Biomimetic and electrolytic calcium phosphate coatings on titanium alloy: physicochemical characteristics and cell attachment

J. Wang^{, , a, b}, P. Layrolle^{a, c}, M. Stigter^a and K. de Groot<sup>a, b

a</sup> Isotis, S.A., Prof. Bronkhorstlaan 10-D, 3723 MB, Bilthoven, The Netherlands
^b Biomaterials Research Group, Leiden University, The Netherlands
^c Inserm EM9903, Research Center on Materials of Biological Interest, Faculty of Dental Surgery, Nantes, France

Received 3 December 2002;  accepted 1 July 2003. ; Available online 2 September 2003.
Biomaterials
Volume 25, Issue 4 , February 2004, Pages 583-592

1. Abstract

Biomimetically deposited octacalcium phosphate (OCP) and carbonate apatite (BCA) as well as electrolytically deposited carbonate apatite (ECA) were considered as promising alternatives to conventional plasma spraying hydroxyapatite. This study compared their physicochemical characteristics and cell attachment behavior. The physicochemical characteristics included scanning electron microscopy observation, X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, surface roughness, coating thickness, dissolution test and scratch test. Cell attachment tests included morphology observation with stereomicroscopy and scanning electron microscopy as well as cell number count with DNA content assay. The OCP coating had 100% crystallinity and was about 40 
m thick, composed of large plate-like crystals of 30 
m, with the lowest surface roughness (R_a=2.33 
m). The BCA coating had 60% crystallinity and was approximately 30 
m in thickness, composed of small crystals of 1-2 
m in size, with the highest surface roughness (R_a=4.83 
m). The ECA coating had intermediate characteristics, with 78% crystallinity, 45 
m thickness, crystals of 5-6 
m and an average roughness of 3.87 
m. All coatings could be seen by eyes dissolving quickly and completely into acidic simulated body fluid (simulated physiological solutions—SPS, pH 3.0) but slowly and incompletely into neutral SPS (pH 7.3). It was suggested that the main factor determining coating dissolution in acidic SPS was the solubility isotherm, while some other factors including crystallinity and crystal size joined to determine coating dissolution in neutral SPS. In regard to adhesive strength, results of scratch test showed the critical load at the first crack of coating (L_c1) was tightly related to crystal size as well as their arrangement, while the critical load at the total delamination of coating (L_c2) was also related to the coating thickness. The ECA coating had the highest values. Owing to higher dissolution rate and globular appearance, BCA coating demonstrated the best goat bone marrow stromal cells attachment at 1 day or 3 days, followed by OCP and ECA coating.

Author Keywords: Biomimetic; Electrolytic deposition; Octacalcium phosphate; Carbonate apatite; Physicochemical characteristic; Cell attachment

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Preparation of biomimetic OCP and CA coatings

2.2. Preparation of electrolytic deposited CA coating

2.3. Physicochemical characteristics

2.4. Dissolution test

2.5. Scratch test

2.6. Cell attachment test

2.7. Statistical analyses

3. Results

3.1. Coating characteristics

3.2. Dissolution test

3.3. Scratch test

3.4. Cell attachment test

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

Calcium phosphate coatings have often been used to increase the bioactivity and success rate of titanium and titanium alloys for some special dental and orthopedic cases, such as dental implants, hip and knee joints, and fracture fixation [1 and 2]. To overcome drawbacks of the commercially provided plasma-sprayed hydroxyapatite (HA) coating, several other deposition methods have been proposed. One of them is the biomimetic coating, where calcium phosphate is deposited on substrates in a simulated body fluid (SBF). Generally, biomimetic deposition processes will last about 7-14 days with daily refreshments [3 and 4]. Although it has been reported that some chemical treatments could reduce soaking time and enhance calcium phosphate deposition [5], these surface treatments might weaken the calcium phosphate/substrate interface. Another idea to shorten immersion time is to increase SBF concentration. Our group has recently reported the biomimetic deposition of carbonate apatite (CA) coating by using five times concentrated SBF within 48 h or octacalcium phosphate (OCP) coating by using supersaturated Ca-P solution within 72 h [6 and 7].

Electrolytic deposition (ELD) is another promising alternative method to plasma-spraying HA [8 and 9]. In the ELD process, electrochemical reactions near the electrode induce local pH increase and thus, calcium phosphate precipitates on the titanium or titanium alloys implants. Most of the ELD are conducted in acidic calcium phosphate solutions and form brushite coating (dicalcium phosphate dihydrate, DCPD). Such brushite coatings normally need aging treatments to convert them into apatite [10, 11 and 12]. Recently, some studies have reported the direct deposition of apatite coating through ELD in SBF [13 and 14]. It has been found that the deposited calcium phosphate coating was composed of CA, or OCP that converted into CA with increasing time and temperature. These calcium phosphate coatings significantly contributed to the early fixation between bone and implants [15, 16 and 17].

The present study aims at comparing different calcium phosphate coatings produced by the biomimetic and ELD methods with respect to their physicochemical characteristics and goat bone marrow stromal cell attachment condition. The physicochemical characteristics include scanning electron microscopy observation, X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, surface roughness, coating thickness, dissolution test and scratch test. Cell attachment test includes morphology observation with stereomicroscopy and scanning electron microscopy as well as cell number count with DNA content assay.

4. 2. Materials and methods

Ti6Al4V plates used in this study were 20×10×1 mm (Smitfort Staal BV, Zwijndrecht, The Netherlands). The titanium plates were sandblasting by alumina particles at a pressure of 4 bar for an average roughness of R_a=4.0 
m. Prior to apply coatings, the samples were ultrasonically cleaned with acetone, ethanol (70%) and demineralized water for 15 min. Then, they were etched in a mixture of 2 ml HF (40%) and 4 ml HNO₃ (66%) in 1000 ml water for 10 min to form a fresh titanium oxide surface.

2.1. Preparation of biomimetic OCP and CA coatings

Biomimetic coatings were prepared in a two-step procedure as previously described [6 and 7]. In the first step, the titanium alloy plates were soaked into supersaturated Ca-P solution with high concentrations of salts and inhibitors of crystal growth to favor heterogeneous nucleation. The concentrations were multiplied by a factor 5 from those in standard SBF excluding the TRIS buffer (see Table 1). The biomimetic solutions were prepared by dissolving reagent grade chemicals in demineralized water under a supply of CO₂ gas at a pH of 5.8. The implants were immersed in this concentrated SBF at 37°C for 24 h with stirring at 250 rounds per minute (rpm). As CO₂ gas evolved out of solution, the pH increased inducing the deposition of a thin and amorphous calcium phosphate (ACP) coating on the titanium samples. In the second step, the thin ACP layer acted as a seed surface to deposit a more crystalline and thicker coating on the plates, and the solution was either highly concentrated with less inhibitors to produce a biomimetic carbonate apatite coating (BCA) or supersaturated and TRIS-buffered at pH 7.4 to deposit a biomimetic OCP coating (OCP). For the BCA coating, the plates were immersed into supersaturated Ca-P solution with lower concentration of Mg and HCO₃ at 50°C for 24 h. For the OCP coating, the plates were placed into TRIS-buffered supersaturated Ca-P solution at 37°C in a shaking water bath for 48 h, with one change of medium at 24 h. After coating, the plates were rinsed with demineralized water and dried at 50°C overnight.

Table 1. Inorganic ion composition (mmol/l) of SBF, supersaturated Ca-P solutions and SPS

SBF—simulated body fluid; ACP—amorphous carbonated calcium phosphate; BCA—biomimetic carbonated apatite; OCP—octacalcium phosphate; ECA—electrolytic deposited carbonate apatite; SPS—simulated physiologic solution.

2.2. Preparation of electrolytic deposited CA coating

Electrolytic deposited CA coating (ECA) was prepared in a supersaturated Ca-P solution (with TRIS buffered at pH 7.0, see Table 1) at 52°C for 10 h. Ti6Al4V plate was placed as cathode and a platinum electrode (10 mm×10 mm×0.1 mm) was placed as anode. The current was maintained at 2.0 mA/cm² by a galvanostat power supply (BioRad PowerPac 1000, USA). The solution was stirred with a magnetic stirring bar at 50 rpm and the calcium phosphate coating was deposited on the Ti6Al4V plates because of increased pH near the plates. After coating, the plates were also rinsed with demineralized water and dried at 50°C overnight.

2.3. Physicochemical characteristics

All coatings were observed with an environmental scanning electron microscope (ESEM, Philips XL-30, The Netherlands). The composition and crystallinity of coatings were determined by X-ray diffraction (XRD, Rigaku Miniflex goniometer, Japan) and Fourier transform infrared spectroscopy (FTIR, PerkinElmer spectrum 1000, UK). The crystallinity index is determined from the XRD pattern and based on the integration of the amorphous wide hump and the crystalline peaks in the range of 2
=25-36°. The crystallinity index was calculated according to the formula [18]:

The thickness was measured in situ with a magnetic induction probe (ElectroPhysik Minitest 2100, Germany). The coating surface profile was scanned by a non-contact Laser Profilometer (UBM measurement system A538, The Netherlands) and the surface roughness was calculated according to DIN (Deutsches Institut für Normung) standards.

2.4. Dissolution test

Two kinds of simulated physiological solutions (SPS) were used for the determination of coating dissolution. One solution was buffered at pH 3.0 with Khydrogenophtalate and HCl, and the other buffered at pH 7.3 with NaOH and HEPES (see Table 1). The coated plates were soaked in 100 ml of SPS maintained at 37°C in a double-jacketed reactor and stirred with a magnetic bar at 200 rpm. The calcium concentration [Ca] in the SPS was measured at fixed interval points with a calcium ion specific electrode and calomel reference electrode calibrated with calcium standards (pH/Ion meter 692, Metrohm, Switzerland).

2.5. Scratch test

The different coatings were tested with an automatic scratch tester (CSEM Revetest, Switzerland). A spherical Rockwell C diamond stylus of 200 
m with a progressive load from 0 to 20 N was used. All scratch traces were observed by stereomicroscopy. The critical loads (L_c) at which the first crack of the coating and the total delamination of the coating occurred were recorded. Three samples were selected for each coating and tests were carried out 10 times for each sample, the value for each coating was determined by averaging the data.

2.6. Cell attachment test

Bone marrow stromal cells were obtained from the iliac crest of young adult goats. The culture medium used was
-MEM supplemented with 15% fetal bovine serum (FBS, Life Technologies, The Netherlands), antibiotics, 0.2 m

-ascorbic acid 2-phosphate (AsAP, Life Technologies, The Netherlands) and 0.01 

-glycerophosphate (
GP, Sigma, The Netherlands). After the first passage, the cells were seeded on coated Ti6Al4V samples placed in 6-well plates at a density of 5000 cells/cm² in 5 ml of medium. The cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ and 95% air. After 1 day and 3 days of culture, the plates (n=2) were rinsed with PBS then fixed and stained with 0.1% methylene blue. The cells attached to the coatings were observed with a stereomicroscopy. Samples were also dehydrated, critical point dried, sputter coated with gold and examined with ESEM. Other parts of plates (n=3) were digested with proteinase K (Sigma, The Netherlands), added with heparin (Leo Pharm, The Netherlands) and Ribonuclease A (Sigma, The Netherlands), then shacked and incubated at 37°C for 30 min. A volume of 100 
l solution of each coating was mixed with 100 
l of 2× Cyquant GRDye (Molecular Probes, Porland), then the fluorescence with fluorimeter (Perkin Helmer) was measured at emission wavelength 520 nm and excitation 480 nm. The DNA content of cells attached on the coating was counted through a pre-made standard DNA curve.

2.7. Statistical analyses

Values of critical load forces and DNA contents were expressed as means±SD. Differences were analysed with ANOVA test, and the statistical significance was defined as P<0.05.

5. 3. Results

3.1. Coating characteristics

Fig. 1 shows ESEM micrographs of the different coatings. The OCP coating exhibited relative flat surface composed of sharp plate-like crystals with 30 
m length and 0.2 
m width (Fig. 1a). The BCA coating exhibited relative rough surface composed of Ca-P globules about 20-30 
m in diameter, which consisted of small crystals approximately 1-2 
m in size (Figs. 1b and c). The ECA coating also exhibited relative rough surface composed of Ca-P globules, with diameter about 50-100 
m, and consisted of crystals approximately 5-6 
m in length (Fig. 1d). The comparison of three coatings is listed in Table 2.

(106K)

Fig. 1. Scanning electron micrograph of different Ca-P coatings. Note (a) relative flat OCP coating composed of 30 
m OCP crystals, (b, c) relative rough BCA coating composed of 20-30 
m Ca-P globules that consisted of 1-2 
m BCA crystals and (d) relatively rough ECA coating composed of 50-100 
m Ca-P globules that consisted of 5-6 
m ECA crystals.

Table 2. Morphology and crystal size of different coatings

The XRD patterns of the three coatings are shown in Fig. 2. The OCP coating exhibited the typical diffraction line at 2
=4.7° corresponding to the (0 1 0) plane. The calculated crystallinity of OCP was 100% (Fig. 2a). The XRD pattern of ECA coating had relatively sharp peaks with a crystallinity of 78% ( Fig. 2b). The XRD pattern of BCA coating exhibited the same diffraction lines but relatively broad peaks and a lower crystallinity of 60% compared with ECA and OCP ( Fig. 2c).

(4K)

Fig. 2. XRD characterization of three coatings. Note (a) typical diffraction line at 2
=4.7° as well as sharp peaks of OCP coating, (b) relatively sharp peaks of ECA coating and (c) relatively broad peaks of BCA coating.

The FTIR spectra of the three coatings are shown in Fig. 3. The OCP coating exhibited sharp P-O bands at 1100, 1070 and 1023 cm⁻¹ and HPO₄²⁻ bands at 906 and 852 cm⁻¹. The two P-O bands at 560 and 600 cm⁻¹ exhibited shoulders at 624 and 526 cm⁻¹ (Fig. 3a). The BCA coating exhibited broad 1041 cm⁻¹ band with two shoulders at 1104 and 960 cm⁻¹. In addition, the FTIR spectrum displayed carbonate bands at 1494, 1476 and 1417 cm⁻¹ (Fig. 3b). The ECA coating had a similar FTIR spectrum as BCA. The positions of carbonate bands were typical of those of AB type carbonate apatite ( Fig. 3c).

(6K)

Fig. 3. FTIR spectra of three coatings. Note (a) OCP exhibited sharp P-O bands at 1100, 1070 and 1023 cm⁻¹ and HPO₄²⁻ bands at 906 and 852 cm⁻¹, (b) BCA and (c) ECA exhibited broad 1041 cm⁻¹ band with two shoulders at 1104 and 960 cm⁻¹ as well as carbonate bands at 1494, 1476 and 1417 cm⁻¹.

The surface roughness (R_a) and coating thickness of each coating are reported in Table 3. The OCP coating had the lowest surface roughness of 2.33 
m, which contrasted with 4.83 and 3.87 
m for BCA and ECA coating, respectively. In addition, the ECA and OCP coating had a comparable thickness about 40-50 
m, but the BCA coating was thinner at 30 
m.

Table 3. Average surface roughness (R_a) and coating thickness of different coatings

The values are present as means±standard deviation (n=3).

3.2. Dissolution test

All coatings quickly dissolved in SPS buffered at pH 3.0 within 30 min (Fig. 4a). During the first 8 min, the OCP coating had the fastest dissolution rate at 6.3 ppm/min cm², while those for ECA and BCA coatings were 5.9 and 5.8 ppm/min cm², respectively. The dissolution continued until no coating could be seen by eyes remaining on the Ti6Al4V substrate within about 1 h. At the end of soaking time (5 h), the total released Ca ion concentration for OCP, ECA and BCA coatings were 15.2, 17.5, and 14.3 ppm/cm².

(8K)

Fig. 4. Cumulative calcium ion release of different coatings (n=3) from (a) SPS at pH 3.0 and from (b) SPS at pH 7.3. Note (a) all coatings dissolved quickly into the acidic SPS but (b) slowly into the neutral SPS.

The three coatings dissolved more slowly at neutral pH than in acidic condition (Fig. 4b). For the first 3 h, the dissolution rates were 1.0, 0.4, and 1.3 ppm/h cm² for OCP, ECA, and BCA coating, respectively. After this initial dissolution, the ECA coating reached a saturation regime with Ca ion concentration at 2.4 ppm/cm² after 12 h soaking. The OCP and BCA coating still slowly dissolved and reached their saturation regime after 12 h of soaking in SPS with Ca ion concentrations at 3.4 and 3.9 ppm/cm², respectively. All coatings still could be seen by eyes remaining on Ti6Al4V substrate after 15 h of soaking in SPS at pH 7.3.

3.3. Scratch test

The scratch test results are shown in Fig. 5. The OCP had the lowest loading force for the first crack (L_c1) but a relatively high loading force for total delamination (L_c2). The ECA coating displayed the highest loading force for both the first crack and total delamination. The critical loads for the first crack of BCA coating was a slightly higher than for OCP, while its total delamination force was inferior to that of OCP. Significant difference was found on the first crack forces among three coatings (P<0.05), but not difference was found among the total delamination forces (P>0.05).

(7K)

Fig. 5. Critical load of different coatings (n=3). L_c1: the first crack of the coating; L_c2: the complete delamination of the coating. Note OCP had the lowest L_c1 but higher L_c2, ECA coating had the highest L_c1 and L_c2, BCA had higher L_c1 but the lowest L_c2. (^*P<0.05; ^**P<0.01)

3.4. Cell attachment test

The BCA coating presented the best surface for goat bone marrow stromal cells attachment. Under stereomicroscopy, large numbers of cells were observed to attach on the BCA coated Ti6Al4V, and these cells differentiated to polygonal shape with extending cytoplasmic processes adhering to the BCA coating after 3 days culture (Figs. 6a and d). In comparison, few cells were found to attach on the OCP coating surface, and these cells presented flat but more regular shape with less cytoplasmic processes extending to the disk after 3 days of culture (Figs. 6b and e). Cells were sparsely observed to attach on the ECA coating, and these cells appeared almost the same shape as those found on the OCP coating after 3 days of culture ( Figs. 6c and f). The DNA content assay confirmed the results of stereomicroscopy and SEM observations ( Table 4). A significant difference was found between the cells attached on BCA and OCP or ECA coatings (P<0.01), while no difference was found between cells attached on OCP and ECA coatings (P>0.05) for both culturing times of 1 and 3 days.

(153K)

Fig. 6. Goat bone marrow stromal cells attachment to the different coatings after 3 days culture. Observed with stereomicroscopy, original magnified 4 times (a-c) and scanning electron microscopy (d-f). Note (a, d) large amounts of cells that present polygonal shape with extending cytoplasmic processes adhered to the BCA coating, (b, e) fewer cells that present flat but more regular shape with less cytoplasmic processes attached to the OCP coating, and (c, f) the fewest cells with the same shape as those on the OCP coating attached on the ECA coating. "C": cells; "CaP": calcium phosphate.

Table 4. DNA content of cells attached on the different coatings

The values are present as means±standard deviation (n=3).

6. 4. Discussion

The results indicated clear differences on the microstructure and composition of the three coatings. The highly crystalline OCP coating has grown to a thickness of 40 
m in supersaturated calcium phosphate solution within 48 h. This relative slow crystal growth rate resulted from the supersaturated regime of the coating solution at a fixed pH of 7.4 [7]. The surface roughness of the OCP coating was lower than other two coatings as the crystals have grown to form a relatively flat surface. As previously reported, the BCA coating presents a composition and crystallinity closely related to those of bone mineral. Both FTIR spectrum and XRD pattern indicated that the BCA coating is composed of a poorly crystallized calcium deficient carbonate apatite. The BCA coating was produced by soaking into a non-buffered and highly concentrated solution with concentration being 5 times higher than in regular SBF solutions. As the CO₂ gas was evolving out of solution, the pH increased due to the buffering capacity of carbonate and phosphate ions and thus led to the precipitation of calcium phosphate on the titanium substrate and in solution. The BCA coating grew suddenly to a thickness of 30 
m when solution reached a pH of 6.5 after 5 h of soaking [19]. Resulting from this fast precipitation mechanism, the BCA coating is composed of relative small crystals within large globules. In a similar way, the ECA coating was produced by soaking into a supersaturated calcium phosphate solution TRIS-buffered at pH 7.0 but with a local increase of pH at the titanium cathode. As a result, the ECA coating was also composed of carbonate apatite as BCA but with higher crystallinity, higher crystal size and a little bit lower surface roughness.

Calcium phosphate coatings deposited from solution at low temperature are different from those produced at several thousands degrees, like in the plasma spraying process. Generally, the plasma-sprayed HA coatings are composed of highly crystallized large particles embedded into a molten amorphous phase, which induced the crystal sizes in the range of hundreds of Ĺ and a quick dissolution of amorphous phase in the early period [20, 21 and 22]. On the contrary, biomimetic and electrochemical calcium phosphate coatings deposited from solutions at ambient temperatures present compositional and structural features closely mimicking those of bone. The deposited crystals are bigger and more uniform with a homogeneous dissolution in the body fluids.

Our study shows that all the coatings were seen by eyes to dissolve completely into the acidic SPS within half an hour, irrespective of their structure, morphology and crystallinity. However, they still have some different parameters determining their dissolution rate. In acidic condition, the OCP coating dissolved faster than both the BCA and ECA coatings because of the higher solubility isotherm of OCP than CA. The ECA coating has a little higher dissolution rate than BCA. Both coatings having almost the same composition, the difference in dissolution behaviors might be caused by the different crystal morphology. The ECA crystals were larger than the BCA crystals and thus, the ECA coating appeared less dense than the BCA. This might result in more space between crystals and higher surface area for the ECA than for the BCA coating. This higher surface area could explain the higher dissolution rate of ECA in acidic SPS.

In neutral conditions (SPS pH 7.3), the dissolution behaviors of the three coatings were different. All the coatings dissolved slowly, but not completely, into SPS until reaching saturation level indicated by a plateau. In contrast with acidic condition, the OCP coating dissolved slower than BCA, but faster than the ECA coating. This difference means that the dissolution behavior of coatings at neutral SPS was not primary derived from their solubility isotherms but also from other factors. The dissolution behaviors are supposed to be the results of a combination of solubility isotherms, crystallinity, crystal size, porosity and specific surface area of the different calcium phosphate coatings [23]. For instance, CA has lower solubility isotherm than OCP, while too lower crystallinity (60%) and smaller crystals (1-2 
m) may induce BCA coating demonstrate a higher dissolution than OCP coating. On the other hand, although still less than that of OCP coating (100% crystallinity and 30 
m crystals), the increase in crystallinity (78%) and crystals size (5-6 
m) of ECA coating, compared with BCA coating, has induced ECA coating demonstrated a lower dissolution than OCP coating.

Adhesion strength is another important characteristic of calcium phosphate coatings, since coatings will exhibit a weight loss when abraded against bone. Plasma spraying HA has been reported a higher critical load (L_c) of 22 N than BCA coating of 7.5 N [24]. However, considering the heterogeneous dissolution behavior of plasma sprayed HA coating, the highly crystalline parts are hardly dissolved and thus a higher bonding strength might be required to sustain long implantation time for coating degradation and bone apposition. On the contrary, the lower strength of coatings deposited from solution might be compensated in fine by their favorable crystal composition, dissolution characteristics and bone integration.

There are several methods to evaluate the interfacial strength, such as pull-off, indentation and shockwave-loading and scratch test. Providing the porous microstructure feature of calcium phosphate coatings deposited from solution, the scratch test might be a convenient method to avoid factitious influence such as glue infiltration. Nevertheless, scratch test is basically a comparison test, and it is difficult to express adhesion quantitatively because the L_c depends on several parameters affected by the testing conditions and the coating-substrate system [25]. In this study, two critical loads were measured. L_c1 and L_c2, respectively, present the first crack and the total delamination of the coating. The results have shown that L_c1 was tightly related to the crystals size as well as their arrangement. For instance, on the one hand, the largest OCP crystals had more space around them and thus featured them the lowest L_c1 value. On the other hand, although ECA crystals were bigger than BCA crystals, the electric current might favor the electrostatic attraction between the substrate and ECA crystals, which demonstrated ECA coating the highest value of L_c1. However, concerning L_c2, the values appear also related to the coating thickness. For example, OCP has higher L_c2 than that of BCA coating. The scratch test results may give me some suggestion on evaluation the behaviors of coatings in vivo. Since the majority of coating weight loss occurred within the first minutes of abrasion [26], we think L_c1 is more meaningful for the coatings. Then, the ECA coating with highest L_c1 might be more stable than other two coatings when implanted.

At last, we use the in vitro cell culture test to evaluate cells attachment condition on different coatings. We demonstrate that the best favorable surface for goat bone marrow stromal cells attachment is BCA coating, and then are OCP and ECA coatings. This result may relate to the physicochemical characteristics of different coatings. It has been suggested that ACP coatings present more rapid bone formation [27]. Because amorphous coatings usually show rapid and extensive dissolution at the early time, the released Ca²⁺ ions will lead to re-precipitation of Ca-P's on the surface that stimulate differentiation of osteogenic cells [28]. In vitro studies also report higher cell differentiation on more soluble calcium phosphate [29 and 30]. Since in our study, despite different crystal phase, BCA coating dissolved faster than OCP and ECA coating, this might induce a better cell attachment on BCA coating. On the other hand, topography and surface roughness of calcium phosphate are also reported to affect bone marrow stromal cells, osteoblasts or osteoblast-like cells adhesion, spreading, proliferation and differentiation [31, 32 and 33]. It is assumed that topography and surface roughness may improve surface wetting properties, which affect cell attachment directly via enhanced formation of focal contacts or indirectly through selectively adsorption of serum proteins required for cell attachment [34]. As we have found, BCA coating was composed of Ca-P globules that consisted of tiny BCA crystals, this globular morphology makes BCA coating possess the highest surface roughness and might also contribute to its favorable cell attachment property. However, although ECA coating also present globular appearance and have higher surface roughness than that of OCP coating, which was composed of plate-like crystals and present a relatively flat surface, the ECA coating demonstrated inferior cell attachment property than OCP coating. We then suggest that coating solubility, surface topography and roughness as well as crystal phase all contribute to the cell attachment property. The effects of coating on cell attachment should be the result of a combination of them.

Nevertheless, in vitro test can just provide information of cell attachment on calcium phosphate coatings. In fact, biomaterials implanted into the bone will encounter many situations that cannot be mimicked in vitro, such as changed pH value induced by tissue inflammation, more and different cell types, local strain produced by loading and movement. Furthermore, besides simple dissolution, coating degradation in vivo also includes osteoclastic resorption. In view of these facts, although BCA coating demonstrates more goat bone marrow cells attachment at early time, its thinner thickness, lower adhesive strength and faster dissolution rate do not necessarily mean a rapid and fixed bone healing when implanted in vivo. Clearly, additional animal experiments in vivo should be performed to understand truly bone responses to these coatings.

7. 5. Conclusions

The paper compares OCP, BCA and ECA coatings on their physicochemical characteristics and cell attachment. OCP coating is composed of big plate-like crystals, which has almost 100% crystallinity and the lowest surface roughness. BCA coating is composed of small crystals, which has the lowest crystallinity but the highest surface roughness. ECA coating lies in the middle of them. All coatings are observed by eyes to dissolve quickly and completely into the acidic SPS (pH 3.0) but slowly and incompletely into the neutral SPS (pH 7.3). It is supposed that the main factor determining coating dissolution in acidic SPS is solubility isotherm, while some other factors, including crystallinity and crystal size, join to determine coating dissolution in neutral SPS. In respect of coatings deposited from solution, scratch test is used to evaluate adhesive strength. Results demonstrate that L_c1 is tightly related to the crystals size, their arrangement and electrostatic attraction, while L_c2 is also related to the coating thickness. Nevertheless, ECA coating has the largest values in either condition. Goat bone marrow cells are used to evaluate cell attachment condition on different coatings. Owing to higher dissolution rate and relative rougher surface, BCA coating represents the best place for cells attachment at 1 day or 3 days. The next are OCP and ECA coating.

8. Acknowledgements

This study was financially supported by IsoTis S.A. The authors gratefully thank Shihong Li, Huiping Yuan, Hongjun Wang and Chang Du for their technical supports and helpful discussion.

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Corresponding author. Isotis, S.A., Prof. Bronkhorstlaan 10-D, 3723 MB , Bilthoven, , The Netherlands. Tel.: [phone]; fax: [phone]

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