Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Accurate real-time feedback of surface EMG during fMRI.

Real-time acquisition of EMG during functional MRI (fMRI) provides a novel method of controlling motor experiments in the scanner using feedback of EMG. Because of the redundancy in the human muscle system, this is not possible from recordings of joint torque and kinematics alone, because these provide no information about individual muscle activation. This is particularly critical during brain imaging because brain activations are not only related to joint torques and kinematics but are also related to individual muscle activation. However, EMG collected during imaging is corrupted by large artifacts induced by the varying magnetic fields and radio frequency (RF) pulses in the scanner. Methods proposed in literature for artifact removal are complex, computationally expensive, and difficult to implement for real-time noise removal. We describe an acquisition system and algorithm that enables real-time acquisition for the first time. The algorithm removes particular frequencies from the EMG spectrum in which the noise is concentrated. Although this decreases the power content of the EMG, this method provides excellent estimates of EMG with good resolution. Comparisons show that the cleaned EMG obtained with the algorithm is, like actual EMG, very well correlated with joint torque and can thus be used for real-time visual feedback during functional studies.

Antennas for over-body-surface communication at 2.45 GHz

In this paper, the on-body performance of a range of wearable antennas was investigated by measuring vertical bar S-21 vertical bar path gain between two devices mounted on tissue-equivalent numerical and experimental phantoms, representative of human muscle tissue at 2.45 GHz. In particular, the study focused on the performance of a compact higher mode microstrip patch antenna (HMMPA) with a profile as low as lambda/20. The 5- and 10-mm-high HMMPA prototypes had an impedance bandwidth of 6.7% and 8.6%, respectively, sufficient for the operating requirements of the 2.45-GHz industrial, scientific, and medical (ISM) band and both antennas offered 11-dB higher path gain compared to a fundamental-mode microstrip patch antenna. It was also dernonstrated that a 7-dB improvement in path gain can be obtained for a fundamental-mode patch through the addition of a shortening wall. Notably, on-body HMMPA performance was comparable to a quarter wave monopole antenna on the same size of ground-plane, mounted normal to the tissue surface, indicating that the low-profile and physically more robust antenna is a promising solution for bodyworn antenna applications.

S-(2-Succinyl)cysteine:a novel chemical modification of tissue proteins by a Krebs cycle intermediate

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

Prevalencia de obesidad y factores de riesgo cardiovascular en estudiantes de ciencias de la salud - universidad del Quindío - 2013

ANTECEDENTES: En Colombia, reportes del año 2010 de la Encuesta Nacional de la Situación en Nutrición ENSIN 2010(2), muestran uno de cada dos colombianos, presentan un índice de masa corporal mayor al esperado (3) METODO: El presente estudio de corte transversal, determino la prevalencia de obesidad y otros factores de riesgo cardiovascular en una población de estudiantes de Ciencias de la Salud de una Universidad regional en el primer periodo académico del año 2013. El tamaño de muestra fue n=113 sujetos que corresponden 60,5% a la carrera de medicina y 39,95% a enfermería. Con el fin de conocer su comportamiento con respecto a hábitos y estilos de vida específicos como el consumo de alcohol, el consumo de tabaco y el sedentarismo, así como su asociación a eventos inflamatorios relacionados con la fisiopatología de los procesos de salud asociados al peso, por medio de instrumentos de medición clínica, antropométrica y sérica, determino un modelo estadístico propicio para entender el comportamiento de la obesidad y la enfermedad Cardiovascular RESULTADOS: La prevalencia estimada de sobrepeso y obesidad por Índice de Masa Corporal (IMC), fue del 27,7% (IC 95%: 19.9%,37.2%); por el perímetro abdominal (OBPABD) se encontró una prevalencia estimada del 27,4% (IC 95%: 19,9% – 36,4%), y la prevalencia con el Índice Cintura Cadera (OBICC) fue de 3,5% (IC 95%:1,3% – 9,3%). CONCLUSIONES: La presencia de hábitos no saludables y la presencia de sobrepeso y obesidad se considera que es necesario en primera instancia una valoración general de estado nutricional de los universitarios de las diferentes facultados y plantear estrategias preventivas ya que la literatura documenta los efectos de los hábitos no saludables sino además documenta los efectos de la prevención de la misma ya que en si se ha encontrado asociación para enfermedades cardiovasculares. Se propone que para obtener mayor información del comportamiento de los factores de riesgo cardiovasculares se deberían realizar estudios retrospectivos en el que intervengan las demás carreras de la universidad y poder evaluar la totalidad de población universitaria

Implementation of a VLSI amplifier interfaced with biomedical sensors for ultra wide band data transmission

This thesis presents a CMOS Amplifier with High Common Mode rejection designed in UMC 130nm technology. The goal is to achieve a high amplification factor for a wide range of biological signals (with frequencies in the range of 10Hz-1KHz) and to reject the common-mode noise signal. It is here presented a Data Acquisition System, composed of a Delta-Sigma-like Modulator and an antenna, that is the core of a portable low-complexity radio system; the amplifier is designed in order to interface the data acquisition system with a sensor that acquires the electrical signal. The Modulator asynchronously acquires and samples human muscle activity, by sending a Quasi-Digital pattern that encodes the acquired signal. There is only a minor loss of information translating the muscle activity using this pattern, compared to an encoding technique which uses astandard digital signal via Impulse-Radio Ultra-Wide Band (IR-UWB). The biological signals, needed for Electromyographic analysis, have an amplitude of 10-100μV and need to be highly amplified and separated from the overwhelming 50mV common mode noise signal. Various tests of the firmness of the concept are presented, as well the proof that the design works even with different sensors, such as Radiation measurement for Dosimetry studies.

Cell Types as Natural Kinds

Talk of different types of cells is commonplace in the biological sciences. We know a great deal, for example, about human muscle cells by studying the same type of cells in mice. Information about cell type is apparently largely projectible across species boundaries. But what defines cell type? Do cells come pre-packaged into different natural kinds? Philosophical attention to these questions has been extremely limited [see e.g., Wilson (Species: New Interdisciplinary Essays, pp 187-207, 1999; Genes and the Agents of Life, 2005; Wilson et al. Philos Top 35(1/2): 189-215, 2007)]. On the face of it, the problems we face in individuating cellular kinds resemble those biologists and philosophers of biology encountered in thinking about species: there are apparently many different (and interconnected) bases on which we might legitimately classify cells. We could, for example, focus on their developmental history (a sort of analogue to a species' evolutionary history); or we might divide on the basis of certain structural features, functional role, location within larger systems, and so on. In this paper, I sketch an approach to cellular kinds inspired by Boyd's Homeostatic Property Cluster Theory, applying some lessons from this application back to general questions about the nature of natural kinds.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Variable Recruitment in Bundles of Miniature Pneumatic Artificial Muscles and Space Qualified Pneumatic Artificial Muscles

The natural compliance and force generation properties of pneumatic artificial muscles (PAMs) allow them to operate like human muscles in anthropomorphic robotic manipulators. Traditionally, manipulators use a single PAM or multiple PAMs actuated in unison in place of a human muscle. However, these manipulators experience efficiency losses when operated outside their target performance ranges. The unidirectional actuation behavior of a miniature PAM bundle and bidirectional actuation behavior of an antagonistic pair of miniature PAM bundles are characterized and modeled. The results are used to motivate the application of a variable recruitment control strategy to a parallel bundle of miniature PAMs as an attempt to mimic the selective recruitment of motor units in a human muscle to improve the operating efficiency of the actuator. Additionally, the fabrication and quasi-static testing results for PAMs assembled from candidate space qualified bladder and braided sleeve materials for use in space robotics are assessed.

Augmentation de l’expression de la chaine α1 de la laminine 111, un potentiel traitement pour la Dystrophie musculaire de Duchenne

La protéine hétérotrimérique laminine-111 permet le lien entre la matrice-extracellulaire et l’intégrine α7β1 du sarcolemme, remplaçant ainsi dans les muscles dystrophiques, des liens normalement assurés par le complexe de la dystrophine. L’injection de laminine-111 dans des souris mdx a permis, entre autre, l’augmentation de l’expression de l’intégrine α7β1, d’empêcher les bris du sarcolemme lors de la contraction musculaire, de restaurer un niveau normal de la créatine kinase sérique, ainsi que d’augmenter la résistance et la force dans les muscles déficients en dystrophine. Ces résultats suggèrent que l’augmentation de la laminine-111 est un potentiel traitement pour la DMD. Les chaines β1 et γ1 de la laminine sont déjà exprimées dans le muscle humain adulte, mais la chaine α1 de la laminine (Lamα1) est exprimée uniquement pendant le stade très précoce 16 cellules de l’embryogenèse. Nous avons donc développé une méthode alternative à l’injection répétée de Laminine-111 en induisant l’expression endogène du gène LAMA1, afin de reformer le complexe trimérique α1β1γ1, la laminine 111. Ceci a été réalisé avec une technologie récente, le système CRISPR/Cas9, dont la Cas9 a été désactivée (dCas9) puis couplée à un domaine d’activation de la transcription, le VP160 (dCas9-VP160). L’utilisation d’un ou plusieurs ARN guides (ARNg) a permis de cibler le promoteur du gène LAMA1. L’ARNm de Lamα1 (qRT-PCR) ainsi que la protéine (immunohistochimie et immunobuvardage) n’ont pas été détecté dans le contrôle négatif, des myoblastes murins (C2C12). Cependant, une expression significative a été observée dans ces myoblastes transfectés avec des plasmides codant pour dCas9-VP160 et un ARNg. L’analyse protéique in vivo, dans des muscles de souris électroporés avec le même plasmide, a démontré une forte augmentation de la chaine α1 de la laminine. Des augmentations plus importantes de l’ARNm de Lamα1 ont été observées en utilisant 2 ARNg, suggérant un effet synergique. L’augmentation de l’expression de Lamα1 par le système de CRISPR/Cas9 devrait être étudiée d’avantage afin de vérifier si cette stratégie pourrait s’avérer efficace dans des cas de myopathies.

Normative reference of standing long jump for Colombian schoolchildren aged 9-17.9 years: The FUPRECOL study

El propósito del presente estudio era generar los valores normativos de salto largo para niños de 9-17.9 años, e investigar las diferencias de sexo y grupo de edad