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Proliferation and Differentiation of MC3T3-E1 Cells on Calcium Phosphate/Chitosan Coatings
1 Key Lab for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, People’s Republic of China, 430079; and Correspondence: * corresponding author, wangjwei{at}hotmail.com
The incorporation of chitosan into electro-deposited calcium phosphate (CaP) coatings increases bone marrow stromal cell attachment. We hypothesized that such electrodeposited CaP/chitosan coatings can also enhance the proliferative ability and differentiation potential of osteoblasts. To verify this hypothesis, we cultured osteoblast-like MC3T3-E1 cells on these CaP coatings. It was found that MC3T3-E1 cells cultured on the electrodeposited CaP/chitosan coatings had cell proliferation rates higher than those on the electrodeposited CaP coatings. At the same time, both alkaline phosphatase activity and collagen expression were increased, and both bone sialoprotein and osteocalcin genes were up-regulated when MC3T3-E1 cells were cultured on the electrodeposited CaP/chitosan coatings. Additionally, within the range of selected chitosan concentrations in solution, no significant difference was found between the CaP/chitosan coatings. Our results suggest that the electrodeposited CaP/chitosan coatings are favorable to the proliferation and differentiation of MC3T3-E1 cells, which may endow them with great potential for future applications.
Key Words: electrodeposition • calcium phosphate coating • chitosan • MC3T3-E1 cells • proliferation • differentiation
Electrolytic deposition has recently attracted more interest in the preparation of calcium phosphate (CaP) coatings (Shirkhanzadeh, 1991; Ban and Maruno, 1998). In contrast to the line-of-sight process of plasma spraying, electrolytic deposition is conducted at ambient temperature and can be applied to complex or porous structures. More importantly, the favorable crystal composition and dissolution characteristics, as well as lower interfacial tension, may overcome the relative weakness of the electrodeposited CaP coatings and thus prevent the coatings’ delamination in vivo. The composition and properties of the coatings strongly depend on the electrolyte. We have reported the electrodeposition of a carbonate apatite coating in a near-neutral supersaturated CaP solution (Wang et al., 2004b). We found that the electro -deposited carbonate apatite coating demonstrated higher strength, but less ability to induce goat bone marrow stromal cell attachment, compared with biomimetically deposited octacalcium phosphate or carbonate apatite coatings. Chitosan has drawn considerable attention in biomedical research for the past 20 yrs. Composites of chitosan and calcium phosphate have also been studied widely as bone substitutes, tissue-engineering scaffolds, or bone cements, demonstrating increased osteoconductivity, biodegradation, and sufficient mechanical strength (Zhang et al., 2003; Hu et al., 2004; Xu and Simon, 2005). To increase the biocompatibility of the coating, we have tried to incorporate chitosan into electrodeposited CaP coatings. The preliminary results confirm that the electrodeposited CaP/chitosan coatings improved bone marrow stromal cell attachment. Generally, osseointegration and bone formation along an implant surface consist of 3 stages: the recruitment and attachment of osteoblasts from the surrounding bone tissue; the proliferation and differentiation of osteoblasts; and, finally, the synthesis and mineralization of a collagenous bone matrix. Since the electrodeposited CaP/chitosan coatings have been demonstrated to improve bone marrow stromal cell attachment (Wang et al., 2004a), we then assumed that the coating can also enhance the proliferation and differentiation of osteoblast cells. To verify this hypothesis, we cultured a pre-osteoblast-like cell line, i.e., the MC3T3-E1 cell line, on the electrodeposited CaP/chitosan coatings. We calculated the cell proliferation rate according to the increased cell number, and investigated cell differentiation at both the protein and gene levels.
Coating Preparation Ti6Al4V plates (20 x 20 x 1 mm; Smitford Staal BV, Zwijndrecht, The Netherlands) with roughness of 4.0 µm were ultrasonically cleaned. We prepared chitosan solution (1 wt%) by dissolving chitosan (more than 85% deacetylation, viscosity > 200 cps; Sigma, St. Louis, MO, USA) into 1% acetic acid, which was then added to a supersaturated CaP solution to obtain 0.1 and 0.2 g/L chitosan. Electrodeposited CaP coatings and CaP/chitosan coatings were prepared on Ti6Al4V plates in different conditions. For the CaP coatings, the deposition was processed at 52°C for 8 hrs in a supersaturated CaP solution (136.8 mM NaCl, 4.0 mM CaCl2·2H2O, 2.0 mM Na2HPO4·2H2O, 50 mM Tris-HCl, pH 7.0), with current maintained at 2.5 mA/cm2 by a galvanostat power supply (BioRad PowerPac 1000, Hercules, CA, USA); for the CaP/chitosan coatings, the deposition was processed at 52°C for 15 hrs (273 mM NaCl, 4.0 mM CaCl2·2H2O, 2.0 mM Na2HPO4·2H2O, 100 mM Tris-HCl, pH 6.8-6.9), with current maintained at 2.0 mA/cm2. All coatings were rinsed with demineralized water and dried at 50°C overnight, and the surface morphology and uniformity were observed by scanning electron microscopy (SEM, Philips XL-30, Eindhoven, The Netherlands).
Cell Culture and Morphology Observation
Cell Proliferation
Cell Differentiation To measure alkaline phosphatase (ALP) activity, we subjected the cell layers to lysis with 1 mL Triton (0.2%), harvested them using a cell scraper, and sonicated them on ice for 15 sec (S250A, Branson Ultrasonics Corp., Danbury, CT, USA). The cell homogenate was incubated with p-nitrophenyl phosphate substrate solution (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 15 min, and then stopped by addition of NaOH (0.1 N). Absorbance was measured at 405 nm (Bio-Tek Elx808, Winooski, VT, USA). The ALP activity was determined as the amount of nitrophenol produced that was normalized to the total cell DNA content. For the measurement of collagen production, cells were first digested with proteinase K at 56°C for 16 hrs. A 100-µL quantity of digested sample was hydrolyzed with 100 µL HCl (6 M) at 110°C for 16 hrs and evaporated under a stream of nitrogen gas at 37°C. The hydrolysate was dissolved in 0.5 mL of demineralized water. A 100-µL quantity of of hydrolysis sample was incubated with 50 µL of chloramine-T (Fluka, Buchs SG, Switzerland) solution for 20 min and 50 µL of p-dimethyl-amino-benzaldehyde (Sigma, St. Louis, MO, USA) solution for 30 min. Absorbance was measured at 570 nm. The collagen content was determined as the hydroxyproline amount relative to total cell DNA content.
To analyze gene expression, we isolated total RNA from MC3T3-E1 cells cultured for 14 days. The primers for bone sialoprotein, osteocalcin, and GAPDH genes are listed in the Table
Statistics Values of cell-doubling index, ALP activity, collagen content, and mRNA level were expressed as means ± standard deviation. Differences were analyzed with a one-way ANOVA test and the Student-Newman-Keuls multiple comparison test. The statistical significance was defined as P < 0.05.
Coating Characteristics and Cell Morphology Both coatings were uniformly deposited across the surfaces of Ti6Al4V plates. Based on the SEM micrographs and light microscopic cross-sectional view, it was found that the electrodeposited CaP coatings were composed of two layers: a layer of regular plate-like crystals about 5–6 µm in length on the coating surface, and a layer of amorphous CaP globules about 50–100 µm in diameter beneath (Figs. 1A, 1B  ). In contrast, the electrodeposited CaP/chitosan coatings were mainly composed of irregular long plate-like crystals, with little amorphous CaP globules beneath. Many chitosan aggregates spread and integrated with CaP crystals (Figs. 1C, 1D). After cultivation, a thin layer of cells was formed on the electrodeposited CaP coating surface (Figs. 1E, 1F); however, there was a thick layer of cells and extracellular matrix on the CaP/chitosan coating surface (Figs. 1G, 1H).
Cell Proliferation Since we had reported previously that a higher chitosan concentration in solution would impede CaP deposition, only 2 chitosan concentrations, i.e., 0.1 g/L and 0.2 g/L, were used in the study. It was found that both CaP and CaP/chitosan coatings provided cell proliferation rates lower than that of tissue culture plastic (Fig. 2 , P < 0.05). However, MC3T3-E1 cells cultured on the CaP/chitosan coatings had cell proliferation rates higher than those on the CaP coatings (except on day 5, P < 0.05). Within the range of selected chitosan concentrations, there was no significant difference in the proliferation rates (P > 0.05).
Cell Differentiation Both ALP activity and collagen production were enhanced when MC3T3-E1 cells were cultured on the electrodeposited CaP/chitosan coatings compared with the CaP coatings at days 7, 10, and 14 (Figs. 3A, 3B ). In contrast to collagen content, the increase in ALP activity was significant (P < 0.05). Additionally, both ALP activity and collagen production were increased with time. For the CaP/chitosan coatings, significantly higher ALP activity was observed from day 7 to day 10, or from day 10 to day 14 (P < 0.05). However, no significant difference in collagen production was found among the 3 timepoints for these coatings (P > 0.05). The chitosan concentration in the solution induced a slight, but not significant, difference between the electrodeposited CaP/chitosan coatings (P > 0.05).
Similarly, both bone sialoprotein and osteocalcin genes were up-regulated when MC3T3-E1 cells were cultured on the electrodeposited CaP/chitosan coatings at day 14. The level of bone sialoprotein mRNA on the CaP/chitosan coatings increased 6.74-fold (0.1 g/L chitosan in solution) and 6.63-fold (0.2 g/L chitosan in solution) (Fig. 3C  , P < 0.05). The level of osteocalcin mRNA on the CaP/chitosan coatings increased 4.68-fold (0.1 g/L chitosan in solution) and 5.59-fold (0.2 g/L chitosan in solution) (Fig. 3D, P < 0.05). No significant difference was found between the CaP/chitosan coatings (P > 0.05).
Although CaP/chitosan composites have been reported to support the attachment, proliferation, and differentiation of osteoblasts (Lee et al., 2000; Zhang et al., 2003; Xu and Simon, 2005; Chesnutt et al., 2007), this is the first paper to present the responses of osteoblasts on the electrodeposited CaP and CaP/chiotsan coatings. Our study indicates clearly that MC3T3-E1 cells on the electrodeposited CaP/chitosan coatings demonstrated greater proliferation rates and a higher differentiation capacity than those on the electrodeposited CaP coatings. Further, within the range of selected chitosan concentrations in solution, no significant difference was found between the CaP/chitosan coatings. Since the cell seeding density and culture conditions were all kept the same, the difference seems to result from the coatings’ composition, solubility, and chitosan. First, the difference might come from the coating characteristics. As we have described previously, the electrodeposited CaP coatings were formed through a rapid electrolytic deposition process, indicated by the precipitation of small crystals and a layer of amorphous CaP coating at the onset, followed by continuous coating formation. The substrate available for electrolytic reactions was reduced, and the local pH increased. The coating precipitation process thus decreased, resulting in a layered coating structure with large, regular, and crystallized crystals on the coating surface. For the electrodeposited CaP/chitosan coatings, however, the existence of chitosan and a relatively lower solution pH greatly decelerated or impeded the deposition process. At the same time, the electrolyte was buffered by a two-times-concentrated HCl-Tris system, which further maintained the stability of local pH values. This mechanism induced a slow and continuous precipitation process. The electrodeposited CaP/chitosan coatings thus formed a non-clearly-layered structure, and the crystals were larger, more irregular, and less crystallized than those of the electrodeposited CaP coatings. Such a deposition process also influenced the coating composition. The electrodeposited CaP coatings were mainly composed of carbonate apatite; however, the CaP/chitosan coatings were characterized by a mixture of carbonate apatite and a little octacalcium phosphate (Wang et al., 2004a). Since the surface composition was important to support the differentiation of osteogenic cells and subsequent apposition of bone matrix (Loty et al., 2001), it was then suggested that such a layered structure, smaller and more regular crystals, and higher crystallized carbonate apatite might endow the MC3T3-E1 cells on the electrodeposited CaP coatings with a lower proliferation rate and differentiation potential. Second, the difference might be reflected in dissolution behavior. Previous studies have demonstrated that, in neutral conditions, the electrodeposited CaP/chitosan coatings dissolved faster and released more calcium ions than the electrodeposited CaP coatings. It is known that MC3T3-E1 cells possess voltage-activated calcium channels in the plasma membrane (Lieberherr, 1987; Yamaguchi et al., 1989). The increased calcium ions might stimulate cell calcium entry and increase intracellular calcium storage. Changes in intracellular protons thus induced higher cell proliferation and differentiation (Mayr-Wohlfart et al., 2001; Ehara et al., 2003). Finally, the enhanced biocompatibility might be attributed to chitosan. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine. The N-acetyl-glucosamine moiety in chitosan has a structural feature also found in glycosaminoglycans (GAGs). Since GAGs show many specific interactions with growth factors, receptors, and adhesion proteins, the analogous structure might endow chitosan with related bioactivities. In contrast, the cationic nature of chitosan itself could electrostatically interact with anionic proteoglycans, GAGs, and other negatively charged molecules. A chitosan-GAG complex on the electrodeposited CaP/chitosan coatings then might adsorb more cytokines or growth factors secreted by colonizing cells—for example, heparin and heparan sulphate (Madihally and Matthew, 1999). In fact, chitosan has been reported to increase the ALP activity of osteoblasts after 3 days’ treatment and to induce a significant increase in BMP-2 mRNA after 7 days’ culture (Ohara et al., 2004). This significant increase was caused by the acceleration of osteoblastic cell proliferation (Yamada et al., 2003). Chitosan has also been reported to support the expression of extracellular matrix proteins in osteoblasts and the preferential attachment of osteoblasts over other cell types (Lahiji et al., 2000; Fakhry et al., 2004). Of course, this effect was not unique to chitosan. Despite the different mechanisms, many proteins, such as BMP-2 or amelogenin, when incorporated into CaP coatings, also increased osteoblast differentiation (Liu et al., 2004; Du et al., 2005). However, the chitosan content within the coatings was limited, since higher chitosan concentration in solution would also impede the CaP coatings’ deposition (Wang et al., 2004a). For the selected chitosan concentrations in this study, we found 3.8% and 4.9% chitosan in the coatings for 0.1 and 0.2 g/L chitosan in solution, respectively (Wang et al., 2006). This might partially explain why a dose-dependent enhancing effect of chitosan was not found between the electrodeposited CaP/chitosan coatings. In summary, the electrodeposited CaP/chitosan coatings demonstrated greater proliferation rates and a higher differentiation potential for MC3T3-E1 cells than did the electrodeposited CaP coatings. The different performances of CaP coatings in vitro might relate to their physicochemical properties and chitosan.
This study was financially supported by IsoTis S.A. The authors are grateful for the technical support of Sanne Both. J. Wang was financially supported by a grant from The Bureau of Science and Technology of Wuhan (20065004116-01). J. de Boer was financially supported by a grant from Senter/Novem. Received for publication August 25, 2007. Revision received February 23, 2008. Accepted for publication March 7, 2008.
Journal of Dental Research, Vol. 87, No. 7,
650-654 (2008)
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ABSTRACT
INTRODUCTION
-MEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 1 mM sodium pyruvate. Cells were cultured (50,000 cells/cm2) on the electrodeposited CaP coatings and CaP/chitosan coatings (0.2 g/L chitosan in solution) for 7 days, fixed in 4% paraformaldehyde, serially dehydrated, critically dried, embedded in resin, and then cross-sectioned with a diamond saw (Leica SP 1600, Wetzlar, Germany). The sections were polished to 100 µm thick, stained with methylene blue, and observed by light microscopy. 
