DNMT3A as an Epigenetic Transcriptional Therapeutic Target in the Progression of Hip Osteoarthritis - Femoroacetabular impingement (FAI) is a pre-arthritic hip condition where abnormal contact between the femur and acetabulum causes cartilage damage and osteoarthritis (OA). It primarily affects individuals aged 20 to 40, and if untreated, can contribute to significant joint damage, accounting for up to 50% of OA cases. The standard treatment, femoral osteochondroplasty (OCP), fails in up to 20% of cases. Additionally, 14% of patients present with moderate OA at diagnosis, making them ineligible for OCP and reliant on total hip replacement (THR), which poses healthcare challenges. Revealing the mechanisms behind FAI-related OA is crucial for developing additional targeted therapies. Our prior studies indicate that in FAI patients, inflammation increases DNMT3A expression, hypermethylates PPARγ CpG region, reduces PPARγ expression, and degrades cartilage, progressing further to late-stage OA. Our goal is to determine if modulating the DNMT3A pathway with siRNA can reduce cartilage degradation and restore cartilage homeostasis and joint integrity. AIM 1: Explore how siRNA DNMT3A promotes PPARγ expression and mediates cartilage catabolism in human chondrocytes. Aim 1A will test the hypothesis that siRNA DNMT3A upregulates PPARγ, thus protecting chondrocytes from catabolism. Primary chondrocytes isolated from healthy donor osteochondral allografts, FAI patients, and OA patients will be divided into four groups: 1) Vehicle; 2) IL-1β; 3) IL-1β + Scramble Control; 4) IL-1β + siRNA DNMT3A. Aim 1B will assess whether siRNA DNMT3A nanoparticles (NP) influence cartilage homeostasis in vitro, using chondrocytes treated with control-NP. PCR and Western Blot will be conducted to assess DNMT3A, PPARγ, COL10A1, MMP13, ADAMTS5, and RUNX2 expression for both Aims 1A and 1B. AIM 2: We will assess the effects of intra-articular siRNA DNMT3A-NP on cartilage catabolism and OA progression using an established rabbit model of FAI-OA. Our hypothesis is that this treatment will slow OA progression compared to controls and will be more effective when combined with OCP surgery. FAI will be induced in rabbits, designating one hip for FAI and the other as a sham control. After four weeks, rabbits will be divided into five groups (n=10 each): 1) Positive control (no treatment), 2) OCP, 3) siRNA DNMT3A-NP, 4) OCP + siRNA DNMT3A-NP, and 5) Scramble Control-NP. After three months, OA will be evaluated using the OARSI score, immunohistochemistry, Fourier transform infrared spectroscopy, and gene expression analyses of IL-1β, MMP13, COL2A1, PPARγ, COL10A1. Histochemical and RNA sequencing analyses of DNMTs and PPARγ will also be conducted. AIM 3: Evaluate siRNA DNMT3A's effect on cartilage mechanics in a rabbit hip FAI-OA model. Hypothesis: Treatment with siRNA DNMT3A-NP will modulate cartilage mechanical properties, attenuating mechanical changes associated with OA progression, with greater benefit observed when combined with OCP. Cartilage samples from Aim 2, along with those from healthy cadavers, FAI patients, and end-stage OA patients undergoing THR will be analyzed using nanoindentation and finite element analysis, to assess mechanical property changes.
