ABSTRACT: The specific objective of this study was to stabilize a simple valid method to prepare pure nanorod hydroxyapatite (HA) mixed with berberine chloride (BER) and Moghat water extract (ME) as composites for incorporation into cellulose acetate (CA) nanofibers to be used as novel bone scaffolds and to determine their efficacy in bone regeneration process In Vitro. Preparation of HA/BER and HA/ME composites were performed by mixing powders using the ball-milling machine. The HA, HA/BER, and HA/ME composites at a concentration of 6.25, 12.5, 25, 50, 100, and 200 mg were mixed with CA solution (13%), then the fiber was formed using electrospinning technique. The properties of the obtained CA fibers were investigated (SEM, TEM, EDX, FTIR, TGA, water uptake, porosity, and mechanical tests). The efficacy of HA and HA composites loaded into CA nanofiber on osteoblast and osteoclast differentiation were measured by tacking ALP, osteocalcin, TRAcP, calcium, and total protein concentration. Moreover, their effects on cell differentiation (CD90 and PARP- ɣ) and death markers (GSK3b, MAPK, Wnt-5 and β-catenin) were evaluated by using ELISA and qPCR. The obtained TEM results indicated that the continuous CA and CA/HA composites electrospun fibers have ultrafine fiber diameters of about 200 nm and uniform distribution of discrete n-HA clusters throughout. In addition, hydrocortisone (HCT) was found to increase the formation of adipocytes and osteoclastic markers CD90 and p38-MAPK which indicated the bone lose process take placed. Treatment with CA loaded with HA, HA/BER or HA/ME decreased CD90, Wnt-5, PARP- ɣ, GSK3b and p38-MAPK associated elevation of osteogenic markers: ALP and osteocalcin. Moreover, HCT overexpressed RANKL and down expressed Osterix gene. Treatment with CA/HA/BER or CA/HA/ME downregulated RANKL and upregulated Osterix associated with a reduction in RANKL/OPG ratio, at p < 0.05. In conclusion, novel CA composite nanofibers (CA/HA/BER and CA/HA/ME) reversed the HCT adverse effect on osteoblast cell death through canonical and non-canonical pathways regulated by Wnt/β-catenin and Wnt/Ca(2+) pathways. Furthermore, our data confirmed that the novel scaffolds create a crosstalk between RUNX-2, RANKL, p38-MAPK, and Wnt signals which positively impact bone regeneration process. Treatment with CA/HA/BER is better compared to the treatment with CA/HA/ME. Nevertheless, both are considered as alternative biomaterial scaffolds with a potential for biomedical applications in the field of bone tissue engineering.
