Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging.

Fracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a non-critical-sized mouse femur defect model. After bridging of the defect, a loading group (n = 10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3 × /week) based on computed strain distribution in the mineralized callus using animal-specific real-time micro-finite element analysis with 2D/3D visualizations and strain histograms. Controls (n = 10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger formation of mineralized callus and higher degree of mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex (n = 1/group). Given these distinct local protein expression patterns induced by cyclic mechanical loading during callus remodelling, the femur defect loading model with individualized load application seems suitable to further understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

Reverse dynamisation: a modern perspective on Stephan Perren's strain theory.

The present review acknowledges the tremendous impact of Stephan Perren's strain theory, considered with respect to the earlier contributions of Roux and Pauwels. Then, it provides further insight by examining how the concept of reverse dynamisation extended Perren's theory within a modern context. A key factor of this more contemporary theory is that it introduces variable mechanical conditions at different time points during bone healing, opening the possibility of manipulating biology through mechanics to achieve the desired clinical outcome. The discussion focusses on the current state of the art and the most recent advances made towards optimising and accelerating bone regeneration, by actively controlling the mechanical environment as healing progresses. Reverse dynamisation utilises a very specific mechanical manipulation regimen, with conditions initially flexible to encourage and expedite early callus formation. Once callus has formed, the mechanical conditions are intentionally modified to create a rigid environment under which the soft callus is quickly converted to hard callus, bridging the fracture site and leading to a more rapid union. The relevant literature, principally animal studies, was surveyed to provide ample evidence in support of the effectiveness of reverse dynamisation. By providing a modern perspective on Stephan Perren's strain theory, reverse dynamisation perhaps holds the key to tipping the balance in favour of a more rapid and reliable union when treating acute fractures, osteotomies, non-unions and other circumstances where it is necessary to regenerate bone.

Skin characteristics associated with foot callus in people with diabetes: A cross-sectional study focused on desmocollin1 in corneocytes.

AIM: The purpose of this study was to investigate the degradation of desmocollin-1 (DSC1), a member of the desmosomal cadherin family in patients with diabetes, as well as the factors associated with the suppression of DSC1 degradation. METHODS: This cross-sectional study included 60 cases of foot callus involving 30 patients with diabetes (DM) and 30 matched volunteers without diabetes (non-DM). DSC1 degradation in samples from debrided calluses was analysed using western blotting. Skin hydration, a factor reported to suppress DSC1 degradation, was measured using a mobile moisture device. RESULTS: Full-length DSC1 (approximately 100 kDa) was detected in six participants only in the DM group, and no relationship was found between the suppression of DSC1 degradation and decreased skin hydration in the DM group. There was no significant difference in skin hydration values between the DM and non-DM groups. CONCLUSION: DSC1 degradation was suppressed in the DM group. There was no relationship between the suppression of DSC1 degradation and decreased skin hydration in the DM group. Current external force callus care would not be sufficient. This study highlights the need to develop novel callus care to enhance the degradation of DSC1.

Green Tea Catechin (-)-Epigallocatechin-3-Gallate (EGCG) Facilitates Fracture Healing.

Green tea drinking can ameliorate postmenopausal osteoporosis by increasing the bone mineral density. (-)-Epigallocatechin-3-gallate (EGCG), the abundant and active compound of tea catechin, was proven to be able to reduce bone loss and ameliorate microarchitecture in female ovariectomized rats. EGCG can also enhance the osteogenic differentiation of murine bone marrow mesenchymal stem cells and inhibit the osteoclastogenesis in RAW264.7 cells by modulation of the receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegrin (OPG) (RANK/RANKL/OPG) pathway. Our previous study also found that EGCG can promote bone defect healing in the distal femur partially via bone morphogenetic protein-2 (BMP-2). Considering the osteoinduction property of BMP-2, we hypothesized that EGCG could accelerate the bone healing process with an increased expression of BMP-2. In this manuscript, we studied whether the local use of EGCG can facilitate tibial fracture healing. Fifty-six 4-month-old rats were randomly assigned to two groups after being weight-matched: a control group with vehicle treatment (Ctrl) and a study group with 10 µmol/L, 40 µL, EGCG treatment (EGCG). Two days after the operation, the rats were treated daily with EGCG or vehicle by percutaneous local injection for 2 weeks. The application of EGCG enhanced callus formation by increasing the bone volume and subsequently improved the mechanical properties of the tibial bone, including the maximal load, break load, stiffness, and Young's modulus. The results of the histology and BMP-2 immunohistochemistry staining showed that EGCG treatment accelerated the bone matrix formation and produced a stronger expression of BMP-2. Taken together, this study for the first time demonstrated that local treatment of EGCG can accelerate the fracture healing process at least partly via BMP-2.

The effects of focal brain damage on fracture healing: An experimental rat study.

OBJECTIVES: This study aims to investigate whether the motor cortex (MC) or the somatosensory cortex (SC) is more active during the course of bone healing after traumatic brain injury (TBI). MATERIALS AND METHODS: Thirty-three male Wistar albino rats (age, 8 to 10 months; weighing, 250 to 300 g) were randomized into three groups as the control group, MC damage group and SC damage group. Two rats from each brain damage group were sacrificed to verify the locations of the cortical injuries. Callus formation, callus/diaphysis ratios, and serum alkaline phosphatase (ALP) levels were measured at one, three and six weeks. RESULTS: The increases in callus masses in the control, MC, and SC groups were statistically significantly different between one and three weeks (p<0.05). Although this increase in the MC and SC groups was significant compared to the control group at the end of one week, no statistically significant difference was found between the MC and SC groups (p>0.05). There was a statistically significant difference in callus/diaphysis ratio between control, MC and SC groups in favor of MC group only at one week (p<0.05). The increase in serum ALP levels at three weeks was statistically significantly different in the MC and SC groups compared to the control group and significantly higher in the MC group compared to the SC group (p<0.05). CONCLUSION: There is a possible relationship between enhanced fracture healing after TBI and damage in the MC. Motor cortex plays a more active role on fracture healing in TBI.

Lowering circulating apolipoprotein E levels improves aged bone fracture healing.

Age is a well-established risk factor for impaired bone fracture healing. Here, we identify a role for apolipoprotein E (ApoE) in age-associated impairment of bone fracture healing and osteoblast differentiation, and we investigate the mechanism by which ApoE alters these processes. We identified that, in both humans and mice, circulating ApoE levels increase with age. We assessed bone healing in WT and ApoE-/- mice after performing tibial fracture surgery: bone deposition was higher within fracture calluses from ApoE-/- mice. In vitro recombinant ApoE (rApoE) treatment of differentiating osteoblasts decreased cellular differentiation and matrix mineralization. Moreover, this rApoE treatment decreased osteoblast glycolytic activity while increasing lipid uptake and fatty acid oxidation. Using parabiosis models, we determined that circulating ApoE plays a strong inhibitory role in bone repair. Using an adeno-associated virus-based siRNA system, we decreased circulating ApoE levels in 24-month-old mice and demonstrated that, as a result, fracture calluses from these aged mice displayed enhanced bone deposition and mechanical strength. Our results demonstrate that circulating ApoE as an aging factor inhibits bone fracture healing by altering osteoblast metabolism, thereby identifying ApoE as a new therapeutic target for improving bone repair in the elderly.

Development of a novel murine delayed secondary fracture healing in vivo model using periosteal cauterization.

INTRODUCTION: Delayed union and nonunion development remain a major clinical problematic complication during fracture healing, with partially unclear pathophysiology. Incidences range from 5 to 40% in high-risk patients, such as patients with periosteal damage. The periosteum is essential in adequate fracture healing, especially during soft callus formation. In this study, we hypothesize that inducing periosteal damage in a murine bone healing model will result in a novel delayed union model. MATERIALS AND METHODS: A mid-shaft femoral non-critically sized osteotomy was created in skeletally mature C57BL/6 mice and stabilized with a bridging plate. In half of the mice, a thin band of periosteum adjacent to the osteotomy was cauterized. Over 42 days of healing, radiographic, biomechanical, micro-computed tomography and histological analysis was performed to assess the degree of fracture healing. RESULTS: Analysis showed complete secondary fracture healing in the control group without periosteal injury. Whereas the periosteal injury group demonstrated less than half as much maximum callus volume (p < 0.05) and bridging, recovery of stiffness and temporal expression of callus growth and remodelling was delayed by 7-15 days. CONCLUSION: This paper introduces a novel mouse model of delayed union without a critically sized defect and with standardized biomechanical conditions, which enables further investigation into the molecular biological, biomechanical, and biochemical processes involved in (delayed) fracture healing and nonunion development. This model provides a continuum between normal fracture healing and the development of nonunions.

Distribution of the Highest Plantar Pressure Regions in Patients with Diabetes and Its Association with Peripheral Neuropathy, Gender, Age, and BMI: One Centre Study.

The abnormal plantar pressure distribution and value play a key role in the formation of plantar calluses and diabetic foot ulcer. The prevalence of the highest pressure different distribution and its association with various factors among patients with diabetes is not well known. The study purpose was to evaluate the prevalence of different regions for the highest pressure on the sole and its association with selected factors among patients with diabetes. Medical records of nonulcer patients were retrospectively analysed. The relationship between pressure patterns on the sole obtained during a pedobarographic test as a semiquantitative assessment with colourful print analysis and neuropathy, gender, age, and BMI was searched. The most common location of the highest pressure was the central part of the forefoot. No association was found between the different highest pressure regions and age, sensory neuropathy, calluses, and foot deformities. The highest pressure on the lateral part of the foot and midfoot was observed more often in females and in patients with a BMI ≥ 35. The prevalence of the highest pressure on the forefoot was more common in patients with a BMI < 35. Conclusions. The most frequent regions of the highest pressure on the sole in patients with diabetes were the central part of the forefoot (2-3 metatarsal heads) with no simple relationship to the assessed variables other than BMI < 35. Female gender and higher BMI seem to be responsible for shifting the place of the highest pressure to other places of the foot.

Establishment of a clinically relevant large animal model to assess the healing of metaphyseal bone.

Despite the high incidence of metaphyseal bone fractures in patients, the mechanisms underlying the healing processes are poorly understood due to the lack of suitable experimental animal models. Hence, the present study was conducted to establish and characterise a clinically relevant large-animal model for metaphyseal bone healing. Six female adult Merino sheep underwent full wedge-shaped osteotomy at the distal left femur metaphysis. The osteotomy was stabilised internally with a customised anatomical locking titanium plate that allowed immediate post-operative full-weight bearing. Bone healing was evaluated at 12 weeks post-fracture relative to the untouched right femur. Histological and quantitative micro-computed tomography results revealed an increased mineralised bone mass with a rich bone microarchitecture. New trabeculae healed by direct intramembranous ossification, without callus and cartilaginous tissue formation. Stiffness at the cortical and trabecular regions was comparable in both groups. Functional morphological analysis of the osteocyte lacunae revealed regularly arranged spherically shaped lacunae along with the canalicular network. Bone surface biochemical analysis using time-of-flight secondary-ion mass spectrometry showed high and homogeneously distributed levels of calcium and collagenous components. Ultrastructure imaging of the new trabeculae revealed a characteristic parallel arrangement of the collagen fibrils, evenly mineralised by the dense mineral substance. The specialised bone cells were also characterised by their unique structural features. Bone remodelling in the fractured femur was evident in the higher expression levels of prominent bone formation and resorption genes. In conclusion, the novel metaphyseal fracture model is beneficial for studying healing and treatment options for the enhancement of metaphyseal bone defects.

Prediction of Callus Subsidence in Distraction Osteogenesis Using Callus Formation Scoring System: Preliminary Study.

OBJECTIVE: To develop the scoring system which describes both quality and quantity of callus formation to predict the callus subsidence. METHODS: Forty-seven bony segments with an average lengthening of 5.17 ± 2.83 cm were included. The score was calculated based on the amount of callus classified in five patterns and the summation with the density of the callus classified in four patterns; the total score was 9. Bony subsidence >10% or >10° angulation were considered significant. We analyzed all of the data to find the most appropriate score that would prevent callus subsidence <10% and prevented angulation of the regeneration bone <10 degrees. Data was analyzed by using the receiver operating characteristic (ROC) curve. An area under the curve of 0.9-1 indicated an excellent test, 0.8-0.9 indicated a good test, 0.7-0.8 indicated a fair test, 0.6-0.7 indicated a poor test, and 0.5-0.6 indicated a fail test. The appropriate score for Ilizarov removal was selected from the highest sensitivity and specificity. RESULTS: Twenty-two tibia segments and 25 femur segments were included. The mean of bone lengthening was 5.17 ± 2.83 cm (range, 1.6-13.5 cm) and the mean of percentage lengthening was 16.58% ± 10.03% (range, 4.63%-56.84%). The mean distraction period was 5 months. The average months of follow-up for measurement of bony subsidence was 4.2 months. Mean subsidence was 21.06% (1.54%-57.44%). The mean of callus subsidence was 1.29 ± 1.17 cm (range, 0.03-4.72 cm). There were 32 segments (68%) with callus subsidence greater than 10% and 15 segments (32%) with subsidence less than 10%. The callus subsidence ranged from 0.3 mm to 4.72 cm, with 68% of bony fragments having significant subsidence. Type 5 callus diameter was statistically significant (P < 0.0001) in preventing callus subsidence compared to the other types. Type 4 callus density was statistically significant in preventing callus subsidence compared to the other types (P < 0.0001). The ROC curve with area under the curve 0.961 and sensitivity 0.933 showed that a callus scoring system score >7.5 was effective in preventing significant callus subsidence. When using score 8 as a result from the ROC curve, 73.3% of bony fragment subsidence was <10% with sensitivity 93.3 and specificity 83.2. CONCLUSION: Callus diameter 81%-100% and callus density type 4 could prevent significant callus subsidence. Based on the results of the present study we suggest using callus score > 8 to determine the time of Ilizarov removal.

Determining the Patient-Specific Optimum Osteotomy Line for Severe Mandibular Retrognathia Patients.

PURPOSE: The purpose of this study is to suggest a patient-specific osteotomy line to optimize the distractor position and thus to minimize the disadvantages of conventional mandibular distraction osteogenesis (MDO) protocols. In addition, this study also aims to compare the conventional MDO protocols with the new MDO protocol proposed in this study in terms of both orthodontic outcomes and mechanical effects of osteotomy level on callus stabilization by means of the finite element method. METHODS: A preoperative patient-specific 3-dimensional bone model was created and segmented by using computed tomography images of an individual patient. Virtual orthodontic set-up was applied to the segmented model prior to the virtual surgery. In order to compare the proposed osteotomy line with the conventional lines used in clinical applications, virtual surgery simulations were performed and callus tissues were modelled for each scenario. The comparison of the success of each osteotomy line was carried out based on the occlusion of the teeth. RESULTS: The osteotomy line determined using the method proposed in this study has resulted in far less malocclusion than the conventional method. Namely, any angular deviation from the optimum osteotomy line determined in this study might result in deep-bite or open-bite. On the other hand, the finite element analysis results have indicated that this deviation also negatively affects the callus stability. CONCLUSION: In order to achieve a better MDO treatment in terms of occlusion of the teeth and the callus stability, the location of the osteotomy line and the distractor position can be computationally determined. The results suggest that MDO protocol developed in this study might be used in clinic to achieve a better outcome from the MDO treatment.

Splenectomy delays fracture healing by affecting the level of tumor necrosis factor alpha, interleukin 6 and bone morphogenetic protein.

BACKGROUND: Abdominal injuries combined with bone fractures are increasing. Splenectomies are often required, but have prolonged healing time for bone fracture. OBJECTIVES: The aim of the study was to explore the molecular mechanism for splenectomy delaying fracture healing. MATERIAL AND METHODS: Eighty-four patients (42 received splenectomy) who received hip fractures operations were recruited in our hospital. One-year follow-up analysis was performed. To ensure the results, an animal model was established. Sprague-Dawley (SD) rats were randomly divided into 5 groups: group A: experimental group, femoral fractures + splenectomy; group B: femoral fractures; group C: splenectomy; group D: femoral fracture + sham splenectomy; group E: sham fracture. After the femoral fracture surgery, the callus status was evaluated by X-ray. RESULTS: After 1-year follow-up, the healing index and bone quality was higher in the fracture-operatedonly group than in the splenectomy group. In contrast, the rate of healing complications was lower in the fracture-operated-only group than in the splenectomy group. Biomarker analysis showed that the serum levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and bone morphogenetic protein (BMP) were higher in the fracture-operated-only group than in the splenectomy group. No difference of the callus status was found among the rats in groups B, D and E (p > 0.05), while there were significant differences of the callus status of the rats in groups A and C at different stages (p < 0.05). On the other hand, the levels of TNF-α, IL-6 and BMP increased, reached peak after 7-day splenectomy surgery, and then decreased significantly in groups A and C (p > 0.05). CONCLUSIONS: Splenectomy delays fracture healing by affecting the levels of TNF-α, IL-6 and BMP.

Using 3D finite element models verified the importance of callus material and microstructure in biomechanics restoration during bone defect repair.

BACKGROUND: There is lack of further observations on the microstructure and material property of callus during bone defect healing and the relationships between callus properties and the mechanical strength. METHODS: Femur bone defect model was created in rabbits and harvested CT data to reconstruct finite element models at 1 and 2 months. Three types of assumed finite element models were compared to study the callus properties, which assumed the material elastic property as heterogeneous (R-model), homogenous (H-model) or did not change from 1 to 2 months (U-model). RESULTS:  The apparent elastic moduli increased at 2 months (from 355.58 ± 132.67 to 1139.30 ± 967.43 MPa) in R-models. But there was no significant difference in apparent elastic moduli between R-models (355.58 ± 132.67 and 1139.30 ± 967.43 MPa) and H-models (344.79 ± 138.73 and 1001.52 ± 692.12 MPa) in 1 and 2 months. A significant difference of apparent elastic moduli was found between the R-model (1139.30 ± 967.43 MPa) and U-model group (207.15 ± 64.60 MPa) in 2 months. CONCLUSIONS: This study showed that the callus structure stability remodeled overtime to achieve a more effective structure, while the material quality of callus tissue is a very important factor for callus strength. At the meantime, this study showed an evidence that the material heterogeneity maybe not as important as it is in bone fracture model.

Severe Hemorrhagic Shock Leads to a Delayed Fracture Healing and Decreased Bone Callus Strength in a Mouse Model.

BACKGROUND: Multiple trauma is frequently associated with hemorrhagic shock and fractures of the extremities. Clinically, the rate of impaired fracture healing (delayed healing and nonunion) seems to be increased in patients with multiple injuries compared with patients with isolated fractures. As the underlying pathogenesis remains poorly understood, we aimed to analyze the biomechanical properties during fracture healing in a murine model. QUESTIONS: The aim of this study was to determine whether fracture healing after severe hemorrhagic shock results in (1) delayed bridging as determined by macroscopic and radiographic assessment, (2) altered conditions of callus components as determined by µCT, and (3) decreased maximum bending moment measured by a three-point-bending test compared with ordinary fracture healing. METHODS: Male C57BL/6NCrl mice were randomly assigned to five groups and four different times (five to 10 mice per group and time). Only the right femur from each mouse was used for analysis: the trauma hemorrhage (TH) group received a pressure-controlled hemorrhagic shock via catheter; the osteotomy (Fx) group underwent osteotomy and implantation of an external fixator on the right femur; the combined trauma (THFx) group received hemorrhagic shock and an external fixator with osteotomy; the sham group underwent implantation of a catheter and external fixator but had no blood loss or osteotomy, and the control group underwent no interventions. After 2, 3, 4, or 6 weeks, five to 10 animals of each group were sacrificed. Bones were analyzed macroscopically and via radiographs, µCT, and three-point-bending test. Statistical significance was set at a probability less than 0.05. Comparisons were performed using the Mann-Whitney U or the Kruskal-Wallis test. RESULTS: In the Fx group, the osteotomy gap was stable and bridged after 2 weeks in contrast to some bones in the THFx group where stable bridging did not occur. No difference was observed between the groups. µCT analysis showed reduced density of bone including callus (THFx: 1.17 g/cm3; interquartile range [IQR], 0.04 g/cm3; Fx: 1.22 g/cm3; IQR, 0.04 g/cm3; p = 0.002; difference of medians [DM], -0.048; 95% CI, -0.073 to -0.029) and increased share of callus per volume of bone mass (%) after 2 weeks in the THFx group compared with the Fx group (THFx: 44.16%; IQR, 8.66%; Fx: 36.73%; IQR, 4.39%; p = 0.015; DM, 7.634; 95% CI, 2.018-10.577). The three-point-bending test established a decreased maximum bending moment in the THFx group compared with the Fx group 2 weeks after surgery (THFx: 7.10 Nmm; IQR, 11.25 Nmm; Fx: 11.25 Nmm; IQR, 5.70 Nmm; p = 0.026; DM, -5.043; 95% CI, -10.867 to -0.74). No differences were observed between the THFx and Fx groups after more than 2 weeks. CONCLUSION: In this in vivo mouse fracture model, we conclude that hemorrhagic shock retards fracture healing during the early phase of the facture healing process. CLINICAL RELEVANCE: A severe hemorrhagic shock in patients could result in initial delayed fracture healing and needs special attention. We plan to conduct a prospective, observational clinical research study to analyze if delayed fracture healing occurs in patients after severe blood loss.

[Basic principles of fracture healing]. / Grundlagen der Knochenbruchheilung.

BACKGROUND: In contrast to other tissues, bone has the remarkable ability to heal without scarring. After union of the fracture, the remodeled bone ideally does not differ from the original bone, especially in terms of biomechanical properties. The healing of a fracture resembles the embryonic development of bone. Depending on the biomechanical properties of the fracture, bone heals directly or indirectly, which refers to the formation of cartilage as a step before new bone appears. Currently, treatment of the patient is often limited to anatomical reduction and optimization of the fracture environment with respect to biomechanics. PROSPECTS: Future treatment strategies, however, could include systemic medication that could be especially beneficial for patients at risk of complications in fracture healing. The aim of this review is to provide an overview on the process of fracture healing and to depict possibilities for current and future treatment strategies.

The effect of a combined thoracic and soft-tissue trauma on blood flow and tissue formation in fracture healing in rats.

INTRODUCTION: Previously, it was found that fracture healing is impaired by blunt chest trauma and an additional soft-tissue trauma. The mechanisms leading to this disturbance are largely unknown. Here, we investigated the effect of thoracic and soft-tissue trauma on blood flow of the injured lower leg and on tissue differentiation and callus formation during fracture healing. MATERIALS AND METHODS: Male Wistar rats received either a mid-shaft fracture of the tibia alone (group A), an additional chest trauma (group B), or additional chest and soft-tissue traumas (group C). Peripheral blood flow was determined by Laser Doppler Flowmetry before and after the injury, and on observation days 1, 3, 7, 14, and 28. Quantitative histological analysis was performed to assess callus size and composition. RESULTS: All groups displayed an initial decrease in blood flow during the first 3 days post-trauma. A recovery of the blood flow that even exceeded preoperative levels occurred in group A and later and to a lesser degree in group B, but not in group C. The amount of callus formation decreased with increasing trauma load. More cartilage was formed after 7 days in groups B and C than in group A. At later healing time points, callus composition did not differ significantly. CONCLUSIONS: An increasing injury burden causes a decreasing blood supply capacity and revascularization, and leads to impaired callus formation and an increasing delay in bone healing.

Effects of local vibration and pulsed electromagnetic field on bone fracture: A comparative study.

The effectiveness of various therapeutic methods on bone fracture has been demonstrated in several studies. In the present study, we tried to evaluate the effect of local low-magnitude, high-frequency vibration (LMHFV) on rat tibia fracture in comparison with pulsed electromagnetic fields (PEMF) during the healing process. Mid-diaphysis tibiae fractures were induced in 30 Sprague-Dawley rats. The rats were assigned into groups such as control (CONT), LMHFV (15 min/day, 7 days/week), and PEMF (3.5 h/day, 7 days/week) for a three-week treatment. Nothing was applied to control group. Radiographs, serum osteocalcin levels, and stereological bone analyses of the three groups were compared. The X-rays of tibiae were taken 21 days after the end of the healing process. PEMF and LMHFV groups had more callus formation when compared to CONT group; however, the difference was not statistically significant (P = 0.375). Serum osteocalcin levels were elevated in the experimental groups compared to CONT (P ≤ 0.001). Stereological tests also showed higher osteogenic results in experimental groups, especially in LMHFV group. The results of the present study suggest that application of direct local LMHFV on fracture has promoted bone formation, showing great potential in improving fracture outcome. Bioelectromagnetics. 38:339-348, 2017. © 2017 Wiley Periodicals, Inc.

Exposure to Secondhand Smoke Impairs Fracture Healing in Rats.

BACKGROUND: Nonsmokers may be affected by environmental tobacco smoke (secondhand smoke), but the effects of such exposure on fracture healing have not been well studied. QUESTIONS/PURPOSES: To explore the possible effects of passive inhalation of tobacco smoke on the healing of a diaphyseal fracture in femurs of rats. We hypothesized that secondhand exposure to tobacco smoke adversely affects fracture healing. METHODS: A mid-diaphyseal fracture was created in the femur of 41 female Wistar rats and fixed with an intramedullary metallic pin; 14 rats were excluded (nine for inadequate fractures and five for K wire extrusion). Tobacco exposure was provided by a smoking machine on a daily basis of four cigarettes a day. Each cigarette yielded 10 mg tar and 0.8 mg nicotine, and was puffed by alternating injections of fresh air for 30 seconds and smoke air for 15 seconds. The smoke exposure was previously adjusted to provide serum levels of cotinine similar to human secondhand tobacco exposure. Cotinine is a predominant catabolite of nicotine that is used as a biological biomarker for exposure to tobacco smoke. In one group (n = 11), the animals were intermittently exposed to tobacco smoke before sustaining the fracture but not afterward. In another group (n = 7), the exposure occurred before and after the fracture. The control group (n = 9) was sham-exposed before and after the fracture. We evaluated the specimens 28 days after bone fracture. The callus quality was measured by dual-energy x-ray absorptiometry (bone mineral density [BMD], bone mineral content [BMC], and callus area), µCT (callus volume and woven bone fraction), and mechanical bending (maximum force and stiffness). RESULTS: Tobacco exposure resulted in delayed bone callus formation, which is represented by decreased BMD (Control: 0.302 ± 0.008 g/cm2 vs Preexposed: 0.199 ± 0.008 g/cm2 and Pre- and Postexposed: 0.146 ± 0.009 g/cm2; mean difference = 0.103 g/cm2, 95% CI, 0.094-0.112 g/cm2and mean difference = 0.156 g/cm2, 95% CI, 0.147-0.167 g/cm2; p < 0.01), BMC (Control: 0.133 ± 0.005 g vs Preexposed: 0.085 ± 0.0034 g and Pre- and Postexposed: 0.048 ± 0.003 g; mean difference = 0.048 g, 95% CI, 0.045-0.052 g and mean difference = 0.085 g, 95% CI, 0.088-0.090 g; p < 0.01), callus volume (Control: 7.656 ± 1.963 mm3 vs Preexposed: 17.952 ± 1.600 mm3 and Pre- and Postexposed: 40.410 ± 3.340 mm3; mean difference = -10.30 mm3, 95% CI, -14.12 to 6.471 mm3 and mean difference, -32.75 mm3, 95% CI, -36.58 to 28.93 mm3; p < 0.01), woven bone fraction (Control: 42.076 ± 3.877% vs Preexposed: 16.655 ± 3.021% and Pre- and Postexposed: 8.015 ± 1.565%, mean difference = 0.103%, 95% CI, 0.094-0.112% and mean difference = 0.156%, 95% CI, 0.147-0.166%; p < 0.01), maximum force (Control: 427.122 ± 63.952 N.mm vs Preexposed: 149.230 ± 67.189 N.mm and Pre- and Postexposed: 123.130 ± 38.206 N.mm, mean difference = 277.9 N.mm, 95% CI, 201.1-354.7 N.mm and mean difference = 304 N.mm, 95% CI, 213.2-394.8 N.mm; p < 0.01) and stiffness (Control: 491.397 ± 96.444 N.mm/mm vs Preexposed: 73.157 ± 36.511 N.mm/mm and Pre- and Postexposed: 154.049 ± 134.939 N.mm/mm, mean difference = 418.2 N.mm/mm, 95% CI, 306.3-530.1 N.mm/mm and mean difference = 337.3 N.mm/mm, 95% CI, 188.8-485.9 N.mm/mm; p < 0. 01). CONCLUSIONS: Rats exposed to tobacco smoke showed delayed fracture healing and callus that was characterized by decreased maturity, density, and mechanical resistance, which was confirmed by all assessment methods of this study. Such effects were more evident when animals were exposed to tobacco smoke before and after the fracture. Future studies should be done in human passive smokers to confirm or refute our findings on fracture callus formation. CLINICAL RELEVANCE: The potential hazardous effects of secondhand smoke on fracture healing in rodents should stimulate future clinical studies in human passive smokers.

Radiological features of healing in newborn clavicular fractures.

OBJECTIVES: Given the frequency of abusive fractures among infants, and the lack of research and or evidence for the phases of fracture healing seen in this age group, this study aims to describe a timetable of radiological features of fracture healing among infants in the first months of life. METHODS: We completed a retrospective cross-sectional time-series study of birth-related clavicle fractures from 2006-2013. A total of 108 digital images were available for review from 61 infants. The presence or absence of four features of healing including periosteal reaction, callus formation, bridging callus and remodelling were scored by three radiologists. RESULTS: The level of agreement between the radiologists was good to high (0.60-0.90). Features of healing were first seen at 7 days (periosteal reaction), 11 days (callus), 20 days (bridging) and 35 days (remodelling), respectively. The peak periods that each feature was present are as follows: periosteal reaction 11-42 days, callus 12-61 days, bridging 22-63 days and remodelling 49-59 days. CONCLUSIONS: Birth-associated clavicle fractures in infants follow a logical progression of healing changes. Understanding the expected progression and timing of fracture healing may be helpful as it pertains to the timing of injury in cases of abuse in infants. KEY POINTS: • Large study describing the time frames of fracture healing in young infants. • Features of fracture healing develop in a logical progression. • Evidence provided for determining fractures are consistent with a proposed time frame. • It is of critical importance to have sound evidence for the dating of fractures.