Facile construction of Mo-based nanozyme system via ZIF-8 templating with enhanced catalytic efficiency and antibacterial performance.

Although Zeolitic Imidazolate Framework-8 (ZIF-8) shows significant promise in chemodynamic therapy of bacterial infections due to its large specific surface area and enzyme-like activity, it still faces a considerable gap compared to natural enzymes. The dependency on low pH and high concentrations of hydrogen peroxide ((H2O2) is a major factor limiting the clinical progress of nanozymes. Single-atom nanozymes (SA-zyme), which exhibit superior catalytic performance, are expected to overcome this limitation. In this study, we used ZIF-8 as a template to prepare structurally regular molybdenum-based single-atom nanozymes (Mo-zyme) by coordinating molybdenum atoms with nitrogen atoms within the zeolitic imidazolate framework and evaporating the zinc element at high temperatures. The cascade catalytic performance of the nanodrugs was enhanced by loading glucose oxidase (GOx) and encapsulating it with a hyaluronic acid (HA) layer to form a composite (Mo/GOx@HA). Upon contact with hyaluronidase from bacteria in infected tissues, the cascade reaction is triggered, resulting in the degradation of the HA shell, and releasing the encapsulated GOx. Once exposed, GOx catalyzes the oxidation of glucose into gluconic acid, resulting in a localized decrease in pH and continuous production of H2O2. The combination of lowered pH and increased H2O2 concentration significantly amplifies the catalytic activity of the Mo-zyme. This enhanced activity facilitates the in situ generation of hydroxyl radicals (•OH) on the bacterial surface, leading to effective and efficient bacterial eradication. Wound infection treatment has demonstrated that the as-prepared Mo/GOx@HA exhibits excellent antibacterial and anti-inflammatory activity. This work provided a promising enzymatic cascade reaction nanoplatform for the treatment of bacteria infected wounds.

Study on the synthesis of iron-based nanomedicine assisted by angelica sinensis polysaccharide with enhanced retention performance and its application in anemia treatment.

Inappropriate treatment of chronic inflammation and infection can lead to serious consequences, with anemia being the most common secondary disease that often requires systematic treatment. However, the complex pathology and gastrointestinal irritation associated with oral iron supplements limit their effectiveness. To address this, a bioactive ingredient derived from natural herbs, Angelica sinensis polysaccharide (ASP), was utilized as an ideal adjuvant for regulating the size and stability of iron oxide nanoparticles (IONPs). Highly hydrophilic ASP-modified IONPs (IONPs@ASP) with a mesoporous structure were developed under the induction of microemulsion.The as-prepared IONPs@ASP exhibited enhanced stability, retention performance and controlled degradation in blood and lysosomal environments, respectively, which is beneficial for long-term intravenous iron maintenance in anemia treatment. After confirming the biosafety of IONPs@ASP, pharmacodynamic results showed that hemoglobin levels increased significantly and rapidly returned to normal levels in anemia model rats treated with IONPs@ASP, even surpassing the effects of IONPs or ASP monotherapy. Additionally, analysis of inflammatory factors in rat serum suggested that ASP effectively upregulated the expression of anti-inflammatory factors, indicating synergistic effects of iron-based nanomedicine and immune regulation in anemia treatment. These findings represent a significant advancement in anemia treatment and open new possibilities for developing versatile nanoparticles based on ASP.

Cartilaginous predictors of residual acetabular dysplasia (RAD) in developmental dysplasia of the hip following closed or open reduction: A systematic review and meta-analysis.

Object: This study was designed to analyze the cartilaginous predictors of residual acetabular dysplasia (RAD) after early treatment of developmental dysplasia of the hip and their diagnostic accuracy. Study design: Databases such as PubMed, Embase, Cochrane, and Web of science were searched to screen the literature. The quality of the literature was assessed by the QUADAS-2 tool. Qualitative and quantitative synthesis of literature were performed based on extracted data. For quantitative synthesis studies, the sensitivity, specificity, diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC) curve with corresponding confidence intervals were calculated. Results: For the cartilaginous acetabular index (CAI) group, the combined values of sensitivity, specificity, and DOR were 0.80 (95% CI = 0.54-0.93), 0.73 (95% CI = 0.57-0.84), and 10.62 (95% CI = 3.96-28.53), respectively. The corresponding values in the cartilaginous center-edge angle (CCE) group were 0.71 (95% CI = 0.57-0.82), 0.78 (95% CI = 0.66-0.87), and 8.64 (95% CI = 3.08-24.25), respectively. The area under the curve (AUC) of SROC was 0.82 (95% CI = 0.78-0.85) and 0.80 (95% CI = 0.76-0.83) for the CAI and CCE groups. The CAI group had higher sensitivity, DOR, and AUC than the CCE group. Conclusion: Both of these two groups have good diagnostic accuracy, and CAI/L-AI has a little edge over CCE/L-CEA. However, there is still more research needed to determine whether they can be used as independent indications for secondary orthopedic surgery.Systematic review registration: [https://www.crd.york.ac.uk/PROSPERO/], identifier: [CRD42022338332].

The diagnosis and treatment of a special rare type of Monteggia equivalent fractures in children.

Purpose: To explore the characteristics, mechanism, treatment, and prognosis of head-neck separation type of Monteggia equivalent fractures in children. Methods: Patients with this injury were reviewed retrospectively. The lesion was characterized by a fracture of the ulnar with radial neck fracture but without dislocation of the radial head. Our classification was based on the direction of displacement and angulation of fractures on radiographs, divided into the extension-valgus type and flexion-varus type. The fractures were treated with reduction and internal fixation, depending on the fracture type. The clinical results were evaluated by using radiology and the Mayo Elbow Performance Score (MEPS). Results: A total of 12 patients were followed up for an average of 40.5 months. The ulnar fractures were treated with closed reduction (CR) and K-wire fixation in one patient, elastic stable intramedullary nail (ESIN) fixation in four patients, open reduction (OR) and plate fixation in five, with no fixation in two. CR with ESIN fixation was successful in 11 patients with radial neck fractures, but one underwent OR and K-wire fixation. All fractures healed on time, with fewer complications (avascular necrosis in one patient, and bulk formation of metaphysis in another). The therapeutic efficacy was evaluated by using MEPS and was found to be excellent in 10 patients, good in one, and fair in another. Conclusions: The head-neck separation type of Monteggia equivalent fractures in children is rare. Its characteristics are different from that of Monteggia fracture. The length and anatomic structure of the ulna should be restored and stabilized first, while the radial neck fracture should be treated with CR and ESIN fixation. Satisfactory clinical results can be achieved with fewer complications.

Phosphorylation of KRT8 (keratin 8) by excessive mechanical load-activated PKN (protein kinase N) impairs autophagosome initiation and contributes to disc degeneration.

Excessive mechanical load (overloading) is a well-documented pathogenetic factor for many mechano stress-induced pathologies, i.e. intervertebral disc degeneration (IDD). Under overloading, the balance between anabolism and catabolism within nucleus pulposus (NP) cells are badly thrown off, and NP cells undergo apoptosis. However, little is known about how the overloading is transduced to the NP cells and contributes to disc degeneration. The current study shows that conditional knockout of Krt8 (keratin 8) within NP aggravates load-induced IDD in vivo, and overexpression of Krt8 endows NP cells greater resistance to overloading-induced apoptosis and degeneration in vitro. Discovery-driven experiments shows that phosphorylation of KRT8 on Ser43 by overloading activated RHOA-PKN (protein kinase N) impedes trafficking of Golgi resident small GTPase RAB33B, suppresses the autophagosome initiation and contributes to IDD. Overexpression of Krt8 and knockdown of Pkn1 and Pkn2, at an early stage of IDD, ameliorates disc degeneration; yet only knockdown of Pkn1 and Pkn2, when treated at late stage of IDD, shows a therapeutic effect. This study validates a protective role of Krt8 during overloading-induced IDD and demonstrates that targeting overloading activation of PKNs could be a novel and effective approach to mechano stress-induced pathologies with a wider window of therapeutic opportunity.Abbreviations: AAV: adeno-associated virus; AF: anulus fibrosus; ANOVA: analysis of variance; ATG: autophagy related; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; CEP: cartilaginous endplates; CHX: cycloheximide; cKO: conditional knockout; Cor: coronal plane; CT: computed tomography; Cy: coccygeal vertebra; D: aspartic acid; DEG: differentially expressed gene; DHI: disc height index; DIBA: dot immunobinding assay; dUTP: 2'-deoxyuridine 5'-triphosphate; ECM: extracellular matrix; EDTA: ethylene diamine tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPS: group-based prediction system; GSEA: gene set enrichment analysis; GTP: guanosine triphosphate; HE: hematoxylin-eosin; HRP: horseradish peroxidase; IDD: intervertebral disc degeneration; IF: immunofluorescence staining; IL1: interleukin 1; IVD: intervertebral disc; KEGG: Kyoto encyclopedia of genes and genomes; KRT8: keratin 8; KD: knockdown; KO: knockout; L: lumbar vertebra; LBP: low back pain; LC/MS: liquid chromatograph mass spectrometer; LSI: mouse lumbar instability model; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMP3: matrix metallopeptidase 3; MRI: nuclear magnetic resonance imaging; NC: negative control; NP: nucleus pulposus; PBS: phosphate-buffered saline; PE: p-phycoerythrin; PFA: paraformaldehyde; PI: propidium iodide; PKN: protein kinase N; OE: overexpression; PTM: post translational modification; PVDF: polyvinylidene fluoride; qPCR: quantitative reverse-transcriptase polymerase chain reaction; RHOA: ras homolog family member A; RIPA: radio immunoprecipitation assay; RNA: ribonucleic acid; ROS: reactive oxygen species; RT: room temperature; TCM: rat tail compression-induced IDD model; TCS: mouse tail suturing compressive model; S: serine; Sag: sagittal plane; SD rats: Sprague-Dawley rats; shRNA: short hairpin RNA; siRNA: small interfering RNA; SOFG: safranin O-fast green; SQSTM1: sequestosome 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VG/ml: viral genomes per milliliter; WCL: whole cell lysate.

Facet joint degeneration-An initial procedure of the cervical spine degeneration.

Objective: This study aims to emphasize the initiating role of facet joint (FJ) degeneration in the process of cervical spine degeneration induced by tangential load, and we further validate it in a novel cervical spine degeneration animal model. Methods: The characteristics of cervical degeneration in patients of different ages were summarized through case collection. In the rat models, Hematoxylin-Eosin, Safranin O staining, and micro-computed tomography were used to show the histopathological changes and bone fiber structure of FJ and the height of intervertebral disc (IVD) space. The ingrowth of nociceptive sensory nerve fibers was observed by immunofluorescence staining. Results: FJ degeneration without IVDs degeneration was more common in people with cervical spondylosis in young patients. The obvious degeneration phenotypes of the FJs preceded the IVDs at the same cervical segment in our animal model. The SP+ and CGRP+ sensory nerve fibers were observed in the articular subchondral bone of degenerated FJs and porous endplates of degenerated IVDs. Conclusion: The FJ degeneration may act as the major contributor to cervical spine degeneration in young people. The dysfunction of functional unit of spine, not a certain part of IVD tissue, results in the occurrence of cervical degeneration and neck pain.

SIRT5-related desuccinylation modification of AIFM1 protects against compression-induced intervertebral disc degeneration by regulating mitochondrial homeostasis.

Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

Targeting clock-controlled gene Nrf2 ameliorates inflammation-induced intervertebral disc degeneration.

BACKGROUND: Intervertebral disc (IVD) is a highly rhythmic tissue, which experiences a diurnal cycle of high/low mechanical loading via the changes of activity/rest phase. There are signs that disruption of the peripheral IVD clock is related to the process of intervertebral disc degeneration (IDD). However, it is still unclear whether inflammation could disturb the IVD clock and thus induce the process of IDD. METHODS AND RESULTS: In this study, we used IL-1ß, a commonly used inflammatory factor, to induce IDD and found that the IVD clock was dampened in degenerated human nucleus pulposus specimens, rat nucleus pulposus (NP) tissues, and cells. In this study, we found that the circadian clock of NP cells was totally disrupted by knockdown of the core clock gene brain and muscle arnt-like protein-1 (Bmal1), which thus induced the dysfunction of NP cells. Next, we explored the mechanism of dampened clock-induced IDD and found that knockdown of Bmal1 decreased the expression of nuclear factor erythroid2-related factor 2 (NRF2), a downstream target gene of Bmal1, and increased inflammatory response, oxidative stress reaction, and apoptosis of NP cells. In addition, NRF2 activation attenuated the dysfunction of NP cells induced by the dampened IVD clock and the degenerative process of NP tissues in an organotypic tissue-explant model. CONCLUSIONS: Taken together, our study extends the relationship between peripheral clock and IVD homeostasis and provides a potential therapeutic method for the prevention and recovery of IDD by targeting the clock-controlled gene Nrf2.

Decoding the annulus fibrosus cell atlas by scRNA-seq to develop an inducible composite hydrogel: A novel strategy for disc reconstruction.

Low back pain is one of the most serious public health problems worldwide and the major clinical manifestation of intervertebral disc degeneration (IVDD). The key pathological change during IVDD is dysfunction of the annulus fibrosus (AF). However, due to the lack of an in-depth understanding of AF biology, the methods to reconstruct the AF are very limited. In this study, the mice AF cell atlas were decoded by single-cell RNA sequencing to provide a guide for AF reconstruction. The results first identify a new population of AF cells, fibrochondrocyte-like AF cells, which synthesize both collagen I and collagen II and are potential functional cells for AF reconstruction. According to the dual features of the AF extracellular matrix, a composite hydrogel based on the acylation of methacrylated silk fibroin with methacrylated hyaluronic acid was produced. To obtain the ability to stimulate differentiation, the composite hydrogels were combined with a fibrochondrocyte-inducing supplement. Finally, reconstruction of the AF defects, by the novel AF stem cell-loaded composite hydrogel, could be observed, its amount of chondroid matrices recovered to 31.7% of AF aera which is significantly higher than that in other control groups. In summary, this study decodes the AF cell atlas, based on which a novel strategy for AF reconstruction is proposed.

Injectable hydrogel with nucleus pulposus-matched viscoelastic property prevents intervertebral disc degeneration.

Background/Objective: Intervertebral disc (IVD) degeneration (IVDD) that greatly affected by regional biomechanical environment is a major cause of low back pain. Injectable hydrogels have been commonly studied for treatment of IVDD due to their capability of mimicking extracellular matrix structure to support cellular behavior and clinical prospects in minimally invasive treatment. However, most hydrogels suffer from complicated chemistry, potential uncertainty and toxicity from in-situ gelation, and mismatch with IVD mechanical environment that limit their therapeutic effects or clinical translation in IVDD or intervertebral disc defect repair. For IVD lesion repair, the study aims to develop a novel hydrogel with shear-thinning enabled injectability, high bio-safety, and mechanical properties adaptable to the IVD environment, using a simple chemistry and method. And therapeutic efficacy of the novel hydrogel in the treatment of IVDD or intervertebral disc defect will be revealed. Methods: A glycerol cross-linked PVA gel (GPG) was synthesized based on multiple H-bonds formation between glycerol molecules and PVA chains. The rheological and mechanical properties were tested. The swelling ratio was measured. The micro-architecture was observed through scanning and transmission electron microscopes. Nucleus pulposus (NP) cells were cultured in GPG-coated plates or silicone chambers treated under hydrostatic or dynamic loading in vitro, and examined for proliferation, vitality, apoptosis, expression of catabolic and anabolic markers. GPG was injected in needle puncture (IDD) or NP discectomy (NPD) models in vivo, and examined through magnetic resonance imaging, micro-computed tomography scanning and histological staining. Results: GPG had a highly porous structure consisting of interconnected pores. Meanwhile, the GPG had NP-like viscoelastic property, and was able to withstand the cyclic deformation while exhibiting a prominent energy-dissipating capability. In vitro cell tests demonstrated that, the hydrogel significantly down-regulated the expression of catabolic markers, maintained the level of anabolic markers, preserved cell proliferation and vitality, reduced apoptotic rate of NP cells under pathologically hydrostatic and dynamic loading environments compared to cells cultured on untreated plate or silicone chamber. In vivo animal studies revealed that injection of GPG efficiently maintained NP structural integrity, IVD height and relative water content in IDD models, and stimulated the fibrous repair in NPD models. Conclusion: This study showed that GPG, with high injectability, NP-like viscoelastic characteristics, good energy-dissipating properties and swelling capacities, preserved NP cells vitality against pathological loading, and had therapeutic effects on IVD repair in IDD and NPD models. The translational potential of this article: Effective clinical strategy for treatment of intervertebral disc degeneration (IVDD) is still lacking. This study demonstrates that injection of a hydrogel with nucleus pulposus-matched viscoelastic property could remarkably prevent the IVDD progress. Prepared with simple chemistry and procedure, the cell/drug-free GPG with high bio-safety and shear-thinning enabled injectability bears great translational potential for the clinical treatment of IVDD via a minimally invasive approach.

Restoring the dampened expression of the core clock molecule BMAL1 protects against compression-induced intervertebral disc degeneration.

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

Quercetin Suppresses Apoptosis and Attenuates Intervertebral Disc Degeneration via the SIRT1-Autophagy Pathway.

Intervertebral disc degeneration (IDD) has been generally accepted as the major cause of low back pain (LBP), which causes an enormous socioeconomic burden. Previous studies demonstrated that the apoptosis of nucleus pulposus (NP) cells and the dyshomeostasis of extracellular matrix (ECM) contributed to the pathogenesis of IDD, and effective therapies were still lacking. Quercetin, a natural flavonoid possessing a specific effect of autophagy stimulation and SIRT1 activation, showed some protective effect on a series of degenerative diseases. Based on previous studies, we hypothesized that quercetin might have therapeutic effects on IDD by inhibiting the apoptosis of NP cells and dyshomeostasis of ECM via the SIRT1-autophagy pathway. In this study, we revealed that quercetin treatment inhibited the apoptosis of NP cells and ECM degeneration induced by oxidative stress. We also found that quercetin promoted the expression of SIRT1 and autophagy in NP cells in a dose-dependent manner. Autophagy inhibitor 3-methyladenine (3-MA) reversed the protective effect of quercetin on apoptosis and ECM degeneration. Moreover, SIRT1 enzymatic activity inhibitor EX-527, suppressed quercetin-induced autophagy and the protective effect on NP cells, indicating that quercetin protected NP cells against apoptosis and prevented ECM degeneration via SIRT1-autophagy pathway. In vivo, quercetin was also demonstrated to alleviate the progression of IDD in rats. Taken together, our results suggest that quercetin prevents IDD by promoting SIRT1-dependent autophagy, indicating one novel and effective therapeutic method for IDD.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.

Association of vitamin D receptor ApaI gene polymorphism with osteoporosis susceptibility in postmenopausal Han Chinese women in Xinjiang.

Osteoporosis is a polygenic disorder and has been demonstrated to be associated with ~30 candidate genes, the majority of which have also been implicated in the regulation of bone mineral density (BMD). Vitamin D receptor (VDR) is the candidate gene that has been most extensively studied. Certain studies have reported that the VDR single nucleotide polymorphism ApaI is associated with the risk of osteoporosis in Caucasian and African women. However, this association has not yet been studied in postmenopausal Han Chinese women in the Xinjiang area. In the present study, ApaI polymorphisms of VDR were defined by polymerase chain reaction-restriction fragment length polymorphism, in order to analyze the distribution of ApaI polymorphisms in postmenopausal Han Chinese women from Xinjiang. BMD was measured by dual energy X-ray absorptiometry at the lumbar spine (L2-4), Ward's triangle, great trochanter and femoral shaft. A total of 336 women were included in this study. The genotype distribution of ApaI was consistent with the Hardy-Weinberg equilibrium (all P>0.05). There were no significant differences in ApaI genotype frequencies between the 90 cases in the osteoporosis group and 246 cases in the non-osteoporosis group (P=0.946). Meanwhile, it was identified that BMD values of the tested locations were negatively correlated with age (P<0.05) and positively correlated with body mass index (BMI; P<0.05). On further attribution risk analysis, BMD was identified as a risk factor [odds ratio (OR): 0.464, 95% confidence interval (CI): 0.372-0.580, P=0.001] and BMI a protective factor (OR: 1.502, 95% CI: 1.008-2.240, P=0.032) in osteoporosis. When BMD was adjusted for confounding factors including age and BMI, it was observed that the ApaI polymorphism was not associated with BMD at the sites tested (P>0.05). In conclusion, the present study identified no significant association of the common VDR polymorphism ApaI with BMD at several skeletal sites in postmenopausal Han Chinese women in the Xinjiang area. Age was negatively correlated with BMD at different sites and identified as a risk factor; while BMI was positively correlated with BMD and identified as a protective factor.

Analysis of short-term treatment with the phosphodiesterase type 5 inhibitor tadalafil on long bone development in young rats.

Cyclic GMP (cGMP) is an important intracellular regulator of endochondral bone growth and skeletal remodeling. Tadalafil, an inhibitor of the phosphodiesterase (PDE) type 5 (PDE5) that specifically hydrolyzes cGMP, is increasingly used to treat children with pulmonary arterial hypertension (PAH), but the effect of tadalafil on bone growth and strength has not been previously investigated. In this study, we first analyzed the expression of transcripts encoding PDEs in primary cultures of chondrocytes from newborn rat epiphyses. We detected robust expression of PDE5 as the major phosphodiesterase hydrolyzing cGMP. Time-course experiments showed that C-type natriuretic peptide increased intracellular levels of cGMP in primary chondrocytes with a peak at 2 min, and in the presence of tadalafil the peak level of intracellular cGMP was 37% greater ( P < 0.01) and the decline was significantly attenuated. Next, we treated 1-mo-old Sprague Dawley rats with vehicle or tadalafil for 3 wk. Although 10 mg·kg-1·day-1 tadalafil led to a significant 52% ( P < 0.01) increase in tissue levels of cGMP and a 9% reduction ( P < 0.01) in bodyweight gain, it did not alter long bone length, cortical or trabecular bone properties, and histological features. In conclusion, our results indicate that PDE5 is highly expressed in growth plate chondrocytes, and short-term tadalafil treatment of growing rats at doses comparable to those used in children with PAH has neither obvious beneficial effect on long bone growth nor any observable adverse effect on growth plate structure and trabecular and cortical bone structure.

The long non-coding RNA NEAT1 contributes to extracellular matrix degradation in degenerative human nucleus pulposus cells.

Intervertebral disc degeneration is a complex disease involving genetic and environmental factors and multiple cellular processes. The role and expression of the lncRNA NEAT1 were assessed in intervertebral disc degeneration. NEAT1 expression was assessed in degenerative and control nucleus pulposus using RT-PCR. Western blotting and RT-PCR were also used to investigate p53 and p21 levels in nucleus pulposus tissues. NEAT1 function in degenerative nucleus pulposus cells was assessed with gain- and loss-of-function experiments. ERK/MAPK signaling was also examined. NEAT1, p53, and p21 were dramatically upregulated in intervertebral disc degeneration. Furthermore, catabolic MMP13 and ADAMTS5 were dysregulated and collagen II and aggrecan were downregulated after NEAT1 overexpression. This effect was reversed by transfection with si-NEAT1 in degenerative nucleus pulposus cells. In addition, NEAT1 was found to affect the activation of the ERK/MAPK pathway. The NEAT1-induced ECM degradation may involve ERK1/2/MAPK signaling. LncRNA NEAT1 may represent a novel molecular target for intervertebral disc degeneration treatment by preventing nucleus pulposus ECM degradation. Impact statement For the first time, our study demonstrates that lncRNA NEAT1 plays a role in the occurrence and development of IDD by participating in extracellular matrix remodeling. This lncRNA regulates catabolic MMP13 and ADAMTS5 and anabolic collagen II and aggrecan by affecting the ERK/MAPK signaling pathway in degenerative human nucleus pulposus (NP) cells. Our research provides a scientific basis for targeting of NEAT1 for the IDD.

Loading-Induced Reduction in Sclerostin as a Mechanism of Subchondral Bone Plate Sclerosis in Mouse Knee Joints During Late-Stage Osteoarthritis.

OBJECTIVE: To establish an unbiased, 3-dimensional (3-D) approach that quantifies subchondral bone plate (SBP) changes in mouse joints, and to investigate the mechanism that mediates SBP sclerosis at a late stage of osteoarthritis (OA). METHODS: A new micro-computed tomography (micro-CT) protocol was developed to characterize the entire thickness of the SBP in the distal femur of a normal mouse knee. Four mouse models of severe joint OA were generated: cartilage-specific Egfr-knockout (Egfr-CKO) mice at 2 months after surgical destabilization of the medial meniscus (DMM), Egfr-CKO mice with aging-related spontaneous OA, wild-type (WT) mice at 10 months after DMM, and WT mice at 14 weeks after DMM plus hemisectomy of the meniscus (DMMH) surgery. As an additional model, mice with knockout of the sclerostin gene (Sost-KO) were subjected to DMMH surgery. Knee joints were examined by micro-CT, histology, and immunohistochemical analyses. RESULTS: Examination of the mouse distal femur by 3-D micro-CT revealed a positive correlation between SBP thickness and the loading status in normal knees. In all 4 mouse models of late-stage OA, SBP sclerosis was restricted to the areas under severely eroded articular cartilage. This was accompanied by elevated bone formation at the bone marrow side of the SBP and a drastic reduction in the levels of sclerostin in osteocytes within the SBP. Unlike in WT mice, no further increase in the thickness of the SBP was observed in response to DMMH in Sost-KO mice. CONCLUSION: Since focal stress on the SBP underlying sites of cartilage damage increases during late stages of OA, these findings establish mechanical loading-induced attenuation of sclerostin expression and elevation of bone formation along the SBP surface as the major mechanisms characterizing subchondral bone phenotypes associated with severe late-stage OA in mice.

EGFR signaling is critical for maintaining the superficial layer of articular cartilage and preventing osteoarthritis initiation.

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.

A comprehensive study of long-term skeletal changes after spinal cord injury in adult rats.

Spinal cord injury (SCI)-induced bone loss represents the most severe osteoporosis with no effective treatment. Past animal studies have focused primarily on long bones at the acute stage using adolescent rodents. To mimic chronic SCI in human patients, we performed a comprehensive analysis of long-term structural and mechanical changes in axial and appendicular bones in adult rats after SCI. In this experiment, 4-month-old Fischer 344 male rats received a clinically relevant T13 contusion injury. Sixteen weeks later, sublesional femurs, tibiae, and L4 vertebrae, supralesional humeri, and blood were collected from these rats and additional non-surgery rats for micro-computed tomography (µCT), micro-finite element, histology, and serum biochemical analyses. At trabecular sites, extreme losses of bone structure and mechanical competence were detected in the metaphysis of sublesional long bones after SCI, while the subchondral part of the same bones showed much milder damage. Marked reductions in bone mass and strength were also observed in sublesional L4 vertebrae but not in supralesional humeri. At cortical sites, SCI induced structural and strength damage in both sub- and supralesional long bones. These changes were accompanied by diminished osteoblast number and activity and increased osteoclast number and activity. Taken together, our study revealed site-specific effects of SCI on bone and demonstrated sustained inhibition of bone formation and elevation of bone resorption at the chronic stage of SCI.

PTH1-34 blocks radiation-induced osteoblast apoptosis by enhancing DNA repair through canonical Wnt pathway.

Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1-34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1-34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/ß-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the ß-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34 or Wnt agonist as possible therapy for radiation-induced osteoporosis.