(M1130-03-17) Growth and Differentiation Factor 5: A Therapeutic Path for Chondrogenic Progenitor Cell-Based Cartilage Regeneration to Prevent Post-Traumatic Osteoarthritis

Purpose: Focal damage to articular cartilage incurred during joint injuries frequently results in progressive joint degeneration, a condition known as post-traumatic osteoarthritis (PTOA). Although cartilage is notoriously incapable of self-repair, evidence suggests that chondrogenic progenitor cells (CPCs) residing within the cartilage superficial zone can be coaxed to repair damaged cartilage, thereby forestalling PTOA. Early interventions that stimulate the recruitment of CPCs and their subsequent chondrogenesis offer a promising regenerative and non-invasive strategy to prevent the progression of PTOA, thus addressing the current lack of effective early clinical therapies. Growth and Differentiation Factor 5 (GDF5) stands out as a key protein involved in cartilage development and homeostasis. In this study, we explored the potential of GDF5 for CPC-based cartilage regeneration by assessing its impact on cell migration, proliferation, chondrogenic differentiation, and anti-catabolic activity in CPCs.

Methods: Bovine CPCs were isolated from the surface of stifle joint cartilage explants by using their migration response to injury-induced stimulation. Then the CPCs were cultured in the medium containing Dulbecco’s Modified Eagle Medium/F12 Medium (1:1) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin at 37 °C, 5% CO₂/O₂. CPC migration triggered by GDF5 was quantified using a Transwell^® migration assay, while CPC proliferation after GDF5 treatment was determined by measuring total double-stranded deoxyribonucleic acid (dsDNA) content. The chondrogenic properties of GDF5 on CPCs were evaluated in two cell culture models: a monolayer model and a pellet model, by analyzing the expression of chondrogenic markers via real-time quantitative PCR (RT-qPCR), glycosaminoglycans (GAGs) production through DMMB staining, pellet wet weight, and histological features with Safranin-O (Saf-O)/fast green staining. The anti-catabolic effect of GDF5 was determined by assessing (via RT-qPCR) the inhibition of ADAMTS5, a key aggrecanase involved in the articular matrix degradation, under either normal or interleukin 1β (IL1β)-induced inflammatory conditions. RNA sequencing was performed to interpret the molecular action of GDF5 on CPCs.

Results: GDF5 demonstrated a robust chemoattractant effect on CPCs, as evidenced by a significantly higher number of migrated CPCs in GDF5-treated groups compared to untreated controls, with migration increasing in a dose-dependent manner (Figure 1A, B). GDF5 treatment significantly promoted CPC proliferation by promoting glycolytic metabolism (Figure 1C, D). In CPC monolayer cultures, GDF5 significantly upregulated chondrogenic markers including SRY-box transcription factor 9 (SOX9), collagen type II alpha 1 (COL2a1), and aggrecan (ACAN) in a dose-dependent manner (Figure 1E, F). Similarly, GDF5 induced chondrogenic differentiation, evidenced by increased pellet wet weight (Figure 2A), elevated GAGs production (Figure 2B), and augmented proteoglycan deposition (Figure 2C, D) in CPC pellet cultures. Furthermore, GDF5 reduced the expression of ADAMTS5 in CPCs under both normal and IL1β-induced inflammatory conditions (Figure 3).

Conclusion: We demonstrated the potential of GDF5 as a therapeutic protein in CPC-based cartilage regeneration. In summary, GDF5 not only acted as an excellent chemoattractant for CPCs and promoted the proliferation of CPCs, but also induced the chondrogenic differentiation of CPCs as evidenced by elevating the expression of key extracellular matrix components. Moreover, GDF5 could suppress the expression of ADAMTS5, indicating a role for GDF5 in preventing cartilage matrix degradation. These findings position GDF5 as a promising candidate for promoting CPC-based cartilage regeneration for preventing the progression of PTOA.

Acknowledgements: This work was supported by the National Institutes of Health (Grant No. 5R21DE031042-02) and the Lyle and Sharon Bighley Chair of Pharmaceutical Sciences.


Figure 1: Effect of GDF5 on CPC migration, proliferation, and chondrogenic differentiation in a monolayer model. (A) Confocal images of migrated CPCs stained with Calcein AM (blue) and (B) quantitative analysis of migrated CPCs in a Transwell® assay (n = 3). (C) CPC proliferation via total dsDNA content (n = 3) and (D) clustering heatmap for key glycolysis-related genes. (E, F) RT-qPCR analysis of chondrogenic marker genes COL2A1, ACAN, and SOX9 in CPC monolayers following GDF5 treatment at days 2 (E) and 7 (F) (n = 3). Data are represented as means ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post-hoc test. P-values of less than or equal to 0.05 were considered significant.


Figure 2: Chondrogenic effects of GDF5 on CPCs in a pellet model. (A) Wet weight of pellets, (B) GAGs production normalized to dsDNA content within the pellets, (C) Saf-O/fast green histological staining, with arrows indicating a notable presence of chondrocyte-like cells in the GDF5-treated groups, and (D) quantitative analysis of Saf-O staining intensity following GDF5 treatment (n = 3). Data are represented as means ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post-hoc test. P-values of less than or equal to 0.05 were considered significant.


Figure 3: GDF5 reduced the expression of ADAMTS5 under both normal and inflammatory conditions. (A) RT-qPCR analysis of ADAMTS5 in CPC monolayers following treatment with GDF5 (n = 3). (B) Dose-dependent response of ADAMTS5 expression in CPCs exposed to indicated concentrations of IL1β, with 1 ng/ml and 5 ng/ml selected for subsequent experiments in inflammatory conditions (n = 3). (C, D) ADAMTS5 expression in CPCs treated with GDF5 with or without inflammation induction using IL1β at 1 ng/ml and 5 ng/ml (n = 3). Data are represented as means ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post-hoc test. P-values of less than or equal to 0.05 were considered significant.