# Comparison of different decellularization protocols for porcine centrum tendineum diaphragmatis and diaphragmatic muscle – a base for effective recellularization

## TL;DR

This study compares three methods to remove cells from pig diaphragm tissues to find the best way to prepare them for tissue engineering and potential clinical use.

## Contribution

The study evaluates decellularization protocols for porcine diaphragm tissues to improve clinical relevance and recellularization potential.

## Key findings

- All three protocols significantly reduced DNA content in decellularized tissues compared to native tissues.
- P3 preserved the highest amount of proteins, followed by P2 and P1.
- P1 and P2 effectively preserved the extracellular matrix without a clear preference between them.

## Abstract

Diaphragmatic dysfunction results from a variety of diseases or post-surgical conditions, leading to impaired lung function and high morbidity and mortality. Current repair materials are limited by poor biocompatibility, functional incompatibility and immune reactions. Tissue engineering via decellularization offers a promising approach by preserving the extracellular matrix while reducing host immune response. However, most studies have focused on rodent models. This study evaluates three decellularization protocols using porcine tissues to increase clinical relevance and optimize diaphragm repair strategies. We compared detergent-enzymatic treatment (DET) adapted from murine diaphragm developed by Andreas et al. (P1) and two decellularization protocols for larger mammalian diaphragm tissues published by Barbon et al. (P2) for human diaphragm and Deeken et al. (P3) for porcine tendinous diaphragm. Decellularized samples were analyzed using histological analysis, SEM, DNA and GAG quantification and proteomic analysis. DNA content was reduced in decellularized tissues significantly between native and decellularized tissues (native: 990.30 ng/mg (IQR = 556.20); P1: 31.92 ng/mg (IQR = 40.38), P2: 32.38 ng/mg (IQR = 20.83), P3: 106.40 ng/mg (IQR = 811.32). Proteomics revealed 4,640 conserved proteins (5.41% classified as matrisomal proteins). The protocols showed a 55.4% concordance of the preserved matrisomal fraction (n = 139). The highest protein preservation was achieved by P3, followed by P2 and P1. The P1 and P2 protocols are effective in preserving the extracellular matrix while removing cellular components, with no clear preference. Within our laboratory setting, P3 showed decellularization, but did not reach current decellularization standards. This study advances the preparation for clinical translation of a decellularized porcine diaphragm.

The online version contains supplementary material available at 10.1186/s13036-025-00602-z.