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.

Hyaluronic Acid Alleviates Oxidative Stress and Apoptosis in Human Tenocytes via Caspase 3 and 7.

Rotator cuff tendinopathy (RCT) is the primary reason for shoulder surgery and its clinical management is still challenging. Hyaluronic acid (HA) has been shown to have anti-inflammatory effects in vitro and in vivo under RCT conditions, characterized by an exaggerated oxidative stress (OS). However, molecular mechanisms underlying HA-related effects are still partially disclosed. With these aims, a cell model of RCT was established by exposing primary human tenocytes to H2O2 for up to 72 h. Four different HAs by molecular weight were administered to measure nitric oxide (NO) and OS, apoptosis, and collagen 1 expression. In parallel, the well-known antioxidant ascorbic acid was administered for comparison. The present study highlights that HAs characterized by a low molecular weight are able to counteract the H2O2-induced OS by decreasing the percentage of apoptotic cells and reversing the activation of caspase 3 and 7. Likewise, NO intracellular levels are comparable to the ones of controls. In parallel, collagen 1 expression was ameliorated by HAs characterized by higher molecular weights compared to AA. These findings confirm that HA plays an antioxidant role comparable to AA depending on the molecular weight, and highlight the molecular mechanisms underlying the HA anti-apoptotic effects.

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.

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.

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.

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.