Digital Theses Archive


Tesi etd-05262021-110609

Type of thesis
Pushing the boundaries of skeletal muscle tissue engineering with multiple biophysical stimulations
Scientific disciplinary sector
Istituto di Biorobotica - BIOROBOTICS
relatore Prof. RICOTTI, LEONARDO
  • biohybrid actuation
  • biophysical stimulation
  • skeletal muscle
  • tissue engineering
Exam session start date
Skeletal muscle tissue engineering is constantly searching for new strategies to direct cell differentiation and mimic natural tissue function and structure. In vitro culture conditions do not accurately reproduce the in vivo microenvironment, characterized by specific biochemical stimuli deriving also from various cellular phenotypes present, from an extracellular matrix stiffness which may vary in time and space, mechanical stimuli deriving from the body movements and tensions. It is therefore crucial to study tissue development conditions and microenvironment, but also the stimuli to which will be subjected in the case of in vivo implantation in healthy or unhealthy conditions. For the creation of large-scale constructs for extensive injury repair or for alternative applications such as biohybrid actuation, a greater focus must be placed on finding new scaffold architectures and technologies enabling the scaling up in dimensions (reaching constructs bigger than 2-3 cm3). The use of compact scaffolds with no vascularization strongly impairs cell survival. Possible solutions can be relying on the use of 3D bioprinting or scaffold with intrinsic or engineered porosity, technologies that allow the fabrication of modular soft scaffolds, capable of showing a promyogenic effect and being compliant to muscular cell contraction.<br>In addition to the scaffold, however, some steps still need to be filled with regards to the choice of the optimal cell source for muscle differentiation, and the achievement of an advanced level of differentiation, which is often related to the functionality of the muscle itself and its contractile capacity (exerted force of natural muscle ~ 1 kN [1] while skeletal muscle-based constructs can reach maximum 5-10 mM[2]). To the best of our knowledge, no studies so far have fabricated a construct that combines an architecture similar to that of muscle tissue, reaching an advanced stage of cellular maturation, and the ability to contract with the same strength as the natural tissue. Since the canonical biochemical protocols used until now do not fulfill these points, of considerable interest is the combination of multiple biophysical stimuli inside these 3D systems. Some studies tended to investigate one or two stimulations, nevertheless, the state of the art lacks thorough investigations on the integration and the interaction between multiple promyogenic stimuli, such as the localized electrical stimulation mediated by focused and controlled ultrasound stimulation of piezoelectric nanomaterials. These multiple approaches must be sought and pursued also to meet the complexity of the in vivo microenvironment.<br>This thesis aimed to bring advances in various areas of skeletal muscle tissue engineering, tackling different needs still unsolved in the field. It has been demonstrated how a porous soft scaffold can support myogenesis for the creation of large-scale muscular constructs, with electrical stimulation highlighted as a necessary stimulus to push the formation of elongated myotubes. Stimulation that was introduced, at a local level, within a printed 3D scaffold thanks to the presence of piezoelectric nanoparticle stimulated with highly controlled low-intensity pulsed ultrasound, and that accelerated the differentiation of the encapsulated myoblasts. <br>A thorough study of muscle tissue development and the canonical protocols used for the differentiation of iPSCs into skeletal muscle led to the writing of a review article, and provided valuable information for understanding how to boost the differentiation of this particular cell type. Little attention has been given to the integration of external stimulations in the differentiation of iPSCs, such as mechanical, electrical stimuli, biomaterial stiffness, that can strongly influence cell fate. This subject assumes more relevance for cell types as iPSCs, whose phenotype strongly relies on the features of the in vivo niche. With preliminary studies on the influence of substrate stiffness, I have demonstrated that there is no influence on the early stages of iPSC differentiation in skeletal muscle cells, but further studies are needed to gain insight into the complete differentiation process.<br>All these efforts can be gathered in the biohybrid actuation field, where several attempts have been made to create proof-of-concept bioactuators, combining artificial and natural elements to generate novel and effective devices [3]. The fabricated devices are able to swim, crawl, and walk inside a canonical culture dish. The field is now ready to focus all the energies to bring these actuators outside the laboratory environment, making them able to operate in air, envisioning their application in real-life scenarios. <br>Concerning the field of biohybrid actuation, a prototype for the application of a porous 3D actuator in real-life scenarios outside the laboratory environment is presented. Subsequently, integrating the results obtained from the present studies on the scaffolds for skeletal muscle tissue engineering, the biophysical promyogenic stimulations, and the studies on the iPSCs, a concept (patent deposited) that would lead to the integration of biohybrid implementation with conventional biomedical devices is described.<br>Some of the above-mentioned activities need to be completed from an experimental viewpoint, and should be evolved in the future to allow their clinical adoption (or industrial adoption, in the case of biohybrid actuators). The results reported in this thesis can be considered a series of interdisciplinary initial seeds toward the implementation of this vision, which may have a high impact on the society, in the long term.<br><br>Skeletal muscle tissue engineering aims at reconstructing the muscle tissue in vitro or directly in vivo, by mimicking the natural tissue structure, physiology, and functionality. The aim of this work is to explore different biomaterials and biophysical stimuli to boost skeletal muscle differentiation. <br>Overall, the thesis is divided into eight chapters. Chapter 2 reports a summary of key concepts on the embryonic development and architecture of the muscular tissue, precious pieces of information to translate into tissue engineering approaches (section 2.1). Afterward, the state of the art is presented, with a literature analysis on the procedures and protocols proposed for muscle tissue reconstruction (section 2.2). Indeed, skeletal muscle is an anisotropic contractile tissue with high metabolic demands. The in vitro reconstruction of these specialized features requires a balance between the choice of the appropriate skeletal muscle source, biomaterial features and fabrication strategy, and biophysical stimulation. In the last part of this chapter, the open challenges in the mentioned fields are highlighted.<br>From chapter 3 onward, the results of the candidate’s research activity are described. Chapter 3 aims at combining biomaterial stiffness, 3D spatial organization and biochemical cues with electrical stimulation, on immortalized myoblasts. To this purpose, a study grounded on a macroporous soft scaffold for the growth and differentiation of immortalized murine myoblasts is described. The porous architecture of the scaffold responds to the high metabolic need of the muscle tissue, and the low stiffness of the elastomeric material (similar to the natural muscle one) mimics the mechanic behavior of the extracellular matrix. The effect of the combination of electrical stimuli and scaffold stiffness is analyzed on the differentiation of myoblasts. Chapter 4 then describes the inclusion of the macroporous scaffold in a structure that isolates it from the outside environment and makes it ideally capable of operating in air, opening possible applications in the biohybrid actuation field. The system is mechanically characterized and the first in vitro results are reported.<br>Chapter 5 still focuses on 3D spatial organization, but it approaches a different fabrication technique, namely 3D bioprinting. Biophysical stimulation consists of an indirect electrical stimulation of myoblasts utilizing piezoelectric barium titanate nanoparticles embedded in the bioink and stimulated with controlled low-intensity pulsed ultrasound (LIPUS). Given the anisotropic and hierarchical structure of muscle tissue, 3D bioprinting fits well with its structural needs. After the validation of the LIPUS system, results of the LIPUS stimulation effect on differentiating myoblasts in a hydrogel solution doped with the piezoelectric nanoparticles are presented.<br>In chapter 6 induced pluripotent stem cells (iPSCs) are introduced and their use in skeletal muscle tissue engineering is discussed. Through an in-depth literature study, the canonical methods for the differentiation of iPSCs in skeletal muscle have been identified, divided into transgenic and non-transgenic, respectively described in sections 6.2.1 and 6.2.2. Experimental results on the effect of substrate stiffness on the myogenic differentiation of these cells are reported in section 6.3.1. <br>In chapter 7, a patented biomedical micro-device controlled by biohybrid actuators derived from patient-specific cells is described, relying on concepts and design considerations partly driven by the efforts described in the previous chapters. Finally, conclusions are drawn in chapter 8. <br><br>[1] M. Zupan, M. F. Ashby, and N. A. Fleck, “Actuator classification and selection - The development of a database,” Adv. Eng. Mater., vol. 4, no. 12, pp. 933–940, Dec. 2002.<br>[2] Y. Morimoto and S. Takeuchi, “Biohybrid Robot Powered by Muscle Tissues,” in Mechanically Responsive Materials for Soft Robotics, Wiley, 2020, pp. 395–416.<br>[3] L. Ricotti et al., “Biohybrid actuators for robotics: a review of devices actuated by living cells,” Sci. Robot., vol. 2, no. 12, Nov. 2017.<br><br><br>