Proteomic composition and immunomodulatory properties of urinary bladder matrix scaffolds in homeostasis and injury

Abstract

Urinary bladder matrix (UBM) is used clinically for management of wounds and reinforcement of surgical soft tissue repair, among other applications. UBM consists of the lamina propria and basal lamina of the porcine urinary bladder, and is decellularized as part of the process to manufacture the medical device. UBM is composed mainly of Collagen I, but also contains a wide variety of fibrillar and basement membrane collagens, glycoproteins, proteoglycans and ECM-associated factors. Upon application of the biomaterial in a traumatic or non-traumatic setting in a mouse model, there is a cascade of immune cells that respond to the damaged tissue and biomaterial. Here, through the use of multicolor flow cytometry, we describe the various cells that infiltrate the UBM scaffold in a subcutaneous and volumetric muscle injury model. A wide variety of immune cells are found in the UBM scaffold immune microenvironment (SIM) including F4/80⁺ macrophages, CD11c⁺ dendritic cells, CD3⁺ T cells and CD19⁺ B cells. A systemic IL-4 upregulation and a local M2-macrophage response were observed in the proximity of the implanted UBM. The recruitment and activation of these cells is dependent upon signals from the scaffold and communication between the different cell types present.

Keywords: Biomaterials; Immunology; Macrophages; T cells.

Fig. 1.

UBM materials characterization. (a) SEM of particulate UBM. (b) Proteomic evaluation of the proteins with highest abundance in UBM and categories of proteins detected. (c) Collagen abundance in UBM (d) Repeatability of iBAQ detection of peptides in 3 different lots of UBM. (e and f) Storage and Loss moduli of 100, 200 and 300 mg/ml ECM pastes. (g) Differential scanning calorimetry hydrated 300 mg/ml paste UBM compared to a milled collagen I control. For rheology, data are means ± SEM, n = 3 samples per group. For proteomics, data are means of 3 lots tested in 3 mass-spectrometry runs per lot.

Fig. 2.

Particulate UBM induces alterations in macrophage phenotype in vitro. (a) RT-PCR analysis of M1 (Tnfa, Il1b, Inos) and M2 (Arg1, Retnla, Il10) genes in bone-marrow derived macrophages cultured on UBM for 24 h in M0 (growth, unstimulated), M1 (inflammatory, LPS+IFNg) or M2 (anti-inflammatory, IL-4) media conditions. (b) Flow cytometric analysis of CD86 (M1), CD206 (M2) and IL4ra (M2) on macrophages at 24 h post-seeding on UBM coated tissue culture plastic (TCP). Data are means ± SEM, n = 4 mice per group.

Fig. 3.

The scaffold immune microenvironment of UBM. C57BL/6 mice received 200 ul of a 300 mg/ml subcutaneous UBM implant. (a) Cross-section of subcutaneous UBM implant at 1 week post-injection. (b) Dorsal (skin), center, and ventral (capsule) sections of UBM implant at 1 week post-injection. (c) FACS analysis of resident immune cells at 1 and 3 weeks post-injection showing high presence of F4/80⁺ macrophages and an increase in CD3⁺ T cells over time. (d) Representative FACS plots from data quantified in (c). (e) Immunofluorescence (IF) staining of implant at 1 week post-injection showing localization of B cells (B220, green), macrophages (F4/80, Red), and T cells (CD3, magenta). (f) Composite cross-section of IF-stained implant at 1 week post-injection.

Fig. 4.

UBM promotes an M2-macrophage phenotype that matures over time. (a) CD206 and CD86 expression on 3 myeloid subtypes detected in the implant at 3 weeks post-injection. (b) CD86 expression quantified as mean fluorescence intensity (MFI) at 1 and 3 weeks post-injection. (c) CD206 expression quantified as MFI. Data are means ± SEM, n = 4 mice per group.

Fig. 5.

UBM-treated muscle wounds recruit a diverse immune cell repertoire. C57BL/6 WT mice received a bilateral 3 mm × 4 mm muscle removal from their quadriceps muscle group which was back filled with 50 ul of a 300 mg/ml UBM paste and analyzed via flow cytometry. (a) Immune cell populations at 1 and 3 weeks post-injury. (b) CD4:CD8 ratio at 3 weeks post-injury. (c) Comparison of cell populations in UBM-treated wound versus saline-treated control at 3 weeks post-injury. Data displayed as fold change over control. (d) Comparison of CD86 (M1 marker) and CD206 (M2 marker) expression in UBM-treated wound versus saline-treated control at 3 weeks post-injury. Data displayed as fold change over control. Data are means ± SEM, n = 4 mice per group.

Fig. 6.

UBM induces a systemic IL-4 upregulation correlated with local antigen-presenting M2-macrophages. (a) RT-PCR of distal lymph nodes (axillary/brachial) at 3 weeks post-injury displayed as RQ to saline treated VML control. (b) Percent (%) of MHCII⁺ cells that are present in the scaffold immune microenvironment at 1 week post-injury. (c) MHCII⁺ expression is selectively detected on CD11b⁺ myeloid cells. (d) MHCII⁺ cells are mainly F4/80⁺CD206⁺ M2 macrophages, representative FACS plot of data quantified in (b). Data are means ± SEM, n = 4 mice per group.