Elucidating Biomaterial-Induced Immunomodulatory Mechanisms of Tissue Remodeling

Introduction: The ability to restore soft tissue is a significant clinical challenge. Soft tissue loss can be congenital, traumatic, or due to tumor resection, and the resulting defects can cause functional, aesthetic, and psychological problems. The available reparative options, both prosthetic or autologous, are often inadequate and can result in donor site defects, infection, tissue necrosis, limited persistence, and fibrosis. We recently developed a nanofiber hydrogel composite (NHC) material which facilitates host cellular infiltration and induces remodeling of the injected volume via the host immune system into vascularized soft tissue with adipocytes. The NHC material triggers remodeling without added cells or exogenous growth factors, making it an ideal material for the study of biomaterial-induced tissue remodeling. This NHC design combines the biocompatibility, biodegradability, and high porosity of a hyaluronic acid (HA) hydrogel with biostimulation and structural reinforcement by covalently conjugating electrospun polycaprolactone (PCL) nanofiber fragments to the HA network. Here, we aim to develop causal mechanistic understanding of the biomaterial-induced local immune modulation and soft tissue remodeling in the injected site.

Materials and

Methods: NHC materials, which consist of hyaluronic acid (HA) covalently bonded to electrospun polycaprolactone (PCL) fibers, were prepared according to a slightly modified version of the previously developed protocol to increase tunability and reproducibility. Gel microparticles were prepared from pre-formed NHC prior to injection. NHC and control hydrogel microparticles were injected subcutaneously in C57BL/6 mice, as well as knockout and targeted perturbation/depletion mouse models of the same background. Explanted materials were analyzed with RT-qPCR, NanoString, histology, multi-parameter flow cytometry, and single-cell RNA sequencing (scRNA-seq). The scRNA-seq data was analyzed with R, using the following packages: Seurat, sctransform, edgeR, DESeq2, apeglm, EnhancedVolcano, & fgsea.

Results, Conclusions, and Discussions: A higher degree of collagen deposition was observed around the HA controls, indicating a fibrosis response, as shown by Masson’s Trichrome staining (Fig. 1A), whereas strands of collagen are speckled throughout the NHC, indicative of tissue remodeling. The gene expression of the responding cells around/inside these materials was analyzed with NanoString at timepoints of 3 days, 7 days, 14 days, 35 days, and 3 months in both the NHC and HA control (Fig. 1B). Sustained higher expression of pro- and anti-inflammatory immune-related genes at 3 months reveals that the NHC induces sustained, controlled immune modulation as it triggers tissue remodeling. The identity of the responding cells was interrogated in more detail using scRNA-seq (Fig. 1C) as well as flow cytometry, revealing the cell subtypes present during this unique response. Immune cell depletions have also yielded mechanistic causality data implicating specific cells in the downstream events of biomaterial-induced tissue remodeling.

The NHC causes soft tissue remodeling through tailored immune cell infiltration and sustained biostimulation, without leading to deleterious inflammation. HA alone does not produce similar results; instead, a fibrotic layer and lack of biostimulation was observed. We have uncovered distinct differences in the host response and tissue remodeling induced by each material by characterizing cellular infiltration, healthy extracellular matrix deposition vs. fibrosis/foreign body response, inflammation, angiogenesis, and adipogenesis. The functional importance of specific infiltrating cell types has also been confirmed with knockout/depletion mouse models, and the subtypes further investigated with scRNA-seq. Together, these assays give unique insights into the microenvironment of matrix-induced tissue remodeling, providing a wealth of information to inform the development and translation of future biomaterials for soft tissue replacement.

Acknowledgements (Optional): We would like to thank the National Science Foundation (NSF GRFP DGE-1746891, National Institute of Dental and Craniofacial Research (NIDCR R01DE031488 & NIDCR F31DE032900), ARCS Foundation, Maryland Stem Cell Research Fund, and Johns Hopkins Biomedical Engineering for their support.