Immunomodulatory extracellular matrix hydrogel induces tissue regeneration in a model of partial glossectomy

Abstract

While oropharyngeal cancer treatment regimens, including surgical resection, irradiation, and chemotherapy, are effective at removing tumors, they lead to muscle atrophy, denervation, and fibrosis, contributing to the pathogenesis of oropharyngeal dysphagia - difficulty swallowing. Current standard of care of rehabilitative tongue strengthening and swallowing exercises is ineffective. Here, we evaluate an alternative approach utilizing an acellular and injectable biomaterial to preserve muscle content and reduce fibrosis of the tongue after injury. Skeletal muscle extracellular matrix (SKM) hydrogel is fabricated from decellularized porcine skeletal muscle tissue. A partial glossectomy injury in the rat is used to induce tongue fibrosis, and SKM hydrogels along with saline controls are injected into the site of scarring two weeks after injury. Tissues are harvested at 3 and 7 days post-injection for gene expression and immunohistochemical analyses, and at 4 weeks post-injection to evaluate histomorphological properties. SKM hydrogel reduces scar formation and improves muscle regeneration at the site of injury compared to saline. SKM additionally modulates the immune response towards an anti-inflammatory phenotype. This study demonstrates the immunomodulatory and tissue-regenerative capacity of an acellular and minimally invasive ECM hydrogel in a rodent model of tongue injury.

Keywords: Biomaterial; Dysphagia; Extracellular matrix; Hydrogel; Immunomodulation; Partial glossectomy; Skeletal muscle regeneration.

Graphical abstract

Fig. 1

SKM hydrogel injection volume optimization after tongue partial glossectomy injury. (A) A range of SKM volumes were injected 2 weeks after partial glossectomy injury, and tissues were harvested 1 week post-injection. Representative fluorescent images show pre-labeled SKM material in red against a DAPI counterstain in blue to indicate cell nuclei. SKM material spread and retention in the target tissue is shown for a range of injection volumes including 50 (B), 100 (C), 200 (D), and 300 μL (C). Scale bar: 2 mm.

Fig. 2

SKM injection after partial glossectomy injury reduces scar formation. (A) Timeline of study to assess therapeutic potential of two doses of SKM hydrogel injection. Tissues were harvested 4 weeks after injection for assessment of scar formation via Masson’s Trichrome stain, in which collagen is blue, denoting the scar region, and muscle fibers are red. (B) High dose SKM group significantly reduced scar formation compared to both the low dose SKM and saline injected groups. Treatment groups were compared using one-way ANOVA with Tukey multiple comparisons test (*p < 0.05; **p < 0.01). Representative images for the saline (C), low dose SKM (D), and high dose SKM (E) injection treatment groups are shown (Scale bar: 1 mm). Scar area was quantified and normalized to the total cross-sectional area for each tissue section including scar tissue, and values for all sections including the scar were averaged for each animal. n = 5–6 animals analyzed/group.

Fig. 3

SKM Injection improves muscle regeneration within scar area. Tongue sections underwent immunohistochemical staining to denote the scar (collagen I, green), myofibers (α-sarcoglycan, red), and nuclei (DAPI, blue). Representative fluorescent images are shown for saline (A) and SKM (B) injection groups (Scale bar: 1 mm). Higher magnification saline (C) and SKM (D) representative images demonstrated differences in fiber size and organization within the region of scar (Scale bar: 500 μm). (E) Fiber counts were normalized to scar area, showing no significant difference between treatment groups. (F) Muscle fiber area distributions are displayed as violin plots, with the dashed line indicating the median. Using a Mann-Whitney test, SKM demonstrated significantly increased fiber areas compared to saline injection (****p < 0.0001). (G) Within the scar area, proportion of fibers with centralized nuclei did not differ between groups. n = 5–6 animals analyzed per group.

Fig. 4

SKM injection induces significant differential transcriptomic expression when compared to both no injection and saline control groups. (A) Experimental timeline for gene expression study using a custom NanoString multiplex gene expression panel. Differentially expressed genes between SKM and either no injection (B–C) or saline controls (D–E) are displayed in a volcano plot for both 3 and 7 day timepoints. n = 11 animals analyzed per group.

Fig. 5

SKM injection stimulates muscle regeneration and immune response related pathways and genes as demonstrated through differential bulk RNA sequencing analysis. RNA isolated from tongue tissues at day 3 and 7 post-injection of SKM or saline and non-injected tissues – previously used for NanoString multiplex gene expression analysis – underwent bulk RNA sequencing. Volcano plots of differentially expressed genes through comparison of SKM and non-injected tissues at day 3 (A) and day 7 (B) post-injection are shown, alongside their respective gene ontology pathways for up- and downregulated biological processes. Similar volcano plots for differential expression between SKM and saline injected time points at day 3 (C) and day 7 (D) post-injection are shown. n = 8 animals analyzed per group.

Fig. 6

SKM injection promotes a transient increase in vasculature development. Tissue sections were stained against smooth muscle (ɑSMA, green), endothelial cells (isolectin, cyan), and nuclei (DAPI). Arterioles, double positive for ɑSMA and isolectin, were counted and assessed for lumen area. Representative images of stained tissue from saline (A) and SKM (B) treated animals at 7 days post-injection are shown (Scale bar: 100 µm). (C) At 7 days post-injection, SKM group demonstrated significantly higher vessel density throughout the whole tissue section (unpaired t-test, ***p<0.001). (D) The vessels in SKM injected animals demonstrated a significantly smaller overall size distribution at 7 days post-injection (Mann-Whitney test, ****p<0.0001). (F) At 4 weeks post-injection, there was a nonsignificant increase in vessel density within the scar area in SKM injected animals (unpaired t-test, p=0.079). (F) At 4 weeks post-injection, the smaller size distribution of vessels in the SKM group was maintained (Mann-Whitney test, ***p<0.001). n = 6 animals per group.

Fig. 7

Fibroadipogenic progenitors are more prominent in the SKM group 3 days post-injection and are colocalized with developing myofibers within the SKM scaffold. Tongue tissues harvested 3 days post-injection were stained against skeletal muscle (ɑ-sarcoglycan, green), fibroadipogenic progenitors (PDGFRɑ, red), and nuclei (DAPI, blue) for both saline (A) and SKM (B) injection groups (Scale bar: 500 µm). PDGFRɑ⁺ nuclei (C, white arrows; Scale bar: 20 µm) within the area of injury were normalized to total nuclei within the injury, and SKM demonstrated an increased percentage of PDGFRɑ⁺ nuclei (D). SKM bolus was visualized with collagen staining (E, dashed oval), and PDGFRɑ⁺ nuclei (orange), representing fibroadipogenic progenitors, were observed infiltrating the SKM bolus (Scale bar: 250 µm). (G, H) Within the bolus, developing myofibers (embryonic myosin heavy chain, green) were observed in regions with high density of fibroadipogenic progenitors (Scale bar: 50 µm). n = 5 animals/group.

Fig. 8

SKM injection mitigates area of injury as early as 7 days post-injection. Tissue sections were stained against hematoxylin and eosin at the 3 day timepoint for saline (A) and SKM (B) treated animals (Scale bar: 500 µm). (C) There was no significant different in percentage of injured area between SKM and saline groups at 3 days post-injection. At the 7 day timepoint, representative images for saline (D) and SKM (E) are shown (Scale bar: 500µm). (F) At 7 days post-injection, SKM group demonstrated significantly reduced percentage of injured area (t-test, *p<0.05). n = 6 animals per group.