚度4μm。切片经二甲苯脱腊，梯度酒精水化，3％H2O2去离子水孵育5分钟灭活内源性过氧化物酶。蒸馏水冲洗，PBS浸泡5分钟，微波修复。滴加山羊血清封闭后，按1：100稀释的兔抗TGF-β1抗体和鼠抗VEGF抗体，4℃过夜。PBS冲洗3分钟，冲洗3次，滴加生物素化二抗室温孵育15分钟。PBS冲洗3分钟，冲洗3次，滴加辣根酶标记链霉卵白素，室温孵育15分钟，PBS冲洗3分钟冲洗3次，用二氨基联苯胺(DAB)显色，自来水冲洗，苏木素复染。梯度酒精脱水，透明后封片。2．3观察指标：血管内皮生长因子(VEGF)、生长转化因子β1(TGF-β1)的表达；实验二1实验材料1．1实验动物：同实验一1．2仪器光镜透射电镜：H-500(日立)由广州军区武汉陆军总医院提供超薄切片机：由武汉大学医学院提供，瑞典LKB-Ⅱ型2．实验方法2．1动物模型制作与分组同实验一2．2观察指标及方法2．2．1大体观察兔子处死后观察外骨痂，内骨痂，桥梁骨痂。2．2．2组织形态观察用耳缘静脉空气栓塞法处死兔子，于术后14天处死第一批兔子，24天处死第二批，34天处死第三批，每次每组5只。将胫骨标本于骨折部上下方各1cm处锯断，10％福尔马林固定备用，5％硝酸溶液脱钙，流水冲洗，梯度酒精脱水，石腊包埋，纵向切片，片厚4um，常规HE染色，再用光学显微镜观察。2．2．3超微结构观察前述方法和时间处死兔子后，立即取1mm3大小的标本，用2．5％戊二醛固定。1％EDTA溶液脱钙，缓冲液(PH7．2-7．4)冲洗三遍，再用1％饿酸后固定2h，缓冲液冲洗，酒精梯度脱水，环氧树脂812包埋，超薄切片，铅铀染色，透射电镜观察。实验三1材料与方法1．1选用18只健康家兔，随机分成A组、B组、C组，动物造模和分组治疗同实验一1．2检测项目1．2．1血清碱性磷酸酶指标：分别于术前及术后第2、4、6周每组取6只兔用9号针头经心脏采血2ml/kg，进行血清碱性磷酸酶(ALP)检测(由湖北中医院测定)。1．2．2生物力学测试抗拉强度试验三组动物在第六周时均采用耳缘静脉空气栓塞处死，解剖出左侧胫骨，清除软组织，纱布包裹，生理盐水浸透。将试验用骨在20±5℃的环境温度下放置不少于3h，，12小时内测量。然后用钢丝一端和兔胫骨固定，一端挂在LJ-500拉力机的夹具上，使用25mm/min拉伸速度进行拉伸试验，直至胫骨拉断，得到拉断力数据，拉断面应在骨折愈合处附近，如果拉断面在其他部位，则此样品试验数据弃除。每个固定方式得到六个有效拉断力数据，按骨外沿面积计算出骨的断面积，用拉断力除以断面积得到抗拉强度值，将六个抗拉强度数据按从大到小排序，第三和第四个数据的平均值为本固定方式的抗拉强度试验值，以MPa表示。2对实验结果作统计学处理实验四1实验材料1．1动物兔18只，体重2-2．5kg。由湖北省科学研究院动物实验中心提供。1．2仪器QDR 2000plus型DEXA2实验方法2．1动物造模与分组选用18只健康家兔，随机分成A组、B组、C组，动物造模和分组治疗同实验一，第6周末处死动物，拆除内外固定，解脱双后肢膝关节，剔除全部软组织，取双侧胫骨，保留骨膜。2．2观察指标：2．2．1 X线摄片观察：每只动物在处死前先行X线摄片检查，以确定没有假关节形成。并观察骨痂生长和骨折愈合情况。2．2．2骨密度测定：采用QDR 2000plus型DEXA，以双能X线吸收法，对胫骨中段骨折区作骨密度(BMD)测定，均在骨折部位取一同等条件兴趣区R1，以骨折为中心由近至远扫描10厘米长距离。使用分析软件分析测定的数据来计算骨折端1cm感兴趣区域(Regions of Interest，ROIs)的BMD。同一动物的未手术侧标本在骨折对应部位扫描测量，作为自体对照数据。每一标本重复测定三次，每次测量后标本重置。DEXA由微机自动分析打印结果。计算每对标本的骨密度比率。3数据处理和统计学分析：参数比率的计算公式为：骨密度比率=对应未手术侧标本的测量值/手术侧标本的测量值×100％。各组骨密度的参数值用均数±标准差(x±s)表示。四、课题研究的结果1．免疫组化结果：在骨折愈合的各个阶段，各组中TGF-β1在细胞中的表达增加，VEGF在细胞中的表达亦增加，二者存在着一定的协调关系。在骨折愈合的早期及中期，血管形成的量及时间A组早于B组和C组，骨痂形成的量亦高于B组和C组，因此阳性表达较B组及C组早，阳性程度明显优于B组和C组。2．大体观察A组：14天骨折段已初步联接，见少量的梭形外骨痂，24天时，骨痂外形呈梭形，骨痂将两骨折段桥接，34天，已由骨痂将骨折段牢固联接在一起，骨折端无活动度。B组：在14天时，处死的5只兔子有1只骨折端有错位；24天时，骨折端有部分联接，但活动度较大，外骨痂较A组少，34天时可见骨痂中等，呈梭形。骨折端未完全由骨痂联接，骨折线仍清晰可见。C组：各时间段骨折端均无错位情况，14天时见纤维连接，24天时见中等量的散在的外骨痂，排列不整齐，未成梭形连接两骨折端，34天时，外骨痂部分成梭形连接两骨折端，但连接的不牢固。3．电镜观察显示A组在骨折愈合过程中，成骨细胞出现早，数量较B组和C组均多，且功能活跃，34天时骨折端见大量功能活跃的骨细胞。4．组织学检测显示，A组骨外膜骨痂，桥梁骨痂能早期加速形成，并逐渐形成连接骨痂，封闭骨痂，骨小梁坚韧，34天时骨痂已连接骨端，骨折端进行编织骨向板层骨转化，明显优于B组和C组。5．血清碱性磷酸酶结果：各组不同时期家兔血清AKP的变化幅度不同，14d时A组血清碱性磷酸酶含量明显高于B组和C组(P<0．01)。6．6周末骨折端生物力学测量显示小夹板固定A组的抗拉强度显著高于小夹板固定B组及钢板固定组(P<0．01，P<0．05)。7．6周末X线检查显示A组骨断端致密骨痂填充，骨折线模糊，外骨痂致密，部份髓腔再通。B组和C组：骨折端骨痂较少，骨折线仍存在。8．6周末骨密度测量显示A组与B组和C组有非常显著差异(P<0．01)；A组BMD值明显优于B组和C组，B组和C组无显著性差异。五课题研究的结论微动能明显促进骨折愈合；A组动物骨折愈合时间最短，其在促进骨折愈合的速度、及质量上有明显的优越性。恰当的小夹板固定能改善骨折局部血循环；增加骨生成细胞的数量，促进骨生成细胞分化、成熟，促进骨折断端矿物质的沉积、提高骨折断端强度，提高骨折愈合质量，缩短骨折愈合时间。更多还原

【Abstract】 一The backgroud and the significance of the researchThe fixation is the important link and basic method to treat the bone fracture, and it is the fundamental condition that the bone fracture can concrescent on time. At present, the main schools of the technique of the fracture fixation in clinic over the world include: AO(Arbeits gemeinschaft for Osbeosynthese fragen) school ; BO ( Biological Osteosynthesis) school, ilizarov external fixation school, that is DO ( distraction Osteosynthesis)school; samiento external fixation clamp stand school; CO (Chinese Osteosynthesis) school.Aomong the total, AO is the method of rigidity fixation, the others are elastic fixation, CO is the classic representative of them.The internal fixation technique of AO established by the Switzerland scholar Muller is founded on the vitodynamics, stuctural and functional anatomy. The AO technique integrate standard fixator design, specialized fixed technique and matched fixing equipment, which is fashionable about 40 years over the world. It can solve effectively the multiple and complicated fracture and fracture in privileged site, such as intraarticular fracture. But at the same time, accompanied by the spreading in clinic and building experience, AO school, which is in governed position in the department of orthopaedics in present ages, make substantial alteration in recent years on the theory and technology system of fracture fixation. The scholars of AO gradually realize the defect of the thought of rigidity fixation which they kept before, such as the negative effect caused by the "stressing block" , etc.So, in recent years, they gradually abandon the opinion that the fracture should "complete rest, absolute fixation" , improve positively the methods of internal and external fixation, consummate and develope technique of the AO internal fixation, thread a needle external fixation and local external fixation.The new opinion of BO is produced on the basis of the AO, whose fundamental content is minute trauma operate, its nucleus is that we do not enforce the the reestablish the anatomic position, but emphasis on the recovery of the line of force and the bone length, and the new idea think highly of the protection of the blood supply of the area of the fracture posion and of the earlier functional exercise after the operation. BO is the improvement of AO, but it is not new theory, and it donot reflect the rich achivement on the bioresearch of the union of the fracture in the 21 century , it cannot explain some important theory problems such as stressing block and the mechanism of delayed healing of the fracture as well.But the producing of the BO makes the research on the union of th fracture go back to the correct track of reconstruction of the effect of the bony callus, which is the regression on more higher level from AO to BO on the history.The raise of BO prognosticate the new trend of the healing of the fracture, The change of the AO school, which is from rigidity fixation to elastic fixation, fit on the more higher request of people to the health, whose medical thought and technique connotat ion is undoubtedly influenced by the principle of elastic fixation deposited in the method of small splint fixation. We should see that, in recent years, there is similar change in spinal column surgery. In international orthopedics area, scholars pay more attentions to the elastic fixation than the rigidity fixation at least in the domain of fixation of bone fracture and spinal column. But until today, BO just is a method of healing, not a theory system, the origin of BO is to make up the shortage of AO, whose opinion influenced by the principle of the "dynamic and static combination, lay equal stress on bones and muscles" . DO technique in the elastic fixation is the result enlightened by the opinion of CO. DO means that draw the bone by the stretch device in invariably velocity and frequency, stimulate the formation of the bony callus in the drawing distance, and then formate the bone. It emphasis on the grasping the dynamic and static relationship. CO technique is origined by the small splint technique from China, which is the result of improvement of the traditional small splint by the mordern vitodynamics, structure and functional anatomy, whose essence is elastic fixation.CO absorb the essence of the traditional Chinese medicine, and tightly combine the development of the mordern science and technology, whose guiding idea is "dynamic and static combination, lay equal stress on bones and muscles, exterior and interior treating, doctor and patient cooperate" , CO methods is primary on no-operate healing, assist with limited operation, which is similar to the idea of BO, that is to say, the idea of CO is advanced, it fit on the new trend of the healing of the bone fracture.So, after a lot of clinical and laboratory research, the international bone and wound department aera mainly recognize that elastic fixation is the most reasonable healing thought and technology , which is most profit for the union of fracture[9]. So called "elastic fixation" means that in the period of the union of fracture, the jog in the broken ends of the fracture is permitted, which is more benefit to the union of fracture, but too more activity between the broken ends is obviously no benefit to the union of fracture, and even make disunion and delayed union, which is the fact received by the commonwealth of learning and confirmed by the clinical practice. The "jog" is a healing method to promote the union of fracture which is admitted by the schollars all over the world, but some experiments are still in the verification and research of its mechanism of the promotion effect to the union to fracture by internal and external fixation clamp stand, and the influence on the union of fracture by the fixation of small flint is still vague.The jog and stress in the broken ends is the one of the most important motive power of the happening and developing of the fracture union. The local external fixation by the flint is not trying to fix the broken ends absolutely, but just keep the corresponding anatomy relationship of the broken ends. The activity of the limbs can make the broken ends accept some pressure and stress, which is a sort of physiological stimulate, it is benefit to the union of fracture.The procedure is sensible to the change of local mechanics environment, the stress change in the broken ends is decided by the loading on the fracture and the organizational characteristics,The fixation of small flint provides the relative mechanics environment of elastic fixation, the small flint, which has reasonable configuration and mechanics superiority, possess keeping bone on axial direction, put the principle of functional activity into effect, which can promote the formate of the bone callus, and can make the bone callus get favourable moulding in the situation of functional status, so the fixation of splint has visible merit in the bone functional adaptation.The splint fixation have no stress block effect, and it has no destruction to the blood supply of the os integumentale, it has no interference to the procedure of the natural union of fracture. But, if the splint is too loose, there is still shear force or shearing stress in the local position of the broken ends, which can interfere the formation of the bony callus, and the aversion can happened in the procedure of the fixation, so the expected result cannot be reached. On the other hand, if the strap is banded too tight, intumesce can be found in the period of the fixation, and then necrosis happens.The healing of long bone shaft fracture by fixation of small splint is the characteristic technique in the Chinese medicine, which has long history of thousands of years, and accumulate abundant of experience too. Small splint has the superiority such as simple operation, low price, easy to functional excise, easy to care for, little damage to the tissue arounding, and having no influence on the blood supply, etc. This superiority lies in the difference with the "rigidity fixation" , the small splint is a kind of elastic fixation of "dynamic and static combination" , which is its essece and soul. The small splint fixation can afford the relative immovable mechanics environment in the broken ends, which permits activity in the broken ends on the longitudinal axis. This activity is benefit to the union of fracture, which can promote the absorb of the blood tumor, periosteum reactive thickening and hyperplasy, bone cells differentiation accelerate, blood vessels regenerate plentifully, bony callus grow and calcify quickly, the accreting time is ahead of schedule. ShangTianyu’ s research demonstrates that the local external fixation by the splint can control the activity that disadvantage to the union of fracture. It balances the the body gravity and the muscle stress which can cause the aversion again in the broken ends by the power of the restrainting of the strap, the lever power of the splint and the effective power of the pressure pad.The mechan-balance is implement by the two main mechanism of the soft tissue of the limb: one is the no compress ibility, the intermission filled with muscle form a invariably system when it is confined by the splint, and the broken ends wrapped by the muscle can get a relative stable situation as well.Besides, this fixation need not decoherence the periosteum, the damage to soft tissue is light, the destroying to the local blood supply is slight too, which is benefit to the union of the fracture; at the same time, the fixation by the small splint do not fix the joint, so it has no influence on the activity of the joint, which make the procedure of the union of the fracture and the functional recovery of the limb at equal pace. The aboving is special fit to the common medical idea of the international medical science community puisue to---take the medical "ending point target" as principle.So we can see that, the science connotation contained in the technique of healing fracture by small splint is very abundant, which is scientific, resonable, advanced, is a shining part of the Chinese medical science, and it is precious cultural and techincal heritage of our nation.However, what is regret that, for thousands years, it still stay in the state of the experience, whose mechanics essence and quantity relation is still not known, it lacks to be systematic and scientific unearthed, arranged, elevated, and be assigned new era connotation, and it has the trend of being drown without knowing. With a sense of responsibility of the history, using the mordern technique, we arrange, dig, improve this traditional technique, interpret the mechanism deeply, succeed and bring new ideas, make this technique have new period characterstic, and spread the clinical application, what is the background and the significance to carry out this research.My research use the traditional Chinese connect bone operation and small splint fixation in the experimental fracture for the first time over the world; we adopt advanced test index to evaluate the auality of the union of the fracture, and to monitor the procedure of the union of fracture and the mechanism how it can promote or be benefit to the union of fracture; we use the baroceptor in the external fixation device, so we can measure the jog extent in the broken ends fairly exactly, and then provide the consideration to design the new external ifxation device; the research result we got afford the experimental and theoretical foundation, which can change the traditional fixation technique of small splint from expirical to sciencetific, This research not only have fairly high practical value to guide the clinical work, but also can help us to dig, arrange, improve the essence of bone and wound technique of our traditional medical science, and it can mix the technique together with the mordern science, and to promote the traditional technique to bring benefit to people, so the research has important academic and theoretical signifcance.This research affords scientific base to the method of healing the bone fracture of four limbs by fixation of the smal1 splint, itestablishes new index to spread this technique, it offers theoretic data and experimental foundation to develop elastic fixed production; it hints the rule we shold followed to explore the new method of fixation of the fracture, so it has prospective guiding significance to prospect of the development of the healing the fracture by the means of combined treatment of traditional Chinese medicine and western medicine.二The purpose of the researchIn the research, we take the animal model of experimental fracture of rabbit tibia as our study object, fixed respectivily by small splint and steel plate. In the fixation of small splint, we let the broken ends accept different pressure by adapt the tightness of the strap. Observe the influence on the union of fracture in broken ends by the action of two different pressure, and compare with the steel fixed group, the main index that we observe are:To detect the express of VEGF and TGF-β1 by the means of immunity classify.To observe the fine structure of the bony callur tissue at different stage of the union of fracture by the technique of the electron microscopy; to observe the histology change at different stage of the union of fracture by the technique of light microscope; observe the vitodynamics influence on the bony callus in the procedure of the union of fracture by the different fixation, and then comprehend the quality of the union of fracture.Observe the influence on the bone density of the bony callus by the different fixation, observe the contents of the S-SAP in the different period after the rabbit experimental fracture, and the radioactivity observation to the local fracure. To evaluate the influence on the union of fracture by the elastic fixation of small splint from different point of view, monitor the procedure of the union of fracture, approach the mechanism how the fixation of small splint can promote the the union of the fracture, and then demonstrate the jog can stimulate the formation of the bony callus, to accelerate the union of fracture. Through this research , we can afford scientific base of healing bone fracture of four limbs by the means of fixation by small splint, so that we can spread this technique and offers theoretic data and experimental foundation to develop elastic fixed production.三Method for the study Experiment one Experiment material1.1Animal 45 rabbits, weigh 2-2. 5KG, afforded by the animal experiment center of hubei science research institute1.2 reagentAnti-VEGF and anti-TGF-β1 are all afforded by Zhongshan Reagent company1. 3 equipment light microscope immunity equipmentSplint: self-made splint is made by Shan tree barks which have good elastic, which is used in defferent place of bone fracture dividely , such as exterior, interior, front and behind. The upward part of the splint is wide and the downward part is narrow. The front splint is 11cm long, and its superior extremity is 2. 4cm wide; The back splint is 10cm long, and its superior extremity is 2. 4cm wide; the interior and exterior splint are both 10 cm long, their superior extremity is 1. 9cm wide. We sting a eyelet of 1.5 mm in the small splint which is used in front and behind part.Steel: It is provided by medical limited company of JiangSu Colden Deer GroupBaroceptor: is afforded by the WuHan ChaoYU detect and control technology company, its structure as follow:The baroceptor is composed by the pressure box, air duct, three way joint, infusing air hope, sensitive element and semiconductor straining piece. Whose principle is that: before measuring the pressure, infuse the air by infusing air hope, then the air duct is filled with air, that means the pressure can be kept balance at two sides of the sensitive element in the baroceptor, so the sensitive element will not be affected by the press. When the pressure affect on the pressure box, semiconductor straining piece pasted on the sensitive element changes the stressing produced by the change of pressure into the change of electric resistance, then establish the fixed quantity relation between the pressure and the electric resistance. We can measure the electric resistance by the measuring electron bridge in order to measure the change of the pressure. When using it, we put the baroceptor into the splint, then we know how tight the bandage is by the number of baroceptor.2. empirical method2.1We select 45 healthy rabbits, get a transverse fracture pattern of 3mm in the meta-infer 1/3 of left shin bone, divide into group A, B and C, In group A and B, we fix the fracture by cypsum, 5 days later, we change the fixation into external fixation by small splint. In group C, the broken bone is fixed by steel plate.Cross a Kirschner wire whose diameter is 1. 5 mm into the rabbit tibial tubercle, and then penetrate into the hole of circa side splint so that the splint cannot slip, the baroceptor is placed in the inner surface of the ventri-splint in the broken ends, and tie up the four splint by four bandage, the tightness of each group is not same, the wag of bandage in group A, B is 3mm and 7mm, and the number of baroceptor is 18kPa in group A, and which in group B is 12kPa.The collection and mamagement of the samplePut the rabbits to death 14 days, 24 days and 34 days dividedly after the operation, saw the sample of shin bone in the place of 1 cm above and below the fracture, hold it by 10% formaldehyde solution, decalcificate by 5% nitric acid, flush by lotic water, dewater by the grad alchol, embed by par, cut sheet in the longth wise, every piece is 4 um thick.The slice is deparaffinaged by the dimethyl benzene, incubated by 3% H2O2 Deionized water For 5 minutes to deactivate the endogenous peroxydase. Washed by the distilled water, soaked in PBS for 5 minutes, repaired by the micro-wave.Dropwise goat setum to block, rabbit anti- by 3% H2O2 Deionized water iAntibody and mice anti-VEGF antibody diluted by the proportion of 1: 100, stay overnight at 4℃. Wash by PBS for 3 minutes, and then dropwise biotinylation anti-2 and incubate for 15 minutes in the room temperature.Wash by PBS for 3 minutes, three times, drop horseradish enzyme labelling strepto-antibiotin, incubate for 15 minutes in the room temperature. Wash by PBS for 3 minutes, three times, colorate by the DAB, wash by the tap water, after stain by hematoxylin. dehydtate by Grad alcoholic, mounting after transpatent.2.3 Observe index: the express of VEGF, TGF-β1 Experiment two1 subject1. 1 Experimental animals: the same as experiment one1.2 equipmentlight microscopeTransmission electron microscope: H-500 (which is produced by Japanese Hitachi company). It is provided by WuHan military area army Gen Hospital.Light microscope (which is produced by Japanese Olympus Company). Transmission electron microscope: H-500 (which is produced by Japanese Hitachi company). It is provided by WuHan military area army Gen Hospital. Ultramicrotome: LKB-2, which is produced by Sweden LKB company, is provided by WuHan University medical college.2. Empirical method2.1 The animal model building and grouping is the same as experiment 12.2 Observe index and method2.2.1 macroscopic observation: We observe external、internal bony callus and bridge bony callus after the death of rabbits.2. 2. 2 histomorphology observation: Put the rabbits to death by means ofaeroembolism in the ear vein, kill the first batch 14 days after theoperation, kill the second batch after 24 days, kill the third batch after34 days, about 5 rabbits every time in each group. saw the sample of shinbone in the place of 1 cm above and below the fracture, hold it by 10%formaldehyde solution, decalcificate by 5% nitric acid, flush by loticwater, dewater by the grad alchol, embed by par, cut sheet in the longthwise, every piece is 5 cm thick, color it by campeachy-eosin in routinemethod, then observe it in light microscope.2. 2. 3 Fine structure observation: We get a sample in the size of 1 mm3in each period, fix it by the 2.5% GA solution, decalcificate it by 1%EATA solution, wash it three times by balanced solution (pH 7. 2-7. 4), thenfix it by 1% osmic acid 2 hours, dehyd it by alchol, Embed it by epoxyresin 812, get extra thin section, color by lead uranium, observe by TEM.experiment 31 Meterial and method1.1 Select 18 healthy rabbit, divided into group A, B and C on random,The animal model building and grouping is the same as experiment 11. 2 Detect item1. 2. 1 S-ALP: get the heart blood 2ml/kg by using number 9 pinhead beforeand 2, 4, 6 weeks after the operation. Detect the S-ALP. (detect by theHUBei Chinese medical hospital)1.2.2 Vitodynamic test tensile strength testThe three group animals are all put to death by the means of aeroemblism from ear vein, dissect left shin bone, get rid of soft tissue, wrapped up by the gauze, soak by the isotornic Na chloride.Put the experimental bone in the temperature of 20±5℃for at least 3h, detect within 12 hours. Then fix the rabbit shin bone with a side of the steel-wire, another side is hanged on the tongs of the LJ-500 tensile force equipment. Carry out the elongation test at the speed of 25mm/min, till pull and break the shin bone, so we can get the number of power of pull and break, the pull break cross-section should around the place of theunion of fracture, otherwise, abandon the data.Every way of fixation can get 6 data of effective pull break power, compute the bone’ s section area by the outside areaof the bone, use pull breal power division the section area, we can get the value of tensible strength, sort the total six data ,2 Make statistic treatment to the experimental result Experiment four1 Experimental meterial1. 1 Animal: 18 rabbits, weigh 2-2. 5KG, afforded by the animal experiment center of hubei science research institute1.2 Equipment: QDR 2000plus type DEXA2 Experiment method2.1 Bulit the animal mode and divide the groupSelect 18 healthy rabbits, devide into group A, B, and C on random, make mode and group the animals just as the experiment one, put the animals to death after the end of 6 weeks, dismantle the exterior and interior fixation, disengage the knee joint of the two posterior limb, reject the whole soft tissue, get the two shin bones, and remain the periosteum.2.2 Objective index2.2.1 Observation from the x-rayTake photographs of x-ray before putting every animal to death, make sure that no pseudpartoculation formates, and observe the condition of the growth of the bony calllus and of the union of fracture.2.2.2 bone densitometryAdopting the QDR 2000plus type DEXA, using the absorption method of the two power x-ray, detect the BMD in the middle tibia of fracture, choose the region of interest R1 in the same condition of the fracture, take the place of fracture as center, scan a distance of 10 cm from near place to the far place. Calculate the BMD of the Regions of Interest ( ROIs) about 1 cm arround the place of fracure using the data measured by the analysis software. Scan the corresponding position of the other side being not operated, the data we get are took as the contrasting data. Every sample are measured three times, we reset the sample every time after the measuring. The microcomputer can automatic analysis and print the result by the DEXA, calculate the ratio of the bone density of every couple of the samples.3 Data processing and statistics analysis: the formula to calculate the ratio of the parameter is that: ratio of bone density=value of corresponding side of being not operated/value of the side of being operated×100%. The value of bone density of every group is expressed as mean±standard deviation (x±s)四The result of the research1. The result of immunity classify: in the every phases of the union of fracture, the cell expression of TGF-β1 increases, and the cell expression of VEGF increases too, and there is invariably rapport between them. In the early phase and the middle phase of the union of fracture, the amount of the blood vessel formation and of the bony callus formation in group A are all more than which in group B and group C, and the time of the blood vessel formation in group A is earlier than which in group B and group C too. So the masculine express in group A is earlier than which in group B and group C, the masculine level in group A surpass obviously than which in group B and group C.2. Macroscopic observation of bony callus in the period of the union of fracture: In group A, the portion of fracture conjugate primarily 14 days after operation, we can see a 1 ittle fusion shape external callus, 24 days later, the callus are in fushion shap, callus bridge the two broken ends, 24 days later, the callus conjugate the two ends firmly, there is no range of motion in the broken ends.Group B: we put 5 rabbits to death at the time of 14 days, dislocation can be seen in one rabbits at the place of fracture; in the time of 24 days, there is partly conjugation in the broken ends, but the range of motion is large, the external callus is less than which in group A, the bony callus is moderate, taking Fusiform shape 34 days later. The broken ends are not connected completely by the bony callus, the fracture line still can be seen clearly.In group C: the broken ends have no dislocation in every period, we can see fibrous joint after 14 days, and we can see sporadic external callus in medium dose in 24 days, it align regularly, joint of Fushion form shape can not be see in the broken end, 34 days later, part of the external callus form Fushion shape, but not very firmly.3. Electron microscope shows that: in group A, in the procedure of union of fracture, osteoblast appears earlier, the quantity of it is more than which in the SF group, besides, its function is active. 34 days later, generous bone cell of active function can be seen in the broken ends. In group B, 24 days later, chondroblast can be seen, and 34 days later, hypertrophic cartilage cells still can be seen.4. The lighe microscope shows that: In group A , periosteumcallus, bridge callus can formate earlier rapidly, then become uniting callus gradually, 34 days later, the callus can connect the broken ends, where the woven bone transformate lamellar bone. Croup B: 14 days later, there is still blood tumor and granulation tissue in the broken ends, the amount of the bony callus is small. 24 days later, there are partly cartilage connection, woven bone is badly to see. 34 days later, the broken ends are not connected completely, and the hypertrophic cartilage cell still can be seen. In group C, 14 days later, a little fibrous callus can be seen in the broken ends, and granulation tissue still can be seen there, 24 days later, slight cartilage connects, 34 days later, the callus step over the broken ends, but not connect completely.5. The vitodynamic measuring in the broken ends6 weeks after the fracture, the tensile atrength in group A is more better than which in group B and the steel fixed group.6. The result of the S-ALP:different extent of jog influences on the the content of S-ALP in the experimental union of the rabbit fracture, the extent of the changing of AKP is different in the different period and in the different group, which is more higher in group A than in group B, C in 14 days.7. The result of the imageology: group A: the broken ends are filled with compact bony callus, the fracture line is vague, external callus is compact, partily pulp cavity is recanalization.Group B: there is many bony callus in the broken ends, pathy shadow can be seen in the broken ends, but the fracture line is still there.Group C: the distance between the broken ends can be seen clearly, there is small raritas bony callus through the broken ends, small external callus formate.8. The measure result of bony densityThere is highly significant deviation between group A and B after 42 days, the value of BMD in group A surpass the value in group B obviously, and there is no significant deviation between group B and C.五The result of the research of this topicThe jog can promote the union of fracture obviously; the time of the union of fracture is shortest in group A which is fixed by the small splint, whose velocity and qualitatively of the union of fracture has conspicuous superiority. Proper fixation by the small splint can improve the blood cycle of the local fracture, increase the amount of the cell, promote the cell differentiation, maturity, facilitate the depositing of the mineral matter in the broken ends of fracture, elavate the quality of union of fracture, shorten the time of union of fracture, and raise the strength of the broken ends.更多还原