Primary Investigator & Professor Purdue University, United States
Introduction: Volumetric muscle loss (VML) injuries are highly prevalent and often result in fibrotic scarring that reduces muscle function and compromises quality of life. Although many biomaterial therapies have been developed to treat VML, an understanding of how biomaterial features affect endogenous fibroblast activation and matrix deposition remains unexplored. One challenge lies in identifying fibroblasts in preserved tissue sections. It has been reported that alpha-smooth muscle actin (α-SMA), a well-known marker of activated myofibroblasts in other tissues, does not accurately stain muscle-resident fibroblasts [1]. To explore this further, we stained mouse tibilas anterior muscle cryosections that had been subjected to VML defects and implanted with bulk or porous granular hydrogels with (α-SMA) or platelet-derived growth factor receptor alpha (PDGFRA) antibodies. PDGFRA is a well-known marker for muscle-resident fibro/adipogenic progenitor cells (FAPs), a cell type believed to play a role in muscle fibrosis after injury. Lastly, Picro Sirius red collagen staining was performed on the cryosections to stain collagen and scar tissue and to examine correlations between cellular components and the extracellular matrix.
Materials and
Methods: Frozen muscle samples were from a bilateral VML injury of the tibialis anterior (TA) muscles study [2]. The muscle samples were of 12-14 week old C57BL/6 mice, which were injected with granular or bulk norbornene-modified hyaluronic acid hydrogels, and euthanized at 4 weeks. The samples were processed by an optimized immunohistochemistry protocol (Fig. 1a) and were stained with Hoechst (nuclei) and either α-SMA or PDGFRA. Samples were permeabilized with 0.1% Triton-X, blocked with 3% Bovine Serum Albumin (BSA), and incubated overnight with the primary antibody. After secondary antibody treatment and counterstaining with Hoechst, sections were mounted using Prolong gold antifade and visualized using fluorescence microscopy. Imaging was performed using a Keyence BZ-X810 inverted fluorescence microscope with a 20x objective lens. The samples were analyzed using ImageJ. Quantification of cell invasion and antibody staining was performed by identifying a region of interest (ROI) bounded by the hydrogel biomaterial which was identified by FITC signal. Then an intensity percentile was determined for the gel, nuclei and each respective staining bound within the ROI.
Results, Conclusions, and Discussions: Results and Discussions: After optimizing antibody concentrations, we successfully stained muscle sections with α-SMA (Fig. 1b) and PDGFRA (Fig. 1c). Intense staining was observed in the VML defect region and in or around the hydrogel compared to the healthy muscle sections which had minimal staining. There was a statistically significant difference in cellular invasion between the bulk and the granular hydrogels, as expected, due to the inherent porosity of granular hydrogels and their ability to support cell recruitment (Fig. 1d-e). Additionally, there was a difference in normalized staining intensity between α-SMA and PDGFRA with α-SMA being higher in normalized. Whether the two antibodies are staining separate cell populations will be investigated in future work. Lastly the Picro Sirius Red staining coupled with polarized microscopy showed the presence of collagen with varying birefringence signals potentially suggestive of a differential fibroblast response based on hydrogel implantation (Fig. f).
Conclusions: Our findings demonstrate the importance of antibody selection to stain for diverse fibroblast populations during muscle repair. Future work will correlate fibroblast presence with collagen staining quantification to determine patterns of fibrosis around or within implanted biomaterials.
