A DIC based technique to measure the contraction of a skeletal muscle engineered tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response.The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based systemallows for an accurate and noninvasive measurement of biological tissues’ spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

A DIC Based Technique to Measure the Contraction of a Skeletal Muscle Engineered Tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response. The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based system allows for an accurate and noninvasive measurement of biological tissues' spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

Additive Manufacturing and Physicomechanical Characteristics of PEGDA Hydrogels: Recent Advances and Perspective for Tissue Engineering

In this brief review, we discuss the recent advancements in using poly(ethylene glycol) diacrylate (PEGDA) hydrogels for tissue engineering applications. PEGDA hydrogels are highly attractive in biomedical and biotechnology fields due to their soft and hydrated properties that can replicate living tissues. These hydrogels can be manipulated using light, heat, and cross-linkers to achieve desirable functionalities. Unlike previous reviews that focused solely on material design and fabrication of bioactive hydrogels and their cell viability and interactions with the extracellular matrix (ECM), we compare the traditional bulk photo-crosslinking method with the latest three-dimensional (3D) printing of PEGDA hydrogels. We present detailed evidence combining the physical, chemical, bulk, and localized mechanical characteristics, including their composition, fabrication methods, experimental conditions, and reported mechanical properties of bulk and 3D printed PEGDA hydrogels. Furthermore, we highlight the current state of biomedical applications of 3D PEGDA hydrogels in tissue engineering and organ-on-chip devices over the last 20 years. Finally, we delve into the current obstacles and future possibilities in the field of engineering 3D layer-by-layer (LbL) PEGDA hydrogels for tissue engineering and organ-on-chip devices

Development of a Biaxial Stretch Bioreactor and Finite Element Models for Mechanobiological Study of Aortic Valve Leaflets

Aortic heart valve disease is a significant cause of mortality worldwide; and replacement surgery is necessary in 70% of cases. Tissue engineered heart valves (TEHVs) are biocompatible and biodegradable, with ability to grow with the patient. However, to date, TEHVs mostly lack ability to withstand native mechanical forces since they are unable to mimic the heterogeneous and anisotropic structure of extracellular matrix (ECM) in native valves. Cyclic stretch is known to modulate ECM fiber synthesis and alignment. However, little tools are available for studying the interaction between aortic tissues and stretch condition. Finite element method is a powerful tool to simulate the complex structure of aortic valve, however, most current simulations modeled the leaflet as a homogenous material, and none of them took the distinctions between two surface layers into account, which were critical for the proper function of the aortic valve.To study the effects of cyclic stretch on extracellular matrix remodeling, the heterogeneous properties of the aortic leaflet, and the effects of heterogeneity on the function of valve, we have 1) Designed, fabricated and validated a biaxial stretch bioreactor; 2) Analyzed train patterns of native aortic leaflets using digital image correlation method; 3) Designed and validated an anisotropic and heterogeneous finite element (FE) model for leaflets. These studies provided insights into the interaction between aortic valve tissue and the mechanical environment, anisotropy and heterogeneity of aortic leaflets ECM due to the distribution of collagen fibers, and detailed distinct strain patterns in fibrosa vs. ventricularis sides and 3 aortic leaflets. Our novel biaxial stretch bioreactor and refined FE model of aortic leaflet will pave path for other scientists to study mechanobiology, design and condition engineered tissues and simulate engineered aortic valve grafts or pathology of calcium deposition

3D microfabrication of biological machines

The burgeoning field of additive manufacturing, or “3D printing”, centers on the idea of creating three-dimensional objects from digital models. While conventional manufacturing approaches rely on modifying a base material via subtractive processes such as drilling or cutting, 3D printing creates three-dimensional objects through successive deposition of two- dimensional layers. By enabling rapid fabrication of complex objects, 3D printing is revolutionizing the fields of engineering design and manufacturing. This thesis details the development of a projection-based stereolithographic 3D printing apparatus capable of high- resolution patterning of living cells and cell signals dispersed in an absorbent hydrogel polymer matrix in vitro. This novel enabling technology can be used to create model cellular systems that lead to a quantitative understanding of the way cells sense, process, and respond to signals in their environment. The ability to pattern cells and instructive biomaterials into complex 3D patterns has many applications in the field of tissue engineering, or “reverse engineering” of cellular systems that replicate the structure and function of native tissue. While the goal of reverse engineering native tissue is promising for medical applications, this idea of building with biological components concurrently brings about a new discipline: “forward engineering” of biological machines and systems. In addition to rebuilding existing systems with cells, this technology enables the design and forward engineering of novel systems that harness the innate dynamic abilities of cells to self-organize, self-heal, and self-replicate in response to environmental cues. This thesis details the development of skeletal and cardiac muscle based bioactuators that can sense external electrical and optical signals and demonstrate controlled locomotive behavior in response to them. Such machines, which can sense, process, and respond to signals in a dynamic environment, have a myriad array of applications including toxin neutralization and high throughput drug testing in vitro and drug delivery and programmable tissue engineered implants in vivo. A synthesis of two fields, 3D printing and tissue engineering, has brought about a new discipline: using microfabrication technologies to forward engineer biological machines and systems capable of complex functional behavior. By introducing a new set of “building blocks” into the engineer’s toolbox, this new era of design and manufacturing promises to open up a field of research that will redefine our world

Experimental and theoretical analyses of compression induced muscle damage : aetiological factors in pressure ulcers

Pressure ulcers form a major problem in health care. They often occur when patients are bedridden, wheelchair bound or wearing prostheses. The ulcers can be very painful for the patient and often lead to prolonged hospitalization. In addition, the huge costs involved with treatment and prevention put a heavy burden on heath care budgets. Pressure ulcers occur often: between 14% and 33% of the patients in health care institutions develop an ulcer, ranging from discolouration of the skin to severe wounds involving necrosis of epidermis, extending to underlying bone, tendon and joints. It is clear that pressure ulcers are caused by prolonged mechanical loading, applied at the interface between skin and support surfaces. However, the aetiology of pressure ulcers is poorly understood. This forms an important obstacle in decreasing the unacceptably high prevalence figures. It is anticipated that a better understanding of the mechanobiological pathways leading to cell and tissue damage can lead to a breakthrough in reducing pressure ulcer prevalence. In addition, a solid scientific base may establish tools for objective risk assessment and judgement of preventive measures. The present study focuses on deep ulcers that initiate in skeletal muscle tissue, since deep ulcers are more extensive and often difficult to prevent. To obtain insight into the aetiology of these deep ulcers, it is necessary to understand the transfer from externally applied loads at the skin, to the local conditions that the cells experience within the tissue. In addition, the question which local conditions are harmful to the cell needs to be investigated. By combining knowledge on "what a cell feels" with knowledge on potentially harmful conditions, a better judgement of dangerous situations may be achieved. Although several causes of cell damage may play a role in the initiation of pressure ulcers, the present study focussed on the impact of cell deformations. To investigate the hypothesis that prolonged cell deformations lead to cell damage at clinically relevant strains, an experimental model system was developed. A key requirement of this experimental model is the possibility to study the role of cell deformation on cell damage independently of other possible causes of damage. To achieve this, in-vitro engineered muscle tissue constructs were developed. These constructs were compressed using a newly developed compression device. A custom made incubator system was developed to allow monitoring of the constructs for extended periods of time. In addition, a novel assay was developed to determine the viability of the cells during compression. This assay provides quantitative and spatial information on cell damage throughout a construct in a non-invasive manner, making use of fluorescent dyes which are visualized by confocal microscopy. The compression of the engineered muscle tissue constructs indicated that a significant increase in cell death occurs within 1-2 hours and that higher strain levels led to an earlier increase in damage. In addition, it was demonstrated that cell damage was uniformly distributed across the indented area of the construct, without a gradient in percentage dead cells between the centre and periphery of the constructs. The results strongly suggest that prolonged cell deformation was the predominant cause of cell damage in these experiments. This puts a new light on observations in literature which suggested that ischaemia is not the sole determinant for the onset of pressure ulcers. Nevertheless, more experiments are needed to clarify the role of prolonged cell deformations on cell damage. First, it is recommended that the actual local cell deformations are quantified during compression of the constructs. Furthermore, from the present experiments it could not be excluded that the compression of the constructs decreased the permeability of the construct and hence affected cellular metabolism. In future, measuring diffusion pathways of both small molecules and larger vital molecules, may indicate whether this change in permeability is significant. A numerical model was developed to predict local cell deformations, in response to tissue compression. Since the local cell deformations cannot be a-priori determined on the basis of homogenized tissue deformations, a multilevel finite element approach was adopted. In this approach, cell deformations are predicted from detailed nonlinear finite element analyses of the local microstructures of the tissue, which consist of an arrangement of cells embedded in a matrix material. To avoid unacceptably large computational times, the multilevel model was designed to run on a parallel computer system. Application of the multilevel model showed that the heterogeneity of the microstructure of the tissue has a profound impact on local cell deformations, which highly exceeded macroscopic tissue deformations. Moreover, microstructural heterogeneity led to complex cell shapes and caused non-uniform deformations within the cells. To investigate the evolution of compression induced damage in skeletal muscle tissue, the multilevel model was extended with a damage law, which was derived from the in-vitro experiments. With this model, the compression of muscle tissue against a bony prominence was simulated. The percentage of cell damage in the microstructure of the tissue was computed, which could be extrapolated to the bulk tissue level. In the present form, a schematic geometry was considered that intended to elucidate general patterns of tissue damage evolution. The simulations confirmed that it is not feasible to predict the onset of tissue damage on the basis of externally applied loading conditions at the skin surface alone, since these externally applied loads are not indicative of the local mechanical conditions that the cells experience within the tissue. In addition, the simulations showed that it is necessary to consider the local load history of the cells, and the tolerance of the tissue. These findings may explain why a strikingly large variability in load/time threshold values was found in animal studies, which attempted to relate external mechanical to tissue damage, thereby ignoring the local mechanical conditions within the tissue. At present, it is premature to utilize the models presented in this thesis in clinical practice, since the extrapolation towards human patients requires more research. Clearly, further extensions and validation of the numerical model with experimental animal models will be required. This should finally lead to the application in more realistic cases, involving patient data on geometry and tissue properties. Nevertheless, the present models provided an essential step towards evidence based risk assessment and prevention

Doctor of Philosophy

dissertationTreatment and management of heart disease is challenging due to the heart's limited ability to self-repair. Although current approaches to manage heart disease, such as pharmacotherapy, medical devices, lifestyle changes, and heart transplantation, have improved and extended the quality of life for millions of individuals, they have inherent shortcomings. Future strategies to manage heart disease will likely be based upon a combination of biological and engineering approaches through cell therapy and tissue engineering strategies, both of which have the potential to regenerate the myocardium and improve cardiac function. However, a key hurdle in applying biological approaches is our limited ability to produce reliable tissue to study disease progression and tissue development, therapeutic intervention, drug discovery, or tissue replacement. Establishing hallmarks of the native myocardium in engineered cardiac tissue is a central goal and appears to be required for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. The objective of this research was to apply an engineering approach to develop tools and methods to produce engineered cardiac tissue and characterize both native and engineered cardiac tissue. Three phases of research included: 1) the development and utilization of a framework to characterize microstructure in living cardiac tissue using confocal microscopy and local dye delivery, 2) the development a next-generation bioreactor capable of continuously monitoring force-displacement in engineered tissue, and 3) the application of confocal imaging and image analysis to quantitatively describe features of the native myocardium, focusing on myocyte geometry and spatial distribution of a major gap junction protein connexin-43, in both engineered tissue and native tissue

The role of mechanical forces in cardiomyocyte differentiation in 3D culture

Heart disease is the leading cause of death in many developing and industrialized countries. The loss of cardiomyocyte (CM) proliferation in the post-natal myocardium is the major barrier to myocardial regeneration, which leads to a loss of functional myocytes and thus contractile function after injury. While significant advances in cardiac tissue engineering as an alternative strategy for treatment have been made in the recent years, the application for repair of the injured myocardium remains to be realized. However, tissue engineering as an in vitro model system for characterizing functional properties of cardiac tissue can be used as a powerful tool now. The overall goal of this doctoral thesis was to determine the role of mechanical strain on CM differentiation within a 3D engineered tissue to use as a system for evaluation of strategies for enhancing directed CM differentiation and tissue contractile properties. Substantial progress towards this goal was made by a combination of testing new strategies for monitoring differential CM differentiation and contractile function, such as using MDSCs in a 3D collagen gel bioreactor to induce CM differentiation and applying mechanical strain to determine the responsive cell type, and by developing new tools and methods for characterizing CM differentiation and cell morphology changes. Our in vitro engineered cardiac tissue from fetal/developing native cardiac cells maintained CM proliferative activity and contractile properties similar to the native myocardium which increased in response to mechanical stretch. The implanted graft maintained CM proliferative activity in vivo, survived as a donor myocardial tissue, and contributed to the cardiac functional recovery of injured myocardium better than a graft with post-natal cardiac cells. Skeletal muscle derived stem cell (MDSC) aggregate formation and 3D collagen gel bioreactor (3DGB) culture (MDSC-3DGB) triggered differentiation of cells with an immature functioning CM phenotype in vitro. In addition, mechanical strain directed cell morphology changes were significant factors in directing CM differentiation from MDSCs within MDSC-3DGB. In conclusion, our 3D collagen gel bioreactor culture, with capabilities for spatial and temporal monitoring, represents a powerful model for elucidating the role of specific environmental factors and their underlying mechanisms on directed cell proliferation and differentiation

Book of Abstracts 15th International Symposium on Computer Methods in Biomechanics and Biomedical Engineering and 3rd Conference on Imaging and Visualization

In this edition, the two events will run together as a single conference, highlighting the strong connection with the Taylor & Francis journals: Computer Methods in Biomechanics and Biomedical Engineering (John Middleton and Christopher Jacobs, Eds.) and Computer Methods in Biomechanics and Biomedical Engineering: Imaging and Visualization (JoãoManuel R.S. Tavares, Ed.). The conference has become a major international meeting on computational biomechanics, imaging andvisualization. In this edition, the main program includes 212 presentations. In addition, sixteen renowned researchers will give plenary keynotes, addressing current challenges in computational biomechanics and biomedical imaging. In Lisbon, for the first time, a session dedicated to award the winner of the Best Paper in CMBBE Journal will take place. We believe that CMBBE2018 will have a strong impact on the development of computational biomechanics and biomedical imaging and visualization, identifying emerging areas of research and promoting the collaboration and networking between participants. This impact is evidenced through the well-known research groups, commercial companies and scientific organizations, who continue to support and sponsor the CMBBE meeting series. In fact, the conference is enriched with five workshops on specific scientific topics and commercial software.info:eu-repo/semantics/draf