Poster N20 - Hyaluronic Acid-Based Cryogel Scaffolds for Treatment of Volumetric Muscle Loss

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kavita Parekh, B.S.

Graduate student
Department of Bioengineering, University of California, Berkeley
Berkeley, California, United States

Co-Author(s)

Sydney Shriver, BS

Graduate student
Department of Biomedical Engineering, University of Virginia, United States
Kiersten Russ

Undergraduate Researcher
Department of Bioengineering, University of California, Berkeley
San Diego, California, United States
AW

Aboubacar Wague

Medical Student
Department of Orthopedic Surgery, University of California, San Francisco, United States
Sankalp Sharma, B.S.

Post Baccalaureate Researcher
Department of Orthopedic Surgery, University of California, San Francisco
Pleasanton, California, United States
Xuhui Liu, MD (he/him/his)

Professor
Department of Orthopedic Surgery, University of California San Francisco, United States
BF

Brian Feeley

Professor
Department of Orthopedic Surgery, University of California, San Francisco, United States
GC

George Christ

Professor of Biomedical Engineering and Orthopaedic Surgery
University of Virginia, United States

Primary Investigator(s)

KH

Kevin E. Healy

Professor
Departments of Bioengineering and Materials Science Engineering, University of California, Berkeley, California, United States

Introduction: Volumetric muscle loss (VML), defined as significant loss of muscle due to a traumatic injury or surgical event leading to persistent functional impairment, is a debilitating condition with limited treatment options that affects both military personnel and civilians. Although there have been efforts to develop biomaterials to treat VML, recent advances are limited by fibrosis and scarring. Hydrogels are robust materials for tissue regeneration, but they are challenging to implement in the VML model and can be difficult to use in certain clinical and austere environments. Cryogels are hydrogels formed below the freezing temperature of the solvent and subsequently lyophilized to produce dry scaffolds with an interconnected microporous structure. These materials provide an exciting, translatable alternative to traditional hydrogels as their pore structure allows immediate cell penetration and greater molecular diffusion within scaffolds. In this work, we describe novel, scalable, degradable hyaluronic acid (HyA)-based cryogel scaffolds to promote muscle regeneration with minimal fibrosis after VML in the masseter muscle (Fig. 1A). To understand cryogel structural and mechanical properties, we developed a novel quantitative three-dimensional confocal scanning laser microscopy (CSLM) technique to quantify the pore size of hydrated cryogel scaffolds, as well as mechanical testing to verify scaffolds’ physiologically relevant moduli. We seeded scaffolds of varying properties with human fibroadipogenic progenitor (hFAP) cells, which are first responder cells for muscle injury, and quantified their promyogenic and profibrotic differentiation. Lastly, we tested the most promising scaffold formulation in vivo in a rat masseter VML model to evaluate regenerated muscle and fibrosis.

Materials and

Methods: Cryogels were composed of modular macromers synthesized according to previously reported protocols (Fig. 1A). Briefly, 60 kDa or 100 kDa molecular weight (MW) HyA derivatives carrying acrylate groups (AcHyA) were synthesized. Then, AcHyA derivatives containing a 15 amino acid RGD (AcHyA-bsp-RGD(15)) were synthesized via Michael-type addition. To create cryogels, macromers, SH-heparin and a bis-cysteine terminated matrix metalloproteinase (MMP)-cleavable crosslinker were dissolved in triethanolamine buffer, combined to initiate gelation, and immediately frozen in stainless steel molds in a freeze dryer at varying temperatures (-5°C, -10°C, -15°C, -20°C, -30°C), then lyophilized. Three-dimensional, quantitative CSLM was employed to analyze the scaffold pore structure. Oscillatory rheology was used to measure the shear moduli (G’, G”), and swelling experiments determined volumetric (Q) and mass (Qm) swelling ratios.
Scaffolds were tested in vitro with hFAPs to assess the impact of scaffold properties on pro-myogenic differentiation. hFAPs were harvested from deltoid muscle biopsies from patients during rotator cuff repair. Dry scaffolds of varying MW and freezing temperature were immersed in 3 million cell/mL cell slurries. After 4 and 7 days, fibrotic (𝛼SMA) and pro-myogenic (UCP-1) differentiation as well as cell spreading were assessed using immunohistochemistry with volumetric segmentation of z-stacks. The most promising scaffold formulation was tested in a VML rat masseter model. Treated animals (N=4) were compared to injured no-treatment controls (N=4). Animals were kept on study for 1, 2, and 4 weeks, after which point animals were sacrificed, and the masseter muscles were explanted and frozen for histologic evaluation by H&E and Masson’s Trichrome staining.

Results, Conclusions, and Discussions: We fabricated cryogels at five distinct temperatures. Existing literature is limited in only quantifying pore structure and metrics of dry scaffolds, which is less biologically relevant since cells interact with a hydrated three-dimensional structure. A quadratic relationship between pore size and freezing temperature in hydrated scaffolds was shown (Fig. 1B). Rheology showed cryogel G’ ranging between 1kPa and 2kPa depending on fabrication temperature, which is within the range of native muscle tissue. Swelling experiments showed ~100% increase in Qm and only ~30% increase in Q, indicating that scaffolds retain large amounts of fluid but maintain their shape (i.e. volume) upon swelling, easing insertion into a VML defect and minimizing compression. The microporous network had pores large enough for cell movement through and penetration into the scaffold (Fig. 1C). The hFAPs showed greatest viability in 100 kDa scaffolds, high penetration, and promyogenic differentiation in scaffolds fabricated at -15°C, (Fig. 1C). Based on our in vitro results, we implanted 100 kDa scaffolds frozen at -15°C in 12-week-old male Lewis rats with ~10% removal (~9x7x5 mm defect) of the masseter mass between the buccal and marginal facial nerves. H&E & Masson’s trichrome staining showed little to no development of a fibrotic capsule as well as ~30% reduction of cross-sectional area of the scaffold over 4 weeks, indicative of cellular remodeling (Fig. 1D).
Collectively, our work presents an understanding of swollen HyA-based cryogel scaffold pore structure and demonstrates that they are excellent materials for muscle regeneration in VML. To our knowledge, our analysis represents the deepest quantification of the hydrated cryogel pore structure performed (~150 µm). This work represents the first cryogel scaffold of its kind for craniofacial VML treatment with minimal fibrosis. We have demonstrated the potential of cryogels as a translatable, tunable biomaterial for muscle regeneration with the additional advantage of a wider range of potential clinical uses, due to its ease of application (no mixing required as with a hydrogel) in more austere environments.

Acknowledgements (Optional):