Rat Fracture Model Research Articles

Bone penetrance of locally administered vancomycin powder in a rat femur fracture model

IntroductionLocally delivered, crystalline vancomycin has been suggested as a potential prophylactic measure against the development of deep and superficial surgical site infection. Clinical expectations regarding the duration and peak of drug concentration in local tissues following administration are unknown. Our goal was to develop concentration vs time curves for locally administered vancomycin powder in a high-energy, open femur fracture rat model in local tissues and to compare that data to two well performed similar, systemic administration studies. MethodsAfter approval for animal research, 24 adult Sprague-Dawley rats sustained closed, midshaft femoral fracture under anesthesia. Fractures were caused via blunt guillotine with 750g metal rod dropped 50cm. Injured hindlimbs were surgically opened at fracture to simulate open injury and stabilized using 0.054 Kirschner wires. Vancomycin powder was administered using weight-based protocol (goal: 25mg/kg). Rats were sacrificed in groups of 4 at 4, 8, 24, 48, 72, 96h. Samples harvested included rat-tail venous blood prior to sacrifice, and femoral bone and anterior thigh soft-tissue were harvested post-mortem. High Performance Liquid Chromatography (HPLC) was performed on all samples. ResultsConcentration vs. time curves demonstrated that the surrounding soft-tissues demonstrated highest maximum concentration (1.5mg vancomycin/g muscle). Bone reached maximum average of 199μg vancomycin/g femur: approximately 13% of maximal soft-tissue absorption. Plasma reached maximum concentration of 1.8μg/mL plasma. All peaks at t=4h. Within 48h, average muscle vancomycin concentration dropped to 3μg/g muscle (0.2% maximum muscle concentration) and the average bone concentration dropped to 1.9μg/g femur (0.9% maximum bone concentration). Vancomycin was undetectable on all samples at 96h. Comparison to classical animal studies suggest local delivery to bone exceeds that of IV dosing for approximately 48h and may peak near concentrations of 102 multiples. ConclusionsLocally administered vancomycin provides drug delivery in excess of IV dosing for approximately 48h after intervention. Exponential decay demonstrates rapid removal of drug to near undetectable levels in bone, plasma, and local soft tissue thereafter in a rat model. Local delivery may generate concentrations exceeding that achievable by steady state systemic dosing for 48h.

Sclerostin Antibody Increases Callus Size and Strength but does not Improve Fracture Union in a Challenged Open Rat Fracture Model.

Open fractures remain a challenge in orthopedics. Current strategies to intervene are often inadequate, particularly in severe fractures or when treatment is delayed. Sclerostin is a negative regulator of bone growth and sclerostin-neutralizing antibodies (Scl-Ab) can increase bone mass and strength. The application of these antibodies to improve orthopedic repair has shown varied results, and may be dependent on the location and severity of the bony injury. We examined Scl-Ab treatment within an established rat osteotomy model with periosteal stripping analogous to open fracture repair. In one study, Scl-Ab was given 25mg/kg bi-weekly, either from the time of fracture or from 3 weeks post-fracture up to an end-point of 12weeks. A second study treated only delayed union open fractures that did not show radiographic union by week 6 post-fracture. Outcome measures included radiographic union, microCT analysis of bone volume and architecture, and histology. In the first study, Scl-Ab given from either 0 or 3weeks significantly improved callus bone volume (+52%, p < 0.05 and +58%, p < 0.01) at 12weeks, as well as strength (+48%, p < 0.05 and +70%, p < 0.05). Despite these improvements, union rate was not changed. In the second study treating only established delayed fractures, bony callus volume was similarly increased by Scl-Ab treatment; however, this did not translate to increased biomechanical strength or union improvement. Sclerostin antibody treatment has limited effects on the healing of challenging open fractures with periosteal stripping, but shows the greatest benefits on callus size and strength with earlier intervention.

Evaluation of the Effect of Platelet-Rich Fibrin on Long Bone Healing: An Experimental Rat Model.

Pseudoarthrosis, or nonunion, of the long bones is a challenging medical condition for orthopedic surgeons to treat. Therefore, healing enhancer materials are commonly used. The authors investigated whether platelet-rich fibrin accelerates long bone healing by comparing radiological and histological findings in a rat model of open femoral fracture. Platelet-rich fibrin is a current biomaterial that contains many growth factors and platelets. There are no studies in the literature investigating the effects of platelet-rich fibrin on fracture healing. Sixteen mature male rats were divided into 2 groups. In both groups, an open femoral fracture was created. The platelet-rich fibrin was obtained by centrifuging blood collected from the rats. Rats in the study group were treated with sterile platelet-rich fibrin, and those in the control group were administered saline. The rats were killed at the end of 4 weeks and examined histologically and radiologically. The radiographic and histological scores of the 2 groups differed significantly (P<.05). These results indicate that platelet-rich fibrin is an efficient biomaterial in fracture healing and that it increases the amount of osseous tissue formation. Platelet-rich fibrin does not cause an allergic reaction, is cost-effective, and is easy to obtain. Additional studies are necessary to determine whether platelet-rich fibrin accelerates the fracture healing process or induces a better quality of fracture healing. [Orthopedics. 2017; 40(3):e479-e484.].

Cystathionine γ-Lyase–Hydrogen Sulfide Induces Runt-Related Transcription Factor 2 Sulfhydration, Thereby Increasing Osteoblast Activity to Promote Bone Fracture Healing

Hydrogen sulfide (H2S) plays an essential role in bone formation, in part, by inhibiting osteoclast differentiation, maintaining mesenchymal stem cell osteogenesis ability, or reducing osteoblast injury. We aimed to investigate the role of H2S in osteoblast function and its possible molecular target. In this study, we found that cystathionine γ-lyase (CSE) majorly contributed to endogenous H2S production in the primary osteoblast. Overexpressed CSE increased osteoblast differentiation and maturation with higher bone morphogenetic protein 2 and osteopontin expression, alkaline phosphatase activity, and calcium nodule formation; in contrast, knockdown of CSE had opposite effects. Runt-related transcript factor 2 (RUNX2) is required for osteoblast biologic function. CSE-H2S increased nuclear RUNX2 accumulation, DNA binding activity, and target gene transcription. Protein sulfhydration is a common signal by H2S. We confirmed that RUNX2 was also sulfhydrated by H2S. This chemical modification enhanced RUNX2 transactivation, which was blocked by dithiothreitol (DTT, sulfhydration remover). Mutation of two cysteine sites in the runt domain of RUNX2 abolished H2S-induced RUNX2 sulfhydration and transactivation. In a bone -fracture rat model, overexpressed CSE promoted bone healing, which confirmed the effect of CSE-H2S on osteoblasts. CSE-H2S is a dominant H2S generation system in osteoblasts and promotes osteoblast activity by the RUNX2 pathway, with RUNX2 sulfhydration as a novel transactivation regulation. CSE-H2S sulfhydrated RUNX2 enhanced its transactivation and increased osteoblast differentiation and maturation, thereby promoting bone healing. Antioxid. Redox Signal. 27, 742-753.

Bisphosphonates Inhibit Pain, Bone Loss, and Inflammation in a Rat Tibia Fracture Model of Complex Regional Pain Syndrome.

Bisphosphonates are used to prevent the bone loss and fractures associated with osteoporosis, bone metastases, multiple myeloma, and osteogenesis deformans. Distal limb fractures cause regional bone loss with cutaneous inflammation and pain in the injured limb that can develop into complex regional pain syndrome (CRPS). Clinical trials have reported that antiresorptive bisphosphonates can prevent fracture-induced bone loss, inhibit serum inflammatory cytokine levels, and alleviate CRPS pain. Previously, we observed that the inhibition of inflammatory cytokines or adaptive immune responses attenuated the development of pain behavior in a rat fracture model of CRPS, and we hypothesized that bisphosphonates could prevent pain behavior, trabecular bone loss, postfracture cutaneous cytokine upregulation, and adaptive immune responses in this CRPS model. Rats underwent tibia fracture and cast immobilization for 4 weeks and were chronically administered either subcutaneously perfused alendronate or oral zoledronate. Behavioral measurements included hindpaw von Frey allodynia, unweighting, warmth, and edema. Bone microarchitecture was measured by microcomputed tomography, and bone cellular activity was evaluated by static and dynamic histomorphometry. Spinal cord Fos immunostaining was performed, and skin cytokine (tumor necrosis factor, interleukin [IL]-1, IL-6) and nerve growth factor (NGF) levels were determined by enzyme immunoassay. Skin and sciatic nerve immunoglobulin levels were determined by enzyme immunoassay. Rats with tibia fractures developed hindpaw allodynia, unweighting, warmth, and edema, increased spinal Fos expression and trabecular bone loss in the lumbar vertebra and bilateral distal femurs as measured by microcomputed tomography, increased trabecular bone resorption and osteoclast surface with decreased bone formation rates, increased cutaneous inflammatory cytokine and NGF expression, and elevated immunocomplex deposition in skin and nerve. Alendronate (60 μg/kg/d subcutaneously [s.c.]) or zoledronate (3 mg/kg/d orally) treatment for 28 days, started at the time of fracture, completely inhibited the development of hindpaw allodynia and reduced hindpaw unweighting by 44% ± 13% and 58% ± 5%, respectively. Orally administered zoledronate (3 mg/kg/d for 21 days) treatment also completely reversed established allodynia and unweighting when started at 4 weeks postfracture. Histomorphometric and microcomputed tomography analysis demonstrated that both the 3 and 60 μg/kg/d alendronate treatments reversed trabecular bone loss (an 88% ± 25% and 188% ± 39% increase in the ipsilateral distal femur BV/TV, respectively) and blocked the increase in osteoclast numbers and erosion surface observed in bilateral distal femurs and in L5 vertebra of the fracture rats. Alendronate treatment inhibited fracture-induced increases in hindpaw inflammatory mediators, reducing postfracture levels of tumor necrosis factor by 43% ± 9%, IL-1 by 60% ± 9%, IL-6 by 56% ± 14%, and NGF by 37% ± 14%, but had no effect on increased spinal cord Fos expression, or skin and sciatic nerve immunocomplex deposition. Collectively, these results indicate that bisphosphonate therapy inhibits pain, osteoclast activation, trabecular bone loss, and cutaneous inflammation in the rat fracture model of CRPS, data supporting the hypothesis that bisphosphonate therapy can provide effective multimodal treatment for CRPS.

Effect of leptin combined with CoCl2 on healing in Sprague Dawley Rat fracture model.

To evaluate the effect of leptin combined with CoCl2 on rat femur fracture healing. 48 male Sprague Dawley rats were randomly divided into two main groups. Then standardized femur fractures were created to all rats. Control group rats were treated with 0.5 mL physiological saline, and experimental group rats were treated with 5 μg/Kg.d leptin and 15 mg/Kg.d CoCl2 along with 0.5 mL physiological saline for 42 days intraperitoneally. Each main group was divided into three subgroups for each evaluation at second, fourth and sixth weeks, each subgroup included eight rats. The radiological evaluation showed that the fracture healing progress of experimental group was superior to control group from second week. At fourth week, experimental group had better fracture healing progress than control group significantly. Results of biomechanics show the ultimate load (N) and deflection ultimate load (mm) of experimental group was significantly increased than that in control group from fourth week. The present result demonstrated that leptin combined with CoCl2 significantly increased the mRNA expression levels of HIF1A, Vegfa, Runx2, Bmp2, Bglap and Alpl. It suggested that leptin combined with CoCl2 have a positive effect on rat femur fracture healing by activating the HIF1A pathway.

Fracture initiates systemic inflammatory response syndrome through recruiting polymorphonuclear leucocytes.

Fracture, a common type injury in trauma patients, often results in the development of the systemic inflammatory response syndrome (SIRS). Though the mechanism of the fracture-initiated SIRS still remains not well characterized, it is well documented that the polymorphonuclear leucocytes (PMN) play an important role in the inflammatory process. We hypothesize that fractures recruit PMN to the local tissue, which is followed by an increase in the number of peripheral PMN and initiation of SIRS. In the current study, we established a closed femoral fracture rat model. We evaluated the levels of MPO, IL-1β and CINC-1 in fractured tissue homogenate, and we measured the levels of IL-6 and IL-10, the biomarkers for systemic inflammatory response, in the rat sera. In clinical part of the study, we collected blood from patients with isolated closed femoral fractures and evaluated PMN-related chemoattractants (IL-8, IL-1β and G-CSF) and the number of peripheral PMN. We further evaluated the level of mitochondrial DNA in the local haematoma of fracture and the circulating plasma of the patients with fracture. In the animal model of closed femoral fracture, we found a significant recruitment of PMN to the local tissue after fracture, which correlates with the elevated MPO level. We also showed that the concentration of IL-1β and CINC-1 in local tissue is significantly increased and might be responsible for the PMN recruitment. Recruitment of PMN to the local tissue was accompanied with a significant increase in the systemic levels of IL-6 and IL-10 in serum. In the patients with closed femoral fracture, we observed an increase in the number of peripheral PMN and PMN-related chemoattractants, including IL-8, IL-1β and G-CSF. The level of mitochondrial DNA in the local haematoma of fracture and the circulating plasma of patients were significantly higher compared to the healthy volunteers. Our data suggest that fracture released mitochondrial DNA into the local haematoma of fracture, which recruited the PMN into the local tissue via chemokines (IL-1β and CINC-1), then increased the numbers of peripheral PMN and SIRS related cytokines in serum (IL-6 and IL-10). This might be the mechanism of the fracture-initiated SIRS.

Histological and biomechanical effects of zoledronate on fracture healing in an osteoporotic rat tibia model.

This study aims to investigate the effects of zoledronate therapy on histological and biomechanical properties of bone healing via a fracture model generated on osteoporotic rat tibiae. Ovariectomized 40 Wistar-Dawley female rats weighing 300 g to 350 g were used in the study. After one week, 2 IU/g heparin injection was started subcutaneously. After four weeks of daily injections, osteoporosis was ensued proven with bone mineral density measurements. Osteoporotic rats were separated into four equal groups randomly as group A (control), group B (calcium and vitamin D), group C (0.1 mg/kg subcutaneous zoledronic acid), and group D (calcium and vitamin D / 0.1 mg/kg subcutaneous zoledronic acid). Six weeks later, all rats were sacrificed, their tibiae were resected, and histopathologic and biomechanical studies were performed. Group C (30.2±1 Nm) and group D (33.3±3 Nm) had significantly higher peak torque values than group A (21.6±6 Nm) and group B (23.6±4 Nm) (p=0.007 and p=0.005, respectively). Group C (1.8) and group D (2.0) had higher stiffness values than group A (1.4) and group B (1.7); however, the difference was not statistically significant (p>0.05 for all). According to histopathological and biomechanical test results, single dose zoledronic acid treatment improves fracture healing in an osteoporotic rat fracture model. Orally administered daily calcium and vitamin D treatment had no effect on fracture healing. There was no additional improvement in fracture healing when calcium and vitamin D treatment was added to zoledronic acid treatment. Positive effects of zoledronic acid treatment on osteoporotic fracture healing and callus quality should be shown by future clinical studies.

Effects of Sclerostin Antibody on the Healing of Femoral Fractures in Ovariectomised Rats.

The inhibition of sclerostin by the systemic administration of a monoclonal antibody (Scl-Ab) significantly increased bone mass and strength in fractured bones in animal models and non-fractured bones in ovariectomised (OVX) rats. In this study, the effects of Scl-Ab on healing were examined in a closed fracture model in OVX rats. Sixty Sprague-Dawley rats underwent an ovariectomy or a sham operation at 4months of age, and a closed fracture of the right femur was performed 3months later. Subcutaneous injections with Scl-Ab (25mg/kg) or saline were then administered on day 1 after the fracture and twice a week for 8weeks (n=20 per group), at which time the fractured femurs were harvested for micro-computed tomography analysis, four-point bending mechanical testing and histomorphometric analysis to examine bone mass, bone strength and dynamic bone formation at the fracture site. The angiogenesis at the fracture site was also examined. Bone marrow stem cells were also isolated from the fractured bone to perform a colony-forming unit (CFU) assay and an alkaline phosphatase-positive (ALP(+)) CFU assay. OVX rats treated with Scl-Ab for 8weeks had significantly increased bone mineral density and relative bone volume compared with OVX rats treated with saline. Similarly, maximum loading, energy to maximum load and stiffness in Scl-Ab-treated OVX rats were significantly higher than those in saline controls. The mineral apposition rate (MAR), mineralising surface (MS/BS) and bone formation rate (BFR/BS) were also significantly increased in Scl-Ab-treated group compared with the saline-treated group in OVX rats. Furthermore, the Scl-Ab-treated group had more CFUs and ALP(+) CFUs than the saline-treated group in OVX rats. No significant difference in angiogenesis at the fracture site was found between the groups. Our study demonstrated that Scl-Ab helped to increase bone mass, bone strength and bone formation at the fracture site in a closed femoral fracture model in OVX rats. Bone marrow stem cells in OVX rats injected with Scl-Ab also had increased CFUs and ALP(+) CFUs.

Combined local and systemic antibiotic delivery improves eradication of wound contamination

Systemic antibiotics reduce infection in open fractures. Local delivery of antibiotics can provide higher doses to wounds without toxic systemic effects. This study investigated the effect on infection of combining systemic with local antibiotics via polymethylmethacrylate (PMMA) beads or gel delivery. An established Staphylococcus aureus contaminated fracture model in rats was used. Wounds were debrided and irrigated six hours after contamination and animals assigned to one of three groups, all of which received systemic antibiotics. One group had local delivery via antibiotic gel, another PMMA beads and the control group received no local antibiotics. After two weeks, bacterial levels were quantified. Combined local and systemic antibiotics were superior to systemic antibiotics alone at reducing the quantity of bacteria recoverable from each group (p = 0.002 for gel; p = 0.032 for beads). There was no difference in the bacterial counts between bead and gel delivery (p = 0.62). These results suggest that local antibiotics augment the antimicrobial effect of systemic antibiotics. Although no significant difference was found between vehicles, gel delivery offers technical advantages with its biodegradable nature, ability to conform to wound shape and to deliver increased doses. Further study is required to see if the gel delivery system has a clinical role.

Rapamycin-induced autophagy activity promotes bone fracture healing in rats.

Autophagy is a crucial mediating process for normal bone cell function and metabolism in physiology or pathology. Rapamycin has been demonstrated to induce the autophagy pathway by inhibiting the mammalian target of rapamycin (mTOR) pathway. However, the contribution of autophagy in orthopedic diseases is rarely reported. The aim of the present study was to evaluate the capacity of pharmacologically induced autophagy to modify disease function in a rat model of bone fracture. A femur fracture model was established via surgery in adult male Sprague-Dawley rats. Rapamycin (n=63 rats) or dimethyl sulfoxide (DMSO) vehicle control (n=63 rats) was administered intraperitoneally for 2, 4 and 6 weeks, and 21 randomly selected rats were sacrificed in each group at each time point. X-ray micro-computed tomography and hematoxylin and eosin staining were used to evaluate the extent of fracture healing in each group. The effects of rapamycin on autophagy, mTOR signaling and the expression levels of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were analyzed using immunohistochemistry, immunofluorescence staining and western blot analysis. Rapamycin affected the mTOR signaling pathway in rats following fracture, as indicated by the inhibition of the phosphorylation of ribosomal protein S6, a target of mTOR, and activation of microtubule-associated protein 2 light chain 3, a key marker of autophagy. Histomorphometry and image examination indicated that the number of osteoblasts in each section was significantly (P<0.01) increased in the rapamycin group compared with the control group, and this was associated with a significant (P<0.05) increase in mineralized callus fraction. Furthermore, rapamycin treatment increased the expression levels of VEGF and PCNA in the rat callus tissue. These results suggest that rapamycin may serve a beneficial function in fracture healing, and that the underlying mechanism may involve the activation of autophagy.

Effect of CoCl₂ on fracture repair in a rat model of bone fracture.

Low oxygen availability is known to activate the hypoxia-inducible factor-1α (HIF-1α) pathway, which is involved in the impairment of fracture healing. However, the role of low oxygen in fracture healing remains to be fully elucidated. In the present study, rats were divided into two groups and treated with CoCl2 or saline, respectively. Mice with tibial fractures were sacrificed at 14, 28 and 42 days subsequent to fracture. Autoradiography was performed to measure healing of the bone tissue. In addition, the effects of cobalt chloride (CoCl2) on the expression of two major angiogenic mediators, HIF‑1α and vascular endothelial growth factor (VEGF), as well as the osteoblast markers runt‑related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) were determined at mRNA and protein levels by reverse transcription‑quantitative polymerase chain reaction, western blot analysis and immunohistochemistry. Systemic administration of CoCl2 (15 mg/kg/day intraperitoneally) significantly promoted fracture healing and mechanical strength. The present study demonstrated that in rats treated with CoCl2, the expression of HIF‑1α, VEGF, Runx2, ALP and OC was significantly increased at mRNA and protein levels, and that CoCl2 treatment enhances fracture repair in vivo.

Can Normal Fracture Healing Be Achieved When the Implant Is Retained on the Basis of Infection? An Experimental Animal Model.

Infection after open fractures is a common complication. Treatment options for infections developed after intramedullary nailing surgery remain a topic of controversy. We therefore used a rat fracture model to evaluate the effects of infection on osseous union when the implant was maintained. In a rat model, (1) does infection alter callus strength; (2) does infection alter the radiographic appearance of callus; and (3) does infection alter the histological properties of callus? An open femoral fracture was created and fixed with an intramedullary Kirschner wire in 72 adult male Sprague-Dawley rats, which were divided into two study groups. In the infection group, the fracture site was contaminated with Staphylococcus aureus (36 animals), whereas in the control group, there was no bacterial contamination (36 animals). No antibiotics were used either for prophylaxis or for treatment. We performed biomechanical (maximum torque causing failure and stiffness), radiographic (Lane and Sandhu scoring for callus formation), and histologic (scoring for callus maturity) assessments at 3 and 6 weeks. The number of bacteria colonies on the femur, wire, and soft tissue inside knee were compared to validate that we successfully created an infection model. The number of bacteria colonies in the soft tissue inside the knee was higher in the infection group after 6 weeks than after the third week, demonstrating the presence of locally aggressive infection. Infection decreased callus strength at 6 weeks. Torque to failure (299.07 ± 65.53 Nmm versus 107.20 ± 88.81, mean difference with 95% confidence interval, 192 [43-340]; p = 0.007) and stiffness at 6 weeks (11.28 ± 2.67 Nmm versus 2.03 ± 1.68, mean difference with 95% confidence interval, 9 [3-16]; p = 0.004) both were greater in the control group than in the group with infection. Radiographic analysis at 6 weeks demonstrated the fracture line was less distinct (Lane and Sandhu score of 2-3) in the infection group and complete union was observed (Lane and Sandhu score of 3-4) in the control group (p = 0.001). Semiquantitative histology scores were not different between the noninfected controls and the rats with infection (score 10 versus 9). Retaining an implant in the presence of an underlying infection without antibiotic treatment leads to weaker callus and impedes callus maturation compared with noninfected controls in a rat model. Future studies might evaluate whether antibiotic treatment would modify this result. This model sets the stage for further investigations that might study the influence of different interventions on fracture healing in implant-associated osteomyelitis. Future observational studies might also evaluate the histological properties of callus in patients with osteomyelitis.

Axial strain enhances osteotomy repair with a concomitant increase in connexin43 expression.

The mechanical environment is known to influence fracture healing. We speculated that connexin43 (Cx43) gap junctions, which impact skeletal homeostasis, fracture healing and the osteogenic response to mechanical load, may play a role in mediating the response of the healing bone to mechanical strain. Here, we used an established rat fracture model, which uses a 2 mm osteotomy gap stabilized by an external fixator, to examine the impact of various cyclical axial loading protocols (2%, 10%, and 30% strain) on osteotomy healing. We examined the presence of Cx43 in the osteotomy-healing environment and assessed how mechanical strain modulates Cx43 expression patterns in the callus. We demonstrated that increased cyclical axial strain results in increased radiographic and histologic bone formation. In addition, we show by immunohistochemistry that Cx43 is abundantly expressed in the healing callus, with the expression most robust in samples exposed to increased cyclical axial strain. These data are consistent with the concept that an increase in Cx43 expression by mechanical load may be part of the mechanisms by which mechanical forces enhances fracture healing.

Novel rat model of nonunion fracture with vascular deficit

Nonunion fractures occur frequently in humans, with profound implications (medical and non-medical). Although there are numerous animal models to study pathogenesis and treatment of nonunion fractures, there is apparently the lack of a definitive model for atrophic nonunion fracture. Therefore, the objective was to develop a low-cost rat model of nonunion fracture with a vascular deficit that enabled standardized quantitative analysis of bone growth and regeneration. The model was developed with two surgeries, performed apart. The first involved osteotomy of the femur diaphysis, removal of periosteum and endosteum, isolation of the fracture site using a latex artefact (Penrose drain tube), and reduction of the fracture using an intramedullary pin, whereas the second surgery was to remove the latex artefact. Based on radiographic imaging, micro-CT and histological analyses done 125 days after the fracture was induced, there was clear evidence of atrophic nonunion fracture, without pin migration or specimen loss. Perceived advantages of this model included low cost, ease of reproducibility, lack of specimen loss, and, finally, the potential to assess bone growth and regeneration under poor vascular conditions.

Traumatic brain injury result in accelerated fracture healing: A Rat Model

Background: In patients who have sustained a traumatic brain injury with an associated extremity fracture there is often a clinical perception that the rate of new bone formation around the fracture site is increased. In this study, we made a rat model of femoral fracture to investigate the serum factors changes in vivo. We examined the effects serum factors on fracture healing and the molecular signaling pathways that might link these two. Methods and Findings: Thirty-Six female Sprague-Dawley rats were randomly allocated into 4 groups; the non-operated control group, traumatic brain injury (TBI) group, bone fracture (Fr) group and the bone fracture/ TBI group (TBI & Fr) (n=9 per group). These animals were sacrificed at 6 weeks after experiments. Volume of callus in fracture was measured weekly with the Perkins volume formula at 2nd week after operation. The radiology imaging was interpreted by two or more senior orthopedic specialists. In vivo chemo taxis assay by serum samples collected at the 1, 3, 7 and 14 days, and 3rd, 4th, 6th week after surgery. Seral NGF, Wnt, RANKL, Dkk-1, ACTH, Leptin titer was measured and analyzed. After 2 weeks operation, the formation of callus was found at the fracture site, callus in TBI & Fr group was slightly higher than Fr group; however, there was no significant difference existed between the TBI & Fr and Fr groups. The ACTH level was significantly elevated in the TBI & Fr group than other groups and attained its peak at 1 week after operation, while the leptin was shown higher levels in Fr groups than TBI and TBI & Fr groups at 2 weeks after operation. The serum insulin level was markedly increased in all experimental groups during 1-3 days after surgery; at the meanwhile, the serum glucose level only significantly increased in the Fx and TBI groups at the 1 and 3 day after operation. The serum of NGF levels in the groups with TBI process (TBI and TBI & Fr groups) was significantly higher than those of the simple fracture group and control group. In the early stage of bone fracture, DKK-1 should be more active than Wnt-3a and NGF; later Wnt-3a and NGF became active to stimulate the osteoblasts for bone formation. After 3 day to 2 week of surgery, the RANKL process was drove to additional bone remodeling in accompany with bone formation. Conclusions: Through this project, the pathogenesis of increased rates of excessive bone healing in the patients with traumatic brain injury will be elucidated for the future study on clinical treatment in both traumatic brain injury and fracture healing. Keywords: Traumatic brain injury, fracture healing,

Sox11-modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs.

Mesenchymal stem cells (MSCs) are a promising cell resource for tissue engineering. Sry-related high-mobility group box 11 (Sox11) plays critical roles in neural development and organogenesis. In the present study, we investigated the role of Sox11 in regulating trilineage differentiation (osteogenesis, adipogenesis, and chondrogenesis) and migration of MSCs, and explored the effect of systemically administrated Sox11-modified MSCs on bone fracture healing using the rat model of open femur fracture. Our results demonstrated that Sox11 overexpression increased the trilineage differentiation and migration of MSCs, as well as cell viability under oxidative stress. The effect of Sox11 on osteogenesis was confirmed by ectopic bone formation assay conducted in nude mice. In addition, we found that Sox11 could activate the bone morphogenetic protein (BMP)/Smad signaling pathway in MSCs. By dual-luciferase reporter assay, we also demonstrated that Sox11 could transcriptionally activate runt-related transcription factor 2 (Runx2) and CXC chemokine receptor-4 (CXCR4) expression. The activation of the BMP/Smad signaling pathway and Runx2, CXCR4 expression may have a synergic effect, which largely contributed to the effect of Sox11 on MSC fate determination and migration. Finally, using an open femur fracture model in rats, we found that a larger number of MSCs stably expressing Sox11 migrated to the fracture site and improved bone fracture healing. Taken together, our study shows that Sox11 is an important regulator of MSC differentiation and migration, and Sox11-modified MSCs may have clinical implication for accelerating bone fracture healing, which can reduce the delayed unions or nonunions.

Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats.

We previously demonstrated that topical cutaneous application of CO2, by means of a hydrogel in which the CO2 readily dissolves, increases blood flow and oxygen dissociation from hemoglobin in the soft tissues surrounding bone. In the present study, we utilized a rat fracture model to test the hypothesis that application of this treatment to fractured limbs would accelerate fracture repair. A closed femoral shaft fracture was created in each rat. Topical cutaneous application of CO2 by means of a hydrogel was performed five times a week for up to four weeks in the CO2/hydrogel group (n = 60). Sham treatments were performed in the control group (n = 60). Radiographic, histological, immunohistochemical, laser Doppler perfusion imaging, real-time polymerase chain reaction, and biomechanical assessments were performed. Radiographic fracture union was evident at week 3 in twelve (86%) of fourteen animals in the CO2/hydrogel group compared with five (36%) of fourteen in the control group (p < 0.05; 95% CI [confidence interval] for the difference in union rate, 2.26% to 99.64%). Histological assessment revealed promotion of endochondral ossification in the CO2/hydrogel group. Immunohistochemical assessment at week 2 showed significantly greater capillary density in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 161 to 258 per mm(2)). Laser Doppler perfusion imaging demonstrated that the blood flow in the fractured limb was significantly greater at weeks 2 and 3 in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 8.4% to 22.4% and 6.7% to 19.0%, respectively). Gene expression of chondrogenic, osteogenic, and angiogenic markers was significantly greater in the CO2/hydrogel group at several time points. Ultimate stress, extrinsic stiffness, and failure energy (relative to the contralateral limb) were significantly greater in the CO2/hydrogel group at week 3 (p < 0.05; 95% CI for the difference, 24.8% to 67.5%, 4.0 % to 22.7%, and 9.6% to 58.8%, respectively). There were no significant differences between the groups with respect to any outcome measure at week 4. Topical cutaneous application of CO2 by means of a hydrogel accelerated fracture repair in association with the promotion of angiogenesis, blood flow, and endochondral ossification. .

Surgical procedures and experimental outcomes of closed fractures in rodent models.

The closed fracture rat model, first described by Bonnarens and Einhorn, has been widely implemented in recent years to characterize various fracture phenotypes and evaluate treatment modalities. Slight modifications in the fixation depth, to reduce surgical error associated with movement/dislocation of the k-wire fixation, were previously described. Here, we describe this method which involves the creation of a medial parapatellar incision, dislocation of the patella, boring an 18 gauge hole through the center of the femur, delivery of an adjunct (if applicable), fixation of the k-wire in the greater trochanter of the femur, suturing of muscle and skin, and finally creation of the mid-diaphyseal fracture with a three-point bending fracture device. Many laboratories routinely perform surgical procedures in which a closed fracture is induced using rat or mouse models. The benefits of such surgical models range from general orthopaedic trauma applications to the assessment of the healing process in genetically modified animals. Other important applications include the assessment of the safety and efficacy of various treatment modalities as well as the characterization of bone repair in metabolic bone diseases or skeletal dysplasia.

Influence of systemic bisphosphonate treatment on mechanical properties of BMP‐induced calluses in a rat fracture model: Comparison of three‐point bending and twisting test

The combination of autograft, BMP and bisphosphonate has been shown to produce strong calluses. In this study, without autograft we investigate the effect of bisphosphonate treatment on BMP-induced calluses, both in bending and in rotation. Sprague-Dawley rats (n = 42) underwent femoral osteotomy and BMP-7 treatment. At 2 weeks an injection of saline or bisphosphonate was administered. The animals were sacrificed after 6 weeks. Both femurs were tested in either three-point bending or twisting. All femurs healed. BMP + bisphosphonate-treatment led to larger calluses (p < 0.05) and in three-point bending, higher ultimate force (p < 0.01) and greater stiffness (p < 0.05) than BMP alone. The BMP + bisphosphonate group was nearly 60% stronger than controls, while the BMP group did not reach the strength (p < 0.05) nor stiffness (p < 0.01) of the controls. In the twisting test, similar trends were found but less pronounced. Three-point bending produced transverse callus associated fractures, whereas the twisting test produced spiral fractures, located in the structurally weaker distal femur. BMP + bisphosphonate-treatment produces calluses that are mechanically superior to calluses induced by BMP alone, when tested both in three-point bending and in twisting. For the mechanical evaluation of pharmacologically enhanced calluses with breaking strengths exceeding the native bone, the bending test is recommended.