Midkine-Deficiency Delays Chondrogenesis during the Early Phase of Fracture Healing in Mice

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.     

Jmjd3和Ezh2拮抗调节小鼠骨折愈合

目的:探讨Jmjd3和Ezh2在小鼠骨折愈合过程中的作用。方法:以软骨细胞条件性基因敲除8-10周龄小鼠为研究对象,按基因型随机分为6组,每组5只:其中实验组基因型为Jmjd3~(fl/fl)/Col2a1-Cre ~(ERT2),Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2)或Jmjd~(3fl/fl)/Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2);对照组基因型为Jmjd3~(fl/fl),Ezh2~(fl/fl)或Jmjd3~(fl/fl)/Ezh2~(fl/fl)。建立骨髓腔中插入固定针的稳定性胫骨骨折模型,于骨折术后3天、5天和7天腹腔注射Tamoxifen 3 mg/次/天。各组于术后3W处死,并于骨折部位取材行X线片及组织学检查。结果:通过连续的X线影像学及HE组织切片观察,骨折术后3周是判断小鼠骨折愈合情况的最佳时间点。X线片发现骨折术后3W时软骨细胞内Jmjd3被敲除小鼠的骨折线较对照组明显且骨化骨痂大小和密度均较低,HE切片显示骨化骨痂面积显著低于对照组,而软骨骨痂面积高于对照组;相反,X线片发现Ezh2被敲除小鼠的骨痂面积明显大于对照组,且密度高于对照组,HE组织切片显示Ezh2被敲除的小鼠的骨化骨痂的钙化程度更高,骨小梁更粗更密集。最后,X线片和HE切片均没有发现软骨细胞Jmjd3和Ezh2同时被敲除的小鼠与对照小鼠之间存在明显差异。结论:以软骨细胞特异基因敲除小鼠为基础,我们首次发现Jmjd3具有促进骨折愈合的作用,而Ezh2具有抑制骨折愈合的作用;并且发现Jmjd3和Ezh2对抗调节小鼠的骨折愈合过程,这些发现为骨折愈合治疗提供了新的分子实验基础。     

Biglycan modulates angiogenesis and bone formation during fracture healing

Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (μCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.     

Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34)

Background

Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH.

Methodology/Principal Findings

Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth ^−/−) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1–4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth ^−/− mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth ^−/− mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth ^−/− mice compared to vehicle-treated wild-type and Pth ^−/− mice.

Conclusions/Significance

Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.     

Bushenhuoxue formula accelerates fracture healing via upregulation of TGF-β/Smad2 signaling in mesenchymal progenitor cells

BackgroundAlthough Bushenhuoxue formula (BSHXF) is successfully used as a non-traumatic therapy in treating bone fracture in China, the molecular mechanism underlying its effects remains poorly understood.PurposeThe present study aims to explore the therapeutic effects of BSHXF on fracture healing in mice and the underlying mechanism.MethodsWe performed unilateral open transverse tibial fracture procedure in C57BL/6 mice which were treated with or without BSHXF. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and quantitative gene expression analysis. Tibial fracture procedure was also performed in Cre-negative and Gli1-CreER; Tgfbr2flox/flox conditional knockout (KO) mice (Tgfbr2^Gli1ER) to determine if BSHXF enhances fracture healing in a TGF-β-dependent manner. In addition, scratch-wound assay and cell counting kit-8 (CCK-8) assay were used to evaluate the effect of BSHXF on cell migration and cell proliferation in C3H10T1/2 mesenchymal stem cells, respectively.ResultsBSHXF promoted endochondral ossification and enhanced bone strength in wild-type (WT) or Cre- control mice. In contrast, BSHXF failed to promote bone fracture healing in Tgfbr2^Gli1ER conditional KO mice. In the mice receiving BSHXF treatment, TGF-β/Smad2 signaling was significantly activated. Moreover, BSHXF enhanced cell migration and cell proliferation in C3H10T1/2 cells, which was strongly attenuated by the small molecule inhibitor SB525334 against TGF-β type I receptor.ConclusionThese data demonstrated that BSHXF promotes fracture healing by activating TGF-β/Smad2 signaling. BSHXF may be used as a type of alternative medicine for the treatment of bone fracture healing.     

Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures

Abstract

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide synthases (NOS) and oxidizes to its stable end-products nitrite (NO₂^-) and nitrate (NO₃^-) We have previously reported in an in vivo rat model that NO is an important regulator for rat bone fracture healing.¹ This study examines the effects of NO on alkaline phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced NO₂^- release and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and the lowest activity on day 14. Inhibition of NOS by co-incubation with an NOS inhibitor,S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU), inhibited ALP activity by an average of 50% at each time point (P <0.01). Supplementation with NO donor 3-morpholino-sydnonomine hydrochloride (SIN-1) at low doses (25 and 0.025 µM) increased ALP activity by 20% (P <0.01). ALP mRNA and histochemical ALP activity were localised to osteoblast-like and chondrocyte-like cells within fracture callus. The current study provides evidence that NO plays a regulatory role in ALP activity during rat fracture healing.     

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.     

Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3 ^-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3 ^-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3 ^-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3 ^-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3 ^-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3 ^-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3 ^-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3 ^-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3 ^-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression of wound closure.     

Genetics of human C4 polymorphism: Detection and segregation of rare and duplicated haplotypes

Applying a combined technology for the detection of allotypec variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B -chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 -chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p. i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p. i. and the C4A1 and C4B51 allotypes as Ch p. i. were genetically inherited. The C4A1 allotype has a unique Rg^- Ch⁺ C4A -chain. Duplicated C4A loci, A ^*3, A ^*2, and A ^*5, A ^*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.Abbreviations used in this paper BF Factor B polymorphism of the alternative pathway of complement activation - C2 second component of complement - C4 fourth component of complement - C4D C4-deficient (C4^*QO/QO) - Ch Chido determinant on C4B^* products - EDTA ethylendiaminetetraacetic acid - GLO I glyoxalase I - HLA human leucocyte antigens, A, B, C and DR (D =related) loci - PAGE polyacrylamide gel electrophoresis - PGM3 phosphoglucomutase, third locus - p. i. partial inhibitor = serological inhibition of some, but not all anti-Ch and anti-Rg sera at selected dilutions - SDS sodium dodecyl sulphate; 94k/96k, 94 000 and 96 000 dalton molecular weight Presented in part at the 1V International Workshop on the Genetics of Complement, July 13–15, 1982, Boston, MA, and the Xth International Complement Workshop, May 25–27,1983 in Mainz, Federal Republic of Germany.     

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     

Fracture Healing Is Delayed in Immunodeficient NOD/scid??IL2Rγ?cnull Mice

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγ_c^null mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγ_c^null mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγ_c^null mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγ_c^null mice. Our results suggest that—under aseptic, uncomplicated conditions—the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγ_c^null mice.     

Identification of circulating murine CD34+OCN+ cells

Background aims

Previous studies identified a circulating human osteoblastic population that expressed osteocalcin (OCN), increased following fracture and pubertal growth, and formed mineralized colonies in vitro and bone in vivo. A subpopulation expressed CD34, a hematopoietic/endothelial marker. These findings led to our hypothesis that hematopoietic-derived CD34⁺OCN⁺ cells exist in the circulation of mice and are modulated after fracture.

Impaired Fracture Healing Caused by Deficiency of the Immunoreceptor Adaptor Protein DAP12

Osteoclasts play an important role in bone metabolism, but their exact role in fracture healing remains unclear. DAP12 is an immunoadaptor protein with associated immunoreceptors on myeloid lineage cells, including osteoclasts. Its deficiency causes osteopetrosis due to suppression of osteoclast development and activation. In this report, we assessed the impact of DAP12 on the fracture healing process using C57BL/6 (B6) and DAP12^–/– mice. Healing was evaluated using radiography, micro-CT, histology, immunohistochemistry and real-time RT-PCR. Radiography showed lower callus volume and lower callus radiolucency in DAP12^–/– mice during later stages. Micro-CT images and quantitative structural analysis indicated that DAP12^–/– mice developed calluses of dense trabecular structures and experienced deteriorated cortical shell formation on the surface. Histologically, DAP12^–/– mice showed less cartilaginous resorption and woven bone formation. In addition, prominent cortical shell formation was much less in DAP12^–/– mice. Immunohistochemistry revealed lower invasion of F4/80 positive monocytes and macrophages into the fracture hematoma in DAP12^–/– mice. The expression levels of Col1a1, Col2a1 and Col10a1 in DAP12^–/– mice increased and subsequently became higher than those in B6 mice. There was a decrease in the gene expression of Tnf during the early stages in DAP12^–/– mice. Our results indicate that DAP12 deficiency impairs fracture healing, suggesting a significant role of DAP12 in the initial inflammatory response, bone remodeling and regeneration.     

Virtual structural analysis of tibial fracture healing from low-dose clinical CT scans

Quantitative assessment of bone fracture healing remains a significant challenge in orthopaedic trauma research. Accordingly, we developed a new technique for assessing bone healing using virtual mechano-structural analysis of computed tomography (CT) scans. CT scans from 19 fractured human tibiae at 12 weeks after surgery were segmented and prepared for finite element analysis (FEA). Boundary conditions were applied to the models to simulate a torsion test that is commonly used to access the structural integrity of long bones in animal models of fracture healing. The output of each model was the virtual torsional rigidity (VTR) of the healing zone, normalized to the torsional rigidity of each patient’s virtually reconstructed tibia. This provided a structural measure to track the percentage of healing each patient had undergone. Callus morphometric measurements were also collected from the CT scans. Results showed that at 12 weeks post-op, more than 75% of patients achieved a normalized VTR (torsional rigidity relative to uninjured bone) of 85% or above. The predicted intact torsional rigidities compared well with published cadaveric data. Across all patients, callus volume and density were weakly and non-significantly correlated with normalized VTR and time to clinical union. Conversely, normalized VTR was significantly correlated with time to union (R² = 0.383, p = 0.005). This suggests that fracture scoring methods based on the visual appearance of callus may not accurately predict mechanical integrity. The image-based structural analysis presented here may be a useful technique for assessment of bone healing in orthopaedic trauma research.     

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.     

Hyperbaric Hyperoxia Accelerates Fracture Healing in Mice

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O₂ at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O₂ at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2′-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.     

Systemic Inhibition of Canonical Notch Signaling Results in Sustained Callus Inflammation and Alters Multiple Phases of Fracture Healing

The Notch signaling pathway is an important regulator of embryological bone development, and many aspects of development are recapitulated during bone repair. We have previously reported that Notch signaling components are upregulated during bone fracture healing. However, the significance of the Notch pathway in bone regeneration has not been described. Therefore, the objective of this study was to determine the importance of Notch signaling in regulating bone fracture healing by using a temporally controlled inducible transgenic mouse model (Mx1-Cre;dnMAML^f/-) to impair RBPjκ-mediated canonical Notch signaling. The Mx1 promoter was synthetically activated resulting in temporally regulated systemic dnMAML expression just prior to creation of bilateral tibial fractures. This allowed for mice to undergo unaltered embryological and post-natal skeletal development. Results showed that systemic Notch inhibition prolonged expression of inflammatory cytokines and neutrophil cell inflammation, and reduced the proportion of cartilage formation within the callus at 10 days-post-fracture (dpf) Notch inhibition did not affect early bone formation at 10dpf, but significantly altered bone maturation and remodeling at 20dpf. Increased bone volume fraction in dnMAML fractures, which was due to a moderate decrease in callus size with no change in bone mass, coincided with increased trabecular thickness but decreased connectivity density, indicating that patterning of bone was altered. Notch inhibition decreased total osteogenic cell density, which was comprised of more osteocytes rather than osteoblasts. dnMAML also decreased osteoclast density, suggesting that osteoclast activity may also be important for altered fracture healing. It is likely that systemic Notch inhibition had both direct effects within cell types as well as indirect effects initiated by temporally upstream events in the fracture healing cascade. Surprisingly, Notch inhibition did not alter cell proliferation. In conclusion, our results demonstrate that the Notch signaling pathway is required for the proper temporal progression of events required for successful bone fracture healing.     

The Wnt Serpentine Receptor Frizzled-9 Regulates New Bone Formation in Fracture Healing

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9 ^−/− mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures.     

Skeletal muscle reperfusion injury is mediated by neutrophils and the complement membrane attack complex

The relative inflammatory roles ofneutrophils, selectins, and terminal complement components areinvestigated in this study of skeletal muscle reperfusion injury. Miceunderwent 2 h of hindlimb ischemia followed by 3 h ofreperfusion. The role of neutrophils was defined by immunodepletion,which reduced injury by 38%, as did anti-selectin therapy withrecombinant soluble P-selectin glycoprotein ligand-immunoglobulin (Ig)fusion protein. Injury in C5-deficient and soluble complement receptortype 1-treated wild-type mice was 48% less than that of untreatedwild-type animals. Injury was restored in C5-deficient micereconstituted with wild-type serum, indicating the effector role ofC5-9. Neutropenic C5-deficient animals showed additive reductionin injuries (71%), which was lower than C5-deficientneutrophil-replete mice, indicating neutrophil activity withoutC5a. Hindlimb histological injury was worse in ischemicwild-type and C5-deficient animals reconstituted with wild-type serum.In conclusion, the membrane attack complex and neutrophils actadditively to mediate skeletal muscle reperfusion injury. Neutrophilactivity is independent of C5a but is dependent on selectin-mediated adhesion.

     

Inactivation of Fam20C in Cells Expressing Type I Collagen Causes Periodontal Disease in Mice

Background

FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues.

Methods

By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C^fl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20C^fl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches.

Results

The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice.

Conclusion

Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.