Impaired Fracture Healing Research Articles

Altered microRNA profile during fracture healing in rats with diabetes

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression. There is increasing evidence that some miRNAs are involved in the pathology of diabetes mellitus (DM) and its complications. We hypothesized that the functions of certain miRNAs and the changes in their patterns of expression may contribute to the pathogenesis of impaired fractures due to DM.MethodsIn this study, 108 male Sprague–Dawley rats were divided into DM and control groups. DM rats were created by a single intravenous injection of streptozotocin. Closed transverse femoral shaft fractures were created in both groups. On post-fracture days 5, 7, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was conducted with miRNA samples from each group on post-fracture days 5 and 11. The microarray findings were validated by real-time polymerase chain reaction (PCR) analysis at each time point.ResultsMicroarray analysis revealed that, on days 5 and 11, 368 and 207 miRNAs, respectively, were upregulated in the DM group, compared with the control group. The top four miRNAs on day 5 were miR-339-3p, miR451-5p, miR-532-5p, and miR-551b-3p. The top four miRNAs on day 11 were miR-221-3p, miR376a-3p, miR-379-3p, and miR-379-5p. Among these miRNAs, miR-221-3p, miR-339-3p, miR-376a-3p, miR-379-5p, and miR-451-5p were validated by real-time PCR analysis. Furthermore, PCR analysis revealed that these five miRNAs were differentially expressed with dynamic expression patterns during fracture healing in the DM group, compared with the control group.ConclusionsOur findings will aid in understanding the pathology of impaired fracture healing in DM and may support the development of molecular therapies using miRNAs for the treatment of impaired fracture healing in patients with DM.

Magnesium Rescues Cystic Fibrosis- and Age-Related Bone Diseases Through an ATF4-Dependent Wnt/β-Catenin Signaling

Bone fragility has emerged as a key clinical manifestation of cystic fibrosis, a genetic disease caused by CFTR mutation, which is not well understood and currently lacks effective therapeutic options. Here we report that CFTR is downregulated in aged wild-type mouse bones; CFTR-deficient mice show early activation of bone cell senescence, abnormal bone micro-architecture, altered osteogenic genes expression and impaired bone fracture healing, mimicking age-related bone disease. We also observed that magnesium ion enters bone cells, activates a transcription factor, ATF4, and promotes Wnt/β-catenin signaling. Magnesium implant improves bone fracture healing in CFTR-mutant mice, which is blocked by Wnt/β-catenin inhibition or ATF4 knockdown. Importantly, oral magnesium or local administration of VX809, a drug that restores CFTR function, improves bone quality in aged wild-type rats/mice. Together, these results collectively show that CFTR-deficiency mimics aging to cause bone defects , while suggesting magnesium supplementation as a possible therapy for CFTR deficiency-associated bone diseases.

The Association of Metal Frame Construct of ESIN and radiographic bone healing of pediatric forearm fractures

IntroductionThe gold-standard surgical procedure of both-bone forearm shaft fracture repair is elastic stable intramedullary nailing (ESIN). Disadvantages effects of ESIN have suggested to be a consequence of inappropriate surgical techniques, while recommendations are not always followed. The purpose of the study was to analyze the effect of inadequate metal frame construct on impaired fracture healing, refracture and changing alignment. Materials and MethodsIt is a population-based study including all consecutive patients, aged less than 16 years, who had been treated for forearm shaft fracture by ESIN during the ten-year period 2009–2018. Altogether 71 patients were included. Non-union, delayed union, and re-fracture during the following 12 months were taken as the main outcome, while inferior metal frame construct of ESIN and the surgical technique characteristics were taken the explanatory factors. Radiographic loss of reduction was a secondary outcome and a change >5° in alignment at any postoperative follow-up exam was recognized. ResultsTwo out of 71 fractures (3%) failed to unite, and ossifying operation was needed. Five cases (7%) showed delayed bone healing, but they ossified in five months without any intervention. These seven patients (10%) had been treated more often with larger nails (> 0.7x MCD) (p = 0.027) and by open reduction (p = 0.02), compared with thinner nails and closed reduction, respectively. Two (3%) patients had a second fracture; however, they happened 2 years after the initial injury. Other surgery or fracture related factors didn't associate with impaired ossifying.Regarding the secondary outcome, altogether 24 (35%) of the analyzed 67 patients showed >5° change in alignment during the postoperative follow-up but only one patient had clinically significant instability that required re-reduction. The alignment changed more usually in distal-third fractures, compared with middle or proximal third fractures (p = 0.019). ConclusionsESIN resulted in good radiographic bone healing in the vast majority (90%) of the patients and completely perfect metal frame construct was not required.

Review of Secondary Causes of Osteoporotic Fractures Due to Diabetes and Spinal Cord Injury.

The aim of this review is to gain a better understanding of osteoporotic fractures and the different mechanisms that are driven in the scenarios of bone disuse due to spinal cord injury and osteometabolic disorders due to diabetes. Despite major advances in understanding the pathogenesis, prevention, and treatment of osteoporosis, the high incidence of impaired fracture healing remains an important complication of bone loss, leading to marked impairment of the health of an individual and economic burden to the medical system. This review underlines several pathways leading to bone loss and increased risk for fractures. Specifically, we addressed the different mechanisms leading to bone loss after a spinal cord injury and diabetes. Finally, it also encompasses the changes responsible for impaired bone repair in these scenarios, which may be of great interest for future studies on therapeutic approaches to treat osteoporosis and osteoporotic fractures.

Cigarette smoke inhalation impairs angiogenesis in early bone healing processes and delays fracture union

AimsCigarette smoking has a negative impact on the skeletal system, causes a decrease in bone mass in both young and old patients, and is considered a risk factor for the development of osteoporosis. In addition, it disturbs the bone healing process and prolongs the healing time after fractures. The mechanisms by which cigarette smoking impairs fracture healing are not fully understood. There are few studies reporting the effects of cigarette smoking on new blood vessel formation during the early stage of fracture healing. We tested the hypothesis that cigarette smoke inhalation may suppress angiogenesis and delay fracture healing.MethodsWe established a custom-made chamber with airflow for rats to inhale cigarette smoke continuously, and tested our hypothesis using a femoral osteotomy model, radiograph and microCT imaging, and various biomechanical and biological tests.ResultsIn the smoking group, Western blot analysis and immunohistochemical staining revealed less expression of vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF). The smoking group also had a lower microvessel density than the control group. Image and biochemical analysis also demonstrated delayed bone healing.ConclusionCigarette smoke inhalation was associated with decreased expression of angiogenic markers in the early bone healing phase and with impaired bone healing.Cite this article: Bone Joint Res. 2020;9(3):99–107.

Propranolol Reverses Impaired Fracture Healing Response Observed With Selective Serotonin Reuptake Inhibitor Treatment.

Selective serotonin reuptake inhibitors (SSRIs) are one of the most commonly prescribed antidepressants worldwide and recent data show significant impairment of fracture healing after treatment with the SSRI fluoxetine in mice. Here, we provide evidence that the negative effects of SSRIs can be overcome by administration of the beta-blocker propranolol at the time of fracture. First, in vitro experiments established that propranolol does not affect osteogenic differentiation. We then used a murine model of intramembranous ossification to study the potential rescue effect of propranolol on SSRI-induced impaired fracture healing. Micro-CT analysis revealed that fluoxetine treatment resulted in a smaller bony regenerate and that this decrease in bone formation can be overcome by co-treatment with propranolol. We then tested this in a clinically relevant model of endochondral ossification. Fluoxetine-treated mice with a femur fracture were treated with propranolol initiated at the time of fracture, and a battery of analyses demonstrated a reversal of the detrimental effect of fluoxetine on fracture healing in response to propranolol treatment. These experiments show for the first time to our knowledge that the negative effects of SSRIs on fracture healing can be overcome by co-treatment with a beta-blocker. © 2019 American Society for Bone and Mineral Research.

Transplantation of IL-10-Overexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorates Diabetic-Induced Impaired Fracture Healing in Mice.

Diabetes mellitus is characterized by hyperglycemia which displays insufficiency or resistance to insulin. One of the complications of diabetes is the increased risk of fracture and the impairment of bone repair and regulation. There have been evidences from previous studies that mesenchymal stem cells (MSCs) from bone marrow promote cartilage and callous formation. In addition, IL-10, an anti-inflammatory cytokine, has been observed to relieve inflammation-related complications in diabetes. In this study, the role of IL-10-overexpressing bone marrow-derived MSCs (BM-MSCs) was examined in the diabetic mice model with femur fracture. MSCs were isolated from the BALB/c mice and IL-10 over expression was conducted with lentivirus transduction. The streptozotocin (STZ)-induced diabetes model with femoral fracture was established. BM-MSCs with IL-10 over expression were transplanted into the fracture area. The expressions of inflammatory factors IL-6, TNF-α and INF-γ were examined by qPCR and immunoblot; the biomechanical strength of the fracture site of the mice was examined and evaluated. Data showed that IL-10 overexpressed BM-MSCs transplantation decreased inflammatory response, promoted bone formation, and increased the strength of the fracture site in STZ-induced diabetic mice with femoral fracture. IL-10 overexpressed BM-MSCs transplantation accelerated fracture repair in STZ-induced diabetic mice, which in turn provides potential clinical application prospects.

Advances and Promises of Nutritional Influences on Natural Bone Repair.

Impaired fracture healing continues to be a significant public health issue. This is more frequently observed in aging populations and patients with co-morbidities that can directly influence bone repair. Tremendous progress has been made in the development of biologics to enhance and accelerate the healing process; however, side-effects persist that can cause significant discomfort and tissue damage. This has been the impetus for the development of safe and natural strategies to hasten natural bone healing. Of the many possible approaches, nutrition represents a safe, affordable, and non-invasive strategy to positively influence each phase of fracture repair. However, our understanding of how healing can be hindered by malnutrition or enhanced with nutritional supplementation has lagged behind the advancements in both surgical management and the knowledge of molecular and cellular drivers of skeletal fracture repair. This review serves to bridge this knowledge gapas well as define the importance of nutrition during fracture healing. The extant literature clearly indicates that pre-existing nutritional deficiencies should be corrected, and nutritional status should be carefully monitored to prevent the development of malnutrition for the best possible healing outcome. It remains unclear, however, whether the provision of nutrients beyond sufficiency has any benefit on fracture repair and patient outcomes. The combined body of pre-clinical studies using a variety of animal models suggests a promising role of nutrition as an adjuvant therapy to facilitate fracture repair, but extensive research is needed, specifically at the clinical level, to clarify the utility of nutritional interventions in orthopedics. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:695-707, 2020.

Current and Future Concepts for the Treatment of Impaired Fracture Healing.

Bone regeneration represents a complex process, of which basic biologic principles have been evolutionarily conserved over a broad range of different species. Bone represents one of few tissues that can heal without forming a fibrous scar and, as such, resembles a unique form of tissue regeneration. Despite a tremendous improvement in surgical techniques in the past decades, impaired bone regeneration including non-unions still affect a significant number of patients with fractures. As impaired bone regeneration is associated with high socio-economic implications, it is an essential clinical need to gain a full understanding of the pathophysiology and identify novel treatment approaches. This review focuses on the clinical implications of impaired bone regeneration, including currently available treatment options. Moreover, recent advances in the understanding of fracture healing are discussed, which have resulted in the identification and development of novel therapeutic approaches for affected patients.

KYNURENINE, AN ENDOGENOUS AHR AGONIST, UPREGULATES CXCL12- AND HDAC3-TARGETING MIRNAS INHIBITING OSTEOGENESIS

Osteoporosis is among the most debilitating disorders for aging men and women. Mechanisms underlying loss of bone mass and impaired fracture healing in aged population are not completely understood. Our lab and others reported increased levels of the oxidized tryptophan metabolite, kynurenine (Kyn), in bone marrow mesenchymal stem cells (BMSCs) and interstitial fluid. Here, we report that Kyn significantly inhibits osteogenic differentiation of BMSCs likely via activating aryl hydrocarbon receptor (AhR). KYN significantly downregulated mRNA levels of pro-osteogenic CXCL12 axis, including CXCL12, and its two main receptors CXCR4 and ACKR3. Secreted protein levels of CXCL12 were significantly reduced with KYN, and were rescued upon the use of AhR antagonist 3’,4’-dimethoxyflavone (DMF). Moreover, KYN upregulated levels of pro-aging and CXCL12-targeting miRNAs miR-29b-1-5p and miR-141-3p. Additionally, KYN significantly inhibited mRNA and protein levels of the epigenetic enzyme Hdac3 which is responsible for activating osteogenesis and inhibiting adipogenesis in BMSCs. Using mutagenesis and luciferase assays, we show evidence that miR-141-3p and miR-29b-1-5p directly target CXCL12 3’-UTR. We also show that miR-29b-1-5p directly targets Hdac3 explaining the significantly increased acetylation levels of H4 with KYN treatment. Finally, we show that mRNA levels of both KYN receptor AhR, and KYN-producing enzyme IDO-1 are regulated by CXCL12. We believe this data explains a novel mechanism of the bone aging phenotype and provides multiple potential clinical intervention targets for controlling osteoporosis.

Type 2 Diabetes-Driven Alterations in Bone Healing and Angiogenesis

Background and Hypothesis: Type 2 diabetes (T2D) is prevalent in the United States. T2D patients are at risk for impaired fracture healing due to decreased angiogenesis, which is required for successful bone regeneration. Bone morphogenetic protein-2 (BMP-2) is often used to help orthopedic surgeons with bone healing in difficult cases. Here, we begin characterizing the mechanism by which T2D alters bone healing with or without BMP-2 treatment. We hypothesize that T2D impairs fracture healing by decreasing angiogenesis and endothelial cell function.
 Project Methods: Using Tie2-CreER;Td-Tomato mice (Tie2CreERT+), we established a high fat diet (HFD)-induced T2D mouse model to compare with control low fat diet (LFD)-fed mice. Mice underwent testing to confirm the T2D-like metabolic phenotype, underwent a femoral critical-size defect surgery that was treated with either saline or BMP-2, and were then assessed biweekly by X-ray imaging over the course of 12 weeks. Finally, bone marrow-derived endothelial cells were collected from these mice to assess changes in endothelial colony and tube formation in vitro.
 Results: Results showed that the HFD mice acquired the T2D metabolic phenotype. Fracture healing was impaired in the HFD mice, even with BMP-2 treatment. The isolation of BMECs was confirmed by visualization of fluorescent Tie2+ cells. Unexpectedly, in vitro tube formation assays indicated that HFD improved vessel-like formation properties. BMP-2 treatment appeared to improve some vessel-like formation properties compared to control treatment.
 Conclusion and Potential Impact: This study is ongoing. Further data will need to be collected to better characterize differences in bone healing and angiogenesis in the healing femurs. Still, these data reveal the mechanisms by which T2D impairs bone healing and demonstrate the important difference between examining endothelial cells in vitro vs. in vivo. Future investigations will examine if thrombopoietin, which our group has previously shown to improve both fracture healing and angiogenesis, may be a more effective treatment than BMP-2 in this model.

A physical model to investigate the acoustic behaviour of microbubbles and nanodroplets within a bone fracture

Impaired fracture healing is a major financial burden for healthcare services; 5%–10% of bone fractures result in non-unions, and there is no clinically approved systemic therapy. This study characterises acoustically stimulated microbubbles (MBs) and nanodroplets (NDs) as non-invasive ultrasound responsive vehicles for the targeted delivery of osteogenic compounds. A microscope-compatible water-tank incorporating a passive cavitation detector was developed to study the acoustic behaviour of MBs and NDs within physical models of bone fractures (gap: 3.5–5.5 mm, angles: 0 deg and 90 deg). The device was designed using COMSOL Multiphysics (Burlington, MA) and tested in-vitro. The bone was simulated using a material with comparable acoustic impedance (Sawbones, WA). Numerical simulations showed that the developed experimental set-up generated a relatively uniform acoustic field at a target plane. It could be operated at either 1 or 2 MHz US frequency, at an acoustic pressure in the range 0–1 MPa. The inclusion of a fracture model caused perturbations to the acoustic field, which were dependent on the architecture of the fracture (i.e., relative to the incident US field). Ongoing studies are investigating how these perturbations affect ND/MB response in-vitro. Further studies will investigate the relationship between MB/ND acoustic response and the release of biologically active compounds.Impaired fracture healing is a major financial burden for healthcare services; 5%–10% of bone fractures result in non-unions, and there is no clinically approved systemic therapy. This study characterises acoustically stimulated microbubbles (MBs) and nanodroplets (NDs) as non-invasive ultrasound responsive vehicles for the targeted delivery of osteogenic compounds. A microscope-compatible water-tank incorporating a passive cavitation detector was developed to study the acoustic behaviour of MBs and NDs within physical models of bone fractures (gap: 3.5–5.5 mm, angles: 0 deg and 90 deg). The device was designed using COMSOL Multiphysics (Burlington, MA) and tested in-vitro. The bone was simulated using a material with comparable acoustic impedance (Sawbones, WA). Numerical simulations showed that the developed experimental set-up generated a relatively uniform acoustic field at a target plane. It could be operated at either 1 or 2 MHz US frequency, at an acoustic pressure in the range 0–1 MPa. The inclusi...

Lowering circulating apolipoprotein E levels improves aged bone fracture healing.

Age is a well-established risk factor for impaired bone fracture healing. Here, we identify a role for apolipoprotein E (ApoE) in age-associated impairment of bone fracture healing and osteoblast differentiation, and we investigate the mechanism by which ApoE alters these processes. We identified that, in both humans and mice, circulating ApoE levels increase with age. We assessed bone healing in WT and ApoE-/- mice after performing tibial fracture surgery: bone deposition was higher within fracture calluses from ApoE-/- mice. In vitro recombinant ApoE (rApoE) treatment of differentiating osteoblasts decreased cellular differentiation and matrix mineralization. Moreover, this rApoE treatment decreased osteoblast glycolytic activity while increasing lipid uptake and fatty acid oxidation. Using parabiosis models, we determined that circulating ApoE plays a strong inhibitory role in bone repair. Using an adeno-associated virus-based siRNA system, we decreased circulating ApoE levels in 24-month-old mice and demonstrated that, as a result, fracture calluses from these aged mice displayed enhanced bone deposition and mechanical strength. Our results demonstrate that circulating ApoE as an aging factor inhibits bone fracture healing by altering osteoblast metabolism, thereby identifying ApoE as a new therapeutic target for improving bone repair in the elderly.

Multiple Fractures and Impaired Bone Fracture Healing in a Patient with Pycnodysostosis and Hypophosphatasia.

Pycnodysostosis (PYCD) is a rare recessive inherited skeletal disease, characterized by short stature, brittle bones, and recurrent fractures, caused by variants in the Cathepsin K encoding gene that leads to impaired osteoclast-mediated bone resorption. Hypophosphatasia (HPP) is a dominant or recessive inherited condition representing a heterogeneous phenotype with dental symptoms, recurrent fractures, and musculoskeletal problems. The disease results from mutation(s) in the tissue non-specific alkaline phosphate encoding gene with reduced activity of alkaline phosphatase and secondarily defective mineralization of bone and teeth. Here, we present the first report of a patient with the coexistence of PYCD and HPP. This patient presented typical clinical findings of PYCD, including short stature, maxillary hypoplasia, and sleep apnoea. However, the burden of disease was caused by over 30 fractures, whereupon most showed delayed healing and non-union. Biochemical analysis revealed suppressed bone resorption and low bone formation capacity. We suggest that the coexistence of impaired bone resorption and mineralization may explain the severe bone phenotype with poor fracture healing.

NLRP3 inflammasome inhibitor glyburide expedites diabetic-induced impaired fracture healing.

Localized inflammation is accompanied by the diabetic-induced fracture. The present study aims to investigate the therapeutic effects of glyburide, an NLRP3 inflammasome inhibitor, in a diabetic-induced fracture model. An animal model of diabetic-induced fracture was established and the mice were administrated with metformin or glyburide for 3 weeks. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to evaluate the relative expressions of IFN-γ, TNF-α, and IL-6. Micro-computed tomography (μCT) scanning was applied to evaluate bone callus formation. Histopathology examinations of fractured femur sections were performed using Tartrate-resistant acid phosphatase (TRAP) staining and Alcian blue and orange G staining. Bone strength was evaluated using Torsional testing. Our results showed that treatment of glyburide significantly decreased the expressions of IFN-γ, TNF-α, and IL-6 in the fracture calluses in diabetic-induced fracture model, while bone callus volume and bone volume fraction were increased. Additionally, our results also demonstrated that treatment of glyburide rescued the increase of osteoclasts in the bone-cartilage interface. Apart from decreasing a percentage of cartilage area and increasing the percentage of bone and fibrotic tissue area, treatment of glyburide increased the maximum torque and yield torque of fractures. These results implied that glyburide might be used as a potential drug candidate for diabetic-induced fracture.

Progranulin promotes diabetic fracture healing in mice with type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor-alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor-like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN-binding receptors. In this study, we employed several in vivo models to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti-inflammatory signaling pathways are involved in PGRN-stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.

Individual Effector/Regulator T Cell Ratios Impact Bone Regeneration.

There is increasing evidence that T lymphocytes play a key role in controlling endogenous regeneration. Regeneration appears to be impaired in case of local accumulation of CD8+ effector T cells (TEFF), impairing endogenous regeneration by increasing a primary “useful” inflammation toward a damaging level. Thus, rescuing regeneration by regulating the heightened pro-inflammatory reaction employing regulatory CD4+ T (TReg) cells could represent an immunomodulatory option to enhance healing. Hypothesis was that CD4+ TReg might counteract undesired effects of CD8+ TEFF. Using adoptive TReg transfer, bone healing was consistently improved in mice possessing an inexperienced immune system with low amounts of CD8+ TEFF. In contrast, mice with an experienced immune system (high amounts of CD8+ TEFF) showed heterogeneous bone repair with regeneration being dependent upon the individual TEFF/TReg ratio. Thus, the healing outcome can only be improved by an adoptive TReg therapy, if an unfavorable TEFF/TReg ratio can be reshaped; if the individual CD8+ TEFF percentage, which is dependent on the individual immune experience can be changed toward a favorable ratio by the TReg transfer. Remarkably, also in patients with impaired fracture healing the TEFF/TReg ratio was higher compared to uneventful healers, validating our finding in the mouse osteotomy model. Our data demonstrate for the first time the key-role of a balanced TEFF/TReg response following injury needed to reach successful regeneration using bone as a model system. Considering this strategy, novel opportunities for immunotherapy in patients, which are at risk for impaired healing by targeting TEFF cells and supporting TReg cells to enhance healing are possible.

Increased Bone Marrow-Specific Adipogenesis by Clofazimine Causes Impaired Fracture Healing, Osteopenia, and Osteonecrosis Without Extraskeletal Effects in Rats.

Mycobacterium leprae infection causes bone lesions and osteoporosis, however, the effect of antileprosy drugs on the bone is unknown. We, therefore, set out to address it by investigating osteogenic differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs). Out of 7 antileprosy drugs, only clofazimine (CFZ) reduced MSCs viability (IC50 ∼ 1 μM) and their osteogenic differentiation but increased adipogenic differentiation on a par with rosiglitazone, and this effect was blocked by a peroxisome proliferator-activated receptor gamma antagonist, GW9662. CFZ also decreased osteoblast viability and resulted in impaired bone regeneration in a rat femur osteotomy model at one-third human drug dose owing to increased callus adipogenesis as GW9662 prevented this effect. CFZ treatment decreased BM MSC population and homing of MSC to osteotomy site despite drug levels in BM being much less than its in vitro IC50 value. In adult rats, CFZ caused osteopenia in long bones marked by suppressed osteoblast function due to enhanced adipogenesis and increased osteoclast functions. A robust increase in marrow adipose tissue (MAT) by CFZ did not alter the hematologic parameters but likely reduced BM vascular bed leading to osteonecrosis (ON) characterized by empty osteocyte lacunae. However, CFZ had no effect on visceral fat content and was not associated with any metabolic and hematologic changes. Levels of unsaturated fatty acids in MAT were higher than saturated fatty acids and CFZ further increased the former. From these data, we conclude that CFZ has adverse skeletal effects and could be used for creating a rodent ON model devoid of extraskeletal effects.

The Effect of Proton Pump Inhibitors on Bone Formation in a Rat Spinal Arthrodesis Model.

Rat posterolateral arthrodesis model. Quantify the impact of administration of a proton pump inhibitor on spine fusion. Proton pump inhibitors (PPIs) are widely used for gastrointestinal disorders and for ulcer prophylaxis in patients taking non-steroidal anti-inflammatory drugs. PPIs cause chronic acid suppression which has been found to result in decreased bone mineral density, increased fracture risk, and impaired fracture healing. Despite advances in surgical techniques, pseudarthrosis still occurs in up to 24% of patients requiring revision surgery following spinal fusion procedures. Thus, there are likely many unidentified risk factors. While PPIs have been hypothesized to impact fracture healing, no study has evaluated their effect on spine arthrodesis rates. Thirty-eight female rats underwent posterolateral lumbar spinal fusion. Rats were divided into two groups: normal saline control and pantroprazole, which was administered by daily intraperitoneal injections. At 8 weeks postoperative spines were evaluated with manual palpation, microCT, histologic analysis, and biomechanical testing. Fusion rates of the control group and PPI group were not significantly different (100% vs. 94%). Average fusion scores were significantly lower in the pantoprazole group. New bone formation identified on microCT imaging of bilaterally fused specimens demonstrated a lower average volume of newly generated bone in the PPI group, but this difference was not significant. Biomechanical testing demonstrated no significant difference in strength or stiffness of the fusion mass between the groups. This study demonstrates that administration of PPIs does not inhibit fusion rates, bone formation, or affect biomechanical integrity of fusion. However, lower fusion scores in the PPI group suggest that a negative impact may still exist. Future studies will explore growth factor and protein expression in the fusion masses as well as utilize higher doses of PPI to fully discern their effect on spine fusion. N/A.

Effect of neurokinin-1-receptor blockage on fracture healing in rats

Neurologic injury and selective blockage of sensory nerve endings is associated with impaired fracture healing, however, the role of specific neurotransmitters has not been sufficiently investigated. Our aim was to investigate the impact of specific Substance P-receptor blockage on fracture healing, since the neuropeptide Substance P has both neurogenic and osteogenic activity. After intramedullary stabilization, an isolated femur fracture was induced in 72 Sprague-Dawley rats. In the NK1-R group, the neurokinin-1-tachykinin receptor for substance P was blocked by a specific antagonist (SR140333) for the first two weeks after fracture induction. The control group only received vehicle. Gene-expression, histology, micro-computed tomography, and biomechanical tests were performed. NK1-receptor blocking suppressed osteocalcin expression at one week, collagen 1A2 expression at one and two weeks and collagen 2A1 expression at 2 weeks after fracture induction. Biomechanical testing revealed a significant reduction in maximal load to failure in the NK1-R group at 6 weeks (69.78 vs. 155.45 N, p = 0.029) and at 3 months (72.50 vs.176.33 N, p = 0.01) of fracture healing. Blocking the NK1-receptor suppresses gene expression in and reduces biomechanical strength of healing bone. Therefore, we assume a potential therapeutic relevance of Substance P in cases of disturbed fracture healing.