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.

Deferoxamine restores callus size, mineralization, and mechanical strength in fracture healing after radiotherapy.

BACKGROUND: Therapeutic augmentation of fracture-site angiogenesis with deferoxamine has proven to increase vascularity, callus size, and mineralization in long-bone fracture models. The authors posit that the addition of deferoxamine would enhance pathologic fracture healing in the setting of radiotherapy in a model where nonunions are the most common outcome. METHODS: Thirty-five Sprague-Dawley rats were divided into three groups. Fracture, irradiated fracture, and irradiated fracture plus deferoxamine. The irradiated fracture and irradiated fracture plus deferoxamine groups received a human equivalent dose of radiotherapy [7 Gy/day for 5 days, (35 Gy)] 2 weeks before mandibular osteotomy and external fixation. The irradiated fracture plus deferoxamine group received injections of deferoxamine into the fracture callus after surgery. After a 40-day healing period, mandibles were dissected, clinically assessed for bony union, imaged with micro-computed tomography, and tension tested to failure. RESULTS: Compared with irradiated fractures, metrics of callus size, mineralization, and strength in deferoxamine-treated mandibles were significantly increased. These metrics were restored to a level demonstrating no statistical difference from control fractures. In addition, the authors observed an increased rate of achieving bony unions in the irradiated fracture plus deferoxamine-treated group when compared with irradiated fracture (67 percent and 20 percent, respectively). CONCLUSIONS: The authors' data demonstrate nearly total restoration of callus size, mineralization, and biomechanical strength, and a threefold increase in the rate of union with the use of deferoxamine. The authors' results suggest that the administration of deferoxamine may have the potential for clinical translation as a new treatment paradigm for radiation-induced pathologic fractures.

Irradiation effect on osteoclastogenesis stimulated by breast cancer cells.

To examine the effects of carbon ion and gamma ray irradiation on cancer-induced osteoclastogenesis, mouse calvaria MC3T3-E1 cells were cultured with conditioned medium from irradiated and non-irradiated MCF7 human breast cancer cells. The authors examined RANKL and OPG mRNA expression in osteoblastic MC3T3-E1 cells following treatment with conditioned MCF7 medium. Co-cultured MC3T3-E1 and bone marrow cells treated with conditioned medium from irradiated MCF7 cells showed decreased numbers of osteoclasts, assessed using TRAP staining. Conditioned medium from control MCF7 cells elevated the RANKL/OPG mRNA ratio in MC3T3-E1 cells; this effect was suppressed by carbon ion irradiation of the MCF7 cells. These data demonstrate that indirect interactions between breast cancer cells and MC3T3-E1 cells induce osteoclastogenesis in vitro through modulation of RANKL expression and that this process is suppressed by carbon ion irradiation.

Effect of low-level laser therapy on the fracture healing process.

Low-level laser therapy (LLLT) is a biophysical form of intervention in the fracture-repair process, which, through several mechanisms, accelerates the healing of fractures and enhances callus formation. The effect of laser on fracture healing is controversial. Some authors affirm that LLLT can accelerate bone formation by increasing osteoblastic activity. The objective of our study was to evaluate the effect of laser therapy on fracture healing. Thirty rabbits were subjected to tibial bone open osteotomies that were stabilized with external fixators. The animals were divided into two study groups: laser group and control group. Callus development and bone mineral density were quantitatively evaluated by CT; the animals were then killed and the fractures were assessed for biomechanical properties. The results demonstrated that the increasing rate of bone mineral density was higher in the laser (L) group than in the control (C) group. CT at 5 weeks revealed a mean callus density of 297 Hounsfield units (HU) for the control group and 691 HU for the L group, which was statistically significant (P = 0.001). In the L group, the mean recorded fracture tension was 190.5 N and 359.3 N for healed and intact bones, respectively, which was statistically significant (P < 0.001). The result of the study showed that the use of laser could enhance callus development in the early stage of the healing process, with doubtful improvement in biomechanical properties of the healing bone; therefore, laser therapy may be recommended as an additional treatment in non-union fractures in humans.

[Effect of low-intensity pulsed ultrasound stimulation on maturation of regenerate bone].

OBJECTIVE: To explore the effect of low intensity pulsed ultrasound stimulation (LIPUS) on the maturation of regenerate bone in a rabbit limb lengthening model. METHODS: Sixty skeletal mature female New Zealand white rabbits were randomly divided into an LIPUS treatment group and a control group. All rabbits were underwent mid-diaphyseal tibial osteotomy and immobilized in an Orthofix M103 Mini lengther. Gradual distraction at 0.5 mm every 12 h for 10 d was performed at day 7 postoperatively. A 4-week course of LIPUS treatment group was applied over the distraction site for 20 min daily starting immediately after the completion of the distraction only for the treatment group. Rabbits were euthanized and the mid-diaphyseal tibia was harvested for evaluation at 4, 8, and 12 wk after the completion of the bone lengthening protocol. Radiographic analysis was performed to study the formation of bone callus using the ImageJ software at 12 wk after the completion of the bone lengthening protocol. Bone mineral density (BMD) of regenerate bone was measured by Dual energy X-ray absorptiometry (DEXA). Torsional testing to failure was performed on the tibia specimens at 8 and 12 wk after the completion of the bone lengthening protocol. RESULTS: Radiographic measurement showed higher relative gray scale of bone callus in the LIPUS group than that in the control group at 12 wk (P<0.05). BMD in the LIPUS group was significantly higher than that in control group at 8 and 12 wk (P<0.05). Biomechanical testing showed that the ultimate torque, ultimate torsional stiffness, and energy absorption at failure of regenerated bone at 8 and 12 wk in the LIPUS treatment group were better than those in the control group (P<0.05). CONCLUSION: LIPUS as a biophysical stimulation may accelerate the formation and maturation of regenerate bone in rabbit tibia lengthening model.

[Low dose X-irradiation promotes callus mineralization: experiment with rats].

OBJECTIVE: To investigate the effect of low dose irradiation (LDI) on callus formation and mineralization. METHODS: 80 SD rats were subjected to standard closed fracture on right femur so as to establish animal models and then were randomly divided into 2 equal groups: LDI group undergoing low dose whole body X-ray irradiation of 1Gy right after fracture induction and control group without irradiation. 1, 2, 3, 4, and 8 weeks later blood samples were collected from the heart to examine the amounts of white blood cells and platelets and alkaline phosphatase (ALP) level. Conventional X-ray films were taken and MicroCT was conducted to evaluate the callus bridging. Then the rats were killed to take out the femurs to undergo histological examination. Peripheral quantitative computed tomography (pQCT) was conducted to quantify the bone mineral content (BMC). Four-point bending test was used to examine the mechanical properties of the callus. RESULTS: The maximum load value and histological score 2 weeks later of the LDI group were both lower than those of the control group, however, the serum ALP, BMC, maximum load level, callus bridging score, and histological score 3 weeks later of the LDI group were all significantly higher than those of the control groups (all P<0.05). 4 and 8 weeks later there were not significant differences in all these parameters between the 2 groups. The WBC amount in the first 2 weeks of experiment of the LDI group was remarkably lower in these 2 groups, however, in the third week, the WBC value of the LDI group was higher than that of the control group (P<0.05). CONCLUSION: LDI promotes the mineralization at the stage of hard callus formation which is probably associated with progenitor cell mobilization.

Low-dose X-irradiation promotes mineralization of fracture callus in a rat model.

OBJECTIVES: This study investigated the hypothesized beneficial effect of low-dose irradiation (LDI) on fracture callus mineralization in a rat model. METHODS: Seventy-two male Sprague-Dawley rats were averagely randomized into LDI group (rats treated with LDI) and SHAM group (rats treated with sham irradiation). Right after either LDI or sham irradiation, a standardized closed fracture on the right femur was established. At 2, 3 and 4 weeks postfracture, 12 rats in each group were euthanized. Fracture callus was assessed by using radiography and MicroCT for callus bridging, peripheral quantitative computed tomography (pQCT) for quantifying bone mineral content (BMC) and cross sectional area (CSA), confocal laser scanning microscopy for measuring area fraction of fluorescence labeling (AFFL) and four-point bending test for examining mechanical properties. RESULTS: The CSA and AFFL were found to be 22 and 33% smaller in the LDI group compared to the SHAM group at 2 weeks (P<0.05 for both), whereas the BMC and AFFL were 15 and 34% higher in the LDI group at 3 weeks (P<0.05 for both). The changing patterns were consistent with the findings in 3-D MicroCT reconstructions. The mechanical parameters (Max-Load, Stiffness and Energy) were also 18, 30 and 24% higher in the LDI group than in the SHAM group at 3 weeks (P<0.05 for all). At 4 weeks, there was no difference found for all assessments between the two groups. CONCLUSION: The results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model.

Low-intensity pulsed ultrasound does not enhance distraction callus in a rabbit model.

Low-intensity pulsed ultrasound has been reported to have a positive effect when applied during the consolidation phase of distraction osteogenesis and bone transportation, but the optimal application time has not been determined. We used a rabbit model to determine whether low-intensity pulsed ultrasound applied during the distraction and early consolidation phases of tibial lengthening would have a positive effect on regenerated bone formation. Radiographic analysis showed no differences in regenerated callus area or in percent of callus mineralization between treated and control tibias immediately after distraction or at 1, 2, or 3 weeks after distraction. Similarly, we observed no differences in structural stiffness or maximal torque to failure at 1.5 or 3 weeks after distraction. We detected no differences in bone mineral appositional rates or percent tissue composition measured histologically between groups. Our data do not support the application of low-intensity pulsed ultrasound to regenerated bone during distraction osteogenesis.

Effect of extracorporeal shock waves on callus formation during bone lengthening.

The effects of extracorporeal shock waves (ESWs) on callus formation during bone lengthening were studied in 25 female Japanese white rabbits. Bone lengthening of 9.8 mm was obtained over 2 weeks using the Orthofix M-100 bone fixator. ESWs were applied 3 weeks after surgery. Pins were removed 7 weeks after surgery, and specimens were prepared after the animals were killed at 9 and 24 weeks. The shock wave setting used was 0.42 mJ/mm(2) with a pulse interval of 2 Hz; 3000 shots each were applied to the central and peripheral areas. The specimens were evaluated using radiography, bone mineral density (BMD) measured by dual energy absorptiometry (DXA), and a three-point bending test to evaluate mechanical strength. Histological examination was performed on the lengthened portion. Radiographs and histological observations revealed no apparent fractures in nonlengthening tibias at the shock wave energy densities used. Radiographic observations revealed no apparent differences between the control group and the ESW group. BMD measurements by DXA revealed significantly increased bone mass in the ESW group 9 weeks after surgery. At 24 weeks after surgery the mean BMD had decreased to 25% and 15% of the values at 9 weeks in the control and ESW groups, respectively. The three-point bending test revealed no significant differences between the groups. Histological observations revealed significant capillary formation and osteoblasts and chondrocytes in the bone marrow as well as bridging of newly formed trabeculae 2 weeks after the bone was lengthened. At 4 weeks after treatment, observations included parts of the lengthened portion with no cortex or immature bone. At 9 weeks after surgery, cortex formation and a normal medullary cavity were clearly observed in the control group, whereas observations in the treated group included areas of the lengthened portion with no cortex and formation of immature trabecular structures and increased cancellous bone in the center of the lengthened portion. At 24 weeks after surgery, more prominent cortex formation and fatty marrow were observed in the ESW group than in the control group.

The effect of extracorporeal shock wave treatment (ESWT) on bone defects. An experimental study.

The aim of this study was to investigate the effects of extracorporeal shock wave therapy (ESWT) on the formation of callus in bone defects created in rabbit radii. this study searches for an answer to whether ESWT may have a therapeutic effect on bone defects. A bone defect with a radius of 1 cm was created in both forelimbs of 20 rabbits. At the 7th, 14th, and 21st days ESWT treatment was applied to the forming callus in the right radius under fluoroscopic control. At the 6th and 12th weeks, the animals were sacrificed and callus analysis was performed by computerized scan, dual energy x-ray absorptiometer. Histological analyses were also performed. The results revealed that the average callus area in the right (ESWT applied) radial defect was greater in both groups and statistically significant at the 12th week (p < 0.05). There was no difference in bone density between defects. Histologically the callus area was greater on the right side (ESWT applied side) in both groups. However in the first group trabeculae were occupying less space on the right side. Granulation tissue areas and chondroid areas were greater on the right side. We conclude that ESWT has a disorganizing and dispersing rather than a direct osteoinductive effect on forming callus. This effect may play a therapeutic role in bone defects and in situations like callus lengthening where a greater amount of callus is necessary.

Autogenous callo-osseous grafts for the repair of osteochondral defects.

We describe a new method of biological repair of osteochondral defects. In rabbit knees an osteochondral defect was reconstructed with a callo-osseous graft made of a superficial sheet of medullary fracture callus attached to a base of cancellous bone. This was taken from the iliac bone of the same animal which had been osteotomised ten days earlier. The reparative tissues were evaluated for 24 weeks by quantitative histology, biochemical analysis of the uronic acid content, and immunohistochemical staining of collagen constituents. The callo-osseous graft provided significantly faster and better repair of the articular surface than an untreated defect or a callo-osseous graft in which the cells had been devitalised by irradiation before transplantation. Our findings indicate that the callo-osseous graft contributes to the repair process by providing both favourable extracellular matrices and pluripotential mesenchymal cells. Our study tested the hypothesis that early medullary callus generates hyaline cartilage instead of bone after transfer to an articular surface.

Low-power electromagnetic stimulation of osteotomized rabbit fibulae. A randomized, blinded study.

The purpose of this study was to determine whether low-power-consuming symmetrical-waveform electromagnetic stimuli could increase the stiffness of fracture sites in a rabbit fibular-osteotomy model. Both active and placebo devices were used in a blinded study protocol. Dose-response studies of pulse amplitude and pulse width were performed by continuous application (twenty-four hours a day) of repetitive (fifteen-hertz), bursted (five-millisecond-long) symmetrical, rectangular electromagnetic stimulus waveforms. The power consumed by these stimuli is approximately one-fifth that consumed by the pulsing electromagnetic field devices that are in current clinical use. Significant increase of callus bending stiffness was produced by pulse widths of five to seven microseconds and pulse amplitudes of fifty to 100 millivolts.

Additive effects of prostaglandin E2 and pulsed electromagnetic fields on fracture healing.

Bone formation of fractured fibulae stimulated by pulsed electromagnetic fields (PEMF), PGE2, and combination of both was assessed with roentgenography and fluorescent labelling compounds, tetracycline, xylenol orange, and calcein. A total of 72 male New Zealand rabbits was osteotomized by creating a 1 mm-gap at fibulae and randomly divided into 8 groups: one control, one treated with PEMF, three treated with PGE2 of various dosages, and three treated with combined treatments of PEMF and PGE2 of specified dosages. PEMF had positive effects on bone formation. Exogenous PGE2 mimicked the effect of PEMF on linear bone growth. The effect of PGE2 on bone formation or bone remodelling was dose-related (5, 15, 50 micrograms/kg), with 5 micrograms/kg body weight as the optimal dosage in this study. Combination of PEMF and PGE2 exhibited a trend of additive effect on bone formation, especially at 15 micrograms/kg of PGE2. It is hypothesized that PEMF may exert its action on bone healing by increasing the endogenous PGE2. We therefore concluded that external stimulation such as PEMF and PGE2 was beneficial and stimulatory towards bone formation and healing in our animal model. However, the effects were somehow specific in electrical waveforms and dosage. Similar to PEMF, PGE2, therefore, may be a potential agent in promoting bone formation in the clinical treatments of fractures or perhaps non-union.

The effect of radiation on the fracture repair process. A biomechanical evaluation of a closed fracture in a rat model.

The effects of a single dose of irradiation on the biomechanical parameters of the fracture healing process were studied in a rat model. Intramedullary pinning was performed before production of a closed femoral midshaft fracture. The experimental group was exposed to 900 rad 3 days after fracture and was compared with a control group with a similar fracture that received no irradiation. Animals were killed at intervals ranging from 2-16 weeks after surgery and the bones were tested until failure in torsion. In the irradiated groups, a delay of 4 weeks was noted in the biomechanical parameters associated with fracture healing (torque to failure, torsional stiffness, angle to failure, and biomechanical stage). Despite this delay in the normal temporal progression, the staging and stiffness approached normal controls within an 8-week period. However, the torque to failure remained below normal levels at the conclusion of this study. These results differ from a previous study using an open fracture model.

Effect of CO2 laser irradiation on experimental fracture healing: a transmission electron microscopic study.

The healing of standardized fracture of rabbit radius was expedited as a result of treatment with low-power CO2 laser irradiation. Observation with transmission electron microscope revealed the following favorable effects of CO2 laser irradiation: The red blood cells were induced to disintegrate, thus promoting the absorption of the hematoma. The macrophages emerged early and increased in number so that debridement of the necrotic tissues was enhanced. The fibroblasts were more active in producing the fibrous callus. The chondrocytes were unusually active in forming bone tissues. The early and sustained appearance of osteoclasts favored the bone remodeling process. Increased capillary formation endowed the fracture healing with rich blood supply. Deposition of calcium salts took place early.