Stefano Testa

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.

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.

Engineering Muscle Networks in 3D Gelatin Methacryloyl Hydrogels: Influence of Mechanical Stiffness and Geometrical Confinement

In this work, the influence of mechanical stiffness and geometrical confinement on the 3D culture of myoblast-laden gelatin methacryloyl (GelMA) photo-crosslinkable hydrogels was evaluated in terms of in vitro myogenesis. We formulated a set of cell-laden GelMA hydrogels with a compressive modulus in the range 1 ÷ 17 kPa, obtained by varying GelMA concentration and degree of cross-linking. C2C12 myoblasts were chosen as the cell model to investigate the supportiveness of different GelMA hydrogels toward myotube formation up to 2 weeks. Results showed that the hydrogels with a stiffness in the range 1 ÷ 3 kPa provided enhanced support to C2C12 differentiation in terms of myotube number, rate of formation, and space distribution. Finally, we studied the influence of geometrical confinement on myotube orientation by confining cells within thin hydrogel slabs having different cross sections: (i) 2,000 μm × 2,000 μm, (ii) 1,000 μm × 1,000 μm, and (iii) 500 μm × 500 μm. The obtained results showed that by reducing the cross section, i.e., by increasing the level of confinement—myotubes were more closely packed and formed aligned myostructures that better mimicked the native morphology of skeletal muscle.

Oxidative stress preconditioning of mouse perivascular myogenic progenitors selects a subpopulation of cells with a distinct survival advantage in vitro and in vivo

Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.

Combination of biochemical and mechanical cues for tendon tissue engineering

Tendinopathies negatively affect the life quality of millions of people in occupational and athletic settings, as well as the general population. Tendon healing is a slow process, often with insufficient results to restore complete endurance and functionality of the tissue. Tissue engineering, using tendon progenitors, artificial matrices and bioreactors for mechanical stimulation, could be an important approach for treating rips, fraying and tissue rupture. In our work, C3H10T1/2 murine fibroblast cell line was exposed to a combination of stimuli: a biochemical stimulus provided by Transforming Growth Factor Beta (TGF‐β) and Ascorbic Acid (AA); a three‐dimensional environment represented by PEGylated‐Fibrinogen (PEG‐Fibrinogen) biomimetic matrix; and a mechanical induction exploiting a custom bioreactor applying uniaxial stretching. In vitro analyses by immunofluorescence and mechanical testing revealed that the proposed combined approach favours the organization of a three‐dimensional tissue‐like structure promoting a remarkable arrangement of the cells and the neo‐extracellular matrix, reflecting into enhanced mechanical strength. The proposed method represents a novel approach for tendon tissue engineering, demonstrating how the combined effect of biochemical and mechanical stimuli ameliorates biological and mechanical properties of the artificial tissue compared to those obtained with single inducement.

High-density ZnO Nanowires as a Reversible Myogenic-Differentiation-Switch

Mesoangioblasts are outstanding candidates for stem-cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells because growth is typically accompanied by differentiation. Here reversible myogenic-differentiation switching during proliferation is achieved by functionalizing the glass substrate with high-density ZnO nanowires (NWs). Specifically, mesoangioblasts grown on ZnO NWs present a spherical viable undifferentiated cell state without lamellopodia formation during the entire observation time (8 days). Consistently, the myosin heavy chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, NWs do not induce any damage while they reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cell removal from the NW-functionalized substrate and replating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch that does not affect the viability. These results can be the first step toward for the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell-based therapy and tissue engineering.

Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction

The presence of tetraplex structures in the promoter region of the myogenic differentiation 1 gene (MyoD1) was investigated with a specific tetraplex-binding porphyrin (TMPyP4), to test its influence on the expression of MyoD1 itself and downstream-regulated genes during myogenic differentiation. TMPyP4-exposed C2C12 myoblasts, blocking MyoD1 transcription, proliferated reaching confluence and fused forming elongated structures, resembling myotubes, devoid of myosin heavy chain 3 (MHC) expression. Besides lack of MHC, upon MyoD1 inhibition, other myogenic gene expressions were also affected in treated cells, while untreated control cell culture showed normal myotube formation expressing MyoD1, Myog, MRF4, Myf5, and MHC. Unexpectedly, the myomaker (Mymk) gene expression was not affected upon TMPyP4 exposure during C2C12 myogenic differentiation. At the genomic level, the bioinformatic comparison of putative tetraplex sites found that three tetraplexes in MyoD1 and Myog are highly conserved in mammals, while Mymk and MHC did not show any conserved tetraplexes in the analysed regions. Thus, here, we report for the first time that the inhibition of the MyoD1 promoter function, stabilizing the tetraplex region, affects downstream myogenic genes by blocking their expression, while leaving the expression of Mymk unaltered. These results reveal the existence of two distinct pathways: one leading to cell fusion and one guaranteeing correct myotube differentiation.