海水浸泡开放性骨折伤愈合过程中组织病理学和血管内皮细胞生长因子(VEGF)表达

目的通过观察普通开放骨折、海水浸泡开放骨折愈合过程的组织学变化和骨痂中血管内皮细胞生长因子(VEGF)的表达,了解海水浸泡开放性骨折愈合过程VEGF的作用与机制。方法新西兰大白兔59只,随机分为普通开放骨折组(对照组)24只和海水浸泡开放骨折组(实验组)35只。造成桡骨横行1.5mm缺损完全开放骨折,普通开放骨折伤组旷置3h,海水浸泡开放骨折伤组海水浸泡伤口3h,之后依次缝合伤口。于第1、3、7、14、21、28、45天处死动物。观察海水浸泡开放骨折伤在骨折愈合中不同时间的病理过程。采用RT-PCR方法检测普通开放骨折、海水浸泡开放骨折不同阶段骨痂中的VEGF的表达及变化。结果海水浸泡开放骨折伤骨痂形成延迟,骨折后第28天,对照组断端间骨痂为骨性骨痂者8例,为软骨者4例,实验组断端间骨痂为骨性骨痂者6例,为软骨者14例。海水浸泡骨折伤愈合过程中新生骨痂的VEGF表达在骨折后逐渐升高,术后14d达到高峰,之后逐渐下降,但在28d时仍保持较高水平,与一般开放骨折愈合过程的VEGF表达无显著差异(P>0.05)。结论海水浸泡使骨折骨痂形成不良率增高,骨折愈合过程有延迟倾向;但骨折愈合过程中VEGF表达无明显变化。     

动态压应力对骨折愈合时骨痂矿化过程的影响

[目的]探讨持续动态压应力对实验性骨折愈合时骨折间隙内骨痂矿化过程的影响。[方法]新西兰兔肱骨干横断截骨，试验组以天鹅记忆接骨器内固定，对照组以4孔加压钢板内固定。分别于术后第2、3、4、8、12周时，在骨折间隙取材，并于术后1、2、3、4、8、12周时抽取血标本，经比色法测定骨痂钙、磷元素含量及血中骨特异性碱性磷酸酶的活性。[结果]试验组和对照组骨折间隙内骨痂钙、磷元素的含量均逐渐升高；血中骨特异性碱性磷酸酶的活性分别于术后4、8周升至峰值，随后缓慢下降。术后3～4周时试验组以上指标均明显高于对照组。[结论]天鹅记忆接骨器产生的持续动态压应力可能促进了骨痂的矿化和软骨内成骨。     

骨折愈合的应力适应性研究

目的 探讨骨折端受力、肌肉动力、骨痂密度与骨折愈合的关系。方法 通过传感器电测技术与X线灰度分析的方法，从三个方面对骨折愈合的应力适应性进行了研究：①分别对14只1岁龄山羊进行了断端受力与骨折愈合的关系的研究；②对10只健康成年家兔进行了肌肉动力与骨折愈合的关系的研究；③对56只健康成年家兔进行了骨痂密度与骨折愈合的关系的研究。结果与结论 ①理想的骨折愈合与最佳的应力状态相适应；②肌肉动力是应力适应的反馈调节因素；③骨痂密度是应力适应的反馈结果。     

Role of mechanical loading in the progressive ossification of a fracture callus

The progressive ossification pattern in a fracture callus was predicted based on a theory that relates the local stimulus for ossification to the tissue mechanical loading history. Two-dimensional finite element analyses of a fracture callus were considered at three different stages of ossification. The sites of callus ossification represented in the initial model were predicted by previous analyses relating mechanical stress and vascularity to the differentiation of mesenchymal tissue in the early callus. The zones of further ossification, bone bridging, and bone consolidation predicted in the present study were found to be similar to the ossification patterns that have been documented by other researchers. The approach used to predict fracture healing is identical to that of previous studies predicting joint morphogenesis, with the exception that fracture healing requires continuous, attached skeletal elements, whereas joint morphogenesis requires discontinuous, articulating skeletal elements.     

血管内皮生长因子在骨折愈合中的自分泌作用

目的探讨血管内皮生长因子（VEGF）在骨折愈合中的自分泌作用。方法利用鼠股骨闭合性骨折模型，应用组织学和逆转录聚合酶链反应（RT-PCR）方法，观察骨折不同的愈合修复阶段中骨痂的组织学变化，检测各种相应骨痂组织中VEGF及其受体VEGFR1（Flt-1）和VEG-FR2（KDR／Flk-1）的mRNA的表达。结果骨折愈合是一个高度有序的组织学变化过程，骨痂内的膜内化骨，软骨形成，软骨内化骨等过程可同时或连续出现。vEGF（251bp）及受体Flt-1（272bp）和KDR／Flk-1（252bp）在骨折后第7、14天的软骨性骨痂（软骨组织）和骨性骨痂（小梁骨-膜内化骨和软骨内化骨）中均同时清晰表达。表达信号均匀，强度较大。结论骨折愈合过程中的软骨性骨痂和骨性骨痂中的软骨-骨细胞系统共表达VEGF及其两种受体，提示VEGF自分泌作用参与骨折愈合过程。     

Effect of recombinant human osteogenic protein-1 on the healing of a freshly closed diaphyseal fracture

Osteogenic protein-1 (OP-1), or bone morphogenetic protein-7, is an osteoinductive morphogen that is involved in embryonic skeletogenesis and in bone repair. In bone defect models without spontaneous healing, local administration of recombinant human OP-1 (rhOP-1) induces complete healing. To investigate the ability of rhOP-1 to accelerate normal physiologic fracture healing, an experimental study was performed. In 40 adult female goats a closed tibial fracture was made, stabilized with an external fixator, and treated as follows: (1) no injection; (2) injection of 1 mg rhOP-1 dissolved in aqueous buffer; (3) injection of collagen matrix; and (4) injection of 1 mg rhOP-1 bound to collagen matrix. The test substances were injected in the fracture gap under fluoroscopic control. At 2 and 4 weeks, fracture healing was evaluated with radiographs, three-dimensional computed tomography (CT), dual-energy X-ray absorptiometry, biomechanical tests, and histology. At 2 weeks, callus diameter, callus volume, and bone mineral content at the fracture site were significantly increased in both rhOP-1 groups compared with the no-injection group. As signs of accelerated callus maturation, bending and torsional stiffness were higher and bony bridging of the fracture gap was observed more often in the group with rhOP-1 dissolved in aqueous buffer than in uninjected fractures. Treatment with rhOP-1 plus collagen matrix did not result in improved biomechanical properties or bony bridging of the fracture gap at 2 weeks. At 4 weeks there were no differences between groups, except for a larger callus volume in the rhOP-1 plus collagen matrix group compared with the control groups. All fractures showed an advanced stage of healing at 4 weeks. In conclusion, the healing of a closed fracture in a goat model can be accelerated by a single local administration of rhOP-1. The use of a carrier material does not seem to be crucial in this application of rhOP-1.     

Repair of segmental bone defects in the rat: an experimental model of human fracture healing.

Bone repair models in animals may be considered relevant to human fracture healing to the extent that the sequence of events in the repair process in the model reflect the human fracture healing sequence. In the present study, the relevance of a recently developed segmental defect model in rat fibula to human fracture healing was investigated by evaluating temporal progression of rigidity of the fibula, mineral content of the repair site, and histological changes. In this model, a surgically created 2-mm-long defect was grafted with a 5-mm-long tubular specimen of demineralized bone matrix (DBM) by inserting it over the cut ends of the fibula. The temporal increase in rigidity of the healing fibula demonstrated a pattern similar to biomechanical healing curves measured in human fracture healing. This pattern was characterized by a short phase of rapidly rising rigidity during weeks 4-7 after surgery, associated with a sharp increase in the mineral content of the repair tissue. This was preceded by a phase of nearly zero rigidity and followed by a phase of slow rate of increase approaching a plateau. Histologically, chondroblastic and osteoblastic blastema originating from extraskeletal and subperiosteal (near fibula-graft junction) regions, infiltrated the DBM graft during the first 2 weeks. The DBM graft assumed the role of a "bridging callus." By weeks 6-8, most of the DBM was converted to new woven and trabecular bone with maximal osteoblastic activity and minimal endochondral ossification. Medullary callus formation started with direct new bone formation adjacent to the cortical and endosteal surfaces in the defect and undifferentiated cells in the center of the defect at 3 weeks. The usual bone repair process in rodents was altered by the presence of the DBM graft to recapitulate the sequential stages of human fracture healing, including the formation of a medullary callus, union with woven and lamellar bone, and recreation of the medullary canal.     

Estrogen receptors in fracture healing.

Estrogen has profound effects on the regulation of bone metabolism, but its role in fracture healing is unknown. Several recent reports have documented the presence of estrogen receptors in vitro and in diseased tissue. The authors investigated estrogen receptors in nonneoplastic skeletal tissues by immunohistochemical and radioligand binding techniques. Using a fibular osteotomy model of fracture healing in New Zealand rabbits, radioligand binding detected specific, high affinity, saturable estradiol binding sites at low levels throughout fracture healing, with a trend towards a bimodal distribution. Peaks occurred three and 16 days after osteotomy. No estrogen receptor was found by either method in later fracture callus, growth plate, or periosteum. These findings suggest a possible role for estrogen in the early inductive phase and later phase of endochondral ossification in fracture healing.     

Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ～6 and 15 weeks post‐fracture in two separate studies using the unilateral transverse mid‐ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre‐treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice‐weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ～6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment‐related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN‐treated rabbits at week‐6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K‐(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre‐ and post‐osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post‐surgery returned callus BMD to Veh. Peak load of ODN‐ or ALN‐treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:72–80, 2016.     

Zoledronic acid suppresses callus remodeling but enhances callus strength in an osteoporotic rat model of fracture healing

Mini-abstractIn this study, we demonstrated that the use of zoledronic acid does not impair fracture healing, but results in superior callus size and resistance at the fracture site, which could be the consequence of a lower rate of bone turnover due to its anti-catabolic effect.ObjectiveTo investigate the effect of inhibition of bone remodeling by the bisphosphonate, zoledronic acid, on callus properties in an osteoporotic rat model of fracture healing.MethodsOvariectomized (OVX) rats were randomly divided into four treatment groups (n = 24 per group): saline control (CNT); and three systemic zoledronic acid-injected groups (0.1 mg/kg), administered 1 day (ZOLD1), 1 week (ZOLW1), and 2 weeks (ZOLW2) after fracture. Rats were killed at either 6 or 12 weeks postoperatively. Postmortem analyses included radiography, microcomputed tomography, histology, histomorphometry, biomechanical tests, and nanoindentation tests.ResultsTreatment with zoledronic acid led to a significant increase in trabecular bone volume within the callus, as well as in callus resistance, compared to those in the saline control rats; delayed administration (ZOLW2) reduced intrinsic material properties, including ultimate stress and elastic modulus, and microarchitecture parameters, including bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and connectivity density (Conn.D), compared with ZOLD1 at 12 weeks after surgery. OVX had a negative effect on the progression of endochondral ossification at 6 weeks. Zoledronic acid administration at an early stage following fracture may bind to early callus, and thus not affect subsequent callus formation and endochondral ossification, while delayed administration (ZOLW2) mildly suppresses bony callus remodeling.ConclusionThe superior results obtained with zoledronic acid (ZOLD1, ZOLW1, and ZOLW2) compared to CNT in terms of callus size and resistance could be the consequence of a lower rate of bone turnover at the fracture site due to the anti-catabolic effect of zoledronic acid. Mild suppression of callus remodeling by delayed administration did not impair the initial phase of the fracture healing process.     

Accelerated bone healing and excessive callus formation in patients with femoral fracture and head injury

The effect of head injury on systemic physiology, including bone healing is still a topic of vivid discussion. Whether the observed changes genuinely represent accelerated fracture healing or are a form of local heterotopic ossification remains unclear. We aimed to investigate whether in patients with long bone fractures the presence of head injury is associated with accelerated bone healing and excessive callus formation. In total 67 patients were studied 17 with head injury and 50 without head injury (25 treated with reamed and the other 25 with the unreamed nailing technique). Both groups were comparable in terms of age, sex, ISS. All underwent stabilisation of their femoral fracture with intramedullary nailing. The quantification of fracture healing response was estimated by taking the radiological ratio of the largest diameter of callus formed into two planes and the adjacent normal diameter of femoral canal. The minimum follow up of the patients was 12 months. In patients with head injury, the mean time to fracture union was significantly shorter than either the reamed or unreamed group (10.5 weeks compared with 20.5 and 26.9 weeks, p<0.001). The difference between the mean callus to diaphyseal ratio was statistically significant for both the AP and Lateral projections (AP: mean difference 0.462, 95% CI 0.312 to 0.602, p<0.0001, LAT: mean difference 0.289, 95% CI 0.142 to 0.436, p<0.001) with the head injured patients having more florid callus compared to the control group.     

高能震波促进骨折愈合机理的探讨

目的 探讨高能震波（ＨＥＳＷ）对兔骨折愈合的影响及其机理。方法 手术造成家兔双侧胫骨标准骨折模型,对１侧胫骨骨折部施以ＨＥＳＷ;另１侧为对照。术后每周进行Ｘ线及组织学检查,并取骨折骨痂切片用骨形态发生蛋白（ＢＭＰ）抗体进行免疫组织化学染色（ＳＡＢＣ法）。结果 Ｘ线摄片及组织学观察显示,实验侧骨痂出现时间及骨折愈合时间均明显早于对照组;骨痂区毛细血管计数、ＢＭＰ表达强度在第２～４周时实验侧均明显大于     

骨折愈合中的软骨骨痂──形态学演变及超微结构观察

通过光镜下不脱钙骨组织学、组织化学和透射电镜对雄性兔桡骨标准缺损骨折模型愈合中软骨骨痂的形成、演变及超微结构进行了观察，结果显示：软骨骨痂由骨折后进入断端间的肉芽组织分化而成，其形成和改建并不完全相同于骺板软骨内化骨。电镜下，骨痂内软骨细胞可分为５个发育阶段：成软骨细胞、软骨细胞、肥大软骨细胞、变性软骨细胞和残存软骨细胞。我们认为１）软骨骨痂由断端周围组织内的间充质细胞分化而成；２）在改建过程中，软骨骨痂能直接形成骨小梁，我们支持肥大软骨细胞能转化为骨细胞的假说；３）软骨骨痂对骨折愈合有重要的作用，能早期填充骨缺损，联接断端，是骨折在重力负载下完成愈合的基础组织之一。     

Cell proliferation and apoptosis during fracture healing.

This study investigated the relation between cell proliferation and apoptosis during fracture healing in a mouse femoral fracture model. Left femoral osteotomies were performed in 30 mature male CFLP mice immobilized with uniplanar external fixators. Six animals were killed on days 2, 4, 8, 16, and 24 postfracture for examination. Localization of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) method. Images of each time-specific specimen were captured. The total callus area, the positively labeled cells by PCNA, and TUNEL per high-power field were quantified. Cell proliferation and apoptosis were found coexisting during the entire period of study. In the early phases of fracture healing (days 2-8), PCNA-positive labeling was predominant and peaked at day 8 and the TUNEL-positive labeling was minimal. In later stages of fracture healing (days 16-24), PCNA expression declined at day 16 as callus ossification and remodeling spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL-positive labeling peaking at day 16 and declining sharply at day 24. These cell activities were reflected by the change of fracture callus, where there was a continuous increase in total callus area to day 16 and subsequent decrease at day 24. This study indicated that cell proliferation and apoptosis are coupled events during fracture repair, cell proliferation is active at the early stages, and apoptosis is active during the phase of callus remodeling.     

Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus.

Low-intensity pulsed ultrasound (LIPUS) has been shown to accelerate fracture healing in both animal models and clinical trials, but the mechanism of action remains unclear. In fracture healing, various consecutive cellular reactions occurred until repair. We investigated whether the advanced effects of LIPUS depended on the duration and timing of LIPUS treatment in a rat closed femoral fracture model to determine the target of LIPUS in the healing process. Sixty-nine Long-Evans male rats that have bilateral closed femoral fractures were used. The right femur was exposed to LIPUS (30 mW/cm2 spatial and temporal average [SATA], for 20 minutes/day), and the left femur was used as a control. Rats were divided into four groups according to timing and duration of treatment (Ph-1, days 1-8; Ph-2, days 9-16; Ph-3, days 17-24; throughout [T], days 1-24 after the fracture). Animals were killed on day 25. After radiographs and microfocus X-ray computed tomography (muCT) tomograms were taken, the hard callus area (HCA), bone mineral content (BMC) at the fracture site, and mechanical torsion properties were measured, and histological analysis was conducted. Interestingly, the maximum torque of the LIPUS-treated femur was significantly greater than that of the controls in all groups without any changes in HCA and BMC. The multiviewing of three-dimensional (3D) muCT reconstructions and histology supported our findings that the partial LIPUS treatment time was able to accelerate healing, but longer treatment was more effective. These results suggest that LIPUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling.     

Transforming growth factor-β enhances fracture healing in rabbit tibiae

The ability of exogenous Transforming Growth Factor-β (TGF-β) to stimulate bone formation in fracture healing was investigated. TGF-β was continuously applied in doses of 1 and 10 μg/day for 6 weeks to 2 groups of adult rabbits with unilateral plated midtibial osteotomies. A group receiving solvent without TGF-β served as control. Fracture healing was evaluated by mechanical tests, bone morphometry and bone densitometry. Increased maximal bending strength and callus formation were demonstrated in the groups receiving TGF-β. TGF-β had no effect on bending-stiffness, bone mineral content, cortical thickness or haversian canal diameter. We conclude that local application of exogenous TGF-β may enhance fracture healing in rabbits.     

Experimental study of effect of stress-relaxation bone plate on fracture healing

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Regional and temporal changes in the acoustic properties of fracture callus in secondary bone healing

Controlled fractures were created in the right femora of 17 male Sprague-Dawley rats. The fractured limbs were harvested at 2, 4, 6, and 8 weeks after fracture, fixed, and embedded in polymethylmethacrylate. Midsagittal sections from each animal were evaluated with a scanning acoustic microscope, a device that generates an acoustic impedance map of the scanned material. The impedance of the fracture callus was measured in six regions on each specimen. These regions were chosen in an effort to distinguish between the impedance of the callus formed through intramembranous or endochondral ossification, and we found that the time course of increasing impedance differed for the fracture callus formed through the two pathways. Additionally, we found a significant difference in the mean impedance of the callus at each time period (p ≤ 0.0013 for all comparisons), which resulted in an extremely linear relationship (r² = 0.999) between mean callus impedance and healing time. This experimental model has become a popular choice for the investigation of fracture healing. As such, an accurate determination of the mechanical properties of the fracture callus is often sought. We propose that the implementation of scanning acoustic microscopy in the study of fracture healing may determine the changes in the material properties more accurately than conventional testing methods.     

Moderate soft tissue trauma delays new bone formation only in the early phase of fracture healing.

The aim of this study was to investigate the effect of a moderate soft tissue trauma to the course of fracture healing in a standardized animal model. Thirty-eight Wistar rats were randomly divided into a fracture group (F, n = 19) and a group with a fracture and a soft tissue trauma (F + STT, n = 19). The fracture and the soft tissue trauma were created using an impact device with a standardized energy. All fractures were stabilized by two Kirschner wires. Three rats were measured for blood flow and sacrificed at days 1, 3, 7, and 14, and seven rats at day 28, from both groups. A three-point bending test was performed on the healed tibia after 28 days. During the first 24 h there was a reduction in blood flow, which was more pronounced in the F + STT group than in the F group. From histological sections, the shape of the callus formation, as well as the tissue distribution of newly formed bone, fibrous cartilage and fibrous connective tissue were determined. Distinctly more periosteal new bone formed and a larger callus formed at days 3 and 7 in group F compared to group F + STT. However, by days 14 and 28, the ossification and overall callus size no longer showed differences between the two groups. A fast recovery of blood flow and callus formation took place in the F + STT group, which led to similar histological and biomechanical results in fracture healing observed after 28 days between the two groups.