During wounding and material implantation there is a disturbance in tissue homeostasis and release of self-antigen. The regulation between tolerance and auto-inflammation in injury is not well understood. We analyzed antigen-presenting cells in biomaterial-treated muscle injury. Pro-regenerative materials enrich Batf3-dependent CD103+ XCR1+CD301b+ dendritic cells associated with cross-presentation and self-tolerance. Muscle trauma was accompanied by CD8+ iTregs and expansion of CD103+XCR1+CD62L- adaptive immune cells. Up-regulation of E-Cadherin (the ligand for CD103) and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Without cross-presenting cells there is an increase in T cell activation, decrease in pro-regenerative macrophage polarization, and defects in muscle healing. These data describe a regulatory communication network through CD103+XCR1+ antigen-presenting and adaptive immune cells resulting in downstream effects on tissue regeneration.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunologic studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes twenty-four distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunologic studies with functional studies.

Tissue clearing of whole intact organs has enhanced imaging by enabling the exploration of tissue structure at a subcellular level in three-dimensional space. Although clearing and imaging of the whole organ have been used to study tissue biology, the microenvironment in which cells evolve to adapt to biomaterial implants or allografts in the body is poorly understood. Obtaining high-resolution information from complex cell-biomaterial interactions with volumetric landscapes represents a key challenge in the fields of biomaterials and regenerative medicine. To provide a new approach to examine how tissue responds to biomaterial implants, we apply cleared tissue light-sheet microscopy and three-dimensional reconstruction to utilize the wealth of autofluorescence information for visualizing and contrasting anatomical structures. This study demonstrates the adaptability of the clearing and imaging technique to provide sub-cellular resolution (0.6 um isotropic) 3D maps of various tissue types, using samples from fully intact peritoneal organs to volumetric muscle loss injury specimens, with and without biomaterials implants. We further apply computational-driven image classification to analyze the autofluorescence spectrum at multiple emission wavelengths to categorize tissue types in the tissue-biomaterial microenvironment.