Many congenital skeletal muscle disorders including muscular dystrophies are caused by genetic mutations that lead to a dysfunction in myocytes effectively binding to the extracellular matrix. This leads to a chronic and continuous cycle of breakdown and regeneration of muscle tissue, ultimately resulting in loss of muscle function and patient mortality. Such disorders lack effective clinical treatments and challenge researchers to develop new therapeutics. The current drug development process often yields ineffective therapeutics due to the lack of genetic homology between pre-clinical animal models and humans. In addition current engineered tissue models using human cells fail to properly emulate native muscle morphology and function due to necrotic tissue cores and an abundance undigested ECM protein. Thus, a more precise benchtop model of 3D engineered human muscle tissue could serve as a better platform for translation to a disease model and could better predict candidate drug efficacy during pre- clinical development. This work presents the methodology for generating a high-content system of contiguous skeletal muscle tissue constructs produced entirely from human cells by using a non-adhesive hydrogel micro-molding technique. Subsequent culture and mold modifications confirmed by morphological and contractile protein analysis improve tissue longevity and myocyte maturation. Finally, mechanical strength and contractile force measurements confirmed that such modulations resulted in skeletal muscle microtissues that were more mimetic of human muscle tissue. This cell self-assembly technique yielded tissues approximately 150um in diameter with cell densities approaching that of native muscle. Modifications including seeding pre-differentiated myoblasts and the addition of ECM producing fibroblasts improved both tissue formation efficiency and cell alignment. Further culture modifications including supplementation of the culture medium with 50ug/ml ascorbic acid and 100ng/ml Insulin-like growth factor-1 coupled with a mold redesign that allowed tissue to passively contract during maturation while still remaining anchored under tension further improved ECM production, myogenic differentiation, and long-term longevity in culture. Further confirmation of the culture improvements were demonstrated by increases in mechanical strength and contractile force production. In conclusion, this approach overcomes cell density limitations with exogenous ECM-based methods and provides a platform for producing 3D models of human skeletal muscle by making tissue entirely using cells. Future work will attempt to translate the methodology used for tissue generation and long-term culture to create benchtop models of disease models of skeletal muscle, streamlining pre- clinical benchtop testing to better predict candidate drug efficacy for skeletal muscle diseases and disorders along with elucidating side effects of non-target drugs.

Cardiovascular disease is the leading cause of non-communicable disease in the whole world killing 17 million people in 2012. Among the many vascular diseases is vascular calcification (VC) which is the mineral build up in the walls of blood vessels. Medial calcification is the plaque buildup in the medial layer of the blood vesicle that is characterized by arterial stiffness and high blood pressure. Current calcification research involves two dimensional (2D) lab methods such as flat petri dishes to investigate the mechanism that causes and inhibits vascular calcification. Research has shown that the use of three-dimensional (3D) models can be beneficial in mimicking the in vitro environment of the human body for lab practices. We aim to create a 3D benchtop model for vascular calcification research from decellularized carotid porcine arteries to understand the more accurate mechanisms that lead to the plaque buildup in the medial layer of the artery wall.

Acute Lymphoblastic Leukemia (ALL) is one of the most common type of hematologic malignancy in children, characterized by an excessive proliferation of unfunctional immature lymphoblasts in the blood and the bone marrow, which leads to a range of severe blood-related complications. Given the remarkable increase in the prevalence of leukemia in the past 20 years, there has been a particular interest in the development of in vitro experimental models for cancer research. Ultra-short self-assembling peptides have shown to be a promising class of synthetic biomaterials due to their biocompatibility, tunable mechanical properties, and the possibility of controlling the scaffold composition. The objective of this study was to create a bioactive but well-defined synthetic 3D model of the bone marrow (BM) microenvironment for the simulation of ALL using biofunctionalized ultrashort self-assembling peptide scaffolds. Different bioactive motifs derived from integral extracellular matrix (ECM) constituents that are known to enhance cell-matrix adhesion, including RGDS from fibronectin, YIGSR from laminin, and GFOGER from collagen, were incorporated into the parent peptide IIZK. These peptides demonstrated to be capable of generating stable hydrogel structures composed of fibrous porous networks, each with unique nanofiber morphology and mechanical properties. All the peptide scaffolds that were investigated in this study exhibited optimal characteristics concerning the cytocompatibility of multiple BM niche cells, including human bone marrow mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), and patient derived ALL cells. The suitability of the scaffolds as drug screening platforms was evaluated, demonstrating their potential as versatile tools for the assessment of drug efficacy.

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