Callus Mineralization Research Articles

Relationship between Callus Growth and Mineral Nutrients Uptake in Salt-Stressed Indica Rice Callus

ABSTRACT One month old rice calli were exposed to 0, 50, and 100 mol m−3 sodium chloride (NaCl) in the liquid LS basal medium supplemented with 2.5 mg L−1 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L−1 kinetin. Callus relative growth rate (RGR; fresh) of both cultivars indicated a progressive decrease; however, callus dry weight increased as the NaCl level increased in the culture medium. Salinity stress increased the callus sodium (Na+), manganese (Mn2+), and magnesium (Mg2+) contents while potassium (K+), calcium (Ca2+), and iron (Fe2+) contents decreased. ‘Basmati-385’ showed less reduction in callus RGR, K+, and Ca2+ contents and a larger increase in callus dry weight, Na+, Mn2+, and Mg2+ contents as compared to ‘Basmati-Karnal’. However, the reverse was true for Fe2+ contents. K+/Na+ and Ca2+/Na+ ratios generally decreased under salt stress. Overall, reduction in callus relative growth rate was found to be inversely correlated with decrease in K+, Ca2+, and Fe2+ uptake and directly correlated with increased Na+ and Mg2+ concentration in callus tissue.

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

To investigate the effect of low dose irradiation (LDI) on callus formation and mineralization. 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. 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). LDI promotes the mineralization at the stage of hard callus formation which is probably associated with progenitor cell mobilization.

Quantitative assessment of mineralization in distraction osteogenesis

The most important decision in distraction osteogenesis is the timing of fixator removal. Various methods have been tried, such as radiographic appearance of callus and bone mineral density (BMD) assessment, but none has acquired gold standard status. The purpose of this study was to develop another objective method of assessment of callus stiffness to help clinicians in taking the most important decision of when to remove the fixator. We made a retrospective study of 70 patients to compare the BMD ratio and pixel value ratio. These ratios were calculated at the time of fixator removal, and Pearson's coefficient of correlation was used to show the comparability. Inter- and intra-observer variability of the new method was also tested. Good correlation was found between BMD ratio and pixel value ratio, with a Pearson's coefficient of correlation of 0.79. The interobserver variability was also low, with high intra-observer reproducibility, suggesting that this test was simple to perform. Pixel value ratio is a good method for assessing callus stiffness, and it can be used to judge the timing of fixator removal.

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

This study investigated the hypothesized beneficial effect of low-dose irradiation (LDI) on fracture callus mineralization in a rat model. 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. 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. The results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model.

Callus mineralization and maturation are delayed during fracture healing in interleukin-6 knockout mice

IL-6 is a pleiotropic cytokine involved in cell signaling in the musculoskeletal system, but its role in bone healing remains uncertain. The purpose of this study was to examine the role of IL-6 in fracture healing. Eight-week-old male C57BL/6 and IL-6 −/− mice were subjected to transverse, mid-diaphyseal osteotomies on the right femora. Sacrifice time points were 1, 2, 4, or 6 weeks post-fracture ( N = 14 per group). Callus tissue properties was analyzed by microcomputed tomography (micro-CT) and Fourier transform infrared imaging spectroscopy (FT-IRIS). Cartilage and collagen content, and osteoclast density were measured histologically. In intact unfractured bone, IL-6 −/− mice had reduced crystallinity, mineral/matrix ratio, tissue mineral density (TMD), and bone volume fraction (BVF) compared to wildtype mice. This suggests that there was an underlying deficit in baseline bone quality in IL-6 −/− mice. At 2 weeks post-fracture, the callus of IL-6 −/− mice had reduced crystallinity and mineral/matrix ratio. These changes were less evident at 4 weeks. At 2 weeks, the callus of the IL-6 −/− mice had an increased tissue mineral density (TMD), an increased cartilage and collagen content, and reduced osteoclast density compared to these parameters in wildtype mice. By 4 and 6 weeks, these parameters were no longer different between the two strains of mice. In conclusion, IL-6 −/− mice had delayed callus maturity, mineralization, and remodeling compared with the callus of the wildtype mice. These effects were transient indicating that the role of IL-6 appears to be most important in the early stages of fracture healing.

Role of parathyroid hormone in the mechanosensitivity of fracture healing

The mechanical environment at a fracture site can influence the course of healing. Intermittent parathyroid hormone (PTH) has been shown to accelerate fracture healing. Intact bone models show that mechanical loading and PTH have a synergistic beneficial effect on osteogenesis. We hypothesized that PTH and mechanical loading would have a similar synergistic effect on fracture healing. Eighty mice underwent surgical osteotomy and intramedullary nailing of the tibia. The mice were divided into four groups: one underwent daily loading, one received daily subcutaneous PTH injections (30 microg/kg/day), one received both loading and PTH, and a control group received sham loading and vehicle injection. Daily loading was applied to the ends of the tibia with an external loading device for 2 weeks. Fracture healing was assessed by microcomputed tomography, histology, and biomechanical testing. The group with both loading and PTH had increased osteoblast and osteoclast activity and was the only group with a significantly larger callus mineral density and bone volume fraction. The PTH only group had significantly more osteoid in the callus compared to the control group, indicating enhanced early osteoblast activity. This group also had a significantly higher bone mineral content and total bone volume compared to controls. The group that received loading as the only intervention had significantly greater osteoclast activity versus controls. The contribution of loading and PTH administration to the fracture healing cascade indicates a synergistic effect. This finding may be of potential clinical utility when weight bearing is utilized to stimulate lower extremity fracture healing.

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.

Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone. Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora. PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.

Impaired Angiogenesis, Early Callus Formation, and Late Stage Remodeling in Fracture Healing of Osteopontin-Deficient Mice

OPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an important role in regulation of angiogenesis, callus formation, and mechanical strength in early stages of healing and facilitates late stage bone remodeling and ECM organization. Osteopontin (OPN) is an extracellular matrix (ECM) protein with diverse localization and functionality that has been reported to play a regulatory role in both angiogenesis and osteoclastic bone remodeling, two vital processes for normal bone healing. Bone repair in wildtype and OPN(-/-) mice was studied using a femoral fracture model. microCT was used for quantitative angiographic measurements at 7 and 14 days and to assess callus size and mineralization at 7, 14, 28, and 56 days. Biomechanical testing was performed on intact bones and on fracture specimens at 14, 28, and 56 days. Histology and quantitative RT-PCR were used to evaluate cellular functions related to ECM formation and bone remodeling. OPN deficiency was validated in the OPN(-/-) mice, which generally displayed normal levels of related ECM proteins. Intact OPN(-/-) bones displayed increased elastic modulus but decreased strength and ductility. Fracture neovascularization was reduced at 7 but not 14 days in OPN(-/-) mice. OPN(-/-) mice exhibited smaller fracture calluses at 7 and 14 days, as well as lower maximum torque and work to failure. At 28 days, OPN(-/-) mice had normal callus size but a persistent reduction in maximum torque and work to failure. Osteoclast differentiation occurred normally, but mature osteoclasts displayed reduced functionality, decreasing late stage remodeling in OPN(-/-) mice. Thus, at 56 days, OPN(-/-) fractures possessed increased callus volume, increased mechanical stiffness, and altered collagen fiber organization. This study showed multiple, stage-dependent roles of OPN during fracture healing. We conclude that OPN deficiency alters the functionality of multiple cell types, resulting in delayed early vascularization, altered matrix organization and late remodeling, and reduced biomechanical properties. These findings contribute to an improved understanding of the role of OPN in vivo and provide new insight into mechanistic control of vascularization and bone regeneration during fracture repair.

Experimentally validated finite element model of a human tibia

Factors which impair fracture healing after intramedullary (IM) nailing of long bone fractures range from surgical and biological factors to mechanical parameters. Mechanical parameters known to prolong bony consolidation are share forces at the site of the fracture. Fracture near press-on interlocking reduces share forces directly at the fracture site and is hypothesised to enhance callus mineralisation. A sheep model of midshaft tibia osteotomies evaluates the technique.Fracture near interlocking was achieved by surfacing a custom made nail with special hutches that enable firm screw seating on top of the nail (“golf ball” structure). Virtual (fine element analysis (FEA)) and biomechanical pilot tests were completed before in vivo application in 12 adult female German black sheep. Midshaft tibia osteotomy was performed creating a subcritical 7 mm gap for delay in union. One group (n = 6) was treated with reamed IM nailing employing the custom made nail and in addition to proximal and distal standard interlocking a fracture near press on interlocking was employed. A second group of six sheep without additional press on interlocking served as control. 10 weeks after operation the quality of fracture healing was determined by micro-CT.The FEA showed that axial loading up to 4000 N did not lead to implant fatigue. Fracture near press on interlocking led to significantly more callus mineralisation compared to the conventional interlocking procedure (0.567 g/cm3 ± 0.106 g/cm3 versus 0.434 g/cm3 ± 0.0836 g/cm3, p = 0.043).Fracture near press on interlocking increases callus mineralisation in a subcritical osteotomy model in sheep. The results indicate that the reduction of share forces at the fracture site after nailing procedures may be effective in reducing the time until bony consolidation.

Osteocyte Apoptosis and Osteoclast Presence in Chicken Radii 0–4 Days Following Osteotomy

Osteocyte apoptosis caused by load-induced microdamage is followed by osteoclastic bone remodeling, and a causal link between apoptosis and repair has been suggested. The objectives of the present study were to use a chick model to examine the incidence of osteocyte apoptosis and the presence of osteoclasts during the first 96 hours following an osteotomy, prior to extensive callus mineralization. Osteotomies were performed on the right radii of 24 chicks at 23-24 days of age. The left radii served as controls. Radii were collected and processed at six time points following surgery (0, 12, 24, 48, 72, and 96 hours). Decalcified bone tissue sections were stained either for apoptosis using a modified TUNEL procedure or for tartrate-resistant acid phosphatase to identify osteoclasts in the intracortical and periosteal envelopes. The percentage of apoptotic osteocytes, as well as osteoclast counts (n/mm or n/mm2) were quantified in four regions (0-1, 1-2, 2-4, and 4-8 mm from the site of the osteotomy; regions 1-4, respectively) in the osteotomized radii and in the same measured areas in the control radii. Data for osteocyte apoptosis and osteoclasts in the control limb were subtracted from the osteotomized limb data to identify differences due to surgical influence. The incidence of osteocyte apoptosis was significantly higher at 12, 24, 48, and 72 hours versus 0 hours following osteotomy, and the response was highest in region 1; however, there was no interaction between time and region. Intracortical osteoclast counts (n/mm2) were elevated after 48 hours, and the response was similar in all regions. The data demonstrate that osteocyte apoptosis occurs within 24 hours in response to an osteotomy and temporally precedes an increase in osteoclast presence. Hence, osteocyte apoptosis may play a role in signaling during the bone healing process.

Effect of Vascularity on Canine Distracted Tibial Callus Consolidation

In the consolidation period of distraction osteogenesis, mineralization occurs before corticalization. We hypothesized that the increased rate of bone mineral density correlates to the density of vascularity in the callus. We unilaterally lengthened the tibia in eight adult beagles. After a waiting period of 7 days, tibiae were lengthened for 30 days. After a consolidation period of an additional 60 days, all animals were euthanized. Just before euthanasia, blood vessels were perfused with 50% w/v barium sulfate solution, and soft radiographs of the distracted callus and the control tibiae were taken. Bone mineral density of the regenerated bones was measured preoperatively by quantitative computed tomography on Days 37, 68, and 98. Increases of the percent bone mineral density (from Day 37 to Day 98 and from Day 68 to Day 98) correlated with the blood vessel volume density ratios on Day 98. Our results suggest that preservation of the higher density of blood vessels in the consolidation period could lead to the better mineralization of the distracted callus.

Use of Peripheral Quantitative Computed Tomography to Monitor Bone Healing After Radial Osteotomy in Three-week-old Chickens (Gallus domesticus)

Peripheral quantitative computed tomography (pQCT), a noninvasive method for measuring bone characteristics, was used to evaluate bone healing in 3 white leghorn chickens (Gallus domesticus) after radial osteotomy at 22 days of age. The time sequence for callus formation and mineralization over a 45-day period was examined by performing serial cross-sectional scans of the osteotomized radius of each bird daily from day 2 to day 29 and on days 42 and 45 after surgery. Complete data were available for 2 birds. Formation of a new marrow cavity was apparent by 4 weeks after surgery, and union of the bone ends appeared complete by day 42. Bone mineral content (BMC), bone mineral density (BMD), and cross-sectional area (CSA) were measured at the radial osteotomy site, at 3 mm proximal to it, and at the corresponding areas on the ulna. The radial osteotomy site began to mineralize by day 4, and BMC and CSA increased approximately 100% in both birds between days 5 and 10. In bird 2, BMC and CSA continued to increase (55% and 74%, respectively). Peak radial levels of both BMC and CSA were followed by plateaus lasting approximately 7 days, then both levels decreased during callus resorption. The radial BMD increased during these plateaus, then decreased as the callus was remodeled. Between days 29 and 42, the BMD of the radial osteotomy site increased and approached that of the corresponding ulna. The sequences of changes in BMC, CSA, and BMD after day 10 were similar in each bird. These results indicate that pQCT is a useful tool to monitor postosteomy bone healing in chicks.

Callus mineralization following distraction osteogenesis of the mandible monitored by scanning acoustic microscopy (SAM)

Scanning acoustic microscopy uses ultrasound to analyse histomorphology of tissues with microscopic resolution and delivers data about physical properties of the specimen. Bony consolidation was monitored by scanning acoustic microscopy in 12 embedded specimens of dog mandibles after distraction osteogenesis. Increasing mineralization was detected by measurements of acoustic impedance (Z). There was a strong correlation between acoustic impedance and time of consolidation. Measurements of the speed of sound (v) provided specific information about non-mineralized zones of the distracted area. Distribution of density in the distracted area could be reconstructed by using the measurements of acoustic impedance and speed of sound. The method seems suitable for studying bone remodelling qualitatively and quantitatively.

Gait evaluation: A tool to monitor bone healing?

Current clinical methods for monitoring fracture healing are often invasive and inaccurate. This paper evaluates the use of a pressure sensitive platform to improve monitoring. Standardised 3 mm diaphyseal bone defects were created in the right tibia of 64 female sheep and stabilised with either a rigid monolateral external fixator or a more flexible variant. Over a nine week healing period gait parameters were measured using a pressure sensitive platform and interfragmentary movements at the fracture site were monitored. Frequency spectra were calculated for the ground reaction forces. The tibiae were tested biomechanically after sacrifice and callus sections were analysed histomorphometrically. All animals unloaded the operated and overloaded the contralateral hindlimb. Callus mineralisation and stiffness, as well as limb loading increased during healing whilst interfragmentary movements were reduced. Larger interfragmentary movements resulted in a slower fracture healing rate as documented histologically and biomechanically. Frequency analysis showed upto 14 dB loss of power at frequencies associated with bone mechanotransduction at four weeks postoperatively, reducing to a 3 dB loss at nine weeks. Gait analysis is a valuable tool for monitoring the course of fracture healing. Different fixation stiffnesses caused different initial interfragmentary movements leading to different healing rates. Ground reaction forces were strongly related to the course of callus mineralisation and thus directly reflected the recovery of stiffness at the fracture site. Reduced levels of loading frequencies that may affect bone healing persist to nine weeks postoperatively.

Effect of pulsed electromagnetic fields on maturation of regenerate bone in a rabbit limb lengthening model

To study the effect of applying pulsed electromagnetic fields (PEMF) during the consolidation phase of limb lengthening, a mid-tibial osteotomy was performed in 18 adult New Zealand White rabbits and an external fixator was applied anteromedially. Animals were randomly assigned to treatment and control groups. After a 7-day latency period, the tibiae were distracted 0.5 mm every 12 h for 10 days. The treatment group received a 20-day course of PEMF for 60 min daily, coinciding with initiation of the consolidation phase. The control group received sham PEMF. Radiographs were performed weekly after distraction. Animals were euthanized 3 weeks after the end of distraction. Radiographic analysis revealed no significant difference in regenerate callus area between treatment and control tibiae immediately after distraction, at 1 week, 2 weeks, or 3 weeks after distraction ( p = 0.71, 0.22, 0.44, and 0.50, respectively). There was also no significant difference in percent callus mineralization ( p = 0.96, 0.69, 0.99, and 0.99, respectively). There was no significant difference between groups with respect to structural stiffness ( p = 0.80) or maximal torque to failure ( p = 0.62). However, there was a significant positive difference in mineral apposition rate between groups during the interval 1-2 weeks post-distraction ( p < 0.05). This difference was no longer evident by the interval 2-3 weeks post-distraction. While PEMF applied during the consolidation phase of limb lengthening did not appear to have a positive effect on bone regenerate, it increased osteoblastic activity in the cortical bone adjacent to the distraction site. Since the same PEMF signal was reported to be beneficial in the rabbit distraction osteogenesis when applied during distraction phase and consolidation phase, application of PEMF in the early phase may be more effective. Further work is necessary to determine optimal timing of the PEMF stimulation during distraction osteogenesis.

Expression of dentin matrix protein 1 (DMP1) during fracture healing

Dentin matrix protein 1 (DMP1) is one of the acidic phosphorylated extracellular matrix proteins called the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family. Recent studies showed that DMP1 is expressed in the mineralized tissues and suggested that DMP1 is involved in the mineralization. We investigated the precise localization of DMP1 messenger RNA (mRNA) and protein during fracture healing. In situ hybridization demonstrated that DMP1 mRNA was strongly expressed in preosteocytes and osteocytes in the bony callus during intramembranous and endochondral ossification while DMP1 mRNA was not detected in osteoblasts and chondrocytes. During endochondral ossification, however, a low number of DMP1-expressing cells were identified in the cluster of hypertrophic chondrocytes. However, these DMP1-expressing cells were not hypertrophic and were likely to be osteoblast-lineage cells, which were embedded in the matrix of bone or cartilage, because type I collagen-expressing cells and invasion of capillary vessels were observed in the same area. Northern blot, in situ hybridization, and immunohistochemical analyses showed that DMP1 mRNA and protein expressions were increased until day 14 postfracture, when bony callus was formed, and then declined to a lower level during remodeling of the bony callus. Therefore, DMP1 is likely to play an important role in the mineralization of the bony callus.

Neuromuscular electrical stimulation enhances fracture healing: results of an animal model

Neuromuscular electrical stimulation (NMES) could simulate physiological muscle functions known to be associated with the normal bone healing process. The object of the present study was to evaluate the effect of NMES on fracture healing, using an animal model. Thirty rabbits received unilateral, transverse, mid-tibial, 3-mm gapped osteotomies that were stabilized with double-bar external fixators. The femoral vein was ligated to induce venous stasis. From the fourth post-operative day, the study group was treated with 1 h daily of NMES for four weeks, while the control group was treated without NMES. For NMES, two surface electrodes were used: one above the patellar tendon and another around the lateral thigh. Callus area and mineral content at the osteotomy gap were measured, biweekly, using computerized tomographic examinations. Biomechanical properties of healing were evaluated with a torsion test, eight weeks after the index operation. Osteotomies treated with NMES exhibited 31% ( p=0.01) higher mineral content and 27% ( p=0.009) larger callus area than control osteotomies at eight weeks. The maximum torque, torsional stiffness, angular displacement at maximum torque, and energy required to failure of specimens in the study group were 62% ( p=0.006), 29% ( p=0.03), 34.6% ( p=0.008), and 124% ( p<0.0001) higher, respectively, than those in the control group at eight weeks. The results of the present study demonstrated that the use of NMES can enhance callus development and mineralization, with the consequent improvement in biomechanical properties of the healing bone.

Effect of intermittent pneumatic soft-tissue compression on fracture-healing in an animal model.

The fracture-healing process is closely related to blood supply. Intermittent pneumatic compression of the surrounding soft tissue may alter blood flow and, therefore, modify the healing process. The object of the present study was to evaluate the effect of intermittent pneumatic compression on fracture-healing in an animal model. Unilateral, transverse, mid-tibial osteotomies with a 3-mm gap were performed in thirty rabbits. The osteotomy site was stabilized with a double-bar external fixator. The femoral vein was ligated to induce venous stasis. Beginning on the fourth postoperative day, fifteen rabbits were treated with intermittent pneumatic compression with use of four rubber balloons, two around the distal part of the calf and two around the midpart of the calf, for one hour daily for four weeks (the study group) and fifteen rabbits were not treated with intermittent pneumatic compression (the control group). Peripheral computerized tomographic examination was performed biweekly to measure callus area and mineral content at the fracture gap. At eight weeks, the rabbits were killed, and the biomechanical properties of the healing fractures were evaluated with a torsional test. An increase in callus area and mineral content at the osteotomy gap was observed in the study group, compared with the values in the control group, starting four weeks after the index procedure. At six weeks, the rabbits treated with intermittent pneumatic compression exhibited, on the average, a 32.2% larger callus area (p = 0.035) and a 49.7% higher mineral content (p = 0.01) at the osteotomy site compared with the values in the control group. The torsional stiffness, maximum torque, angular displacement at maximum torque, and energy required to failure of specimens in the study group were an average of 27.0% (p = 0.05), 61.5% (p = 0.0001), 35.4% (p = 0.0003), and 110.8% (p = 0.0001) higher, respectively, than those in the control group at eight weeks. Intermittent pneumatic compression enhanced callus mineralization and development and it improved the biomechanical properties of a healing osteotomy site in the rabbit tibia.

Biomechanical evaluation of healing in a non-critical defect in a large animal model of osteoporosis

Current methods for fracture treatment in osteoporosis are not always sufficient. To develop new fixation strategies (both mechanical and biological) requires pre-clinical testing utilizing appropriate models. The aim of this study was to apply a recently developed sheep model of osteoporosis to the study of healing in a non-critical long bone defect. A standardized transverse mid-shaft tibial osteotomy (with a fracture gap of 3 mm) was performed in seven osteoporotic and seven normal sheep and stabilized with a special external fixator for 8 weeks. The fixator was used for weekly in vivo bending stiffness measurements. Ex vivo bending stiffness and torsional stiffness of the callus zone were also determined. Callus area, callus density, and osteoporosis status were determined at 0, 4, and 8 weeks using peripheral quantitative computed tomography. The increase of in vivo bending stiffness of the callus was delayed approximately 2 weeks in osteoporotic animals. A significant difference (33%) in torsional stiffness was found between the osteotomized and contralateral intact tibia in osteoporotic animals, but no significant difference occurred in normal sheep (2%). In osteoporotic animals, ex vivo bending stiffness was reduced 21% ( p=0.05). Bending stiffness was correlated with callus density ( r=0.76, r=0.53); torsional stiffness was correlated with callus area ( r=0.60) and to a lesser extent with callus density ( r=0.53). This study demonstrated a delay of fracture healing in osteoporotic sheep tibiae with respect to callus formation, mineralization, and mechanical properties.