Synthesis and Characterization of Melatonin-Loaded Chitosan Microparticles Promote Differentiation and Mineralization in Preosteoblastic Cells
In terms of a novel scaffold with well good osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation. The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro. The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy. The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on preosteoblastic MC3T3-E1 cells was characterized in vitro. An in vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period. In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced alkaline phosphatase (ALP) mRNA expression and ALP activity compared with the control group. Meanwhile, the osteoblast-specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), collagen I (Col I), and osteocalcin (Ocn), were also significantly upregulated. Furthermore, quantificational alizarin red–based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared with controls. In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of preosteoblast cells in vitro.
The morphology of chitosan microparticles (CS MPs) and melatonin (Mel)-loaded CS MPs. Scanning electron microscopy photographs of (a, c) surfaces and (b, d) cross sections of CS MPs and Mel-loaded CS MPs. The CS used was 156 and 330 kDa at a concentration of 2% and 3% prepared by ionic cross-linking (IC) (upper panel, IC and melatonin-loaded IC CS MPs) and oil-in-water emulsion (Em) (lower panel, Em and melatonin-loaded Em CS MPs) methods, respectively. Morphologic characterization and diameter measurements were conducted using a field emission scanning electron microscopy system at an accelerating voltage of 15 kV. The magnification of the representative images is ×2000.
Fourier transform infrared spectroscopy analysis and the stability analysis and accumulated in vitro release profile of melatonin (Mel) from Mel-loaded chitosan microparticles (CS MPs). (a) The Fourier transform infrared spectroscopy was used to examine the spectra of tripolyphosphate (TPP), CS, ionic cross-linking (IC), and oil-in-water emulsion (Em) methods, which the wave number (−1) of representative groups, such as PO43− (710 899 cm−1), N-H (1576 cm−1), and C=O (1659 cm−1), is illustrated. (b) The stability analysis was performed by analyzing the weight remaining of IC and Em and melatonin-loaded IC CS MPs (IC-M) and melatonin-loaded Em CS MPs (Em-M) as a function of degradation time. The accumulated in vitro release kinetics of Mel from the (c) IC-M and (d) Em-M methods. Data represent mean ± SD of at least 4 independent experiments.
Melatonin (Mel)-loaded microparticles (MPs) facilitate cell adhesion and enhance alkaline phosphatase activity of MC3T3-E1 cells. MC3T3-E1 cells (1 × 104 cells/well) were incubated, and cell viability was quantified by (3-(4,5-dimethylthiazol-2yl)-,5-diphenyl-2H-tetrazoliumbromide) calorimetric assay with (a) ionic cross-linking (IC) and (b) oil-in-water emulsion (Em) directly or with leaching solution (IC leaching solution and Em leaching solution) for 72 hours. Scanning electron microscopy observation of MC3T3-E1 morphology after a 2-day culture in direct contact with the (c) melatonin-loaded IC CS MPs (IC-M) and (d) melatonin-loaded Em CS MPs (Em-M) methods. (e) MC3T3-E1 cells were induced toward osteogenic differentiation in cell culture medium (noninduced group, medium alone) or osteogenic-induced medium (induced group, osteogenic-induced medium), Mel (17.2 mM), IC, Em, IC-M, and Em-M. After 7 days, the cells were lysed, and the clear supernatant was used to measure ALP activity. Data represent mean ± SD of at least 5–7 independent experiments. *P < .05, **P < .01, ***P < .001: difference from values between IC-M (osteogenic medium) and Mel (osteogenic medium). #P < .05, ##P < .01, ###P < .001: difference from values between IC-M (osteogenic medium) and IC (osteogenic medium) (1-way ANOVA).
Melatonin (Mel)-loaded microparticles (MPs) upregulate the expression of markers of osteoblast differentiation and accelerate mineralization matrix formation of MC3T3-E1 cells. MC3T3-E1 cells were cultured with samples including blank control group (Control, medium alone), osteogenic-induced medium (OS), Mel (17.2 mM), ionic cross-linking (IC) and oil-in-water emulsion (Em), melatonin-loaded IC CS MPs (IC-M), and melatonin-loaded Em CS MPs (Em-M) in the presence or absence of osteogenic-induced medium to initiate differentiation. (a) Expressions of runt related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteocalcin (Ocn), bone morphogentic protein-2 (Bmp2), and collagen I (Col I) were measured by qRT-PCR and normalized to β-actin expression at 7 and 11 days, respectively. Relative expression levels of each gene were calculated using the 2–ΔΔCt method. The mineralization matrix formation was measured by Alizarin red S staining at day 11. (b) Entire plate view of the Alizarin red S staining in 24-well plates. (c) Optical images of Alizarin red S staining for each sample in the presence or absence of osteogenic-induced medium. (d) Absorbance was quantified by spectrophotometry at 562 nm. Data represent mean ± SD of at least 3–5 independent experiments. *P < .05, **P < .01, ***P < .001: difference from values of OS in the 11-day group. #P < .05, ##P < .01, ###P < .001: difference from values of Mel in the 11-day group (1-way ANOVA).
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