# 4-Hydroxybutyrate (4HB) released from poly-4-hydroxybutyrate scaffolds does not impact hallmark phenotypes of cancer in malignant or non-malignant breast cells

## TL;DR

This study shows that 4HB, a byproduct of a medical implant material, does not promote cancer-like behaviors in breast cells at concentrations up to ten times higher than those found in the body.

## Contribution

The study provides in vitro evidence that 4HB released from P4HB scaffolds does not enhance cancer-related cell behaviors.

## Key findings

- 4HB concentrations up to 10X the clinically relevant level did not increase metabolic activity, proliferation, migration, or colony formation in breast cancer or non-cancer cells.
- No significant differences were observed between 4HB-treated cells and controls in multiple in vitro assays.
- The peri-implant concentration of 4HB in a rabbit model was measured at 163 µM after a simulated 1-year implantation.

## Abstract

Poly-4-hydroxybutyrate (P4HB) scaffolds are increasingly used to reinforce soft tissue during implant-based reconstruction after mastectomy. P4HB undergoes hydrolytic degradation to a natural metabolite, 4-hydroxybutyrate (4HB). Understanding the direct effects of 4HB on cancer cells is essential for assessing the oncologic safety of P4HB scaffolds used in breast reconstruction surgery. The aim of this study was to evaluate the effects of sodium 4-hydroxybutyrate (Na4HB) on multiple, relevant human breast cancer and epithelial cell types using a panel of well-established in vitro assays aligned with several hallmarks of cancer.

First, the clinically relevant tissue concentration of 4HB was determined via a rabbit model to quantify 4HB in the peri-implant tissue of P4HB scaffolds. Second, human breast cell types, including non-malignant HMEC and MCF-10A, and cancerous MCF-7, BT-474 and MDA-MB-231, were exposed to Na4HB at up to 10X the clinically relevant tissue concentration. Cells were then evaluated for cancer related phenotypes: metabolic activity (MTT assay), proliferation (BrdU assay), migration (Scratch and Transwell assays), and colony formation (soft agar assays). Specific inhibitory control compounds for each assay were included to confirm assay performance.

The average peri-implant concentration of 4HB was found to be 163 µM after a simulated 1-year implantation in a rabbit model. Across the five assays and all three Na4HB concentrations, ranging from below to over 10X the peri-implant level (70, 350, 1750 µM), there was no statistically significant increase in any cancer-related phenotype including metabolic activity, proliferation, migration and colony formation in either malignant or non-malignant cell types compared to controls treated with either the PBS vehicle or sodium acetate as determined by one-way ANOVA followed by Tukey’s multiple comparison test (p > 0.05).

This comprehensive, in vitro evaluation suggests that 4HB does not modify growth or activity of malignant or non-malignant breast cells at concentrations up to 10X the peri-implant level. While these findings suggest that 4HB released from degrading P4HB Scaffolds is unlikely to promote oncogenic behavior in vitro, further co-culture systems, in vivo studies and long-term clinical data can be used to further assess the oncologic safety in breast reconstruction.

The online version contains supplementary material available at 10.1186/s13058-026-02234-7.