Characterization of titanium surfaces with calcium and phosphate and osteoblast adhesion

B. Feng ^{, , a, c}, J. Weng ^{a, c}, B. C. Yang ^{a, c}, S. X. Qu ^{a, c} and X. D. Zhang <sup>b, c

a</sup> School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
^b Engineering Research Center in Biomaterials, Sichuan University, Chengdu 610064, China
^c National Engineering Research Center for Biomaterials, Chengdu 610064, China

Received 15 July 2003;  accepted 11 October 2003.  Available online 27 November 2003.
Biomaterials
Volume 25, Issue 17 , August 2004, Pages 3421-3428

1. Abstract

The titanium surfaces containing calcium, phosphate ions and the carbonate apatite were characterized. The effect of surface chemistry on the initial rabbit osteoblast response on these surfaces was investigated. The cell count and alkaline phosphatase (ALP) specific activity assay were used for biochemical analyses. Scanning electron microscopy was used for morphology observation and in particular X-ray photoelectron spectroscopy (XPS) for surface chemistry characterization. The number of cells adhering to the apatite coating surface was the maximum, the number of cells on the surface containing calcium without phosphate ions was higher than that containing phosphate without calcium, and the number on the unmodified titanium surface was the least. The osteoblasts cultured on the apatite surface exhibited the highest ALP specific activity, next were the ones on the surface containing solely calcium, the lowest were on the unmodified titanium surface. On the substrate surfaces removed of adhered cells, the order of nitrogen amounts detected by XPS was consistent with ones of ALP specific activity and cell number, except for the unmodified titanium surface. For the substrate surfaces removed of adhered osteoblasts, XPS analysis showed that calcium and phosphorous amounts decreased during cell adhesion. After cell culture the Ca2p binding energy (BE) values for apatite coating and the surface containing solely calcium were similar to those of the two surfaces adsorbed bovine serum albumin (BSA). The P2p BE values for the surfaces containing phosphate ions, including the apatite coating and the surface containing solely phosphate ions, showed the same change. But after cell culture the decrease of the P2p BE value for the coating surface was larger than the one for the surface containing solely phosphate ions. Considering the bovine serum albumin adsorption on the same samples, these results indicated that calcium ions on titanium surfaces play a more important role than phosphate ions in initial interactions among culture medium, osteoblasts and titanium surfaces. On the apatite coating surface, calcium ions are active sites for osteoblast adhesion, while calcium and phosphate ions co-exist on titanium surfaces, the former promotes the osteoblast adhesion onto the phosphate sites on titanium surfaces. The cell adhesion was a complicated biological and chemical process relating to surface several elements similar to protein adsorption.

Author Keywords: Author Keywords: Osteoblast; Titanium; Surface characterization; Calcium; Phosphate ion

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Pretreatment and surface characterization

2.2. Cell culture and analyses

3. Results

3.1. Surface characteristics for pretreated samples

3.2. Cell morphology and count

3.3. ALP specific activity

3.4. Analysis of surface chemistry after cell culture

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

The development of bone-implant interfaces depends on direct interactions of bone matrix and osteoblasts with biomaterials. The cell adhesion and spreading belong to the first phase of cell/material interactions and surface characteristics of materials play an essential part in this first phase [1, 2, 3 and 4]. In vitro studies on cell attachment and proliferation are generally concerned with the influence of surface topography [4, 5 and 6], next with surface charge and surface energy [7, 8, 9 and 10]. Effects of surface chemistry on cell behaviors also are coming to be noticed. Initial attachment and spreading of human trabecular osteoblasts were much lower on carbonate apatite compared to stoichiometric hydroxyapatite [10]. When a carbonate apatite layer formed on a bioactive apatite-wollastonite glass-ceramic, the osteoblastic activity increased by 30%, and mineralized bone nodules attached in larger amounts onto the surface [11]. Cell morphology was modified by CoCrMo or titanium [12]. Osteoblast-like cells appeared to be very sensitive to minor variations in surface composition and topography of commercially pure (cp) titanium grades 1 and 4 [13]. The growth of human bone-derived cells on commercially pure Ti and Ti6Al4V expressed different protein levels. The cytoskeletal organization by neonatal rat calvarial osteoblasts attaching to and spreading on 316 steel, Ti6Al4V, CoCrMo, Synamel (hydroxyapatite), alumina and borosilicate glass showed different behaviors [14]. These studies reveal influence of surface chemistry on cell adhesion to different extents.

Titanium and its alloys with apatite coatings have been increasingly used clinically for bone-substitute biomaterials, since they combine good bioactivity of apatite and favorable bulk mechanical properties of titanium. However, there were fewer reports about response of cells to apatite coatings on titanium, especially roles of calcium and phosphate ions in the interaction between cells and coatings [15 and 16]. In this present paper, the initial cell adhesion on titanium surfaces containing, respectively, calcium, phosphate and apatite coating were investigated, and effects of calcium and phosphate ions on initial interactions among culture medium, osteoblasts and the material surfaces were studied. Rabbit osteoblast was chosen as a test cell. The surface characterization, focusing on surface chemistry, was carried out using X-ray photoelectron spectroscopy (XPS).

4. 2. Materials and methods

2.1. Pretreatment and surface characterization

Commercially pure titanium plates 10×10×2 mm³ in size were wet ground with 120-grid metallographic alumina paper and then washed ultrasonically in turn with actone, ethyl alcohol and deionized water, and dried at room temperature. The plates were subjected to three kinds of pretreatment, which yielded samples S, C, P, A according to the following description [17 and 18]:

• S: titanium plates;

• C (the titanium surface containing solely calcium): titanium plates were immersed in a boiling saturated Ca(OH)₂ solution for 30-40 min (pre-calcification);

• P (the titanium surface containing solely phosphate ions): titanium plates were immersed in a 20% H₃PO₄ solution at pH=2.0-2.4 for 30 min at 85-95°C (pre-phosphatization);

• A (the titanium-carbonate apatite coating (CHA)): pre-calcificied titanium plates (C) were immersed in 20 ml supersaturated calcium phosphate solution (SCP) in a seal polystyrene vial at 37°C for 1 week. The SCP was refreshed every 2 days. The SCP had the following ion concentration: Ca²⁺ 3.10 m
, HPO₄²⁻ 1.86 m
, Na⁺ 136.8 m
, Cl⁻ 144.5 m
and K⁺ 3.71 m
. The solution was buffered at pH 7.4 with tris-hydroxymethylaminomethane and hydrochloric acid at room temperature.

Except for pre-calcification, after each of the above treatments, the samples were rinsed with abundant deionized water and then dried at room temperature. After pre-calcification, the titanium plates were supersonically washed.

For pre-treated samples, the surface morphologies, crystal structures and chemical compositions were characterized by scanning electron microscopy (SEM, S-450, Hitachi, Japan), X-ray diffractometer (XRD, D/max-IIIA X-ray diffraction analyzer, Japan) and XPS (XPS, XSAM-8000, England) with MgK
radiation [17, 18 and 19].

2.2. Cell culture and analyses

Rabbit osteoblastic cells were isolated from bone samples obtained in normal individuals undergoing local surgery. The third passage cells were seeded onto the six samples per treatment in 2×10⁶ /ml density. All the samples were incubated in an F-12 culture medium with 100 IU/ml penicillin, 100 IU/ml streptomycin and 10% bovine serum albumin at 37°C in 5% CO₂ atmosphere for 24 h. Corresponding to S, C, P and A, after cell culture, there were samples SCE, CCE, PCE and ACE.

After cell culture for 24 h, the samples were gently washed twice with phosphate buffer saline (PBS) to remove non-adhered cells. The adhered cells were counted using a phase-contract microscope at a magnification of ×250. Five to six fields per sample were evaluated on three separate samples.

The adhered cells on three samples were trypsinized with 0.01% trypsin/0.5 m
ethylenediaminetetraacetic acid (EDTA) and then washed twice with DHanks' solution and PBS, respectively. After the trypsinized cells were lysed with 1 ml 0.1% Triton X-100 overnight at 4°C, the ALP specific activity was evaluated with biochemical analyzer (7170A Automatic Analyzer, Hitachi, Japan).

The sample substrates on which cells were removed were rinsed thoroughly with double-distilled water and then dried at 50°C. The surface chemistry of the substrates before and after cell culture was evaluated using XPS. The surface atomic concentration was derived from a multiplex channel. Binding energy peak areas were determined using a Gauss curve-fitting routine.

The other samples with cells were gently washed with PBS and double-distilled water, respectively, and fixed with 2.5% glutaradehyde buffered by PBS. Then the samples were dehydrated in a graded series of alcohol (50%, 70%, 90% and 100%) and amyl acetate, critical-point dried and finally coated with a thin layer of gold. SEM was used for the morphological examination.

5. 3. Results

3.1. Surface characteristics for pretreated samples

The characteristics of pretreated samples have been reported in our previous papers [17, 18 and 19]. The main results are given in the following. The oxide film on titanium surface (S) was anatase. On the pre-calcified titanium surface (C), the predominant was Ca(OH)₂, next hydrated TiO₂, CaCO₃ as well as CaTiO₃. The pre-phosphatized titanium surface (P) consisted of titanium phosphate composites containing PO₄³⁻, HPO₄²⁻ and H₂PO⁻. After pre-calcification and then immersion in SCP (A), a coating which consisted of carbonate hydroxyapatite (CHA) and a little octacalcium phosphate formed on the titanium surface. The surface components of these samples are summarized in Table 1.

Table 1. Surface components on pre-treated titanium

3.2. Cell morphology and count

Fig. 1 shows that osteoblasts cultured for 24 h were capable of adhering onto all the sample surfaces in flattened and elongated morphology. On the CHA coating surface (ACE), plate-like crystal grains were difficult to be observed. This might be attributed to re-precipitation of Ca²⁺ and PO₄³⁻ ions in the CHA coating during cell attachment, which changed CHA crystal into finer grains. For the pre-calcified titanium, flattened and elongated cells oriented approximately in surface groves in their long axis and there were unspread and spherical cells (CCE). Cells on the PCE surface were less than those on ACE and CCE surfaces, and a few irregular shapes (on the right below in Fig. 1 PCE) might result from dead cells. Cells on SCE were few. That is, for cell attachment and growth, the CHA coating was the best, and the surface containing calcium was better than the one containing phosphate ions. Cell count analysis was corresponding to the SEM morphology ( Fig. 2). The number of cells that adhered to ACE surface was maximum, and the number on CCE is higher than that on PCE.

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Fig. 1. Morphology of adhered osteoblasts on the sample surfaces after cell culture for 24 h.

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Fig. 2. Numbers of adhered osteoblasts on the sample surfaces after cell culture for 24 h.

3.3. ALP specific activity

ALP specific activity is used as a biochemical marker for expressing osteoblast activity, since ALP can mediate bone mineralization by decomposing phosphate compounds and stimulating the combination of phosphate and calcium in extracellular matrix [20 and 21]. The ALP specific activity of the osteoblast is shown in Fig. 3. The cultured cell on ACE exhibited the highest ALP specific activity, next on CCE, finally on SCE. This result also reveals the agreement with the SEM and cell count analyses.

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Fig. 3. ALP specific activity of adhered osteoblasts on the sample surfaces after cell culture for 24 h.

3.4. Analysis of surface chemistry after cell culture

Nitrogen detected by XPS has been assumed to be indicative of the presence of protein [22 and 23]. Cell attachment at initial stage involves the interaction between proteins and the biomaterial surfaces. So the presence of nitrogen at interfaces and its amount could also be used as an indirect index of interacting ability between cells and surfaces in the initial cell adhesion. As shown in Fig. 4, the nitrogen amounts on the substrate surfaces have the following order: ACE>CCE>PCE~SCE. This coincides with the analyses of the cell count and ALP specific activity, except for SCE. After cell culture, the Ca amount on the CCE substrate surface and phosphorous amount on the PCE substrate surface significantly decreased, which was probably related to dissolutions and re-precipitation of Ca²⁺ and PO₄³⁻ ions. When the samples were immersed in culture medium, there should be a dynamic equilibrium that Ca²⁺ or PO₄³⁻ ions dissolve from the material surfaces into the solution and reprecipitate from the solution onto the material surfaces. Fig. 5 indicates that the dissolutions of Ca²⁺ or PO₄³⁻ ions were faster than their reprecipitation within 24 h. An important reason might be high concentrations of Ca²⁺ and PO₄³⁻ ions on the surfaces; next that Ca²⁺ ions at surfaces mediated the interaction between osteoblasts and materials, resulting in its consummation. Comparing the ACE to A substrate surfaces (Fig. 5. A-ACE), the concentration reductions of Ca²⁺ and PO₄³⁻ ions on their surfaces were very small. This suggests that solubility of carbonate apatite is very low and the equilibrium of dissolution and reprecipitation of Ca²⁺ and PO₄³⁻ ions easily builds. In addition, phosphorous was detected on the CCE surface (not shown), which implied the ability of Ca²⁺ to richen PO₄³⁻ ions.

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Fig. 4. Concentration of N on the substrate surfaces after cell culture for 24 h and then removed of osteoblast obtained by XPS.

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Fig. 5. Concentration reduction of Ca and P on substrate surfaces after cell culture for 24 h and then removing osteoblast obtained by XPS.

Fig. 6 shows N1s XPS spectra for the substrate surfaces removed of cells, and as control, also gives XPS spectra for the samples immersed in bovine serum albumin (BSA) for 24 h. The substrate surface containing phosphate ions (Fig. 6, PCE) exhibits a value different N1s BE value from that after protein adsorption ( Fig. 6, PB). For the samples containing calcium, i.e., C and A, after BSA adsorption and cell culture, respectively, the surface and the substrate surface removed of the adhered cells show similar N1s spectra ( Fig. 6, ACE and AB1; CCE and CB). But the N1s peaks in co-existence of Ca²⁺ and PO₄³⁻ ions (the ACE substrate and AB1) are different from those of the samples containing solely Ca (CCE and CB) and could be deconvoluted to three subpeaks located at 398.3, 399.6 and 400.9 eV. Similar to AB1 [23], Fig. 7 gives a N1s curve fitting for the ACE substrate, which suggests the existence of many kinds of N chemical states on the substrate surfaces containing both Ca²⁺ and PO₄³⁻ ions.

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Fig. 6. N1s XPS spectra for the substrate surfaces of samples after cell culture for 24 h and then removed of osteoblast. As control, the N1s XPS spectra of samples after immersion in BSA for 24 h are shown. PB—pre-phosphatized sample (P) was immersed in BSA solution for 24 h. CB—pre-calcified sample (C) was immersed in BSA solution for 24 h. AB1—Ti-CHA coating (A) was immersed in BSA solution for 24 h.

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Fig. 7. Representative deconvolution of N1s XPS envelope for the substrate surfaces of Ti-CHA coating after cell culture for 24 h and then removed of osteoblast.

Fig. 8 is Ca2p XPS spectra of the samples. For the titanium surfaces containing calcium including pre-calcified surface (C) and CHA coating (A), either after cell culture or protein adsorption, the Ca2p BE changes (ΔBEs) were approximately equal. Fig. 9 is P2p XPS spectra of the samples. The surfaces containing solely phosphate ions (P), after cell culture and BSA adsorption, exhibited similar P2p BE values (Fig. 8, PB and PCE), and so did the CHA coating ( Fig. 8, ACE and AB1). These results suggest that Ca²⁺ and PO₄³⁻ ions on the surfaces situated in a similar chemical environment in the two test conditions and support a generally acknowledged concept that proteins mediate cell response to biomaterial surfaces. However, after BSA adsorption and cell culture, the change of P2p BE values for the surfaces containing solely phosphate were less than those for the CHA coating, which might relate to function of Ca²⁺ on the CHA surface. Furthermore, for the four samples with respect to protein adsorption (PB and AB) and cell culture (ACE and AB), broader P2p peaks suggest that there were more than one P-group. Fig. 10 shows a curve fitting taking the ACE substrate as example. The osteoblast adhesion also resulted obviously in change of C1s and O1s XPS spectra of the samples. The deconvolution for these spectra revealed many kinds of C-groups and O-groups as possible products from osteoblast adhesion (not shown).

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Fig. 8. Ca2p XPS spectra for the substrate surface of samples after cell culture for 24 h and then removed of osteoblast. As control, the Ca2p XPS spectra of samples after immersion in BSA are shown. CB—pre-calcified sample (C) was immersed in BSA solution for 24 h. AB1—Ti-CHA coating (A) was immersed in BSA solution for 24 h.

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Fig. 9. P2p XPS spectra for the substrate surface of samples after cell culture for 24 h and then removed of osteoblast. As control, the P2p XPS spectra of samples immersed in BSA are shown. PB—pre-phosphatized sample (P) was immersed in BSA solution for 24 h. AB1—Ti-CHA coating (A) was immersed in BSA solution for 24 h.

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Fig. 10. Representative deconvolution of P2p XPS envelope for the substrate surfaces of Ti-CHA coating after cell culture for 24 h and then removed of osteoblast.

6. 4. Discussion

The titanium surfaces containing calcium including pre-calcified and CHA surfaces adsorbed more proteins than the surface containing solely phosphate ions, which suggests that the existence of calcium on the surfaces lead to the stronger adsorption ability of proteins as osteoblast ligants. The N1s and Ca2p BE values of the surfaces containing calcium appeared obviously changes after cell culture for 24 h. Thus the initial cell adhesion should be chemical interactions rather than physical ones. The BE change of the surfaces containing solely phosphate ions after cell culture was obviously smaller than that containing calcium, i.e., the interactions were much weaker. On the CHA surface containing Ca²⁺ and PO₄³⁻ ions, both Ca²⁺ and PO₄³⁻ sites are capable of adsorbing proteins as cell ligands; but Ca²⁺ ions probably work in chemical synergism with PO₄³⁻ ions, and stimulate adsorption of proteins and osteoblast adhesion via PO₄³⁻ ions onto the surfaces. The slight difference in the N amounts on the CCE and ACE surfaces implied that other components such as PO₄³⁻ ions do not significantly affect the stronger interactions between Ca²⁺ ions and proteins as cell ligands. Moreover, the small phosphorous amount on the CCE substrate surface might be attributed to the formation of calcium phosphate, which resulted from the reaction of Ca²⁺ ions on the surface with free PO₄³⁻ ions decomposed by ALP. Combining the cell count and ALP analysis, it can be deduced that Ca²⁺ ions on titanium surfaces played more important role than PO₄³⁻ ions in osteoblast adhesion. Except for chemical reactions, surface electricity may also be a factor.

During cell culture, proteins, mainly extracellular matrix (ECM) proteins adsorb to material surfaces, and the adsorbed proteins as cell ligants coordinate in covalence to proteins as cell receptors, resulting in cell attachment onto material surfaces. For osteoblast culture in serum, a routine culture medium, fibronectin (FN) and vitronectin (VN) in the ECM are important cell attachment-promoting proteins and influence over cell attachment and spreading. By coordinating FN and VN to the receptors at the cell membrane, osteoblasts adhere onto material surfaces [24 and 25]. Both FN and VN easily adsorb onto positively charged surfaces of materials [4 and 26]. In other words, the positively charged surfaces are favorable to FN and VN adsorption, resulting in cell attachment and spreading. After pre-calcification, because there were a number of Ca²⁺ ions and stronger basicity on the surfaces, the surfaces should be positively charged in the biological solution at pH=7.2-7.4 [27 and 28]. Thus, Ca²⁺ ions formed the surface active sites with positive charges. FN and VN would easily adsorb onto the positive charged sites, and then interact and bond with substrate surfaces, promoting the attachment of osteoblasts onto the surfaces. The pre-phosphatized surface on which Ti₃(PO₄)₄, Ti(HPO₄)₂ and Ti(H₂PO₄)₄ covered would be negatively charged in the biological solution at pH=7.2-7.4. Due to the similar electricity, the Coulomb repel force between the surface and FN or VN would not favor FN and VN to approach the surface and strongly combine with the surface. As a result, osteoblasts difficultly attached onto the surface, though other combined ways such as weaker covalence than static electrical force were possible. Therefore, for the combination of osteoblasts on titanium-CHA coating, FN and VN probably easily adsorbed onto the surface via the Ca²⁺ ion active sites with positive electricity; and then FN and VN on the surface coordinated to cell receptors, resulting in osteoblast adhesion onto the surface, spread and differentiation. Furthermore, the XPS analyses, including the curve fitting, suggest that the initial cell adhesion onto the surfaces involves many kinds of biological and chemical interactions relating to Ca, P, C, O and N elements or their groups.

In addition, Ca²⁺ ions also mediate cell-cell conjunction and cell-cell communication via gap conjunction [29]. The dissolution of Ca²⁺ ions on the surface containing calcium increased Ca²⁺ ion concentration in the culture medium and stimulated these functions, which might also be beneficial to cell adhesion and higher cell activity on the surfaces containing calcium including pre-calcified and CHA coating surfaces. Of course, to understand the mechanism in detail, further work should be based on investigations from materials, physical chemistry and biology, especially molecular biology.

7. 5. Conclusions

The osteoblast amount and activity on the surfaces containing calcium are higher than those on the surface containing solely phosphate ions.

Ca²⁺ ion sites on the material surfaces favor protein adsorption, such as FN and VN as ligands of osteoblast, onto the surface due to positive electricity, chemical and biological function. On the apatite surfaces, Ca²⁺ ions are the active sites of the osteoblast adhesion and also promote the cell adhesion on PO₄³⁻ ion sites.

The cell adhesion is a complicated biological and chemical process relating to surface several elements similar to protein adsorption.

8. Acknowledgements

This work was supported by Ministry of Science and Technology of P.R. China under 863 program grant 2001AA 326010.

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