Fortified Withaferin A accelerates transition from fibrovascular to bone remodeling phase during endochondral bone formation to promote ossification

Kunal Chutani ¹ Nikhil Rai ¹ Anirban Sardar ¹ ANUPAMA YADAV ¹ Divya Rai ¹ Anuj Raj ¹ Bhaskar Maji ¹ Shikha Verma ¹ Ashish Kumar Tripathi ¹ Geeta Dhaniya ¹ Lal Hingorani ² Prabhat Ranjan Mishra ¹ Ritu Trivedi ^1*

• ¹ Central Drug Research Institute (CSIR), Lucknow, India
• ² Pharmanza Herbals Pvt Ltd, Gujarat, India

This study shows that Fortified Withaferin A (FWA, 10% w/w) accelerates bone healing, advancing the fibrovascular to bone remodeling phase within 12 days, compared to the typical 23-24-day healing time. Administered orally at 125 mg.kg -1 , FWA (10% w/w) effectively downregulated bone-resorbing genes, promoted anabolic responses, and reduced inflammation. In a transverse osteotomy model, FWA (10% w/w) enhanced post-natal bone regeneration, likely via Runx-2 activation and modulation of osteogenic genes, alongside suppression of E3 ubiquitin-ligases Smurf1 and 2, resulting in significantly enhanced callus formation and healing speed. FWA (10% w/w) outperformed parathyroid hormone (PTH) in osteoclast regulation and reduced recovery time, highlighting its potential as a therapeutic agent for bone health.Micro-CT results revealed that the enhanced callus area ~95.14% elicited by FWA (10% w/w) within 12 days of treatment outperformed ~72.87% of those levels associated with normal bone-healing observed over the same period. In an estrogendeficient model, FWA l(10% w/w) ed to an ~83.88% bone volume fraction at 23 days, exceeding the ~76.80% in controls (normal healing) and matching PTH effects.Improved bone-regeneration was validated through calcein incorporation and gene expression analyses. Additionally, the material stiffness revealed significant gains in cortical bone stiffness, with the average Young's modulus rising from ~54 ±1.03 MPa to ~63 ± 2.54 MPa following FWA (10% w/w) treatment. Pharmacokinetic profiling indicated plasma exposure at 226 ng/ml*hr, and higher trough concentration at 24-hr facilitated optimum therapeutic effectiveness leading to early bone-healing transition compared to normal healing. Generally, these results demonstrate that FWA (10% w/w) could significantly enhance bone mineralization and be a potent intervention for delayed healing in osteoporotic fractures.