CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model.

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs.

Conjugation with Methylsulfonylmethane Improves Hyaluronic Acid Anti-Inflammatory Activity in a Hydrogen Peroxide-Exposed Tenocyte Culture In Vitro Model.

Rotator cuff tears (RCTs) and rotator cuff disease (RCD) are important causes of disability in middle-aged individuals affected by nontraumatic shoulder dysfunctions. Our previous studies have demonstrated that four different hyaluronic acid preparations (HAPs), including Artrosulfur® hyaluronic acid (HA) (Alfakjn S.r.l., Garlasco, Italy), may exert a protective effect in human RCT-derived tendon cells undergoing oxidative stress damage. Recently, methylsulfonylmethane (MSM) (Barentz, Paderno Dugnano, Italy) has proven to have anti-inflammatory properties and to cause pain relief in patients affected by tendinopathies. This study aims at evaluating three preparations (Artrosulfur® HA, MSM, and Artrosulfur® MSM + HA) in the recovery from hydrogen peroxide-induced oxidative stress damage in human tenocyte. Cell proliferation, Lactate Dehydrogenase (LDH) release, and inducible nitric oxide synthases (iNOS) and prostaglandin E2 (PGE2) modulation were investigated. In parallel, expression of metalloproteinases 2 (MMP2) and 14 (MMP14) and collagen types I and III were also examined. Results demonstrate that Artrosulfur® MSM + HA improves cell escape from oxidative stress by decreasing cytotoxicity and by reducing iNOS and PGE2 secretion. Furthermore, it differentially modulates MMP2 and MMP14 levels and enhances collagen III expression after 24 h, proteins globally related to rapid acceleration of the extracellular matrix (ECM) remodelling and thus tendon healing. By improving the anti-cytotoxic effect of HA, the supplementation of MSM may represent a feasible strategy to ameliorate cuff tendinopathies.

Expression profiling of microRNAs and isomiRs in conventional central chondrosarcoma.

Conventional central chondrosarcoma (CCC) is a malignant bone tumor that is characterized by the production of chondroid tissue. Since radiation therapy and chemotherapy have limited effects on CCC, treatment of most patients depends on surgical resection. This study aimed to identify the expression profiles of microRNAs (miRNAs) and isomiRs in CCC tissues to highlight their possible participation to the regulation of pathways critical for the formation and growth of this type of tumor. Our study analyzed miRNAs and isomiRs from Grade I (GI), Grade II (GII), and Grade III (GIII) histologically validated CCC tissue samples. While the different histological grades shared a similar expression profile for the top abundant miRNAs, we found several microRNAs and isomiRs showing a strong different modulation in GII + GIII vs GI grade samples and their involvement in tumor biology could be consistently hypothesized. We then in silico validated these differently expressed miRNAs in a larger chondrosarcoma public dataset and confirmed the expression trend for 17 out of 34 miRNAs. Our results clearly suggests that the contribution of miRNA deregulation, and their targeted pathways, to the progression of CCC could be relevant and strongly indicates that when studying miRNA deregulation in tumors, not only the canonical miRNAs, but the whole set of corresponding isomiRs should be taken in account. Improving understanding of the precise roles of miRNAs and isomiRs over the course of central chondrosarcoma progression could help identifying possible targets for precision medicine therapeutic intervention.

Extracellular vesicles from rat-bone-marrow mesenchymal stromal/stem cells improve tendon repair in rat Achilles tendon injury model in dose-dependent manner: A pilot study.

Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries.

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.

Combined ascorbic acid and T3 produce better healing compared to bone marrow mesenchymal stem cells in an Achilles tendon injury rat model: a proof of concept study.

BACKGROUND: This pilot study aimed to ascertain whether the local application of ascorbic acid (AA), of T3, and of rat (r) bone marrow mesenchymal stem cells (BMSCs), alone or in all possible combinations, promoted healing after an Achilles tendon injury in a rat model. METHODS: An Achilles tendon defect was produced in 24 6-8-week-old male inbred Lewis rats. The animals were then randomly divided into eight groups of three rats each. The tendon defect was filled with 50 µL of phosphate-buffered saline (PBS) containing (1) 50 µg/mL AA (AA group), (2) 10-7 M T3 (T3 group), (3) 4 × 106 rBMSCs (rBMSC group), (4) 50 µg/mL AA + 10-7 M T3 (AA + T3 group), (5) 4 × 106 rBMSCs + 50 µg/mL AA (rBMSC + AA group), (6) 4 × 106 rBMSCs + 10-7 M T3 (rBMSC + T3 group), (7) 4 × 106 rBMSCS + 50 µg/mL AA + 10-7 M T3 (rBMSC + AA + T3 group), and (8) PBS only (control group: CTRL). All treatments were administered by local injection immediately after the tendons had been damaged; additionally, AA was injected also on the second and fourth day from the first injection (for groups 1, 4, 5, and 7), and T3 was injected again every day for 4 days (for groups 2, 4, 6, and 7). At 30 days from initial treatment, tendon samples were harvested, and the quality of tendon repair was evaluated using histological and histomorphological analysis. The structure and morphology of the injured Achilles tendons were evaluated using the modified Svensson, Soslowsky, and Cook score, and the collagen type I and III ratio was calculated. RESULTS: The group treated with AA combined with T3 displayed the lowest Svensson, Soslowsky, and Cook total score value of all tissue sections at histopathological examination, with fiber structure close to regular orientation, normal-like tendon vasculature, and no cartilage formation. AA + T3 also showed the highest collagen I and the lowest collagen III values compared to all other treatments including the CTRL. CONCLUSION: There are potential benefits using a combination of AA and T3 to accelerate tendon healing.

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.

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.