| Sign In to gain access to subscriptions and/or personal tools. |
Preparation and Characterization of Electrodeposited Calcium Phosphate/Chitosan Coating on Ti6Al4V Plates
1 IsoTis S.A., Prof. Bronkhorstlaan 10-D, 3723 MB Bilthoven, The Netherlands; Correspondence: * corresponding author, jiawei.wang{at}isotis.com
Electrolytically deposited carbonate apatite coating demonstrates higher strength but weaker support for bone marrow stromal cell attachment than do biomimetically deposited coatings. It is hypothesized that the incorporation of chitosan will increase the biocompatibility of electrolytic coating while maintaining its original strength. To verify this hypothesis, we formed a hybrid calcium phosphate/chitosan coating through electrodeposition. We found that the incorporation of chitosan influenced calcium phosphate formation and crystallization. Moreover, coating thickness and surface roughness decreased with increasing chitosan concentration. Hybrid coating exhibited an increased dissolution rate in both acidic and neutral simulated physiologic solution, whereas no significant difference on adhesive strength was found between the hybrid and original coatings (P > 0.05). Most importantly, the calcium phosphate/chitosan coating proved to be a more favorable surface for goat bone marrow stromal cell attachment than an unincorporated coating (P < 0.01). Considering its economic and simple production, a hybrid calcium phosphate/chitosan coating is thought to be an attractive candidate for future applications.
Key Words: electrodeposition • chitosan • carbonate apatite • octacalcium phosphate • cell attachment
Calcium phosphate coatings on dental or orthopedic implants are known to accelerate bone growth and enhance bone fixation (Clemens et al., 1998; Moroni et al., 1998). However, plasma-sprayed hydroxyapatite, the only commercially available coating, does have some drawbacks. A few other methods, such as biomimetic deposition or electrolytic deposition, have been proposed to prepare calcium phosphate coatings in simulated body fluid or supersaturated Ca-P solution at ambient temperature. These coatings, generally composed of octacalcium phosphate (OCP) or carbonate apatite (CA), are suited to complex and porous structure (Ban and Maruno, 1993; Habibovic et al., 2002). Moreover, their lower interfacial tension as well as higher degradability may help to prevent coating delamination in vivo. Electrolytically deposited CA is found to demonstrate higher strength than coatings biomimetically deposited from solution; however, a problem is that this type of coating has less bone marrow stromal cell attachment (Wang et al., 2004). Analogous to co-deposition of bioactive agents in biomimetic coating (Liu et al., 2001, 2003), we also intend to increase the biocompatibility of the electrolytic coating by incorporation of some bioactive agents. Chitosan is derived from partially deacetylated chitin and consists of copolymers of glucosamine and N-acetyl glucosamine. As a linear polymer, chitosan has many amino groups attached on the polysaccharide main chain that are readily available for chemical reaction and salt formation with acids (Singla and Chawla, 2001). In the past 20 yrs, chitosan has drawn considerable attention in biomedical areas, such as wound dressings (Kratz et al., 1997), cholesterol-lowering agent (Sugano et al., 1988), hemostatic agent (Malette et al., 1983), skin-grafting template (Ma et al., 2001), and drug delivery systems (Aiedeh et al., 1997; Zhang and Zhang, 2002). Composites of chitosan and calcium phosphate have also demonstrated increased osteoconductivity and biodegradation, together with sufficient mechanical strength (Muzzarelli et al., 1993, 1994; Yamaguchi et al., 2001; Xu et al., 2002). Furthermore, chitosan is found to potentiate the differentiation of osteoprogenitor cells and support the expression of extracellular matrix proteins by human osteoblasts and chondrocytes (Klokkevold et al., 1996; Lahiji et al., 2000). Therefore, we assume that incorporating chitosan into electrolytically deposited CA will improve the coating biocompatibility while maintaining its original mechanical properties. To verify this hypothesis, we prepared an electrolytically deposited calcium phosphate/chitosan coating on Ti6Al4V plates. We investigated the hybrid coating with scanning electron microscopy (SEM) and x-ray diffractometer (XRD). Some physicochemical parameters, such as coating thickness, surface roughness, dissolution rate, and adhesive strength, were also investigated. Finally, biocompatibility was studied with the use of goat bone marrow stromal cells.
Preparation of Calcium Phosphate/Chitosan Coating Ti6Al4V plates (20 mm x 10 mm x 1 mm, Smitford Staal BV, Zwijndrecht, The Netherlands) with roughness of 4.0 µm were ultrasonically cleaned by acetone, ethanol (70%), and demineralized water. We prepared 1 wt% chitosan solution by dissolving chitosan (more than 85% deacetylation; Sigma, St. Louis, MO, USA) into 1% acetic acid, which was then added into supersaturated Ca-P solution to obtain final chitosan concentrations of 0.05, 0.1, 0.15, and 0.2 g/L. Electrolytically deposited calcium phosphate coating (ELD coating) and calcium phosphate/chitosan coating (ELDC coating) were prepared on cathodic Ti6Al4V plates in different conditions. For the ELD coating, the deposition was processed at 52°C for 10 hrs in supersaturated Ca-P solution (pH buffered at 7.0, see Table  ) with current maintained at 2.0 mA/cm2 by a galvanostat power supply (BioRad PowerPac 1000, Hercules, CA, USA); for the ELDC coating, the deposition was processed at 52°C for 15 hrs in supersaturated Ca-P solution with chitosan (pH buffered at 6.8~6.9; see Table), with current maintained at 2.0 mA/cm2. The plates were then rinsed with demineralized water and dried at 50°C overnight.
Characterization of Calcium Phosphate/Chitosan Coating All coatings were observed with SEM (Philips XL-30, Eindhoven, The Netherlands) and determined by XRD (Rigaku Miniflex, Tokyo, Japan). The crystallinity index was calculated from the XRD pattern (Tsui et al., 1998). The thickness was measured in situ with a magnetic induction probe (ElectroPhysik Minitest 2100, Cologne, Germany). The coating surface profile was scanned with a Laser Profilometer (UBM A538, Sunnyvale, CA, USA), and surface roughness was determined according to DIN 4768 (Deutsches Institut für Normung) standards. We conducted a dissolution test by soaking coatings at 37°C in 100 mL simulated physiological solutions with pH buffered at 5.0 and 7.3, respectively (see Table  ). We measured the dissolution rate by recording Ca ion release through a pH/Ion meter (Metrohm 692 pH/Ion meter, Herisau, Switzerland) at fixed interval points. The coating adhesive strength was measured with an automatic scratch tester (CSEM REVETEST, Neuchâtel, Switzerland). All scratch traces were observed with stereo-optical microscopy (Nikon SMZ-10A, Kanagawa, Japan). We noted the critical load forces when the first crack and the total delamination of the coating occurred.
Cell Attachment Test
Statistics
Coating Characteristics Both the ELD and the ELDC coatings are uniformly deposited across the surfaces of titanium alloy plates. The ELD coating was composed of Ca-P globules with diameters of 50–100 µm, which were in turn formed by small plate-like crystals about 5–6 µm long (Fig. 1a ). On the ELDC coatings, many chitosan aggregates were seen around the calcium phosphate globules (Fig. 1b). These chitosan aggregates, increased in the coatings with increasing chitosan concentration in solution, spread and integrated with calcium phosphate crystals, or sometimes interconnected to form nets (Figs. 1c, 1d). The XRD patterns of the two coatings were also different (data not shown). The ELD coating demonstrated typical carbonate apatite peaks with a crystallinity of about 78%, whereas a little mixing phase was found in the ELDC coatings at 2 = 4.7° corresponding to the (010) plane of OCP. The crystallinity of these coatings was about 60~70%.
The coating thickness and surface roughness decreased gradually with increasing chitosan concentration in solution. The ELD coating had the highest thickness (45 ± 4 µm, n = 3), which decreased followed by the increasing chitosan concentration at 0.05 g/L (40 ± 3 µm, n = 3), 0.1 g/L (37 ± 4 µm, n = 3), 0.15 g/L (33 ± 4 µm, n = 3), and 0.2 g/L (28 ± 3 µm, n = 3). In contrast, the surface roughness of the ELD coating was 3.9 ± 0.6 µm (n = 3), but that of the ELDC coating (0.05 g/L chitosan) increased to 4.7 ± 0.4 µm (n = 3), then it decreased to 4.2 ± 0.3 µm (n = 3), 4.0 ± 0.3 µm (n = 3), and 3.7 ± 0.3 µm (n = 3) at 0.1, 0.15, and 0.2 g/L chitosan, respectively. The coating dissolution behavior was also different. In neutral conditions, for the first 3 hrs, the ELD and ELDC coatings (0.2 g/L chitosan in solution) demonstrated similar dissolution rates at 0.4 ± 0.1 ppm/hr cm2 (n = 3). After that, the ELD coating reached a saturation regime with Ca ion concentration at 2.4 ± 0.3 ppm/cm2 (n = 3) within 15 hrs. The ELDC coating continued dissolving slowly and reached a final concentration of 4.3 ± 0.5 ppm/cm2 (n = 3) after 15 hrs. However, in acidic conditions, for the first 3 hrs, the ELDC coating dissolved faster at 2.8 ± 0.4 ppm/hr cm2 (n = 3) than the ELD coating at 1.7 ± 0.3 ppm/hr cm2 (n = 3). Then, the ELDC coating slowed its dissolution rate, and both coatings reached a final concentration of 9.3 ± 0.5 ppm/cm2 (n = 3) and 8.0 ± 0.5 ppm/cm2 (n = 3), respectively, after 15 hrs.
The scratch test results are shown in Fig. 2
Cell Attachment Test Under light microscopy, large amounts of bone marrow stromal cells were observed to attach to the ELDC coating, in contrast to only a few cells on the ELD coating after 1 day’s or 3 days’ culture (data not shown). The cells on the ELDC coating had a polygonal shape and connected with each other through extending cytoplasmic processes (Figs. 3c , 3d). At higher magnification, these cells were found to attach favorably with incorporated chitosan (Figs. 3e, 3f). In contrast, cells on the ELD coating were flat with a more regular shape (Figs. 3a, 3b). A significant difference (P < 0.01) was also found on the DNA contents of cells attached to coatings at both 1 day’s (7.4 ± 0.9 ng, n = 3 for ELD; and 63.8 ± 20.9 ng, n = 3 for ELDC) and 3 days’ culture (9.0 ± 1.5 ng, n = 3 for ELD; and 194.6 ± 35.9 ng, n = 3 for ELDC).
Calcium phosphate incorporated with chitosan has been extensively studied as bone substitutes, tissue engineering scaffolds, or bone cements. In the present study, for the first time, we have incorporated chitosan into calcium phosphate coatings through an electrodeposition method. Chitosan, with more than 85% deacetylation, is known to be insoluble in alkaline conditions but dissolves easily in organic acid and takes a positive charge. When chitosan is dissolved into acidic supersaturated Ca-P solution in the presence of an electric field, two events will take place: On the one hand, calcium phosphate will precipitate on the cathodic substrate through locally increased pH, which is called electrolytic deposition (Ban and Maruno, 1998). On the other hand, positively charged chitosan will also move to the cathode by electric attraction, which is called electrophoretic deposition (Zhitomirsky, 2000). Thus, through different deposition mechanisms, a hybrid calcium phosphate/chitosan coating will be formed on the cathodic substrate. Considering the independence of these two processes, we hypothesize that chitosan will deposit adjoining the calcium phosphate crystals and fill the spaces among them. SEM micrographs have shown that chitosan is tightly entrapped within surface calcium phosphate crystals. However, further studies are required to elucidate the coating’s inner structure. XRD analysis indicates little OCP phase present in the CA crystals and a decreased crystallinity of the coating. It has been reported that electrolytically deposited calcium phosphate was initially OCP, which later transferred into CA (Ban and Maruno, 1998). Therefore, we assume that the incorporation of chitosan influences calcium phosphate formation and crystallization, with the result that OCP is inhibited to transfer into CA and crystallinity is decreased. This inhibitive action of chitosan is comparable with that of some proteins, such as serum albumin and osteocalcin, which are known as crystallization inhibitors in solution (Hlady and Furedi-Mihofer, 1979; Hauschka and Carr, 1982). However, it is still not clear how chitosan affects calcium phosphate crystallization. One explanation might be that the positively charged chitosan molecules competitively bind with negatively charged phosphate ions, thus inhibiting the calcium phosphate crystals’ formation. Other possibilities may be that chitosan attaches to the plates to inhibit the subsequent deposition of calcium phosphate, or that viscous chitosan molecules prevent calcium and phosphate ions from moving to the cathode. In any event, this inhibitive effect seems related to the chitosan concentration in solution. Also, both coating thickness and surface roughness decrease with increasing chitosan concentration. Compared with the ELD coating, our results show that the ELDC coating exhibited an increased dissolution rate in both acidic and neutral simulated physiological solutions. This change may mainly be caused by the incorporation of chitosan. Due to the inhibitive effect of chitosan, the lower-crystallized ELDC coating might exhibit more rapid and extensive dissolution (Maxian et al., 1993). On the other hand, previous reports have demonstrated that calcium phosphate/chitosan composites exhibited higher yielding strength (Yamaguchi et al., 2001; Xu et al., 2002). Therefore, we assume that chitosan aggregates will also enhance coating strength by virtue of their enwrapping of calcium phosphate globules. However, we find no significant differences between the strengths of the ELD and the ELDC coatings. This is possibly due to the lower amount of chitosan in the coatings. After all, since the ELD coating has demonstrated higher strength than those biomimetically deposited from solutions (Wang et al., 2004), the relatively higher strength of the ELDC coating may benefit its future application. Turning to the coatings’ biocompatibility, our results demonstrate that more bone marrow stromal cells attach to the ELDC coating after 1 day’s or 3 days’ culture. Furthermore, we are interested to find that cells favorably attach to the incorporated chitosan. These findings clearly indicate that the ELDC coating is more effective for bone morrow stromal cell attachment than the ELD coating. It also confirms our hypothesis that biocompatibility is indeed increased by the incorporation of chitosan. Two factors are thought to contribute to this enhancement. The first one comes from the structure change of calcium phosphate coating. Since amorphous coatings usually show rapid and extensive dissolution at early time points, the released Ca ions will lead to reprecipitation of Ca-P, thus stimulating the differentiation of osteogenic cells (Maxian et al., 1993; Zeng et al., 1999). This point corresponds to the lower crystallinity and higher dissolution rate of the ELDC coating. The second one comes from the incorporated chitosan. It is believed that the chemical structure of chitosan resembles that of glycosaminoglycan, which is the key molecule in the extracellular matrix to modulate cell morphology and function (Lahiji et al., 2000). So, this similarity may help chitosan to enhance cell attachment. This is in line with our finding that bone marrow stromal cells favorably attached to incorporated chitosan. In summary, the ELDC coating demonstrates improved biocompatibility while simultaneously maintaining its original strength. Considering its economic and simple production, we think it to be an attractive candidate for future clinical applications.
This study was financially supported by IsoTis S.A., which owns the IP rights of this work. The authors gratefully thank Pierre Layrolle, Shihong Li, Huiping Yuan, Hongjun Wang, and Chang Du for their technical support and helpful discussions. Received for publication October 17, 2003. Revision received February 10, 2004. Accepted for publication February 17, 2004.
Journal of Dental Research, Vol. 83, No. 4,
296-301 (2004)
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
-MEM medium supplemented with 15% fetal bovine serum (Life Technologies, Breda, The Netherlands), antibiotics, 0.2 mM L-ascorbic acid 2-phosphate (Life Technologies, Breda, The Netherlands), and 0.01 M β-glycerophosphate (Sigma, Zwijndrecht, The Netherlands). The second-passage cells were seeded on Ti6Al4V plates with the ELD and ELDC coatings (0.2 g/L chitosan in solution) at 5000 cells/cm2 and cultured at 37°C with 5% CO2 and 95% air. After 1 day or 3 days, some plates were rinsed with PBS and fixed with 1.5% glutaraldehyde. The cells were either stained with 0.1% methylene blue to be observed with stereo-optical microscopy or serial-dehydrated, critical-point-dried, and sputter-coated with gold to be examined with SEM. Other plates were digested with proteinase K (Sigma, Zwijndrecht, The Netherlands) at 56°C for 16 hrs. The DNA content of cells attached to the coatings was counted with a cell proliferation assay kit (CyQuant, Leiden, The Netherlands). 
