Screening of MMP-13 Inhibitors Using a GelMA-Alginate Interpenetrating Network Hydrogel-Based Model Mimicking Cytokine-Induced Key Features of Osteoarthritis In Vitro

Abstract

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. Current clinical treatment options lack effective pharmaceutical interventions targeting the disease's root causes. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, very few drugs within this class are in preclinical or clinical trial phases. Hydrogel-based 3D in vitro models have shown promise as preclinical testing platforms due to their resemblance to native extracellular matrix (ECM), abundant availability, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). The results demonstrate a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. However, inconsistencies in human cartilage explant samples led to inconclusive results. Nonetheless, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening.

Keywords: GelMA; IPN hydrogel; MMP-13 inhibitors; cartilage explants; drug screening; sodium alginate.

Figure 1

Synthesis of GelMA-alginate IPN hydrogels. (a) Schematic diagram of the synthesis of a GelMA-alginate IPN hydrogel. GelMA polymer chains, sodium alginate, Na⁺, and Ca²⁺ are represented by green, red, yellow, and blue colors, respectively. (b) Increasing opacity of GelMA-alginate IPN hydrogels corresponds to incremental increases in sodium alginate concentration.

Figure 2

Characteristics of GelMA-alginate hydrogels with variation in sodium alginate content. (a) Microstructure of GelMA-alginate hydrogels. The scale bar is 100 μm. (b) Compressive modulus of GelMA-alginate hydrogels. (c) Swelling degree of GelMA-alginate hydrogels. Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 3

Evaluation of cell viability of TC28a2 chondrocytes in GelMA-alginate hydrogels on days 1 and 7. (a) Live/dead staining of chondrocytes. Green fluorescence indicates viable cells whereas red fluorescence indicates dead cells. The scale bar is 100 μm. (b) Metabolic activity of chondrocytes assessed by alamarBlue™ assay. Data are presented as mean ± SD, with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 4

Evaluation of different chondrogenic differentiation media. (a) Staining of Alcian blue (upper row) and Sirius red (lower row) on days 7, 14, and 21. The culture medium in each group from left to right is CCM + AA2P + DXM, CCM + AA2P + DXM + TGF-β1, CCM + AA2P + DXM + TGF-β1 + ITS Premix, and CCM only (control). (b) Quantification of Alcian blue staining and Sirius red staining. Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 5

Confirmation of chondrogenesis in GelMA-alginate hydrogel. (a) qRT-PCR analysis of gene expression of chondrogenic markers. Gene expression is normalized to GAPDH and expressed relative to the control group (Day 0). Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01. (b) Immunofluorescence staining of type II collagen (green) in cryosections. The scale bar is 100 μm. (c) Alcian blue/nuclear fast red staining of sulfated glycosaminoglycans (sGAGs) in paraffin sections. Nuclei, cytoplasm, and sGAGs are stained dark pink to red, pale pink, and blue, respectively. The scale bar is 100 μm.

Figure 6

Evaluation of cytokines for MMP-13 induction in monolayer TC28a2 chondrocytes. (a) Cell viability using MTT assay 24 h post-treatment with inflammatory cytokines. (b) Measurement of MMP-13 concentration by ELISA in cell supernatant 2 d post-cytokine induction. (c) Immunofluorescence staining of MMP-13 2 d post-cytokine induction. The scale bar is 100 μm. Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 7

Evaluation of MMP-13 inhibitors. (a) Cell viability of TC28a2 chondrocytes using MTT assay 24 h post-treatment with MMP-13 inhibitors. (b) Comparison of inhibitory effects of inhibitors using a fluorogenic substrate assay. Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 8

Screening of MMP-13 inhibitors using GelMA-alginate cartilage constructs with OA-like conditions. (a) Evaluation of MMP-13 expression in TC28a2 chondrocytes using immunofluorescence staining 3 d post-treatment with IL-1β and TNF-α. The scale bar is 100 μm. (b) Fluorescence intensity of MMP-13 expression in TC28a2 chondrocytes analyzed by ImageJ (version 1.54 g). (c) Measurement of active MMP-13 concentration by ELISA in cell supernatant. (d) Measurement of C2C concentration for type II collagen cleavage by ELISA in cell supernatant. Data are presented as mean ± SD with statistical significance indicated as * p < 0.05 and ** p < 0.01.

Figure 9

Histological analysis of human articular cartilage by staining of H&E, Safranin O, and Alcian blue, and IHC staining of type II collagen. The scale bar is 100 μm.