# Glycosaminoglycan-functionalized hydrogels for sustained delivery of tissue inhibitor of metalloproteinase-3 mediating matrix metalloprotease inhibition and extracellular matrix stabilization

## TL;DR

This study develops a hydrogel that delivers TIMP-3 to reduce tissue damage by inhibiting harmful enzymes and stabilizing the extracellular matrix.

## Contribution

A novel GAG-functionalized hydrogel system for sustained TIMP-3 delivery with controlled release and bioactivity is introduced.

## Key findings

- sHAc-functionalized hydrogels sustained TIMP-3 release for 28 days and reduced ECM degradation.
- TIMP-3 released from the hydrogels retained bioactivity and inhibited MMP-9 in vitro and ex vivo.
- Murine models showed reduced inflammation and altered tissue signatures with sHAc-functionalized implants.

## Abstract

Excessive protease activity and impaired tissue regeneration are hallmarks of many disease states. Elevated matrix metalloproteinase-9 (MMP-9) plays a key role in adverse tissue remodeling by excessively degrading extracellular matrix (ECM) components and growth factors. Tissue inhibitor of metalloproteinase-3 (TIMP-3) regulates ECM turnover, and its bioavailability is influenced by glycosaminoglycans (GAGs). This study aimed to develop a methacrylated gelatin (GelMA)-based hydrogel functionalized with acrylated sulfated hyaluronan (sHAc) as a TIMP-3 delivery system to decrease ECM degradation under pathophysiological conditions. sHAc incorporation enhanced hydrogel stiffness, reduced degradation rates and yielded sustained TIMP-3 release for up to 28 days. Molecular modeling and surface plasmon resonance demonstrated preferential binding of TIMP-3 to sHAc over hyaluronan methacrylates, together providing a molecular rationale for the reduced and sustained release of TIMP-3 from sHAc-containing hydrogels. Angiogenesis-related functional assays, supported by molecular modeling studies, indicate that sHAc modulates the anti-angiogenic activity of TIMP-3 by altering vascular endothelial growth factor receptor-associated signaling, while preserving metalloproteinase inhibition. Released TIMP-3 from GelMA/sHAc hydrogels retained bioactivity, effectively inhibiting MMP-9 activity and mitigating ECM degradation in-vitro and in human ex-vivo models. In a murine subcutaneous implantation model, sHAc-functionalized TIMP-3-loaded hydrogels were associated with reduced inflammatory cell presence and altered vascular- and matrix-related tissue signatures compared with GelMA controls. These findings underscore the potential of sHAc-functionalized GelMA hydrogels as biomaterials for therapeutics delivery, offering controlled TIMP-3 release and sustained bioactivity to promote ECM stability and on-demand MMP inhibition. This system represents a promising strategy for addressing the challenges of excessive MMP activity.

Image 1

•sHAc functionalization controls TIMP-3 binding and sustained release.•sHAc modulates TIMP-3 anti-angiogenic activity via VEGFR-associated signaling.•GelMA/sHAc hydrogels deliver bioactive TIMP-3 in-vitro and ex-vivo.•GelMA/sHAc implants show reduced inflammation in a murine subcutaneous model.•The platform attenuates excessive protease-driven ECM degradation ex-vivo.

sHAc functionalization controls TIMP-3 binding and sustained release.

sHAc modulates TIMP-3 anti-angiogenic activity via VEGFR-associated signaling.

GelMA/sHAc hydrogels deliver bioactive TIMP-3 in-vitro and ex-vivo.

GelMA/sHAc implants show reduced inflammation in a murine subcutaneous model.

The platform attenuates excessive protease-driven ECM degradation ex-vivo.