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Tissue Engineering

Tissue Engineering - Poster Session E

Poster L5 - The Effect of Extracellular Matrix Hydrogel Treatments on Fibrosis Production in a Canine Model of Volumetric Muscle Loss.

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Wound healing can be thought of in terms of overlapping phases namely; the inflammatory, proliferative and regenerative phases. While each phase is vital to the promotion of healthy tissue regrowth, the dysregulation of any one phase can lead to pathologic outcomes. Generally, for small wounds, the body is well adapted to promote regenerative healing. Exercise injuries and soft-tissue injuries are examples where regenerative healing occurs. Typically, fibrosis is generally beneficial during the proliferation phase of normal wound healing. In contrast, with significant muscle loss, as in volumetric muscle loss (VML), the body prioritizes wound closure and reducing infection risk. This primarily leads to deposition of fibrotic collagenous tissue in place of injured muscle. Further, a prolonged inflammatory phase can also contribute to a more fibrotic phenotype. This fibrosis has multiple deleterious impacts, primarily causing muscle weakness and loss of function. While a variety of treatments and physical therapies have been utilized to treat VML, this study focuses on an extracellular matrix (ECM) hydrogel as its primary treatment modality. ECM, which is essential for structural support, has been shown to have regenerative properties. ECM hydrogel can be injected into the wound site to provide endogenous biochemical signaling and provide additional structural support to promote the healing and regenerative tissue process.

Materials and

Methods:  Four canine treatment groups were used, a no treatment negative control, and animals which received ECM hydrogel treatments in varying quantities, thus labelled ECM 1x, ECM 3x and ECM 5x. Samples from these canines were taken longitudinally at key time points to view the progression of the healing around the wound site. These samples were then stained with a Masson’s Trichrome stain and subsequently imaged.

For the purpose of assessing the levels of fibrosis in the stained samples, a pixel classifier was trained using four representative images from across different treatment groups and time points. Qupath, an open-source platform, was used to accomplish this. The top six millimeters from the wound cap of each tissue sample were selected as the region of interest. Then, the folds in the samples were removed (de-selected) to reduce the likelihood of false positives and artificially increasing the percentage of mature fibrotic tissue. An artificial neural network machine learning program (ANN_MLP) was trained to differentiate between the pixels present as Dark Blue (Fibrosis), Light Blue (Young Collagen), Red (Not Collagen) and Background. In addition to quantifying the %fibrosis in the top 6 mm of the biopsies, scar tissue depth analysis was performed, from the wound cap down to the transition zone, a region defined as the boundary between healthy muscle and dying or regenerating myofibrils.

To analyze the resultant data, GraphPad Prism 10 was used to perform a two-way ANOVA test followed by Tukey’s multiple comparison test.

Results, Conclusions, and Discussions: A general trend towards increased fibrosis was observed over the course of the study, as seen in Figure 1. At the terminal time point of day 42, the mean %fibrosis values of 38%, 35% and 46% for untreated, ECM 3x and ECM 5x groups were observed respectively. Interestingly, while the untreated and ECM 5x groups showed continual increase in fibrosis, the ECM 3x group resulted in decreased fibrosis at day 42 compared to that observed at day 21.

The depth of the scar tissue, or transition zone, as seen in Figure 2, is significantly lowered in ECM-treated animals at both day 14 and 42. Further, ECM 3x treated dogs have significantly smaller scar tissue than both untreated and ECM 5x dogs. As visualized in Figures 2 and 3, while they have the smallest transition zones, they also have comparable quantities of mature collagenous tissue. This could point to a delay in the maturation of the scar tissue in untreated animals and a resurgence of inflammation in ECM 5x-treated dogs.

All the quantifications related to the project that include cellular infiltration, muscle regeneration as well as protein analysis and RNA sequencing, were performed using 6mm biopsies. Therefore, for cross-study comparisons, the fibrosis quantification was restricted to the 6mm depth. However, for the scar depth analysis, it was imperative to carry it to the transition zone.

In summary, the data from this study show that ECM hydrogel treatments in instances of volumetric muscle loss have an impact upon scar tissue depth and quality within the remodeling injury site.

Additional studies are needed to gain a better understanding of the mechanisms which contributed to the observed biological responses and further long-term studies are needed to fully assess the disparate remodeling outcomes with treatment modalities.

Acknowledgements (Optional):  Thank you to Dr. Brown for his oversight, guidance and support for this project, Mangesh Kulkarni, Shanae Butler and Clint Skillen for their assistance in protocol development and general lab support. This work was funded by the Swanson School of Engineering and the Department of Bioengineering.