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 on 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) 2000×2000 m, ii) 1000×1000 m and iii) 500×500 m. The obtained results showed that by reducing the cross-section—i.e., by increasing the level of confinement—myotubes were more likely restrained and formed aligned myostructures that better mimicked the native morphology of skeletal muscle

Evidence for a quadruplex structure in the polymorphic hs1.2 enhancer of the immunoglobulin heavy chain 3’ regulatory regions and its conservation in mammals

Regulatory regions in the genome can act through a variety of mechanisms that range from the occurrence of histone modifications to the presence of protein-binding loci for self-annealing sequences. The final result is often the induction of a conformational change of the DNA double helix, which alters the accessibility of a region to transcription factors and consequently gene expression. A similar to 300 kb regulatory region on chromosome 14 at the 3' end (3'RR) of immunoglobulin (Ig) heavy-chain genes shows very peculiar features, conserved in mammals, including enhancers and transcription factor binding sites. In primates, the 3'RR is present in two copies, both having a central enhancer named hs1.2. We previously demonstrated the association between different hs1.2 alleles and Ig plasma levels in immunopathology. Here, we present the analysis of a putative G-quadruplex structure (tetraplex) consensus site embedded in a variable number tandem repeat (one to four copies) of hs1.2 that is a distinctive element among the enhancer alleles, and an investigation of its three-dimensional structure using bioinformatics and spectroscopic approaches. We suggest that both the role of the enhancer and the alternative effect of the hs1.2 alleles may be achieved through their peculiar three-dimensional-conformational rearrangement

High-density ZnO nanowires for cellular biointerfaces: a new role as myogenic differentiation switch

The design of artificial platforms for expanding undifferentiated stem cells is of tremendous importance for regenerative medicine [1]. We have recently demonstrated that a ZnO nanowires (NWs) decorated glass support permits to obtain a differentiation switch during proliferation for mesoangioblasts (MABs)– i.e. multipotent progenitor cells which are remarkable candidates for the therapy of muscle diseases [2]. We have optimized the ZnO NWs synthesis on glass surfaces by numerical simulations and experimental systematic investigations, considering zinc speciation and supersaturation [3]. In particular, we demonstrated by numerical simulations that the ligand ethylenediamine, at the isoelectric point of the ZnO NWs tips, can effectively control – at 1:1 stoichiometric ratio with zinc – both speciation and supersaturation of zinc in the nutrient solution. In this regard, we employed ethanolamine (a safer precursor) for in-situ producing ethylenediamine by means of a zinc-catalysed amination reaction of ethanolamine by ammonia. The obtained highquality ZnONWs-cells biointerface allows cells to maintain viability and a spherical viable undifferentiated state during the 8 days observation time. Simulations of the interface by theoretical models [4] and our experimental investigations by SEM and confocal microscopy demonstrate that NWs do not induce any damage on the cellular membrane, whilst blocking their differentiation. More specifically, the myosin heavy chain, typically expressed in differentiated myogenic progenitors, is completely absent. Interestingly, the differentiation capabilities are completely restored upon cell removal from the NW-functionalized substrate and regrowing onto a standard culture glass dish. These results open the way towards unprecedented applications of ZnO NWs for cell-based therapy and tissue engineering [5]. References [1] G. Cossu, P. Bianco, Curr. Opin. Genet. Dev. 2003, 13, 537-542. [2] V. Errico, G. Arrabito, E. Fornetti, C. Fuoco, S. Testa, G. Saggio, S. Rufini, S. M. Cannata, A. Desideri, C. Falconi, C. Gargioli, ACS Appl. Mater. Interfaces, 2018, 10, 14097- 14107. [3] G. Arrabito, V. Errico, Z. Zhang, W. Han, C. Falconi, Nano Energy, 2018, 46, 54-62. [4] N. Buch-Månson, S. Bonde, J. Bolinsson, T. Berthing, J. Nygård, K.L. Martinez, Adv. Funct. Mater. 2015, 25, 3246-3255. [5] Y. Su, I. Cockerill, Y. Wang, Y.-X. Qin, L. Chang, Y. Zheng, and D. Zhu, Trends in Biotechnology, 2019, 37, 428-441

Muscle tissue engineering

Skeletal muscle tissue engineering is relatively new field exploiting knowledge of histology, cell biology, medicine, chemistry and engineering to regenerate, reconstruct and replace damaged or lost muscles. The detailed picture of the muscle histophysiology, the characteristic organization of the muscular tissue and the influence of specific location (cell niche) on muscle stem cell behaviour explain the rationale of limited results obtained so far to mimic this complex architecture organization of the skeletal muscle tissue. Nevertheless the novel approaches through new muscle progenitor/stem cell consciousness together with modern three-dimensional cell culture systems relying on innovative material scaffold guaranteeing versatility, cell adhesion, cell survival and muscle differentiation, offer a completely new scenario for the future application of these biotechnology for the treatment of muscle degenerating affected individual

Intramuscular Transplantation of Muscle Precursor Cells over-expressing MMP-9 improves Transplantation Success

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin in muscles. A therapeutic approach to restore dystrophin expression in DMD patient’s muscles is the transplantation of muscle precursor cells (MPCs). However, this transplantation is limited by the low MPC capacity to migrate beyond the injection trajectory. Matrix metalloproteases (MMPs) are key regulatory molecules in the remodeling of extracellular matrix (ECM) components. MPCs over-expressing MMP-9 were tested by zymography, migration and invasion assays in vitro and by transplantation in mouse muscle. In vitro, MPCs over-expressing MMP-9 have a better invasion capacity than control MPCs. When these cells are transplanted in mouse muscles, the transplantation success is increased by more than 50% and their dispersion is higher than normal cells. MMP-9 over-expression could thus be an approach to improve cell transplantation in DMD patients by increasing the dispersion capacity of transplanted cells

Tissue engineering for skeletal muscle regeneration

Stem cells and regenerative medicine have obtained a remarkable consent from the scientific community for their promising ability to recover aged, injured and diseased tissue. However, despite the noteworthy potential, hurdles currently hinder their use and clinical application: cell survival, immune response, tissue engraftment and efficient differentiation. Hence a new interdisciplinary scientific approach, such as tissue engineering, is going deep attempts to mimic neo-tissue-genesis as well as stem cell engraftment amelioration. Skeletal muscle tissue engineering represents a great potentiality in medicine for muscle regeneration exploiting new generation injectable hydrogel as scaffold supporting progenitor/stem cells for muscle differentiation reconstructing the natural skeletal muscle tissue architecture influenced by matrix mechanical and physical property and by a dynamic environmen

Autologous progenitor cells in a hydrogel form a supernumerary and functional skeletal muscle in vivo

Stem cells and regenerative medicine have obtained a remarkable consent from the scientific community for their promising ability to recover aged, injured and diseased tissue. However, despite the noteworthy potential, hurdles currently hinder their use and clinical application: cell survival, immune response, tissue engraftment and efficient differentiation. Hence a new interdisciplinary scientific approach, such as tissue engineering, is going deep attempts to mimic neo-tissue-genesis as well as stem cell engraftment amelioration. Skeletal muscle tissue engineering represents a great potentiality in medicine for muscle regeneration exploiting new generation injectable hydrogel as scaffold supporting progenitor/stem cells for muscle differentiation reconstructing the natural skeletal muscle tissue architecture influenced by matrix mechanical and physical property and by a dynamic environment