Introduction. The fracture healing outcome is often evaluated via ex vivo testing of the fracture callus. However, there is only a small time window, where the callus stiffness is significantly different, i.e. a delayed fracture healing might be undetected if the time point of sacrifice is improper. The aim of this study was to develop an in vivo monitoring concept, which allows determining the fracture callus stiffness in vivo over the whole healing time in rats. Hypothesis. The fracture callus stiffness can be monitored by measuring the deformation of the external fixation device during gait analysis at several healing time points. Materials & Methods. The right femurs of sixteen wistar rats were osteotomized and stabilized with an external fixation device (stiffness 119 N/mm or 32 N/mm). The fixator body was instrumented with a stain gauge to measure the deformation. Gait analysis was performed once per week in a gait wheel equipped with a ground reaction force measuring device. Results. The deformation of the fixation devices decreased over the healing time indicating an increase of the callus stiffness. The flexible fixated group showed a later increase of the callus stiffness indicating a delay in fracture healing. Discussion & Conclusion. Measuring the deformation of the fixator and gait analysis provides a powerful tool to monitor the fracture healing process in rats. With this, it is possible to detect a delayed fracture healing process more reliable than with ex vivo analyses

Purpose of this study is to create an experimental model of electrophysologic evaluation of the supraspinatus muscle on rats, after traumatic rupture of its tendon. The population of this study consisted of 10 male Sprague Dawley rats weighting 300–400g. Under general anaesthesia we proceeded with traumatic rupture of the supraspinatus tendon and exposure of the muscle. The scapula was immobilized, and the supraspinatus tendon was attached to a force transducer using a 3–0 silk thread. A dissection was performed in order to identify the suprascapular nerve, which was then stimulated with a silver electrode. Stimulations were produced by a stimulator (Digitimer Stimulator DS9A) and were controlled by a programmer (Digitimer D4030). Fiber length was adjusted until a single stimulus pulse elicited maximum force during a twitch under isometric conditions. Rectangular pulses of 0.5 ms duration were applied to elicit twitch contractions. During the recordings, muscles were rinsed with Krebs solution of approximately 37 8C (pH 7.2–7.4) and aerated with a mixture of 95% O2 and 5% CO2. The output from the transducer was amplified and recorded on a digital interface (CED). The following parameters were measured at room temperature (20–21 8C): single twitch tension; time to peak; half relaxation time; tetanic tensions at 10, 20, 40, 80 and 100 Hz; and fatigue index, which was evaluated using a protocol of low frequency (40 Hz) tetanic contraction, during 250 ms in a cycle of 1 s, for a total time of 180 s. The fatigue index value was then calculated by the formula [fatigue index=(initial tetanic tension − end tetanic tension) ∗ 100/(initial tetanic tension)]. In the end, the transducer was calibrated with standard weights and tensions were converted to grams. The mean single twitch was 8.2, the time to peak 0.034 msec and the half relaxation time 0.028 msec. The strength of titanic muscle contractures was 5.7 msec at 10Hz and 17.7 at 100Hz. Finally, the fatigue index was calculated at 48.4. We believe that electrophysiologic evaluation of the supraspinatus muscle in rats will help us understanding the pathology of muscle atrophy after rotator cuff tears and possibly the functional restoration after cuff repair

Introduction. Nowadays, autologous platelet-rich plasma is used commonly in wound treatment. However, platelet gel, which was derived from allogeneic platelet-rich plasma (PRP) [1,2], has never been studied about efficacy in vivo or animal models. We aimed to determine efficacy of allogeneic platelet-gel on wound healing in rats by comparing with untreated, antibiotic-gel (Mupirocin 2%) treated and gel (sodium carboxymethylcellulose(NaCMC))-treated control. Methods. Fresh frozen plasma was centrifuged at 1200-G for 15 minutes to extract PRP which would be freeze-dried at −70°c, sterilized with gamma ray of Cobalt source 25 kGy and stored at −70°c. Then, processed freeze-dried PRP was mixed with gel base (NaCMC) as in form of allogeneic platelet-gel concentrated 30 mg/1g by sterilization process (table 1). Full-thickness of 6-mm-diameter skin punch biopsies were performed on 18 female Wistar rats which each rat had four wounds at back. Each wound was applied with untreated care, antibiotic-gel, NaCMC-gel and platelet-gel, respectively. Wound healing was studied from day 0–12. Animals were sacrificed with wound tissues removal on day 3, 7, 12 post-biopsy. Digital planimetric measurement device (VISITRAK, Smith and Nephew) was used in evaluation of total wound area on day 0, 3, 7, 12 post-biopsy. Histopathological changes of wound healing were studied, using 4-μm thickness section with haematoxylin-eosin (H&E) and Masson's trichrome-stain, under light microscope. Results. Platelet-gel reduced wound size more rapidly on day 3, 7 than other groups with statistical significance (p<0.05), although no statistically significant difference compared to antibiotic-treated wounds. Histological study confirmed earlier granulation forming and more collagen fibers in platelet-gel treated group when compared with others. discussion & Conclusions. Allogeneic platelet gel produced the satisfactory efficacy on acute wound healing in rat. This platelet gel needs further study in human for efficacy and safety that might be developed for using in acute wound treatment in the future

In polytrauma patients invasive surgeries can potentiate the posttraumatic systemic inflammation thus increasing the risk of multi organ dysfunction. Therefore, fractures are initially treated by external fixators, which later are replaced by intramedullary nails. We showed that a severe trauma impaired the healing of fractures stabilized by external fixation. Here we studied, whether the conversion to an intramedullary nail increases posttraumatic inflammation and leads to further impairment of healing. 44 rats received a femur osteotomy stabilized by an external fixator (FixEx). Half of the rats underwent a thoracic trauma (TXT) at the same time. After 4 days the fixator was replaced by an intramedullary nail (IMN) in half of the rats of each group. The rats were killed after 40 and 47 days. C5a serum levels were measured 0, 6, 24, and 72h after the 1st as well as the 2nd surgery. The calli were evaluated by three-point-bending test, μCT and histomorphometry. The TXT significantly increased serum C5a levels after the 2nd surgical intervention. After 40 days the switch from FixEx to IMN significantly decreased bending stiffness in rats with and without TXT. After 47 days flexural rigidity in rats subjected to conversion was significantly decreased compared to rats treated only with a FixEx, particularly in combination with TXT. This study showed that after a severe trauma the conversion of the fixation could provoke a second hit and contribute to delayed fracture healing

The role of mesenchymal stem cells (MSCs) in enhancing healing process has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union. Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilised by intramedullary nail fixation with a 1mm gap maintained by a spacer. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5×106 and were injected into the fracture site. At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value =<0.05, unpaired t-test, corrected by Benjamini & Hochberg. We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus

There is evidence that fracture healing is delayed in severely injured patients. We recently demonstrated that a blunt chest trauma, which induced posttraumatic systemic inflammation, considerably impaired fracture healing in rats. Because the complement anaphylatoxin C5a is an important trigger of systemic inflammation, we tested the hypothesis, whether the impairment of fracture healing observed after a severe trauma resulted from systemically activated complement. 16 male Wistar rats received a thoracic trauma and a femur osteotomy stabilized by an external fixator. Immediately and 12 h after the trauma, half of the animals received a C5aR-antagonist to prevent the C5a-dependent systemic inflammation. Control rats received a nonsense peptide, which does not provoke any biological effect. The animals were killed after 35 days and the calli were analyzed by three point bending testing, μCT and histomorphometry. Statistics: Mann-Whitney U test, level of significance to p<0.05. The treatment with the C5aR-antagonist increased flexural rigidity significantly by 55%, improved bony bridging of the fracture gap and led to a slightly larger and qualitatively improved callus as evaluated by μCT and histological measurements. This study shows, that the immunomodulation by a C5aR-antagonist significantly reduced the deleterious effects of a thoracic trauma on fracture healing. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter. . Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment. Cite this article: Bone Joint J 2015; 97-B:1144–51

Objectives. One commonly used rat fracture model for bone and mineral research is a closed mid-shaft femur fracture as described by Bonnarens in 1984. Initially, this model was believed to create very reproducible fractures. However, there have been frequent reports of comminution and varying rates of complication. Given the importance of precise anticipation of those characteristics in laboratory research, we aimed to precisely estimate the rate of comminution, its importance and its effect on the amount of soft callus created. Furthermore, we aimed to precisely report the rate of complications such as death and infection. Methods. We tested a rat model of femoral fracture on 84 rats based on Bonnarens’ original description. We used a proximal approach with trochanterotomy to insert the pin, a drop tower to create the fracture and a high-resolution fluoroscopic imager to detect the comminution. We weighed the soft callus on day seven and compared the soft callus parameters with the comminution status. Results. The mean operating time was 34.8 minutes (. sd. 9.8). The fracture was usable (transverse, mid-shaft, without significant comminution and with displacement < 1 mm) in 74 animals (88%). Of these 74 usable fractures, slight comminution was detected in 47 (63%). In 50 animals who underwent callus manipulation, slight comminution (n = 32) was statistically correlated to the amount of early callus created (r = 0.35, p = 0.015). Two complications occurred: one death and one deep infection. Conclusions. We propose an accurate description of comminution and complications in order to improve experiments on rat femur fracture model in the field of laboratory research. Cite this article: Bone Joint Res 2013;2:149–54

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. Cite this article: Bone Joint J 2015;97-B:1423–7

Objectives. Small animal models of fracture repair primarily investigate indirect fracture healing via external callus formation. We present the first described rat model of direct fracture healing. Methods. A rat tibial osteotomy was created and fixed with compression plating similar to that used in patients. The procedure was evaluated in 15 cadaver rats and then in vivo in ten Sprague-Dawley rats. Controls had osteotomies stabilised with a uniaxial external fixator that used the same surgical approach and relied on the same number and diameter of screw holes in bone. Results. Fracture healing occurred without evidence of external callus on plain radiographs. At six weeks after fracture fixation, the mean stress at failure in a four-point bending test was 24.65 N/mm. 2. (. sd. 6.15). Histology revealed ‘cutting-cones’ traversing the fracture site. In controls where a uniaxial external fixator was used, bone healing occurred via external callus formation. Conclusions. A simple, reproducible model of direct fracture healing in rat tibia that mimics clinical practice has been developed for use in future studies of direct fracture healing

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing. A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague–Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology. The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm. -2. (. sd. 7.63) vs 24.65 Nmm. -2. (. sd. 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (. sd. 0.75) vs 4.6 mmAl (. sd. 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007). . Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing. Cite this article: Bone Joint J 2013;95-B:1263–8

Introduction. It is well known that blood flow is a critical key component of fracture repair. Previously, we demonstrated that transcutaneous application of CO2 increased blood flow in the human body. To date, there has been no report investigating the effect of the carbonated therapy on fracture repair. Hypothesis. We hypothesized that the transcutaneous application of CO2 to fracture site would accelerate fracture repair. Materials & Methods. A closed femoral shaft fracture was produced in rats. Transcutaneous CO2 absorption enhancing hydrogel and CO2 adaptor that sealed the body surface and retained the gas inside were used for CO2 treatment. Rats without CO2 treatment served as control. Radiographic, biomechanical and histological analysis was performed to assess the fracture repair. Gene expression of chondrogenic, hypertrophic, osteogenic and angiogenic markers was measured by real-time PCR at 1, 2, 3, and 4 weeks post-fracture. Results. Union rate, biomechanical properties, and gene expression of chondrogenic, hypertrophic, osteogenic and angiogenic markers was significantly higher in CO2 group compared to control group. Histological evaluation demonstrated that enchondral ossification was promoted in CO2 group. Discussion & Conclusions. Our study indicate that transcutaneous application of CO2 accelerates fracture repair via acceleration of endochondral ossification and vascularization, and may become a novel and useful therapy for promoting fracture repair

Clinical evidence that patients with type 2 diabetes mellitus (T2DM) have increased risk of fractures is reported. Furthermore, thiazolidinediones, used to treat T2DM increases the risk of secondary osteoporosis & subsequent fractures. The osteogenic potency of metformin is reported in vitro, few studies have investigated the effects of metformin on bone mass and fracture healing in vivo. We aimed to investigate the effects of metformin on fracture healing in vivo. Method. 20 female Wistar rats aged 3 months were randomly divided in two groups, one group receiving saline, the other group receiving metformin administered orally via the drinking water at a concentration of 2mg/ml. After 4 weeks of metformin treatment, a mid-diaphyseal, open External fixation fracture was performed. Rats were sacrified 4 weeks later. Right contralateral tibia and left osteotomised femora were excised, bone architecture analysed by micro-CT in the right tibia. Results. No significant differences were noted between the two groups. Fracture callus volume and mineral content after 4 weeks were similar in metformin and saline groups. Discussion Our results indicate that while metformin has no adverse effects on bone, it does not promote bone mass, as suggested by in vitro studies. This confirms clinical data which have not shown direct links between metformin and decreased fracture risk

Introduction. Failures in fracture healing are mainly caused by a lack of neovascularization. We have previously demonstrated that G-CSF-mobilized peripheral blood (GM-PB) CD34+ cells, an endothelial progenitor enriched cell population, contributed to fracture healing via vasculogenesis and osteogenesis. We postulated the hypothesis that local transplantation of culture expanded bone marrow (cEx-BM) CD34+ cells could exhibit therapeutic potential for fracture healing. Materials. BM CD34+ cells were cultured in specific medium with 5 growth factors for 1week. A reproducible model of femoral fracture was created in nude rats with periosteum cauterization, which leads to nonunion at 8 weeks post-fracture. Rats received local administration of the following cells or PBS alone(1)cEx-BM, (2)BM, (3)GM-PB CD34+ cells or (4)PBS. Results. Our 7-day culture expansion technique allowed us to obtain 23 times of BM CD34+ cells maintaining 60% purity of CD34 positivity. cEx-BM CD34+ cells exhibited striking therapeutic efficacy for unhealing fracture promoting neovascularization and osteogenesis in sites of fracture. Moreover, cEx-BM CD34+ cells showed high capacity of colony formation and osteogenic differentiation. Conclusion. BM CD34+ cells can be obtained from the fracture site at the time of primary operation and stored for further use, autologous culture expanded BM CD34+ cell transplantation therapy would be not only a simple but also powerful therapeutic strategy for unhealing fracture

Common cell based strategies for treating bone defects require time-consuming and expensive isolation and expansion of autologous cells. We developed a novel expedited technology creating gene activated muscle grafts. We hypothesized that BMP-2 activated muscle grafts provide healing capabilities comparable to autologous bone grafting, the clinical gold standard. Two male, syngeneic Fischer 344 rats served as tissue donors. Muscle tissue was harvested from hind limbs and incubated with an adenoviral vector carrying the cDNA encoding BMP-2. Bone tissue was harvested from the iliac crest. Segmental bone defects were created in the right femora of 12 rats and were filled with either BMP-2 activated muscle tissue or bone grafts. After 8 weeks, femora were evaluated by radiographs, microCT, and biomechanical tests. BMP-2 activated muscle grafts and autologous bone grafts resulted in complete mineralization and healing, as documented by radiographs and microCT. Bone volume in the muscle graft defects (33+/-12mm3) was similar to autologous bone graft defects (39+/-5mm3). Torque at failure of the two groups was statistically indistinguishable (240+/-115 Nmm vs. 232+/-108Nmm). In previous experiments we demonstrated that the large segmental defect model in this study will not heal with either empty defects or non-activated muscle grafts. Our findings therefore demonstrate that BMP-2 gene activation of muscle tissue effectively stimulates defect healing similar to autologous bone grafts

Introduction. Nonunion is a common and costly fracture outcome. Intricate reciprocity between angiogenesis and osteogenesis means vascular cell-based therapy offers a novel approach to stimulating bone regeneration. Hypothesis. The current study compared early and late outgrowth endothelial progenitor cell subtypes (EPCs vs OECs) for fracture healing potential in vitro and in vivo. Methods. Primary cell cultures were isolated and characterized by endothelial assays, immunosorbent assays, and multi-color flow cytometry. Co-cultures of EPC subtypes with/without primary osteoblasts (pObs) were analyzed for tube length and connectivity. In vivo, EPCs or OECs (1×10. 6. ) seeded on a gelfoam scaffold were implanted in a rat model of nonunion. Radiography was used to monitor callus formation. Results. OECs expressed more BMP-2 and less VEGF than EPCs (p<0.05). Analysis of surface markers showed decreased CD34+/CD133+/Flk-1+, CD133+ and CD45+ populations in OECs while CD34+/CD31+/Flk-1+ cells increased. pObs significantly inhibited the strong tubulogenesis of OECs while enhancing connectivity and sprout length of EPCs. In vivo, 0/6 scaffold-control and 1/5 OEC rats achieved union at 10 weeks. In comparison, all EPC rats achieved full or partial union. Discussion and Conclusion. Despite favorable tubulogenic and osteoconductive profiles of OECs, EPCs display enhanced fracture healing in vivo. Differences in CXCR4 expression and cell-mediated effects may contribute to this result

Introduction. Traumatized musculoskeletal tissue often exhibits prolonged time to healing, mostly due to low blood flow and innervation. Intermittent Pneumatic Compression (IPC) increases blood flow and decreases thromboembolic event after orthopedic surgery,[1] however little is known about healing effects.[2] We hypothesized that IPC could stimulate tissue repair: 1.) blood flow 2.) nerve ingrowth 3.) tissue proliferation and during immobilisation enhance 4.) biomechanical tissue properties. Methods. Study 1: In 104 male Sprague Dawley (SD) rats the right Achilles tendon was ruptured and the animals freely mobilized. Half the group received daily IPC-treatment, using a pump and cuff over the hindpaw that inflates/deflates cyclicly, 0–55mmHg (Biopress SystemTM, Flexcell Int.), and the other half received sham-treatment. Healing was assessed at 1,3,6 weeks by perfusion-analysis with laser doppler scanner (Perimed, Sweden), histology and biomechanical testing. Study 2: 48 male SD-rats were ruptured as above. Three groups of each 16 rats were either mobilized, immobilized or immobilized with IPC treatment. Immobilization was performed by plaster cast. Healing was assessed at 2 weeks with histology and biomechanical testing. Results. Study 1: At 3 and 6 weeks reperfusion increased by 21% and 23% (p< 0.05) after IPC-treatment, strengthened by the observation of elevated numbers of blood vessels and nerves. Fibroblast density was at all time points significantly increased in the IPC group. At three and six weeks the IPC treated tendons displayed an increased tissue organization confirmed by higher collagen I/III ratio in the IPC group. No differences (p = 0.10) were found regarding biomechanical strength. Study 2: Compared to mobilization, immobilization caused a downregulation (p<0.05) of all biomechanical and histological parameters, eg. maximum force decreased 80% and collagen III occurrence by 83%. However when immobilization was combined with IPC biomechanical and histological healing increased significantly compared to pure immobilization, eg. maximum force increased 63% and collagen III occurrence by 150%. Conclusion. This study demonstrated that IPC treatment can counteract biomechanical and morphological deficits caused by immobilization by enhancing proliferative soft tissue repair. Thus, IPC promotes tissue repair by stimulating tissue perfusion and nerve ingrowth as well as accelerating both fibroblast proliferation and collagen organization

Purpose. The purpose of this study was to evaluate the effects of implantation of mesenchymal stem cell derived condrogenic cells (MSC-DC) on bone healing in segmental defects in rat femur. Methods. Five-millimeter segmental bone defects were produced in the mid-shaft of the femur of Fisher 344 rats and stabilized with external fixator. The Treatment Group received MSC-DC, seeded on a PLGA scaffold, locally at the site of the bone defect, and Control Group received scaffold only. The healing processes were monitored radiographically (Softex), and studied radiographically (Micro-CT) and histologically. Results. All the bone defects in the Treatment Group healed radiographically with bridging callus formation at 4 weeks after the procedure, while none of the Control Group had achieved bone union. Micro-CT showed that newly formed bone volume in the Treatment Group at 16 weeks was 1.5 times that of unaffected side. Histological examination showed that the implanted scaffold of the Treatment Group were covered with periosteum-derived bridging callus and filled with cancellous bone-like tissue derived from enchondral ossification. Conclusion. The results of this study suggest that implantation of MSC-DC surprisingly enhances bone healing in segmental bone defects in rat much better than previously reported similar therapy using MSC

Introduction. what size of defect is optimal for creating an atrophic nonunion animal model has not been well defined. Our aim in this study was to establish a clinically relevant model of atrophic nonunion in rat femur by creation of a bone defect to research fracture healing and nonunion. Materials and methods. We used 30 male Fischer 344 rats (aged 10–11 weeks), which were equally divided into six groups. The segmental bone defects to a single femur in each rat were performed by double transverse osteotomy, and different sized defects were created by group for each group (1 mm, 2 mm, 3 mm, 4 mm, 5 mm and 6 mm). The defects were measured and maintained strictly by using an original external fixator. The periosteum for each defect was stripped both proximally and distally. Thereafter, these models were evaluated by radiology and histology. Radiographs were taken at baseline and at intervals of two weeks over a period of 8 weeks. Atrophic nonunion was defined as a lack of continuity and atrophy of both defect ends radiologically and histologically at eight weeks. Results. In the 1 mm defect group, all defects had healed. In the 2 mm group, one-fifth remained atrophic nonunion. In the 3 mm group, three-fifths had atrophic nonunion, and all of the defects of groups of 4 mm and over were atrophic nonunion. Conclusion. This study showed that we were able to predictively produce an atrophic nonunion animal model by creating defects of at least 4 mm in the Fischer 344 rat femur

Introduction. Despite the routine use of irrigation, debridement and systemic antibiotics, there is a high incidence of infection in severe open fractures. The synergistic use of local and systemic antibiotics appreciably reduces infection rates although the time window within which this is effective is unknown. The aim was to determine if delaying treatment of wounds causes higher levels of infection. Methods. A defect was created in the femurs of 90 Sprague-Dawley rats and inoculated with 105CFUs Staphylococcus aureus. At 2, 6 and 24 hours following contamination, the defect was irrigated and debrided. The experimental groups had either vancomycin or tobramycin impregnated PMMA beads placed within the segmental defect. The controls received no further treatment. Two weeks after wound closure, the bacteria within the femur were quantified. Results. Delaying irrigation and debridement resulted in significantly more bacteria (p<0.01) within the control group (2 hr < 6 hr <24 hr). Both locally delivered tobramycin and vancomycin significantly reduced the bacteria (p<0.05) when administered at the earlier time points (2 and 6 hours). Locally-delivered antibiotics were ineffective when delivered at 24 hours. Conclusion. Delaying treatment of contaminated defects reduces its effectiveness to eradicate infection. This is presumably because of the biofilm formation by the bacteria. Biofilms begin to form within a couple of hours and are mature within 12 hours. Early treatment of the wound allows the surgeon to physically remove the bacteria or have antibiotics present before a mature biofilm protects the bacteria