A comparison of stereology, structural rigidity and a novel 3D failure surface analysis method in the assessment of torsional strength and stiffness in a mouse tibia fracture model

In attempting to develop non-invasive image based measures for the determination of the biomechanical integrity of healing fractures, traditional μCT based measurements have been limited. This study presents the development and evaluation of a tool for assessment of fracture callus mechanical properties through determination of the geometric characteristics of the fracture callus, specifically along the surface of failure identified during destructive mechanical testing. Fractures were created in tibias of ten male mice and subjected to μCT imaging and biomechanical torsion testing. Failure surface analysis, along with previously described image based measures was calculated using the μCT image data, and correlated with mechanical strength and stiffness. Three-dimensional measures along the surface of failure, specifically the surface area and torsional rigidity of bone, were shown to be significantly correlating with mechanical strength and stiffness. It was also shown that surface area of bone along the failure surface exhibits stronger correlations with both strength and stiffness than measures of average and minimum torsional rigidity of the entire callus. Failure surfaces observed in this study were generally oriented at 45° to the long axis of the bone, and were not contained exclusively within the callus. This work represents a proof of concept study, and shows the potential utility of failure surface analysis in the assessment of fracture callus stability.     

Micro-computed tomography prediction of biomechanical strength in murine structural bone grafts

Correlating massive bone graft strength to parameters derived from non-invasive imaging is important for pre-clinical and clinical evaluation of therapeutic adjuvants designed to improve graft repair. Towards that end, univariate and multivariate regression between measures of graft and callus geometry from micro-CT imaging and torsional strength and rigidity were investigated in a mouse femoral graft model. Four millimeter mid-diaphyseal defects were grafted with live autografts or processed allografts and allowed to heal for 6, 9, 12, or 18 weeks. We observed that allograft remodeling and incorporation into the host remained severely impaired compared to autografts mainly due to the extent of callus formation around the graft, the rate and extent of the graft resorption, and the degree of union between the graft and host bone as judged by post-mechanical testing analysis of the mode of failure. The autografts displayed greater ultimate torque and torsional rigidity compared to the allografts over time. However the biomechanical properties of allografts were equivalent to autografts by 9 weeks but significantly decreased at 12 and 18 weeks. Multivariate regression analysis demonstrated significant statistical correlations between combinations of the micro-CT parameters (graft and callus volume and cross-sectional polar moment of inertia) with the measured ultimate torque and torsional rigidity (adjusted R(2)=44% and 50%, respectively). The statistical correlations approach used in this mouse study could be useful in guiding future development of non-invasive predictors of the biomechanical properties of allografts using clinical CT.     

不同植入物内固定治疗四肢创伤骨折后骨不连的临床对比研究

目的:探讨不同植入物内固定治疗四肢创伤骨折后骨不连的临床疗效。方法:选择我科2010年2月~2013年2月四肢创伤骨折后骨不连患者38例,按照随机数表法将38例患者随机分为两组,分别为LC-DCP组以及LCP组,每组各19例,观察两组患者的平均手术持续时间、骨折临床愈合时间、X线骨痂评分以及并发症。结果:LC-DCP组平均手术持续时间为(134.73±12.91)min,LCP组为(129.54±14.87)min,两组比较不存在统计学差异(P0.05)。LC-DCP组患者平均骨折临床愈合时间为(3.94±0.83)月,LCP组为(3.81±0.69)月,两组间不存在统计学显著性差异(P0.05)。LC-DCP组患者X线骨痂评价标准平均评分值为(2.73±0.51)分,LCP组为(2.86±0.49)分,两组间差异不存在统计学意义(P0.05)。结论:两种钢板联合植骨治疗四肢创伤骨折后骨不连均能够取得良好的治疗效果,均可以作为治疗四肢创伤骨折后骨不连患者的有效方法。     

Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing

Beyond its role in the regulation of red blood cell proliferation, the glycoprotein erythropoietin (EPO) has been shown to promote cell regeneration and angiogenesis in a variety of different tissues. In addition, EPO has been indicated to share significant functional and structural homologies with the vascular endothelial growth factor (VEGF), a cytokine essential in the process of fracture healing. However, there is complete lack of information on the action of EPO in bone repair and fracture healing. Therefore, we investigated the effect of EPO treatment on bone healing in a murine closed femur fracture model using radiological, histomorphometric, immunohistochemical, biomechanical and protein biochemical analysis. Thirty-six SKH1-hr mice were treated with daily i.p. injections of 5000 U/kg EPO from day 1 before fracture until day 4 after fracture. Controls received equivalent amounts of the vehicle. After 2 weeks of fracture healing, we could demonstrate expression of the EPO-receptor (EPOR) in terminally differentiating chondrocytes within the callus. At this time point EPO-treated animals showed a higher torsional stiffness (biomechanical analysis: 39.6+/-19.4% of the contralateral unfractured femur) and an increased callus density (X-ray analysis (callus density/spongiosa density): 110.5+/-7.1%) when compared to vehicle-treated controls (14.3+/-8.2% and 105.9+/-6.6%; p<0.05). Accordingly, the histomorphometric examination revealed an increased fraction of mineralized bone and osteoid (33.0+/-3.0% versus 28.5+/-3.6%; p<0.05). Of interest, this early effect of the initial 6-day EPO treatment had vanished at 5 weeks after fracture. We conclude that EPO-EPOR signaling is involved in the process of early endochondral ossification, enhancing the transition of soft callus to hard callus.     

Is callus calcium content an indicator of the mechanical strength of healing fractures? An experimental study in rat metatarsals

Changes in the mechanical properties and the calcium content of healing fracture callus were followed, using rat metatarsals. By 24 weeks post-fracture the mean ultimate tensile stress and elastic modulus were still less than half that of the contralateral unfractured bone, whereas the mean torsional modulus had almost reached that of the unfractured bone. The calcium content of the callus formed immediately between the fractured ends of the bone showed changes which coincided with the increases in mechanical strength and the moduli, thus measurement of callus calcium content would enable the prediction of the strength of a healing fracture.     

Mineral and matrix contributions to rigidity in fracture healing

The purpose of this study was to investigate the relationships among selected properties of fracture callus: bending rigidity, tissue density, mineral density, matrix density and mineral-to-matrix ratio. The experimental model was an osteotomized canine radius in which the development of the fracture callus was modified by electrical stimulation with various levels of direct current. This resulted in a range of values for the selected properties of the callus, determined post mortem at 7 weeks after osteotomy. We found that the rigidity (R) of the bone-callus combination obeyed relationships of the form R = axb, where x is the tissue density, mineral density, matrix density or the mineral-to-matrix ratio of the repair tissue. These are analogous to power-law relationships found in studies of compact and cancellous bone. The results suggest that fracture callus at 7 weeks after osteotomy in canine radius behaves more like immature compact bone than cancellous bone in its mechanical and physicochemical properties. The present study demonstrates the feasibility of developing non-invasive in vivo densitometric methods to monitor fracture healing, since models may be developed that can predict mechanical properties from densitometric data. Further studies are needed to develop a refined model based on experimental data on the mechanical and physicochemical properties and microstructure of fracture callus at different stages of healing.     

Fetal fracture healing in a lamb model.

A large animal model to assess fetal fracture repair and the ability to close excisional bony defects is presented. Incisional and excisional ulnar fractures were made in 14 midgestation fetal lambs, harvested at serial time points, and subjected to high-resolution low-kilovolt magnification radiographs, magnetic resonance imaging scans, and histologic analysis. Fetal fracture healing was characterized by early closure of excisional defects and rapid fracture healing with minimal or no soft-tissue inflammation or callus formation. Magnetic resonance imaging scans of the fractures revealed a characteristic pattern compatible with the histologic findings, namely, minimal inflammation in soft tissue adjacent to the fracture site. Histologic and magnification radiographic findings indicated that complete bony repair occurred within 21 days in incisional defects and within 40 days in excisional defects. In both cases, healed fetal bone resembled normal bone matrix. Excisional defects, including periosteum, of greater than three times the width of the bony cortex closed rapidly with virtually normal-appearing bony matrix and with minimal or no callus formation.     

Predicting the external formation of a bone fracture callus: an optimisation approach

The formation of a fracture callus in vivo tends to form in a structurally efficient manner distributing tissues where mechanical stimulus persists. Therefore, it is proposed that the formation of a fracture callus can be modelled in silico by way of an optimisation algorithm. This was tested by generating a finite element model of a transversal bone fracture embedded in a large tissue domain which was subjected to axial, bending and torsional loads. It was found that the relative fragment motion induced a compressive strain field in the early callus tissue which could be utilised to simulate the formation of external callus structures through an iterative optimisation process of tissue maintenance and removal. The phenomenological results showed a high level of congruence with in vivo healing patterns found in the literature. Consequently, the proposed strategy shows potential as a means of predicting spatial bone healing phenomena for pre-clinical testing.     

NGF与Neuritin在脑外伤伴四肢骨折病人血清中的表达及意义

目的：通过检测脑外伤患者、脑外伤合并骨折患者、骨折患者及正常人外周血中NG3F,neuritin不同时间段的表达,根据其含量的变化,判断与骨折愈合速度的相关性,寻找骨折愈合的关键因子。方法：收集单纯脑外伤、单纯骨折患者各80例、脑外伤合并骨折患者60例、健康体检人群20例。选取外伤后3天、10天、2周抽取所有实验对象的静脉血,应用ELISA技术测定标本中NGF与Neuritin的含量。结果：损伤后每个时间段里,患者血清中NGF、neuritin含量有不同程度升高,均高于正常对照组,其中又以脑外伤合并骨折组最高。血清NGF在骨折合并脑外伤组伤后第3天含量明显升高,为（0．86±0．21）,伤后10天为（1．47±0．29）。14天为（2．07±0．21）,脑外伤合并四肢骨折组neuritin血清含量在伤后第3天略有升高为（83．47±18．85）,10天（108．50±31．65）,2周（91．86±21．12）．脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,差别均有统计学意义（P〈0．05）。结论：脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,说明与骨折愈合有着密切的相关性,两种因子可能在骨折愈合修复过程中共同起作用,从而,推测可能是脑外伤后骨折愈合加速的重要因素。     

Predicting the external formation of a bone fracture callus: an optimisation approach

The formation of a fracture callus in vivo tends to form in a structurally efficient manner distributing tissues where mechanical stimulus persists. Therefore, it is proposed that the formation of a fracture callus can be modelled in silico by way of an optimisation algorithm. This was tested by generating a finite element model of a transversal bone fracture embedded in a large tissue domain which was subjected to axial, bending and torsional loads. It was found that the relative fragment motion induced a compressive strain field in the early callus tissue which could be utilised to simulate the formation of external callus structures through an iterative optimisation process of tissue maintenance and removal. The phenomenological results showed a high level of congruence with in vivo healing patterns found in the literature. Consequently, the proposed strategy shows potential as a means of predicting spatial bone healing phenomena for pre-clinical testing.     

Experimental study in rat metatarsals to determine whether callus vascularity is an indicator of the mechanical strength of healing fractures

Changes in the mechanical properties and percentage area of blood vessels of healing fracture callus were followed using rat metatarsals. By 24 weeks after fracture the mean ultimate tensile stress and elastic modulus were still less than half that of the contralateral unfractured bone, whereas the mean torsional modulus had almost reached that of the unfractured bone. The percentage area of blood vessels declined from five days post-fracture and showed no changes which coincided with the increases in mechanical strength or moduli. We conclude that studies of vascularity would not justify a prediction of the strength of a healing fracture.     

An improved method to assess torsional properties of rodent long bones

Torsion is an important testing modality commonly used to calculate structural properties of long bones. However, the effects of size and geometry must be excluded from the overall structural response in order to compare material properties of bones of different size, age and species. We have developed a new method to analyze torsional properties of bones using actual cross-sectional information and length-wise geometrical variations obtained by micro-computed topographic (μCT) imaging. The proposed method was first validated by manufacturing three rat femurs through rapid prototyping using a plastic with known material properties. The observed variations in calculated torsional shear modulus of the hollow elliptical model of mid-shaft cross-section (Ekeland et al.), multi-prismatic model of five true cross-sections (Levenston et al.) and multi-slice model presented in this study were 96%, ?7% and 6% from the actual properties of the plastic, respectively. Subsequently, we used this method to derive relationships expressing torsional properties of rat cortical bone as a function of μCT-based bone volume fraction or apparent density over a range of normal and pathologic bone densities. Results indicate that a regression model of shear modulus or shear strength and bone volume fraction or apparent density described at least 81% of the variation in torsional properties of normal and pathologic bones. Coupled with the structural rigidity analysis technique introduced by the authors, the relationships reported here can provide a non-invasive tool to assess fracture risk in bones affected by pathologies and/or treatment options.     

A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing

Mouse models are of increasing interest to study the molecular aspects of fracture healing. Because biomechanical factors greatly influence the healing process, stable fixation of the fracture is of interest also in mouse models. Unlike in large animals, however, there is a lack of mouse models which provide stable osteosynthesis. The purpose of this study was therefore to develop a technique for a more stable fixation of femoral fractures in mice and to analyze the impact of stability on the process of fracture healing. The new technique introduced herein includes an intramedullary pin and an extramedullary metallic clip. Ex vivo biomechanical analysis revealed a significantly higher implant stiffness of our pin-clip technique when compared with previously described intramedullary fixation techniques. In vivo, we studied the course of healing after the more stable fixation with our pin-clip technique and compared the results with that observed after unstable fixation with the pin-clip technique after cutting the clip. After 2 and 5 weeks of fracture healing radiological analysis demonstrated that the more stable fixation with the pin-clip technique results in a significantly higher union rate compared to the unstable fixation. Torsional stiffness at 5 weeks was almost 3-fold of that measured after unstable fixation. Histomorphological analysis further showed that fractures stabilized with the pin-clip technique healed with a smaller periosteal callus area, an increased fraction of bone and a reduced amount of fibrous tissue. Of interest, the pin-clip fixation showed reliable union after 5 weeks, whereas the unstable pin fixation did not regularly achieve adequate fracture healing. In conclusion, we introduce a novel, easily applicable internal osteosynthesis technique in mice, which provides rotational stability after femoral fracture fixation. We further show that a more stable osteosynthesis significantly improves the process of fracture healing also in mice.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.     

Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography

Although fracture healing is frequently studied in pre-clinical models of long bone fractures using rodents, there is a dearth of objective quantitative techniques to assess successful healing. Biomechanical testing is possibly the most quantitative and relevant to a successful clinical outcome, but it is a destructive technique providing little insight into the cellular mechanisms associated with healing. The advent of X-ray computed tomography (CT) has provided the opportunity to quantitatively and non-destructively assess bone structure and density, but it is unknown how measurements derived using this technology relate to successful healing. To examine possible relationships, we used a pre-clinical model to test for statistically significant correlations between quantitative characteristics of the callus by micro-CT (μCT) and the bending strength, stiffness, and energy-to-failure of the callus as assessed by three-point bending of excised bones. A closed, transverse fracture was generated in the mid-shaft of rat femurs by impact loading. Shortly thereafter, the rats received a one-time, local injection of either the vehicle or one of four doses of lovastatin. Following sacrifice after 4 weeks of healing, fractured femurs were extracted for μCT analysis and then three-point bending. Setting the region of interest to be 3.2 mm above and below the fracture line, we acquired standard and new μCT-derived measurements. The mineralized callus volume and the mineral density of the callus correlated positively with callus strength (r_xy=?0.315, p=0.016 and r_xy=0.444, p<0.0005, respectively) and stiffness (r_xy=?0.271, p=0.040 and r_xy=0.325, p=0.013, respectively), but the fraction of the callus that mineralized and the moment of inertia of the callus did not. This fraction did correlate with energy-to-failure (r_xy=?0.343, p=0.0085). Of the μCT-derived measurements, quantifying defects within the outer bridging cortices of the callus produced the strongest correlation with both callus strength (r_xy=0.557, p<0.0001) and stiffness (r_xy=0.468, p=0.0002). By both reducing structural defects and increasing mineralization, lovastatin appears to increase the callus strength.     

Rigidity and stress analyses of external fracture fixation devices—A theoretical approach

The rigidity and stresses in external fracture fixation devices were studied by means of the finite element method. Different geometries and material parameters were simulated using a beam element model. Axial, bending and torsional loads were applied through the bone ends and the displacement obtained at the fracture sites was used to calculate the fracture fixation stiffness. The key parameters which increased fixation rigidity were identified. High pin stresses were predicted under certain application conditions. Possible clinical implications for the use of such apparatus are discussed in the light of bone fracture healing. The present results are expected to have a significant impact on future design modifications and clinical applications of this popular instrument in orthopedic surgery and traumatology.     

A surrogate long-bone model with osteoporotic material properties for biomechanical testing of fracture implants

In vitro comparative testing of fracture fixation implants is limited by the highly variable material properties of cadaveric bone. Bone surrogate specimens are often employed to avoid this confounding variable. Although validated surrogate models of normal bone (NB) exist, no validated bone model simulating weak, osteoporotic bone (OPB) is available. This study presents an osteoporotic long-bone model designed to match the lower cumulative range of mechanical properties found in large series of cadaveric femora reported in the literature. Five key structural properties were identified from the literature: torsional rigidity and strength, bending rigidity and strength, and screw pull-out strength. An OPB surrogate was designed to meet the low range for each of these parameters, and was mechanically tested. For comparison, the same parameters were determined for surrogates of NB. The OPB surrogate had a torsional rigidity and torsional strength within the lower 2% and 16%, respectively, of the literature based cumulative range reported for cadaveric femurs. Its bending rigidity and bending strength was within the lower 11% and 8% of the literature-based range, respectively. Its pull-out strength was within the lower 2% to 16% of the literature based range. With all five structural properties being within the lower 16% of the cumulative range reported for native femurs, the OPB surrogate reflected the diminished structural properties seen in osteoporotic femora. In comparison, surrogates of NB demonstrated structural properties within 23-118% of the literature-based range. These results support the need and utility of the OPB surrogate for comparative testing of implants for fixation of femoral shaft fractures in OPB.     

Biomechanical consequences of callus development in Hoffmann, Wagner, Orthofix and Ilizarov external fixators.

A theoretical analysis by a finite elements model (FEM) of some external fixators (Hoffmann, Wagner, Orthofix and Ilizarov) was carried out. This study considered a logarithmic progress of callus elastic characteristics. A standard configuration of each fixator was defined where design and application characteristics were modified. A comparison among standard configurations and influence of every variation was made with regard to displacement and load transmission at the fracture site. An experimental evaluation of standard configurations was performed with a testing machine. After experimental validation of the theoretical model was achieved, an application of physiological loads which act on a fractured limb during normal gait was analysed. A minimal contribution from an external fixator to the total rigidity of the bone-callus-fixator system was assessed when a callus showing minimum elastic characteristics had just been established. Insufficient rigidity from the fixation devices to assure an adequate immobilization during the early stages of fracture healing was verified. However, regardless of the external fixator, callus development was the overriding element for the rigidity of the fixator-bone system.     

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.KEY WORDS: Whole-body vibration, LMHFV, Fracture healing, Oestrogen receptor signalling, Wnt signalling