Background and Rationale
Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is widely used for osteoporosis and bone metastasis, but it induces severe cytotoxicity in oral mucosal cells and is a major contributor to MRONJ. Polydeoxyribonucleotide (PDRN), known for its regenerative and anti-inflammatory effects, may counteract ZA-induced toxicity. This study aimed to elucidate the molecular mechanism of ZA-induced growth suppression in gingival fibroblasts and to determine whether PDRN can prevent or reverse these effects.

Objectives
1. To identify the molecular pathways by which ZA suppresses growth in human gingival fibroblasts (HGF-1).
2. To evaluate the protective and therapeutic effects of PDRN against ZA-induced cytotoxicity.
3. To clarify the involvement of A2A receptor signaling and the ROS-TBK1 axis in the cytoprotective actions of PDRN.

Methods (Summary)
• Cell line: HGF-1
• Treatments: ZA (1–50 μM), PDRN (1–100 μg/mL)
• Assays:
o Cell viability
o Western blot for TBK1, PKB(Akt), STAT-3
o siRNA knockdown (TBK1, PKB, STAT-3)
o ROS measurement via DCFH-DA
o A2AR blockade (ZM241385)
• Additional test: PDRN post-treatment following 48h ZA pre-exposure

Key Findings
1. ZA significantly inhibits HGF-1 growth in a dose-dependent manner and induces marked morphological damage.
2. ZA causes:
o Reduced PKB and STAT-3 phosphorylation
o Strong activation of TBK1
3. TBK1 knockdown partially rescues ZA-treated cells, whereas PKB/STAT-3 knockdown further decreases viability.
4. PDRN (100 μg/mL) effectively prevents and reverses ZA-induced cytotoxicity, associated with:
o Suppression of TBK1 hyperphosphorylation
o Partial restoration of PKB activity
o Minimal restoration of STAT-3
5. ZA induces a sharp ROS increase at 8h; both PDRN and NAC effectively reduce ROS.
6. PDRN post-treatment after prior ZA exposure rescues cell growth and reduces TBK1 activation.

Conclusion
ZA suppresses the growth of gingival fibroblasts through ROS elevation, TBK1 activation, and inhibition of PKB/STAT-3. PDRN counteracts these effects primarily by reducing oxidative stress and inhibiting TBK1 activation, offering a mechanistic basis for its potential use in preventing or managing MRONJ-related soft-tissue toxicity.