Bioprinting Extracellular Matrix Scaffolds as a Potential Treatment for Volumetric Muscle Loss Público

Yu, Austin (Spring 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/kk91fm71r?locale=es
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Abstract

Volumetric Muscle Loss is characterized by a loss of at least 20% muscle mass and muscle functionality. Because current treatments do not replace muscle function, the need for tissue regeneration therapies persists. Decellularized extracellular matrix (dECM) derived from host tissue-type offers a potential solution to providing a tissue environment suitable for myoblast differentiation but lacks the proper stiffness and physical organization for functional muscle growth. Gelatin Methacrylate (GelMA) along with 3D-bioprinting has been used to create a stiffer, printable biomaterial that facilitates nutrient transport and cell alignment. We hypothesized that GelMA biomaterial would contain similar structural protein content and stiffness to native muscle tissue and serve as a biomaterial scaffold for myoblast proliferation. Our overall objective was to develop a new dECM-GelMA hydrogel biomaterial that contained key structural cures for C2C12 myoblast proliferation.  DNA quantification confirmed the removal of nucleic material following decellularization at <0.005 ng/mg, and mechanical testing found the stiffness of dECM-GelMA at 5.4 kPa to be similar to muscle tissue at 4.9 kPa. Immunohistochemical staining for collagen I and laminin in dECM and dECM-GelMA detected collagen I in dECM-GelMA. Laminin was detected in dECM but not in the hydrogel, prompting the need for future protein quantification. Cell proliferation assays measuring cell density in dECM and dECM-GelMA coated wells demonstrated similar proliferation to a collagen I positive control after 24 hours. A proliferation assay in dECM-GelMA printed patches also displayed increased proliferation after 72 hours. These promising data demonstrate the potential efficacy of dECM-GelMA as a scaffold for muscle progenitor cells and could be used in future work as a treatment for VML injury.

Table of Contents

Introduction . . . . . . . . 1

Volumetric Muscle Loss . . . . . . . . 1     

Muscle Tissue Regeneration . . . . . . . . 1    

dECM as a Biomaterial Treatment Option for VML . . . . . . . . 2   

3D Bioprinting using dECM . . . . . . . . 3   

Gelatin Methacrylate as a Biocompatible Support . . . . . . . . 4   

Printing with dECM-GelMA . . . . . . . . 5   

Objectives . . . . . . . . 6

Methods . . . . . . . . 7  

Preparation of Biomaterials . . . . . . . . 7         

Characterization of dECM-GelMA . . . . . . . . 9   

Proliferation of C2C12 cells in Non-Printed dECM-GelMA . . . . . . . . 10   

Proliferation of C2C12 cells in dECM-GelMA Constructs . . . . . . . . 11 

Statistics . . . . . . . . 12

Results . . . . . . . . 13   

DNA Quantification . . . . . . . . 13   

Mechanical Testing . . . . . . . . 15   

dECM-GelMA Content Characterization . . . . . . . . 17   

Proliferation of C2C12 cells in dECM and dECM-GelMA . . . . . . . . 22   

Proliferation of C2C12 cells in dECM-GelMA Printed Constructs . . . . . . . . 26   

Discussion . . . . . . . . 27   

Conclusion and Future Directions . . . . . . . . 29   

References . . . . . . . . 30   

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