An alternative osteo-healing formulation with osteo-healing properties was formulated by combining gentamicin and Nigella sativa oil (NSO) in a form of gentamicin-N. sativa fusion emulsion (GNFE). This work aims to formulate a stable emulsion and to study the effects of GNFE on UMR-106 osteoblast-like rat osteosarcoma cell line in vitro and its related mechanisms of bone healing and regeneration. Emulsion A, B, C and D had been formulated, with final concentration of gentamicin was made constant at 0.1%, whereas NSO concentration was varied at 32.5%, 35.0%, 40.2% and 46.4% in all formulations respectively. Stability studies of emulsion A, B, C and D were performed at different storage conditions (8°C, 25°C and 50°C), followed by in vitro study of MTT assay, Alizarin Red S (ARS) staining, von Kossa staining and quantification, alkaline phosphatase (ALP) quantification and quantitation of collagen type-1 and osteocalcin (qPCR). Results showed that all emulsions were stable at storage temperature of 8°C. In vitro results showed that emulsion D produced the highest cell viability (97.1%) at 72 hours of post-incubation. The highest mineral deposits (2.64 ± 0.05) and ALP activity (2.19 ± 0.3 nmol) was produced by emulsion D at day 21. Lastly, the highest expression of collagen type-1 (29.4 ± 1.01 folds) and osteocalcin (1.8 ± 0.51 folds) were expressed by the cells treated with emulsion C. Thus, stable GNFE may have the ability to promote bone formation.

Local treatment of osteomyelitis using gentamicin-loaded PMMA beads has several challenges such as (i) requires invasive surgery to remove the beads from the implant site, (ii) can become the site of adherence for biofilm producing bacteria and (iii) has reduced effectiveness against biofilm bacteria. Therefore, in this study, gentamicin was fused with Nigella sativa oil (NSO) to be formulated as an optimised emulsion. The emulsion was then combined with clay-chitosan beads to improve gentamicin’s efficacy and reduce the risk of toxicity. During the pre-formulation phase, the physical mixtures of gentamicin, NSO, and other excipients were subjected to compatibility test using differential scanning calorimetry (DSC) and attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR). The excipients used were Span 20, Span 80, Tween 20, Tween 40, Tween 80, Triton X-100 (TX100), polyvinyl alcohol (PVA), and polyethylene glycol 400 (PEG400). Design of experiment (DOE) was employed to develop the formulation of gentamicin-N. sativa oil emulsion (GNE). Plackett-Burman design (PBD) was used to identify the significant factor(s) and understand the effect of several factor(s) on emulsion droplet size (Y1), and polydispersity index (PDI) (Y2). These factors include (i) NSO concentration (X1), (ii) emulsifier concentration (X2-X12), (iii) type of machine (X13), (iv) homogenisation time (X14) and (v) rate (X15). Central composite design (CCD) was utilised to predict the best formulation of GNE. The stability of GNE was further characterised based on droplet size, PDI, and zeta potential after centrifugation 4000 rpm for 20 min. Antimicrobial test of the emulsion was conducted against osteomyelitic P. aeruginosa, S. aureus, and S. epidermidis strains. GNE-loaded clay-chitosan beads were then fabricated. Furthermore, scanning electron microscope (SEM) was utilised to observe the morphology of the beads. To quantify gentamicin in the beads, a quantification method validation was carried out using ATR-FTIR following ICH Q2 (R1) guideline. From the compatibility test, the result indicated that GS interacted physically with NSO and the excipients, but lacked in the chemical interactions that may compromise the efficacy and safety of GS in further formulation. The main effect analysis of each significant factors showed that adding NSO into the emulsion would significantly increase the droplet size, whereas the presence of Tween 40 and TX100 was able to decrease the droplet size. Stable emulsion with low PDI value (<0.5) was achieved by using sonicator and increasing the concentration of Tween 20, 40, and 80. NSO and Tween 80 were selected to be further investigated during GNE optimisation. The formulation with NSO concentration of 50% v/v and Tween 80 6% v/v was selected to be the optimum formulation with 292.00 ± 36.52 nm, 0.21 ± 0.04, and -31.23 ± 1.24 mv on droplet size, PDI, and zeta potential, respectively. Antimicrobial test showed that the combination of gentamicin and NSO in the optimised emulsion was able to enhance its efficacy of antimicrobial effect. The optimised GNE was successfully incorporated with clay-chitosan to form beads with the average diameter size of 4.27 ± 0.14 mm. SEM image showed the morphology of the beads appeared as a nonporous surface. The method validation for quantification of gentamicin using ATR-FTIR successfully met the acceptance criteria underlined by ICH Q2 (R1) guideline. These findings will serve as baseline research to develop new strategy in treating osteomyelitis.