Decellularization protocol-dependent damage-associated molecular patterns in rat uterus scaffolds differentially affect the immune response after transplantation

Scaffolds derived from decellularized tissue possess many advantages for bioengineering applications, including for novel infertility treatments. However, the decellularization process results in allogenic-independent damage-associated molecular patterns (DAMPs). This field is poorly studied, in particular for uterus bioengineering applications. An increased knowledge concerning the immune system activation after transplantation of decellularized tissue will enable safer construct development and thereby accelerate translation from research to clinic. We therefore transplanted rat uterus scaffolds produced by three different decellularization protocols based on Triton X-100 (P1 and P2) or sodium deoxycholate (P3) in a syngeneic animal model and assessed the immune response towards DAMPs exposed by the decellularization process. Biopsies were retrieved on day 5, 15, and 30 post transplantation and immunohistochemistry-stained CD45⁺ (leucocytes), CD4⁺ (T-cells), CD8a⁺ (cytotoxic T-cells), CD22⁺ (B-cells), NCR1⁺ (NK-cells), CD68⁺ (pan-macrophages), and CD163⁺ (M2 macrophages) cells within the grafts were quantified. The gene expression for interferon γ, interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF) eotaxin-2, RANTES, MCP-1, MIP-1α, MIP-3α, IL-8 were also measured. Scaffolds from P1 induced a rapid cell infiltration after transplantation, presumably induced by DNA-based DAMPs. However, this response was only transient. Protocol 3 derived scaffolds induced an early pro-inflammatory cytokine response at the transcript level which remained high throughout the study. This response may be caused by the stronger decellularization detergent that could expose more extracellular matrix-related DAMPs. However, earlier proteomics analysis also identified significantly more abundant heat shook proteins-related DAMPs in this scaffold type. Protocol 2 caused the least immunogenic scaffolds and should thus be the future focus for in vivo uterus bioengineering applications.