An in vitro study to assess the effect of hyaluronan-based gels on muscle-derived cells: Highlighting a new perspective in regenerative medicine

Abstract

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H 2 O 2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity

“…The primers used to amplify each PCR product are reported in Table 3. Values are expressed as a fold change (2 −∆∆Ct method) in treated cells vs. untreated cells (the control) and normalized to transcript levels of housekeeping gene [35].…”

Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation

“…After a 24-h incubation, the cells were harvested and lysated using TRIzol ® Reagent (Invitrogen, Milan, Italy) in order to isolate cellular RNA [ 47 ]. For each sample, 1 µg of total RNA was reverse transcribed into cDNA following the manufacturer’s protocol (Reverse Transcription System Kit, Promega, Milan, Italy).…”

“…Following that, a solution of Triton X-100 at 0.2% v / v in PBS was used as a cellular permeabilizer. Immunofluorescence was performed following the experimental protocol previously reported [ 47 ]. In this context, human primary antibodies against COMP-2 (diluted 1:100), and HAS-1 (diluted 1:100) were incubated overnight at 4 °C.…”

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair

“…Total RNA was extracted from control and treated HaCaT, HDF monolayers and FT-Skin model at different time points (4 and 24 h for 2D culture and 24 and 7 days for FT-Skin) using TRIzol ® (Invitrogen, Milan, Italy), according to the manufacturer's procedures described in literature [39,40]. Briefly, extracted RNA was quantified with a Nanodrop spectrophotometer (Celbio, Milan, Italy) and 1 μg of DNase-digested total RNA was used (DNA-free kit; Ambion-Applied Biosystems) to synthesize cDNA using the Reverse Transcription System Kit (Promega, Milan, Italy).…”