Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

CHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation.

Circulating microRNAs (miRNA) alterations have been reported in severe trauma patients but the pathophysiological relevance of these changes is still unclear. miRNAs are critical biologic regulators of pathological events such as hypoxia and inflammation, which are known to induce endoplasmic reticulum (ER) stress. ER stress is emerging as an important process contributing to the development of single and/or multiple organ dysfunction after trauma hemorrhagic shock (THS) accompanied by impaired tissue microcirculation and inflammation. Here, we aim to bring new insights into the involvement of miRNAs associated with ER stress in THS. THS was induced in rats by a median laparotomy and blood withdrawal until mean arterial pressure (MAP) dropped to 30-35 mmHg followed by a restrictive (40 min) and full reperfusion (60 min) with Ringer's solution. Tunicamycin was used to induce ER stress. Blood samples were collected 24 h after THS for the determination of pathological changes in the blood (PCB) and circulating miRNAs. Plasma levels of circulating miRNAs were compared between THS, tunicamycin, and sham groups and correlated to biomarkers of PCB. MiRNA profile of THS animals showed that 40 out of 91 (44%) miRNAs were significantly upregulated compared to sham (p < 0.01). The data showed a very strong correlation between liver injury and miR-122-5p (r = 0.91, p < 0.00001). MiR-638, miR-135a-5p, miR-135b-5p, miR-668-3p, miR-204-5p, miR-146a-5p, miR-200a-3p, miR-17-5p, miR-30a-5p, and miR-214-3p were found positively correlated with lactate (r > 0.7, p < 0.05), and negatively with base excess (r ≤ 0.8, p < 0.05) and bicarbonate (r ≤ 0.8, p < 0.05), which are clinical parameters that reflected the shock severity. Tunicamycin significantly modified the microRNA profile of the animals, 33 out of 91 miRNAs were found differentially expressed. In addition, principal component analysis revealed that THS and tunicamycin induced similar changes in plasma miRNA patterns. Strikingly, the data showed that 15 (25.9%) miRNAs were regulated by both THS and tunicamycin (p < 0.01). This included miR-122-5p, a liver-specific microRNA, but also miR-17-5p and miR-125b-5p which are miRNAs remarkably involved in unfolded protein response (UPR)-mediating pro-survival signaling (IRE1α). Since miRNAs associated with ER stress are clearly correlated with THS, our data strongly suggest that interaction between miRNAs and ER stress is an important pathologic event occurring during THS. Overall, we consider that the miRNA profile developed in this study can provide a rationale for the development of bench-to-bedside strategies that target miRNAs in critical care diseases or be used as biomarkers in the prognosis of trauma patients.

Treatment of degenerating tendons still presents a major challenge, since the aetiology of tendinopathies remains poorly understood. Besides mechanical overuse, further known predisposing factors include rheumatoid arthritis, diabetes, obesity or smoking all of which combine with a systemic inflammation.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.

Vertebral fractures are the hallmark of osteoporosis. MicroRNAs (miRNAs) are a prominent class of gene regulators likely to affect bone homeostasis, including bone remodelling and fracture healing by altering gene expression in bone cells.

MicroRNA‑21 (miR‑21) is a small non‑coding RNA that is differentially expressed during tooth development, particularly during amelogenesis. Although orthodontic tooth movement and the innate immune response are impaired, miR‑21 knockout mice demonstrate no obvious skeletal phenotype. However, the consequence of miR‑21 knockout on tooth phenotype and corresponding alveolar bone is unknown. The current study utilized landmark‑based geometric morphometrics to identify anatomical dissimilarities of the three lower and upper molars, and the corresponding alveolar bone, in miR‑21 knockout and wild‑type control mice. The anatomical structures were visualized by microcomputer tomography. A total of 36 and 38 landmarks were placed on mandibular and maxillary molars, respectively. For the alveolar bone, 16 landmarks were selected on both anatomical sites. General Procrustes analysis revealed significantly smaller molars and dimensions of the alveolar bone in the mandible of the miR‑21 knockout mice when compared with wild‑type controls (P=0.03 and P=0.04, respectively). The overall dimension of the mandible was reduced by the lack of miR‑21 (P=0.02). In the maxilla, the dimension of the alveolar bone was significant (P=0.02); however, this was not observed in the molars (P=0.36). Based on principal component analysis, no changes in shape for any of the anatomical sites were observed. Dental and skeletal jaw length were calculated and no prognathism was identified. However, the fluctuating asymmetry of the molars in the mandible and the maxilla was reduced in the miR‑21 knockout mice by 38 and 27%, respectively. Taken together, the results of the present study revealed that the molars in the mandible and the dimension of the respective alveolar bone were smaller in miR‑21 mice compared with wild‑type littermates, suggesting that miR‑21 influences tooth development.

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from the Chinese Hamster (CGR) and Chinese hamster ovary (CHO) cells provide new tools for the discovery of novel miRNAs in this important production system. Version 20 of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences for CGR/CHO. We searched for evolutionary conserved miRNAs from miRBase v20 in recently published genomic data, derived from Chinese hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several hundred miRNA sequences in the genome. For several of these, the expression in CHO cells could be verified from multiple next-generation sequencing experiments. In addition, several hundred unexpressed miRNAs are awaiting further confirmation by testing for their transcription in different Chinese hamster tissues.

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21(CIP1) and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21(CIP) (1) , and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood-CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood-CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

MicroRNAs (miRNAs) are small noncoding RNAs demonstrated as critical post-transcriptional modulators in dental tissues and bone regeneration, particularly miR-21-5p. However, the role of miR-21-5p in the healing of alveolar sockets following tooth extraction remains unknown. In this study we evaluated the influence of miR-21-5p in the healing of alveolar socket after tooth extraction.

Platelets are involved in a variety of diseases, making their adequate functional assessment is essential. However, due to their easily activatable nature this has some methodological pitfalls. Therefore, the availability of stable, easily measurable surrogate markers would be beneficial. In this regard, some evidence suggests that certain microRNAs (miRNAs) circulating in plasma might be useful. We aimed to corroborate their suitability by analyzing plasma samples obtained in a randomized controlled trial, which assessed the effects of periodontal treatment on platelet function. We hypothesized that miRNA levels mirror changes of platelet activation and -function. Both platelet function and miRNA abundance were quantified using state-of-the-art flow cytometry and qPCR methods. The following miRNAs were quantified: 223-3p, 150-5p, 197-3p, 23a-3p, 126-3p, 24-3p, 21-5p, 27b-3p, 33a-5p, 320a, 191-5p, 28-3p, 451a, 29b-3p, and 1-3p. However, periodontal treatment did not affect the abundance of any investigated miRNAs to a relevant extent. Platelet activation and reactivity indices did neither correlate with any tested miRNA at baseline, nor after the treatment period. In addition, there was no evidence that investigated miRNAs were released by platelets, as suggested previously. In conclusion, our data suggest that in patients suffering from periodontal disease the investigated miRNAs are unlikely to be suitable biomarkers for platelet function. Our data aim to raise awareness that previously determined platelet activation dependent circulating miRNAs are not suitable as platelet biomarkers in all cohorts.

Blood circulating microRNAs (c-miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti-aging therapies. Based on a cross-sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c-miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR-19a-3p and miR-19b-3p, were found increased at old age but decreased at extreme age, as confirmed by RT-qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT-qPCR, but only with a high-resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR-19a/b-3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age-related increase of plasma miR-19a/b-3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c-miRs and isomiRs as additional parameter to track the onset of aging and age-related diseases using new potential biomarkers.

MicroRNAs (miRNAs)-short, single-stranded, noncoding RNAs-regulate several biological processes, including bone metabolism.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from Schistosoma mansoni, has been shown to influence the fate of host T-cells through manipulation of the NF-κB pathway. We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated manipulation of the host immune system. We used a fragment-based screening approach by means of nuclear magnetic resonance (NMR) to identify binders to the precursor of the parasite sma-microRNA-10 present in their extracellular vesicles. The small fragments identified were used to select larger molecules. These molecules were shown to counteract the inhibition of NF-κB activity by sma-microRNA-10 in cell-based assays.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

The assessment of bone quality and the prediction of fracture risk in idiopathic osteoporosis (IOP) are complex prospects as bone mineral density (BMD) and bone turnover markers (BTM) do not indicate fracture-risk. MicroRNAs (miRNAs) are promising new biomarkers for bone diseases, but the current understanding of the biological information contained in the variability of miRNAs is limited. Here, we investigated the association between serum-levels of 19 miRNA biomarkers of idiopathic osteoporosis to bone microstructure and bone histomorphometry based upon bone biopsies and µCT (9.3 μm) scans from 36 patients. Four miRNAs were found to be correlated to bone microarchitecture and seven miRNAs to dynamic histomorphometry (p < 0.05). Three miRNAs, namely, miR-29b-3p, miR-324-3p, and miR-550a-3p showed significant correlations to histomorphometric parameters of bone formation as well as microstructure parameters. miR-29b-3p and miR-324-p were found to be reduced in patients undergoing anti-resorptive therapy. This is the first study to report that serum levels of bone-related miRNAs might be surrogates of dynamic histomorphometry and potentially reveal changes in bone microstructure. Although these findings enhance the potential value of circulating miRNAs as bone biomarkers, further experimental studies are required to qualify the clinical utility of miRNAs to reflect dynamic changes in bone formation and microstructure.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional control of gene expression and might be used as biomarkers for diabetes-related complications. The aim of this case-control study was to explore potential differences in circulating miRNAs in young individuals with long-duration type 1 diabetes (T1D) compared to healthy controls, and how identified miRNAs are expressed across different tissues. Twelve adolescents, age 15.0-17.9 years, with T1D duration of more than 8 years (mean 11.1 years), were enrolled from the Swedish diabetes quality registry. An age-matched control group was recruited. Circulating miRNAs (n = 187) were analyzed by quantitative PCR. We observed that 27 miRNAs were upregulated and one was downregulated in T1D. Six of these miRNAs were tissue-enriched (blood cells, gastrointestinal, nerve, and thyroid tissues). Six miRNAs with the largest difference in plasma, five up-regulated (hsa-miR-101-3p, hsa-miR-135a-5p, hsa-miR-143-3p, hsa-miR-223-3p and hsa-miR-410-3p (novel for T1D)) and one down-regulated (hsa-miR-495-3p), with P-values below 0.01, were selected for further in-silico analyses. AKT1, VEGFA and IGF-1 were identified as common targets. In conclusion, 28 of the investigated miRNAs were differently regulated in long-duration T1D in comparison with controls. Several associations with cancer were found for the six miRNAs with the largest difference in plasma.