# Optimizing Cellular Metabolism Through Mass Balance Analysis to Improve Skin Wound Healing

## TL;DR

This study identifies glycine as a promising supplement to improve skin wound healing by enhancing cellular metabolism.

## Contribution

The study uses computational modeling and in vitro experiments to show that glycine improves wound healing processes.

## Key findings

- Glycine supplementation significantly increased cellular proliferation in cultured keratinocytes.
- Glycine accelerated wound closure in scratch assays compared to base media.
- Glutamine and valine did not improve wound healing parameters and sometimes suppressed glycine's benefits.

## Abstract

Chronic wound healing remains a significant clinical challenge despite years of intense research. This study leverages insights from known cellular biochemistry using computational modeling to identify potential metabolic supplements that could improve chronic skin wounds. In particular, the Recon database was analyzed computationally to identify glycine and glutamine as the most likely metabolites to enhance ATP and biomass synthesis during both unrestrained and restrained oxygen uptake, which more closely mimics hypoxia in chronic skin wounds. This finding was subsequently tested in vitro on cultured immortalized keratinocytes, and revealed that glycine significantly increased cellular proliferation and accelerated wound closure. Glutamine and valine, used to control for nitrogen load increase, showed no improvement in these parameters.

Accelerating healing is a clinical goal in both acute and chronic non-healing skin wounds. We leveraged the public Recon database, which seeks to aggregate all of the metabolic pathways in the human body, to uncover whether increasing the supply of specific metabolites can bolster cellular metabolism and, in turn, enhance wound healing. The database was reduced to a set of 357 reactions and 339 metabolites that were better suited for human cells in culture. Monte Carlo simulations were performed to identify the impact of 25 different inputs on the metabolic fluxes within the cellular biochemical network. Biomass and ATP production were used as surrogate markers for cell proliferation and cell migration (an energy-intensive process), respectively, both of which are critical to wound healing. The subset of simulations yielding the highest ATP production or biomass production were those where glycine and/or glutamine uptake was increased. Maximizing ATP and biomass also generally increased oxygen uptake. Due to its low availability in chronic wounds, another set of simulations was carried out in which oxygen uptake was held constant to mimic the effect of a limited oxygen supply. However, even with this constraint, glycine and glutamine remained the most promising interventions. The predictions were tested in vitro using immortalized human keratinocytes. Amino acid uptake was tentatively increased by supplementing the base culture media with additional glycine and/or glutamine, with valine supplementation with a similar nitrogen load as a control. Glycine supplementation significantly increased cellular proliferation above the base media and accelerated wound closure rate in wound scratch assay. However, glutamine and valine supplementation did not improve these parameters above base media, and glutamine even suppressed the benefit of glycine in cultures supplemented with both amino acids. In conclusion, glycine supplementation enhances cellular processes that are associated with wound healing.