Biomaterials direct functional B cell response in a material-specific manner

Abstract

B cells are an adaptive immune target of biomaterials development in vaccine research but, despite their role in wound healing, have not been extensively studied in regenerative medicine. To probe the role of B cells in biomaterial scaffold response, we evaluated the B cell response to biomaterial materials implanted in a muscle wound using a biological extracellular matrix (ECM), as a reference for a naturally derived material, and synthetic polyester polycaprolactone (PCL), as a reference for a synthetic material. In the local muscle tissue, small numbers of B cells are present in response to tissue injury and biomaterial implantation. The ECM materials induced mature B cells in lymph nodes and antigen presentation in the spleen. The synthetic PCL implants resulted in prolonged B cell presence in the wound and induced an antigen-presenting phenotype. In summary, the adaptive B cell immune response to biomaterial induces local, regional, and systemic immunological changes.

Fig. 1.. Biomaterial implants have diverging influences on B cells in quad tissue and in LN.

(A) Flow cytometry counts of a subset of B cells (defined as CD19⁻B220⁺) in quad over time after injury. B cell number in tissue peaks at day 5 in ECM and at weeks 1 and 3 in PCL. (B) Flow cytometry assessment of B cells in the LN (represented as a percentage of CD45⁺Live⁺ cells) following injury and biomaterial implant. (C) Histology of LNs at 3 weeks after injury. Data are means ± SD, n = 4, two-way analysis of variance (ANOVA) with subsequent multiple comparison testing. ANOVA [(A and B): ****P < 0.0001, ***P < 0.001, **P < 0.01]. VML indicates injury with saline, ECM indicates injury implanted with ECM, and PCL indicates injury implanted with PCL.

Fig. 2.. ECM induces germinal center formation.

(A) Dimensional reduction projection of B cells onto two dimensions using UMAP. Cells are colored by cluster. Violin plots of cluster gene expression of surface markers identified by differential expression analysis demonstrating genes highly expressed in cluster 3 associated with germinal center formation. (B) Flow cytometry counts of GL7⁺ B cells (defined as GL7⁺CD19⁺) in the draining LN following injury ± biomaterial shown as bar graphs for each time point. (C) Flow cytometry plots of day 5 GL7 on B cells comparing VML, ECM, and PCL. (D) Gene expression using qRT-PCR at 1-week expression of Aicda and Jchain compared to VML. (E) Serum analysis of IgG1 at 6 weeks after injury. (F) Heatmap of NanoString pathway scores in which the scale reflects the z score of each pathway at 3 weeks after injury in the spleen. (G) Volcano plot of PCL to ECM gene expression differences, where S1008a and Cd22 are divergently regulated. Data are means ± SD, n = 4, one-way ANOVA with subsequent multiple comparison testing. (C) ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. VML indicates injury with saline, ECM indicates injury with ECM, and PCL indicates injury with PCL.

Fig. 3.. PCL induces B cell antigen presentation in the quad tissue.

(A) Flow cytometry counts of B cell subsets: CD19⁺B220⁻, CD19⁻B220⁺, and CD19⁺B220⁺ in the quad tissue at day 5 after injury. (B) MHCII⁺B220⁺ cell population in quadricep tissue at day 5 after injury. (C) Flow cytometry counts of B cell subsets: CD19⁺B220⁻, CD19⁻B220⁺, and CD19⁺B220⁺ in the quad tissue at week 3 after injury. Data from CD19⁻B220⁺ are also used in Fig. 1A. Data are means ± SD, n = 4, two-way ANOVA with subsequent multiple comparison testing [(B and C): *P < 0.05, **P < 0.01]. VML indicates injury with saline, ECM indicates injury with ECM, and PCL indicates injury with PCL.

Fig. 4.. muMt⁻ reduces PCL-mediated fibrosis.

(A) Histological staining of 6-week PCL implants in BL6-WT and muMt⁻ with Masson’s trichrome for collagen fibrils. Scale bar, 400 μm. (B) Gene expression analysis of fibrosis-related genes including Col1a1, S100a4, p21, Tgfb1, Il23a, Col3a1 via qRT-PCR. (C) Immunofluorescence staining of CD20⁺ cells (yellow) and the implant barrier (red) in human tissue samples surrounding breast capsule implants of acellular adipose tissue (AAT) and (D) of silicone. Scale bar, 100 μm. Data are means ± SD, n = 3 for NT controls, n = 4 for PCL biomaterial, two-way ANOVA with subsequent multiple comparison testing [(C): ***P < 0.001, **P < 0.01, *P < 0.05]. muMt⁻ indicates mice that lack mature B cells, BL6 indicates C57BL6 mice, VML indicates injury with saline, ECM indicates injury with ECM, and PCL indicates injury with PCL.