Early Phase Of Fracture Healing Research Articles

Inflammatory response toward a Mg-based metallic biomaterial implanted in a rat femur fracture model

The immune system plays an important role in fracture healing, by modulating the pro-inflammatory and anti-inflammatory responses occurring instantly upon injury. An imbalance in these responses can lead to adverse outcomes, such as non-union of fractures. Implants are used to support and stabilize complex fractures. Biodegradable metallic implants offer the potential to avoid a second surgery for implant removal, unlike non-degradable implants. However, considering our dynamic immune system it is important to conduct in-depth studies on the immune response to these implants in living systems. In this study, we investigated the immune response to Mg and Mg-10Gd in vivo in a rat femur fracture model with external fixation. In vivo imaging using liposomal formulations was used to monitor the fluorescence-related inflammation over time. We combine ex vivo methods with our in vivo study to evaluate and understand the systemic and local effects of the implants on the immune response. We observed no significant local or systemic effects in the Mg-10Gd implanted group compared to the SHAM and Mg implanted groups over time. Our findings suggest that Mg-10Gd is a more compatible implant material than Mg, with no adverse effects observed in the early phase of fracture healing during our 4-week study. Statement of SignificanceDegradable metallic implants in form of Mg and Mg-10Gd intramedullary pins were assessed in a rat femur fracture model, alongside a non-implanted SHAM group with special respect to the potential to induce an inflammatory response. This pre-clinical study combines innovative non-invasive in vivo imaging techniques associated with multimodal, ex vivo cellular and molecular analytics. The study contributes to the development and evaluation of degradable biometals and their clinical application potential. The study results indicate that Mg-10Gd did not exhibit any significant harmful effects compared to the SHAM and Mg groups.

Inflammatory response toward a Mg-based metallic biomaterial implanted in a rat femur fracture model

The immune system plays an important role in fracture healing, by modulating the pro-inflammatory and anti-inflammatory responses occurring instantly upon injury. An imbalance in these responses can lead to adverse outcomes, such as non-union of fractures. Implants are used to support and stabilize complex fractures. Biodegradable metallic implants offer the potential to avoid a second surgery for implant removal, unlike non-degradable implants. However, considering our dynamic immune system it is important to conduct in-depth studies on the immune response to these implants in living systems. In this study, we investigated the immune response to Mg and Mg-10Gd in vivo in a rat femur fracture model with external fixation. In vivo imaging using liposomal formulations was used to monitor the fluorescence-related inflammation over time. We combine ex vivo methods with our in vivo study to evaluate and understand the systemic and local effects of the implants on the immune response. We observed no significant local or systemic effects in the Mg-10Gd implanted group compared to the SHAM and Mg implanted groups over time. Our findings suggest that Mg-10Gd is a more compatible implant material than Mg, with no adverse effects observed in the early phase of fracture healing during our 4-week study. Statement of SignificanceDegradable metallic implants in form of Mg and Mg-10Gd intramedullary pins were assessed in a rat femur fracture model, alongside a non-implanted SHAM group with special respect to the potential to induce an inflammatory response. This pre-clinical study combines innovative non-invasive in vivo imaging techniques associated with multimodal, ex vivo cellular and molecular analytics. The study contributes to the development and evaluation of degradable biometals and their clinical application potential. The study results indicate that Mg-10Gd did not exhibit any significant harmful effects compared to the SHAM and Mg groups.

Age-related changes to macrophage subpopulations and TREM2 dysregulation characterize attenuated fracture healing in old mice.

Fracture healing complications increase with age, with higher rates of delayed unions and nonunions and an associated increase in morbidity and mortality in older adults. Macrophages have a dynamic role in fracture healing, and we have previously demonstrated that age-related changes in macrophages are associated with attenuated fracture repair in old mice. Here, we provide a single cell characterization of the immune cells involved in the early phase of fracture healing. We show that there were multiple transcriptionally distinct macrophage subpopulations present simultaneously within the healing tissue. Fracture healing was attenuated in old mice compared to young, and macrophages from the fracture callus of old mice demonstrated a pro-inflammatory phenotype compared to young. Interestingly, Trem2 expression was decreased in old macrophages compared to young. Young mice lacking Trem2 demonstrated attenuated fracture healing and inflammatory dysregulation similar to old mice. Trem2 dysregulation has previously been implicated in other age-related diseases, but its role in fracture healing is unknown. This work provides a robust characterization of the macrophage subpopulations involved in fracture healing, and further reveals the important role of Trem2 in fracture healing and may be a potential driver of age-related inflammatory dysregulation. Future work may further examine macrophages and Trem2 as potential therapeutic targets for management of fracture repair in older adults.

Diclofenac, a NSAID, delays fracture healing in aged mice

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (μCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling.

Ultrasonic-controlled “explosive” hydrogels to precisely regulate spatiotemporal osteoimmune disturbance

Spatiotemporal Immune disorder is a key factor leading to the failure of bone tissue healing. It is of vital importance to accurately suppress excessive peak immune response within 24-48 h of the injury and so regulate the spatiotemporal osteoimmune disturbance of bones. In this study, Ultrasound Controlled “Explosive” (UCE) hydrogels were prepared from gelatin-hyaluronic acid methacrylate hydrogels loaded with resveratrol nanobubbles produced by double emulsification through a condensation reaction. Such materials innovatively enable ultrasound-controlled RES release for precise regulation of spatiotemporal osteoimmune disorders. Under an ultrasonic power level of 1.5 W/cm2, the rate of effectively released RES through the blast of UCE hydrogels reached 38.14 %. And compared with the control group, the in vivo inhibition of inflammation and osteogenesis effects of UCE hydrogels were more effective, respectively. As suggested by the results, the excessive local inflammatory response was inhibited by the release of resveratrol, the temporospatial disorder of bone immune was precisely regulated, and as a result, the process of bone repair was accelerated. Altogether, this study confirms that the newly created UCE Hydrogels effectively promote bone repair by intervening peak inflammation during the early phase of fracture healing.

The specialist in regeneration—the Axolotl—a suitable model to study bone healing?

While the axolotl’s ability to completely regenerate amputated limbs is well known and studied, the mechanism of axolotl bone fracture healing remains poorly understood. One reason might be the lack of a standardized fracture fixation in axolotl. We present a surgical technique to stabilize the osteotomized axolotl femur with a fixator plate and compare it to a non-stabilized osteotomy and to limb amputation. The healing outcome was evaluated 3 weeks, 3, 6 and 9 months post-surgery by microcomputer tomography, histology and immunohistochemistry. Plate-fixated femurs regained bone integrity more efficiently in comparison to the non-fixated osteotomized bone, where larger callus formed, possibly to compensate for the bone fragment misalignment. The healing of a non-critical osteotomy in axolotl was incomplete after 9 months, while amputated limbs efficiently restored bone length and structure. In axolotl amputated limbs, plate-fixated and non-fixated fractures, we observed accumulation of PCNA+ proliferating cells at 3 weeks post-injury similar to mouse. Additionally, as in mouse, SOX9-expressing cells appeared in the early phase of fracture healing and amputated limb regeneration in axolotl, preceding cartilage formation. This implicates endochondral ossification to be the probable mechanism of bone healing in axolotls. Altogether, the surgery with a standardized fixation technique demonstrated here allows for controlled axolotl bone healing experiments, facilitating their comparison to mammals (mice).

Wealth index is significantly associated with the early phase of fracture healing among fractured patients at the University of Gondar Specialized Hospital, Northwest Ethiopia

Bone fracture is a cause of 5 million annual injuries globally. It contributes to profound physical impairment, a reduction in quality of life, long hospitalization time, and reduced institutional care. The socioeconomical impact of bone fracture will be high in the next 50 years in developing countries, including Ethiopia. This study aimed to assess the factors associated with the early phase of fracture healing among fractured adult patients at the University of Gondar Specialized hospital, Northwest Ethiopia. A secondary data analysis was done from the previous institution-based prospective follow-up study from March to June 2015. An interviewer-administered pretested and structured questionnaire was used to collect the data. Early ossification of fractured bone was defined when the bone was ossified/calcified within 1 month of the fracture using X-ray findings. Data were entered into EpiInfo version 3.5.3 software and exported to SPSS version 20 for Windows for further analysis. Binary logistic regression analysis was employed to find factors associated with early ossification of a fractured bone. In the multivariable model, variables that had a p–value of < 0.05 were considered statistically significant. Also, a 95% CI for odds ratios was calculated to see the degree of association. In total, 118 participants with a bone fracture participated in this study. Of all, 36.4% of them had ossified bone during the early phase of bone healing. Dietary diversity score (DDS) [AOR = 18.6, 95% CI (4.10–84.34)], the type of fractured bone [AOR: = 0.13, 95% CI (0.03–0.50)], and household wealth index [AOR = 8.6, 95% CI (1.51–49.05)] were independently associated with the early phase of fractured bone healing/ossification. Bone ossification during the first month of fracture was low. Dietary diversity score, type of fractured bone, and household wealth index were the factors associated with the early phase of bone ossification. So, improving dietary practice after the incident is essential. Also, health care providers will have to consider the fractured bone type and household assets during the clinical intervention for fractured bone.

FOXO1 expression in chondrocytes modulates cartilage production and removal in fracture healing.

Fracture healing is a multistage process characterized by inflammation, cartilage formation, bone deposition, and remodeling. Chondrocytes are important in producing cartilage that forms the initial anlagen for the hard callus needed to stabilize the fracture site. We examined the role of FOXO1 by selective ablation of FOXO1 in chondrocytes mediated by Col2α1 driven Cre recombinase. Experimental mice with lineage-specific FOXO1 deletion (Col2α1Cre+FOXO1L/L) and negative control littermates (Col2α1Cre-FOXO1L/L) were used for in vivo, closed fracture studies. Unexpectedly, we found that in the early phases of fracture healing, FOXO1 deletion significantly increased the amount of cartilage formed, whereas, in later periods, FOXO1 deletion led to a greater loss of cartilage. FOXO1 was functionally important as its deletion in chondrocytes led to diminished bone formation on day 22. Mechanistically, the early effects of FOXO1 deletion were linked to increased proliferation of chondrocytes through enhanced expression of cell cycle genes that promote proliferation and reduced expression of those that inhibit it and increased expression of cartilage matrix genes. At later time points experimental mice with FOXO1 deletion had greater loss of cartilage, enhanced formation of osteoclasts, increased IL-6 and reduced numbers of M2 macrophages. These results identify FOXO1 as a transcription factor that regulates chondrocyte behavior by limiting the early expansion of cartilage and preventing rapid cartilage loss at later phases.

The Long Pentraxin PTX3 in Bone Homeostasis and Pathology.

The innate immune system is equipped with a number of germ-line encoded soluble pattern recognition molecules (PRMs) that collectively mediate the humoral host response to infection and damage in cooperation with cells and tissues of the immune and non-immune compartments. Despite the impressive diversity in structure, source, and regulation across PRMs, these all share remarkably similar functions inasmuch as they recognize microbes and damaged tissues, activate complement, exert opsono-phagocytic activities, and regulate inflammation. The long pentraxin 3 (PTX3) is a prototypic soluble PRM. Long known as a major player in innate immunity, inflammation and matrix remodeling, only recently has PTX3 emerged as a mediator of bone homeostasis in rodents and humans. Ptx3-targeted mice exhibit reduced trabecular volume during bone development, and impaired callus mineralization following experimental fracture. The murine gene is expressed in vivo by non-hematopoietic periosteal cells in the early phases of fracture healing, and in vitro by maturing osteoblasts. Human osteoblasts do express the PTX3 protein, whose levels positively correlate with bone density in vivo and osteoblast proliferation and maturation in vitro, thus pointing to a role in bone deposition. Contrasting evidence, however, suggest osteoclastogenesis-promoting effects of PTX3, where its expression has been associated with periodontitis, arthritis, and bone metastasis, conditions hallmarked by inflammation and bone resorption. Here, we review past and recent literature on the functions exerted by this long pentraxin in bone biology, with major emphasis on physiological skeletal remodeling, fracture healing, and chronic diseases of the bone.

A PDGFRβ-PI3K signaling axis mediates periosteal cell activation during fracture healing.

Insufficient and delayed fracture healing remain significant public health problems with limited therapeutic options. Phosphoinositide 3-kinase (PI3K) signaling, a major pathway involved in regulation of fracture healing, promotes proliferation, migration, and differentiation of osteoprogenitors. We have recently reported that knock-in mice with a global increase in PI3K signaling (gCblYF) show enhanced femoral fracture healing characterized by an extraordinary periosteal response to injury. Interestingly, of all growth factor receptors involved in fracture healing, PI3K directly binds only to PDGFR. Given these findings, we hypothesized a PDGFR-PI3K interaction is necessary for mediating robust periosteal cell activation following fracture. In this study, we isolated primary periosteal cells from gCblYF mice to analyze cross-talk between the PDGFRβ and PI3K signaling pathways. We found PDGFRβ signaling contributes to robust Akt phosphorylation in periosteal cells in comparison with other growth factor signaling pathways. Additionally, we performed femoral fractures on gCblYF mice with a conditional removal of PDGFRβ in mesenchymal progenitors using inducible alpha smooth muscle actin (αSMA) CreERT2 mice. Our studies showed that depletion of PDGFRβ signaling within these progenitors in the early phase of fracture healing significantly abrogates PI3K-mediated periosteal activation and proliferation three days after fracture. Combined, these results suggest that PDGFRβ signaling through PI3K is necessary for robust periosteal activation in the earliest phases of fracture healing.

Pharmacologic targeting of \u03b2-catenin improves fracture healing in old mice

β-catenin protein needs to be precisely regulated for effective fracture repair. The pace of fracture healing slows with age, associated with a transient increase in β-catenin during the initial phase of the repair process. Here we examined the ability of pharmacologic agents that target β-catenin to improve the quality of fracture repair in old mice. 20 month old mice were treated with Nefopam or the tankyrase inhibitor XAV939 after a tibia fracture. Fractures were examined 21 days later by micro-CT and histology, and 28 days later using mechanical testing. Daily treatment with Nefopam for three or seven days but not ten days improved the amount of bone present at the fracture site, inhibited β-catenin protein level, and increased colony forming units osteoblastic from bone marrow cells. At 28 days, treatment increased the work to fracture of the injured tibia. XAV939 had a more modest effect on β-catenin protein, colony forming units osteoblastic, and the amount of bone at the fracture site. This data supports the notion that high levels of β-catenin in the early phase of fracture healing in old animals slows osteogenesis, and suggests a pharmacologic approach that targets β-catenin to improve fracture repair in the elderly.

Enhancement of bone consolidation using high-frequency pulsed electromagnetic fields (HF-PEMFs): An experimental study on rats.

In vitro studies showed that high-frequency pulsed electromagnetic fields (HF-PEMFs) increase the activity/expression of early and late osteogenic markers and enhance bone mineralization. The main aim of this study was to investigate the in vivo effects of HF-PEMFs on fracture healing using a rat model. A femur fracture was established by surgery in 20 male Wistar rats. Titanium nails were implanted to reduce and stabilize the fracture. After surgery, 20 rats were equally divided into untreated control and treated group (from the first postoperative day HF-PEMFs at 400 pulses/sec [pps] were applied for 10 minutes/day, for two weeks). Quantitative and qualitative assessment of bone formation was made at two and eight weeks following surgery and included morphological and histological analysis, serological analysis by ELISA, micro-computed tomography (micro-CT), and three-point bending test. At two weeks in HF-PEMF group, soft callus was at a more advanced fibrocartilaginous stage and the bone volume/total tissue volume (BV/TV) ratio in the callus area was significantly higher compared to control group (p = 0.047). Serum concentration of alkaline phosphatase (ALP) and osteocalcin (OC) was significantly higher in HF-PEMF group (ALP p = 0.026, OC p = 0.006) as well as the mechanical strength of femurs (p = 0.03). At eight weeks, femurs from HF-PEMF group had a completely formed woven bone with dense trabeculae, active bone marrow, and had a significantly higher BV/TV ratio compared to control (p = 0.01). HF-PEMFs applied from the first postoperative day, 10 minutes/day for two weeks, enhance bone consolidation in rats, especially in the early phase of fracture healing.

Notch in skeletal physiology and disease.

Notch (Notch1 through 4) are transmembrane receptors that play a fundamental role in cell differentiation and function. Notch receptors are activated following interactions with their ligands in neighboring cells. There are five classic ligands termed Jagged (Jag)1 and Jag2 and Delta-like (Dll)1, Dll3, and Dll4. Recent work has established Notch as a signaling pathway that plays a critical role in the differentiation and function of cells of the osteoblast and osteoclast lineages and in skeletal development and bone remodeling. The effects of Notch are cell-context dependent, and the four Notch receptors carry out specific functions in the skeleton. Gain- and loss-of-function mutations of components of the Notch signaling pathway result in a variety of congenital disorders with significant craniofacial and skeletal manifestations. The Notch ligand Jag1 is a determinant of bone mineral density, and Notch plays a role in the early phases of fracture healing. Alterations in Notch signaling are associated with osteosarcoma and with the metastatic potential of carcinoma of the breast and of the prostate. Controlling Notch signaling could prove useful in diseases of Notch gain-of-function and in selected skeletal disorders. However, clinical data on agents that modify Notch signaling are not available. In conclusion, Notch signaling is a novel pathway that regulates skeletal homeostasis in health and disease.

Hemorrhagic shock alters fracture callus composition and activates the IL6 and RANKL/OPG pathway in mice.

Fracture and hemorrhagic shock often lead to impaired fracture healing. To elucidate underlying pathogenesis, this study aimed to analyze histological properties during fracture healing after hemorrhagic shock and involved signaling pathways in mice. Male C57BL/6NCrl mice were assigned into five groups. Control group underwent no interventions. Sham group had a catheter and external fixator but neither blood loss nor osteotomy. Trauma-hemorrhage (TH) group received a pressure-controlled hemorrhagic shock; osteotomy (Fx) group, an osteotomy and fixator; and combined trauma (THFx) group, both hemorrhagic shock and externally fixed osteotomy. After 1, 2, 3, and 4 weeks, the animals were killed. Undecalcified bones were analyzed histologically and signaling pathways relevant for fracture healing by polymerase chain reaction and Western blot. Statistical significance was set at 0.05 or less. Comparisons were performed using the Mann-Whitney U or Kruskal-Wallis test. In the THFx group, a decreased bone formation after 3 weeks, a reduction of both bone and cartilage after 2 weeks, and an enhanced activation of the RANKL/OPG and IL6 signaling pathway after 1 week were shown in comparison to Fx. Hemorrhagic shock has a retarding effect on fracture healing in the early phase of fracture healing and leads to activation of the IL6 and RANKL/OPG signaling pathways.

N-Acetyl Cysteine Treatment Restores Early Phase Fracture Healing in Ethanol-Fed Rats.

Fracture healing in alcoholics is delayed and often associated with infections resulting in prolonged rehabilitation. It has been reported that binge drinking of alcohol increases oxidative stress and delays fracture healing in rats, which is prevented by treatment with the antioxidant n-acetyl cysteine (NAC). Oxidative stress is a significant factor in pathologies of various organs resulting from chronic alcoholism. Therefore, we hypothesize that treatment with NAC reduces oxidative stress and restores fracture healing in chronic alcoholics. Rats (10months old) were pair-fed the Lieber-DeCarli ethanol (EtOH) diet or control diet for 16weeks. A closed fracture was performed and rats allowed to recover for 72hours. Rats were divided into 4 groups-control, control+NAC, EtOH, and EtOH+NAC-and injected intraperitoneally with 200mg/kg of NAC daily for 3days. Serum and bone fracture callus homogenates were collected and assayed for traditional markers of inflammation, oxidative stress, and bone regeneration. The oxidative stress marker malondialdehyde (MDA) was increased in both serum and bone tissue in EtOH-fed animals compared to controls. NAC treatment significantly (p<0.01) reduced MDA to near normal levels and dramatically increased the index of antioxidant efficacy (catalase/MDA ratio) (p<0.01). Inflammatory markers tumor necrosis factor-α, interferon-γ, and interleukin-6 were significantly decreased in serum and callus following NAC treatment. NAC treatment reduced EtOH-induced bone resorption as evidenced by significant decreases in C-telopeptide of type-I-collagen levels (p<0.05) and band-5 tartrate-resistant acid phosphatase levels in the tissue (p<0.001). Oxidative stress and excessive inflammation are involved in the inhibition of fracture healing by EtOH. In this study, early short-term treatment of EtOH-fed animals with the antioxidant NAC reduced oxidative stress and normalized the innate immune response to fracture in the early phase of fracture healing, thereby restoring the normal onset of bone regeneration.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506

BackgroundHeightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration.MethodsWe hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle).ResultsThe salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque productionConclusionCollectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion.

Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5'Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1α independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1α-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. Stem Cells 2016;34:2342-2353.

Study of Serum Vascular endothelial growth factor level in Patients with Simple Diaphyseal Fractures healing

Background & Objectives: Vascular endothelial growth factor (VEGF) plays an important role in the bone repair process by acting as a potent mediator of angiogenesis. In the present study, we aim to analyse the temporal expression of serum VEGF during the early phase of fracture healing and further correlate it with the fracture healing outcomes. Methods: In this longitudinal cohort study, as per inclusion- exclusion criteria, total 76 adult patients with simple, fresh traumatic diaphyseal fractures of both bones of leg managed conservatively; were enrolled. The VEGF examination was carried out by taking venous blood (2ml) at 4th, 7th, 10th, 15th and 20th day after trauma and clinico-radiological examination was done at 6th, 10th 16th, 20th and 24th week after post trauma. As per clinico-radiological outcomes, these patients were divided into normal healing and impaired healing groups. These healing outcomes then were correlated with serum VEGF level in both groups. Results: Difference between the demographic variables of both groups was statistically insignificant. The mean RUST Score in each follow up except the baseline value shows statistically significant difference. The serum VEGF level seem to higher in impaired healing patients (Group-II) as compared to the normal healing patients (Group-I) in each follow-ups, however stastically, they shows insignificant difference. Further the correlation between healing outcomes with serum VEGF level was also found to be insignificant. Interpretation & Conclusions: The serum VEGF act as an potent mediator of angiogenesis during the early phase of healing and can be used as a biomarker to predict the progress of fracture healing outcomes. Key Word: Vascular endothelial growth factor, Diphyseal tibial fracture, simple fracture

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

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.