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.     

In-vivo imaging of the fracture healing in medaka revealed two types of osteoclasts before and after the callus formation by osteoblasts

The fracture healing research, which has been performed in mammalian models not only for clinical application but also for bone metabolism, revealed that generally osteoblasts are induced to enter the fracture site before the induction of osteoclasts for bone remodeling. However, it remains unknown how and where osteoclasts and osteoblasts are induced, because it is difficult to observe osteoclasts and osteoblasts in a living animal. To answer these questions, we developed a new fracture healing model by using medaka. We fractured one side of lepidotrichia in a caudal fin ray without injuring the other soft tissues including blood vessels. Using the transgenic medaka in which osteoclasts and osteoblasts were visualized by GFP and DsRed, respectively, we found that two different types of functional osteoclasts were induced before and after osteoblast callus formation. The early-induced osteoclasts resorbed the bone fragments and the late-induced osteoclasts remodeled the callus. Both types of osteoclasts were induced near the surface on the blood vessels, while osteoblasts migrated from adjacent fin ray. Transmission electron microscopy revealed that no significant ruffled border and clear zone were observed in early-induced osteoclasts, whereas the late-induced osteoclasts had clear zones but did not have the typical ruffled border. In the remodeling of the callus, the expression of cox2 mRNA was up-regulated at the fracture site around vessels, and the inhibition of Cox2 impaired the induction of the late-induced osteoclasts, resulting in abnormal fracture healing. Finally, our developed medaka fracture healing model brings a new insight into the molecular mechanism for controlling cellular behaviors during the fracture healing.     

A hybrid bioregulatory model of angiogenesis during bone fracture healing

Bone fracture healing is a complex process in which angiogenesis or the development of a blood vessel network plays a crucial role. In this paper, a mathematical model is presented that simulates the biological aspects of fracture healing including the formation of individual blood vessels. The model consists of partial differential equations, several of which describe the evolution in density of the most important cell types, growth factors, tissues and nutrients. The other equations determine the growth of blood vessels as a result of the movement of leading endothelial (tip) cells. Branching and anastomoses are accounted for in the model. The model is applied to a normal fracture healing case and subjected to a sensitivity analysis. The spatiotemporal evolution of soft tissues and bone, as well as the development of a blood vessel network are corroborated by comparison with experimental data. Moreover, this study shows that the proposed mathematical framework can be a useful tool in the research of impaired healing and the design of treatment strategies.     

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.     

Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13-/- mice is intrinsic to cartilage and bone. Mmp13-/- mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.     

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing: a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.     

Recent advances in bone regeneration using adult stem cells

Bone is a highly vascularized tissue reliant on the close spatial and temporal association between bloodvessels and bone cells. Therefore, cells that participate in vasculogenesis and osteogenesis play a pivotal role in bone formation during prenatal and postnatal periods. Nevertheless, spontaneous healing of bone fracture is occasionally impaired due to insufficient blood and cellular supply to the site of injury. In these cases, bone regeneration process is interrupted, which might result in delayed union or even nonunion of the fracture. Nonunion fracture is difficult to treat and have a high financial impact. In the last decade, numerous technological advancements in bone tissue engineering and cell-therapy opened new horizon in the field of bone regeneration. This review starts with presentation of the biological processes involved in bone development, bone remodeling, fracture healing process and the microenvironment at bone healing sites. Then, we discuss the rationale for using adult stem cells and listed the characteristics of the available cells for bone regeneration. The mechanism of action and epigenetic regulations for osteogenic differentiation are also described. Finally, we review the literature for translational and clinical trials that investigated the use of adult stem cells(mesenchymal stem cells, endothelial progenitor cells and CD34+ blood progenitors) for bone regeneration.     

Podoplanin Immunopositive Lymphatic Vessels at the Implant Interface in a Rat Model of Osteoporotic Fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.     

Development of a locking femur nail for mice

We herein report on a novel locking intramedullary nail system in a murine closed femur fracture model. The nail system consists of a modified 24-gauge injection needle and a 0.1-mm-diameter tungsten guide wire. Rotation stability was accomplished by flattening the proximal and distal end of the needle. Torsional mechanical testing of the implants in osteotomized cadaveric femora revealed a superiority of the locking nail (3.9+/-1.0 degrees rotation at a torque of 0.9 Nmm, n=10) compared to the unmodified injection needle (conventional nail; 52.4+/-3.2 degrees, n=10, p<0.05). None of the implants, however, achieved the rotation stability of unfractured femora (0.3+/-0.5 degrees, n=10). In a second step, we tested the feasibility of the in vivo application of the locking nail to stabilize a closed femoral midshaft fracture in C57BL/6 mice. Of interest, none of the 10 animals showed a dislocation of the locking nail over a 5-week period, while 3 of 4 animals with conventional nail fracture stabilization showed a significant pin dislocation within the first 3 days (p<0.05). Mechanical testing after 5-weeks stabilization with the locking nail revealed an appropriate bone healing with a torque at failure of 71.6+/-3.4% and a peak rotation before failure of 68.4+/-5.3% relative to the unfractured contralateral femur. With the advantage that closed fractures can be fixed with rotation stability, the herein introduced model may represent an ideal tool to study bone healing in transgenic and knockout mice.     

Locally administrated perindopril improves healing in an ovariectomized rat tibial osteotomy model

Angiotensin-converting enzyme inhibitors are widely prescribed to regulate blood pressure. High doses of orally administered perindopril have previously been shown to improve fracture healing in a mouse femur fracture model. In this study, perindopril was administered directly to the fracture area with the goal of stimulating fracture repair. Three months after being ovariectomized (OVX), tibial fractures were produced in Sprague-Dawley rats and subsequently stabilized with intramedullary wires. Perindopril (0.4 mg/kg/day) was injected locally at the fractured site for a treatment period of 7 days. Vehicle reagent was used as a control. Callus quality was evaluated at 2 and 4 weeks post-fracture. Compared with the vehicle group, perindopril treatment significantly increased bone formation, increased biomechanical strength, and improved microstructural parameters of the callus. Newly woven bone was arranged more tightly and regularly at 4 weeks post-fracture. The ultimate load increased by 66.1 and 76.9% (p<0.01), and the bone volume over total volume (BV/TV) increased by 29.9% and 24.3% (p<0.01) at 2 and 4 weeks post-fracture, respectively. These findings suggest that local treatment with perindopril could promote fracture healing in ovariectomized rats.     

Short-term load bearing capacity of osteochondral autografts implanted by the mosaicplasty technique: an in vitro porcine model

Articular surface congruency and graft stability are considered essential factors in the success of osteochondral grafting; however, quantitative measures of short-term load bearing capacity of grafts implanted by the mosaicplasty technique have not been reported. The purpose of this study was to develop a live tissue in vitro model to examine short-term fixation strength of mosaicplasty autografts immediately after and 1 week following graft implantation. Cylindrical osteochondral autografts were implanted in vitro by the mosaicplasty technique on five pairs of porcine femoral condyles within one and a half hours of animal sacrifice. Immediately following the surgical procedure, graft push-in and pull-out strength tests as well as indentation tests to determine modulus of the surrounding cancellous bone were performed on half of the specimens from the distal femurs of each animal. The remaining specimens, matched for location in the contralateral leg, were incubated in culture medium for 7 days prior to performing the same set of mechanical tests. Averaged push-in and pull-out graft fixation strength decreased 44% from 135.7 to 75.5N over the 7-day period, while no change in modulus was detected in the surrounding cancellous bone. These in vitro results demonstrate a substantial deterioration of short-term fixation strength of mosaicplasty grafts from the immediate post-operative state. Such a reduction in short-term graft load bearing capacity may pose a threat to the surgically established articular surface congruency and blood vessels formed during the early stages of the healing response.     

The effects of low laser irradiation on angiogenesis in injured rat tibiae

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm(-2) were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm(-2), but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.     

Continual measurement of intramedullary blood perfusion with laser Doppler flowmetry in intact and ostectomized tibiae of rabbits

Blood flow is important for the healing of bone fractures. Until now, however, there have been no publications on the daily, continual measurement of intramedullary blood perfusion using laser Doppler flowmetry (LDF) in the conscious animal. In this study, a model for the daily, continual measurement of intramedullary blood perfusion by LDF and the temperature near the cortex both in intact and ostectomized tibiae in the conscious rabbit is described. The probes for blood perfusion and temperature measurement were implanted permanently at three different localizations into the right tibia of 10 adult New Zealand White rabbits. The probes were held in place by a bilateral, single-plane external fixator. In five of these animals, a midshaft tibial ostectomy was created in order to simulate a fracture. Intramedullary blood perfusion and temperature were measured daily over 49 days. While in intact tibiae no significant (P > 0.05) differences were found in blood perfusion readings taken at various time points, for mean values or for blood perfusion over time, in ostectomized tibiae the differences were significant: various time points (P = 0.0056), mean values (P = 0.0034) and blood perfusion over time (P = 0.0337). Blood perfusion readings at the centre probe were elevated compared with those at the proximal and distal probes. Thus, a revascularization in the ostectomy gap during the fracture healing was proven by means of the LDF. No influence of the blood perfusion on the temperature in the ostectomy area could be determined during healing of the ostectomy. The described model seems suitable for the continual measurement of intramedullary blood perfusion both in intact and ostectomized tibiae in the conscious rabbit.     

DEXA对骨髓炎骨缺损治疗中骨痂密度的评价

目的：探讨DEXA对骨髓炎骨缺损治疗中骨痂密度的评价及意义。方法：严格按照纳入排除标准,选取21例骨髓炎清创后伴大段皮质骨缺损一期植骨的病人。术后4,6,8,10个月后对骨折端骨痂行双能X线骨密度仪检测,并进行X摄片以及Enneking评分,从而明确植骨区愈合骨痂的密度变化趋势,骨愈合情况以及症状改善情况。结果：（1）X线摄片结果显示：4个月后：骨缺损区依然清晰可见,内有少量稀疏骨痂通过,少量外骨痂形成。6个月后：植骨区内骨痂含量明显增多,且外骨痂膨大。8个月：缺损区模糊,有较致密骨痂生成,且外骨痂逐渐减少。10个月：植骨区骨痂更加致密,且部份髓腔再通。（2）Enneking评分：患者术后第10个月功能恢复情况评估正常功能20例,20分以下的患者1例。（3）BMD测定：骨折端的骨密度及骨密度比率随时间延长而增加,植骨10个月后患侧的骨密度已可基本上达到正常对照侧的骨密度水平。结论：双能X线骨密度测量从一定程度上反映出骨痂的力学强度特性。在感染性骨缺损治疗中可以作为检测植骨区的恢复情况的参考。     

Organic Gallium Treatment Improves Osteoporotic Fracture Healing Through Affecting the OPG/RANKL Ratio and Expression of Serum Inflammatory Cytokines in Ovariectomized Rats

This study aimed to investigate the impact of organic gallium (OG) on osteoporotic fracture healing in ovariectomized female Sprague-Dawley rats, as well as study the mechanisms of OG on osteoporotic fracture healing. Forty-five female Sprague-Dawley rats were divided into three groups: sham operation group (Sxas control group), ovariectomized group (Ovx), and Ovx treated with OG group (Ovx + OG). Rat femoral fractures were studied using a standardized fracture-healing model utilizing bone fixation with an intramedullary pin. Six weeks later, analyses of micro-CT, histomorphometric, RNA extraction, RT-qPCR, and serum were performed following sacrifice of all mice. In comparison with Ovx group, OG can significantly increase bone volume (BV), tissue volume (TV), BV/TV radio, bone strength, callus bony area, and as similar to BMP-2 expression. OG treatment elevated OPG messenger RNA (mRNA) and inhibited RANKL mRNA, and showed an effect on OPG/RANKL ratio. OG treatment can inhibit the expression of TNF-α and IL-6. In conclusion, current study results indicate that organic OG can positively affect the OPG/RANKL ratio and inhibit the expression of serum inflammatory cytokines; thus, it can improve osteoporotic fracture healing.     

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.     

Longitudinal Analysis of Osteogenic and Angiogenic Signaling Factors in Healing Models Mimicking Atrophic and Hypertrophic Non-Unions in Rats

Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group) to an open osteotomy (hypertrophy group) led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group) decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.     

The enhancement of bone regeneration by ultrasound

Millions of fractures occur every year worldwide, with nearly 6.2 million fractures reported annually in the United States alone. Even though treatment methods have improved over the last few decades, 5–10% of fractures still show delayed healing. A significant subpopulation of these delayed healings do not heal by nine months and are thus termed non-unions. Experimental studies have shown some evidence that low intensity pulsed ultrasound stimulation (LIPUS) results in enhanced bone regeneration during fracture healing and callus distraction. LIPUS treatment has led to increased callus area and accelerated return of bone strength following fracture. Histological studies suggest that LIPUS influences all major cell types involved in bone healing, including osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells. The affect of LIPUS seems to be limited to cells in soft tissue, whereas cells in calcified bone seem not to be effected. In vitro cell culture studies as well as tissue culture studies have shown some effects on cell differentiation and protein synthesis. Even though the energy used by LIPUS treatment is extremely low, the effects are evident. The most probable source of the therapeutic benefits observed with LIPUS treatment involves nonthermal mechanisms that influence cell membrane permeability and increase cellular activity. Despite clinical and experimental studies demonstrating the enhancing effect of LIPUS on bone regeneration, the biophysical mechanisms involved in the complex fracture healing process remain unclear and requires further research.     

Derby intramedullary nail — A biomechanical comparison

This paper describes the design and function of the Derby intramedullary nail and its biomechanical testing, comparing it with the k nail and reamed unfractured femurs. The Derby nail is designed to transmit torsional loads and apply compression to the fracture site using a sliding proximal collar and distal extendable ‘wings’. Femurs fixed with Derby, K and Zickel nails and AO plates were tested in torsion and the rotation and torsional load at failure recorded and compared with unfractured control femurs. The results show that the strength of the Derby nail in torsion is ≈4 times and the energy to failure 2.3 times that of the k nail.