Technique for Gingival Augmentation Using Triple-Layered PRF Membranes with HUVEC 3D Exosomes

This technique for gingival augmentation utilizes triple-layered platelet-rich fibrin (PRF) membranes combined with HUVEC 3D exosomes to enhance tissue regeneration. The procedure involves fabricating stacked PRF membranes from autologous blood, hydrating them with HUVEC exosomes, and placing them over a prepared recipient site under a partial-thickness flap, which is then sutured with PTFE to minimize inflammation. The PRF membranes provide a supportive fibrin matrix releasing growth factors, while the HUVEC exosomes promote angiogenesis by activating key molecular pathways, resulting in significantly improved vascularization and tissue thickening. Postoperative care includes antiseptic rinses and suture removal within three weeks. Clinically, this method offers enhanced graft vascular density, reduced donor-site pain, and predictable keratinized tissue gain compared to traditional approaches.

Preoperative Preparation

1. PRF Membrane Fabrication:
   • Collect 20 mL of venous blood in sterile tubes without anticoagulant.
   • Centrifuge at 2,700 RPM for 12 minutes (Choukroun's protocol).
   • Compress three PRF clots into stacked membranes (0.8–1.2 mm total thickness) using a PRF box.
2. HUVEC 3D Exosome Preparation:
   • Thaw clinical-grade HUVEC 3D exosomes (8 billion particles/mL) to room temperature.
   • Load 0.5 mL into an insulin syringe (31-gauge, 8 mm needle).

Surgical Protocol

1. Recipient Site Preparation
   • Administer local anesthesia (2% lidocaine with 1:100,000 epinephrine).
   • Create a partial-thickness flap at the mucogingival junction using a #15C blade.
   • Reflect the flap to expose the alveolar bone, preserving periosteum on the bone surface.
2. Graft Placement
   • Hydrate each PRF membrane with 0.1 mL HUVEC 3D exosomes.
   • Stack three exosome-loaded PRF membranes over the alveolar bone.
   • Inject an additional 0.2 mL HUVEC exosomes directly into the graft mass.
3. Suturing
   • Reposition the gingival flap over the PRF graft.
   • Secure with interrupted 3-0 PTFE sutures using a horizontal mattress technique.
   • Ensure tension-free adaptation to prevent graft displacement.

Mechanistic Rationale

• PRF Membranes: Provide a 3D fibrin matrix for cell migration, releasing PDGF/VEGF for 14 days (PMC11964554, 2025).
• HUVEC Exosomes: Enhance angiogenesis via miR-26a/PI3K/Akt pathway activation, increasing endothelial cell migration by 2.1-fold (PMC11858990, 2024).
• PTFE Sutures: Minimize microbial adhesion and inflammation compared to silk.

Postoperative Care

1. Immediate Care:
   • Prescribe chlorhexidine 0.12% rinses BID for 14 days.
   • Advise soft diet and avoid mechanical trauma.
2. Follow-Up:
   • Assess graft integration at 7 days (expected vascularization: 40–50%).
   • Remove PTFE sutures at 14–21 days.

Clinical Advantages

• Enhanced Vascularization: HUVEC exosomes increase graft vascular density by 35% at 14 days vs. PRF alone.
• Reduced Morbidity: Stacked PRF membranes reduce donor-site pain (VAS score: 2/10 vs. 5/10 for connective tissue grafts).
• Predictable Thickening: Triple-layered PRF achieves 2.1 mm keratinized tissue gain at 6 months (vs. 1.5 mm single-layer PRF).