Hormones and tendinopathies: the current evidence.

BACKGROUND: Tendinopathies negatively affect the quality of life of millions of people, but we still do not know the factors involved in the development of tendon conditions. SOURCES OF DATA: Published articles in English in PubMed and Google Scholar up to June 2015 about hormonal influence on tendinopathies onset. One hundred and two papers were included following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. AREAS OF AGREEMENT: In vitro and in vivo, tenocytes showed changes in their morphology and in their functional properties according to hormonal imbalances. AREAS OF CONTROVERSY: Genetic pattern, sex, age and comorbidities can influence the hormonal effect on tendons. GROWING POINTS: The increasing prevalence of metabolic disorders prompts to investigate the possible connection between metabolic problems and musculoskeletal diseases. AREAS TIMELY FOR DEVELOPING RESEARCH: The influence of hormones on tendon structure and metabolism needs to be further investigated. If found to be significant, multidisciplinary preventive and therapeutic strategies should then be developed.

Influence of Thyroid Hormones on Tendon Homeostasis.

Tendinopathies have a multifactorial etiology driven by extrinsic and intrinsic factors. Recent studies have elucidated the importance of thyroid hormones in the alteration of tendons homeostasis and in the failure of tendon healing after injury. The effects of thyroid hormones are mediated by receptors (TR)-α and -ß that seem to be ubiquitous. In particular, T3 and T4 play an antiapoptotic role on tenocytes, causing an increase in vital tenocytes isolated from tendons in vitro and a reduction of apoptotic ones; they are also able to influence extra cellular matrix proteins secretion in vitro from tenocytes, enhancing collagen production. From a clinical point of view, disorders of thyroid function have been investigated only for rotator cuff calcific tendinopathy and tears. In this complex scenario, further research is needed to clarify the role of thyroid hormones on the onset of tendinopathies.

Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Hyaluronic acid increases tendon derived cell viability and collagen type I expression in vitro: Comparative study of four different Hyaluronic acid preparations by molecular weight.

BACKGROUND: Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III. METHODS: An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture. RESULTS: All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs. CONCLUSIONS: HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Knock-down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC-derived dendritic cells.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal ß-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of ß-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of ß-N-acetyl-hexosaminidase A isoenzyme within this mechanism.

Dual Acting Carbon Monoxide Releasing Molecules and Carbonic Anhydrase Inhibitors Differentially Modulate Inflammation in Human Tenocytes.

Sustained oxidative stress and inflammation have been reported as the major factors responsible for the failure of tendon healing during rotator cuff tears (RCTs) and rotator cuff disease (RCD). Although, their therapeutic management remains still challenging. Carbonic anhydrases (CAs) are involved in many pathological conditions, and the overexpression of both CA9 and 12 in inflamed joints has been recently reported. Consequently, a selective CA9/12 inhibition could be a feasible strategy for improving tendon recovery after injury. In addition, since carbon monoxide (CO) has been proven to have an important role in modulating inflammation, CO releasing molecules (CORMs) can be also potentially suitable compounds. The present study aims at evaluating five newly synthesized dual-mode acting CA inhibitors (CAIs)-CORMs compounds, belonging to two chemical scaffolds, on tendon-derived human primary cells under H2O2 stimulation in comparison with Meloxicam. Our results show that compounds 2 and 7 are the most promising of the series in counteracting oxidative stress-induced cytotoxicity and display a better profile in terms of enhanced viability, decreased LDH release, and augmented tenocyte proliferation compared to Meloxicam. Moreover, compound 7, as a potent superoxide scavenger, exerts its action inhibiting NF-ĸB translocation and downregulating iNOS, whereas compound 2 is more effective in increasing collagen I deposition. Taken together, our data highlight a potential role of CA in RCTs and RCD and the prospective effectiveness of compounds acting as CAI-CORM during inflammation.

Design of novel three-phase PCL/TZ-HA biomaterials for use in bone regeneration applications.

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(epsilon-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL-HA composite and PCL/TZ blend, the three-phase PCL/TZ-HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.

Nrf2-mediated cytoprotective effect of four different hyaluronic acids by molecular weight in human tenocytes.

Non-traumatic rotator cuff tears (RCTs) are a frequent and potentially disabling injury. There is growing evidence that hyaluronic acid (HA) is effective for pain relief and to counteract inflammation in RCTs, however, its effective role in tendinopathies remains poorly studied. This study aims to disclose a possible molecular mechanism underlying the cytoprotective effects of four different HA preparations (Artrosulfur HA®, Synolis VA®, Hyalgan® and Hyalubrix®) under H2O2-induced oxidative stress. Expression-levels of Lactate dehydrogenase (LDH) released were quantified in cell supernatants, CD44 expression levels were analysed by fluorescence microscopy, the mitochondrial membrane depolarisation (TMRE assay) was measured by flow cytometry and the role of the transcription factor Nrf2 was investigated as a potential therapeutic target for RCT treatment. The modulation of extracellular matrix- (ECM) related protein expression (Integrin ß1, pro-collagen 1A2 and collagen 1A1) and autophagy occurrence (Erk 1/2 and phosphoErk 1/2 and LC3B), were all investigated by Western Blot. Results demonstrate that Artrosulfur HA, Hyalubrix and Hyalgan improve cell escape from H2O2-induced oxidative stress, decreasing cytotoxicity, reducing Nrf2 expression and enhancing catalase recovery. This study lays the grounds for further investigations insight novel pharmaceutical strategies targeting key effectors involved in the molecular cascade triggered by HA.

Evaluation of the psychometric properties of the Barthel Index in an Italian ischemic stroke population in the acute phase: a cross-sectional study.

The objective of this study was to assess and validate the psychometric properties of the Italian culturally adapted Barthel Index (IcaBI) in a cohort of people with ischemic stroke. The validation process was conducted in an Italian cohort of 99 stroke inpatients to whom the IcaBI was administered in order to test its structural validity, and inter-and intrarater reliability. The internal consistency (Cronbach's alpha) was 0.901. Factor analysis revealed a two-factor structure. The interclass correlation coefficient 3,1 (ICC) for intra-rater reliability was estimated at 0.987 (95% CI: 0.975-0.993), while the ICC for inter-rater reliability was 0.909 (95% CI: 0.852-0.948). This study demonstrates the psychometric properties of the IcaBI in an Italian stroke population, and therefore shows that the scale can be considered a valid and reliable assessment tool for measuring functional disability in Italian acute ischemic stroke survivors.

Up-regulation of EphB and ephrin-B expression in rhabdomyosarcoma.

BACKGROUND: Increased expression of Eph receptors and their ephrin ligands has been implicated in promoting angiogenesis and tumour progression in several malignancies. Here the expression of mRNA for ephrin-B and EphB receptors in rhabdomyosarcoma (RMS) cell lines and primary tumours was investigated. MATERIALS AND METHODS: Expression of mRNA for ephrin-B and EphB receptors in RMS cell lines and primary tumours was measured by real-time RT-PCR and compared with the expression in normal striated muscle. RESULTS: A dysregulation of both ligands and receptors was found in all cell lines. In embryonal tumours, overexpression of ephrin-B1 correlated with overexpression of EphB1 (r = 0.97, p < 0.01) and EphB3 (r = 0.94, p < 0.05); overexpression of ephrin-B2 correlated with overexpression of EphB1 (r = 0.94, p < 0.05), EphB2 (r = 0.88, p < 0.01) and EphB4 (r = 0.76, p < 0.01). In alveolar tumours, no similar correlations were found. A correlation between EphB2 and EphB4 receptors was demonstrated in both tumour types, being positive in embryonal cases (r = 0.81, p < 0.01) and negative in alveolar (r = -1.00, p < 0.01). CONCLUSION: A global up-regulation of ephrin-B and EphB receptors in RMS tumours was found. The correlation between EphB2 and EphB4 receptors suggests a possible role for ephrin-B and EphB receptors in RMS development.

CD34 human hematopoietic progenitor cell line, MUTZ-3, differentiates into functional osteoclasts.

OBJECTIVE: CD14(+) monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34(+) stem cell and CD14(+) cell populations, we investigated the capacity of the stem/progenitor CD34(+) population to differentiate into functional OCs. MATERIALS AND METHODS: Sorted MUTZ-3-CD34(+) and MUTZ-3-CD14(+) cells were cultured in presence of M-CSF, RANK-L, and TNF-alpha to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34(+)OC and -CD14(+)OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis. RESULTS: MUTZ-3-CD34(+) differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34(+) generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R(+)/RANK(+) that at the same time co-expressed CD14 and CD34. CONCLUSIONS: These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14(+)CD34(+)M-CSF-R(+)RANK(+) population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Hyaluronic acid increases tendon derived cell viability and proliferation in vitro: comparative study of two different hyaluronic acid preparations by molecular weight.

BACKGROUND: Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate the effects of two different HA on human rotator cuff tendon derived cells in terms of cell viability, proliferation and apoptosis. METHODS: An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of two different HA preparations: Sinovial HL® (High-Low molecular weight) (MW: 80-100 kDa) and KDa Sinovial Forte SF (MW: 800-1200), at various concentrations. Tendon derived cells morphology was evaluated after 0, 7 and 14 d of culture. Viability and proliferation were analyzed after 0, 24, and 48 h of culture and apoptosis occurrence was assessed after 24 h of culture. RESULTS: All the HAPs tested here increased viability and proliferation, in a dose-dependent manner and they reduced apoptosis at early stages (24 h) compared to control cells (without HAPs). CONCLUSIONS: HAPs enhanced viability and proliferation and counteracted apoptosis in tendon derived cells.

Combined supplementation of ascorbic acid and thyroid hormone T3 affects tenocyte proliferation. The effect of ascorbic acid in the production of nitric oxide.

BACKGROUND: Tissue engineering is now increasingly focusing on cell-based treatments as promising tools to improve tendon repair. However, many crucial aspects of tendon biology remain to be understood before adopting the best experimental approach for cell-tissue engineering. METHODS: The role played by Ascorbic Acid (AA) alone and in combination with thyroid hormone T3 in the viability and proliferation of primary human tendon-derived cells was investigated. Human tenocyte viability was detected by Trypan blue exclusion test and cellular proliferation rate was evaluated by CFSE CellTrace™. In addition, the potential role of the AA in the production of Nitric Oxide (NO) was also examined. RESULTS: In this in vitro model, an increase in tenocyte proliferation rate was observed as a consequence of progressively increased concentrations of AA (from 10 to 50 µg/ml). The addition of the T3 hormone to the culture further increased tenocyte proliferation rate. In detail, the most evident effect on cellular growth was achieved using the combined supplementation of 50 µg/ml AA and 10-7 M T3. CONCLUSION: We showed that the highest concentration of AA (100 and 500 µg/ml) caused cytotoxicity to human tenocytes. Moreover, it was shown that AA reduces NO synthesis. These results show that AA is a cell proliferation inducer that triggers tenocyte growth, while it reduces NO synthesis.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines.

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species.

Osteogenic differentiation of CD271(+) cells from rabbit bone marrow cultured on three phase PCL/TZ-HA bioactive scaffolds: comparative study with mesenchymal stem cells (MSCs).

Tissue engineering is one of the major challenges of orthopedics and trauma surgery for bone regeneration. Biomaterials filled with mesenchymal stem cells (MSCs) are considered the most promising approach in bone tissue engineering. Furthermore, our previous study showed that the multi-phase poly [ε-caprolactone]/thermoplastic zein-hydroxyapatite (PCL/TZ-HA) biomaterials improved rabbit (r) MSCs adhesion and osteoblast differentiation, thus demonstrating high potential of this bioengineered scaffold for bone regeneration. In the recent past, CD271 has been applied as a specific selective marker for the enrichment of MSCs from bone marrow (BM-MSCs). In the present study, we aimed at establishing whether CD271-based enrichment could be an efficient method for the selection of rBM-MSCs, displaying higher ability in osteogenic differentiation than non-selected rBM-MSCs in an in vitro system. CD271(+) cells were isolated from rabbit bone marrow and were compared with rMSCs in their proliferation rate and osteogenic differentiation capability. Furthermore, rCD271(+) cells were tested in their ability to adhere, proliferate and differentiate into osteogenic lineage, while growing on PCL/TZ-HA scaffolds, in comparison to rMSCs. Our result demonstrate that rCD271(+) cells were able to adhere, proliferate and differentiate into osteoblasts when cultured on PCL/TZ-HA scaffolds in significantly higher levels as compared to rMSCs. Based on these findings, CD271 marker might serve as an optimal alternative MSCs selection method for the potential preclinical and clinical application of these cells in bone tissue regeneration.

A new human cell line, PDSS-26, from poorly differentiated synovial sarcoma, with unique chromosomal anomalies.

Permanent synovial sarcoma cell lines are invaluable tools for understanding of the biology of this tumor. The present study reports the establishment of a new human cell line, PDSS-26, derived from a surgical specimen of a poorly differentiated synovial sarcoma. PDSS-26 has a doubling time of a 72 hours and grows as a monolayer of spindle cells that retain immunoreactivity for bcl-2 and vimentin. Karyotypic analysis revealed a rearrangement involving chromosomes 17 and 18, at the breakpoints q11.2 and q11.2, respectively, as the only structural aberrations. Analysis by reverse transcriptase polymerase chain reaction showed the presence of the SYT-SSX1 fusion transcript in both the primary tumor and the cell line. Cytoplasmic PTEN staining was detected by immunohistochemistry in both the PDSS-26 cell line and in original tumor, whereas no mutation was identified by automatic sequencing. Thus, PDSS-26 cells could be useful for future functional studies.

Thyroid hormones increase collagen I and cartilage oligomeric matrix protein (COMP) expression in vitro human tenocytes.

BACKGROUND: we previously demonstrated the presence of high levels of thyroid hormones (THs) receptors isoforms in healthy tendons, their protective action during tenocyte apoptosis, and the capability to enhance tenocyte proliferation in vitro. In the present study we tested the ability of THs to influence ECM protein tenocyte secretion in an in vitro system. METHODS: primary tenocyte-like cells were cultivated for 1, 7 and 14 days in the presence of T3 or T4 individually or in combination with ascorbic acid (AA). RESULTS: THs (T3 or T4) in synergism with AA increase significantly the total collagen production after 14 days. THs in synergism with AA increase significantly the expression of collagen I,biglycan and COMP, after some days. CONCLUSION: THs play a role on the extra cellular matrix of tendons, enhancing in vitro the production of several proteins such as collagen I, biglycan and COMP. THs receptors are active on human tenocytes, and can play a role in tendon ailments.