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Functionalization of hyaluronic acid hydrogels with ECM-derived peptides to control myoblast behavior.

ABSTRACT: Volumetric muscle loss (VML) occurs when skeletal muscle injury is too large for the body to fully self-repair. Typically, fibrotic tissue fills the void, which reduces muscle functionality and limb movement. Although a wide variety of natural and synthetic scaffolds have been studied with the purpose of providing the appropriate structural support, to date no scaffold has significantly restored muscle functionality after VML. Satellite cells, adult stem cells within the muscle capable of restoring smaller injuries, are sensitive to the stiffness and composition of the surrounding environment. Scaffolds that only address structural support are not sufficient to restore functionality and instead need to be designed to both promote satellite cell activation and prevent excessive fibroblast recruitment. The objective of this study was to design a scaffold that mimicked the regenerative environment and determine how the biomechanical properties differentially influence myogenic precursor and connective tissue cells. One of the main extracellular matrix (ECM) molecules upregulated during regeneration is hyaluronic acid (HA). Therefore, thiol-modified HA and poly(ethylene glycol) diacrylate hydrogels were generated and functionalized with peptides based on ECM known to influence regeneration, including fibronectin, laminin and tenascin-C. Scaffolds with different stiffness were created by varying HA content. The influence of HA stiffness and peptide functionalization on myogenic precursor and connective tissue cell proliferation, migration and gene expression was quantified. Our results indicated that HA hydrogels functionalized with the laminin peptide, IKVAV, show potential due to the enhanced promotion of myogenic cell behaviors including migration, proliferation and an increase in relevant transcription factors. STATEMENT OF SIGNIFICANCE: The goal of this study was to identify hyaluronic acid (HA) hydrogels with peptide and stiffness combinations that will direct muscle-derived cells towards regenerating phenotypes. While the interaction of skeletal muscle with RGD-functionalized HA hydrogels has been investigated, none of the other peptides described in this study had been used in the context of HA-based scaffolds and skeletal muscle-derived cells. Notably, the response of cells to variations in mechanics was dependent on ECM coating and lineage. The 3% HA functionalized with the laminin peptide, IKVAV, showed the most promise for future in vivo studies, as these hydrogels best promoted myoblast cell proliferation, attachment and spreading, enhanced migration over connective tissue cells and upregulated transcription factors associated with activated satellite cells.

SUBMITTER: Silva Garcia JM

PROVIDER: S-EPMC6326884 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Functionalization of hyaluronic acid hydrogels with ECM-derived peptides to control myoblast behavior.

Silva Garcia Juan Martin JM Panitch Alyssa A Calve Sarah S

Acta biomaterialia 20181201

Volumetric muscle loss (VML) occurs when skeletal muscle injury is too large for the body to fully self-repair. Typically, fibrotic tissue fills the void, which reduces muscle functionality and limb movement. Although a wide variety of natural and synthetic scaffolds have been studied with the purpose of providing the appropriate structural support, to date no scaffold has significantly restored muscle functionality after VML. Satellite cells, adult stem cells within the muscle capable of restor ...[more]

PMID: 30508655

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Project description:Hyaluronic acid (HA) is an extracellular matrix (ECM) component that has been shown to play a significant role in regulating muscle cell behavior during repair and regeneration. For instance, ECM remodeling after muscle injury involves an upregulation in HA expression that is coupled with skeletal muscle precursor cell recruitment. However, little is known about the role of HA during skeletal muscle development. To gain insight into the way in which HA mediates embryonic myogenesis, we first determined the spatial distribution and gene expression of CD44, RHAMM and other HA related proteins in embryonic day (E)10.5 to E12.5 murine forelimbs. While HA and CD44 expression remained high, RHAMM decreased at both the protein (via immunohistochemistry) and RNA (via qPCR) levels. Next, we determined that 4-methylumbelliferone-mediated knockdown of HA synthesis inhibited the migration and proliferation of E11.5/E12.5 forelimb-derived cells. Then, the influence of CD44 and RHAMM on myoblast and connective tissue cell behavior was investigated using antibodies against these receptors. Anti-RHAMM, but not anti-CD44, significantly decreased the total distance myogenic progenitors migrated over 24?h, whereas both inhibited connective tissue cell migration. In contrast, anti-CD44 inhibited the proliferation of connective tissue cells and muscle progenitors, but anti-RHAMM had no effect. However, when myoblasts and connective tissue cells were depleted of CD44 and RHAMM by shRNA, motility and proliferation were significantly inhibited in both cells indicating that blocking cell surface-localized CD44 and RHAMM does not have as pronounced effect as global shRNA-mediated depletion of these receptors. These results show, for the first time, the distribution and activity of RHAMM in the context of skeletal muscle. Furthermore, our data indicate that HA, through interactions with CD44 and RHAMM, promotes myogenic progenitor migration and proliferation. Confirmation of the role of HA and its receptors in directing myogenesis will be useful for the design of regenerative therapies that aim to promote the restoration of damaged or diseased muscle.

Project description:The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. STATEMENT OF SIGNIFICANCE: Factors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland's ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.

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Functionalization of hyaluronic acid hydrogels with ECM-derived peptides to control myoblast behavior.

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Functionalization of hyaluronic acid hydrogels with ECM-derived peptides to control myoblast behavior.

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