Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Age-related secretion of grancalcin by macrophages induces skeletal stem/progenitor cell senescence during fracture healing.

Skeletal stem/progenitor cell (SSPC) senescence is a major cause of decreased bone regenerative potential with aging, but the causes of SSPC senescence remain unclear. In this study, we revealed that macrophages in calluses secrete prosenescent factors, including grancalcin (GCA), during aging, which triggers SSPC senescence and impairs fracture healing. Local injection of human rGCA in young mice induced SSPC senescence and delayed fracture repair. Genetic deletion of Gca in monocytes/macrophages was sufficient to rejuvenate fracture repair in aged mice and alleviate SSPC senescence. Mechanistically, GCA binds to the plexin-B2 receptor and activates Arg2-mediated mitochondrial dysfunction, resulting in cellular senescence. Depletion of Plxnb2 in SSPCs impaired fracture healing. Administration of GCA-neutralizing antibody enhanced fracture healing in aged mice. Thus, our study revealed that senescent macrophages within calluses secrete GCA to trigger SSPC secondary senescence, and GCA neutralization represents a promising therapy for nonunion or delayed union in elderly individuals.

Bone marrow mesenchymal stem cells derived exosomal miRNAs can modulate diabetic bone-fat imbalance.

Background: Diabetes mellitus is a chronic metabolic disease with systemic complications. Patient with diabetes have increased risks of bone fracture. Previous studies report that diabetes could affect bone metabolism, however, the underlying mechanism is still unclear. Methods: We isolated exosomes secreted by bone marrow mesenchymal stem cells of normal and diabetic mice and test their effects on osteogenesis and adipogenesis. Then we screened the differential microRNAs by high-throughput sequencing and explored the function of key microRNA in vitro and in vivo. Results: We find that lower bone mass and higher marrow fat accumulation, also called bone-fat imbalance, exists in diabetic mouse model. Exosomes secreted by normal bone marrow mesenchymal stem cells (BMSCs-Exos) enhanced osteogenesis and suppressed adipogenesis, while these effects were diminished in diabetic BMSCs-Exos. miR-221, as one of the highly expressed miRNAs within diabetic BMSCs-Exos, showed abilities of suppressing osteogenesis and promoting adipogenesis both in vitro and in vivo. Elevation of miR-221 level in normal BMSCs-Exos impairs the ability of regulating osteogenesis and adipogenesis. Intriguingly, using the aptamer delivery system, delivery normal BMSCs-Exos specifically to BMSCs increased bone mass, reduced marrow fat accumulation, and promoted bone regeneration in diabetic mice. Conclusion: We demonstrate that BMSCs derived exosomal miR-221 is a key regulator of diabetic osteoporosis, which may represent a potential therapeutic target for diabetes-related skeletal disorders.

Different protein expression patterns in rat spinal nerves during Wallerian degeneration assessed using isobaric tags for relative and absolute quantitation proteomics profiling.

Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the di?erences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China (approval No. 2016-x9-07) in September 2016.

Technological Platform for Vertical Multi-Wafer Integration of Microscanners and Micro-Optical Components.

We describe an original integration technological platform for the miniaturization of micromachined on-chip optical microscopes, such as the laser scanning confocal microscope. The platform employs the multi-wafer vertical integration approach, combined with integrated glass-based micro-optics as well as micro-electro-mechanical systems (MEMS) components, where the assembly uses the heterogeneous bonding and interconnecting technologies. Various heterogeneous components are disposed in vertically stacked building blocks (glass microlens, MEMS actuator, beamsplitter, etc.) in a minimum space. The platform offers the integrity and potential of MEMS microactuators integrated with micro-optics, providing miniaturized and low cost solutions to create micromachined on-chip optical microscopes.

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.

Efficacy of mesenchymal stem cells in treating patients with osteoarthritis of the knee: A meta-analysis.

To assess the clinical efficacy and safety of mesenchymal stem cell (MSC) treatment for osteoarthritis of the knee (KOA), a systematic electronic literature search was performed on PubMed, EMBASE and Web of Science. Studies published in English from the earliest record to December 2014 were searched using the following keywords: Cartilage defect, cartilage repair, osteoarthritis, KOA, stem cells, MSCs, bone marrow concentrate (BMC), adipose-derived mesenchymal stem cells, synovial-derived mesenchymal stem cells and peripheral blood-derived mesenchymal stem cells. The effect sizes of selected studies were determined by extracting pain scores from the visual analog scale and functional changes from International Knee Documentation Committee and Lysholm and Western Ontario and McMaster Universities Osteoarthritis Index before and after MSCs or reference treatments at 3, 6, 12, and 24 months. The factors were analyzed and the outcomes were modified after comparing the MSC group pooled values with the pretreatment baseline or between different treatment arms. A systematic search identified 18 clinical trials on this topic, including 10 single-arm prospective studies, four quasi-experimental studies and four randomized controlled trials that used BMCs to treat 565 patients with KOA in total. MSC treatment in patients with KOA showed continual efficacy for 24 months compared with their pretreatment condition. Effectiveness of MSCs was improved at 12 and 24 months post-treatment, compared with at 3 and 6 months. No dose-responsive association in the MSCs numbers was demonstrated. However, patients with arthroscopic debridement, activation agent or lower degrees of Kellgren-Lawrence grade achieved improved outcomes. MSC application ameliorated the overall outcomes of patients with KOA, including pain relief and functional improvement from basal evaluations, particularly at 12 and 24 months after follow-up.

Investigation of Surface Pre-Treatment Methods for Wafer-Level Cu-Cu Thermo-Compression Bonding.

To increase the yield of the wafer-level Cu-Cu thermo-compression bonding method, certain surface pre-treatment methods for Cu are studied which can be exposed to the atmosphere before bonding. To inhibit re-oxidation under atmospheric conditions, the reduced pure Cu surface is treated by H2/Ar plasma, NH3 plasma and thiol solution, respectively, and is covered by Cu hydride, Cu nitride and a self-assembled monolayer (SAM) accordingly. A pair of the treated wafers is then bonded by the thermo-compression bonding method, and evaluated by the tensile test. Results show that the bond strengths of the wafers treated by NH3 plasma and SAM are not sufficient due to the remaining surface protection layers such as Cu nitride and SAMs resulting from the pre-treatment. In contrast, the H2/Ar plasma⁻treated wafer showed the same strength as the one with formic acid vapor treatment, even when exposed to the atmosphere for 30 min. In the thermal desorption spectroscopy (TDS) measurement of the H2/Ar plasma⁻treated Cu sample, the total number of the detected H2 was 3.1 times more than the citric acid⁻treated one. Results of the TDS measurement indicate that the modified Cu surface is terminated by chemisorbed hydrogen atoms, which leads to high bonding strength.

Effect of lentivirus-mediated uPA silencing on the proliferation and apoptosis of chondrocytes and the expression of MMPs.

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1ß (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.

Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate.

Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.

Icariin and icaritin stimulate the proliferation of SKBr3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway.

Icariin (ICA) and icaritin (ICT), with a similar structure to genistein, are the important bioactive components of the genus Epimedium, and regulate many cellular processes. In the present study, using the estrogen receptor (ER)-negative breast cancer cell line, SKBr3, as a model, we examined the hypothesis that ICA and ICT at low concentrations stimulate SKBr3 cell proliferation in vitro through the functional membrane, G protein­coupled estrogen receptor 1 (GPER1), mediated by the epithelial growth factor receptor (EGFR)­mitogen-activated protein kinase (MAPK) signaling pathway. MTT assay revealed that ICA and ICT at doses of 1 nM to 1 µM markedly stimulated SKBr3 cell proliferation in a dose-dependent manner. The ICA- and ICT-stimulated cell growth was completely suppressed by the GPER1 antagonist, G-15, indicating that the ICA­ and ICT-stimulated cell proliferation was mediated by GPER1 activation. Semi-quantitative RT-PCR analysis revealed that treatment with ICA and ICT enhanced the transcription of c-fos, a proliferation-related early gene. The ICA- and ICT-stimulated mRNA expression was markedly attenuated by G-15, AG-1478 (an EGFR antagonist) or PD98059 (a MAPK inhibitor). Our data also demonstrated that ICA and ICT increased the phosphorylation of ERK1/2. The ICA- and ICT-stimulated ERK1/2 phosphorylation was blocked by pre-treatment of the cells with G-15 and AG-1478 or PD 98059. Flow cytometric analysis confirmed that the ICA- and ICT-stimulated SKBr3 cell proliferation involved the GPER1-mediated modulation of the EGFR­MAPK signaling pathway. To the best of our knowledge, our current findings demonstrate for the first time that ICA and ICT promote the progression of ER-negative breast cancer through the activation of membrane GPER1.

[Oct4 methylation in induced differentiation of bone mesenchymal stem cells].

OBJECTIVE: To investigate the methylation Oct4 in orientation induced differentiation in bone marrow mesenchymal stem cells METHODS: Mice BMSCs were isolated and purified from bone marrow by adherent culture,and then identified by morphology and immunocytochemistry.Mouse osteoblastic cells were cultured by bone fragments inoculation,and then identified by alkaline phosphatase(AKP)staining and alizarin red staining.BMSCs were induced to differentiate into osteoblasts in vitro. Indirect immunofluorescence staining and reverse transcription polymerase chain reaction(RT PCR)were used to detect the expressions of Oct4 in BMSCs before and after induction.The methylation status of Oct4 gene in mouse BMSCs was explored by a methylation specific PCR before and after induction RESULTS: The isolated mice BMSCs massively proliferated in vitro and formed cell colones with uniform morphology.Positive expressions of CD29,cKit,and CD44 and negative expression of CD34 were found in the isolated cells.After 10 days[DK]'[DK] induction,both AKP and the alizarin red were positive in cells and osteoblastic cells isolated from mice skull bones.The indirect immunoinfluorescence staining and RT-PCR also showed that the Oct4 expression in the directed differentiation of mouse BMSCs was down-regulated.The CpG island of Otc4 gene promoter in mouse BMSCs became methylated during the induced differentiation. CONCLUSIONS: Mice BMSCs and osteoblasts were successfully cultured in vitro in this studyOct4 may be involved in the maintenance of adult stem cell pluripotency.The down regulated expression of Oct4 gene in mouse BMSCs during the directed differentiation may contribute to the methylation of CpG island in Otc4 gene promoter.

A ZnO nanorod-based SAW oscillator system for ultraviolet detection.

A high-precision ultraviolet (UV) detector combining ZnO nanostructure and a dual delay line surface acoustic wave (SAW) oscillator system is presented. The UV detector is made of ZnO nanorods on a 128 degrees YX-LiNbO(3)-based two-port SAW oscillator. The ZnO nanorod synthesized by chemical solution method is used as a UV sensing material. The center frequency of the SAW device is at 145 MHz. A dual delay line SAW oscillator system was constructed to eliminate external environmental fluctuations. Under illumination of a UV source consisting of an Xe lamp and a monochromator, frequency shifts of the UV detector were measured. A maximum frequency shift of over 40 kHz was observed under 365 nm illumination for several on-off cycles, indicating the ZnO nanorod-based detector was sensitive to UV light and with good repeatability. Moreover, frequency shifts reached a value of 19 kHz after 365 nm was turned on for 10 s, which implies a real-time high-sensitivity UV sensor was successfully fabricated. Results show a ZnO nanostructure-based SAW oscillator system is a promising candidate for a real-time, fast-response, high-precision UV detector.

[A comprehansive evaluation on stroke prevalence among elderly in urban and rural areas of Beijing, 2002].

OBJECTIVE: To describe the prevalence and disability of stroke as well as the stroke-related diseases among elderly in urban and rural regions of Beijing. METHODS: In 2002, three communities were selected from urban, suburb and rural regions from Beijing areas, respectively. Twenty percent of the elderly were randomly selected from three communities. The information about history of stroke, hypertension, heart diseases and diabetes, self-rated health (SRH), activity of daily living (ADL) and instrumental ADL (IADL), smoking and drinking habits, knowledge about cardiovascular diseases prevention were collected. RESULTS: A total of 2487 elderly were interviewed and the prevalence of stroke was 12.9% (321/2481). Eighty-seven of the stroke patients were diagnosed by CT/MRI. 19.9% of stroke patients had experienced 2 or more attacks. The highest prevalence of stroke was in the urban region and the lowest in the rural region (16.9% vs. 8.5%, P for trend < 0.01) while it was higher in males than in females (P < 0.05). The prevalence of stroke tended to increase with age in urban and 34.6% of the stroke patients had recovered completely. The proportions of poor SRH, ADL and IADL dependence, as well as the prevalences of hypertension, heart diseases and diabetes were higher among the elderly with stroke than those without. Although rates of awareness and treatment of hypertension were at the high levels among the elderly with stroke , the control rate was low, especially in the rural region (as low as 4.3%). The level of knowledge on the prevention of cardiovascular diseases, and the rates of smoking and drinking were similar between the elderly with or without stroke. CONCLUSION: The prevalence of stroke had increased dramatically during the past decade in Beijing. The proportion of poor SHR, ADL and IADL dependence, prevalence rates of stroke related diseases were higher among the elderly with stroke than those without. Secondary prevention of stroke among Beijing elderly called for urgent action.