不同时期注射富血小板血浆对牵张成骨的影响

目的确定富血小板血浆(platelet-rich plasma,PRP)在牵张成骨中的最佳注射时期。方法将36只大白兔随机分为4组,每组9只。Ⅰ组为对照组不注射PRP,其余3组分别在延迟期、牵张期和固定期注射0.5ml PRP于牵张间隙中。牵张结束后2、4、8周每组各处死3只动物取材。双能量X线骨密度仪(dualenergy X-ray absorptionmetry,DEXA)测量新生骨骨密度,采用方差分析法对数据进行统计分析;HE染色光镜下观察新骨生成情况。结果实验组新骨生成速率高于对照组;牵张结束后2、4周时,牵张期注射PRP组骨密度测量和组织学观察与其余两实验组间差异有统计学意义(P<0.05)。结论PRP对牵张成骨具有促进作用,牵张期注射PRP可缩短牵张成骨过程。     

Upper leg lengthening on an intramedullary nail: a preliminary report

Current methods of treatment for anisomelia are based on distraction osteogenesis. This involves creating callus during gradual distraction using an external fixator. The lengthening of long bones by Ilizarov's method requires keeping the external fixator in place for a long period of time, which increases the risk of complications, such as pin - track infection, stiffness of the joint, misalignment, and fracture of the regenerate. In order to shorten the period during which the external fixator must remain in place, we have introduced a method which enables the external fixator to be removed when the distraction phase is completed. During the period 1999-2001 we carried out femoral lengthening in 9 patients, using a combination of external fixation and intramedullary nail. After osteotomy of the femur and fixation by intramedullary nail, an external fixator was also applied, allowing for gradual distraction. After the requisite lengthening had been obtained by distraction at the rate of 1 mm per day, the nail was locked and the external fixator was removed. The operated patients ranged in age from 13 to 23; the average age at the moment of surgery was 16,2 years. The mean femoral anisomelia was 4,5 cm (range: 2,5-8,3). In seven cases the etiology of the anisomelia was congenital, in one case avascular necrosis of the femur, and in one case a history of femur fracture. The average duration of external fixation was 11 days for 1 cm of bone lengthening. All the lengthening femurs healed without bone union complications. In 8 cases we obtained complete isomelia, wile in 1 case there remained an inequality of 6 mm due to premature callus maturation and bone union. This is turn was caused by the patient's voluntary termination of distraction in conjunction with a two-week delay in the scheduled clinical follow-up. The removal of the external apparatus upon completion of distraction made it possible to institute early rehabilitation and enabled a quick restoration of physiological mobility in the knee joint. The chief advantages of limb lengthening using an intramedullary nail are the significant reduction in the duration of external fixation, protection of the bone regenerate against fracture, and a shorter period of time for rehabilitation. The disadvantages of this method include the extended surgical procedure and the risk of nail wedging in the intramedullary canal, which prevents lengthening.     

脂联素对兔下颌快速牵张成骨的影响

背景:脂联素可参与骨代谢及成血管过程,但目前关于脂联素对牵张成骨有无促进作用尚不清楚。目的:通过建立兔下颌快速牵张动物模型,探讨局部应用脂联素对骨牵张新骨再生的影响。方法:16只新西兰大白兔随机摸球法均分为对照组及实验组,建立兔下颌单侧快速牵张模型,牵张速率为2mm/d。在牵张开始的1,3,5d,对照组及实验组分别于牵张间隙注入200μL磷酸盐缓冲液或含有2μg重组人脂联素的磷酸盐缓冲液。结果与结论:两组动物牵张间隙内均可观察到新骨生成,组织学及显微CT检查显示实验组的新骨生成与钙化明显高于对照组。实验结果显示局部应用脂联素可有效促进兔下颌快速骨牵张的新骨再生。     

Distraction-resisting force during tibial diaphyseal lengthening and consolidation--a study on a rabbit model

BACKGROUND: Distraction-resisting force is generated in the soft tissues and callus during limb lengthening. Monitoring this force may offer a method of studying the behaviour of soft tissue and detecting the distraction osteogenesis related problems, and help to prevent complications. Changes in the post distraction period have not been previously investigated and there are no reports on the contribution of gastrocnemius to the distraction-resisting force. METHODS: Sixteen immature New Zealand White rabbits underwent 30% (left) tibial diaphyseal lengthening at a rate of two 0.4 mm increments per day. Using an instrumented bilateral fixator, the passive distraction-resisting force and the contribution made by gastrocnemius were measured at the end of lengthening and 5 weeks after lengthening. FINDINGS: The distraction-resisting force at the end of lengthening (mean 44 N (SD 10)) was statistically higher (p < 0.01) than that five weeks after lengthening (mean 20 N (SD 8)), so was the contribution of the gastrocnemius to the force (mean 11 N (SD 5 N) or 25% (SD 7) at the end of lengthening and 3 N (SD 1) or 13% (SD 5.2) five weeks later). INTERPRETATION: The callus rather than the surrounding muscles generates most of the passive DRF and its share of the force increased during consolidation period.     

The impact of selected biological factors on bone regeneration in distraction osteogenesis

Background. Comparatively few reports have analyzed the biological factors influencing the course of distraction osteogenesis, such as the etiology and magnitude of shortening or the patient's age at surgery. The aim of our study was to assess the impact of selected biological factors on the course of bone regeneration in distraction osteogenesis of the lower leg. Material and method. We examined 126 patients, on whom 138 lower leg lengthening operations were performed. Congenital shortening was the most numerous cause (54.9%), followed by postinfectious (16.6%), posttraumatic (14.3%), neurogenic (7.9%), and achondroplastic (6.3%). The course of distraction osteogenesis was analyzed on the basis of duration of treatment, expressed by the lengthening index. Results. The highest lengthening index values were found in neurogenic patients, somewhat lower in congenital. The lowest lengthening index values were observed in postinfectious and posttraumatic shortenings, and in the group of bi-level lengthenings in achondroplastic patients. The lengthening index decreased along with increasing elongation. Lower lengthening index values were found in patients younger than 20 years, regardless of the method and number of osteotomy levels. Conclusions. There is clinical evidence for the impact of etiology, age, and magnitude of shortening on the course of distraction osteogenesis. The shortest treatment duration was found in shortening of acquired etiology and achondroplasia, and in patients older than 20 years. The values of the lengthening index were the lowest in lengthenings over 6 cm, whereas the greatest values were observed in lengthenings less than 4 cm.     

Enhancement of bone regeneration with the accordion technique via HIF‐1α/VEGF activation in a rat distraction osteogenesis model

Axial micromotion of bone fragments promotes callus formation and bone healing during the process of distraction osteogenesis (DO). This study investigated the effects of the combined axial compression and distraction (accordion) technique on bone regeneration in rat DO model. Male Sprague–Dawley rats (n = 62) underwent right tibial transverse osteotomy and were randomly divided into four groups after lengthening: control (no manipulation) and three experimental groups assigned on the basis of the period of accordion manoeuvres in the consolidation phase (Groups 1, 2, and 3 with accordion technique applied at Weeks 1, 3, and 5, respectively). Animals were terminated at 1 week after each accordion phase (i.e., Weeks 2, 4, and 6). Callus formation was monitored by X‐ray radiography; new bone quality was evaluated by microcomputed tomography, histological analysis, and mechanical testing. Serum levels of hypoxia‐inducible factor (HIF)‐1α and vascular endothelial growth factor (VEGF) were measured. Callus formation after accordion manoeuvre at Week 3 (Group 2) increased significantly over time of consolidation. The microcomputed tomography and mechanical analysis revealed Group 2 had more newly formed bone and superior mechanical properties in contrast to the other groups at termination. Histomorphological and immunohistochemical analyses confirmed a greater degree of osteogenesis and angiogenesis corresponding to increased serum levels of HIF‐1α and VEGF in Group 2. The accordion technique was effective in promoting bone consolidation via activation of HIF‐1α/VEGF during DO. The accordion technique may be used in the middle phase of bone consolidation to promote bone formation in patients undergoing DO treatment.     

骨形态形成蛋白在下颌骨快速牵张成骨中的应用

背景:牵张成骨已经成为治疗不同类型颅面畸形和骨缺损的有效的方法,但是牵张成骨的主要缺点是牵张期和稳定期比较长,可能导致牵张过程中严重的并发症。目的:总结骨形态形成蛋白在快速牵张成骨过程中作用的研究现状。方法:电子检索计算机Pubmed数据库(1989/2011)收录的骨形态形成蛋白和牵张成骨相关综述和论文报告。结果与结论:共纳入骨形态形成蛋白在快速牵张成骨中的作用相关文献32篇。骨形态形成蛋白具有很强的成骨活性,能促进骨再生和骨改建。目前的研究显示应用骨形态形成蛋白能加快牵张成骨过程中新骨形成和缩短治疗的疗程。但是骨形态形成蛋白应用于临床还需要进一步的研究。     

下颌骨牵张成骨和牵张器拆除时机的评价

目的:牵张成骨是治疗颌骨畸形和骨缺损的新方法,但力学研究及由此而确立的牵张器拆除时机的研究甚少。通过建立了山羊下颌骨牵张模型,观察下颌骨牵张后的物理、机械特性,探索牵引器拆除的时间。方法:8只山羊单侧下颌骨2次/d,1mm/d,共8d,后以牵开器继续固定至4周,行放射学、组织学、骨密度及力学测试。结果:牵拉术后下颌骨成骨明显,牵拉后2周,X线示骨间隙内新骨已基本连接骨缺损,4周时骨化明显。其骨密度与正常松质骨无明显差别,极限载荷为正常侧的61%。结论:生长期山羊为一良好的下颌骨牵张模型动物,牵拉后4周可以考虑去除牵开器。     

Computed radiographic and ultrasonic evaluation of bone regeneration during tibial distraction osteogenesis in rabbits

Computed radiography (CR) and a combined ultrasound (US) approach involving two-dimensional (2-D) and three-dimensional (3-D) ultrasonography with ultrasonometry were employed to evaluate their respective efficacies in monitoring bone regeneration during rabbit tibial distraction osteogenesis (DO). Results demonstrated that 2-D and 3-D ultrasonography depicted bone callus growth changes during distraction while CR could not. Evaluation of callus speed of sound, acoustic reflection and attenuation showed significant linear changes over time during early DO stage (p < 0.05). However, surrogate measure of callus density by CR only showed such significant linear changes during consolidation (p < 0.05). Also, callus speed of sound and acoustic reflection during early DO stage showed strong predictions to the bone mineral density and microstructural properties (adjusted-R² = 0.43–0.67) of consolidated bone callus measured at the treatment end-point by microcomputed tomography. Findings of the present study indicated a preferred use of the combined US approach over CR in the early monitoring of bone regeneration during DO treatment.     

下颌骨牵引成骨过程中基因干预对牵引区转化生长因子β1表达的影响

背景:局部基因治疗能促进牵引区新骨的生成,但关于基因治疗后对局部生长因子表达的影响目前尚不清楚。目的:观察电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中转化生长因子β1表达的影响。方法:新西兰大白兔双侧下颌骨截骨后3d开始下颌骨牵引,0.8mm/d,连续牵引7d后,随机分为5组,分别在牵引区注射2μg(0.1g/L)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165、空质粒pIRES及相同剂量的生理盐水。之后施加电穿孔刺激。结果与结论:免疫组织化学染色发现转化生长因子β1主要在细胞胞浆中表达,给药7d时骨端骨细胞、编织骨痂骨细胞、骨痂表面成骨细胞呈转化生长因子β1染色阳性;14d时新生成的编织骨痂骨细胞、骨痂表面成骨细胞、肉芽组织中的间质细胞、单核巨细胞、多核巨细胞转化生长因子β1染色阳性;28d时转化生长因子β1阳性细胞明显减少。其中注射重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165后转化生长因子β1的表达明显多于注射空质粒pIRES及生理盐水(P<0.05或P<0.01)。说明基因治疗能促进转化生长因子β1的表达,促进牵引区细胞基质的形成和新骨生成。     

固定期放疗对兔下颌骨牵张成骨术中新骨形成影响的实验研究

目的研究固定期放疗对兔下颌骨牵张成骨新骨形成的影响。方法 30只成年新西兰大耳白兔随机分成A、B、C三组,A组实验动物行双侧下颌骨皮质骨切开术并植入牵张器,5d后开始骨牵引,速率为0.5mm/次,2次/d,连续10d,共延长下颌骨10mm,固定10周;B组实验动物行双侧下颌骨皮质骨切开、植入牵张器,进行骨牵张,牵张结束后固定10周,并在固定4周后开始用直线加速器照射双侧下颌骨,5.4Gy/次,隔日1次,共5次,总剂量为27Gy;C组动物为对照组。固定期结束处死动物后,取各组动物牵张区新生骨痂行X线检查、组织学观察、骨形态计量学分析、骨密度测定及三点弯曲试验测试牵引区抗弯强度。结果大体观察和X线检查显示所有进行下颌骨牵张的实验动物牵张间隙均有新骨形成,骨小梁沿牵张方向排列,骨密度较高;组织学观察显示A组实验动物牵张区均充满排列整齐的新生编织骨,B组可见较多的胶原纤维成发及软骨岛,新生骨小梁不及A组致密、成熟;骨形态计量学分析和机械力学分析结果显示B组的新生骨在骨密度和新生骨小梁数目上与A组比较无统计学差异,但是在新骨矿化程度方面,固定期放疗组较差,其机械强度也较低(P<0.05)。结论在牵张成骨术的固定期进行放疗仍可以出现牵张区的新生骨形成,但软骨成分较多,机械强度较低。牵张成骨术用于颌骨恶性肿瘤切除术后颌骨早期重建是可行的。     

胫骨牵引成骨过程中骨形态形成蛋白4及其受体的表达

背景：骨形态形成蛋白4及其受体在骨再生和修复过程中起着重要的作用,但具体机制尚不清楚。目的：建立小鼠胫骨牵引成骨模型,分析骨形态形成蛋白4及其受体在牵引成骨过程中的表达,探讨机械牵张力转化为生物信号从而调节骨再生过程的机制。方法：健康雄性8周龄CD-1小鼠36只,按照手术时间随机分成术后第5,9,13,17,24和31天组,每组6只。所有小鼠均接受左胫骨中上段低能截骨,安置体外延长固定架,截骨后5d为静止期;截骨后第6天起开始每天进行胫骨延长,速率为0.2mm,2次/d,共12d,为牵引期;自第18天停止牵引,为固塑期。于术后第5,9,13,17,24和31天分别处死动物,采集胫骨标本,作组织学检查、RT-PCR和原位杂交实验分析骨形态形成蛋白4及其受体激活素样激酶3以及骨钙素的表达。结果与结论：组织学检查显示静止期修复过程基本与骨折愈合过程相似。小鼠骨折断端在持续牵引下有明显的骨痂形成,骨形态形成蛋白4及其受体激活素样激酶3以及骨钙素的mRNA的表达明显增强。结果表明,牵引成骨是一种持续的骨再生过程,机械张力可通过刺激骨形态形成蛋白4及其受体以及骨钙素的持续高表达维持骨痂的不断形成和再塑,以充填连续延长的骨折间隙。     

Effects of hyperbaric oxygen therapy on distraction osteogenesis

Investigators in the present study explored the effects of hyperbaric oxygen (HBO) therapy on distraction osteogenesis in a rabbit limb-lengthening model. HBO treatment was provided to participants in a small animal pressure chamber once a day during the latent period of distraction osteogenesis at 2.5 absolute atmospheres of pressure. Bone mineral density measurements were obtained through torsional testing. The control group received no HBO treatment. Animals were killed 8 wk postoperatively for biomechanical testing. There was a statistically significant increase in bone mineral density in the HBO group compared with that in the non-HBO group, but no statistically significant differences were observed between biomechanical parameters of the 2 groups. The investigators suggest that these results must be further studied through histologic examination to determine the stepwise effects of HBO during and after collagen synthesis.     

人骨形态发生蛋白2基因修饰自体骨髓间充质干细胞移植促进兔下颌骨牵张成骨实验的组织形态学观察

背景:如何提高牵张成骨过程中新骨形成的速度和质量,缩短牵张成骨治疗时间,减少并发症的发生是目前该领域的研究热点。目的:观察人骨形态发生蛋白2基因修饰自体骨髓间充质干细胞移植对兔下颌骨牵张成骨新骨形成的促进作用。方法:36只新西兰白兔随机摸球法分为3组。建立牵张成骨动物模型,在固定期第2天,实验组于牵张间隙注射人骨形态发生蛋白2基因修饰的自体骨髓间充质干细胞;对照组注射等量自体骨髓间充质干细胞;空白组注射等量生理盐水。结果与结论:在固定期2周及6周实验组牵张区骨小梁形成质量明显好于对照组和空白组。证实骨形态发生蛋白2基因修饰的自体骨髓间充质干细胞移植能有效促进兔下颌骨牵张成骨新骨形成。     

Interleukin-1 and tumor necrosis factor antagonists attenuate ethanol-induced inhibition of bone formation in a rat model of distraction osteogenesis

Chronic ethanol exposure inhibits rapid bone formation during distraction osteogenesis (DO; fracture and limb lengthening) and decreases volumetric bone mineral density (BMD) in a model of intragastric dietary infusion [total enteral nutrition (TEN)] in the rat. The hypothesis tested herein was that overexpression of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha mediates these deleterious effects of ethanol on the rat skeleton. Two studies (study 1, female rats; study 2, male rats) were performed to test the potential protective effects of the IL-1 and TNF antagonists: IL-1 receptor antagonist (IL-1ra) and 30-kDa polyethylene glycol-conjugated soluble TNF receptor type 1 (sTNFR1). All rats were infused with a liquid diet +/- ethanol (EtOH) and underwent tibial fractures and DO. During distraction, the animals received a combination of IL-1ra (1.8-2.0 mg/kg/day) and sTNFR1 (2.0 mg/kg/2 days) or vehicle. A comparison of distracted tibial histological sections demonstrated 1) significant antagonist-related increases in bone column formation over the EtOH controls (studies 1 and 2), and 2) restoration of new bone equivalent to that of the TEN controls (study 2). In contrast, examination of intact proximal tibial metaphyses by peripheral quantitative computerized tomography revealed decreases in volumetric BMD of both EtOH control and EtOH antagonist groups (study 2). These results demonstrate that short-term systemic administration of IL-1 and TNF antagonists together protect rapid bone formation during DO from the deleterious effects of chronic ethanol but are ineffective in regard to intact bone homeostasis.     

电穿孔介导基因治疗下颌骨牵引过程中血管内皮细胞生长因子的表达

背景:课题组前期实验已证明基因治疗能促进下颌骨牵引区新骨的生成。目的:探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中血管内皮细胞生长因子表达的影响。方法:45只新西兰大白兔双侧下颌骨截骨,建立下颌骨牵引模型。牵引7d后将模型兔随机摸球法均分为pIRES-hVEGF165-hBMP2组、pIRES-hBMP2组、pIRES-hVEGF165组、空质粒载体pIRES组、生理盐水对照组,分别于牵引区注射相应质粒或生理盐水。各组动物均施加电穿孔刺激。结果与结论:血管内皮细胞生长因子主要在骨周围结缔组织成纤维细胞、血管内皮细胞、骨组织成骨细胞和骨细胞表达,也可见炎细胞如单核细胞表达。各组均在固定期第7天表达最强,14d下降,28d时表达较弱,但在各时点基因治疗组明显强于对照组。说明电穿孔介导的基因治疗能使血管内皮细胞生长因子持续表达,并使其表达时限延长,促进牵引区新生血管生成和新骨形成。     

自体富血小板血浆促进兔上颌缝的牵张成骨

背景：富血小板血浆内含多种高浓度的生长因子，具有促进新骨生成，加速愈合的作用，对牵张成骨的作用尚无公识性结论。目的：观察自体富血小板血浆对免上颌骨牵张成骨的影响。设计、时间及地点：随机对照动物实验，于200707／08在中山大学北校区何母实验楼生理实验室完成。材料：3～5月龄健康家兔16只，雌雄各半，体质量1．4～1．7kg，随机分为实验组和对照组，每组8只。自制上切牙带环、外置式前牵引面具、牵引橡皮圈。凝结剂由1000U牛凝血酶溶于1mL100肌氯化钙制成。方法：分别在家兔左侧前颌缝的两侧置入钛钉（直径1．5ram），戴自制前牵引装置，试验组在牵张开始时，注射V（富血小板血浆）：V（凝结剂）=9：1混合的凝胶样物质至左侧前颌缝内。两组分别持续牵引1周和3周，每个时间点4只家兔。主要观察指标：牵引结束后测量骨缝两侧钛钉间距离的增加值以及组织学观察结果。结果：对照组和试验组家兔上颌均向前移位。同对照组相比，实验组骨缝标志钉间距离增加值较大，新骨生成与矿化较快，血管分布较多，牵张间隙中骨小粱较为粗壮和成熟。结论：富血小板血浆有助于骨组织再生，对兔上颌牵张成骨具有促进作用。     

Zoledronic acid improves the mechanical properties of normal and healing bone

Objective. To determine the effects of the bisphosphonate zoledronic acid on the mechanical properties of normal and regenerating bone in a rabbit model of distraction osteogenesis.

Background. Bisphosphonate therapy is used for treating osteoporosis and, more recently, to enhance bone healing and reduce stress-shielding osteoporosis in distraction osteogenesis.

Methods. Thirty eight rabbits underwent 14 days of distraction osteogenesis on the right rear limb. They received either zero, one or two doses of intravenous zoledronic acid. Four point bending tests were performed to collect mechanical data.

Results. The peak load capacity of the regenerating bone was significantly increased by bisphosphonate therapy, but a similar trend in normal bone was not significant. The energy absorbed to failure did not vary significantly in either group.

Conclusions. These data suggest that bisphosphonate therapy has a beneficial effect on the load capacity of regenerating bone, without increasing the brittleness of either new or existing bone.Relevance

Zoledronic acid administration may be a potentially valuable adjunct to distraction osteogenesis treatment, to enhance bone strength, thus reducing refracture complications.     

骨延长患者诱导型一氧化氮合酶的表达

背景:骨延长的牵张刺激如何转变为骨质再生的生物学信息至今不明确。目的:观察骨延长患者诱导型一氧化氮合酶的动态表达。方法:单侧胫腓骨延长组患者8例,分别于手术前第3天、术后第3,7,8,11,30天、停止延长时、停止延长第3,15,30天、拆除外固定器时、拆除外固定器后30d,检测血清诱导型一氧化氮合酶水平。并设立年龄相匹配的对照组8例。结果与结论:骨延长组牵张操作开始第1天时(术后第8天),血清诱导型一氧化氮合酶即开始升高,停止延长时达高峰,这一时段各个时相骨延长组血清诱导型一氧化氮合酶均显著高于对照组(P<0.01),停止延长3d时的血清诱导型一氧化氮合酶亦高于对照组(P<0.05)。提示诱导型一氧化氮合酶的高表达是骨延长的分子生物学机制之一。     

Biomechanical assessment of a novel bone lengthening plate system — A cadaveric study

Background

Although many types of external fixators have been developed for distraction osteogenesis, all have some drawbacks. We recently developed a novel bone lengthening plate to overcome these problems. The purpose of this study is to conduct biomechanical analyses using cadavers to assess the stability of the bone lengthening plate in relation to distraction length and femoral bone mineral density.

Methods

We used human cadaveric femurs (n = 18) to assess the effects of distraction length and bone mineral density on the biomechanical stability of the bone lengthening plate. After establishing control (n = 6, 0 mm lengthening) and experimental groups (n = 12, 30 mm lengthening), we measured biomechanical stability (structural stiffness, ultimate load, and displacement) under a compressive load. The experimental group was subdivided into a group with normal bone mineral density (n = 6) and a group with osteoporosis (n = 6), and the biomechanical stability of these groups was compared.

Finding

Structural stiffness differed significantly between the control (417.6 N/mm) and combined experimental groups (185.6 N/mm, p = 0.002). Ultimate load also differed significantly between the control (1327.8 N) and combined experimental (331.4 N, p = 0.002) groups. Bone mineral density was unrelated to structural stiffness (p = 0.204), ultimate load (0.876), or displacement (0.344). In all cases, failure of the bone lengthening plate occurred at the longitudinal connectors, such as the connecting columns between the upper and lower plates, and the lengthening shaft of the bone lengthening plate.

Interpretation

The biomechanical stability of the bone lengthening plate was affected by the lengthening length but not by bone mineral density. In addition, biomechanical stability during lengthening was most strongly influenced by the longitudinal connectors.