Osteogenic effects of microRNA-335-5p/lipidoid nanoparticles coated on titanium surface.

OBJECTIVE: In this study, we aimed to investigate the therapeutic potential of miR-335-5p lipidoid nanocomplexes coated on Titanium (Ti) SLActive surface by lyophilization. DESIGN: In our model, we coated miR-335-5p/Lipidoid nanoparticles on titanium implant, seeded GFP-labelled mouse bone marrow stromal cells (BMSCs) onto the functionalized Ti implant surface, and analyzed the transfection efficiency, cell adhesion, proliferation, and osteogenic activity of the bone-implant interface. RESULTS: The Ti SLActive surface displayed a suitable hydrophilicity ability and provided a large surface area for miRNA loading, enabling spatial retention of the miRNAs within the nanopores until cellular delivery. We demonstrated a high transfection efficiency of miR-335-5p lipidoid nanoparticles in BMSCs seeded onto the Ti SLActive surface, even after 14 days. Alkaline phosphatase (ALP) activity and cell vitality were significantly increased in BMSCs transfected with miR-335-5p at 7 and 14 days as opposed to cells transfected with negative controls. When miR-335-5p transfected BMSCs were induced to undergo osteogenic differentiation, we detected increased mRNA expression of osteogenic markers including Alkaline phosphatase (ALP), collagen I (COL1), osteocalcin (OCN) and bone sialoprotein (BSP) at 7 and 14 days as compared with negative controls. CONCLUSION: MiR-335-5p lipidoid nanoparticles could be used as a new cost-effective methodology to increase the osteogenic capacity of biomedical Ti implants.

Effects of moving cupping therapy on hip and knee range of movement and knee flexion power: a preliminary investigation.

Cupping therapy has become more popular in the sports setting because of its simplicity in application, minimal adverse effects, and reduction in pain and muscle tenderness, yet there is little research on its effectiveness for range of movement and power.Objectives: The primary aim of this study was to investigate if a single session of moving cupping to the posterior aspect of the lower limb effects hip and knee range of movement and knee flexion power. The secondary aim was to consider participants' views and perceptions of moving cupping therapy.Methods: Twenty-one healthy participants (12 male and 9 female) aged between 19 and 31 years volunteered to take part in the study. All participants received 15 min of moving cupping therapy to their dominant posterior lower extremity. Hip and knee range of movement and knee flexion isokinetic power measurements were taken prior to and immediately after the moving cupping intervention. Participants also completed a questionnaire based on their experience and perceptions of cupping.Results: Results showed a significant increase (p = <.05) in hip and knee range of movement measurements by 7% in the straight leg raise and 4% in a popliteal angle test. However, no significant changes were seen in the knee flexion power measures. Data from the questionnaire suggest that despite moving cupping being reported as 'uncomfortable' it is considered acceptable.Discussion: Moving cupping therapy may have short-term changes to range of movement but not power, though the limitations of this study mean that rigorous studies are required before the effectiveness of moving cupping can be determined.

Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1.

Central adiponectin (APN) in either the globular (gAPN) or full-length forms decreases sympathetic tone and increases trabecular bone mass in mice through the hypothalamus. It is known that cannabinoid type-1 (CB1) receptors are expressed in the hypothalamic ventromedial nucleus and participate in energy metabolism by controlling sympathetic activity. However, whether central APN could influence endocannabinoid signaling through CB1 receptor to regulate bone metabolism has not been characterized. Here we demonstrate that gAPN downregulated CB1 expression in embryonic mouse hypothalamus N1 cells in vitro. gAPN intracerebroventricular (icv) infusions also decreased hypothalamic CB1 expression and bone formation parameters in APN-knockout (APN-KO) and wild-type mice. Most importantly, mice pretreated with icv infusions with the CB1 receptor agonist arachidonyl-2'-chloroethylamine or antagonist rimonabant attenuated or enhanced respectively central APN induction of bone formation. We then investigated whether epigenetic signaling mechanisms were involved in the downregulation of hypothalamic CB1 expression by gAPN. We found gAPN enhanced expression levels of various histone deacetylases (HDACs), especially HDAC5. Furthermore, chromatin immunoprecipitation assays revealed HDAC5 bound to the transcriptional start site transcription start site 2 region of the CB1 promoter. In summary, our study identified a possible novel central APN-HDAC5-CB1 signaling mechanism that promotes peripheral bone formation through epigenetic regulation of hypothalamic CB1 expression.

MicroRNA-99a is a novel regulator of KDM6B-mediated osteogenic differentiation of BMSCs.

Skeletal tissue originates from mesenchymal stem cells (MSCs) with differentiation potential into the osteoblast lineage regulated by essential transcriptional and post-transcriptional mechanisms. Recently, miRNAs and histone modifications have been identified as novel key regulators of osteogenic differentiation of MSCs. Here, we identified miR-99a and its target lysine (K)-specific demethylase 6B (KDM6B) gene as novel modulators of osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Microarray profiling and further validation by quantitative real-time RT-PCR revealed that miR-99a was up-regulated during osteoblastic differentiation of BMSCs, and decreased in differentiated osteoblasts. Transfection of miR-99a mimics inhibited osteoblastic commitment and differentiation of BMSCs, whereas inhibition of miR-99a by inhibitors enhances these processes. KDM6B was determined as one of important targets of miR-99a, which was further confirmed by luciferase assay of 3'-UTR of KDM6B. Moreover, HOX gene level decreased after transfection of miR-99a mimics in BMSCs, which indicated that KDM6B is a bona fide target of miR-99a. Furthermore, in a model of in vivo bone regeneration, osteoblast-specific gain- and loss-of-function experiments performed using cranial bone defects revealed that miR-99a mimics-transfected BMSCs reduced bone formation, and conversely, miR-99a inhibitors-transfected BMSCs increased in vivo bone formation. Tissue-specific inhibition of miR-99a may be a potential novel therapeutic approach for enhancing BMSCs-based bone formation and regeneration.

Exercise-induced irisin in bone and systemic irisin administration reveal new regulatory mechanisms of bone metabolism.

Irisin is a polypeptide hormone derived from the proteolytic cleavage of fibronectin-type III domain-containing 5 (FNDC5) protein. Once released to circulation upon exercise or cold exposure, irisin stimulates browning of white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression, leading to an increase in total body energy expenditure by augmented UCP1-mediated thermogenesis. It is currently unknown whether irisin is secreted by bone upon exercise or whether it regulates bone metabolism in vivo. In this study, we found that 2 weeks of voluntary wheel-running exercise induced high levels of FNDC5 messenger RNA as well as FNDC5/irisin protein expression in murine bone tissues. Increased immunoreactivity due to exercise-induced FNDC5/irisin expression was detected in different regions of exercised femoral bones, including growth plate, trabecular bone, cortical bone, articular cartilage, and bone-tendon interface. Exercise also increased expression of osteogenic markers in bone and that of UCP1 in WAT, and led to bodyweight loss. Irisin intraperitoneal (IP) administration resulted in increased trabecular and cortical bone thickness and osteoblasts numbers, and concurrently induced UCP1 expression in subcutaneous WAT. Lentiviral FNDC5 IP administration increased cortical bone thickness. In vitro studies in bone cells revealed irisin increases osteoblastogenesis and mineralization, and inhibits receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. Taken together, our findings show that voluntary exercise increases irisin production in bone, and that an increase in circulating irisin levels enhances osteogenesis in mice.

Overexpression of MiR-335-5p Promotes Bone Formation and Regeneration in Mice.

MicroRNAs (miRNAs) and the Wnt signaling pathway play critical roles in regulating bone development and homeostasis. Our previous study revealed high expression of miR-335-5p in osteoblasts and hypertrophic chondrocytes in mouse embryos and the ability of miR-335-5p to promote osteogenic differentiation by downregulating Wnt antagonist Dickkopf-1 (DKK1). The purpose of this study was to investigate the effects of miR-335-5p constitutive overexpression on bone formation and regeneration in vivo. To that end, we generated a transgenic mouse line specifically overexpressing miR-335-5p in osteoblasts lineage by the osterix promoter and characterized its bone phenotype. Bone histomorphometry and µCT analysis revealed higher bone mass and increased parameters of bone formation in transgenic mice than in wild-type littermates. Increased bone mass in transgenic mice bones also correlated with enhanced expression of osteogenic differentiation markers. Upon osteogenic induction, bone marrow stromal cells (BMSCs) isolated from transgenic mice displayed higher mRNA expression of osteogenic markers than wild-type mice BMSCs cultures. Protein expression of Runx2 and Osx was also upregulated in BMSC cultures of transgenic mice upon osteogenic induction, whereas that of DKK1 was downregulated. Most important, BMSCs from transgenic mice were able to repair craniofacial bone defects as shown by µCT analysis, H&E staining, and osteocalcin (OCN) immunohistochemistry of newly formed bone in defects treated with BMSCs. Taken together, our results demonstrate constitutive overexpression of miR-335-5p driven by an osterix promoter in the osteoblast lineage induces osteogenic differentiation and bone formation in mice and support the potential application of miR-335-5p-modified BMSCs in craniofacial bone regeneration. © 2017 American Society for Bone and Mineral Research.

Effect of a Price Transparency Intervention in the Electronic Health Record on Clinician Ordering of Inpatient Laboratory Tests: The PRICE Randomized Clinical Trial.

Importance: Many health systems are considering increasing price transparency at the time of order entry. However, evidence of its impact on clinician ordering behavior is inconsistent and limited to single-site evaluations of shorter duration. Objective: To test the effect of displaying Medicare allowable fees for inpatient laboratory tests on clinician ordering behavior over 1 year. Design, Setting, and Participants: The Pragmatic Randomized Introduction of Cost data through the electronic health record (PRICE) trial was a randomized clinical trial comparing a 1-year intervention to a 1-year preintervention period, and adjusting for time trends and patient characteristics. The trial took place at 3 hospitals in Philadelphia between April 2014 and April 2016 and included 98 529 patients comprising 142 921 hospital admissions. Interventions: Inpatient laboratory test groups were randomly assigned to display Medicare allowable fees (30 in intervention) or not (30 in control) in the electronic health record. Main Outcomes and Measures: Primary outcome was the number of tests ordered per patient-day. Secondary outcomes were tests performed per patient-day and Medicare associated fees. Results: The sample included 142 921 hospital admissions representing patients who were 51.9% white (74 165), 38.9% black (55 526), and 56.9% female (81 291) with a mean (SD) age of 54.7 (19.0) years. Preintervention trends of order rates among the intervention and control groups were similar. In adjusted analyses of the intervention group compared with the control group over time, there were no significant changes in overall test ordering behavior (0.05 tests ordered per patient-day; 95% CI, -0.002 to 0.09; P = .06) or associated fees ($0.24 per patient-day; 95% CI, -$0.42 to $0.91; P = .47). Exploratory subset analyses found small but significant differences in tests ordered per patient-day based on patient intensive care unit (ICU) stay (patients with ICU stay: -0.16; 95% CI, -0.31 to -0.01; P = .04; patients without ICU stay: 0.13; 95% CI, 0.08-0.17; P < .001) and the magnitude of associated fees (top quartile of tests based on fee value: -0.01; 95% CI, -0.02 to -0.01; P = .04; bottom quartile: 0.03; 95% CI, 0.002-0.06; P = .04). Adjusted analyses of tests that were performed found a small but significant overall increase in the intervention group relative to the control group over time (0.08 tests performed per patient day, 95% CI, 0.03-0.12; P < .001). Conclusions and Relevance: Displaying Medicare allowable fees for inpatient laboratory tests did not lead to a significant change in overall clinician ordering behavior or associated fees. Trial Registration: clinicaltrials.gov Identifier: NCT02355496.

Runx2/DICER/miRNA Pathway in Regulating Osteogenesis.

DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21-25 nucleotide duplex in cell lineage differentiation, identity, and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt-related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 -/-) compared with that of Runx2 +/- and Runx2 +/+ mice. We further established the calvarial bone critical-size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post-surgery, micro-CT analysis revealed impaired bone formation, and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR-335-5p and miR-17-92 cluster to form mature miRNAs, which target and decrease the Dickkopf-related protein 1 (DKK1), and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/ß-catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage-specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study, also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. J. Cell. Physiol. 232: 182-191, 2017. © 2016 Wiley Periodicals, Inc.

Residents' self-report on why they order perceived unnecessary inpatient laboratory tests.

Resident physicians routinely order unnecessary inpatient laboratory tests. As hospitalists face growing pressures to reduce low-value services, understanding the factors that drive residents' laboratory ordering can help steer resident training in high-value care. We conducted a qualitative analysis of internal medicine (IM) and general surgery (GS) residents at a large academic medical center to describe the frequency of perceived unnecessary ordering of inpatient laboratory tests, factors contributing to that behavior, and potential interventions to change it. The sample comprised 57.0% of IM and 54.4% of GS residents. Among respondents, perceived unnecessary inpatient laboratory test ordering was self-reported by 88.2% of IM and 67.7% of GS residents, occurring on a daily basis by 43.5% and 32.3% of responding IM and GS residents, respectively. Across both specialties, residents attributed their behaviors to the health system culture, lack of transparency of the costs associated with health care services, and lack of faculty role models that celebrate restraint. Journal of Hospital Medicine 2015;11:869-872. © 2015 Society of Hospital Medicine.

Adiponectin regulates bone marrow mesenchymal stem cell niche through a unique signal transduction pathway: an approach for treating bone disease in diabetes.

Adiponectin (APN) is an adipocyte-secreted adipokine that exerts well-characterized antidiabetic properties. Patients with type 2 diabetes (T2D) are characterized by reduced APN levels in circulation and impaired stem cell and progenitor cell mobilization from the bone marrow for tissue repair and remodeling. In this study, we found that APN regulates the mobilization and recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to participate in tissue repair and regeneration. APN facilitated BMSCs migrating from the bone marrow into the circulation to regenerate bone by regulating stromal cell-derived factor (SDF)-1 in a mouse bone defect model. More importantly, we found that systemic APN infusion ameliorated diabetic mobilopathy of BMSCs, lowered glucose concentration, and promoted bone regeneration in diet-induced obesity mice. In vitro studies allowed us to identify Smad1/5/8 as a novel signaling mediator of APN receptor (AdipoR)-1 in BMSCs and osteoblasts. APN stimulation of MC3T3-E1 osteoblastic cells led to Smad1/5/8 phosphorylation and nuclear localization and increased SDF-1 mRNA expression. Although APN-mediated phosphorylation of Smad1/5/8 occurred independently from adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1, it correlated with the disassembly of protein kinase casein kinase 2 and AdipoR1 in immunoprecipitation experiments. Taken together, this study identified APN as a regulator of BMSCs migration in response to bone injury. Therefore, our findings suggest APN signaling could be a potential therapeutic target to improve bone regeneration and homeostasis, especially in obese and T2D patients.

Central adiponectin administration reveals new regulatory mechanisms of bone metabolism in mice.

Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.

Sustained release of adiponectin improves osteogenesis around hydroxyapatite implants by suppressing osteoclast activity in ovariectomized rabbits.

Lack of estrogen could lead to decreased bone mass and increased risk for osteoporosis, which has a negative influence on biomaterial implantation. Adiponectin (APN), an adipose-derived hormone, has been shown to increase bone density by inhibiting osteoclast formation and promoting the formation of osteoblasts. This study was designed to investigate the direct effects of APN released from the Matrigel controlled-release system on the activity of rabbit mature osteoclasts and osteoclast precursor RAW264.7 cells in vitro, and to determine its effects by improving osteogenesis around the hydroxyapatite (HA) implant in ovariectomized (OVX) rabbits. APN+Matrigel+HA, APN+HA, Matrigel+HA and HA were implanted into mandibular defects of OVX rabbits. At 4 weeks after implantation, the mandibles were examined by histology, microcomputed tomography and biomechanical testing. The results demonstrated that Matrigel extended the length of APN released to achieve long-term persistence. The sustained release of APN suppressed the osteoclastic activity both in vitro and in vivo, and improved the peri-implant osteogenesis in OVX rabbits, while the short-term APN treatment did not. Sustained release of APN may be an effective strategy to improve the restoration of bone defects by the use of HA materials under osteoporotic conditions in which osteoclasts are highly activated.