Semi-synthetic Hyaluronic Acid Hydrogels Promote Muscle Regeneration by Inducing BAT Differentiation of Fibro-adipogenic Progenitor Cells (FAPs)

Introduction:: Skeletal muscle has a remarkably efficient innate regenerative capacity after injury up to a critical sized defect, after which regeneration is insufficient to fully repair the tissue. This condition, known as volumetric muscle loss, can lead to permanent muscle weakness and limb impairment. Prior studies have identified fibro-adipogenic progenitor cells (FAPs) as key regulators in repair of injured skeletal muscles. In uninjured muscles, FAPs are present in a quiescent state; however, they rapidly expand in response to muscle injury, adopting a brown-adipose tissue (BAT) phenotype to support satellite cell myogenesis.^[1] To take advantage of this promyogenic capacity, we have developed tunable semi-synthetic hyaluronic acid (HyA) hydrogels for tissue regeneration and stem cell transplantation.^[2,3,4] This highly tunable system allows us to explore a range of parameters to support muscle regeneration including modulus, degradation rate, cell specificity, and growth factor sequestration (Fig 1).^[5] We hypothesize that our HyA hydrogel can promote beige adipogenesis of human FAPs (hFAPs), thereby inducing muscle regeneration. Having already demonstrated upregulation of the BAT-specific marker (UCP1) after 7 days of hFAP culture within our hydrogel, we sought to further optimize our system using a variety of cell adhesion motifs specific to integrins associated with hFAP-BAT differentiation.

Materials and Methods:: The HyA hydrogels were synthesized using previously reported methods.[2][3] Briefly, an HyA derivative carrying hydrazide groups (HyA-ADH) was synthesized, and N-acryloxysuccinimide was subsequently reacted with the HyA-ADH solution to generate acrylate groups on the HyA (AcHyA). For growth factor sequestration, SH-heparin was also synthesized to bind AcHyA via Michael-type addition. hFAPs were harvested from deltoid muscle biopsies (10 mg samples) performed on patients undergoing arthroscopic rotator cuff repair. Cells were encapsulated by mixing the cells into the macromer precursor solution containing one of four cell binding peptides (Fig. 2A - bsp-RGD(15), C16, Ag73, and P3). Gelation was initiated by addition of a bis-cysteine terminated peptide-cleavable crosslinker of varying degradation rates (e.g., different Michaelis-Menten kinetics). BAT phenotype was assessed by immunostaining for UCP-1 and 𝛼SMA after 1, 3 and 7 days. ELISA for IL-10 was performed on the media at 1 and 7 day timepoints. For in vivo experiments, ten 9-month-old NSG mice underwent unilateral right suprascapular nerve denervation and combined supraspinatus and infraspinatus tendon transection, described previously.^[6] The mice were then randomized to treatment with 10μL saline (PBS) (n=5) or 10μL HyA-hydrogel (n=5). Mice were humanely sacrificed at 6 weeks and bilateral supraspinatus muscles were harvested and analyzed using immunohistochemistry.

Results, Conclusions, and Discussions:: Conjugation of different integrin engaging peptides impacted spreading and protein expression of FAPs after 7 days in culture as indicated by immunostaining (Fig 2A). Quantitative analysis of immunostaining demonstrated increased expression of UCP1 in the HyA-bsp-RGD(15), C16, and P3 peptide hydrogels compared to HyA-T1 (Fig. 2B). By measuring promyogenic cytokine secretion in the media of hFAPs encapsulated within HyA-bsp-RGD(15) hydrogels we found that IL-10 secretion was significantly increased after 7 days (Fig 2C). In vivo, HyA-bsp-RGD(15) hydrogel implantation in a rotator cuff injury model resulted in a significant decrease in relative muscle weight loss compared to the PBS control (Fig 2D). Trichrome staining showed fibrosis was also significantly decreased in the HyA-bsp-RGD(15) hydrogel group compared to PBS (Fig 2E). Furthermore, fatty infiltration was reduced to the level of the control group in the HyA-bsp-RGD(15) hydrogel treatment group compared to PBS. To measure vascularization, laminin and CD31 expression were quantified via immunostaining, revealing a significant increase of both markers in the HyA-bsp-RGD(15) hydrogel group compared to PBS treatment. Together, our results suggest that hydrogel-driven FAP-BAT differentiation may contribute to the positive regenerative outcomes after treatment of muscular injury with our hydrogel, highlighting the potential for this material to serve as an effective therapeutic for rotator cuff diseases. In conclusion, our in vitro work demonstrates the ability of our HyA hydrogels to support BAT differentiation of hFAPs as well as to increase promyogenic cytokine secretion over time. In vivo, the HyA-bsp-RGD(15) hydrogel reduces fibrosis, fatty infiltration, and muscle atrophy while supporting vascularization of injured muscle tissue, and ultimately muscle regeneration.

Acknowledgements (Optional): : This work was supported in part by California Institute for Regenerative Medicine, NIH-NIDCR (C-Doctor) U24 DE026914, NIH-NHLBI R01HL130417, DODCDMRP W81XWH-17-PRORP-ARA

References (Optional): : [1] Liu X. et al.,MusclesLigamentsTendonsJ.2016,6(1),6-15

[2] Jha et al., Biomaterials. 2015, 47, 1-12

[3] Jha et al., Biomaterials. 2016, 89, 113-147

[4] Browne et al., ACS Biomater. Sci. Eng. 2020, 6(2), 1135-1143

[5] Dienes et al., ACS Biomater. Sci. Eng. 2021, 7(4), 1587-1599

[6] Liu X et al. J Bone Joint Surg Am. 2012, 6(1):6-15