Adaptive hydrogel loaded with pre-coordinated stem cells for enhanced osteoarthritis therapy

Abstract

Osteoarthritis (OA) is a prevalent chronic joint disease with no currently available cure. Despite the promise of mesenchymal stromal cells (MSCs) in promoting OA management, direct intra-articular administration of MSCs faces several critical challenges, including rapid cell clearance from the joint cavity, limited survival in the hostile inflammatory environment, and insufficient control over their differentiation. In this study, we present a strategy that enhances the functionality of MSCs via pre-coordinated with Mg²⁺ and hypoxia-mimicking agent dimethyloxalylglycine (DMOG) integrated within an adaptive hydrogel for OA treatment. Mg²⁺ regulates macrophage polarization toward an anti-inflammatory phenotype, inhibits osteoclast activation, and preserves subchondral bone integrity by activating the PI3K-Akt signaling pathway. Concurrently, DMOG, activates the HIF-1α pathway, mimicking hypoxic microenvironment that support chondrocyte repair and stimulate cartilage matrix synthesis. MSCs pre-coordinated with Mg²⁺ and DMOG exhibit enhanced chondrogenic differentiation and immunomodulatory capacity, thus improving their regenerative potential in OA. To facilitate localized and sustained delivery, a self-healing tissue-adhesive hydrogel composed of phenylboronic acid and methacrylate-modified hyaluronic acid (HAMA-PBA) is synthesized to encapsulate the pre-coordinated MSCs. This hydrogel ensures cellular retention and functionality at the injury site. In vivo, the system significantly reduces joint inflammation, enhances cartilage regeneration, and improves joint function. Overall, these findings demonstrate a synergistic and effective stem cell-based therapeutic strategy for OA treatment through biochemical preconditioning and biomaterial-enabled delivery.

Keywords: Cell therapy; Chondrogenic; Hypoxia; Injectable hydrogel; Osteoarthritis.

Graphical abstract

Scheme 1

Schematic illustration showing the dual-crosslinking network hydrogel embedded with pre-coordinated BMSCs, in conjunction with Mg²⁺ and DMOG injection, exhibit synergistic effects for cellular regulation in OA joint. (A) Pre-coordination of BMSCs with Mg²⁺ and DMOG. (B) Formation and functional overview of the adaptive hydrogel, featuring a dual-crosslinked network composed of covalent and dynamic interactions. (C) Intra-articular administration of the hydrogel encapsulating pre-coordinated BMSCs to promote cartilage regeneration and mitigate OA progression in the OA rat model.

Fig. 1

Comparative analysis of Mg²⁺ in combination with three hypoxia mimics on chondrocyte phenotype in vitro. (A) Schematic showing the rat articular chondrocyte isolation. (B) The impact of defined concentrations of Mg²⁺, DMOG, Co²⁺, and DFO on the morphology of chondrocyte. (C) Immunofluorescence staining of HIF-1α, SOX9 and COL-II in chondrocytes. (D) Analysis of aspect ratio based on Fig. 1B. (E and F) ELISA quantitative analysis of HIF-1α and COL-II in chondrocytes. (G to J) The changes in mRNA levels of chondrogenesis and apoptosis related indicators (SOX9, ACAN, BCL-2, and AGO) in chondrocytes for 14 days. (K) Schematic illustration depicting the synergistic role of Mg²⁺ and hypoxia mimics (DMOG, Co²⁺, DFO) in promoting chondrogenic differentiation of BMSCs. (L) Immunofluorescence staining of HIF-1α and SOX9 in BMSCs. (M) Heatmap presenting the quantitative analysis of gene expression (HIF-1α, ACAN, OCT4, and TNF-α) in BMSCs cultured with Mg²⁺ and hypoxia mimics conditioned media. (N) Schematic illustrating the synergistic regulation of Mg²⁺ and DMOG on chondrocytes and BMSCs. Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 2

Chondrogenic differentiation of BMSCs regulated by Mg²⁺ and DMOG. (A) Differential gene Wayne plots. (B) Volcano plot distributions of differential genes in Mg²⁺ group compared to DMOG treated BMSCs. (C) KEGG pathway analysis in Mg²⁺ group compared to DMOG group. (D) GO pathway analysis in Mg²⁺, DMOG, and M + D groups. (E to G) Enrichment analysis of GSEA genes in M + D groups regarding the signaling pathway that maintains stemness, HIF-1α signaling pathway, and TGF-β signaling pathway. (H) The expression ratios of the candidate genes within the PI3K-Akt signaling pathway, Proteoglycans, Rap 1 signaling pathway, MAPK signaling pathway, ECM-receptor interaction, Ras signaling pathway and Focal adhesion, motivated by the M + D. (I) The expression levels of genes associated with chondrogenic differentiation and hypoxic pathway in the Mg²⁺, DMOG, and M + D groups. Results are presented as mean ± SD (n ≥ 3).

Fig. 3

M + D treatment reinstates chondrogenic phenotypes in OA-derived chondrocytes by suppressing inflammatory responses. (A) Immunofluorescence staining for RAW264.7 morphology, iNOS, and Arg-1. (B) Semi-quantitative analysis of fluorescence staining for iNOS and Arg-1 based on Fig. 3A. (C) Flow cytometry for RAW264.7 expression of CD206 and CCR7. (D) Analysis of the ring distribution plot statistics of flow cytometry results. (E to H) qRT-PCR for gene expression of relevant anti-inflammatory and pro-inflammatory factors (IL-1β, IL-6, IL-4, and IL-10). (I) Schematic of the extraction of human-derived OA chondrocytes. (J) Immunofluorescence staining for cellular morphology, HIF-1α, and MMP13 in OA-derived chondrocytes. (K and L) Statistics of cellular aspect ratio and spreading area based on Fig. 3J. (M to T) qRT-PCR quantification of the expression of factors associated with inflammation (CCL2, IL-1β, and TNF-α), cartilage matrix degradation (MMP13), and cartilage matrix synthesis (COL-II, SOX9, ACAN and HIF-1α). Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 4

Cell proliferation and chondrogenic phenotype maintenance in dual network HP-DMEM hydrogel. (A) Schematic illustration of cell growth in a dual network hydrogel with covalent and dynamic bonds. (B) Self-healing properties of HP-DMEM hydrogel. (C) Injectability and adhesion of HP-DMEM hydrogel. (D and E) Rheological testing. (F) Haemolysis assay. (G) Schematic illustration of cell growth in elastic and viscoelastic networks. (H) Live-dead staining of BMSCs incubated in HAMA and HP-DMEM hydrogels for 3 days in 3D mode. (I) Cell size distribution. (J and K) BMSCs are loaded in HAMA and HP-DMEM hydrogel with or without M + D pre-coordination for immunofluorescence labeling of COL-II and HIF-1α after 4 weeks of culture in vitro. (The yellow arrow in (K) shows cell aggregates forming within the dule-network hydrogel.) (L) Schematic diagram of subcutaneous implantation for evaluating ectopic chondrogenesis. (M) At week 4 post-implantation, hydrogels are retrieved for immunofluorescence staining on HIF-1α and COL-II, as well as, H&E and Alcian Blue staining. (N) Semi-quantitative analysis of HIF-1α and COL-II staining fluorescence intensity based on Fig. 4M. Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 5

HP + Sti-C hydrogel associated with M + D injection into the OA modeled joint cavity for synergistic therapy to inhibit subchondral bone sclerosis and cartilage degradation at week 8 post-operation. (A) Flow chart illustrating the timeline of the OA model induced in rats by ACLT and DMM surgery, followed by the subsequent therapeutic treatment and characterization. (B) Micro-CT and T2-weighted MRI images used to detect the changes in knee cartilage and subchondral bone tissues with OA. (C and D) The articular space width and osteophytes volume number quantified from 3D reconstructed micro-CT images. (E to H) Trabecular bone parameters, including BV/TV, MD, Tb. Th, and Tb. N, are quantitatively assessed. (I) Paraffin-embedded sections of rat knee joints are stained using H&E staining, and Safranin O-Fast green staining (Saf O/Fast green). Representative immunohistochemistry staining for COL-II and MMP13 within the articular cartilage is shown. (J and K) The OARSI score and Mankin score are analyzed and assigned accordingly. Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 6

Picrosirius red staining on collagen fibers in cartilage tissues collected from OA modeled knee joints with different treatments. (A) Histological sections stained with picrosirius red at week 4 and 8 post-operation, and imaged using Polarized light microscope. The lower panel shows the higher magnification images of the yellow box in the upper panel. (B) Orientation and dispersion of collagen fibers in the ROI-1 and ROI-2 analyzed for stained samples at week 8. (C) The orientation and dispersion of collagen fibers in different groups are analyzed using polar coordinate statistics. Collagen fibers running parallel to the articulating surface have an orientation of 0°, whereas fibers perpendicular to the surface have an orientation of 90°. Results are presented as mean ± SD (n ≥ 4).

Fig. 7

In vivo anti-inflammatory effects and inhibition of resorption in subchondral bone. (A and B) Immunofluorescence staining for the iNOS and Arg-1. (C) Micro-CT reconstructed images of subchondral bone tissues at week 4 with various treatments in OA modeled rat joints. (D) TRAP staining for osteoclasts. (E) The proportion of TRAP-stained positive cells. (F and G) Semiquantitative analysis of immunofluorescence staining for iNOS and Arg-1. Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 8

Mg²⁺ and DMOG synergize to inhibit osteoclast activation and modulate the cross-talk between chondrocytes and osteoclasts. (A) CLSM images showing F-actin in bone marrow mononuclear cells (BMMCs) after treatment with osteoclastic medium supplemented with Mg²⁺, DMOG, and M + D for 5 days, respectively. (B) Quantitative assay of TRAP activity at days 3 and 5. (C) TRAP staining of BMMCs. (D to F) Relative expression of mRNA (TRAP, MMP9, and Cathepsin K) of BMMCs. (G) SEM images showing the resorption lacunae formed on bovine dental slices at day 5 with different treatments. (H) Bone resorption area calculated based on Fig. 8G using ImageJ software. (I) Schematic illustration of the suppression of BMMCs differentiation into osteoclasts by Mg²⁺ and DMOG. (J) Schematic representation of the interaction between osteoclasts and chondrocytes in Transwell culture. (K) TRAP staining of osteoclasts in the upper chambers. (L) Quantification of TRAP activity expressed by osteoclasts. (M) Analysis of the number of osteoclasts based on the staining of TRAP in Fig. 8K. (N) Staining for ALP, COL-X, and COL-II in chondrocytes located in the lower chambers. (O and P) Based on the relative fluorescence intensity distribution of COL-X and COL-II for the single cell in Fig. 8N. Results are presented as mean ± SD (n ≥ 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 9

Schematic representation of the function of M + D in vivo and in vitro. (A) In vivo, within the OA modeled joint cavity, the combined treatment results in the suppression of pro-inflammatory cytokines release, reduced osteoclast activation, while augmenting anti-inflammatory phenotypes, stimulating cartilage matrix synthesis, and decreasing cartilage degradation and chondrocyte hypertrophy. (B) In vitro, Mg²⁺ synergizes with DMOG to inhibit the expression of inflammatory genes and enhance cartilage-related genes expression at the intracellular level.