Development of a Multifunctional Hydrogel Platform for Investigating and Enhancing Muscle Regeneration

Introduction:: Volumetric muscle loss causes muscle to lose its intrinsic ability to regenerate, resulting in scarring. The fibrotic tissue that forms can lead to functional impairment, greatly impacting a person’s mobility and quality of life. Current treatment relies on physical therapy and autologous tissue grafts that result in limited functional recovery. Tissue engineered constructs, such as decellularized scaffolds, biomaterials for cell delivery, and in vitro tissue mimetic scaffolds, are a promising avenue to repairing skeletal muscle tissue; however, mimicking the complex architecture of muscle to provide appropriate biophysical and biochemical cues remains a challenge. Our overall goal is to enhance muscle tissue repair by generating a library of multifunctional hydrogel-containing scaffolds that can be customized to match the tissue environment and serve as a platform for therapeutic protein delivery. We are currently investigating a dynamic covalent hydrazone-crosslinked hyaluronic acid (HA) hydrogel platform. We optimized the physicochemical properties of our HA hydrogel to closely match the muscle tissue environment using design of experiments (DOE), which is a statistical modeling tool that increases the amount of information obtained while reducing the number of experiments required. We determined the molecular weight of HA and polymer concentrations that yielded hydrogels with a desired gelation time, compression modulus, and mass change over time. We then combined our HA hydrogel with poly-ε-caprolactone (PCL) melt-electrowritten (MEW) 3D-printed scaffolds to develop a composite scaffold that could increase the attachment and anisotropic alignment of myoblasts and promote myotube formation while simultaneously serving as a platform for therapeutic protein delivery.

Materials and Methods:: HA (40kDa or 100kDa) was functionalized to contain aldehyde (HA-OX) or adipic acid dihydrazide (HA-ADH) functional groups to produce a dynamic covalent hydrazone crosslinked hydrogel. MODDE^® Pro software was used to perform D-optimal DOE to determine the concentration and molecular weight of HA that results in a hydrogel that resembles muscle tissue. We mixed equal volumes of HA-ADH and HA-OX at various polymer concentrations and determined the gelation timing, compression modulus, and mass change. Compression modulus was evaluated by rheology, mass change was evaluated by weighing hydrogels incubated in saline at various time points, and gelation time was evaluated by an inverted tube test. MEW PCL scaffolds were printed onto a tubular or flat print bed with a fiber diameter 10µm or 27µm and fiber spacing of 500µm in the x-direction and 250µm in the y-direction. Scaffolds were etched with 0.1M NaOH for 30min, sterilized with ethanol, and incubated in high serum media (10% FBS) overnight. We dip-coated the scaffolds into a 100kDa 2wt% HA-ADH solution followed by a 100kDa 2wt% HA-OX solution and allowed crosslinking for 30min. We seeded C2C12 murine myoblasts on top of the scaffold and allowed attachment and proliferation for 24h before exchanging the media to low serum media (1% FBS). Cells were allowed to proliferate for an additional 48h before they were fixed with 3.75% formaldehyde in saline and stained with Phalloidin 647 for F-actin and DAPI for cell nuclei. Samples were imaged using spinning disk confocal microscopy, and images were processed using IMARIS software.

Results, Conclusions, and Discussions:: We successfully modified HA with aldehyde and adipic acid dihydrazide functional groups to obtain a dynamic covalent hydrazone-crosslinked hydrogel (Fig 1A). To optimize our HA hydrogel, we varied the molecular weight of the HA and concentrations of HA-OX and HA-ADH solutions, formed hydrogels, and measured the gelation time, compression modulus, and mass change, which was used to train a D-optimal DOE statistical model. We achieved a large range of physicochemical properties by tuning the polymer concentrations and molecular weight of HA (Fig 1B). We obtained surface response curves that allowed us to predict the optimal weight percent of HA-ADH and HA-OX and molecular weight of HA that produced a hydrogel with a maximal compression modulus, while simultaneously demonstrating a minimal gelation timing (1-2 min) and mass change (±10%). We analyzed confocal images for our cell-seeded PCL-HA composite scaffold to determine the number of nuclei to evaluate cellular attachment, the nuclei directionally to show cellular alignment, and the nuclei aspect ratio, which corresponds to the nuclei elongation. Spherical nuclei (aspect ratio of 1) indicate undifferentiated cells, while elongated nuclei (aspect ratio of 0) indicate the early phase of myogenic differentiation. Our PCL-HA composite scaffold increased the attachment and alignment of C2C12 myoblasts, and nuclei were uniformly in the same direction and elongated (Fig 1C). We will further optimize our PCL-HA composite scaffold to increase anisotropic alignment by varying the MEW PCL fiber diameters and spacing. We will also perform myoblast differentiation assays to confirm that our scaffold supports and enhances myotube formation. In the future, we will incorporate sustained delivery of therapeutic proteins into our scaffold to enhance muscle regeneration. In conclusion, we have developed a HA hydrogel and composite PCL-HA scaffold that resembles the muscle tissue environment by mimicking the anisotropic architecture of muscle tissue and has the potential to promote muscle repair.


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