Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 μg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.     

A fuzzy logic model of fracture healing

A quantitative biomechanical model describes the tissue transformation during healing of a transverse osteotomy of a sheep metatarsal. The model predicts bridging of the bone ends through cartilage, followed by the growth of a callus cuff, and finally, the resorption of callus after ossification of the interfragmentary gap. We suggest bone density or the modulus of elasticity do not sufficiently characterize healing tissue for predictive purposes. In addition to the stimulus reflected by strain energy density we introduce a new osteogenic factor based upon stress gradients and which predicts areas of a high osteogenic capacity. Our model distinguishes three basic types of tissue, namely bone, cartilage and fibrous tissue. A fuzzy controller is proposed to model the tissue reaction. A set of fuzzy rules derived from medical knowledge has been implemented to describe tissue transformation such as intramembraneous or chondral ossification, atrophy or destruction. Fuzzy logic is able to model tissue transformation processes within the numerical simulation of remodeling processes. This approach improves the simulation tools and affords the potential to optimize planning of animal experiments and conduct parametric studies.     

Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing

Beyond its role in the regulation of red blood cell proliferation, the glycoprotein erythropoietin (EPO) has been shown to promote cell regeneration and angiogenesis in a variety of different tissues. In addition, EPO has been indicated to share significant functional and structural homologies with the vascular endothelial growth factor (VEGF), a cytokine essential in the process of fracture healing. However, there is complete lack of information on the action of EPO in bone repair and fracture healing. Therefore, we investigated the effect of EPO treatment on bone healing in a murine closed femur fracture model using radiological, histomorphometric, immunohistochemical, biomechanical and protein biochemical analysis. Thirty-six SKH1-hr mice were treated with daily i.p. injections of 5000 U/kg EPO from day 1 before fracture until day 4 after fracture. Controls received equivalent amounts of the vehicle. After 2 weeks of fracture healing, we could demonstrate expression of the EPO-receptor (EPOR) in terminally differentiating chondrocytes within the callus. At this time point EPO-treated animals showed a higher torsional stiffness (biomechanical analysis: 39.6+/-19.4% of the contralateral unfractured femur) and an increased callus density (X-ray analysis (callus density/spongiosa density): 110.5+/-7.1%) when compared to vehicle-treated controls (14.3+/-8.2% and 105.9+/-6.6%; p<0.05). Accordingly, the histomorphometric examination revealed an increased fraction of mineralized bone and osteoid (33.0+/-3.0% versus 28.5+/-3.6%; p<0.05). Of interest, this early effect of the initial 6-day EPO treatment had vanished at 5 weeks after fracture. We conclude that EPO-EPOR signaling is involved in the process of early endochondral ossification, enhancing the transition of soft callus to hard callus.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Callus stimulation in distraction osteogenesis

Distraction osteogenesis has been described as in vivo tissue engineering. The ability to stimulate this process for the repair of bony defects or lengthening of congenitally shortened facial structures is likely to significantly impact the field of craniofacial surgery. The purpose of this study was to determine whether mechanical stimulation of the distracted rabbit mandible would accelerate the maturation of the bony callus when applied during the early consolidation period. Twenty adult New Zealand White rabbits underwent unilateral mandibular osteotomy. A uni-directional internal distractor device (Synthes, Paoli, Pa.) was positioned along a plane perpendicular to the line of osteotomy. After a 7-day latency period, distraction was commenced at a rate of 1.0 mm/day for 12 days in all animals. In a control group of 10 rabbits, a consolidation period of 8 weeks was observed before they were killed. In the experimental group of 10 rabbits, daily alternate compression and distraction of 1 mm (sequential compression and distraction) was performed for 3 weeks followed by a 5-week period of rigid fixation. Each animal received a dose of a fluorescent label at three different time points during the study: at the end of the distraction period, 3 weeks after the completion of the distraction phase, and 3 days before it was killed. All animals were killed 8 weeks after the completion of the distraction phase. Undecalcified histologic analysis and 3-point bending tests to failure were performed on the extracted mandibles. The results of the experimental and control groups were compared.Four animals in the control group and three animals in the experimental group were excluded from the study because of screw loosening resulting in distractor dislodgment or because of infection. On histologic analysis, cortical thickness at the center of the callus was found to be significantly greater in the experimental group compared with the control group when normalized to the contralateral hemimandible (83 percent versus 49 percent, respectively; p < 0.007). The ratio of cortical to cancellous bone in the distracted callus was uniformly found to be greater in the experimental specimens. The mineral apposition rate was calculated by using fluorescence microscopy and found to be significantly greater in the experimental group both during the period of sequential compression and distraction (3.2 microm/day versus 2.1 microm/day, p = 0.02) and after the period of sequential compression and distraction (1.4 microm/day versus 1.1 microm/day, p = 0.006). Mechanical testing revealed no significant differences in bending strength or stiffness between experimental or control groups (p = 0.54 and 0.47, respectively). This study has demonstrated that daily alternating compression and distraction of 1 mm amplitude during the early consolidation period has a stimulatory impact on callus formation with respect to osteoblastic activity, remodeling, and maturation of bone. Optimal timing and amplitude of sequential movement, long-term biomechanical differences, and molecular pathways have yet to be elucidated.     

Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing

A new quantitative tissue differentiation theory which relates the local tissue formation in a fracture gap to the local stress and strain is presented. Our hypothesis proposes that the amounts of strain and hydrostatic pressure along existing calcified surfaces in the fracture callus determine the differentiation of the callus tissue. The study compares the local strains and stresses in the callus as calculated from a finite element model with histological findings from an animal fracture model. The hypothesis predicts intramembranous bone formation for strains smaller approximately +/- 5% and hydrostatic pressures smaller than +/- 0.15 MPa. Endochondral ossification is associated with compressive pressures larger than about -0.15 MPa and strains smaller than +/- 15%. All other conditions seemed to lead to connective tissue or fibrous cartilage. The hypothesis enables a better understanding of the complex tissue differentiation seen in histological images and the mechanical conditions for healing delayed healing or nonunions.     

CD133: Enhancement of Bone Healing by Local Transplantation of Peripheral Blood Cells in a Biologically Delayed Rat Osteotomy Model

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction.     

Mechanical induction of critically delayed bone healing in sheep: radiological and biomechanical results

This study aimed to mechanically produce a standardized ovine model for a critically delayed bone union. A tibial osteotomy was stabilized with either a rigid (group I) or mechanically critical (group II) external fixator in sheep. Interfragmentary movements and ground reaction forces were monitored throughout the healing period of 9 weeks. After sacrifice at 6 weeks, 9 weeks and 6 months, radiographs were taken and the tibiae were examined mechanically. Interfragmentary movements were considerably larger in group II throughout the healing period. Unlike group I, the operated limb in group II did not return to full weight bearing during the treatment period. Radiographic and mechanical observations showed significantly inferior bone healing in group II at 6 and 9 weeks compared to group I. After 6 months, five sheep treated with the critical fixator showed radiological bridging of the osteotomy, but the biomechanical strength of the repair was still inferior to group I at 9 weeks. The remaining three animals had even developed a hypertrophic non-union. In this study, mechanical instability was employed to induce a critically delayed healing model in sheep. In some cases, this approach even led to the development of a hypertrophic non-union. The mechanical induction of critical bone healing using an external fixation device is a reasonable attempt to investigate the patho-physiological healing cascade without suffering from any biological intervention. Therefore, the presented ovine model provides the basis for a comparative evaluation of mechanisms controlling delayed and standard bone healing.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.     

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.KEY WORDS: Whole-body vibration, LMHFV, Fracture healing, Oestrogen receptor signalling, Wnt signalling     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Effect of isolation of periosteum and dura on the healing of rabbit calvarial inlay bone grafts

Little is understood about the role of the recipient site in the revascularization and incorporation of autogenous inlay bone grafts in the craniofacial skeleton. Clinical experience demonstrates that secondary complex cranial vault reconstruction performed with scarred avascular dura or poor soft-tissue coverage may undergo significant resorption, thus compromising the aesthetic outcome. This study was designed to determine the effect of isolating autogenous orthotopic inlay calvarial bone grafts from the surrounding dura and/or periosteum on graft revascularization, healing, and volume maintenance in the adult rabbit. Adult rabbits were randomized into four groups (n = 10 per group); in each rabbit, the authors created a circular, 15-mm in diameter, full-thickness cranial defect followed by reconstruction with an autogenous calvarial bone graft, which was replaced orthotopically and held with microplate fixation. Silicone sheeting (0.5 mm thickness) was used to isolate the dura (group II), the periosteum (group II), or both dura and periosteum (group IV) from the graft interface. No silicone was placed in group I. Animals were killed 10 weeks postoperatively, and calvaria were harvested to assess graft surface area, morphology, quantitative histology, fluorochrome staining, and revascularization. Grafts isolated from both the dura and periosteum exhibited significant decreases in total bone (cortical and trabecular) surface area, blood vessel count, and interface healing compared with nonisolated control grafts. Isolation of either the dura or periosteum significantly (p < 0.05) decreased blood vessel count but had no significant effect on interface healing. Isolation of the dura alone was associated with a significant (p < 0.05) decrease in graft cross-sectional surface area and dural cortical thickness compared with nonisolated control grafts, but this effect was not observed when the periosteum alone was isolated. Quantitative histology performed 10 weeks after surgery indicated that graft isolation was associated with increased marrow fibrosis and necrosis compared with nonisolated controls; it also demonstrated evidence of increased activity in bone remodeling (osteoblast and osteocyte count, new trabecular bone, and surface resorption). Triple fluorochrome staining suggested increased bone turnover in the nonisolated grafts compared with isolated grafts at 1 and 5 weeks postoperatively. This study demonstrates that isolating a rabbit calvarial inlay autogenous bone graft from the dura and/or periosteum results in significantly (p < 0.05) decreased revascularization, interface healing, and cross-sectional areas of amount of mature bone compared with nonisolated control grafts 10 weeks after surgery. At this time point, histologic examination demonstrates a paradoxical increase in bone remodeling in isolated bone grafts compared with controls. It is possible that the inhibition of revascularization results in a delayed onset of the remodeling phase of graft incorporation. However, in the model studied, it is not known whether the quantitative histologic and morphometric parameters measured in these isolated grafts exhibit a "catch-up" phenomenon at time points beyond 10 weeks after surgery. The results of this study emphasize the importance of a healthy recipient site in the healing and incorporation of calvarial bone grafts but stress the need for further investigation at later time points.     

Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

Osteogenic growth peptide enhances the rate of fracture healing in rabbits

The discovery of growth factors, such as osteogenic growth peptide (OGP), that stimulate bone formation led to experiments to discover whether they can accelerate fracture healing. To determine whether OGP enhances the rate of healing in rabbits, fractures were made in the tibiae of New Zealand White rabbits and immobilized with either a plastic plate (unstable mechanical conditions), or a dynamic compression plate (stable mechanical conditions). OGP was administered to experimental animals by intravenous injection from day 4 until the day before sacrifice; control animals were not injected. After treatment with OGP, callus development under unstable mechanical conditions was accelerated. At 7 days, the cartilage in the centre of the callus was covered by bone and endochondral ossification had started; these events occur at 10 days in control fractures. Subsequently, endochondral ossification is completed earlier which allows the invasion of the fracture gap by cells, so that cortical union is complete by 21 to 28 days. In control fractures, bone is only beginning to form in the gaps at 28 days. There was no increase in the size of the callus in any of the experimental fractures compared to the untreated controls. Treatment with OGP has no observable effect on the rate of healing of fractures under stable mechanical conditions. These observations suggest that under unstable mechanical conditions only, the rate of callus formation and subsequent cortical healing is enhanced by treatment with OGP, but that the size of the callus is determined by mechanical and other factors.     

Lentivirus-mediated Wnt10b overexpression enhances fracture healing in a rat atrophic non-union model

Lentivirus vectors encoding Wnt10b gene (LV–Wnt10b) or luciferase gene (LV-luc) were constructed to determine whether Wnt10b overexpression improved fracture healing in a rat atrophic non-union model. After fracture, rats were injected with LV-Wnt10b or LV-luc. Luciferase signals were clearly detected. At 2 and 4 weeks, LV-Wnt10b group had 107 and 98 % more proliferating cell nuclear antigen (PCNA) positive cells, respectively, and promoted expression of bone morphogenetic protein-2 (BMP-2) in the callus compared with controls. LV-Wnt10b injection significantly increased bone mass density and bone mineral content: 46–84 and 96–193 %, respectively, at the site of fracturein the LV-Wnt10b group compared with controls. At 8 weeks, fractured femora were healed in the LV-Wnt10b group compared with atrophic non-unions formed in controls. Thus, Wnt10b overexpression associated with lentiviral gene therapy is effective in healing atrophic non-unions in rats.     

Assessment of a mechano-regulation theory of skeletal tissue differentiation in an in vivo model of mechanically induced cartilage formation

Mechanical cues are known to regulate tissue differentiation during skeletal healing. Quantitative characterization of this mechano-regulatory effect has great therapeutic potential. This study tested an existing theory that shear strain and interstitial fluid flow govern skeletal tissue differentiation by applying this theory to a scenario in which a bending motion applied to a healing transverse osteotomy results in cartilage, rather than bone, formation. A 3-D finite element mesh was created from micro-computed tomography images of a bending-stimulated callus and was used to estimate the mechanical conditions present in the callus during the mechanical stimulation. Predictions regarding the patterns of tissues—cartilage, fibrous tissue, and bone—that formed were made based on the distributions of fluid velocity and octahedral shear strain. These predictions were compared to histological sections obtained from a previous study. The mechano-regulation theory correctly predicted formation of large volumes of cartilage within the osteotomy gap and many, though not all patterns of tissue formation observed throughout the callus. The results support the concept that interstitial fluid velocity and tissue shear strain are key mec- hanical stimuli for the differentiation of skeletal tissues.     

Local Application of Ibandronate/Gelatin Sponge Improves Osteotomy Healing in Rabbits

Delayed healing or non-union of skeletal fractures are common clinical complications. Ibandronate is a highly potent anti-catabolic reagent used for treatment of osteopenia and fracture prevention. We hypothesized that local application of ibandronate after fracture fixation may improve and sustain callus formation and therefore prevent delayed healing or non-union. This study tested the effect of local application of an ibandronate/gelatin sponge composite on osteotomy healing. A right-side distal-femoral osteotomy was created surgically, with fixation using a k-wire, in forty adult male rabbits. The animals were divided into four groups of ten animals and treated by: (i) intravenous injection of normal saline (Control); (ii) local implantation of absorbable gelatin sponge (GS); (iii) local implantation of absorbable GS containing ibandronate (IB+GS), and (iv) intravenous injection of ibandronate (IB i.v.). At two and four weeks the affected femora were harvested for X-ray photography, computed tomography (CT), biomechanical testing and histopathology. At both time-points the results showed that the calluses in both the ibandronate-treated groups, but especially in the IB+GS group, were significantly larger than in the control and GS groups. At four weeks the cross sectional area (CSA) and mechanical test results of ultimate load and energy in the IB+GS group were significantly higher than in other groups. Histological procedures showed a significant reduction in osteoclast numbers in the IB+GS and IB i.v. groups at day 14. The results indicate that local application of an ibandronate/gelatin sponge biomaterial improved early osteotomy healing after surgical fixation and suggest that such treatment may be a valuable local therapy to enhance fracture repair and potentially prevent delayed or non-union.     

Mineral and matrix contributions to rigidity in fracture healing

The purpose of this study was to investigate the relationships among selected properties of fracture callus: bending rigidity, tissue density, mineral density, matrix density and mineral-to-matrix ratio. The experimental model was an osteotomized canine radius in which the development of the fracture callus was modified by electrical stimulation with various levels of direct current. This resulted in a range of values for the selected properties of the callus, determined post mortem at 7 weeks after osteotomy. We found that the rigidity (R) of the bone-callus combination obeyed relationships of the form R = axb, where x is the tissue density, mineral density, matrix density or the mineral-to-matrix ratio of the repair tissue. These are analogous to power-law relationships found in studies of compact and cancellous bone. The results suggest that fracture callus at 7 weeks after osteotomy in canine radius behaves more like immature compact bone than cancellous bone in its mechanical and physicochemical properties. The present study demonstrates the feasibility of developing non-invasive in vivo densitometric methods to monitor fracture healing, since models may be developed that can predict mechanical properties from densitometric data. Further studies are needed to develop a refined model based on experimental data on the mechanical and physicochemical properties and microstructure of fracture callus at different stages of healing.     

The spatio-temporal arrangement of different tissues during bone healing as a result of simple mechanobiological rules

During secondary bone healing, different tissue types are formed within the fracture callus depending on the local mechanical and biological environment. Our aim was to understand the temporal succession of these tissue patterns for a normal bone healing progression by means of a basic mechanobiological model. The experimental data stemmed from an extensive, previously published animal experiment on sheep with a 3?mm tibial osteotomy. Using recent experimental data, the development of the hard callus was modelled as a porous material with increasing stiffness and decreasing porosity. A basic phenomenological model was employed with a small number of simulation parameters, which allowed comprehensive parameter studies. The model distinguished between the formation of new bone via endochondral and intramembranous ossification. To evaluate the outcome of the computer simulations, the tissue images of the simulations were compared with experimentally derived tissue images for a normal healing progression in sheep. Parameter studies of the threshold values for the regulation of tissue formation were performed, and the source of the biological stimulation (comprising e.g. stem cells) was varied. It was found that the formation of the hard callus could be reproduced in silico for a wide range of threshold values. However, the bridging of the fracture gap by cartilage on the periosteal side was observed only (i) for a rather specific choice of the threshold values for tissue differentiation and (ii) when assuming a strong source of biological stimulation at the periosteum.     

Organic Gallium Treatment Improves Osteoporotic Fracture Healing Through Affecting the OPG/RANKL Ratio and Expression of Serum Inflammatory Cytokines in Ovariectomized Rats

This study aimed to investigate the impact of organic gallium (OG) on osteoporotic fracture healing in ovariectomized female Sprague-Dawley rats, as well as study the mechanisms of OG on osteoporotic fracture healing. Forty-five female Sprague-Dawley rats were divided into three groups: sham operation group (Sxas control group), ovariectomized group (Ovx), and Ovx treated with OG group (Ovx + OG). Rat femoral fractures were studied using a standardized fracture-healing model utilizing bone fixation with an intramedullary pin. Six weeks later, analyses of micro-CT, histomorphometric, RNA extraction, RT-qPCR, and serum were performed following sacrifice of all mice. In comparison with Ovx group, OG can significantly increase bone volume (BV), tissue volume (TV), BV/TV radio, bone strength, callus bony area, and as similar to BMP-2 expression. OG treatment elevated OPG messenger RNA (mRNA) and inhibited RANKL mRNA, and showed an effect on OPG/RANKL ratio. OG treatment can inhibit the expression of TNF-α and IL-6. In conclusion, current study results indicate that organic OG can positively affect the OPG/RANKL ratio and inhibit the expression of serum inflammatory cytokines; thus, it can improve osteoporotic fracture healing.