Platelet-rich plasma affects gap junctional features in myofibroblasts in vitro via vascular endothelial growth factor (VEGF)-A/VEGF receptor.

NEW FINDINGS: What is the central question of this study? It is a challenge to discover effective therapies for fibrosis. Increasing evidence supports the antifibrotic potential of platelet-rich plasma (PRP) as a source of bioactive molecules, such as vascular endothelial growth factor (VEGF)-A. However, the effects and mechanisms of action of PRP need to be clarified. What is the main finding and its importance? This report clarifies the mechanisms mediating the antifibrotic action of PRP, strengthening the role of VEGF-A/VEGF receptor, and identifies gap junction currents and connexin 43 as novel targets of this pathway in the fibroblast-to-myofibroblast transition induced by the transforming growth factor-ß1. ABSTRACT: Despite increasing experimental evidence, the antifibrotic potential of platelet-rich plasma (PRP) remains controversial, and its mechanisms of action are not fully clarified. This short report extends our previous research on the capability of PRP to prevent the in vitro differentiation of fibroblasts toward myofibroblasts, the key effectors of fibrosis, induced by the profibrotic agent transforming growth factor-ß1 (TGF-ß1). In particular, we focused on the involvement of signalling mediated by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) in the PRP-induced fibroblast response, highlighting gap junction features. Electrophysiological and morphological analyses revealed that PRP hindered morphofunctional differentiation of both murine NIH/3T3 and human primary adult skin fibroblasts toward myofibroblasts as judged by the analysis of membrane phenomena, α-smooth muscle actin and vinculin expression and cell morphology. Neutralization of VEGF-A by blocking antibodies or pharmacological inhibition of VEGFR by KRN633 in TGF-ß1-treated fibroblasts prevented the PRP-promoted effects, such as the reduction of voltage-dependent transjunctional currents in cell pairs and a decreased expression of connexin 43, the typical connexin isoform forming voltage-dependent connexons. The role of VEGF-A in inhibiting these events was confirmed by treating TGF-ß1-stimulated fibroblasts with soluble VEGF-A. The results obtained when cells were differentiated using KRN633 alone suggest an antagonistic cross-talk between TGF-ß1 and VEGFR. In conclusion, this study identifies, for the first time, gap junction currents as crucial targets in the VEGF-A/VEGFR-mediated antifibrotic pathway and provides new insights into mechanisms behind the action of PRP in preventing differentiation of fibroblasts to myofibroblasts.

Bone Marrow-Mesenchymal Stromal Cell Secretome as Conditioned Medium Relieves Experimental Skeletal Muscle Damage Induced by Ex Vivo Eccentric Contraction.

Bone marrow-mesenchymal stem/stromal cells (MSCs) may offer promise for skeletal muscle repair/regeneration. Growing evidence suggests that the mechanisms underpinning the beneficial effects of such cells in muscle tissue reside in their ability to secrete bioactive molecules (secretome) with multiple actions. Hence, we examined the effects of MSC secretome as conditioned medium (MSC-CM) on ex vivo murine extensor digitorum longus muscle injured by forced eccentric contraction (EC). By combining morphological (light and confocal laser scanning microscopies) and electrophysiological analyses we demonstrated the capability of MSC-CM to attenuate EC-induced tissue structural damages and sarcolemnic functional properties' modifications. MSC-CM was effective in protecting myofibers from apoptosis, as suggested by a reduced expression of pro-apoptotic markers, cytochrome c and activated caspase-3, along with an increase in the expression of pro-survival AKT factor. Notably, MSC-CM also reduced the EC-induced tissue redistribution and extension of telocytes/CD34+ stromal cells, distinctive cells proposed to play a "nursing" role for the muscle resident myogenic satellite cells (SCs), regarded as the main players of regeneration. Moreover, it affected SC functionality likely contributing to replenishment of the SC reservoir. This study provides the necessary groundwork for further investigation of the effects of MSC secretome in the setting of skeletal muscle injury and regenerative medicine.

The Amphipathic GM1 Molecule Stabilizes Amyloid Aggregates, Preventing their Cytotoxicity.

Amyloid aggregates have been demonstrated to exert cytotoxic effects in several diseases. It is widely accepted that the complex and fascinating aggregation pathway involves a series of steps during which many heterogeneous intermediates are generated. This process may be greatly potentiated by the presence of amphipathic components of plasma membrane because they may serve as interaction, condensation, and nucleation points. However, there are few data regarding structural alterations induced by the binding between the amyloid fibrils and membrane components and its direct effects on cell integrity. In this study, we found, by 1-anilinonaphthalene 8-sulfonic acid and transmission electron microscopy/fast Fourier transform, that yeast prion Sup35 oligomers showed higher structural uniformity and altered surface properties when grown in the presence of monosialotetrahexosylganglioside, a component of the cell membrane. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and confocal/sensitized Förster resonance energy transfer analyses revealed that these fibrils showed low cytotoxicity and affinity to plasma membrane. Moreover, time-lapse analysis of Sup35 oligomer fibrillation on cells suggested that the amyloid aggregation process per se exerts cytotoxic effects through the interaction of amyloid intermediates with plasma membrane components. These data provide, to our knowledge, new insights to understand the mechanism of amyloid growth and cytotoxicity in the pathogenesis of amyloid diseases.

Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Aging and neurodegenerative diseases share a condition of neuroinflammation entailing the production of endogenous cell debris in the CNS that must be removed by microglia ( i.e., resident macrophages), to restore tissue homeostasis. In this context, extension of microglial cell branches toward cell debris underlies the mechanisms of microglial migration and phagocytosis. Amoeboid morphology and the consequent loss of microglial branch functionality characterizes dysregulated microglia. Microglial migration is assisted by another glial population, the astroglia, which forms a dense meshwork of cytoplasmic projections. Amoeboid microglia and disrupted astrocyte meshwork are consistent traits in aged CNS. In this study, we assessed a possible correlation between microglia and astroglia morphology in rat models of chronic neuroinflammation and aging, by 3-dimensional confocal analysis implemented with particle analysis. Our findings suggest that a microglia-astroglia interaction occurs in rat hippocampus via cell-cell contacts, mediating microglial cell branching in the presence of inflammation. In aged rats, the impairment of such an interaction correlates with altered distribution, morphology, and inefficient clearance by microglia. These data support the idea that generally accepted functional boundaries between microglia and astrocytes should be re-evaluated to better understand how their functions overlap and interact.-Lana, D., Ugolini, F., Wenk, G. L., Giovannini, M. G., Zecchi-Orlandini, S., Nosi, D. Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Sphingosine 1-Phosphate (S1P)/ S1P Receptor Signaling and Mechanotransduction: Implications for Intrinsic Tissue Repair/Regeneration.

Tissue damage, irrespective from the underlying etiology, destroys tissue structure and, eventually, function. In attempt to achieve a morpho-functional recover of the damaged tissue, reparative/regenerative processes start in those tissues endowed with regenerative potential, mainly mediated by activated resident stem cells. These cells reside in a specialized niche that includes different components, cells and surrounding extracellular matrix (ECM), which, reciprocally interacting with stem cells, direct their cell behavior. Evidence suggests that ECM stiffness represents an instructive signal for the activation of stem cells sensing it by various mechanosensors, able to transduce mechanical cues into gene/protein expression responses. The actin cytoskeleton network dynamic acts as key mechanotransducer of ECM signal. The identification of signaling pathways influencing stem cell mechanobiology may offer therapeutic perspectives in the regenerative medicine field. Sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signaling, acting as modulator of ECM, ECM-cytoskeleton linking proteins and cytoskeleton dynamics appears a promising candidate. This review focuses on the current knowledge on the contribution of S1P/S1PR signaling in the control of mechanotransduction in stem/progenitor cells. The potential contribution of S1P/S1PR signaling in the mechanobiology of skeletal muscle stem cells will be argued based on the intriguing findings on S1P/S1PR action in this mechanically dynamic tissue.

Influence of Platelet-Rich and Platelet-Poor Plasma on Endogenous Mechanisms of Skeletal Muscle Repair/Regeneration.

The morpho-functional recovery of injured skeletal muscle still represents an unmet need. None of the therapeutic options so far adopted have proved to be resolutive. A current scientific challenge remains the identification of effective strategies improving the endogenous skeletal muscle regenerative program. Indeed, skeletal muscle tissue possesses an intrinsic remarkable regenerative capacity in response to injury, mainly thanks to the activity of a population of resident muscle progenitors called satellite cells, largely influenced by the dynamic interplay established with different molecular and cellular components of the surrounding niche/microenvironment. Other myogenic non-satellite cells, residing within muscle or recruited via circulation may contribute to post-natal muscle regeneration. Unfortunately, in the case of extended damage the tissue repair may become aberrant, giving rise to a maladaptive fibrotic scar or adipose tissue infiltration, mainly due to dysregulated activity of different muscle interstitial cells. In this context, plasma preparations, including Platelet-Rich Plasma (PRP) and more recently Platelet-Poor Plasma (PPP), have shown advantages and promising therapeutic perspectives. This review focuses on the contribution of these blood-derived products on repair/regeneration of damaged skeletal muscle, paying particular attention to the potential cellular targets and molecular mechanisms through which these products may exert their beneficial effects.

Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-ß1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis.

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-ß1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-ß1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated "from bench to bedside," could potentially offer advantages over the single treatments.

The contribution of leucocytes to the antimicrobial activity of platelet-rich plasma preparations: A systematic review.

The infection of a wound is one of the major contributors to delays in healing and tissue regeneration. As multi-drug resistance to antibiotics is becoming a serious threat, research in this field has focused on finding new agents and strategies to fight infection and additionally to reduce healing times. The topical use of autologous Platelet Rich Plasma (PRP) as a biological accelerator of the healing process, has been safely used as a form of treatment for wounds since the 1990s. Although the presence or absence of leucocytes in PRP preparation was previously neglected, in the last decade more attention has been paid to their role and several studies have been conducted to explore both their immuno-metabolic effects and their antimicrobial properties. In this review, we aim to summarise the literature on the contribution of leucocytes included in PRP preparations in terms of their antimicrobial properties. This should help to inform clinical practice and additional research in this promising field.

Red (635 nm), Near-Infrared (808 nm) and Violet-Blue (405 nm) Photobiomodulation Potentiality on Human Osteoblasts and Mesenchymal Stromal Cells: A Morphological and Molecular In Vitro Study.

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm² energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration.

Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as ß-amyloid (Aß) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aß autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aß aggregation (+300 and +800% of aggregated Aß peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aß clearance (-45% of Aß deposits on APJs, and +33% of Aß deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aß deposits and are involved in novel Aß-mediated adhesions between neurons. These data define the effects of Aß deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

Mesenchymal stromal cell and osteoblast responses to oxidized titanium surfaces pre-treated with λ = 808 nm GaAlAs diode laser or chlorhexidine: in vitro study.

Preservation of implant biocompatibility following peri-implantitis treatments is a crucial issue in odontostomatological practice, being closely linked to implant re-osseointegration. Our aim was to assess the responses of osteoblast-like Saos2 cells and adult human bone marrow-mesenchymal stromal cells (MSCs) to oxidized titanium surfaces (TiUnite®, TiU) pre-treated with a 808 ± 10 nm GaAlAs diode laser operating in non-contact mode, in continuous (2 W, 400 J/cm2; CW) or pulsed (20 kHz, 7 µs, 0.44 W, 88 J/cm2; PW) wave, previously demonstrated to have a strong bactericidal effect and proposed as optional treatment for peri-implantitis. The biocompatibility of TiU surfaces pre-treated with chlorhexidine digluconate (CHX) was also evaluated. In particular, in order to mimic the in vivo approach, TiU surfaces were pre-treated with CHX (0.2%, 5 min); CHX and rinse; and CHX, rinse and air drying. In some experiments, the cells were cultured on untreated TiU before being exposed to CHX. Cell viability (MTS assay), proliferation (EdU incorporation assay; Ki67 confocal immunofluorescence analysis), adhesion (morphological analysis of actin cytoskeleton organization), and osteogenic differentiation (osteopontin confocal immunofluorescence analysis; mineralized bone-like nodule formation) analyses were performed. CHX resulted cytotoxic in all experimental conditions. Diode laser irradiation preserved TiU surface biocompatibility. Notably, laser treatment appeared even to improve the known osteoconductive properties of TiU surfaces. Within the limitations of an in vitro experimentation, this study contributes to provide additional experimental basis to support the potential use of 808 ± 10 nm GaAlAs diode laser at the indicated irradiation setting, in the treatment of peri-implantitis and to discourage the use of CHX.

Effects of photodynamic laser and violet-blue led irradiation on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide attached to moderately rough titanium surface: in vitro study.

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This study investigates the in vitro efficacy of photodynamic treatment (PDT) with methylene blue (MB) photoactivated with λ 635 nm diode laser and of λ 405 nm violet-blue LED phototreatment for the reduction of bacterial biofilm and lipopolysaccharide (LPS) adherent to titanium surface mimicking the bone-implant interface. Staphylococcus aureus biofilm grown on titanium discs with a moderately rough surface was subjected to either PDT (0.1% MB and λ 635 nm diode laser) or λ 405 nm LED phototreatment for 1 and 5 min. Bactericidal effect was evaluated by vital staining and residual colony-forming unit count. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, discs coated with Escherichia coli LPS were treated as above before seeding with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Both PDT and LED phototreatment induced a statistically significant (p < 0.05 or higher) reduction of viable bacteria, up to -99 and -98% (5 min), respectively. Moreover, besides bactericidal effect, PDT and LED phototreatment also inhibited LPS bioactivity, assayed as nitrite formation, up to -42%, thereby blunting host inflammatory response. Non-invasive phototherapy emerges as an attractive alternative in the treatment of peri-implantitis to reduce bacteria and LPS adherent to titanium implant surface without causing damage of surface microstructure. Its efficacy in the clinical setting remains to be investigated.

Low intensity 635 nm diode laser irradiation inhibits fibroblast-myofibroblast transition reducing TRPC1 channel expression/activity: New perspectives for tissue fibrosis treatment.

BACKGROUND AND OBJECTIVE: Low-level laser therapy (LLLT) or photobiomodulation therapy is emerging as a promising new therapeutic option for fibrosis in different damaged and/or diseased organs. However, the anti-fibrotic potential of this treatment needs to be elucidated and the cellular and molecular targets of the laser clarified. Here, we investigated the effects of a low intensity 635 ± 5 nm diode laser irradiation on fibroblast-myofibroblast transition, a key event in the onset of fibrosis, and elucidated some of the underlying molecular mechanisms. MATERIALS AND METHODS: NIH/3T3 fibroblasts were cultured in a low serum medium in the presence of transforming growth factor (TGF)-ß1 and irradiated with a 635 ± 5 nm diode laser (continuous wave, 89 mW, 0.3 J/cm(2) ). Fibroblast-myofibroblast differentiation was assayed by morphological, biochemical, and electrophysiological approaches. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 and of Tissue inhibitor of MMPs, namely TIMP-1 and TIMP-2, after laser exposure was also evaluated by confocal immunofluorescence analyses. Moreover, the effect of the diode laser on transient receptor potential canonical channel (TRPC) 1/stretch-activated channel (SAC) expression and activity and on TGF-ß1/Smad3 signaling was investigated. RESULTS: Diode laser treatment inhibited TGF-ß1-induced fibroblast-myofibroblast transition as judged by reduction of stress fibers formation, α-smooth muscle actin (sma) and type-1 collagen expression and by changes in electrophysiological properties such as resting membrane potential, cell capacitance and inwardly rectifying K(+) currents. In addition, the irradiation up-regulated the expression of MMP-2 and MMP-9 and downregulated that of TIMP-1 and TIMP-2 in TGF-ß1-treated cells. This laser effect was shown to involve TRPC1/SAC channel functionality. Finally, diode laser stimulation and TRPC1 functionality negatively affected fibroblast-myofibroblast transition by interfering with TGF-ß1 signaling, namely reducing the expression of Smad3, the TGF-ß1 downstream signaling molecule. CONCLUSION: Low intensity irradiation with 635 ± 5 nm diode laser inhibited TGF-ß1/Smad3-mediated fibroblast-myofibroblast transition and this effect involved the modulation of TRPC1 ion channels. These data contribute to support the potential anti-fibrotic effect of LLLT and may offer further informations for considering this therapy as a promising therapeutic tool for the treatment of tissue fibrosis.

The effects of diode laser on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study.

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-µm fiber. Setting parameters were 2 W (400 J/cm2) for continuous wave mode; 22 µJ, 20 kHz, 7 µs (88 J/cm2) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.

MSCs seeded on bioengineered scaffolds improve skin wound healing in rats.

Growing evidence has shown the promise of mesenchymal stromal cells (MSCs) for the treatment of cutaneous wound healing. We have previously demonstrated that MSCs seeded on an artificial dermal matrix, Integra (Integra Lifesciences Corp., Plainsboro, NJ) enriched with platelet-rich plasma (Ematrix) have enhanced proliferative potential in vitro as compared with those cultured on the scaffold alone. In this study, we extended the experimentation by evaluating the efficacy of the MSCs seeded scaffolds in the healing of skin wounds in an animal model in vivo. It was found that the presence of MSCs within the scaffolds greatly ameliorated the quality of regenerated skin, reduced collagen deposition, enhanced reepithelization, increased neo-angiogenesis, and promoted a greater return of hair follicles and sebaceous glands. The mechanisms involved in these beneficial effects were likely related to the ability of MSCs to release paracrine factors modulating the wound healing response. MSC-seeded scaffolds, in fact, up-regulated matrix metalloproteinase 9 expression in the extracellular matrix and enhanced the recruitment of endogenous progenitors during tissue repair. In conclusion, the results of this study provide evidence that the treatment with MSC-seeded scaffolds of cutaneous wounds contributes to the recreation of a suitable microenvironment for promoting tissue repair/regeneration at the implantation sites.

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells.

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7(+) satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.

Label-free three-dimensional imaging and quantitative analysis of living fibroblasts and myofibroblasts by holotomographic microscopy.

Holotomography (HT) is a cutting-edge fast live-cell quantitative label-free imaging technique. Based on the principle of quantitative phase imaging, it combines holography and tomography to record a three-dimensional map of the refractive index, used as intrinsic optical and quantitative imaging contrast parameter of biological samples, at a sub-micrometer spatial resolution. In this study HT has been employed for the first time to analyze the changes of fibroblasts differentiating towards myofibroblasts - recognized as the main cell player of fibrosis - when cultured in vitro with the pro-fibrotic factor, namely transforming growth factor-ß1. In parallel, F-actin, vinculin, α-smooth muscle actin, phospho-myosin light chain 2, type-1 collagen, peroxisome proliferator-activated receptor-gamma coactivator-1α expression and mitochondria were evaluated by confocal laser scanning microscopy. Plasmamembrane passive properties and transient receptor potential canonical channels' currents were also recorded by whole-cell patch-clamp. The fluorescence images and electrophysiological results have been compared to the data obtained by HT and their congruence has been discussed. HT turned out to be a valid approach to morphologically distinguish fibroblasts from well differentiated myofibroblasts while obtaining objective measures concerning volume, surface area, projection area, surface index and dry mass (i.e., the mass of the non-aqueous content inside the cell including proteins and subcellular organelles) of the entire cell, nuclei and nucleoli with the major advantage to monitor outer and inner features in living cells in a non-invasive, rapid and label-free approach. HT might open up new research opportunities in the field of fibrotic diseases. RESEARCH HIGHLIGHTS: Holotomography (HT) is a label-free laser interferometric imaging technology exploiting the intrinsic optical property of cells namely refractive index (RI) to enable a direct imaging and analysis of whole cells or intracellular organelles. HT turned out a valid approach to distinguish morphological features of living unlabeled fibroblasts from differentiated myofibroblasts. HT provided quantitative information concerning volume, surface area, projection area, surface index and dry mass of the entire fibroblasts/myofibroblasts, nuclei and nucleoli.

Photoactivation of bone marrow mesenchymal stromal cells with diode laser: effects and mechanisms of action.

Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low-level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch-1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up-regulation and activation of Notch-1 pathway, and with increased membrane conductance through voltage-gated K(+) , BK and Kir, channels and T- and L-type Ca(2+) channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch-1 up-regulation were severely decreased by the pre-treatment with the channel inhibitor Ba(2+) (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation.

HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.