(H-309) Evaluation of Decellularized Porcine Dermal Hydrogel for Biointegration of Acellular Dermal Matrix in an Irradiated Murine Model

Introduction:: Functional wound healing requires the careful coordination of several complex cellular and molecular processes. The modulation of the inflammatory immune response and regulation of the extracellular matrix (ECM) and microenvironment depends on the spatiotemporal interplay between macrophages and fibroblasts. Recently, the use of decellularized ECM products have demonstrated great promise in controlling the immune response. One such product, a sheet of decellularized dermis known as an Acellular Dermal Matrix (ADM), has become commonplace in post-mastectomy implant-based breast reconstructive surgeries for providing mechanical support to the implant and soft tissue support to the overlying skin. Cellular infiltration of the ADM determines surgical outcomes, and incomplete incorporation can cause surgical complications such as flap necrosis, seroma formation, and fibrosis. The irregular topologies of the ADM and the overlying skin may cause a physical gap that hinders cell migration and subsequent matrix remodeling; as such, we developed a flowable decellularized porcine dermal (DPD) hydrogel to ease ADM integration by enabling persistent tissue contact. In a healthy mouse model, DPD hydrogel exhibited no adverse effects on fibrotic capsule formation while positively modulating the macrophage response compared to ADM alone. However, many patients seeking breast reconstructive surgeries have also undergone radiation therapy. We have previously shown that history of irradiation influences the macrophage response to ADM implantation. Here, we present a characterization of the immunomodulatory effects of concurrent DPD hydrogel and ADM use in an irradiated murine model.


Materials and Methods:: To generate DPD hydrogel, porcine dermis was mechanically processed to remove the epidermis and subcutaneous fat before undergoing a non-ionic detergent-based decellularization and lyophilization prior to enzymatic solubilization and subsequent gelation. C57BL/6J mice received 0 Gy or 35 Gy of dorsal skin radiation 12 weeks prior to implantation. Bilateral implants of 5mm diameter ADM were placed subcutaneously, with one side receiving 0.5 mL of DPD hydrogel before closure of the surgical pocket. Implants and surrounding tissue were harvested from the skin to the muscle fascia. Tissue digestion preceded flow cytometry for characterization of the macrophage response at 1 (n=7), 3 (n=8), and 6 weeks (n=8) using a robust panel of pro-inflammatory and pro-regenerative proteins. Percent change in Median Fluorescent Intensity (MFI) induced by DPD hydrogel was calculated for each protein using contralateral ADM-only controls and changes greater than 10% were considered to be of interest. Statistical testing of MFI shifts was performed with paired t-tests (or nonparametric Wilcoxon matched-pairs signed rank test where appropriate) with Holm-Sidak multiple hypothesis correction. Hematoxylin and eosin (H&E) staining of implant cross sections at 3 and 6 weeks was performed for pathological analysis of fibrosis via capsule formation (n=4). Various capsule endpoints such as thickness and cellularity were scored on a Likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe) by a pathologist blinded to sample condition.


Results, Conclusions, and Discussions:: The percentage of recovered cells expressing F4/80, a murine pan-macrophage marker, showed no significant differences between DPD hydrogel and control groups at each timepoint but did decrease over time. DPD hydrogel caused MFI shifts greater than 10% in CD9, CD38, CD86, CCR2, Arg1, CX3CR1, CD163, and CXCR4, but all were statistically insignificant (Figure 1).



We previously observed a temporal decrease in CD9 expression with ADM alone in irradiated mice compared to healthy mice. Interestingly, the use of DPD hydrogel in irradiated mice causes a 42% increase at 3 weeks and a 17% increase at 6 weeks in CD9 expression compared to ADM alone. This trend is consistent with our previous observations in healthy mice.



Pathology review of the fibrotic capsule revealed significant differences. At 3 weeks, DPD hydrogel decreased capsule thickness (p=0.0095), neutrophils (p=0.0300), lymphocytes (p=0.0427), plasma cells, (p=0.0063), mast cells (p=0.0007), and foreign body giant cells (p=0.0026). By 6 weeks, only the difference in mast cells was retained (p=0.0106).



DPD hydrogel addresses a limitation in the use of ADM during implant-based breast reconstructive surgeries. Previously, we showed the strong wound healing potential of DPD hydrogel within normal mice, and that radiation like that received by patients undergoing breast reconstruction has early pro-inflammatory and complex late-stage effects on the immune response to ADM implantation. Here, we explore the effect of DPD hydrogel within a more clinically relevant irradiated microenvironment. Shifts in expression of 8 out of the 12 proteins on our flow cytometry panel indicate the immunomodulatory effects of DPD hydrogel within an irradiated environment. In CD9, where radiation with ADM implantation alone caused a temporal decrease in expression significant at 3 and 6 weeks, DPD hydrogel caused 42% and 17% increases in MFI respectively. DPD hydrogel decreases fibrotic capsule endpoints compared to ADM alone at the earlier 3-week timepoint, and the effect resolves by 6 weeks, although the decreased mast cell count suggests an attenuated long-term inflammation response, which is corroborated by alterations to pro- and anti-inflammatory proteins. Taken collectively, supplementing ADM with DPD hydrogel displays heightened immune and matrix modulation – both critical elements of wound healing.


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