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Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration

BackgroundCell-transplantation therapies have attracted attention as treatments for skeletal-muscle disorders; however, such research has been severely limited by poor cell survival. Tissue engineering offers a potential solution to this problem by providing biomaterial adjuvants that improve survival and engraftment of donor cells.MethodsIn this study, we investigated the use of intra-muscular transplantation of mesoangioblasts (vessel-associated progenitor cells), delivered with an injectable hydrogel biomaterial directly into the tibialis anterior (TA) muscle of acutely injured or dystrophic mice. The hydrogel cell carrier, made from a polyethylene glycol-fibrinogen (PF) matrix, is polymerized in situ together with mesoangioblasts to form a resorbable cellularized implant.ResultsMice treated with PF and mesoangioblasts showed enhanced cell engraftment as a result of increased survival and differentiation compared with the same cell population injected in aqueous saline solution.ConclusionBoth PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle.

3D hydrogel environment rejuvenates aged pericytes for skeletal muscle tissue engineering

Skeletal muscle tissue engineering is a promising approach for the treatment of muscular disorders. However, the complex organization of muscle, combined with the difficulty in finding an appropriate source of regenerative cells and in providing an adequate blood supply to the engineered tissue, makes this a hard task to face. In the present work, we describe an innovative approach to rejuvenate adult skeletal muscle-derived pericytes (MP) based on the use of a PEG-based hydrogel scaffold. MP were isolated from young (piglet) and adult (boar) pigs to assess whether aging affects tissue regeneration efficiency. In vitro, MP from boars had similar morphology and colony forming capacity to piglet MP, but an impaired ability to form myotubes and capillary-like structures. However, the use of a PEG-based hydrogel to support adult MP significantly improved their myogenic differentiation and angiogenic potentials in vitro and in vivo. Thus, PEG-based hydrogel scaffolds may provide a progenitor cell “niche” that promotes skeletal muscle regeneration and blood vessel growth, and together with pericytes may be developed for use in regenerative applications.

In vivo generation of a mature and functional artificial skeletal muscle

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro‐generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG‐fibrinogen hydrogel. Once engineered to express placental‐derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG‐fibrinogen‐embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient‐specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

PlGF–MMP9-engineered iPS cells supported on a PEG–fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)–fibrinogen scaffold. When assessing optimal stiffness of the PEG–fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

The niche-derived glial cell line-derived neurotrophic factor (GDNF) induces migration of mouse spermatogonial stem/progenitor cells.

In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

Purification and characterization of a fibrinogenolytic and hemorrhagic metalloproteinase isolated from Vipera lebetina venom.

Serious clinical problems such as hemorrhage, edema and tissue necrosis are observed following viperid envenoming. A proteinase (VLH2) was isolated from Vipera lebetina by combination of two chromatographic steps of gel filtration on Sephadex G-75 followed by DEAE Sephadex A-50. This acidic proteinase, with a molecular mass of about 55 kDa and isoelectric point of 5.4, displayed a fibrinogenolytic and hemorrhagic activities. VLH2 hydrolyses rapidly the Aalpha-chain of fibrinogen, followed, more slowly, by the Bbeta-chain, leaving the gamma-chain unaffected. The proteolytic and hemorrhagic activities of VLH2 were inhibited by EDTA, EGTA and 1-10 phenanthroline. However, these activities were not affected by AEBSF, Aprotinine, and E64, suggesting that VLH2 is a metalloproteinase with an alpha-fibrinogenase activity, requiring calcium and zinc for its activity. The enzyme VLH2 did not have proteolytic activity towards extracellular components gelatin, laminin and fibronectin. The hemorrhagic metalloproteinase VLH2 has a myotoxic activity, as determined by serum CK level and histological observation of muscle tissue. Furthermore, VLH2 is able to induce apoptosis of C2C12 myotubes. These results indicate that VLH2 is implicated in the local and systemic bleeding, contributing thus in the toxicity of V. lebetina venom.

S-Nitrosoglutathione Reductase Deficiency-Induced S-Nitrosylation Results in Neuromuscular Dysfunction

AIMS Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. RESULTS We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. INNOVATION Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. CONCLUSION These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

Activation of the Pro-Oxidant PKCβII-p66Shc Signaling Pathway Contributes to Pericyte Dysfunction in Skeletal Muscles of Diabetic Patients with Critical Limb Ischemia

Critical limb ischemia (CLI), foot ulcers, former amputation, and impaired regeneration are independent risk factors for limb amputation in subjects with diabetes. The present work investigates whether and by which mechanism diabetes negatively impacts on functional properties of muscular pericytes (MPs), which are resident stem cells committed to reparative angiomyogenesis. We obtained muscle biopsy samples from patients with diabetes who were undergoing major limb amputation and control subjects. Diabetic muscles collected at the rim of normal tissue surrounding the plane of dissection showed myofiber degeneration, fat deposition, and reduction of MP vascular coverage. Diabetic MPs (D-MPs) display ultrastructural alterations, a differentiation bias toward adipogenesis at the detriment of myogenesis and an inhibitory activity on angiogenesis. Furthermore, they have an imbalanced redox state, with downregulation of the antioxidant enzymes superoxide dismutase 1 and catalase, and activation of the pro-oxidant protein kinase C isoform β-II (PKCβII)-dependent p66Shc signaling pathway. A reactive oxygen species scavenger or, even more effectively, clinically approved PKCβII inhibitors restore D-MP angiomyogenic activity. Inhibition of the PKCβII-dependent p66Shc signaling pathway could represent a novel therapeutic approach for the promotion of muscle repair in individuals with diabetes.

Metformin protects skeletal muscle from cardiotoxin induced degeneration.

The skeletal muscle tissue has a remarkable capacity to regenerate upon injury. Recent studies have suggested that this regenerative process is improved when AMPK is activated. In the muscle of young and old mice a low calorie diet, which activates AMPK, markedly enhances muscle regeneration. Remarkably, intraperitoneal injection of AICAR, an AMPK agonist, improves the structural integrity of muscles of dystrophin-deficient mdx mice. Building on these observations we asked whether metformin, a powerful anti-hyperglycemic drug, which indirectly activates AMPK, affects the response of skeletal muscle to damage. In our conditions, metformin treatment did not significantly influence muscle regeneration. On the other hand we observed that the muscles of metformin treated mice are more resilient to cardiotoxin injury displaying lesser muscle damage. Accordingly myotubes, originated in vitro from differentiated C2C12 myoblast cell line, become more resistant to cardiotoxin damage after pre-incubation with metformin. Our results indicate that metformin limits cardiotoxin damage by protecting myotubes from necrosis. Although the details of the molecular mechanisms underlying the protective effect remain to be elucidated, we report a correlation between the ability of metformin to promote resistance to damage and its capacity to counteract the increment of intracellular calcium levels induced by cardiotoxin treatment. Since increased cytoplasmic calcium concentrations characterize additional muscle pathological conditions, including dystrophies, metformin treatment could prove a valuable strategy to ameliorate the conditions of patients affected by dystrophies.

Matrix scaffolding for stem cell guidance toward skeletal muscle tissue engineering

Extracellular matrix (ECM) is composed of many types of fibrous structural proteins and glycosaminoglycans. This important cell component not only provides a support for cells but is also actively involved in cell-cell interaction, proliferation, migration, and differentiation, representing, therefore, no longer only a mere static structural scaffold for cells but rather a dynamic and versatile compartment. This aspect leads to the need for investigating new bio-inspired scaffolds or biomaterials, able to mimic ECM in tissue engineering. This new field of research finds particular employment in skeletal muscle tissue regeneration, due to the inability of this complex tissue to recover volumetric muscle loss (VML), after severe injury. Usually, this is the result of traumatic incidents, tumor ablations, or pathological states that lead to the destruction of a large amount of tissue, including connective tissue and basement membrane. Therefore, skeletal muscle tissue engineering represents a valid alternative to overcome this problem.Here, we described a series of natural and synthetic biomaterials employed as ECM mimics for their ability to recreate the correct muscle stem cell niche, by promoting myogenic stem cell differentiation and so, positively affecting muscle repair.