# A method for temporal-spatial multivariate genomic analysis of acute wound healing via tissue stratification: a porcine negative pressure therapy pilot study

## TL;DR

This study introduces a new method to analyze wound healing by examining gene activity in specific tissue layers, revealing hidden patterns missed by traditional methods.

## Contribution

The novel approach of tissue stratification and spatial RNAseq provides precise genomic insights into wound healing that were previously undetectable.

## Key findings

- Different gene expression profiles were observed between individual tissue layers within a single wound group.
- Tissue stratification revealed unique differentially expressed genes hidden in bulk analysis.
- Signaling inversion or amplification between tissue layers was detected, which could be canceled out in bulk analysis.

## Abstract

Introduction: Wound therapies are capable of modulating the complex molecular signaling profile of tissue regeneration. However traditional, bulk tissue analysis results in nonspecific expressional profiles and diluted signaling that lacks temporal-spatial information.

Methods: An acute incisional porcine wound model was developed in the context of negative pressure wound therapy (NPWT). Dressing materials were inserted into wounds with or without NPWT exposure and evaluated over 8-hours. Upon wound explantation, tissue was stratified and dissected into the epidermis, dermis, or subcutaneous layer, or left undissected as a bulk sample and all groups processed for RNAseq. RNAseq of stratified layers provided spatial localization of expressional changes within defined tissue regions, including angiogenesis, inflammation, and matrix remodeling.

Results: Different expressional profiles were observed between individual tissue layers relative to each other within a single wound group and between each individual layer relative to bulk analysis. Tissue stratification identified unique differentially expressed genes within specific layers of tissue that were hidden during bulk analysis, as well as amplification of weak signals and/or inversion of signaling between two layers of the same wound, suggesting that two layers of skin can cancel out signaling within bulk analytical approaches.

Discussion: The unique wound stratification and spatial RNAseq approach in this study provides a new methodology to observe expressional patterns more precisely within tissue that may have otherwise not been detectable. Together these experimental data offer novel insight into early expressional patterns and genomic profiles, within and between tissue layers, in wound healing pathways that could potentially help guide clinical decisions and improve wound outcomes.