Enhancing Bone Healing Using Carvacrol on Calcium Phosphate Substrates

Carvacrol has therapeutic potential due to its anti-inflammatory, antioxidant, and antimicrobial properties. Here, Carvacrol was incorporated on three-dimensionally printed tricalcium phosphate (3DP TCP), hydroxyapatite-pressed (HAP) discs, and plasma-sprayed HA-coated Ti6Al4V (HA-Ti64) implants to evaluate its localized release and impact on bone regeneration. Carvacrol release from HA-Ti64 reaches a ∼100% within 3 days in acidic pH, whereas from HAP discs, it releases 60% over 14 days. <i>In vitro</i> studies using osteoblasts show a 20% increase in cell viability on Carvacrol-treated HAP substrates than control. Osteoclast assays show a 64% reduction in TRAP activity and disrupted actin ring formation in carvacrol loaded substrate. Coculture of hMSCs and THP-1 monocytes shows enhanced osteogenic gene expression, a 4.7-fold increase in <i>RUNX2</i>, and a 0.3-fold reduction in <i>RANKL</i> expression than the control, by day 21. Angiogenesis assays show a ∼28% increase in tubular segment formation for Carvacrol-loaded scaffold than control. <i>In vivo</i>, Carvacrol-loaded HA-Ti64 implants in a rat distal femur model exhibit a 30% increase in new bone formation compared to control. These results confirm that Carvacrol-loaded calcium phosphate substrates enhance osteogenesis, suppress osteoclastogenesis, and promote angiogenesis, offering a promising strategy for localized, plant-based therapies in bone tissue engineering.