Osteogenic Potential of Human Dental Pulp Stem Cells Co-Cultured with Equine Bone Substitute Combined with Melatonin.

Bone blocks are proposed in oral bone regeneration for their biocompatibility and osteoconductivity. Human dental pulp stem cells (hDPSCs) have been used with bone substitutes as a biocomplex. Melatonin, produced by the pineal gland, has specific functions in the oral cavity in bone remodeling and enhancing the dual actions on osteoblasts and osteoclasts, the genic expression of bone markers. This study evaluated the osteogenic differentiation of hDPSCs, stimulated by melatonin on equine bone blocks. hDPSCs were cultured in growth medium (GM) or differentiation medium (DM) with or without the presence of equine bone blocks and 100 µm melatonin. After 7, 14, and 21 days of culture, expression of miRNAs (miR-133a, miR-133b, miR-135a, miR-29b, miR-206, and miR- let-7b) and genes (RUNX2, SMAD5, HDAC4, COL4a2, and COL5a3), osteocalcin levels and histolgic analyses were evaluated. Melatonin and equine blocks increased the osteogenic potential of hDPSCs even in GM, regulated miRNA and gene expression related to osteogenesis, and increased osteocalcin. hDPSCs cultured in DM showed a significantly higher osteogenic potential compared to GM. This study suggests that equine bone blocks and melatonin enhanced osteogenesis, stimulating early stages of cell differentiation. hDPSCs/equine bone block and melatonin represent a promising, useful biocomplex in bone regeneration with a potential for a possible clinical application.

Corrigendum to "Superoxide Anion Production and Bioenergetic Profile in Young and Elderly Human Primary Myoblasts".

[This corrects the article DOI: 10.1155/2018/2615372.].

Neuromuscular Electrical Stimulation Induces Skeletal Muscle Fiber Remodeling and Specific Gene Expression Profile in Healthy Elderly.

Skeletal muscle aging is a multifactorial process strictly related to progressive weakness. One of the results that were focused on was the fiber phenotype modification and their loss. The physiological muscle recruitment to contraction, basically prosecuted under volitional control, can also be engaged by means of Neuromuscular Electrical Stimulation (NMES). Knowing that the NMES is effective in improving muscle strength in active healthy elderly, the aim was to investigate which physiological modifications were able to produce in the Vastus lateralis muscle and the pathways involved. It was found that NMES increased the cross sectional area and the isometric strength of type II myofibers together with the activated myogenic pathway in order to shift glycolytic toward the oxidative phenotype II myofibers, at a molecular level and with an increase of maximal voluntary contraction (MVC) at a functional level. Using the TaqMan low density array on 48 different genes, we found that NMES specific gene regulation highlighted: (i) increased protein synthesis with respect to protein degradation; (ii) the activation of an apoptotic pathway involved in the differentiation process; (iii) increased regeneration signals; (iv) oxidative enzyme regulation. These pathways were validated via confirmatory RT-PCR for genes involved in the regeneration process as well as Myosin isoforms. We also investigated the oxidative stress status analyzing superoxide anion levels, the protein expression of two different superoxide dismutase and the activity of both catalase and superoxide anion dismutase, being two main antioxidant enzymes. In conclusion, data demonstrates that NMES is effective in producing physiological adaptation on Vastus Lateralis of active healthy elderly as well as providing new insights for further research on elderly who experienced muscle detriment for periodic or permanent immobility.

Superoxide Anion Production and Bioenergetic Profile in Young and Elderly Human Primary Myoblasts.

Sarcopenia is the age-related loss of skeletal muscle mass, strength, and function. It is associated with regenerative difficulties by satellite cells, adult muscle stem cells, and alteration of oxidative management, mainly the increase in superoxide anions (O2•-). We aimed to investigate the relation between regenerative deficit in elderly and increase in O2•- production along with mitochondrial alterations. Myoblasts and myotubes from skeletal muscle of young and elderly healthy subjects (27.8 ± 6 and 72.4 ± 6.5 years old) were measured: (1) superoxide dismutase activity and protein content, (2) mitochondrial O2•- production levels, (3) O2•- production variability, and (4) mitochondrial bioenergetic profile. Compared to young myoblasts, elderly myoblasts displayed decreased SOD2 protein expression, elevated mitochondrial O2•- baseline levels, and decreased oxidative phosphorylation and glycolysis. Additionally, elderly versus young myotubes showed elevated mitochondrial O2•- levels when stressed with N-acetyl cysteine or high glucose and higher glycolysis despite showing comparable oxidative phosphorylation levels. Altogether, the elderly may have less metabolic plasticity due to the impaired mitochondrial function caused by O2•-. However, the increased energy demand related to the differentiation process appears to activate compensatory mechanisms for the partial mitochondrial dysfunction.

Neuromuscular electrical stimulation improves skeletal muscle regeneration through satellite cell fusion with myofibers in healthy elderly subjects.

The aim of this study was to determine whether neuromuscular electrical stimulation (NMES) affects skeletal muscle regeneration through a reduction of oxidative status in satellite cells of healthy elderly subjects. Satellite cells from the vastus lateralis skeletal muscle of 12 healthy elderly subjects before and after 8 wk of NMES were allowed to proliferate to provide myogenic populations of adult stem cells [myogenic precursor cells (MPCs)]. These MPCs were then investigated in terms of their proliferation, their basal cytoplasmic free Ca2+ concentrations, and their expression of myogenic regulatory factors (PAX3, PAX7, MYF5, MYOD, and MYOG) and micro-RNAs (miR-1, miR-133a/b, and miR-206). The oxidative status of these MPCs was evaluated through superoxide anion production and superoxide dismutase and glutathione peroxidase activities. On dissected single skeletal myofibers, the nuclei were counted to determine the myonuclear density, the fiber phenotype, cross-sectional area, and tension developed. The MPCs obtained after NMES showed increased proliferation rates along with increased cytoplasmic free Ca2+ concentrations and gene expression of MYOD and MYOG on MPCs. Muscle-specific miR-1, miR-133a/b, and miR-206 were upregulated. This NMES significantly reduced superoxide anion production, along with a trend to reduction of superoxide dismutase activity. The NMES-dependent stimulation of muscle regeneration enhanced satellite cell fusion with mature skeletal fibers. NMES improved the regenerative capacity of skeletal muscle in elderly subjects. Accordingly, the skeletal muscle strength and mobility of NMES-stimulated elderly subjects significantly improved. NMES may thus be further considered for clinical or ageing populations.NEW & NOTEWORTHY The neuromuscular electrical stimulation (NMES) effect on skeletal muscle regeneration was assessed in healthy elderly subjects for the first time. NMES improved the regenerative capacity of skeletal muscle through increased myogenic precursor cell proliferation and fusion with mature myofibers. The increased cytoplasmic free Ca2+ concentration along with MYOD, MYOG, and micro-RNA upregulation could be related to reduced O2·- production, which, in turn, favors myogenic regeneration. Accordingly, the skeletal muscle strength of NMES-stimulated lower limbs of healthy elderly subjects improved along with their mobility.