Synthesis and Characterization of Melatonin-Loaded Chitosan Microparticles Promote Differentiation and Mineralization in Preosteoblastic Cells

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.

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.

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.

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).

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 

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 (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.

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.

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.

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.

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.

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).

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).

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.

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).

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).

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

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.