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.

Guided bone regeneration was introduced as a therapeutic modality in recent years, which consists of using bone substitutes to facilitate bone healing and to enhance bone regeneration.1  Numerous studies have provided substantial evidence on bone substitutes stimulating osteogenic differentiation in vitro and enhancing bone formation in vivo. Now, bone substitutes are considered a crucial factor in bone regeneration.1,2 

Chitosan (CS) has excellent performance as a biodegradable scaffold in tissue engineering because of its biocompatibility and functional versatility.3,4  Various forms of CS scaffold have been developed and engineered, which have the capability to control the release of growth factors derived from the integrated scaffold, such as microspheres and microparticles (MPs).5,6 

Melatonin (N-acetyl-5-methoxytryptamine), well known for its wide range of beneficial effects, has been discussed for several decades.7  In terms of a novel scaffold with good osteoinductive and osteoconductive capacity, melatonin (Mel) possesses an advantageous effect on chemical linkage in scaffold structures, which may allow osteogenic differentiation.7,8  By controlling and prolonging the release of Mel from the scaffold, the microenvironment with a relatively stable concentration of Mel may optimize the advantageous effects on bone regeneration.7 

The aim of this study is to fabricate Mel-loaded CS MPs as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs and evaluating its impact on the osteogenic capacity of preosteoblasts MC3T3-E1 cells in vitro.

Chitosan was purchased from a commercial company with a molecular weight 156 and 330 kDa and deacetylation degree ≥ 75% (Sigma Chemical Company, St Louis, Mo). The MC3T3-E1 cells, a clonal preosteoblastic cell line, was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). All the other chemicals and laboratory consumables were of reagent grade and were purchased from Sigma Chemical unless stated otherwise.

Preparation of CS MPs

The CS MPs were prepared by 2 different methods: ionic cross-linking and oil-in-water emulsion methods. For the ionic cross-linking method, the CS solution (2% and 3%, wt/vol) was prepared by dissolving CS (60 and 90 mg) in acetic acid (0.5 M) at room temperature. The CS solution was dropped into 10% tripolyphosphate solution, and the CS MPs were washed and lyophilized accordingly.9  For the oil-in-water emulsion method, Tween 80 (60 μL) was added into the CS solution to be prepared as aqueous phase. The oil phase, 300 μL dicholoromethane, was mixed with the aqueous phase (CS solution) by homogenizer and centrifuged (5000 rpm) for 1 minute. The oil-in-water emulsion was dropped into tripolyphosphate solutions, separated, washed with double distilled water, and lyophilized accordingly.10  The CS MPs prepared by ionic cross-linking and oil-in-water emulsion methods were named as IC and Em, respectively.

Preparation of Mel-loaded CS MPs

To fabricate Mel-loaded CS MPs using the ionic cross-linking method, Mel (13.5 g) was dispersed in CS solution and then followed the procedure for ionic cross-linking CS MPs accordingly. For the oil-in-water emulsion method, the same weight of Mel was dissolved in dicholoromethane solution, and then the oil phase was mixed with the aqueous phase. Subsequently, the procedure was followed by the oil-in-water emulsion CS MPs. The ionic cross-linking and oil-in-water emulsion CS MPs (IC and Em) that were Mel loaded were named IC-M and Em-M, respectively.

Characterization of CS MPs, Mel-loaded CS MPs, and MC3T3-E1 cell attachment morphology

To observe the morphology of fabricated CS MPs and MC3T3-E1 cell attachment, specimens were dehydrated in graded alcohols and then sputter-coated with gold-palladium; a scanning electron microscope (SEM; JSM-7600F; JEOL Ltd, Japan) was operated at an accelerating voltage of 15 kV.11 

Fourier transform infrared spectrometer (FTIR; Bio-Rad, Richmond, Calif) were used to analyze the chemical structure of CS MPs and to detect evidence of cross-link formation.12  The in vitro degradation of the CS MPs was performed according to American Society for Testing and Materials (F-1635-95).

To evaluate the encapsulation efficiency of Mel in Mel-loaded CS MPs, the amount of free Mel in supernatants was quantified by reading its absorbance at 220 nm using a spectrophotometer (SpectraMax 340PC384; Molecular Devices, San Jose, Calif). The encapsulation efficiency of Mel was determined using the following equation13:
formula

In vitro release profile and Mel release kinetics of Mel-loaded CS MPs

The Mel-loaded CS MPs were suspended in phosphate-buffered saline (PBS) containing 0.02% Tween 80 and then incubated at 37°C with agitation. At specified intervals, 2 mL supernatant was extracted and replenished with fresh PBS, and the concentration of Mel was determined by a spectrophotometer at 220 nm.14 

To determine the drug release mechanism of Mel-loaded CS MPs in the formulation by fitting release profiles with the classical equation15:
formula
where Mt and M represented the amount of drug released at time t and saturation, n is a diffusion exponent, and k is a characteristic constant. The n values for Fickian diffusion, anomalous diffusion, and case II transport were 0.43, 0.43–0.85, and 0.85, respectively.

Cell viability assay

The cytotoxicity of Mel and CS MPs to MC3T3-E1 cell proliferation was evaluated using MTT (3-(4,5-dimethylthiazol-2yl)-,5-diphenyl-2H-tetrazoliumbromide) assays described previously.16  The leaching solutions were prepared by mixing CS MPs prepared by ionic cross-linking and oil-in-water emulsion methods with 100 mL α-MEM and then centrifuged to extract supernatant. The MC3T3-E1 cells (1 × 104 cells/well) were cultured with MPs directly or with the leaching solution for 72 hours. The medium was removed, and 20 μL MTT solution was added to each well for an additional 2 hours. After the removal of solutions, dimethyl sulfoxide was added to dissolve formazan products, and the plates were analyzed with a spectrophotometer at 570 nm.

Alkaline phosphatase activity

Alkaline phosphatase (ALP) activity was determined as described previously.14  The cells were incubated with samples including the blank control group, Mel (17.2 mM), CS MPs, and Mel-loaded CS MPs in the presence or absence of osteogenic-induced (OS) medium to initiate differentiation. After 7 days, the cells were lysed and centrifuged with 0.2% Triton X-100. The supernatant was used to measure ALP activity, which was determined by spectrophotometer at 405 nm using p-nitrophenyl phosphate as the substrate.

Quantitative real-time polymerase chain reaction

To quantitatively assess osteoblastic-specific genes, such as runt related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteocalcin (Ocn), bone morphogentic protein-2 (Bmp2), and collagen I (Col I), quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. The β-actin gene was chosen as the housekeeping gene. The special forward primers and reverse primers of the representative specific osteoblastic genes were designed (Supplemental Table 1). The value 2–ΔΔCt (a logarithm of Ct value that presents the threshold of the gene amplification circles) demonstrated the fluorescence expression level of these characteristic genes relative to the control group.

Mineralization matrix formation assay

The extent of mineralized matrix was determined by Alizarin red S staining as described previously.14  Cells were fixed in 70% ethanol, washed with PBS, and stained with Alizarin red S (40 mM at pH 4.2) for 10 minutes at room temperature. The staining was released from the cell matrix by incubation in 10% (wt/vol) cetylpyridinium chloride for 15 minutes. The plate containing stained matrix was photographed, and the amount of dye released was quantified by spectrophotometry at 562 nm.

Statistical analysis

All values are presented as the mean ± SD for 4 measurements. Differences between treated and untreated control groups were assessed by Student's t test. Multiple comparisons were evaluated by 1-way analysis of variance (ANOVA), followed by Scheffe's F test. Statistical analysis was performed with the statistical software package (PASW statistics 22.0; SPSS Inc, Munich, Germany). P < .05 was considered to indicate statistical significance.

Characterization of CS MPs and Mel-loaded CS MPs

The CS particle size was positively correlated to its molecular weight and concentration (Figure 1). When the molecular weight or concentration of CS increased, the pore size of both ionic cross-linking and oil-in-water emulsion fabricated CS MPs opposingly decreased. Notably, regardless of fabrication method, similar morphologic findings (ie, particle size and surface appearance) were also observed in Mel-loaded CS MPs (Figure 1). However, the internal pore size was smaller in the CS MPs with loaded Mel (Figure 1d) than without (Figure 1b). The characteristic peaks of CS, tripolyphosphate, and CS MPs (ionic cross-linking and oil-in-water emulsion) were presented by FTIR spectroscopy (Figure 2), with CS MPs possessing a superposed band of the −PO43− group on tripolyphosphate and the amine/amide groups on CS, revealing that CS MPs were effectively fabricated.

Figure 1.

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.

Figure 1.

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.

Close modal
Figure 2.

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.

Figure 2.

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.

Close modal

Encapsulation efficiency of fabricated CS MPs and Mel-loaded CS MPs

The encapsulation efficiency is increased along with increasing molecular weight of CS (Table 1). Additionally, higher CS concentration resulted in higher encapsulation efficiency of Mel. However, there is no significant difference of encapsulation efficiency between different preparation methods (Table 1).

Table 1

The encapsulation efficiency (%) of melatonin in melatonin-loaded chitosan microparticles fabricated by ionic cross-linking (IC-M) and oil-in-water emulsion (Em-M) methods

The encapsulation efficiency (%) of melatonin in melatonin-loaded chitosan microparticles fabricated by ionic cross-linking (IC-M) and oil-in-water emulsion (Em-M) methods
The encapsulation efficiency (%) of melatonin in melatonin-loaded chitosan microparticles fabricated by ionic cross-linking (IC-M) and oil-in-water emulsion (Em-M) methods

Stability and in vitro release profile and kinetics of Mel-loaded MPs

For the stability analysis, oil-in-water emulsion fabricated CS MPs degraded faster than the ones prepared using the ion cross-linking. After encapsulating Mel, both Mel-loaded CS MPs had similar degradation rates, following 9 days of incubation (Figure 2b).

The in vitro release profiles of Mel were similar in IC-M (Figure 2c), and EM (Figure 2d). For the concentration of CS of 2%, both 156- and 330-kDa Mel was released constantly for 216 hours, whereas the linear curve exhibited a zero-order delivery of Mel in both systems when the CS molecular weight is 330 kDa and concentration is at 3% (Figure 2). The Mel released 216 hours later was 44.27% and 37.35% of IC-M and Em-M, respectively (Figure 2). The estimated parameters are release kinetics of 156-kDa CS MPs are Fickian diffusion and 330-kDa CS MPs are anomalous transport (Table 2).

Table 2

Estimated parameters and drug release mechanism of the prepared microparticles at physiologic environment*

Estimated parameters and drug release mechanism of the prepared microparticles at physiologic environment*
Estimated parameters and drug release mechanism of the prepared microparticles at physiologic environment*

Mel-loaded MPs facilitate cell attachment and enhance ALP activity of MC3T3-E1 cells

Specifically, there were no apparent enhancement of cellular proliferation when CS MPs (IC and Em) were added either after exposure to an osteogenic-induced medium or as a supplement (Figure 3a and 3b), indicating that fabricated CS MPs had no apparent inhibitory effect on the growth of osteoblastic cells.

Figure 3.

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).

Figure 3.

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).

Close modal

MC3T3-E1 had an almost spread out appearance, with multiple peripheral filopodia and cytoplasmic extensions both on IC-M (Figure 3c) and Em-M (Figure 3d). Moreover, there was no significant enhancement in the ALP activity of cells exposed to Mel for 7 days at a concentration of 17.2 mM, which is equivalent to the concentration of the in vitro release profile. However, sustained released Mel from IC-M, but not Em-M, supplemented with OS medium significantly increased ALP activity compared with IC and Mel (OS medium) groups (Figure 3e).

Mel-loaded MPs upregulate the expression of osteoblast-specific mRNA and accelerate calcium mineralization formation of MC3T3-E1 cells

Sustained release of Mel from MPs exhibited notable induction in expression levels for all investigated osteoblast-specific genes (Figure 4a). Compared with the osteogenic-induced medium alone, there is a noticeable increase in calcium deposition in MC3T3-E1 cultured in the osteogenic-induced medium with the addition of Mel-loaded CS MPs (Figure 4b and 4c). Furthermore, calcium deposition significantly increased in groups with Mel-loaded CS MPs (IC-M and Em-M) compared with the Mel group (Figure 4; P < .001).

Figure 4.

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).

Figure 4.

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).

Close modal

Bone tissue engineering has developed rapidly with the use of three-dimensional (3D) scaffolds to promote osteoblastic cell attachment, proliferation, and differentiation, and stimulating new tissue growth, which should be free of antigenic effects, biocompatible, biodegradable, have space-making capacity, and have osteoinductive properties, which serve as a suitable microstructure (pore size and porosity) for tissue engineering.2,8,17  In the present study, the physical-chemical properties of fabricated MPs, including morphology and interconnective microstructures, were characterized by scanning electron microscopy (Figure 1) and FTIR spectroscopy (Figure 2). Although the size of interconnective pores reduced when fabricated CS MPs are encapsulated with Mel (Figure 1), our findings revealed that bone-forming osteoblasts were well proliferated, readily attached to the scaffold surface (Figure 4), enhanced osteoblast-specific genes expression, and deposited mineralized matrix (Figure 4) on the fabricated Mel-loaded CS MPs, which are all essential parameters to promote the bone regeneration process.

A series of reports has demonstrated that Mel has been recognized to facilitate osteogenic differentiation in several kinds of cells in vitro and to reduce bone loss and enhance bone formation in vivo.8,14,18,19  In this study, Mel is effectively encapsulated into CS MPs (Table 1), Mel sustained release systems were prepared by ionic cross-linking and oil-in-water emulsion(Figure 2), and the characteristics of promoting osteogenesis were verified (Figure 4), indicating Mel might provide promising roles in bone regeneration in the clinical setting.

This study also attempted to identify the sustained release mechanism in the formulation by fitting release profiles with the classic equation.15  Notably, for the comparison of CS molecular weight, CS MPs with relative lower molecular weight (156 kDa) exhibited larger interconnective pore size than those with higher molecular weight (330 kDa; Figure 1), which resulted in a higher diffusion rate of Mel (Table 2), indicating that Fickian diffusion, a diffusion-controlled release mechanism, is presented. When CS MPs were 330 kDa, the smaller interconnective pore size slowed down the diffusion rate of Mel (Figure 1), suggesting a combination of diffusion and relaxation release mechanism (Table 2). In general, the release rates of Mel from IC-M exceed than those from Em-M (Figure 2). The obvious difference between the 2 fabrication systems was that Mel is homogeneously dispersed in the whole MPs by using the ionic cross-linking method; however, Mel was mainly encapsulated in the center of MPs by using the oil-in-water emulsion method. This might explain, at least in part, why Mel in Mel-loaded ionic cross-linking CS MPs may diffuse into the medium more easily than in oil-in-water emulsion CS MPs, resulting in a higher release rate. Moreover, the biodegradation rate of MPs may be another key factor affecting Mel release (Figure 2).

Osteoblast proliferation and differentiation are crucial steps during bone regeneration, and the activity of ALP has been known as a characteristic early marker for differentiation of osteoblasts during osteogenesis.2  Our finding is in line with previous studies that constant administration of Mel is needed for ALP activity and calcium deposition.14,18  Therefore, Mel-loaded CS MPs, a novel Mel sustained release system, was capable of stimulating osteogenic differentiation in vitro.

Based on current results, the supplementation with pharmacologic doses of Mel are beneficial to treat bone-related disorders, including osteoporosis,20  fracture healing,21  inflammatory bone resorption,22,23  and osseointegration of dental implants.7  Topical application of Mel may minimize harmful effects than systemic administration.7  On the contrary, the major disadvantages of topical application would be that, although a higher initial dosage of Mel may offer a greater therapeutic effect, it is associated with increased toxicity and with shorter duration, which may be restrictive in clinical situations.14,24  Therefore, to keep an adequate pharmacologic concentration of Mel in the local microenvironment, long-term, controlled release of Mel is especially warranted to develop in biomedical devices and the pharmaceutical industry.

In this study, this sustained controlled release system provides scientific rationale that Mel-loaded CS MPs may possess beneficial effects for better bone tissue regeneration. However, there were some limitations concerning this Mel sustained controlled release system. First, Mel-loaded CS MPs can accelerate osteogenic differentiation of preosteoblast cells in vitro through controlled release of Mel over a period of time. Further in vivo studies would be needed to create a sound data-driven foundation for successful product advancement, which may lead to new findings in tissue engineering and the potential for clinical applications in the future. Additionally, this sustained release system may potentially be used as a bone growth stimulator in the future; manipulating and facilitating the application of Mel-loaded CS MPs during bone remodeling process (ie, anabolic and catabolic effects) would be essential before clinical practice. With the best chance to achieve predictable and desired clinical results, objectives regarding systemic approaches to assess developability potential in advancing structural and chemical properties of bone graft substitutes would be an attractive concept worthy of further consideration in bone tissue engineering.1,5,6 

Mel-loaded CS MPs, a simple Mel sustained release system, can release Mel in a controlled manner to generate a microenvironment with relatively stable concentrations of Mel, thus accelerating osteogenic differentiation of preosteoblast cells in vitro.

Abbreviations

Abbreviations
ALP

alkaline phosphatase

Bmp2

bone morphogentic protein-2

Col I

collagen I

CS

chitosan

Em

oil-in-water emulsion

Em-M

melatonin-loaded oil-in-water emulsion CS microparticles

FTIR

fourier transform infrared spectrometer

IC

ionic cross-linking

IC-M

melatonin-loaded ionic cross-linking CS microparticles

Mel

melatonin

MPs

microparticles

MTT

(3-(4,5-dimethylthiazol-2yl)-,5-diphenyl-2H-tetrazoliumbromide)

Ocn

osteocalcin

qRT-PCR

quantitative reverse transcriptase-polymerase chain reaction

Runx2

runt related transcription factor 2

The authors acknowledge Dr Wu-Chien Chien (Department of Public Health, National Defense Medical Center, Taipei, Taiwan) as an independent statistician for reviewing statistical analysis, materials and methods, results, and conclusion sections. The authors also thank Yu-Fang Huang (School of Dentistry, National Defense Medical Center) for help in English editing. This project was supported by the International Team for Implantology Foundation, Switzerland (Grant 880_2012). This study was also partially supported by the Ministry of Science and Technology (MOST 104-2221-E-019-017 -MY3), Taiwanese Ministry of National Defense (D101-12-3, MAB-105-091, MAB-107-096), Tri-Service General Hospital (TSGH-C108-030, TSGH-C108-184, TSGH-D-109042, TSGH-D-109169), Chi Mei Medical Center (CLFHR10627, CLFHR10726, CMNDMC10909), and Teh-Tzer Study Group for Human Medical Research Foundation (B1031087, B1051039).

The authors have no conflicts of interest relevant to this article.

1. 
Wessing
B,
Lettner
S,
Zechner
W.
Guided bone regeneration with collagen membranes and particulate graft materials: a systematic review and meta-analysis
.
Int J Oral Maxillofac Implants
.
2018
;
33
:
87
100
.
2. 
Gruber
R,
Stadlinger
B,
Terheyden
H.
Cell-to-cell communication in guided bone regeneration: molecular and cellular mechanisms
.
Clin Oral Implants Res
.
2017
;
28
:
1139
1146
.
3. 
Ribeiro
JCV,
Vieira
RS,
Melo
IM,
Araujo
VMA,
Lima
V.
Versatility of chitosan-based biomaterials and their use as scaffolds for tissue regeneration
.
Sci World J
.
2017
;
2017
:
8639898
.
4. 
Ahsan
SM,
Thomas
M,
Reddy
KK,
et al.
Chitosan as biomaterial in drug delivery and tissue engineering
.
Int J Biol Macromol
.
2018
;
110
:
97
109
.
5. 
Boukari
Y,
Qutachi
O,
Scurr
DJ,
et al.
A dual-application poly (dl-lactic-co-glycolic) acid (PLGA)-chitosan composite scaffold for potential use in bone tissue engineering
.
J Biomater Sci Polym Ed
.
2017
;
28
:
1966
1983
.
6. 
Meng
D,
Dong
L,
Wen
Y,
Xie
Q.
Effects of adding resorbable chitosan microspheres to calcium phosphate cements for bone regeneration
.
Mater Sci Eng C Mater Biol Appl
.
2015
;
47
:
266
272
.
7. 
Arora
H,
Ivanovski
S.
Melatonin as a pro-osteogenic agent in oral implantology: a systematic review of histomorphometric outcomes in animals and quality evaluation using ARRIVE guidelines
.
J Periodontal Res
.
2017
;
52
:
151
161
.
8. 
Clafshenkel
WP,
Rutkowski
JL,
Palchesko
RN,
et al.
A novel calcium aluminate-melatonin scaffold enhances bone regeneration within a calvarial defect
.
J Pineal Res
.
2012
;
53
:
206
218
.
9. 
Shu
XZ,
Zhu
KJ.
A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery
.
Int J Pharm
.
2000
;
201
:
51
58
.
10. 
Ko
JA,
Park
HJ,
Hwang
SJ,
Park
JB,
Lee
JS.
Preparation and characterization of chitosan microparticles intended for controlled drug delivery
.
Int J Pharm
.
2002
;
249
:
165
174
.
11. 
Huang
Y-C,
Yang
C-Y,
Chu
H-W,
Wu
W-C,
Tsai
J-S.
Effect of alkali on konjac glucomannan film and its application on wound healing
.
Cellulose
2015
;
22
:
737
747
.
12. 
Jayasuriya
AC,
Bhat
A.
Fabrication and characterization of novel hybrid organic/inorganic microparticles to apply in bone regeneration
.
J Biomed Mater Res A
.
2010
;
93
:
1280
1288
.
13. 
Grenha
A,
Seijo
B,
Remunan-Lopez
C.
Microencapsulated chitosan nanoparticles for lung protein delivery
.
Eur J Pharm Sci
.
2005
;
25
:
427
437
.
14. 
Zhang
L,
Zhang
J,
Ling
Y,
et al.
Sustained release of melatonin from poly (lactic-co-glycolic acid) (PLGA) microspheres to induce osteogenesis of human mesenchymal stem cells in vitro
.
J Pineal Res
.
2013
;
54
:
24
32
.
15. 
Kim
SW,
Bae
YH,
Okano
T.
Hydrogels: swelling, drug loading, and release
.
Pharm Res
1992
;
9
:
283
290
.
16. 
Choi
EM.
Magnolol protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity through activation of mitochondrial function
.
Inflammation
2012
;
35
:
1204
1212
.
17. 
Titorencu
I,
Albu
MG,
Nemecz
M,
Jinga
VV.
Natural polymer-cell bioconstructs for bone tissue engineering
.
Curr Stem Cell Res Ther
.
2017
;
12
:
165
174
.
18. 
Lai
M,
Jin
Z,
Tang
Q,
Lu
M.
Sustained release of melatonin from TiO2 nanotubes for modulating osteogenic differentiation of mesenchymal stem cells in vitro
.
J Biomater Sci Polym Ed
.
2017
;
28
:
1651
1664
.
19. 
Xu
L,
Zhang
L,
Wang
Z,
et al.
melatonin suppresses estrogen deficiency-induced osteoporosis and promotes osteoblastogenesis by inactivating the NLRP3 inflammasome
.
Calcif Tissue Int
.
2018
;
103
:
400
410
.
20. 
Li
T,
Jiang
S,
Lu
C,
et al.
Melatonin: another avenue for treating osteoporosis?
J Pineal Res.
2018
:
e12548.
21. 
Dong
P,
Gu
X,
Zhu
G,
et al.
Melatonin induces osteoblastic differentiation of mesenchymal stem cells and promotes fracture healing in a rat model of femoral fracture via neuropeptide Y/neuropeptide Y receptor Y1 signaling
.
Pharmacology
.
2018
;
102
:
272
280
.
22. 
Arabaci
T,
Kermen
E,
Ozkanlar
S,
et al.
Therapeutic effects of melatonin on alveolar bone resorption after experimental periodontitis in rats: a biochemical and immunohistochemical study
.
J Periodontol
.
2015
;
86
:
874
881
.
23. 
Huang
CC,
Chiou
CH,
Liu
SC,
et al.
Melatonin attenuates TNF-alpha and IL-1beta expression in synovial fibroblasts and diminishes cartilage degradation: implications for the treatment of rheumatoid arthritis
.
J Pineal Res.
2019
:
e12560.
24. 
Altindal
DC,
Gumusderelioglu
M.
Melatonin releasing PLGA micro/nanoparticles and their effect on osteosarcoma cells
.
J Microencapsul
.
2016
;
33
:
53
63
.

Author notes

† 

These authors contributed equally to this work.

Supplementary data