R13 - Damaged Tissue Mimicking Hydrogels for Immunomodulatory Tissue Regeneration

Introduction: : Damage-associated molecular patterns (DAMPs) are molecules expressed on damaged extracellular matrix (ECM) or released by damaged cells to facilitate an immune response in a sterile environment. DAMPs are recognized by pattern recognition receptors on macrophages and initiate the secretion of inflammatory cytokines and chemokines. DAMPs have been associated with many inflammatory diseases such as osteoarthritis, atherosclerosis, and Parkinson’s disease, and are generally regarded as one of the major underlying causes. As DAMPs are often associated with these pro-inflammatory diseases, it is easy to understand why DAMPs have yet to be explicitly leveraged in tissue engineering, but the inflammatory response initiated by DAMPs are critical in the wound healing response. Thus, our group’s past work has isolated damaged ECM as an immunomodulatory cue for wound healing, and here we successfully incorporate the said ECM motifs (mECM) into a 3D scaffold and evaluate changes in tissue regeneration both in vitro and in vivo.

Materials and

Methods: : To evaluate any changes in collagen I hydrogel properties after mECM incorporation, gel fraction and degree of swelling were evaluated. Hydrogel topography was imaged via scanning electron microscopy (SEM). mECM hydrogel effects on primary rat fibroblast morphology was determined by encapsulating cells within the hydrogels, and staining for actin and nuclei at 1, 3, and 5 days post encapsulation. Actin area, nuclei area, and % elongated cells were quantified at all timepoints. Proliferation of the fibroblasts within the gels was evaluated via nuclei count at 1, 3, and 5 days post-encapsulation. To determine the efficacy of mECM hydrogels for calvarial bone defect regeneration, 10%, 20%, and 30% mECM hydrogels were implanted in 2mm critical-sized defects in C57BL6 mice. At 1-week post-surgery, the defect tissue was isolated and analyzed via single-cell RNA sequencing to determine cell population and gene expression within the defect. At 3 and 6 weeks post-surgery, the tissue within the defect will be analyzed for bone growth via micro-computed tomography and for bone morphology via Masson’s trichrome staining. For all in vitro and in vivo studies pure collagen (0% mECM) hydrogels were utilized as controls.

Results, Conclusions, and Discussions:: mECM incorporation affected gel structure and pore size, as demonstrated in figure 1A. Changes in gel fraction and degree of swelling were observed as mECM concentration increased. Gel fraction, the fraction of the total initial gel that is incorporated within the hydrogel network and not part of the soluble fraction, increased with an increase in mECM concentration, as shown in figure 1B. Although not statistically significant, this suggests that mECMs are part of the hydrogel network and are able to be crosslinked with collagen to form cross-linkable hydrogel networks. Degree of swelling was also increased with an increase in mECM concentration (figure 1C). When evaluating morphology of the fibroblasts, there was a significant increase in actin area in fibroblasts exposed to 20% mECM hydrogels at day 5 (figure 1D) indicating cell spreading, while no changes were observed in nuclei area across all groups (figure 1E). Also, at the day 5 timepoint, higher concentrations of mECM induced increased % of elongated cells, indicative of mechanical stress, compared to the control and 10% mECM hydrogels. When evaluating proliferation of the cells, After 5 days of exposure, there was a significant reduction in nuclei count in fibroblasts exposed to higher concentration of mECM hydrogels compared to the control. Both a decrease in proliferation and cell elongation resemble a myofibroblast-like phenotype, an important contributor in wound healing. Finally, our group has started investigating the efficacy of mECM hydrogels in calvarial bone regeneration as outlined in figure 1I. Hydrogels have successfully been implanted and we have started harvesting tissues at 1, 3, and 6, weeks post-implantation to evaluate immune cell population and their gene-expression at early timepoints, and determine bone growth and morphology at 3 and 6 weeks. Our work has shown that immunomodulatory mECMs can successfully be integrated into collagen hydrogel networks, while having significant effects on fibroblast morphology by learning them towards a myofibroblast-like phenotype. The efficacy of the hydrogels are further being evaluated in an in vivo model, and we will gain further insight on the impact of the DAMP induced immune response and how it can be leveraged for tissue regeneration.

Acknowledgements and/or References (Optional):: This work is supported by The University of Texas System Trauma Research and Combat Casualty Care Collaborative (175143) and the National Institutes of Health (K12 GM111726).