Volumetric muscle loss injuries overwhelm the endogenous regenerative capacity of skeletal muscle, and the associated oxidative damage can delay regeneration and prolong recovery. This study aimed to investigate the effect of silicon-ions on C2C12 skeletal muscle cells under normal and excessive oxidative stress conditions to gain insights into its role on myogenesis during the early stages of muscle regeneration. In vitro studies indicated that 0.1 mM Si-ions into cell culture media significantly increased cell viability, proliferation, migration, and myotube formation compared to control. Additionally, MyoG, MyoD, Neurturin, and GABA expression were significantly increased with addition of 0.1, 0.5, and 1.0 mM of Si-ion for 1 and 5 days of C2C12 myoblast differentiation. Furthermore, 0.1–2.0 mM Si-ions attenuated the toxic effects of H2O2 within 24 h resulting in increased cell viability and differentiation. Addition of 1.0 mM of Si-ions significantly aid cell recovery and protected from the toxic effect of 0.4 mM H2O2 on cell migration. These results suggest that ionic silicon may have a potential effect in unfavorable situations where reactive oxygen species is predominant affecting cell viability, proliferation, migration, and differentiation. Furthermore, this study provides a guide for designing Si-containing biomaterials with desirable Si-ion release for skeletal muscle regeneration.
The aim of this systematic review was to perform qualitative and quantitative analysis on the toxic effects of chloroquine (CQ) and hydroxychloroquine (HCQ) on skeletal muscles. We designed the study according to PRISMA guidelines. Studies for qualitative and quantitative analyses were selected according to the following inclusion criteria: English language; size of sample (> 5 patients), adult (> age of 18) patients, treated with CQ/HCQ for inflammatory diseases, and presenting and not presenting with toxic effects on skeletal muscles. We collected data published from 1990 to April 2020 using PubMed, Cochrane Library, EMBASE, and SciELO. Risk of bias for observational studies was assessed regarding the ROBIN-I scale. Studies with less than five patients (case reports) were selected for an additional qualitative analysis. We used the software Comprehensive Meta-Analysis at the confidence level of 0.05. We identified 23 studies for qualitative analysis (17 case-reports), and five studies were eligible for quantitative analysis. From case reports, 21 patients presented muscle weakness and confirmatory biopsy for CQ/HCQ induced myopathy. From observational studies, 37 patients out of 1,367 patients from five studies presented muscle weakness related to the use of CQ/HCQ, and 252 patients presented elevated levels of muscle enzymes (aldolase, creatine phosphokinase, and lactate dehydrogenase). Four studies presented data on 34 patients with confirmatory biopsy for drug-induced myopathy. No study presented randomized samples. The chronic use of CQ/HCQ may be a risk for drug-induced myopathy. There is substantiated need for proper randomized trials and controlled prospective studies needed to assess the clinical and subclinical stages of CQ/HCQ -induced muscle myopathy.
Introduction Traumatic injuries lead to volumetric muscle loss (VML) and nerve damage that cause chronic functional deficits. Due to the inability of mammalian skeletal muscle to regenerate the damaged tissue in VML injuries, muscle flap procedures have been used. Functional muscle transfer, including vasculature and innervation, has been shown to improve strength to an injured muscle. However, most muscle flap procedures are not intended to restore muscle function except in limited circumstances. Thus, these procedures have not adequately addressed this issue and novel engineered materials that regenerate the damaged tissue and restore the functionality is crucially needed. Here, we introduce novel bioactive amorphous silica‐base biomaterials that accelerate muscle, nerve, and vascular healing for fully functional tissue regeneration. Materials and Methods Micro‐patterned scaffolds were designed and developed on Si‐wafer using photolithography, then 200 nm layer of SiONx was deposited by Plasma Enhanced Chemical Vapor Deposition technique. C2C12 mouse myoblast and NG108‐15 neuroblastoma x rat glioma hybrid cell lines were used for in‐vitro studies. In‐vitro studies using different ionic silicon concentration (0.1, 0.5, and 1.0 mM) were performed to determine the optimal concentration for muscle and nerve cells. MTS assay and fluorescence microscopy were used to study the cell viability, growth, and differentiation. Also, qRT‐PCR was used to investigate the targeted gene expression. Results In‐vitro studies revealed that 0.1 mM of Si4+ significantly enhance the C2C12 cell growth, fusion index (1.5 fold), and the area covered by myotubes (*** p<0.001) compared to the control (Fig. 1). Also, 0.1 mM of Si4+ significantly increase the NG108‐15 neural cell viability after 12 hours in growth media. qRT‐PCR data revealed that 0.1 mM of Si4+ increases MyoD gene expression 3‐folds compared to the control by day 1, and MyoG expression 2‐folds by day 5. Also, 0.5 mM of Si4+ significantly increases the GAP43 gene expression 3‐folds by day 1 of neural cell culture. Intriguingly, 0.5 mM of Si4+ significantly increase Neurturin (Myokine) protein concentration at day 3 of C2C12 cell culture. Most intriguingly, the formed myotubes and axons were perfectly aligned (Fig. 2) on the surface of micro‐patterned SiONx scaffolds (Without laminin coating). Conclusion This study confirms that 0.1 mM of Si4+ is the optimal concentration for muscle and nerve cells that enhance the cell differentiation and increase the myogenic/neurogenic gene expression as well as myogenic/neurogenic markers. Surface pattern successfully support axons and myotubes alignment on the SiONx surface. Support or Funding Information National Institutes of Health (Grant No. 1R56DE027964‐01A1‐01)University of Texas STARS award Program Si‐ions (0.1 mM) enhance Fusion index, total number of cells, and area covered by myotubes in C2C12 cells after 4 days of differentiation. aligned axons and myotubes on the patterned SiONx. A) DAPI and MHC Stained myotubes, b) R...
Introduction The traumatic or surgical loss of skeletal muscle with resultant functional impairment (volumetric muscle loss (VML)) is a challenging clinical problem. The current standard treatment care for VML is autologous tissue grafts and physical therapy, which still faces the challenge of donor site morbidity in large defect cases. 3D printing techniques for tissue engineering allows the precise control of micropatterning by determining the dimensions of filaments, the size and shape of pores and the percentage of porosity of the scaffold. Our hypothesis is using 3D printing of biopolymer‐nanosilicate scaffolds in‐situ will enhance muscle regeneration in VML defects. Materials and Methods Methacrylated gelatin (MAG) biopolymer Laponite (Lp) nano‐silicate scaffolds were prepared as a bio‐ink using a solution‐precipitation process. Our study includes designing and optimization of 3D printing parameters for printing MAG‐Lp scaffolds and in‐vitro evaluation of cell viability (live‐dead), proliferation, and differentiation of mouse myoblast precursor cells on these scaffolds. We further analyzed the in‐situ (directly in the defect) 3D printing (using a robocaster) in a rat VML model in tibialis anterior muscle to evaluate the feasibility, precision, flexibility and attachment of the scaffold to the defect. Results Silicon ion enhanced cell proliferation and differentiation (fusion index by 1.5 times and overexpression of myogenic factors) in mouse myoblast precursor cells as compared to control. Qualitative live/dead analysis showed more abundance of live myoblast precursor cells (green) and decreased number of dead cells (red) on MAG‐Lp surface as compared to MAG (Fig. 1). We also show successful printing abilities of MAG‐Lp scaffolds in the volumetric muscle loss defect (Fig. 2) along with increased attachment to the adjacent tissues. The animal also showed ability to stand on/flex the surgical limb, with sufficient grip strength to perform normal activity, immediately after recovering from the surgery. Conclusion Our preliminary findings conclude that 3D printing of MAG‐Lp can enhance myogenesis in myoblasts. We further conclude that successful proof of concept of an in‐situ bioprinting method of reconstructive scaffolds can be used for immediate repair and loading of muscles for VML injury treatment. Support or Funding Information NIH/NIDCR (R03DE023872‐01, 1R56DE027964‐01A1‐01), Texas STARs award, Departmental Start‐up Funds, University of Texas at Arlington, College of Nursing and Health Innovation, Arlington, TX showing live/dead assay on MAG and MAG‐Lp scaffolds. Green represents live cells, whereas red represents dead cells. showing In‐situ 3D printing biopolymer scaffold in a rat volumetric muscle loss model in tibialis anterior muscle (20–25% muscle loss) along with UV/photo curing
Introduction : Skeletal muscle constitutes about 40% of the total body mass and plays a significant role in the movement of the human body. Although skeletal muscles have remarkable endogenous regenerative capacity, this capacity is overwhelmed following acute severe traumatic injuries. Furthermore, the associated oxidative damage can delay regeneration process and prolong recovery. These traumatic injuries result from combat‐ and/or trauma‐induced muscle injuries and often lead to irreversible tissue damage and impaired vascularization. Volumetric muscle loss (VML) is a severe traumatic injury that results in a critical loss (≥ 20%) of the native muscle mass leading to permanent disability. Our hypothesis is that using 3D bioprinted hydrogel modified with silica‐based nanoparticles (NPs) laden human skeletal muscle cells will provide the required architecture and enhance muscle regeneration in VML defects where high levels of reactive oxygen species (ROS) is predominant. Materials and Methods Sodium metasilicate powder was used to adjust and optimize the effective concentration of ionic silicon, while hydrogen peroxide was used as sources of ROS to simulate the oxidative damage conditions of VML injuries. Silica based nanoparticles were embedded into GelMA‐based hydrogels for 3D printing of scaffolds that mimic the skeletal muscle architecture. Results Our preliminary data using ionic silicon indicated that Si‐ions are not cytotoxic to myoblast cells under tested concentrations (0.1‐2.0 mM) (Figure 1). Furthermore, Si‐ions significantly enhanced myoblast cell viability, proliferation, and differentiation into myotubes as indicated by a higher fusion index compared to the control. In‐vitro studies indicated that0.4 mM of H2O2 into the growth media significantly decreases the cell viability after 6 and 24 hr. compared to the control (**p < 0.01, n=4 per group). Addition of 0.5‐1.0 mM of Si into the growth media significantly enhances the cell viability under conditions that mimic high ROS (i.e., H202 treated group). The 3D printed scaffolds of GelMA + NPs indicated a higher myoblast cell viability significantly reducing cell death compared to the bare GelMA scaffolds as shown at Figure 2. Conclusion Our preliminary findings conclude that 0.1 mM Si‐ions enhance myoblast viability, proliferation, and differentiation of C2C12 myoblast cells. Using 0.4 mM of H2O2 can simulate the oxidative damage condition in myoblast cells in‐vitro. Using 0.1‐0.5 mM silicon ions can attenuate the oxidative damage (0.4 mM H2O2) on C2C12 myoblast cells. 3D printed hydrogels loaded with silica‐based nanoparticles are promising materials for 3D printing of muscle constructs.
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