METHODS: MC3T3-E1 cells were seeded on HA and treated with recombinant IL-6 or rIL-17A or combination of the two cytokines. Cell proliferation and differentiation activity were measured by MTS and alkaline phosphatase assays respectively. Observation of cell adhesion and proliferation was examined by scanning electron microscopy. Gene and protein expressions were performed on RANKL and OPG using qPCR, Western blot and ELISA.

AIM OF THE STUDY: To evaluate the effects of EL on the time-mannered sequential proliferative, differentiative, and morphogenic modulation in osteoblasts compared with testosterone.

MATERIALS AND METHODS: Cell proliferation was analysed using MTS assay and phase contrast microscopy. Osteogenic differentiation of MC3T3-E1 cells was assessed through a series of characteristic assays which include crystal violet staining, alkaline phosphatase (ALP) activity and Van Gieson staining. Taken together, the bone mineralization of extra cellular matrix (ECM) was estimated using alizarin red s (ARS) staining, von kossa staining, scanning electron microscopic (SEM) and energy dispersive x-ray (EDX) analysis.

RESULTS: The cell proliferation data clearly revealed the efficiency of EL particularly at a dose of 25µg/mL, in improving the growth of MC3T3-E1 cells compared with the untreated cells. Data also showed the prominence of EL in significantly promoting ALP activity throughout the entire duration of treatment compared with the testosterone-treated cells. The osteogenic differentiation potential of EL was further explored by analysing mineralization data which revealed that the calcified nodule formation (calcium deposition) and phosphate deposition was more pronounced in cells treated with 25µg/mL concentration of EL at various time points compared with the untreated and testosterone treated cells. The scanning electron microscopic (SEM) analysis also revealed highest globular masses of mineral deposits (identified as white colour crystals) in the ECM of cultured cells treated with 25µg/mL concentration of EL.

Materials and methods: QOS collagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%-10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts.

Results and discussion: The study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution - at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed.

METHODS: Prior to analyzing the ability of this novel combined herbal therapy to promote aspects of bone regeneration, its cytotoxicity was determined using MC3T3-E1 cells (pre-osteoblast model). Cell proliferation was evaluated using phase-contrast microscopy and cell differentiation was estimated using alkaline phosphatase activity. The effect of the combined herbal therapy (CUR + FLL) was also assessed in terms of mineralization in the extracellular matrix (ECM) of cultured cells. Further, to explore the molecular mechanisms of bone formation, time-dependent expression of bone-regulating protein biomarkers was also evaluated.

RESULTS: Combined herbal therapy (CUR + FLL) significantly upregulated the viability, proliferation and differentiation of MC3T3-E1 cells compared to the monotherapy of CUR or FLL. The magnitude of ECM mineralization (calcium deposition) was also higher in MC3T3-E1 cells treated with combined therapy. The time-dependent expression of bone-forming protein biomarkers revealed that the tendency of expression of these bone-regulating proteins was remarkably higher in cells treated with combined therapy.

MATERIALS AND METHODS: Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001-1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively.

RESULTS: The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner (P<0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups (P<0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 (P<0.05).