牵张成骨术在延长下颌骨中新骨生成方式的研究

目的　探讨山羊下颌骨牵张成骨过程中新骨生成方式及其影响因素。方法　用口外牵张器 ,按1mm/天的牵张速率将 12只成年山羊的双侧下颌骨延长 10 m m,牵张结束后固定至第 2、4和 8周分别处死 4只动物 ,拍摄下颌骨 X线片后取牵张区新生骨痂作组织学观察。结果　牵张间隙内新骨组织沿牵张方向向心性生长 ,成骨方式主要是膜内成骨 ,在牵张器松动的标本早期仍可观察到散在的软骨岛。结论　牵张延长下颌骨过程中新骨生成方式主要为膜内成骨 ,软骨化骨只是在牵张器固定不良时发生。     

兔下颌骨牵拉成骨动物模型的建立及初步观察

目的　建立兔下颌骨牵拉成骨动物模型,研究了解颅面部牵拉骨生成的规律。方法　将12 只新西兰大白兔右侧下颌骨第一磨牙前完全截骨后用牵拉器固定,左侧不做手术为对照侧。1周后以每天一次0.9 m m 的速度逐步牵拉,连续10 天。牵拉完成后1 天,2、4 和8 周每组处死3只兔,取下完整下颌骨进行大体测量,X线摄片,新骨组织学观察。结果　12只兔右侧下颌骨平均延长8.3 m m ,与对照侧比较有显著性差异(P< 0.01),无骨不连及畸形愈合。X线摄片发现,延长完成后2周牵拉间隙已被骨痂桥接,8 周时,X线片上很难分辨新骨和正常骨。组织学观察牵拉早期即有胶原束形成,随后钙化成骨,未发现软骨中介体。结论　运用牵拉成骨技术可成功地延长兔下颌骨,无骨不连和畸形愈合等并发症,新骨以膜内成骨方式生成     

Effect of distraction frequency on bone formation during bone lengtheningA study in chickens

We compared the effects of two distraction frequencies on bone formation during tibial lengthening by evaluating radiographs, bone mineral density, and histological findings. In 15 mature White Leghorn chickens, both tibiae were distracted at a rate of 0.75 mm/day for 10 days. The distraction frequency was 2 steps (0.375 mm/12 hour) by hand on the right side and 120 steps (0.00625 mm/12 min) by autodistractor on the left. Serial radiographs showed faster bone formation on the 120-step side than on the 2-step side. Bone mineral density on the 120-step side was also higher than that on the 2-step side at all times. On the 2-step side, endochondral ossification was marked in the early stage of distraction; then intramembranous ossification became the main mechanism of bone formation. On the 120-step side, however, intramembranous bone formation predominated throughout the study. Our findings support the contention that, at least in skeletally mature chickens, an increase in the distraction frequency improves osteogenesis during bone lengthening.     

Effect of distraction frequency on bone formation during bone lengthening: a study in chickens

We compared the effects of two distraction frequencies on bone formation during tibial lengthening by evaluating radiographs, bone mineral density, and histological findings. In 15 mature White Leghorn chickens, both tibiae were distracted at a rate of 0.75 mm/day for 10 days. The distraction frequency was 2 steps (0.375 mm/12 hour) by hand on the right side and 120 steps (0.00625 mm/12 min) by autodistractor on the left. Serial radiographs showed faster bone formation on the 120-step side than on the 2-step side. Bone mineral density on the 120-step side was also higher than that on the 2-step side at all times. On the 2-step side, endochondral ossification was marked in the early stage of distraction; then intramembranous ossification became the main mechanism of bone formation. On the 120-step side, however, intramembranous bone formation predominated throughout the study. Our findings support the contention that, at least in skeletally mature chickens, an increase in the distraction frequency improves osteogenesis during bone lengthening.     

Rat model of distraction osteogenesis

Prior studies of distraction osteogenesis in dog and rabbit models have shown predominantly intramembranous bone formation. Other models of fracture healing normally display mixtures of both endochondral and intramembranous bone formation. We have established a rat model of tibial lengthening that reliably reproduces the pattern of zonal osteogenesis previously observed in dog and rabbit models. A distraction rate of 0.25 mm twice a day with a 0-day latency period produced intramembranous bone with zones of progressive mineralization from collagen. With this protocol, rats bridged the distraction gap with a 25% increase in the tibial bone length. After 20 days of distraction and 50 days of consolidation, the three-point bending stiffness, as a percentage of the contralateral control, reached a level equivalent to that measured in the canine model for a 15% lengthening (28-day distraction and 84-day consolidation). Radiodensitometric analysis of the regenerate bones measured 97% of the unaffected contralateral tibial densities, and mineral analyses demonstrated that calcium and phosphorus levels in the regenerate bone reached 78% of contralateral tibial levels by day 70. We concluded that a rat model of distraction ostegenesis will be useful for a wide range of studies involving rapid intramembranous bone formation.     

The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction

To assess the influence of both the rate and the frequency of distraction on osteogenesis during limb elongation, a canine tibia was used with various combinations of distraction rates (0.5 mm, 1.0 mm, or 2.0 mm per day) and distraction frequencies (one step per day, four steps per day, 60 steps per day). The distractions were performed after both open osteotomy and closed osteoclasis. Histomorphic and biochemical studies were conducted on the elongated osseous tissue, fascia, skeletal muscle, smooth muscle, blood vessels, nerves, and skin. It was determined that distraction at a rate of 0.5 mm per day often led to premature consolidation of the lengthening bone, while a distraction rate of 2.0 mm per day often resulted in undesirable changes within elongating tissues. A distraction rate of 1.0 mm per day led to the best results. It was also observed that the greater the distraction frequency, the better the outcome. With optimum preservation of periosseous tissues, bone marrow, and blood supply at the time of osteotomy, stability of external fixation, and 1.0 mm per day of distraction in four steps, osteogenesis within the distraction gap of an elongating bone takes place by the formation of a physislike structure, in which new bone forms in parallel columns extending in both directions from a central growth zone. The growth plate that forms under the influence of tension-stress has features of both physeal and intramembranous ossification, yet is neither; instead, the distraction regenerated bone is unique, providing numerous applications in clinical traumatology, orthopedics, and other medical disciplines.     

Distraction osteogenesis by Ilizarov and unilateral external fixators in a canine model

We studied distraction osteogenesis in canine experimental model using two types of external fixators, Ilizarov (n=6) or AO unilateral (n=9) external fixator. Distraction started 1 week after surgery (2 x 0.5 mm/day) and lasted for 3 weeks. Specimens were harvested from weeks 7 through 12. The outcome was assessed by X-ray, histology, histomorphometry and microradiography. Bone regeneration as observed by X-rays was satisfactory and similar in both groups. Both endochondral ossification and intramembranous ossification were found simultaneously in both groups. In both groups, bone formation parameters were significantly higher in the area of consolidating bone. No differences in histomorphometric parameters existed between the groups. In the study period, the bone formation was enhanced and prevailed in the distraction area. This study demonstrated the utility of the canine experimental model for the study of distraction osteogenesis.     

Histomorphometry of distraction osteogenesis during experimental tibial lengthening

OBJECTIVES: The aim of the current study was to analyze distraction osteogenesis histomorphometrically to investigate the amount of enchondral ossification adjacent to intramembranous ossification. STUDY DESIGN: Species-specific, longitudinal time study.METHODS Lengthening of 25% of the right tibia was performed in 24 beagle dogs by callus distraction after osteotomy and application of a ring fixator. Distraction was started at the fifth postoperative day with a distraction rate of 0.5 mm twice a day. Twelve dogs were killed at the end of the distraction phase of 25 days (group A) and the remaining 12 dogs after an additional consolidation period of 25 days (group B). The tibia was removed from the distracted right leg and from the left control side of each animal, and longitudinal sections were cut and stained with Pentachrome. In all tibiae, an area of 0.5 x 2.4 cm within the endosteal bone was evaluated histomorphometrically with a Merz grid, and the results were compared between distraction and control side as well as between the dogs of group A and group B. RESULTS: On the distraction side, the typical signs of an increased de novo bone formation with a significant increase of osteoblasts and osteoid in group A, as well as an additional significant increase of bone volume and trabecular thickness in group B, were found. In all distraction cases, islands of cartilage formation, which underwent enchondral ossification, were found, besides membranous ossification. The ratio between membranous and enchondral ossification was found to be five to one. CONCLUSION: Bone formation during distraction osteogenesis results from both membranous and enchondral ossification.     

Novel approach to calvarial bone transport using a rabbit model

Calvarial defects sometimes require cranioplasty to protect the brain. Alloplastic materials, such as acrylic resin, hydroxyapatite ceramics, and titanium, involve various problems, such as vulnerability, infection, deformity resulting from growth, and high cost. We devised a new bone transport model in the rabbit based on the distraction osteogenesis theory of Ilizarov. Twelve Japan white rabbits with a mean body weight of 2.5 kg aged 12 weeks were used. Craniectomy (7 x 14 mm) was performed in 12 rabbits. Trapezoid bone osteotomy was performed anterior to the calvarial defect in 10 rabbits. The distraction device (Extension-plates) was fixed between the trapezoid bone island and the skull. Distraction was initiated 5 days postoperatively. The device was activated once every other day, with approximately 0.75 mm or 0.5 mm per activation. Bone distraction was continued until the rod could not be moved. The lengths of distraction were 4 mm in two cases, 5 mm in one case, 6 mm in one case, and 7 mm in two cases, with a mean of 5.5 +/- 0.56 mm. Both radiographic and histological findings showed osteogenesis by intramembranous ossification and trans-chondroid bone formation. Distraction osteogenesis has potential clinical applications in cranioplasty, especially in children because usage of autogenous bone is difficult if not impossible in most cases.     

Development of tensile strength during distraction osteogenesis in a rat model.

These studies were designed to determine the reliability of in vitro tensile testing to measure the temporal development of regenerate bone strength in rats during limb lengthening (distraction osteogenesis, DO). External fixators were placed on the right tibiae of 36 virus-free, 400-450 g male Sprague Dawley rats, and osteotomies (n = 33) were performed. Distraction was initiated the following morning (0 day latency) at 0.4 mm/day and continued to day 20. The 8 mm gap was allowed to consolidate for up to 50 days (day 70 postop). Contralateral unoperated and operated (fixator only) controls were included. On days 20, 30, 50 and 70 postop, the rats were anesthetized, and their tibiae were radiographed prior to undergoing sacrifice for histological or tensile analysis. On day 70, an additional group was tested by three-point bending. Radiodensity measurements demonstrated progressive mineralization of the DO gap, and histology confirmed typical intramembranous ossification of collagen bundles oriented parallel to the distraction force. Tensile stiffness increased significantly between days 20 and 30 postop, this increase correlated with initial radiographic and histologic bridging of the DO gap. Energy to failure and ultimate tensile strength increased progressively to day 70. At day 70, the force to failure for three-point bending was 65% of control tibiae. In conclusion, in vitro tensile testing provides a reliable method to test the development of structural integrity during the early stages of DO. Therefore, the biomechanical effects of postulated modulators of bone repair can be measured during early stages (bone formation, bridging, early consolidation) of DO in a rat model.     

ADAM10在小鼠颅面部膜内成骨过程中的表达

目的观察解聚素样金属蛋白酶10(A disintegrin and metalloprotease 10,ADAM10)在小鼠颅面部骨骼膜发育过程中的表达变化。方法以野生型C57BL/6小鼠为实验对象,阿辛蓝-茜素红染色显示小鼠膜内成骨区域,结合免疫组织荧光,检测ADAM10在骨组织中的表达。三标免疫荧光观察ADAM10在MC3T3细胞系中亚细胞定位。蛋白免疫印迹检测ADAM10在小鼠出生前后的表达量变化。结果组织阿辛蓝茜素红染色显示小鼠前颅骨、鼻旁软骨、上颌骨、腭板和下颌骨成骨活跃,并结合免疫组织荧光检测,发现ADAM10在小鼠前颌骨、鼻旁软骨、上颌骨、腭板和下颌骨广泛表达,且主要表达在成骨活跃的区域。MC3T3细胞三标免疫荧光检测进一步定位ADAM10广泛分布在胞浆中,在质膜和细胞核附近表达高;其胞核附近的高表达信号与高尔基体共标,提示其可能在高尔基体中加工后至细胞膜发挥功能。同时,蛋白免疫印迹结果证实,ADAM10在小鼠出生前后表达高,成年后表达明显降低。结论 ADAM10广泛表达于小鼠早期颅面部膜内成骨活跃区域,提示其可能调控小鼠颅面部膜内成骨过程。     

Temporal and spatial expression of bone morphogenetic protein-2, -4, and -7 during distraction osteogenesis in rabbits

The Ilizarov method of limb lengthening makes use of the fact that osteogenesis is induced at an osteotomy site when distraction is applied. It is unknown at present how the mechanical forces created by distraction are translated into biological signals. Because bone morphogenetic proteins (BMPs) are potent inducers of osteogenesis in many experimental systems, they are obvious candidates for playing a role in this process. In this study, we investigated the temporal and spatial expression of BMP-2, -4, and -7 proteins during distraction osteogenesis using immunohistochemistry. An osteotomy was performed on the right tibiae of white New Zealand rabbits. After a delay of 7 days, distraction was started at a rate of 0.25 mm/12 h for 3 weeks, followed by a 3 week consolidation phase. Each week after osteotomy one rabbit was killed for immunohistochemical studies. Staining for BMP-2, -4, and -7 was evident before distraction was applied and was mainly localized to mesenchymal cells and osteoblastic cells in the periosteal region. After distraction was started, the typical fibrous interzone developed between the osteotomy fragments, where both intramembranous and endochondral ossification were noted. In this area, cells resembling fibroblasts and chondrocytes, but not mature osteoblasts, showed intense staining for all three BMPs. This high level of expression was maintained during the entire distraction phase and then gradually disappeared during the consolidation phase. These results are compatible with the hypothesis that BMPs play an important role in the signaling pathways that link the mechanical forces created by distraction to biological responses.     

Temporal and spatial expression of bone morphogenetic protein-2, -4, and -7 during distraction osteogenesis in rabbits

The Ilizarov method of limb lengthening makes use of the fact that osteogenesis is induced at an osteotomy site when distraction is applied. It is unknown at present how the mechanical forces created by distraction are translated into biological signals. Because bone morphogenetic proteins (BMPs) are potent inducers of osteogenesis in many experimental systems, they are obvious candidates for playing a role in this process. In this study, we investigated the temporal and spatial expression of BMP-2, -4, and -7 proteins during distraction osteogenesis using immunohistochemistry. An osteotomy was performed on the right tibiae of white New Zealand rabbits. After a delay of 7 days, distraction was started at a rate of 0.25 mm/12 h for 3 weeks, followed by a 3 week consolidation phase. Each week after osteotomy one rabbit was killed for immunohistochemical studies. Staining for BMP-2, -4, and -7 was evident before distraction was applied and was mainly localized to mesenchymal cells and osteoblastic cells in the periosteal region. After distraction was started, the typical fibrous interzone developed between the osteotomy fragments, where both intramembranous and endochondral ossification were noted. In this area, cells resembling fibroblasts and chondrocytes, but not mature osteoblasts, showed intense staining for all three BMPs. This high level of expression was maintained during the entire distraction phase and then gradually disappeared during the consolidation phase. These results are compatible with the hypothesis that BMPs play an important role in the signaling pathways that link the mechanical forces created by distraction to biological responses.     

A murine model of distraction osteogenesis

Distraction osteogenesis is both a valuable clinical technique and a useful tool for investigating the basic mechanisms involved in bone tissue regeneration. Here we describe the development of a murine model of this procedure that can be used in transgenic animals to investigate the role of specific genes in tissue regeneration. Ring fixators were applied to the lower leg of 12 normal adult male mice. An osteotomy was made in the diaphysis of the tibia, and 7 days after the operation the bone fragments were distracted by 0.25 mm twice a day for 10 days. Specimens were examined immediately at the end of distraction and after 14-70 days of consolidation. At the end of distraction, the distraction gap was filled with fibroblast-like cells arranged longitudinally. After 14 days of consolidation, there was radiographical evidence of bone formation in the distraction gap and, after 28 days of consolidation, the bone fragments were fused with regenerated bone. By 70 days of consolidation, the regenerated bone had been almost completely remodeled and the intramedullary canal reestablished. This study is the first to report consolidation of the distraction gap with regenerated bone in a murine model of distraction.     

长骨干骨痂延长骨愈合成骨方式实验研究

目的 探讨长骨干骨痂延长骨愈合的成骨方式及其细胞学机制。方法 健康成年山羊10只,左胫骨中段骨膜下横形截骨,以四环式外固定架固定。2周后骨延长,每日延长1mm,连续30天,延长开始至延长结束后16周分期处死动物取材,以X线摄片、光镜及电镜作连续观察。结果 骨延长开始时,骨外膜侧巳有小梁骨形成,纤维组织呈纵向排列,与牵拉方向一致;成纤维细胞合成,分泌大量胶原纤丝。早期骨形成始于骨外膜侧及两截骨端,逐渐向纤维组织深入。骨延长结束后,纤维组织最终为成熟骨组织所替代。电镜观察显示上述新骨形成和改建活动均有成纤维细胞的参与。结论 长骨干骨痂延长骨愈合过程主要通过膜内骨化方式完成,成纤维细胞可能起着重要作用。     

The histology of distraction osteogenesis using different external fixators

The local biology of distraction osteogenesis using Ilizarov and Wagner external fixators was compared in unilateral tibial lengthenings in two groups of eight dogs. Serial roentgenograms with histological correlation revealed some basic similarities as well as distinct differences between the two groups. All dogs fully bridged the experimental gap by intramembranous ossification. Subtle histological changes in the alignment of collagen bundles and subsequent ossification reflected the mechanical configuration of each fixator. The multiplanar, full-pin Ilizarov system delivered concentric load to the site of osteogenesis, inducing perfectly parallel columns of new bone. The uniplanar, one-half pin Wagner system induced angulation of the collagen and subsequent bone columns on the basis of eccentric load to the osteogenic interface. Despite a significant difference in axial rigidity, the Ilizarov and Wagner external fixators induced osteogenesis of equal volume.     

Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.

Salter's type III and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibial transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-X immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-alpha1) on day 3, along with some cbf-alpha1+ osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphatase-immunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.     

rhBMPs对山羊下颌骨较大速率DO成骨的影响

目的研究在下颌骨较大速率牵引成骨术(DO)中应用重组人骨形成蛋白(rhBMPs)对成骨的影响,探讨下颌骨较大速率DO的可行性。方法12只山羊双侧下颌骨行DO,山羊及左右下颌骨随机化分为实验侧、对照侧,一只山羊作为正常标本。术中预置牵开间隙3.5mm,实验侧应用rhBMPs,延迟期为3d,牵引速率为1.5mm/d,分3次牵引,延长幅度为20mm。3、5、7、12周行大体、X线、骨密度、生物力学和组织学观察。结果延长幅度达到20mm,自固定期3周开始观察,牵引间隙新骨的再生形成及成熟改建仍是连续的骨愈合过程。固定期5周时,大体及X线观察表明实验侧已形成良好的骨皮质连续性,密度近于正常骨,而对照侧牵引间隙中间区却存在骨不连或间隙存在。组织学显示DO过程仍然是膜内成骨,对照侧牵引间隙中央可见中央区纤维连接、软骨改变,周边区可见新骨组织形成。生物力学三点弯曲实验显示实验侧在5周时接近正常骨,与对照侧有显著差异。骨密度随时间递增,实验侧在各时间点均高于对照侧。本实验条件下在5周后拆除牵引器较为可靠。结论较大速率DO的骨再生形成也是一个连续的膜内成骨过程。rhBMPs在较大速率和较大幅度DO中的应用能够加速骨的再生和愈合速...     

Bone lengthening osteogenesis,a combination of intramembranous and endochondral ossification: an experimental study in sheep

We evaluated the morphological features of the newly formed tissue in an experimental model of tibial callotasis lengthening on 24 lambs, aged from 2 to 3 months at the time of operation. A unilateral external fixator prototype Monotube Triax^® (Stryker Howmedica Osteonics, New Jersey) was applied to the left tibia. A percutaneous osteotomy was performed in a minimally traumatic manner using a chisel. Lengthening was started 7 days after surgery and was continued to 30 mm. The 24 animals were randomly divided into three groups of 8 animals each: in Group 1, lengthening took place at a rate of 1 mm/day for 30 days; in Group 2, at a rate of 2 mm/day for 15 days; in Group 3, at a rate of 3 mm/day for 10 days. In each group, 4 animals were killed 2 weeks after end of lengthening, and the other 4 animals at 4 weeks after end of lengthening. To assess bony formation in the distraction area, radiographs were taken every 2 weeks from the day of surgery. To study the process of vascularization, we used Spalteholz’s technique. After killing, the tibia of each animal was harvested, and sections were stained with hematoxylin and eosin, Masson’s trichrome, and Safranin-O. Immunohistochemistry was performed, using specific antibodies to detect collagens I and II, S100 protein, and fibronectin. A combination of intramembranous and endochondral ossification occurred together at the site of distraction. Our study provides a detailed structural characterization of the newly formed tissue in an experimental model of tibial lengthening in sheep and may be useful for further investigations on callotasis.