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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.

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.

In article described research the results of the prevalence of the genetic polymorphism of the gene Methylentetrahydrofolatereductase C677T (MTHFR) in 130 patients with pseudarthrosis of long bones and in those with consolidated fractures. The incidence of allele-T among patients with pseudarthrosis was 1.4 times higher than among those with consolidated fractures. Pathological genotype MTHFR 677-TT was associated with the development avital types of pseudarthrosis and increase the proportion of people with hyperhomocysteinemia, high content of inflammatory mediators and development refracture.

The failure of an osseous fracture to heal (development of a non-union) is a common and debilitating clinical problem. Mice lacking the tumor suppressor Pten in osteoblasts have dramatic and progressive increases in bone volume and density throughout life. Since fracture healing is a recapitulation of bone development, we investigated the process of fracture healing in mice lacking Pten in osteoblasts (Ocn-cre(tg/+;)Pten(flox/flox) ). Mid-diaphyseal femoral fractures induced in wild-type and Ocn-cre(tg/+;)Pten(flox/flox) mice were studied via micro-computed tomography (µCT) scans, biomechanical testing, histological and histomorphometric analysis, and protein expression analysis. Ocn-cre(tg/+;)Pten(flox/flox) mice had significantly stiffer and stronger intact bones relative to controls in all cohorts. They also had significantly stiffer healing bones at day 28 post-fracture (PF) and significantly stronger healing bones at days 14, 21, and 28 PF. At day 7 PF, the proximal and distal ends of the Pten mutant calluses were more ossified. By day 28 PF, Pten mutants had larger and more mineralized calluses. Pten mutants had improved intramembranous bone formation during healing originating from the periosteum. They also had improved endochondral bone formation later in the healing process, after mature osteoblasts are present in the callus. Our results indicate that the inhibition of Pten can improve fracture healing and that the local or short-term use of commercially available Pten-inhibiting agents may have clinical application for enhancing fracture healing.

Understanding of the biology of bone regeneration has been increasing rapidly, with greater appreciation for the importance of biochemical aspects as well as the mechanical requirements for bone to heal. There are a number of situations where there is difficulty in bone healing such as fracture non-union; or growth such as osteogenesis imperfecta; or a requirement for surplus bone to reconstruct defects such as following surgery for tumour excision or limb lengthening. There is a greater understanding of the complex interplay between osteoblasts and osteoclasts, and the chemical mediators that provide signalling along complex pathways. Although we have known about substances such as Bone Morphogenic Proteins and Growth Hormones for some time, their application in clinical practice is still not widespread, and we need to study them more to understand their role in bone healing. With newer technologies such as stem cells and gene therapy being developed there is the potential for vast improvement in bone regenerative techniques, although we are not at a stage where we can be confident that these techniques will work. In this review article we discuss the basic healing process of bone and how our understanding of this has led to improved techniques as well as the potential for future developments in new technologies.

Objectives: This study focuses upon the morphologic and molecular features of the layer of cells, termed the "biomembrane," which forms around methacrylate spacers in bone segmental defects. The objective of this research was to assess the biomembrane formed in a novel rodent femoral segmental defect model at 4, 8, and 16 weeks with histologic and molecular studies., Methods: Following Institutional Animal Care and Use Committee approval, a segmental defect was created in the rat femur and stabilized with the AO LockingRatNail and analyzed at 4, 8, and 16 weeks postsurgery using digital radiologic imaging, morphological and immunohistochemical studies, and genomewide gene expression studies employing microarray analysis., Results: The biomembrane formed around the methacrylate spacer was rich in vasculature, which showed vascular endothelial growth factor immunolocalization. The biomembrane supported development of foci of bone and cartilage within it. Bone morphogenetic protein 2 immunolocalization and gene expression were positive within developing osseous and chondrocyte foci. Microarray analysis showed significant expression of key genes related to bone and cartilage formation and angiogenesis., Conclusions: This rat bone model was effective in creation of the biomembrane. Bone and cartilage foci were formed within the vascularized biomembrane with associated expression of genes critical for bone and cartilage development/formation and vascularization. The polymethyl methacrylate-induced biomembrane offers an exciting potential solution for segmental defects; the biomembrane, may act as a receptive bed and also serve as a source for mesenchymal stem cells, which could be recruited/directed for the healing process.

This study sought to determine the cellular and molecular mechanisms of cyclooxygenase-2 (COX2) gene therapy to accelerate fracture repair in a mouse multiple tibial fractures model. The lenti-COX2 (or lenti-gfp control vector) was injected into fractures on day 1 post-fracture. At days 3-7, the COX2 treatment increased Sdf1-, Cxcr4-, Nes-, and Podxl-expressing mesenchymal stem cells (MSCs) within fracture calluses, suggesting an enhanced MSC recruitment or expansion. The COX2-treated mice formed smaller cartilaginous calluses that had less cartilage tissues than control mice. The expression of Sox9 mRNA was 7-fold less in COX2-treated than in control calluses at day 14, implying that COX2 reduces chondrocytic differentiation of MSCs. The therapy also enhanced angiogenesis as reflected by increased immunostaining of CD31, vWF, and α-SMA over controls in the cartilaginous callus at day 14-21. At which time, the COX2 gene therapy promoted bony remodeling of the cartilaginous callus to bridge the fracture gap that was accompanied by 2-fold increase in osteoclasts along the surface of the woven bone and an onset of osteogenesis. Blocking angiogenesis with daily injection of endostatin from day 4 to day 10 into fracture sites blocked the COX2-mediated reduction of callus size that was associated with an increase in hypertrophic chondrocytes and concomitant reduction in osteoclasts. In conclusion, COX2 accelerates fracture healing in part through three biological actions: 1) increased recruitment/expansion of MSCs; 2) decreased cartilaginous callus formation; and 3) increased angiogenesis-dependent cartilage remodeling. These effects were associated with an earlier onset of bony bridging of the fracture gap., (Published by Elsevier Inc.)

Intertrochanteric fractures occur most commonly in elderly patients. Osteoblasts and osteoclasts have been reported to be regulated by the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), during bone modeling and remodeling, respectively. Based on these observations, we hypothesized that the serum levels of RANKL, OPG and the RANKL/OPG ratio are important in the healing of intertrochanteric fractures in elderly patients. Enzyme-linked immunosorbent assays were used to measure the serum concentrations of RANKL and OPG in 36 elderly patients with intertrochanteric fractures and 30 age-matched healthy control subjects, at baseline and 4, 8 and 12 weeks following injury. The RANKL/OPG ratio in the two groups was also evaluated. Similar trends in RANKL and OPG levels were detected during the fracture healing process. The serum levels of RANKL and OPG were higher in the fracture group than in the controls, and were significantly higher at baseline and 4 weeks following injury (P<0.05). Notably, although the RANKL/OPG ratio gradually increased during healing, it was lower in the fracture group than in the control group. The RANKL/OPG ratio was significantly lower immediately after and 4 weeks after injury in the fracture group (P<0.05). Our data suggest a close correlation between higher serum levels of RANKL and OPG and the fracture healing process, indicating that RANKL and OPG are involved in fracture healing. The serum RANKL/OPG ratio also appears to be significant in the healing of intertrochanteric fractures in elderly patients.

Objective: To examine the relationship between inflammation and posttraumatic arthritis (PTA) in a murine intraarticular fracture model., Methods: Male C57BL/6 and MRL/MpJ "superhealer" mice received tibial plateau fractures using a previously established method. Mice were killed on day 0 (within 4 hours of fracture) and days 1, 3, 5, 7, 28, and 56 after fracture. Synovial tissue samples, obtained prior to fracture and on days 0, 1, 3, 5, and 7 after fracture, were examined by reverse transcription-polymerase chain reaction for gene expression of proinflammatory cytokines and chemokines. Synovial fluid and serum samples were collected to measure cytokine concentrations, using enzyme-linked immunosorbent assay. Whole joints were examined histologically for the extent of synovitis and cartilage degradation, and joint tissue samples from all time points were analyzed immunohistochemically to evaluate the distribution of interleukin-1 (IL-1)., Results: Compared to C57BL/6 mice, MRL/MpJ mice had less severe intraarticular and systemic inflammation following joint injury, as evidenced by lower gene expression of tumor necrosis factor α and IL-1β in the synovial tissue and lower protein levels of IL-1α and IL-1β in the synovial fluid, serum, and joint tissues. Furthermore, after joint injury, MRL/MpJ mice had lower gene expression of macrophage inflammatory proteins and macrophage-derived chemokine (CCL22) in the synovial tissue, and also had reduced acute and late-stage infiltration of synovial macrophages., Conclusion: C57BL/6 mice exhibited higher levels of inflammation than MRL/MpJ mice, indicating that MRL/MpJ mice are protected from PTA in this model. These data thus suggest an association between joint tissue inflammation and the development and progression of PTA in mice., (Copyright © 2013 by the American College of Rheumatology.)

The differentiation of skeletal tissue phenotypes is partly regulated by mechanical forces. This mechanoregulatory aspect of tissue differentiation has been the subject of many experimental and computational investigations. However, little is known about what factors promoted the emergence of mechanoregulated tissue differentiation in evolution, even though mechanoregulated tissue differentiation, for example during development or healing of adult bone, is crucial for vertebrate phylogeny. In this paper, we use a computational framework to test the hypothesis that the emergence of mechanosensitive genes that trigger endochondral ossification in evolution will stabilise in the population and create a variable mechanoregulated response, if the endochondral ossification process enhances fitness for survival. The model combines an evolutionary algorithm that considers genetic change with a mechanoregulated fracture healing model in which the fitness of animals in a population is determined by their ability to heal their bones. The simulations show that, with the emergence of mechanosensitive genes through evolution enabling skeletal cells to modulate their synthetic activities, novel differentiation pathways such as endochondral ossification could have emerged, which when favoured by natural selection is maintained in a population. Furthermore, the model predicts that evolutionary forces do not lead to a single optimal mechanoregulated response but that the capacity of endochondral ossification exists with variability in a population. The simulations correspond with many existing findings about the mechanosensitivity of skeletal tissues in current animal populations, therefore indicating that this kind of multi-level models could be used in future population based simulations of tissue differentiation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purposes: The objective of this study was to investigate the role of stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, on bone healing and whether SDF-1 contributes to accelerating bone repair in traumatic brain injury (TBI)/fracture model., Materials and Methods: Real-time polymerase chain reaction and immunohistochemical analysis were used to detect the expression of SDF-1 during the repair of femoral bone in TBI/fracture model. The TBI/fracture model was treated with anti-SDF-1 neutralizing antibody or AMD3100, an antagonist for CXCR4, and evaluated by histomorphometry. In vitro and in vivo migration assays were used to evaluate the functional effect of SDF-1 on primary mesenchymal stem cells., Results: The expression of SDF1 and CXCR4 messenger RNA was increased during the bone healing in TBI/fracture model but was less increased in fracture only model. High expression of SDF-1 protein was observed in the surrounding tissue of the damaged bone. Treated with anti-SDF-1 antibody or AMD3100 could inhibit new bone formation. SDF-1 increased mesenchymal stem cell chemotaxis in vitro in a dose-dependent manner. The in vivo migration study demonstrated that mesenchymal stem cells recruited by SDF-1 participate in endochondral bone repair., Conclusion: The SDF-1/CXCR4 axis plays a crucial role in the accelerating fracture healing under the condition of TBI and contributes to endochondral bone repair.

Atrophic non-union represents a complex clinical condition and research is ongoing in an effort to elucidate its pathophysiology and to offer new and more efficient treatment modalities. Differences seen in fracture healing responses and final outcome may be attributed among other factors to biological variations between patients resulting in a "disturbed" signalling pathway and an "inert or deficient local biology with reduced potentials for bone regeneration". The genetic contribution with or without the interaction of other exogenous factors in cases of impaired fracture healing, is yet to be elucidated. However, preliminary animal and human studies demonstrate the molecular basis of fracture non-unions and correlate genetic variants of the molecules regulating fracture healing and their expression patterns with impaired bone healing and fracture non-union. Further research is needed to clarify the genetic component and its role and interaction with other risk factors that may result in increased susceptibility of a patient to develop this complication., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The differentiation of osteoblasts from their precursors, mesenchymal stem cells, is an important component of bone homeostasis as well as fracture healing. The A2B adenosine receptor (A2BAR) is a Gα(s)/α(q)-protein-coupled receptor that signals via cAMP. cAMP-mediated signaling has been demonstrated to regulate the differentiation of mesenchymal stem cells (MSCs) into various skeletal tissue lineages. Here, we studied the role of this receptor in the differentiation of MSCs to osteoblasts. In vitro differentiation of bone marrow-derived MSCs from A2BAR KO mice resulted in lower expression of osteoblast differentiation transcription factors and the development of fewer mineralized nodules, as compared with WT mice. The mechanism of effect involves, at least partially, cAMP as indicated by experiments involving activation of the A2BAR or addition of a cAMP analog during differentiation. Intriguingly, in vivo, microcomputed tomography analysis of adult femurs showed lower bone density in A2BAR KO mice as compared with WT. Furthermore, A2BAR KO mice display a delay in normal fracture physiology with lower expression of osteoblast differentiation genes. Thus, our study identified the A2BAR as a new regulator of osteoblast differentiation, bone formation, and fracture repair.

Objective: Although several systemic and local factors are known to impair fracture healing, there is still no explanation, why some patients with sufficient fracture stability, showing none of the existing risk factors, still fail to heal normally. An investigation of local gene expression patterns in the fracture gap of patients with non-unions could decisively contribute to a better understanding of the pathophysiology of impaired fracture healing. For the first time, this study compares the expression of a large variety of osteogenic and chondrogenic genes in patients with regular and failed fracture healing., Methods: Between March 2006 and May 2007, a total of 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. Tissue samples of patients with normal and failed fracture healing were collected intraoperatively. Patients were divided into groups depending on the fracture date, and only patients with fractures two to four weeks old and patients with non-unions more than 9 months old were included in the final analysis. For the gene expression analysis, a customised cDNA array - containing 226 genes involved in osteo- and chondrogenesis - was used., Results: In the cDNA array analysis, the expression of eight genes was significantly elevated two-fold or more in the group with failed fracture healing relative to the normal controls. Conversely, no genes were found to be expressed at a higher level in the control group. The identified genes are supposed to be involved in extracellular matrix assembly, cytoskeletal structure, and differentiative and proliferative processes., Conclusions: The differences in gene expression pattern indicate a change in the composition and structure of the extracellular matrix, and a possible turn in the healing programme towards fibrous scar tissue formation, leading to non-union., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The 1,25-(OH)(2)D metabolite mediates the endocrine actions of vitamin D by regulating in the small intestine the expression of target genes that play a critical role in intestinal calcium absorption. The major role of the vitamin D hormone on bone is indirect and mediated through its endocrine function on mineral homeostasis. However, genetic manipulation of the expression of Cyp27b1 or the VDR in chondrocytes strongly support a direct role for locally synthesized 1,25(OH)(2)D, acting through the VDR, in vascular invasion and osteoclastogenesis during endochondral bone development. Cells from the growth plate respond to the 24,25-(OH)(2)D and 1,25-(OH)(2)D metabolites in a cell maturation-dependent manner and the effects of 1,25-(OH)(2)D are thought to be mediated through binding to the membrane-associated receptor PDIA3 (protein disulfide isomerase associated 3). The physiological relevance of membrane-mediated 1,25-(OH)(2)D signaling is emerging and is discussed. Finally, preliminary results suggest that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing and support a role for 24,25-(OH)(2)D in mammalian fracture repair., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Insulin-like growth factors (IGF-I/II) are important growth factors in bone, and their actions are regulated by six IGF binding proteins (IGFBPs). However, little is known about their exact functions in fracture healing. The aim of this study was to compare the gene expression and immunolocalization of IGFs and IGFBPs between standard healing fractures and nonunions using rat experimental models. Standard healing fractures and nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of the healing fractures and the fibrous tissue of the nonunions, and gene expression were analyzed by real-time PCR. Additionally, immunolocalization of these proteins was studied by immunohistochemistry at postfracture days 7, 14, and 21. In nonunions, the gene expression of IGF-I/II and IGFBP-6 was significantly higher, and that of IGFBP-5 was significantly lower at several time points. The immunolocalization of IGF-I/II and IGFBP-5 was widely distributed in both models. In contrast, that of IGFBP-6 was barely detected in the fracture callus. In conclusion, our results suggest that IGFs/IGFBPs may have important roles not only in fracture healing but also in nonunion formation., (Copyright © 2011 Orthopaedic Research Society.)

It is well recognized by orthopedic surgeons that fractures of alcoholics are more difficult to heal successfully and have a higher incidence of non-union, but the mechanism of alcohol's effect on fracture healing is unknown. In order to give direction for the study of the effects of alcohol on fracture healing, we propose to identify gene expression and microRNA changes during the early stages of fracture healing that might be attributable to alcohol consumption. As the inflammatory stage appears to be the most critical for successful fracture healing, this paper focuses on the events at day three following fracture or the stage of inflammation. Sprague-Dawley rats were placed on an ethanol-containing or pair-fed Lieber and DeCarli diet for four weeks prior to surgical fracture. Following insertion of a medullary pin, a closed mid-diaphyseal fracture was induced using a Bonnarens and Einhorn fracture device. At three days' post-fracture, the region of the fracture calluses was harvested from the right hind-limb. RNA was extracted and microarray analysis was conducted against the entire rat genome. There were 35 genes that demonstrated significant increased expression due to alcohol consumption and 20 that decreased due to alcohol. In addition, the expression of 20 microRNAs was increased and six decreased. In summary, while it is recognized that mRNA levels may or may not represent protein levels successfully produced by the cell, these studies reveal changes in gene expression that support the hypothesis that alcohol consumption affects events involved with inflammation. MicroRNAs are known to modulate mRNA and these findings were consistent with much of what was seen with mRNA microarray analysis, especially the involvement of smad4 which was demonstrated by mRNA microarray, microRNA and polymerase chain reaction.

Fractures initiate one round of endochondral bone formation in which callus cells differentiate in a synchronous manner that temporally phenocopies the spatial variation of endochondral development of a growth plate. During fracture healing C57BL/6J (B6) mice initiate chondrogenesis earlier and develop more cartilage than bone, whereas C3H/HeJ (C3H) mice initiate osteogenesis earlier and develop more bone than cartilage. Comparison of the transcriptomes of fracture healing in these strains of mice identified the genes that showed differences in timing and quantitative expression and encode for the variations in endochondral bone development of the two mouse strains. The complement of strain-dependent differences in gene expression was specifically associated with ontologies related to both skeletal and vascular formation. Moreover, the differences in gene expression associated with vascular tissue formation during fracture healing were correlated with the underlying differences in development and function of the cardiovascular systems of these two strains of mice. Significant differences in gene expression associated with bone morphogenetic protein/transforming growth factor β (BMP/TGF-β) signal-transduction pathways were identified between the two strains, and a network of differentially expressed genes specific to the MAP kinase cascade was further defined as a subset of the genes of the BMP/TGF-β pathways. Other signal-transduction pathways that showed significant strain-specific differences in gene expression included the RXR/PPAR and G protein-related pathways. These data identify how bone and vascular regeneration are coordinated through expression of common sets of transcription and morphogenetic factors and suggest that there is heritable linkage between vascular and skeletal tissue development during postnatal regeneration., (Copyright © 2011 American Society for Bone and Mineral Research.)

Fracture healing is a well-organized process between several molecules and mediators. As known from other diseases, genetic polymorphisms may exhibit different expression patterns in these mediators. Concerning fracture healing, this may lead to an extended healing process or non-union. We investigated the incidence of polymorphisms in patients with aseptic non-unions after femoral and tibial shaft fractures as compared to patients with uneventful healing. Exclusion criteria were smoking, diabetes, bilateral fractures, systemic corticoid therapy, and septic non-unions. Analysis of allele frequencies and genotype distribution of various mediators were carried out following PCR. Clinical parameters such as injury severity and in-hospital were analyzed. Fifty patients following non-union (group NU) were enrolled, the control group consisted of 44 patients (group H). A significant association of a PDGF haplotype and non-unions following fracture could be observed. There was a significantly increased in-hospital time and amount of surgical procedures in group NU. Polymorphisms within the PDGF gene seem to be a genetic risk factor for the development of non-unions of the lower extremity following fracture. The early identification of high risk patients could result in an adapted therapeutical strategy and might contribute to a significant decrease of posttraumatic non-unions., (Copyright © 2011 Orthopaedic Research Society.)

Small animal fracture models have gained increasing interest in fracture healing studies. To achieve standardized and defined study conditions, various variables must be carefully controlled when designing fracture healing experiments in mice or rats. The strain, age and sex of the animals may influence the process of fracture healing. Furthermore, the choice of the fracture fixation technique depends on the questions addressed, whereby intra- and extramedullary implants as well as open and closed surgical approaches may be considered. During the last few years, a variety of different, highly sophisticated implants for fracture fixation in small animals have been developed. Rigid fixation with locking plates or external fixators results in predominantly intramembranous healing in both mice and rats. Locking plates, external fixators, intramedullary screws, the locking nail and the pin-clip device allow different degrees of stability resulting in various amounts of endochondral and intramembranous healing. The use of common pins that do not provide rotational and axial stability during fracture stabilization should be discouraged in the future. Analyses should include at least biomechanical and histological evaluations, even if the focus of the study is directed towards the elucidation of molecular mechanisms of fracture healing using the largely available spectrum of antibodies and gene-targeted animals to study molecular mechanisms of fracture healing. This review discusses distinct requirements for the experimental setups as well as the advantages and pitfalls of the different fixation techniques in rats and mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A new type of endoplasmic reticulum (ER) stress transducer, Old Astrocyte Specifically Induced Substance (OASIS), which is induced by bone morphogenetic protein-2 (BMP2), has been reported to activate the transcription of type I collagen and contribute to the secretion of bone matrix proteins in osteoblasts. Here, we examined the role of OASIS in fracture healing using the fracture models in wild-type (WT) and OASIS(-/-) mice. We found that the expression of OASIS mRNA was induced after fracture. Micro-computed tomography indicated that the callus density of OASIS(-/-) mice was less than that of WT mice, and the newly formed bone in OASIS(-/-) mice exhibited a decrease of the bone volume by bone morphometric analysis. Biomechanically, the callus bone strength of OASIS(-/-) mice was inferior to that of WT mice. Based on RT-PCR, in situ hybridization, immunohistochemical, and electrophoretic analyses, it was clarified that the synthesis of type I collagen was impaired in OASIS(-/-) mice. Electron microscopic analysis revealed that OASIS(-/-) osteoblasts in the fracture callus contained the abnormal expansion of the ERs, similar to OASIS(-/-) osteoblasts in the normal skeletal development. Thus, OASIS may play a role in bone formation through the expression of type I collagen and the secretion of bone matrix proteins in fracture healing., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study examined the impact of an interleukin-6 (IL-6) knockout on fracture healing in terms of histological and biomechanical responses. Following IACUC approval, tibial fractures were produced in 4- to 6-week-old IL-6 knockouts (n = 35) and wild-type mice (n = 36) and harvested along with contralateral limbs at 2 and 6 weeks postsurgery. Histology quantified stage of healing, lymphocyte infiltration, TRAP+ cells, and osteocalcin deposition. Bend testing established maximum load and stiffness. Based on normality assessments, Mann-Whitney U or independent t-tests were used for data analysis using a p-value threshold of 0.05. Stage of healing, lymphocyte infiltration, and osteocalcin deposition were similar for all time points (p ≥ 0.243). TRAP+ cell counts were reduced approximately 10-fold in the knockout at 2 weeks (p = 0.015) but were similar at 6 weeks (p = 0.689). Force-to-failure in knockouts was approximately 40% that of wild-type mice at 2 weeks (p = 0.040) but similar at 6 weeks (p = 0.735). Knockout bone was about 25% less stiff at 2 weeks but approximately 60% stiffer at 6 weeks (p ≥ 0.110). The absence of IL-6 during early fracture healing significantly reduced osteoclastogenesis and impaired callus strength. By 6 weeks, most histological and biomechanical parameters were similar to fractures in wild-type bone., (Copyright © 2011 Orthopaedic Research Society.)

Over the last decades, the medical world has advanced dramatically in the understanding of fracture repair. The three components needed for fracture healing are osteoconduction, osteoinduction and osteogenesis. With newly designed scaffolds, ex vivo produced growth factors and isolated stem cells, most of the challenges of critical size bone defects have been resolved in vitro, and in some cases in animal models as well. However, there are still challenges needed to be overcome before these technologies can be fully converted from the bench to the bedside. These technological and biological advancements need to be converted to mass production of affordable products that can be used in every part of the world. Vascularity, full substation of scaffolds by native bone, and bio-safety are the three most critical steps to be challenged before reaching the clinical setting., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The enhancement of bone regeneration with biological agents including osteogenic growth factors and mesenchymal stem cells (MSCs) is becoming a clinical reality. Many exciting findings have been obtained following MSC implantation in animal models, and the data demonstrating their clinical efficacy in humans are promising. The overwhelming majority of experimental work has been performed with MSCs "amplified"in vitro. The nature of native MSCs in skeletal tissues however, remains poorly understood. This review summarizes recent findings pertaining to the definition and characterisation of MSCs in skeletal tissues and discusses the mechanisms of their actions in regenerating of bone in vivo. In respect to traditional tissue engineering paradigm, we bring together literature showing that the ways MSCs are extracted, expanded and implanted can considerably affect bone formation outcomes. Additionally, we discuss current animal models used in MSC research and highlight recent experiments showing important contribution of the host, and not only donor MSCs, in bone tissue formation. This knowledge provides a platform for novel therapy development for bone regeneration based on pharmacologically manipulated endogenous MSCs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Rib fractures are a frequent traumatic injury associated with a relatively high morbidity. Currently, the treatment of rib fractures is symptomatic. Since it has been reported that pulsed ultrasounds accelerates repair of limb fractures, we hypothesized that the application of pulsed ultrasounds will modify the course of healing in an animal model of rib fracture., Methods: We studied 136 male Sprague-Dawley rats. Animals were randomly assigned to different groups of doses (none, 50, 100, and 250 mW/cm(2) of intensity for 3 minutes per day) and durations (2, 10, 20, and 28 days) of treatment with pulsed ultrasounds. In every subgroup, we analyzed radiologic and histologic changes in the bone callus. In addition, we examined changes in gene expression of relevant genes involved in wound repair in both control and treated animals., Results: Histologic and radiologic consolidation was significantly increased by pulsed ultrasound treatment when applied for more than 10 days. The application of 50 mW/cm(2) was the most effective dose. Only the 100 and 250 mW/cm(2) doses were able to significantly increase messenger RNA expression of insulin-like growth factor 1, suppressor of cytokine signaling-2 and -3, and vascular endothelial growth factor and decrease monocyte chemoattractant protein-1 and collagen type II-alpha 1., Conclusions: Our findings indicate that pulsed ultrasound accelerates the consolidation of rib fractures. This study is the first to show that pulsed ultrasound promotes the healing of rib fractures. From a translational point of view, this easy, cheap technique could serve as an effective new therapeutic modality in patients with rib fractures., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Ex-vivo regional gene therapy with bone marrow cells (BMCs) overexpressing bone morphogenetic protein-2 (BMP-2) has demonstrated efficacy in healing critical sized bone defects in preclinical studies. The purpose of this preclinical study was to compare the osteoinductive potential of a novel "same day" ex-vivo regional gene therapy versus a traditional two-step approach, which involves culture expansion of the donor cells before implantation. In the "same day" strategy buffy coat cells were harvested from the rat bone marrow, transduced with a lentiviral vector-expressing BMP-2 for 1 hour and implanted into a rat femoral defect in the same sitting. There was no significant difference (P = 0.22) with respect to the radiographic healing rates between the femoral defects treated with the "same day" strategy (13/13; 100%) versus the traditional two-step approach (11/14; 78%). However, the femoral defects treated with the "same day" strategy induced earlier radiographic bone healing (P = 0.004) and higher bone volume (BV) [micro-computed tomography (micro-CT); P < 0.001]. The "same day" strategy represents a significant advance in the field of ex-vivo regional gene therapy because it offers a solution to limitations associated with the culture expansion process required in the traditional ex vivo approach. This strategy should be cost-effective when adapted for human use.

Background: The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences., Results: Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity., Conclusions: Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

Background: Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients., Methodology/principal Findings: Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture., Conclusions: The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The molecular and cellular mechanisms involved in bone development provide an insight into the nature of bone regeneration. Sox9 is a key transcription factor for chondrogenesis and is also expressed in osteochondroprogenitors during embryonic bone development. However, it has not been determined whether Sox9-expressing cells appear during fracture repair other than in the cartilaginous callus. On the other hand, the difference between bone development and repair is that the motion of the fractured segments is associated with the subsequent fate decision of osteochondrogenic precursors between osteogenesis or chondrogenesis, but the underlying mechanism of this still has to be elucidated. We herein evaluate whether Sox9-expressing cells appear during osseous regeneration in the initial stages of fracture healing in vivo. We also investigated the association between Sox9 induction and mechanical stress and the role of Runx1 expression. As a result, Sox9- and Runx1-expressing cells were detected in the periosteal callus together with Runx2 expression. Their expression levels were significantly downregulated during its ossification, as observed in embryonic bone development. The application of cyclic tension to isolated and cultured stromal cells resulted in the upregulation and maintenance of Sox9 mRNA expression in vitro. These results showed that as in early skeletal development, Sox9- and Runx1-expressing precursor cells first appear in the periosteal callus as an early fracture repair response. Our findings also suggested that the mechanical environment modulates Sox9 expression levels in osteochondrogenic precursors and consequently influences their fate decision between osteogenic and chondrogenic lineage commitment., (Copyright © 2011 S. Karger AG, Basel.)

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery., (Copyright © 2010. Published by Elsevier Inc.)

While the essential role of periosteum in cortical bone repair and regeneration is well established, the molecular pathways that control the early osteogenic and chondrogenic differentiation of periosteal stem/progenitor cells during repair processes are unclear. Using a murine segmental bone graft transplantation model, we isolated a population of early periosteum-callus-derived mesenchymal stem cells (PCDSCs) from the healing autograft periosteum. These cells express typical mesenchymal stem cell markers and are capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Characterization of these cells demonstrated that activation of the hedgehog (Hh) pathway effectively promoted osteogenic and chondrogenic differentiation of PCDSCs in vitro and induced bone formation in vivo. To determine the role of the Hh pathway in adult bone repair, we deleted Smoothened (Smo), the receptor that transduces all Hh signals at the onset of bone autograft repair via a tamoxifen-inducible RosaCreER mouse model. We found that deletion of Smo markedly reduced osteogenic differentiation of isolated PCDSCs and further resulted in a near 50% reduction in periosteal bone callus formation at the cortical bone junction as determined by MicroCT and histomorphometric analyses. These data strongly suggest that the Hh pathway plays an important role in adult bone repair via enhancing differentiation of periosteal progenitors and that activation of the Hh pathway at the onset of healing could be beneficial for repair and regeneration.

Background and Purpose: Cyclooxygenase-2 (COX-2) promotes inflammation by synthesizing pro-inflammatory prostaglandins from arachidonic acid. Inflammation is an early response to bone fracture, and ablation of COX-2 activity impairs fracture healing. Arachidonic acid is also converted into leukotrienes by 5-lipoxygenase (5-LO). We hypothesized that 5-LO is a negative regulator of fracture healing and that in the absence of COX-2, excess leukotrienes synthesized by 5-LO will impair fracture healing., Methods: Fracture healing was assessed in mice with a targeted 5-LO mutation (5-LO(KO) mice) and control mice by radiographic and histological observations, and measured by histomorphometry and torsional mechanical testing. To assess effects on arachidonic acid metabolism, prostaglandin E2, F2α, and leukotriene B4 levels were measured in the fracture calluses of control, 5-LO(KO) COX-1(KO), and COX-2(KO) mice by enzyme linked immunoassays., Results: Femur fractures in 5-LO(KO) mice rapidly developed a cartilaginous callus that was replaced with bone to heal fractures faster than in control mice. Femurs from 5-LO(KO) mice had substantially better mechanical properties after 1 month of healing than did control mice. Callus leukotriene levels were 4-fold higher in mice homozygous for a targeted mutation in the COX-2 gene (COX-2(KO)), which indicated that arachidonic acid was shunted into the 5-LO pathway in the absence of COX-2., Interpretation: These experiments show that 5-LO negatively regulates fracture healing and that shunting of arachidonic acid into the 5-LO pathway may account, at least in part, for the impaired fracture healing response observed in COX-2(KO) mice.

We previously identified osteoactivin (OA) as a novel secreted osteogenic factor with high expression in developing long bones and calvaria, and that stimulates osteoblast differentiation and matrix mineralization in vitro. In this study, we report on OA mRNA and protein expression in intact long bone and growth plate, and in fracture calluses collected at several time points up to 21 days post-fracture (PF). OA mRNA and protein were highly expressed in osteoblasts localized in the metaphysis of intact tibia, and in hypertrophic chondrocytes localized in growth plate, findings assessed by in situ hybridization and immunohistochemistry, respectively. Using a rat fracture model, Northern blot analysis showed that expression of OA mRNA was significantly higher in day-3 and day-10 PF calluses than in intact rat femurs. Using in situ hybridization, we examined OA mRNA expression during fracture healing and found that OA was temporally regulated, with positive signals seen as early as day-3 PF, reaching a maximal intensity at day-10 PF, and finally declining at day-21 PF. At day-5 PF, which correlates with chondrogenesis, OA mRNA levels were significantly higher in the soft callus than in intact femurs. Similarly, we detected high OA protein immunoexpression throughout the reparative phase of the hard callus compared to intact femurs. Interestingly, the secreted OA protein was also detected within the newly made cartilage matrix and osteoid tissue. Taken together, these results suggest the possibility that OA plays an important role in bone formation and serves as a positive regulator of fracture healing., (© 2010 Wiley-Liss, Inc.)

Objectives: Heterotopic Ossification (HO) is a significant complication after trauma occurring in 12% to 25% of fractures. Although clinical predictors have been studied to determine the likelihood of developing HO, the results have been inconsistent. This study examines genetic predictors of the HO phenotype to identify the "at-risk" patient and increase the understanding of the genetic contribution to the formation of HO., Methods: We examined the frequency of 61 single nucleotide polymorphisms (SNPs) in 1095 consecutive trauma patients with fractures. Radiographic studies of these patients were examined for HO in follow-up. Ten percent of the patients in the study demonstrated radiographic evidence of HO. Multivariate logistic regression was used to analyze each SNP independently while adjusting for severity of injury (as measured by the Trauma and Injury Severity Score)., Results: Three SNPs (beta2-adrenergic receptor, toll-like receptor 4, complement factor H) were identified that were associated with an increased or decreased frequency of HO. The less common polymorphism of the beta2-adrenergic receptor gene was associated with increased risk of HO. For toll-like receptor 4 and complement factor H, the less common polymorphism was associated with a decreased risk of HO., Conclusions: The SNPs identified as predictors of HO formation are representative of the adrenergic system, immune system, and the alternative complement system. This represents the interplay of multiple pathways that affect bone remodeling, aberrations of which may be found in the genome.

The development of new technologies to enhance skeletal healing after fracture or surgery is an important goal of musculoskeletal regenerative medicine. Although the bone morphogenetic proteins have shown some efficacy in this area, there is a need for more effective and less expensive therapies for bone repair and regeneration. A recent report demonstrating that Wnt signaling could be used to stimulate bone healing may provide a new direction for designing anabolic therapies for the skeleton. The identification of human phenotypes demonstrating robust bone formation as a result of mutations in Wnt signaling provides a strong basis for pursuing this area of investigation.

While the osteoinductive activity of recombinant bone morphogenetic protein 7 (BMP7) is well established, evaluation of the role of endogenous BMP7 in bone formation and fracture healing has been hampered by perinatal lethality in BMP7 knockout mice. Here we employ conditional deletion of BMP7 from the embryonic limb prior to the onset of skeletogenesis to create limb bones lacking BMP7. We find that the absence of locally produced BMP7 has no effect on postnatal limb growth, articular cartilage formation, maintenance of bone mass, or fracture healing. Our data suggest that other BMPs present in adult bone are sufficient to compensate for the absence of BMP7.

Traumatic bone defects and nonunion represent a significant source of morbidity and socioeconomic burden in trauma patients. The treatment of these conditions is currently hampered by inadequate therapies. This has prompted a new era of investigation into biologic therapies for augmenting fracture healing. Within this body of research, gene therapy has arisen as a novel and effective method of delivering therapeutic proteins at a site of desired bone regeneration. Gene therapy has shown tremendous potential in preclinical studies of fracture healing, but to date, no clinical trials have occurred. This article reviews the scientific basis for gene therapy in fracture healing, provides an overview of important preclinical studies that have been performed to date, and discusses the current barriers and future directions of gene therapy as it applies to fracture healing.

Loading of the rat ulna is an ideal model to examine stress fracture healing. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by fatigue loading of the rat ulna. Ulnae were harvested 1, 2, 4, 6, 8, and 10 weeks following creation of a stress fracture. Stress fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Histomorphometry demonstrated rapid progression of basic multicellular units from 1 to 4 weeks with significant slowing down of healing by 10 weeks after loading. Quantitative PCR was performed at 4 hours, 24 hours, 4 days, 7 days, and 14 days after loading. Gene expression was compared to an unloaded control group. At 4 hours after fracture, there was a marked 220-fold increase (P<0.0001) in expression of IL-6. There were also prominent peak increases in mRNA expression for OPG, COX-2, and VEGF (all P<0.0001). At 24 hours, there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days, there was a significant increase in mRNA expression for Bcl-2, COX-1, IGF-1, OPN, and SDF-1. At 7 days, there was significantly increased mRNA expression of RANKL and OPN. Prominent, upregulation of COX-2, VEGF, OPG, SDF-1, BMP-2, and SOST prior to peak expression of RANKL indicates the importance of these factors in mediating directed remodelling of the fracture line. Dramatic, early upregulation of IL-6 and IL-11 demonstrate their central role in initiating signalling events for remodelling and stress fracture healing., ((c) 2009 Elsevier Inc. All rights reserved.)

Unlabelled: We previously reported the presence of the bacterial genetic material (16S rRNA) and viable pathogens in fracture gaps specimens, which suggests an impaired pathogen recognition and/or elimination. The aim of study was to validate the hypothesis that patients with delayed bone fracture healing express the higher frequency of TLR4 mutations. Observations were performed in 295 patients treated due to closed fractures of the long bones of the lower extremity; in 151 with delayed bone union (group A), and in 144 with uneventful healing (group B). Control group consisted of 125 healthy blood donors from ethnically the same as investigations groups polish population. Fracture gaps and deep tissue biopsies served for microbiological studies, and DNA isolated from venous blood leukocyteswas used for analysis of mutations of TLR4 gene at Asp299Gly (1/W) and Thr399Ile (2/W)., Results: Microbiological studies revealed positive isolates in 31.5% fracture gaps in group A and 16.4% in group B (p < 0.05). The most frequent isolates were S. epidermidis, S. aureus and S. warneri, capitis, sciuri and lentus, in lower percentage micrococci and enterococci. Amplification of 16S rRNA was positive in 56.8 and 65.2% of fracture gaps in both groups respectively. The frequency of occurrence of 1/W was significantly higher (p <0.05) in subgroups of patients with non-healing infected vs. sterile fractures. In all subgroups with viable pathogens isolated from fracture gaps the frequency of 1/W allele was higher when compared with subgroups, where fracture gaps occurred sterile., Discussion: Performed investigations supported our previously reported observations that gaps of closed bone fractures are not sterile and are positive for 16S rRNA. Genetic predisposal to infection and inflammatory response evoked by a single TLR4 mutation may be one of the factors affecting bone union. Observed coexistence of bacterial colonization with decreased inflammatory reaction observed in individuals bearing TLR4 mutations have to be mentioned as a possible, etiologic factor responsible for delayed healing

Delays in bone healing or even the development of a nonunion could be related to the concentrations and/or functions of the bone morphogenetic proteins (BMPs). The RNA expression profile of the BMPs within fracture nonunion tissue is unknown. This preliminary descriptive study was performed to define the RNA profiles of the BMPs, their receptors, and their inhibitors within human fracture nonunion tissue and correlate them to matched healing bone. All patients had hypertrophic nonunions. Tissue samples taken from the nonunion site of 15 patients undergoing surgical treatment for an established nonunion were analyzed. The RNA expression patterns of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8; BMP receptor Types IA, IB, and II; and the BMP inhibitors chordin, Noggin, Drm (Gremlin), and follistatin were determined in the nonunion (fibrous tissue) and healing bone (callus tissue) using quantitative real-time PCR. Comparison between the nonunion and healing bone samples revealed substantially elevated concentrations of BMP-4, Drm/Gremlin, follistatin, and Noggin in nonunion tissue when compared to healing bone. In contrast, BMP-7 concentration was higher in the healing bone. Our data suggest inhibition of BMP-7, by Drm (Gremlin), follistatin, and Noggin and upregulation of BMP-4 may play an integral role in the development of nonunions.

The osteoinductive capability of BMPs appears diminished in the setting of acute infection. We applied rhBMP-2 to a segmental defect in a rat femur and measured the expression of key bone formation genes in the presence of acute infection. Types I and II collagen, osteocalcin, and BMP Type II receptor mRNA expression were characterized in 72 Sprague-Dawley rats, which received either bovine collagen carrier with 200 mug rhBMP-2 plus Staphylococcus aureus, carrier with bacteria only, carrier with rhBMP-2 only, or carrier alone. Six animals from each group were euthanized at 1, 2, and 4 weeks. Total RNA was isolated and extracted, and mRNA was determined by real-time comparative quantitative PCR. Infected defects had little expression of collagen I and II and osteocalcin mRNAs, while BMP receptor II expression with infection was greater than carrier-only controls at weeks 2 and 4. Notably, all four genes were upregulated in infected defects in the presence of rhBMP-2. Thus, in a clinical setting with a high risk of infection and nonunion, such as a compound fracture with bone loss, rhBMP-2 may increase the rate and extent of bone formation. Even if infection does occur, rhBMP-2 may allow a quicker overall recovery time.

The transcription regulator CITED2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is known to suppress genes mediating angiogenesis and extracellular matrix (ECM) remodeling. However, it is unclear whether CITED2 has a role in controlling skeletal repair or remodeling. We tested the hypothesis that CITED2 functions in bone fracture healing by suppressing the expression of genes critical to ECM remodeling, angiogenesis and osteogenesis, importantly the matrix metalloproteinases (MMPs). Three hours following mandibular osteotomy or sham surgery of adult rats, osteotomy fronts were harvested and the expression of CITED2 and genes associated with fracture healing was ascertained by quantitative PCR. In parallel, gain-of-function studies examined the effect of overexpressing CITED2 on the expression and activity of several MMPs. In the fractured mandible, CITED2 expression was inversely related to the expression of MMP-2, -3, -9, -13, VEGF, HIF-1alpha, M-CSF, RANK-L, and OPG. Consistent with this, the over-expression of CITED2 in osteoblasts inhibited the expression and activity of MMP-2, -3, -9, and -13. Taken together, the studies suggest that CITED2 is a critical upstream regulator of fracture healing. The suppression of CITED2 early after fracture may allow an optimal initiation of the healing response.

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2(-/-) mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2(-/-) mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2(-/-) callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2(-/-) mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2(-/-) fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2(-/-) mice.

Genetic studies have identified a high bone mass of phenotype in both human and mouse when canonical Wnt signaling is increased. Secreted frizzled related protein 1 (sFRP1) is one of several Wnt antagonists and among the loss-of-function mouse models in which 32-week-old mice exhibit a high bone mass phenotype. Here we show that impact fracture healing is enhanced in this mouse model of increased Wnt signaling at a physiologic level in young (8 weeks) sFRP1(-/-) mice which do not yet exhibit significant increases in BMD. In vivo deletion of sFRP1 function improves fracture repair by promoting early bone union without adverse effects on the quality of bone tissue reflected by increased mechanical strength. We observe a dramatic reduction of the cartilage callous, increased intramembranous bone formation with bone bridging by 14 days, and early bone remodeling during the 28-day fracture repair process in the sFRP1(-/-) mice. Our molecular analyses of gene markers indicate that the effect of sFRP1 loss-of-function during fracture repair is to accelerate bone healing after formation of the initial hematoma by directing mesenchymal stem cells into the osteoblast lineage via the canonical pathway. Further evidence to support this conclusion is the observation of maximal sFRP1 levels in the cartilaginous callus of a WT mouse. Hence sFRP1(-/-) mouse progenitor cells are shifted directly into the osteoblast lineage. Thus, developing an antagonist to specifically inhibit sFRP1 represents a safe target for stimulating fracture repair and bone formation in metabolic bone disorders, osteoporosis and aging.

Fibroblast growth factor-2 (FGF-2) is a crucial regulator of bone metabolism with strong stimulatory effects on both bone formation and resorption. The potent bone anabolic action of FGF-2 has been demonstrated in several animal models including rodents and non-human primates. Our recent clinical trial has revealed that recombinant human (rh) FGF-2 in gelatin hydrogel dose dependently accelerated bone union of a surgical osteotomy with an excellent safety profile, suggesting the efficacy of this agent for bone repair.

Fracture healing is a process comprising of a local bleeding followed by inflammation and differentiation of mesenchymal cells that lead to formation of soft extracellular matrix tissue, cartilage and bone. This pathway includes endo-chondral bone formation and in part intra-membranous bone formation. During this process several sets of cytokines are involved in the regulation of the progress in fracture healing. This paper reviews the molecules involved in fracture healing.

When bone tissue, which plays a central role in locomotive organs, is broken by trauma and decreased in its own function of support, it is lead to be remarkable decline in the activity of daily life (ADL). Recently, in a clinical situation, various treatments have been tried for prophylaxis of fractures, and these treatments have been rewarded with good results. However, fractures would be occurred in a certain amount and there would be serious problems in ADL if the treatment fails to repair fractures. Furthermore, fractures could spontaneously heal in several weeks, it can be achieved in early rehabilitation and improvement of ADL, if we can accelerate fracture healing. Bone morphogenetic proteins, which were advocated by Dr. Urist, would be expected to be a key to establish much better society especially for the old.

Objective: To study the correlation of intermittent low-dose administration of recombinant human parathyroid hormone (1-34) [rhPTH (1-34)] and the expression of Runx2 during early stage of fracture healing., Method: Sixty 2-month old male Sprague-Dawley rats underwent close unilateral femoral fracture and intramedullary nail fixation, and then were randomly divided into 2 equal groups: treatment group undergoing subcutaneous injection of rhPTH (1-34) 10 microgxkg(-1)xd(-1) immediately after the operation, and control group undergoing subcutaneous injection of normal saline of the same dose. Six rats from each group were killed with their bilateral femurs taken out on days 2, 4, 7, 14, and 21 after the operation to undergo X-ray photography Tissue RNA and protein were extracted from the bone tissues and the levels of Runx2 mRNA and protein expression were evaluated through real time quantitative PCR and Western-blotting. Blood samples were collected from the abdominal aorta before the rats were killed to undergo detection of serum calcium, phosphorus, and alkaline phosphatase (AKP)., Results: The Runx2 mRNA levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were 2.6 and 3.8 times as those of the control group respectively (both P<0.05). The Runx2 protein levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were significantly higher than those of the control group respectively too. Since day 14 fracture healing was seen, and the status of fracture healing was better in the rhPTH (1-34) group than in the control group on days 14 and 21. The levels of serum calcium ion on days 14 and 21 of the rhPTH (1-34) group were both significantly higher than those of the control group (both P<0.05). There were not significant differences in the serum levels of AKP, osteocalcin and collagen type Iat any time point between the 2 groups., Conclusion: Intermittent low-dose administration of rhPTH (1-34) up-regulates the levels of osteogenesis-specific Runx2 mRNA and protein expression to accelerate the early stage fracture healing during the early stage.

Fibroblast growth factors (FGFs) are important signaling molecules that regulate many stages of endochondral bone development. During the healing of a skeletal fracture, several features of endochondral bone development are reactivated. To better understand the role of FGFs in skeletal fracture healing, we quantitatively evaluated the temporal expression patterns of Fgfs, Fgf receptors (Fgfrs), and molecular markers of bone development over a 14-day period following long bone fracture in a mouse model. These studies identify distinct groups of FGFs that are differentially expressed and suggest active stage-specific roles for FGF signaling during the fracture repair process., ((c) 2009 Wiley-Liss, Inc.)