Osteoimmunology of Fracture Healing.

PURPOSE OF REVIEW: The purpose of this review is to summarize what is known in the literature about the role inflammation plays during bone fracture healing. Bone fracture healing progresses through four distinct yet overlapping phases: formation of the hematoma, development of the cartilaginous callus, development of the bony callus, and finally remodeling of the fracture callus. Throughout this process, inflammation plays a critical role in robust bone fracture healing. RECENT FINDINGS: At the onset of injury, vessel and matrix disruption lead to the generation of an inflammatory response: inflammatory cells are recruited to the injury site where they differentiate, activate, and/or polarize to secrete cytokines for the purposes of cell signaling and cell recruitment. This process is altered by age and by sex. Bone fracture healing is heavily influenced by the presence of inflammatory cells and cytokines within the healing tissue.

Histone deacetylase1 promotes TGF-ß1-mediated early chondrogenesis through down-regulating canonical Wnt signaling.

Cartilage formation during both embryonic development and bone repairing processes involves mesenchymal stem cells (MSCs) differentiation. Wnt/ß-catenin signaling pathway inhibits early chondrogenesis and is down-regulated during Transforming growth factor-ß1 (TGF-ß1)-induced chondrogenesis. However, the regulatory molecules that participate in the process is unknown. This study was designed to investigate the underlying mechanisms that down-regulate Wnt/ß-catenin pathway during chondrogenesis. TGF-ß1-induced micromass cultures of C3H10T1/2 were used as chondrocyte differentiation model. Gene expression profile was detected by realtime-PCR. Regulatory role of HDAC1 on ß-catenin was investigated by luciferase assay, chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation (Co-IP) assay and in vitro ubiquitination assay. In this study, we showed that HDAC1 was induced and suppressed ß-catenin gene expression through direct binding to its promoter. Besides, HDAC1 could also interact with deacetylate ß-catenin protein through its deacetylase domain, which causes degradation of ß-catenin. Our results indicate that HDAC1 plays an important role in chondrogenesis and may represent a therapeutic target for modulation of cartilage development.

The impact of age and sex on the inflammatory response during bone fracture healing.

Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory "M1" and anti-inflammatory "M2" macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1ß, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice, whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice - thus we termed these "age-accumulated" cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as "fracture response" cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: (1) age-accumulated cytokines, (2) female-specific age-accumulated cytokines, and (3) fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.

Adsorption Performance and Mechanisms of Tetracycline on Clay Minerals in Estuaries and Nearby Coastal Areas.

Clay minerals in sediments have strong adsorption capacities for pollutants, but their role in the distribution of antibiotics in estuaries and nearby coastal areas is unclear. We evaluated the clay mineral montmorillonite (SWy-2) adsorption capacity for tetracycline (TC). We assessed the adsorption capacity of SWy-2 for TC by measuring the removal percentage of 30 mg/L TC over time. The effects of pH and ionic strength on the TC adsorption onto SWy-2 were investigated. We analyzed the kinetics of TC adsorption using a pseudo-second-order model and determined the adsorption isotherm using the Langmuir equation. SWy-2 particles were characterized using zeta potential, Fourier transform infrared (FTIR), and X-ray diffraction (XRD) analyses before and after TC adsorption. The removal percentage of 30 mg/L TC by SWy-2 reached 70.76% within 0.25 h and gradually increased to 78.64% at 6 h. TC adsorption was influenced by pH and ionic strength, where low pH enhanced and high ionic strength reduced the adsorption. The kinetics of TC adsorption followed a pseudo-second-order model, and the adsorption isotherm adhered to the Langmuir equation. The saturated adsorption capacity (qmax) of SWy-2 for TC was 227.27 mg/g. Zeta potential, FTIR, and XRD analyses confirmed that electrostatic interactions and chemical bonds played a significant role in the TC adsorption by SWy-2. SWy-2 clay mineral exhibits a substantial adsorption capacity for TC, indicating its potential as an effective sorbent to mitigate antibiotic contamination in estuaries and nearby coastal areas. The observed effects of pH and ionic strength on TC adsorption have implications for the environmental fate and transport of antibiotics. The pseudo-second-order kinetic model and Langmuir isotherm equation provide valuable insights into the adsorption behavior and capacity of TC on SWy-2. Characterization analyses support the involvement of electrostatic interactions and chemical bonds in the SWy-2-TC adsorption mechanism.

Vagus nerve stimulation rescues persistent pain following orthopedic surgery in adult mice.

ABSTRACT: Postoperative pain is a major clinical problem imposing a significant burden on patients and society. In a survey 2 years after orthopedic surgery, 57% of patients reported persisting postoperative pain. However, only limited progress has been made in the development of safe and effective therapies to prevent the onset and chronification of pain after orthopedic surgery. We established a tibial fracture mouse model that recapitulates clinically relevant orthopedic trauma surgery, which causes changes in neuropeptide levels in dorsal root ganglia and sustained neuroinflammation in the spinal cord. Here, we monitored extended pain behavior in this model, observing chronic bilateral hindpaw mechanical allodynia in both male and female C57BL/6J mice that persisted for >3 months after surgery. We also tested the analgesic effects of a novel, minimally invasive, bioelectronic approach to percutaneously stimulate the vagus nerve (termed percutaneous vagus nerve stimulation [pVNS]). Weekly pVNS treatment for 30 minutes at 10 Hz for 3 weeks after the surgery strongly reduced pain behaviors compared with untreated controls. Percutaneous vagus nerve stimulation also improved locomotor coordination and accelerated bone healing. In the dorsal root ganglia, vagal stimulation inhibited the activation of glial fibrillary acidic protein-positive satellite cells but without affecting microglial activation. Overall, these data provide novel evidence supportive of the use of pVNS to prevent postoperative pain and inform translational studies to test antinociceptive effects of bioelectronic medicine in the clinic.

Global trends in research on oxidative stress related to heart failure from 2012 to 2021: a bibliometric analysis and suggestion to researchers.

Background: Oxidative stress leads to an increase in reactive oxygen in the body. During heart failure (HF), when the body's antioxidant defense system fails to remove excessive reactive oxygen species, myocardial cells will be damaged or even die. Over the past ten years, the number of research publications on oxidative stress related to HF has increased. Methods: We searched publications published in 2012-2021 and the Web of Science Core Collection (WoSCC) recording information. Based on the VOSviewer and CiteSpace, we conducted a bibliometric analysis of the overall distribution of journals, keywords, authors, major countries, annual output, active institutions, and cocited literature. The Global Citation Score (GCS) was used to evaluate the impact and quality of highly cited papers. Results: We retrieved 5,616 articles and reviews. Over the past ten years, the number of annual publications on oxidative stress related to HF has increased. USA has published the largest number of articles and obtained the highest number of citations (NC) and H-index. The University of California and PLoS One are the most productive affiliations and journals in terms of publications on oxidative stress related to HF. The GCS of articles written by Paulus WJ in 2013 was 1,632, which was the top ranking. The most frequent keywords are "oxidative stress", "heart failure", "inflammation", "dysfunction" and "apoptosis". The top three authors are Kang Yuming, Ren Jun and Okoshi Katashi. "Impact", "induced myocardial infarction", "cardiovascular outcome", "empagliflozin", "sglt2 inhibitor", "protect", and "Na+/H+ exchanger" have become popular research topics. Conclusions: Our research shows the research focus and development trends of oxidative stress related to HF in the past decade. Understanding the most important indicators of oxidative stress related to HF and the hot spots in the field of oxidative stress research related to HF can assist scholars, countries and policy-makers in the field in better understanding oxidative stress related to HF and can also lead to better decisions in oxidative stress treatment.

Vagus nerve stimulation rescues persistent pain following orthopedic surgery in adult mice.

Postoperative pain is a major clinical problem imposing a significant burden on our patients and society. Up to 57% of patients experience persistent postoperative pain 2 years after orthopedic surgery [49]. Although many studies have contributed to the neurobiological foundation of surgery-induced pain sensitization, we still lack safe and effective therapies to prevent the onset of persistent postoperative pain. We have established a clinically relevant orthopedic trauma model in mice that recapitulates common insults associated with surgery and ensuing complications. Using this model, we have started to characterize how induction of pain signaling contributes to neuropeptides changes in dorsal root ganglia (DRG) and sustained neuroinflammation in the spinal cord [62]. Here we have extended the characterization of pain behaviors for >3 months after surgery, describing a persistent deficit in mechanical allodynia in both male and female C57BL/6J mice after surgery. Notably, we have applied a novel minimally invasive bioelectronic approach to percutaneously stimulate the vagus nerve (termed pVNS) [24] and tested its anti-nociceptive effects in this model. Our results show that surgery induced a strong bilateral hind-paw allodynia with a slight decrease in motor coordination. However, treatment with pVNS for 30-minutes at10 Hz weekly for 3 weeks prevented pain behavior compared to naïve controls. pVNS also improved locomotor coordination and bone healing compared to surgery without treatment. In the DRGs, we observed that vagal stimulation fully rescued activation of GFAP positive satellite cells but did not affect microglial activation. Overall, these data provide novel evidence for the use of pVNS to prevent postoperative pain and may inform translational studies to test anti-nociceptive effects in the clinic.

Quanduzhong capsules for the treatment of grade 1 hypertension patients with low-to-moderate risk: A multicenter, randomized, double-blind, placebo-controlled clinical trial.

Background: Duzhong [DZ (Eucommia ulmoides Oliv.)] is regarded as a traditional Chinese medicine with a history dating back more than 2000 years. This herb is considered a nourishing herb in China and is commonly used as a tonic to strengthen muscles and bones, nourish the kidneys and liver, and soothe miscarriages. Moreover, there is evidence that DZ is capable of regulating blood pressure (BP), and several compounds isolated from DZ have been shown to have a BP-lowering effect. Quanduzhong capsules contain an extract of DZ [Eucommia ulmoides Oliv. (Eucommiaceae; Eucommiae cortex)] that is effective in treating hypertension. This multicenter, randomized, double-blind, placebo-controlled clinical trial sought to evaluate the clinical efficacy of Quanduzhong capsules in the treatment of low-to-moderate risk grade 1 hypertension patients. Materials and methods: A total of 60 patients from 3 centers with documented low-to-moderate risk grade 1 hypertension were randomly assigned in a 1:1 ratio to the test group or the control group. After a 1 week lead-in period using sham Quanduzhong capsules, all patients who met the entry criteria (29 cases in the test group and 29 cases in the control group) entered the 4 week test period. The test group took Quanduzhong capsules, and the control group continued to take sham Quanduzhong capsules. The primary endpoints [24-h mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) determined via 24-h ambulatory blood pressure monitoring (ABPM); office SBP and DBP] and secondary endpoints [mean arterial pressure; mean pulse; daytime mean SBP and DBP; nocturnal mean SBP and DBP; SBP and DBP load; area under the blood pressure (BP) curve; morning peak BP; early morning SBP and DBP; smoothness index of SBP and DBP; 24 h BP mean coefficient of variation (CV); percentage of patients with circadian restoration in ABPM; home BP; quality of life evaluated by WHO Quality of Life-BREF questionnaire; grading and quantitative evaluation of hypertension symptoms; values of plasmatic renin activity, angiotensin II, aldosterone, ß-2 microglobulin and homocysteine] were assessed following the treatment. Drug-related adverse events and adverse drug reactions were also compared. Results: After a 4 week test period, a significant difference in the DBP CV between the two groups was observed (-2.49 ± 4.32 vs. 0.76 ± 4.3; p < .05). Moreover, the mean office SBP change was -7.62 ± 9.32 mmHg, and the mean DBP change was -4.66 ± 6.03 (p < .05). Among the three subjects with abnormal homocysteine levels in the test group, homocysteine levels decreased by 6.23 ± 9.15 µmol/L after treatment. No differences were observed between the two groups in any other indicators. After 4 weeks of treatment, there were no significant differences between the groups in terms of safety indicators (p > .05). No abnormal vital signs (except BP) or severe liver or renal function impairment were observed during the treatment periods; in addition, adverse events and drug reactions were mild. Conclusion: Treatment with Quanduzhong capsules reduced office SBP and DBP as well as DBP CV determined by 24-h ambulatory BP monitoring in patients with grade 1 hypertension at low-to-moderate risk. Clinical Trial Registration: https://www.chictr.org.cn/showproj.aspx?proj=32531, identifier ChiCTR1900021699.

IL-23, but not IL-12, plays a critical role in inflammation-mediated bone disorders.

Interleukin-12 (IL-12) and IL-23 are thought to have central roles in inflammation and are critical to pathologies associated with inflammation-induced bone disorders. The deletion of IL-12p40 (a common subunit of IL-12 and IL-23) can improve bone regeneration. However, the relative roles of IL-12 and IL-23 in bone disorders are largely unknown. Methods: Ectopic bone formation and skull defect models were established to evaluate the relative roles of IL-12 and IL-23 in inflammatory bone disorders. Differences in bone mass among WT, IL-12p35-/-, and IL-12p40-/- mice (young and elderly) were detected by micro-CT. Osteogenic and osteoclastic activities were explored using ELISA, qRT-PCR, and histological analysis. Moreover, the mechanisms by which IL-12 and IL-23 regulated the differentiation of BMMSCs and RAW264.7 cells were explored using Alizarin Red and tartrate-resistant acid phosphatase staining in vitro. Apilimod was used to inhibit IL-12 and IL-23 production in vivo. Results: Mice deficient in IL-12p40 promoted bone formation and protected against aging-related bone loss. By contrast, bone loss was aggravated in IL-12-/- mice, suggesting that IL-23 may play a dominant role in inflammation-related bone disorders. Mechanistically, IL-12 and IL-23 coupled osteogenesis and osteoclastic activities to regulate bone homeostasis and repair. IL-23 deficiency increased bone formation and inhibited bone resorption. Finally, apilimod treatment significantly improved bone regeneration and calvarial defect repair. Conclusion: These data collectively uncover a previously unrecognized role of IL-23 in skeletal tissue engineering. Thus, IL-23 can act as a biomarker to predict diseases and treatment efficacy, and apilimod can be used as an effective therapeutic drug to combat inflammatory bone disorders.

High-Dose TGF-ß1 Impairs Mesenchymal Stem Cell-Mediated Bone Regeneration via Bmp2 Inhibition.

Transforming growth factor-ß1 (TGF-ß1) is a key factor in bone reconstruction. However, its pathophysiological role in non-union and bone repair remains unclear. Here we demonstrated that TGF-ß1 was highly expressed in both C57BL/6 mice where new bone formation was impaired after autologous bone marrow mesenchymal stem cell (BMMSC) implantation in non-union patients. High doses of TGF-ß1 inhibited BMMSC osteogenesis and attenuated bone regeneration in vivo. Furthermore, different TGF-ß1 levels exhibited opposite effects on osteogenic differentiation and bone healing. Mechanistically, low TGF-ß1 doses activated smad3, promoted their binding to bone morphogenetic protein 2 (Bmp2) promoter, and upregulated Bmp2 expression in BMMSCs. By contrast, Bmp2 transcription was inhibited by changing smad3 binding sites on its promoter at high TGF-ß1 levels. In addition, high TGF-ß1 doses increased tomoregulin-1 (Tmeff1) levels, resulting in the repression of Bmp2 and bone formation in mice. Treatment with the TGF-ß1 inhibitor SB431542 significantly rescued BMMSC osteogenesis and accelerated bone regeneration. Our study suggests that high-dose TGF-ß1 dampens BMMSC-mediated bone regeneration by activating canonical TGF-ß/smad3 signaling and inhibiting Bmp2 via direct and indirect mechanisms. These data collectively show a previously unrecognized mechanism of TGF-ß1 in bone repair, and TGF-ß1 is an effective therapeutic target for treating bone regeneration disability. © 2019 American Society for Bone and Mineral Research.

lncRNA ADAMTS9-AS2 Controls Human Mesenchymal Stem Cell Chondrogenic Differentiation and Functions as a ceRNA.

Long noncoding RNAs (lncRNAs) have emerged as key regulators of cell differentiation and development. However, potential roles for lncRNAs in chondrogenic differentiation have remained poorly understood. Here we identify lncRNA ADAMTS9 antisense RNA 2, ADAMTS9-AS2, which controls the chondrogenic differentiation by acting as a competing endogenous RNA (ceRNA) in human mesenchymal stem cells (hMSCs). We screen out ADAMTS9-AS2 of undifferentiated and differentiated cells during chondrogenic differentiation by microarrays. Suppression or overexpression of lncRNA ADAMTS9-AS2 correlates with inhibition and promotion of hMSC chondrogenic differentiation, respectively. We find that ADAMTS9-AS2 can sponge miR-942-5p to regulate the expression of Scrg1, a transcription factor promoting chondrogenic gene expression. Finally, we confirm the function of ADAMTS9-AS2 to cartilage repair in the absence of transforming growth factor ß (TGF-ß) in vivo. In conclusion, ADAMTS9-AS2 plays an important role in chondrogenic differentiation as a ceRNA, so that it can be regarded as a therapy target for cartilage repair.

MiR-146a Deletion Protects From Bone Loss in OVX Mice by Suppressing RANKL/OPG and M-CSF in Bone Microenvironment.

MicroRNAs play important roles in osteoporosis and show great potential for diagnosis and therapy of osteoporosis. Previous studies have demonstrated that miR-146a affects osteoblast (OB) and osteoclast (OC) formation. However, these findings have yet to be identified in vivo, and it is unclear whether miR-146a is related to postmenopausal osteoporosis. Here, we demonstrated that miR-146a knockout protects bone loss in mouse model of estrogen-deficient osteoporosis, and miR-146a inhibits OB and OC activities in vitro and in vivo. MiR-146a-/- mice displayed the same bone mass as the wild type (WT) but exhibited a stronger bone turnover than the WT did under normal conditions. Nevertheless, miR-146a-/- mice showed an increase in bone mass after undergoing ovariectomy (OVX) compared with those subjected to sham operation. OC activities were impaired in the miR-146a-/- mice exposed to estrogen deficiency, which was diametrically opposite to the enhanced bone resorption ability of WT. Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) from a bone microenvironment affect this extraordinary phenomenon. Therefore, our results implicate that miR-146a plays a key role in estrogen deficiency-induced osteoporosis, and the inhibition of this molecule provides skeleton protection. © 2019 American Society for Bone and Mineral Research.

Meta-analysis of urinary C-terminal telopeptide of type II collagen as a biomarker in osteoarthritis diagnosis.

OBJECTIVE: This study aims to discover that the urinary C-terminal telopeptide of type II collagen (uCTX-II) levels differ between osteoarthritis (OA) patients and healthy individuals (controls). According to this difference, we may conclude that uCTX-II can be a biomarker for OA diagnosis. METHODS: We searched MEDLINE and EMBASE databases updated to 2014 to find literature on OA biomarkers. We retrieved the publications that met the required criterion. Literature quality was assessed according to the Newcastle-Ottawa Scale. Publication bias was assessed by Begg's test and Egger's test with the software STATA version 12.0. The weighted mean difference (WMD) was calculated, and the subgroup analysis was completed using STATA 12.0. RESULTS: Six publications were included in our analysis. The WMD for OA patients versus the controls was 83.05, which was within the 95% confidence interval. For subgroup analysis, the WMD of patients with severe OA was 119.92, whereas that of patients with mild OA was 28.07. CONCLUSIONS: uCTX-II levels were higher in OA patients than in controls, subgroup analysis revealed that the uCTX-II levels rised with the OA severity, the heterogeneity originated from different levels of OA severity, These results showed that uCTX-II would be a promising clinical biomarker in OA diagnosis.