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Enhancing osteoblast functions on a magnesia partially stabilised zirconia bioceramic by means of laser irradiation
Materials Science and Engineering C 25 (2005) 496 – 502 www.elsevier.com/locate/msec
Enhancing osteoblast functions on a magnesia partially stabilised zirconia bioceramic by means of laser irradiation L. Haoa,*, D.R. Mab, J. Lawrencec, X. Zhud a
Rapid Manufacturing Research Group, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Leicestershire, LE11 3TU, United Kingdom b Department of Plastic Surgery, Singapore General Hospital, Outram Road, Singapore 169608, Singapore c Manufacturing Engineering Division, School of Mechanical and Production Engineering, Nanyang Technological University (NTU), Nanyang Avenue, Singapore 639798, Singapore d Singapore Eye Research Institute, Singapore General Hospital, Singapore 168759, Singapore Received 7 April 2004; received in revised form 11 October 2004; accepted 7 March 2005 Available online 31 May 2005
Abstract In order to promote and sustain osteointegration of surrounding bone, a CO2 laser has been used to modify a magnesia partially stabilised zirconia (MgO – PSZ) for improved osteoblast cells’ functions. The surface characterisation revealed that the increase in surface roughness, surface oxygen content and surface energy brought about the higher wettability characteristics of the MgO – PSZ following CO2 laser irradiation. The alkaline phosphatase synthesis, osteocalcin production and proliferation of human fetal osteoblasts (hFOB) were investigated using in vitro cellular models. It was found that the osteoblasts performed better on the CO2 laser treated sample than on the untreated sample in terms of these functions of osteoblasts. Wettability characteristics, which could influence protein adsorption and initial cell response, could be predominant mechanism active in improvement of the cell functions. The results of the study provided evidence that laser irradiation could be a promising surface processing tool of bioceramic for improved bonding with bone. D 2005 Elsevier B.V. All rights reserved. Keywords: CO2 laser; Magnesia partially stabilised zirconia (MgO – PSZ); Osteoblast; Alkaline phosphatase synthesis; Osteocalcin
1. Introduction The extent of osteointegration between bone and a newly implanted material is influenced by many factors including a number of biological and surrounding tissue responses of the hosts. Properties of the biomaterial surface (such as topography and wettability) control the type and magnitude of cellular and molecular events at the tissue-implant interface. The key physical properties of a biomaterial can be retained while only the outermost surface is modified to tailor to the biointeractions. Hence, if surface modification is properly carried out, the mechanical properties and functionality of the device will be unaffected, but the tissue
* Corresponding author. Tel.: +44 1509 227565; fax: +44 1509 227549. E-mail address: [email protected] (L. Hao). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.03.003
interface-related biocompatibility can be improved [1]. It is, therefore, of paramount importance to fabricate biomaterials with adequate bulk properties followed by a special treatment to enhance the surface property [2]. Magnesia partially stabilised zirconia (MgO –PSZ) is a bioinert ceramic used as orthopaedic and dental implant that exhibits high mechanical strength, excellent corrosion resistance, and good biocompatibility. However, bioinert ceramics have often clinically failed due to lack of direct bonding with bone, that is, insufficient osteointegration that depends on the adhesion of anchorage-dependent cells. Lately, several publications have investigated the modification of biocompatibility of biomaterials’ surface following the laser irradiation. A CO2 pulsed laser was used to graft a polymer [3] and a rubber [4]. The results showed a marked reduction of the platelet adhesion and aggregation for the modified polymer surface and cell
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
attachment with greater degree of spreading and flattening on the unmodified rubber surface. L929 fibroblast cells attached and proliferated extensively on the CO2 and KrF laser treated films [5] in comparison with the unmodified PET, with surface morphology and wettability being found to affect cell adhesion and spreading. Improvement in fibroblast cell adhesion [6] and osteoblast cell adhesion [7] were found to result from the higher wettability of magnesia partially stabilised zirconia (MgO – PSZ) following CO2 laser treatment. In this study, the osteoblast cell functions in terms of alkaline phosphatase synthesis, osteocalcin production and proliferation of human fetal osteoblast cells (hFOB) were investigated on CO2 laser modified MgO – PSZ using in vitro cellular model. The effects of wettability characteristics on the osteoblast function were analysed. This provided the first evidence of enhanced osteoblast cell functions and the mechanisms active on MgO – PSZ following CO2 laser treatment.
2. Experimental procedures 2.1. Material and CO2 laser surface treatment The material investigated was a 4% magnesia partially stabilised zirconia (MgO –PSZ). The material was obtained in sheet form with dimension of 50 50 2.15 mm3 (Goodfellow, Ltd). For experimental convenience it was cut into blocks of 50 12 2.15 mm3 with a diamond rimmed cutting blade. The material was used as received prior to laser treatment. A 3 kW CO2laser (Trumpf, Ltd) emitting with a wavelength of 10.6 Am was used in this study. The laser produced a transverse electromagnetic mode (TEM01) beam and was operated in the continuous wave (CW) mode. A series of optical units were used to deliver the CO2 laser beam to the workpiece through the laser head, which was positioned by means of 2 linear axes ( y- and z-axis) and 2 rotary axes (b- and c-axis). The defocused CO2 laser beam was traversed in single time across the surface of the MgO –PSZ samples placed on the state using the x-axis. Beams with power densities of 0.9 and 1.6 kW/cm2 and an 11 mm spot diameter were applied, whilst the traverse speed was set at 2000 mm/min with O2 process gas of 2 bars pressure and 10 l/min gas flow rate. 2.2. Characterisation of the MgO –PSZ The surface roughness (R a ) of the MgO – PSZ was measured by a profilometer (Surface Tester, SV-600). Five measurements were made at different positions of each sample and the mean values were obtained. The surface microstructure and oxygen content of the MgO – PSZ were analysed by scanning electron microscopy (SEM) and X-ray photoemission spectroscopy (XPS). For cross-section analysis, the samples were cold mounted in resin before
497
grinding on increasing finer SiC grinding papers followed by 1 Am finish using diamond past. The polished samples were then etched with 10 ml HNO3 and 20 ml HF (48%) for 20 min to affect the ceramic layer. To investigate the effects of laser irradiation on the wetting and surface energy properties of the MgO –PSZ, a set of sessile drop control experiments were carried out using glycerol, formamide, ethyleneglycol, polyglycol E-200, and polyglycol 15-200 with known total surface energy (c lv), depressive (c lvd), and polar (c lvp) component values [8]. The contact angles, h, of the test liquids on the untreated and CO2 laser treated MgO – PSZ were determined in normal atmospheric conditions at 25 -C using a sessile drop testing machine (First Ten Angstroms, Inc). 2.3. Cell culture and evaluation 2.3.1. Cell culture The human osteoblast cell line hFOB 1.19 was obtained from the American Type Culture Collection (Manassas, Inc). The cells were cultured with a medium containing a 1:1 mixture of Dulbecco’s Modified Eagle’s medium without phenol red and Ham’s F12 medium with 2.5 mM l-glutamine (d-MEM/F-12 Medium), supplemented with 10% fetal bovine serum (ATCC) and 0.3 mg/ml G418 (Calbiochem) in a 37 -C, 5% CO2/95% air incubator. Osteoblast cells at passages 2 – 3 were used in this experiment. 2.3.2. Alkaline phosphatase assay For the staining of human osteoblast cells an alkaline phosphatase assay kit (Sigma diagnostics, Inc) was used. After rinsing with PBS, the samples with cells were fixed by immersing in citrate buffered acetone for 30 s and rinsed gently with deionised water for 45 s. After alkaline – dye mixture was added to the samples and they were incubates at 24 -C for 30 min protected from direct light. After rinsing thoroughly in deionised water for 2 min and place in Mayer’s Hematoxylin solution for 10 min, positive staining for alkaline phosphatase (red –violet) was identified by optical microscopy and evaluated by scoring cell rating and count. 2.3.3. Osteocalcin assay For the quantification of osteocalcin in the cell culture supernatant of human maxillar osteoblast cells, the Tissue Tacklei DAB multispecies immunohistochemistry system (Calbiochem, Inc) was applied performed according to the manufacturer’s instructions. In brief, the standards, the curve control and the cell culture supernatants were premixed with biotinylated osteocalcin, incubated in microwells precoated with anti-osteocalcin (Calbiochem, Inc) for 1 h, washed and incubated for 15 min with peroxidaseconjugated streptavidin, which binds strongly to the biotinylated osteocalcin. After a further washing step the chromogenic substrate was added and incubated for 30 min.
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The reaction was stopped by 2 M H2SO4 and the staining of osteocalcin evaluated. Osteocalcin is exclusively synthesized by osteoblasts and is believed to prevent premature mineralization of newly formed, but disorganized bone matrix [9].
content of the MgO – PSZ increased with increasing the power density. The surface oxygen content was 41.6 at.% T 3.9 (untreated), 44.2 T 3.2 at.% (0.9 kW/cm2), and 64.3 at.% T5.2 (1.6 kW/cm2). Furthermore, the analysis revealed that the surface microstructure varied with the different CO2 laser power densities applied. The defined microstructures [10] according to the main structure of the MgO –PSZ were hexagonal structure (0.9 kW/cm2) and cellular formation (1.6 kW/cm2).
2.3.4. Cell proliferation Cell viability and proliferation on each specimen was measured by MTT assay. After cultured for 7 days, the osteoblast cells on each specimen were gently washed with phosphate-buffered solution and were measured by MTT assay using 3(4,5-dimethyl-thiazole-2-yl) 2,5-diphenyl tetrazolium bromide (MTT, Sigma, Inc). The MTT solution was added to each specimen and the cells were incubated for 4 h at 37 -C, then the medium was replaced with dimethylsulfoxide. Absorbance of the solution was measured by a multiplate reader (EL312, Bio-Tek Instrument, Inc) at 490 nm.
3.2. Wettability and surface energy When a drop of liquid is brought into contact with a flat solid surface, the final shape taken by the drop is expressed by the contact angle, h. An increased value of cos h indicates higher wettability of material. As evident from Table 1, h of the test liquids decrease after the laser treatment, indicating that the wettability of the MgO – PSZ increased with increasing power density. In order to state concisely, the glycerol was used as a typical liquid expressing the change in h and thus the wettability of the MgO – PSZ, due to the similar trend of the change in h of the test liquids for the samples before and after CO2 laser treatment. In solid– liquid systems where both dispersion forces and polar forces are present cos h can be related to the surface energies of the respective liquid and solid by [11]
2.3.5. Statistics Statistical analysis was performed with a SPSS v.12 software package (SPSS/PC Inc., Chicago, IL). Data are reported as mean T S.D. at a significance level of p < 0.05. After having verified normal distribution and homogeneity of variances, one-way ANOVA and Scheffe´’s post hoc multiple comparison tests were done.
cos h ¼
3. Results
2 cdsv cdlv
1=2
þ 2 cpsv cplv clv
1=2 1
ð1Þ
where c dlv and c plv are the dispersive and polar components of the surface energy, c lv, of the liquid, and, c dsv and c psv are the dispersive and polar components of the surface energy, c sv, of the solid. Previous work [12] has described the detailed calculation procedure and determined the surface energy of the untreated and CO2 laser treated MgO – PSZ as shown in Table 1. The CO2 laser treatment increased c sv of the MgO – PSZ by primarily increasing c psv, since c dsv was similar for all the samples.
3.1. The depth of melting and heat affected zone (HAZ) and surface roughness By means of cross-sectional SEM analysis it was possible to determine the depth of melting and heat affected zone (HAZ) in CO2 laser treated samples following the polishing and etching. It was found that the mean value of the melt depth was 18.4 T 3.3 Am (0.9 kW/cm2) and 60.7 T 4.9 Am (1.6 kW/cm2), while the mean value of the HAZ was 63.5 T4.6 Am (0.9 kW/cm2) and 243 T18.2 Am (1.6 kW/cm2) for the CO2 laser treated samples. CO2 laser treatment brought about a consistently rougher surface of the MgO – PSZ compared with on untreated sample, with the R a increasing as the laser power density increased. The R a was 0.295 T 0.217 Am (untreated), 0.333 T 0.274 Am (0.9 kW/cm2), and 0.717 T0.453 Am (1.6 kW/cm2). XPS analysis showed that the surface oxygen
3.3. Synthesis of alkaline phosphatase The cell rating was determined on the basis of the quantity and intensity of precipitated dye within the cytoplasm of these cells. As shown in Fig. 1, the granules on the untreated sample are small and show faint to moderate intensity of staining. On the other hand, the
Table 1 The h of the test liquids and determined surface energy of the untreated and laser treated MgO – PSZ Laser power density
Untreated 0.9 kW/cm2 1.6 kW/cm2
Surface energy (mJ/cm2)
h of the test liquids Glycerol
Formamide
Ethylene-glycol
Polyglycol E-200
Polyglycol 15-200
c sv
c Psv
c dsv
79 62 40
73 57 36
61 48 29
53 40 26
35 28 19
52.8 66.3 108.9
10.1 21.9 60.7
42.8 44.4 48.2
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
499
100µm
100µm
(a)
(b)
100µm
50µm
(c)
(d)
Fig. 1. Optical image of the positive staining for alkaline phosphatase on the untreated (a) and CO2 laser treated sample at 0.9 kW/cm2 (b) (c) and sample at 1.6 kW/cm2 (d).
granules are medium to large and show a strong intensity of staining in case of the laser treated sample at 0.9 kW/cm2, and a brilliant intensity of staining on the laser treated sample at 1.6 kW/cm2. One granule expressed the spreading of the cells on the laser treated sample at 0.9 kW/cm2 as shown in Fig. 1(c). Hence, the leukocyte alkaline phospha-
3.4. Osteocalcin determination Similarly, the cell rating was determined based on the quantity and intensity of precipitated dye within the cytoplasm of these cells. As shown in Fig. 3, the granule size and intensity of staining reveal the same trend as for alkaline phosphatase. The osteocalcin score, determined by quantity and intensity of the staining, is shown in Fig. 4.
120
100
LAPA Score
tase activity (LAPA) score, determined by the number of cells counted multiplied the value of cell rating, increases with laser power density as shown in Fig. 2.
80
3.5. Cell proliferation 60
As shown in Fig. 5, the MTT assay indicated that there was higher cell viability and proliferation on the CO2 laser treated samples than on the untreated samples. The cell viability measured by recording the optical density, increases slightly for the laser treated sample at 0.9 kW/cm2 and considerably for the laser treated sample at 1.6 kW/cm2.
40
20
0
Untreated
CO2 laser 0.9 kW/cm2
CO2 laser 1.6 kW/cm2
Fig. 2. Leukocyte alkaline phosphatase activity (LAPA) score of the untreated and CO2 laser treated MgO – PSZ (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
4. Discussion As Table 1 shows, the CO2 laser treatment brought about a higher wettability of MgO –PSZ surface. The increase in
500
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
100µm
100µm
(a)
(b)
100µ µm
(c) Fig. 3. Optical image of the positive staining for osteocalcin on the untreated (a) and CO2 laser treated sample at 0.9 kW/cm2 (b) and sample at 1.6 kW/cm2 (c).
wettability is caused by the considerable increase in c sv and c svp after CO2 laser treatment. The changes of the surface energy of the MgO – PSZ are thought to be due to the microstructural change occasioned by melting of the surface during laser treatment, a transition that is known to effect an increase in c svp [13 – 15], and thus improvement of the wettability and an increase in the adhesion at the interface in contact with the control liquids. In addition, the previous
study has revealed that changes in surface energy of the MgO –PSZ were attributed to the changes in microstructures by the form of crystal size and phase changes [16]. The surface energy increase as the crystal size and tetragonal phase increase in the MgO – PSZ surface after CO2 laser treatment. Such changes in the surface energy were, along with other things, brought about as a result of changes in the 0.12
100 0.10
Optical Density
Osteocalcin Score
80
60
40
0.08
0.06
0.04
0.02
20
0.00
Untreated
0
Untreated
CO2 laser 0.9 kW/cm
2
CO2 laser 1.6 kW/cm
CO2 laser
2
0.9 kW/cm
CO2 laser
2
1.6 kW/cm
2
Fig. 4. Osteocalcin score of the untreated and CO2 laser treated MgO – PSZ (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
Fig. 5. The MTT optical density measured after 7-day cell culture for untreated and CO2 laser treated samples (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
Optical Density Osteocalcin Score LPPA Score
microstructure of the MgO –PSZ after CO2 laser interaction. Hexagonal and cellular microstructural pattern appeared at the MgO – PSZ surface following CO2 laser treatment, revealing that solidification of the individual melted particulates had occurred. Such a solidification structure indicates that exposure of the MgO – PSZ to CO2 laser irradiation resulted in rapid heating of the surface, which consequently leaded to the melting and resolidification of the MgO – PSZ surface. The local melting and resolidification of a small portion of the surface of a material with a laser beam provides unique opportunity for controlled crystal growth under high temperature gradients and high cooling rates [17]. Furthermore, the observed increase in the wetting performance of the MgO – PSZ could have certainly been influenced by the increase in the surface oxygen content of the MgO – PSZ surface as a result of the laser treatment, since this is known to increase the likelihood of wetting [18,19]. The oxygen enrichment of the MgO – PSZ surface is active in promoting wetting and adhesion. After CO2 laser treatment, the roughness of the MgO – PSZ surface increased with power density. This is likely to be the result of turbulent convection in the melt pool caused by the TEM01 mode of the CO2 laser beam. Indeed, the power density distribution of the CO2 laser appears to be multimode [17]. When h < 90-, the rougher the surface is, the lower is h. It is reverse when h > 90- [20]. In this study, the rougher surface generated by the CO2 laser treatment could be attributed to the higher wettability characteristics on the condition of h <90-. As one shown in Fig. 6, the enhanced cell functions represented by the cell proliferation (optical density), osteocalcin, and LAPA scores increase with the increasing wettability. Hence, it is believed that the wettability of the MgO – PSZ was the primary factor governing the cell response. The effects of wettability on cell functions could 90 60 30 0 90 60
501
result from its influence on the protein adsorption and cell adhesion. The adsorption of the proteins is the net result of various types of interactions which depend on the nature of the protein aqueous solution. The difference in cellular response of different materials suggests that there are differences in the organization of the adsorbed protein layer. Protein adsorption mediated cell behaviours are regarded as fundamental reactions at biomaterial/tissue interfaces [21]. Indeed, the CO2 laser treatment brought about a lower amount of adsorbed albumin and a higher amount of the adsorbed fibronectin on the modified MgO – PSZ compared with the untreated sample [22]. Such interactions would benefit the osteoblast cell adhesion since albumin is a non-cell adhesive protein [23] while fibronectin is a cell adhesive protein [24]. Furthermore, it had been shown that the enhancement in wettability generated by the CO2 laser treatment was the predominant mechanism that influences the improvement of human skin fibroblast adhesion [6] and human fetal osteoblast cell adhesion and growth [7]. It was found that after 1 day of culture, the osteoblast cells spread well and covered more surface areas on the higher surface energy substrates than on the lower surface energy substrates of the MgO –PSZ [7]. Initial events during cell-biomaterial interactions (such as cell adhesion and concomitant morphology) affect functions, such as cell population motility, proliferation, and synthesis of extracellular matrix proteins of anchoragedependent cells [25 – 27]. Increased osteoblast adhesion coupled with enhanced proliferation and synthesis of alkaline phosphatase has been observed on surfaces (for example, borosilicate glass and titanium) modified with immobilized peptide sequences (such as arginine– glycine– aspartic acid –serine (RGDS) and lysine –arginine– serine– arginine (KRSR) [28,29] contained in extracellular matrix proteins such as vitronectin and collagen. The enhanced cell adhesion [30] on nanophase ceramics brought about the enhanced cell functions of these materials [31]. The results of the present in vitro study provided the first evidence of enhanced functions (specifically, cell proliferation, synthesis of alkaline phosphatase, and osteocalcin in the extracellular matrix) of osteoblasts on CO2 laser treated MgO – PSZ. These improved functions would certainly promote and sustain osteointegration of surrounding bone that are needed to improve implant efficacy.
30 0
5. Conclusions
0.12 0.08 0.04 0.00 0.1
0.2
0.3
0.4
0.5
0.6
Wettability, cos θ (Glycerol)
0.7
0.8
Fig. 6. The relationship between optical density (cell proliferation), osteocalcin score, LAPA score, and wettability of the MgO – PSZ.
The results of the present in vitro study provided the first evidence of enhanced functions (specifically, cell proliferation, synthesis of alkaline phosphatase, and osteocalcin in the extracellular matrix) of osteoblasts on CO2 laser treated MgO – PSZ. The CO2 laser treatment of the MgO – PSZ brought about changes in surface energy, surface roughness, surface oxygen content, and microstructure that resulted in higher wettability of the MgO – PSZ compared with
502
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untreated specimen. Wettability, which could influence protein adsorption and initial cell response, could be a predominant mechanism active in improvement of the cell functions. These improved functions would certainly promote and sustain osteointegration of surrounding bone that is needed to improve implant efficacy.
References [1] B.D. Ratner, Biomaterials Science: An Introduction to Materials in Medicine, Academic Press, San Diego, 1996, p. 484. [2] R.N.S. Sodhi, J. Electron Spectroscopy Related Phen. 81 (1996) 269 – 284. [3] H. Mirzadeh, A.A. Katbab, R.P. Burford, Radiation Phys. Chem. 46 (1995) 859 – 862. [4] H. Mirzadeh, A.A. Katbab, M.T. Khorasani, R.P. Burford, E. Gorgin, A. Golestani, Biomaterials 16 (1995) 641 – 648. [5] M. Dadsetan, H. Mirzadeh, N. Sharifi-Sanjani, M. Daliri, J. Biomed. Mater. Res. 57 (2001) 183 – 189. [6] L. Hao, J. Lawrence, Mater. Sci. Eng. C. 23 (2003) 627 – 639. [7] L. Hao, J. Lawrence, K.S. Chian, J. Biomaterials Application (2004) (accepted for publication). [8] A.J. Kinloch, Adhesion and Adhesives: Science and Technology, Chapman and Hall, 1987, p. 30. [9] L. Hyldstrup, I. Clemmensen, B.A. Jensen, I. Transbol, Scandinavian J. Clinical Lab. Investigation 48 (1988) 611 – 619. [10] L. Hao, J. Lawrence, Proc. IMechE Part B, J. Eng. Manufacture (2003) (Accepted for publication). [11] F.M. Fowkes, Ind. Eng. Chem. 56 (1964) 40 – 52. [12] L. Hao, J. Lawrence, J. Phys. D. 36 (2003) 1292 – 1299. [13] R.J. Good, L.A. Girifalco, J. Phys. Chem. 64 (1960) 561 – 565.
[14] J. Lawrence, L. Li, J.T. Spencer, Appl. Surf. Sci. 138 – 139 (1999) 388 – 393. [15] J. Lawrence, L. Li, J. Phys. D. 32 (1999) 1075 – 1082. [16] L. Hao, J. Lawrence, Mater. Sci. Eng. A. 364 (2003) 171 – 181. [17] L. Hao, J. Lawrence, G.C. Lim, H.Y. Zheng, Optics Lasers Eng. 42 (2004) 355 – 374. [18] Q. Song, A.N. Netravali, J. Adhesion Sci. Tech. 12 (1998) 957 – 982. [19] J. Lawrence, L. Li, Laser modification of the wettability characteristics of engineering materials, Professional Engineering, London, 2001. [20] R.N. Wenzel, Ind. Eng. Chem. 28 (1936) 988 – 994. [21] J.E. Davies, The importance and measurement of surface charge species in cell behaviour at the biomaterial interface, in: B.D. Ratner (Ed.), Surface Characterization of Biomaterials, Elsevier, Amsterdam, 1988, pp. 219 – 234. [22] L. Hao, J. Lawrence, J. Biomed. Mater. Res. 69A (2004) 748 – 756. [23] D.A. Puleo, R. Bizios, Bone Mineral 18 (1992) 215 – 226. [24] F. Grinnell, M.K. Feld, J. Biological Chem. 257 (1982) 4888 – 4893. [25] K.C. Olbrich, T.T. Andersen, F.A. Blumenstock, R. Bizios, Biomaterials 17 (1996) 759 – 764. [26] J.Y. Martin, Z. Schwartz, T.W. Hummert, D.M. Schraub, J. Simpson, J. Lankford Jr., D.D. Dean, D.L. Cochran, B.D. Boyan, J. Biomed. Mater. Res. 29 (1995) 389 – 401. [27] K. Kieswetter, Z. Schwartz, T.W. Hummert, D.L. Cochran, J. Simpson, D.D. Dean, B.D. Boyan, J. Biomed. Mater. Res. 32 (1996) 55 – 63. [28] K.E. Healy, A. Rezania, R.A. Stile, Annals of the New York Academy of Sciences 875 (1999) 24 – 35. [29] K.C. Dee, T.T. Andersen, R. Bizios, J. Biomed. Mater. Res. 40 (1998) 371 – 377. [30] T.J. Webster, R.W. Siegel, R. Bizios, Biomaterials 20 (1999) 1221 – 1227. [31] T.J. Webster, C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios, Biomaterials 21 (2000) 1803 – 1810.
Enhancing osteoblast functions on a magnesia partially stabilised zirconia bioceramic by means of laser irradiation L. Haoa,*, D.R. Mab, J. Lawrencec, X. Zhud a
Rapid Manufacturing Research Group, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Leicestershire, LE11 3TU, United Kingdom b Department of Plastic Surgery, Singapore General Hospital, Outram Road, Singapore 169608, Singapore c Manufacturing Engineering Division, School of Mechanical and Production Engineering, Nanyang Technological University (NTU), Nanyang Avenue, Singapore 639798, Singapore d Singapore Eye Research Institute, Singapore General Hospital, Singapore 168759, Singapore Received 7 April 2004; received in revised form 11 October 2004; accepted 7 March 2005 Available online 31 May 2005
Abstract In order to promote and sustain osteointegration of surrounding bone, a CO2 laser has been used to modify a magnesia partially stabilised zirconia (MgO – PSZ) for improved osteoblast cells’ functions. The surface characterisation revealed that the increase in surface roughness, surface oxygen content and surface energy brought about the higher wettability characteristics of the MgO – PSZ following CO2 laser irradiation. The alkaline phosphatase synthesis, osteocalcin production and proliferation of human fetal osteoblasts (hFOB) were investigated using in vitro cellular models. It was found that the osteoblasts performed better on the CO2 laser treated sample than on the untreated sample in terms of these functions of osteoblasts. Wettability characteristics, which could influence protein adsorption and initial cell response, could be predominant mechanism active in improvement of the cell functions. The results of the study provided evidence that laser irradiation could be a promising surface processing tool of bioceramic for improved bonding with bone. D 2005 Elsevier B.V. All rights reserved. Keywords: CO2 laser; Magnesia partially stabilised zirconia (MgO – PSZ); Osteoblast; Alkaline phosphatase synthesis; Osteocalcin
1. Introduction The extent of osteointegration between bone and a newly implanted material is influenced by many factors including a number of biological and surrounding tissue responses of the hosts. Properties of the biomaterial surface (such as topography and wettability) control the type and magnitude of cellular and molecular events at the tissue-implant interface. The key physical properties of a biomaterial can be retained while only the outermost surface is modified to tailor to the biointeractions. Hence, if surface modification is properly carried out, the mechanical properties and functionality of the device will be unaffected, but the tissue
* Corresponding author. Tel.: +44 1509 227565; fax: +44 1509 227549. E-mail address: [email protected] (L. Hao). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.03.003
interface-related biocompatibility can be improved [1]. It is, therefore, of paramount importance to fabricate biomaterials with adequate bulk properties followed by a special treatment to enhance the surface property [2]. Magnesia partially stabilised zirconia (MgO –PSZ) is a bioinert ceramic used as orthopaedic and dental implant that exhibits high mechanical strength, excellent corrosion resistance, and good biocompatibility. However, bioinert ceramics have often clinically failed due to lack of direct bonding with bone, that is, insufficient osteointegration that depends on the adhesion of anchorage-dependent cells. Lately, several publications have investigated the modification of biocompatibility of biomaterials’ surface following the laser irradiation. A CO2 pulsed laser was used to graft a polymer [3] and a rubber [4]. The results showed a marked reduction of the platelet adhesion and aggregation for the modified polymer surface and cell
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
attachment with greater degree of spreading and flattening on the unmodified rubber surface. L929 fibroblast cells attached and proliferated extensively on the CO2 and KrF laser treated films [5] in comparison with the unmodified PET, with surface morphology and wettability being found to affect cell adhesion and spreading. Improvement in fibroblast cell adhesion [6] and osteoblast cell adhesion [7] were found to result from the higher wettability of magnesia partially stabilised zirconia (MgO – PSZ) following CO2 laser treatment. In this study, the osteoblast cell functions in terms of alkaline phosphatase synthesis, osteocalcin production and proliferation of human fetal osteoblast cells (hFOB) were investigated on CO2 laser modified MgO – PSZ using in vitro cellular model. The effects of wettability characteristics on the osteoblast function were analysed. This provided the first evidence of enhanced osteoblast cell functions and the mechanisms active on MgO – PSZ following CO2 laser treatment.
2. Experimental procedures 2.1. Material and CO2 laser surface treatment The material investigated was a 4% magnesia partially stabilised zirconia (MgO –PSZ). The material was obtained in sheet form with dimension of 50 50 2.15 mm3 (Goodfellow, Ltd). For experimental convenience it was cut into blocks of 50 12 2.15 mm3 with a diamond rimmed cutting blade. The material was used as received prior to laser treatment. A 3 kW CO2laser (Trumpf, Ltd) emitting with a wavelength of 10.6 Am was used in this study. The laser produced a transverse electromagnetic mode (TEM01) beam and was operated in the continuous wave (CW) mode. A series of optical units were used to deliver the CO2 laser beam to the workpiece through the laser head, which was positioned by means of 2 linear axes ( y- and z-axis) and 2 rotary axes (b- and c-axis). The defocused CO2 laser beam was traversed in single time across the surface of the MgO –PSZ samples placed on the state using the x-axis. Beams with power densities of 0.9 and 1.6 kW/cm2 and an 11 mm spot diameter were applied, whilst the traverse speed was set at 2000 mm/min with O2 process gas of 2 bars pressure and 10 l/min gas flow rate. 2.2. Characterisation of the MgO –PSZ The surface roughness (R a ) of the MgO – PSZ was measured by a profilometer (Surface Tester, SV-600). Five measurements were made at different positions of each sample and the mean values were obtained. The surface microstructure and oxygen content of the MgO – PSZ were analysed by scanning electron microscopy (SEM) and X-ray photoemission spectroscopy (XPS). For cross-section analysis, the samples were cold mounted in resin before
497
grinding on increasing finer SiC grinding papers followed by 1 Am finish using diamond past. The polished samples were then etched with 10 ml HNO3 and 20 ml HF (48%) for 20 min to affect the ceramic layer. To investigate the effects of laser irradiation on the wetting and surface energy properties of the MgO –PSZ, a set of sessile drop control experiments were carried out using glycerol, formamide, ethyleneglycol, polyglycol E-200, and polyglycol 15-200 with known total surface energy (c lv), depressive (c lvd), and polar (c lvp) component values [8]. The contact angles, h, of the test liquids on the untreated and CO2 laser treated MgO – PSZ were determined in normal atmospheric conditions at 25 -C using a sessile drop testing machine (First Ten Angstroms, Inc). 2.3. Cell culture and evaluation 2.3.1. Cell culture The human osteoblast cell line hFOB 1.19 was obtained from the American Type Culture Collection (Manassas, Inc). The cells were cultured with a medium containing a 1:1 mixture of Dulbecco’s Modified Eagle’s medium without phenol red and Ham’s F12 medium with 2.5 mM l-glutamine (d-MEM/F-12 Medium), supplemented with 10% fetal bovine serum (ATCC) and 0.3 mg/ml G418 (Calbiochem) in a 37 -C, 5% CO2/95% air incubator. Osteoblast cells at passages 2 – 3 were used in this experiment. 2.3.2. Alkaline phosphatase assay For the staining of human osteoblast cells an alkaline phosphatase assay kit (Sigma diagnostics, Inc) was used. After rinsing with PBS, the samples with cells were fixed by immersing in citrate buffered acetone for 30 s and rinsed gently with deionised water for 45 s. After alkaline – dye mixture was added to the samples and they were incubates at 24 -C for 30 min protected from direct light. After rinsing thoroughly in deionised water for 2 min and place in Mayer’s Hematoxylin solution for 10 min, positive staining for alkaline phosphatase (red –violet) was identified by optical microscopy and evaluated by scoring cell rating and count. 2.3.3. Osteocalcin assay For the quantification of osteocalcin in the cell culture supernatant of human maxillar osteoblast cells, the Tissue Tacklei DAB multispecies immunohistochemistry system (Calbiochem, Inc) was applied performed according to the manufacturer’s instructions. In brief, the standards, the curve control and the cell culture supernatants were premixed with biotinylated osteocalcin, incubated in microwells precoated with anti-osteocalcin (Calbiochem, Inc) for 1 h, washed and incubated for 15 min with peroxidaseconjugated streptavidin, which binds strongly to the biotinylated osteocalcin. After a further washing step the chromogenic substrate was added and incubated for 30 min.
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The reaction was stopped by 2 M H2SO4 and the staining of osteocalcin evaluated. Osteocalcin is exclusively synthesized by osteoblasts and is believed to prevent premature mineralization of newly formed, but disorganized bone matrix [9].
content of the MgO – PSZ increased with increasing the power density. The surface oxygen content was 41.6 at.% T 3.9 (untreated), 44.2 T 3.2 at.% (0.9 kW/cm2), and 64.3 at.% T5.2 (1.6 kW/cm2). Furthermore, the analysis revealed that the surface microstructure varied with the different CO2 laser power densities applied. The defined microstructures [10] according to the main structure of the MgO –PSZ were hexagonal structure (0.9 kW/cm2) and cellular formation (1.6 kW/cm2).
2.3.4. Cell proliferation Cell viability and proliferation on each specimen was measured by MTT assay. After cultured for 7 days, the osteoblast cells on each specimen were gently washed with phosphate-buffered solution and were measured by MTT assay using 3(4,5-dimethyl-thiazole-2-yl) 2,5-diphenyl tetrazolium bromide (MTT, Sigma, Inc). The MTT solution was added to each specimen and the cells were incubated for 4 h at 37 -C, then the medium was replaced with dimethylsulfoxide. Absorbance of the solution was measured by a multiplate reader (EL312, Bio-Tek Instrument, Inc) at 490 nm.
3.2. Wettability and surface energy When a drop of liquid is brought into contact with a flat solid surface, the final shape taken by the drop is expressed by the contact angle, h. An increased value of cos h indicates higher wettability of material. As evident from Table 1, h of the test liquids decrease after the laser treatment, indicating that the wettability of the MgO – PSZ increased with increasing power density. In order to state concisely, the glycerol was used as a typical liquid expressing the change in h and thus the wettability of the MgO – PSZ, due to the similar trend of the change in h of the test liquids for the samples before and after CO2 laser treatment. In solid– liquid systems where both dispersion forces and polar forces are present cos h can be related to the surface energies of the respective liquid and solid by [11]
2.3.5. Statistics Statistical analysis was performed with a SPSS v.12 software package (SPSS/PC Inc., Chicago, IL). Data are reported as mean T S.D. at a significance level of p < 0.05. After having verified normal distribution and homogeneity of variances, one-way ANOVA and Scheffe´’s post hoc multiple comparison tests were done.
cos h ¼
3. Results
2 cdsv cdlv
1=2
þ 2 cpsv cplv clv
1=2 1
ð1Þ
where c dlv and c plv are the dispersive and polar components of the surface energy, c lv, of the liquid, and, c dsv and c psv are the dispersive and polar components of the surface energy, c sv, of the solid. Previous work [12] has described the detailed calculation procedure and determined the surface energy of the untreated and CO2 laser treated MgO – PSZ as shown in Table 1. The CO2 laser treatment increased c sv of the MgO – PSZ by primarily increasing c psv, since c dsv was similar for all the samples.
3.1. The depth of melting and heat affected zone (HAZ) and surface roughness By means of cross-sectional SEM analysis it was possible to determine the depth of melting and heat affected zone (HAZ) in CO2 laser treated samples following the polishing and etching. It was found that the mean value of the melt depth was 18.4 T 3.3 Am (0.9 kW/cm2) and 60.7 T 4.9 Am (1.6 kW/cm2), while the mean value of the HAZ was 63.5 T4.6 Am (0.9 kW/cm2) and 243 T18.2 Am (1.6 kW/cm2) for the CO2 laser treated samples. CO2 laser treatment brought about a consistently rougher surface of the MgO – PSZ compared with on untreated sample, with the R a increasing as the laser power density increased. The R a was 0.295 T 0.217 Am (untreated), 0.333 T 0.274 Am (0.9 kW/cm2), and 0.717 T0.453 Am (1.6 kW/cm2). XPS analysis showed that the surface oxygen
3.3. Synthesis of alkaline phosphatase The cell rating was determined on the basis of the quantity and intensity of precipitated dye within the cytoplasm of these cells. As shown in Fig. 1, the granules on the untreated sample are small and show faint to moderate intensity of staining. On the other hand, the
Table 1 The h of the test liquids and determined surface energy of the untreated and laser treated MgO – PSZ Laser power density
Untreated 0.9 kW/cm2 1.6 kW/cm2
Surface energy (mJ/cm2)
h of the test liquids Glycerol
Formamide
Ethylene-glycol
Polyglycol E-200
Polyglycol 15-200
c sv
c Psv
c dsv
79 62 40
73 57 36
61 48 29
53 40 26
35 28 19
52.8 66.3 108.9
10.1 21.9 60.7
42.8 44.4 48.2
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
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100µm
100µm
(a)
(b)
100µm
50µm
(c)
(d)
Fig. 1. Optical image of the positive staining for alkaline phosphatase on the untreated (a) and CO2 laser treated sample at 0.9 kW/cm2 (b) (c) and sample at 1.6 kW/cm2 (d).
granules are medium to large and show a strong intensity of staining in case of the laser treated sample at 0.9 kW/cm2, and a brilliant intensity of staining on the laser treated sample at 1.6 kW/cm2. One granule expressed the spreading of the cells on the laser treated sample at 0.9 kW/cm2 as shown in Fig. 1(c). Hence, the leukocyte alkaline phospha-
3.4. Osteocalcin determination Similarly, the cell rating was determined based on the quantity and intensity of precipitated dye within the cytoplasm of these cells. As shown in Fig. 3, the granule size and intensity of staining reveal the same trend as for alkaline phosphatase. The osteocalcin score, determined by quantity and intensity of the staining, is shown in Fig. 4.
120
100
LAPA Score
tase activity (LAPA) score, determined by the number of cells counted multiplied the value of cell rating, increases with laser power density as shown in Fig. 2.
80
3.5. Cell proliferation 60
As shown in Fig. 5, the MTT assay indicated that there was higher cell viability and proliferation on the CO2 laser treated samples than on the untreated samples. The cell viability measured by recording the optical density, increases slightly for the laser treated sample at 0.9 kW/cm2 and considerably for the laser treated sample at 1.6 kW/cm2.
40
20
0
Untreated
CO2 laser 0.9 kW/cm2
CO2 laser 1.6 kW/cm2
Fig. 2. Leukocyte alkaline phosphatase activity (LAPA) score of the untreated and CO2 laser treated MgO – PSZ (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
4. Discussion As Table 1 shows, the CO2 laser treatment brought about a higher wettability of MgO –PSZ surface. The increase in
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100µm
100µm
(a)
(b)
100µ µm
(c) Fig. 3. Optical image of the positive staining for osteocalcin on the untreated (a) and CO2 laser treated sample at 0.9 kW/cm2 (b) and sample at 1.6 kW/cm2 (c).
wettability is caused by the considerable increase in c sv and c svp after CO2 laser treatment. The changes of the surface energy of the MgO – PSZ are thought to be due to the microstructural change occasioned by melting of the surface during laser treatment, a transition that is known to effect an increase in c svp [13 – 15], and thus improvement of the wettability and an increase in the adhesion at the interface in contact with the control liquids. In addition, the previous
study has revealed that changes in surface energy of the MgO –PSZ were attributed to the changes in microstructures by the form of crystal size and phase changes [16]. The surface energy increase as the crystal size and tetragonal phase increase in the MgO – PSZ surface after CO2 laser treatment. Such changes in the surface energy were, along with other things, brought about as a result of changes in the 0.12
100 0.10
Optical Density
Osteocalcin Score
80
60
40
0.08
0.06
0.04
0.02
20
0.00
Untreated
0
Untreated
CO2 laser 0.9 kW/cm
2
CO2 laser 1.6 kW/cm
CO2 laser
2
0.9 kW/cm
CO2 laser
2
1.6 kW/cm
2
Fig. 4. Osteocalcin score of the untreated and CO2 laser treated MgO – PSZ (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
Fig. 5. The MTT optical density measured after 7-day cell culture for untreated and CO2 laser treated samples (there was significant statistical difference between the untreated sample and samples CO2 laser treated at 0.9, 1.6 kW/cm2, p <0.05).
L. Hao et al. / Materials Science and Engineering C 25 (2005) 496 – 502
Optical Density Osteocalcin Score LPPA Score
microstructure of the MgO –PSZ after CO2 laser interaction. Hexagonal and cellular microstructural pattern appeared at the MgO – PSZ surface following CO2 laser treatment, revealing that solidification of the individual melted particulates had occurred. Such a solidification structure indicates that exposure of the MgO – PSZ to CO2 laser irradiation resulted in rapid heating of the surface, which consequently leaded to the melting and resolidification of the MgO – PSZ surface. The local melting and resolidification of a small portion of the surface of a material with a laser beam provides unique opportunity for controlled crystal growth under high temperature gradients and high cooling rates [17]. Furthermore, the observed increase in the wetting performance of the MgO – PSZ could have certainly been influenced by the increase in the surface oxygen content of the MgO – PSZ surface as a result of the laser treatment, since this is known to increase the likelihood of wetting [18,19]. The oxygen enrichment of the MgO – PSZ surface is active in promoting wetting and adhesion. After CO2 laser treatment, the roughness of the MgO – PSZ surface increased with power density. This is likely to be the result of turbulent convection in the melt pool caused by the TEM01 mode of the CO2 laser beam. Indeed, the power density distribution of the CO2 laser appears to be multimode [17]. When h < 90-, the rougher the surface is, the lower is h. It is reverse when h > 90- [20]. In this study, the rougher surface generated by the CO2 laser treatment could be attributed to the higher wettability characteristics on the condition of h <90-. As one shown in Fig. 6, the enhanced cell functions represented by the cell proliferation (optical density), osteocalcin, and LAPA scores increase with the increasing wettability. Hence, it is believed that the wettability of the MgO – PSZ was the primary factor governing the cell response. The effects of wettability on cell functions could 90 60 30 0 90 60
501
result from its influence on the protein adsorption and cell adhesion. The adsorption of the proteins is the net result of various types of interactions which depend on the nature of the protein aqueous solution. The difference in cellular response of different materials suggests that there are differences in the organization of the adsorbed protein layer. Protein adsorption mediated cell behaviours are regarded as fundamental reactions at biomaterial/tissue interfaces [21]. Indeed, the CO2 laser treatment brought about a lower amount of adsorbed albumin and a higher amount of the adsorbed fibronectin on the modified MgO – PSZ compared with the untreated sample [22]. Such interactions would benefit the osteoblast cell adhesion since albumin is a non-cell adhesive protein [23] while fibronectin is a cell adhesive protein [24]. Furthermore, it had been shown that the enhancement in wettability generated by the CO2 laser treatment was the predominant mechanism that influences the improvement of human skin fibroblast adhesion [6] and human fetal osteoblast cell adhesion and growth [7]. It was found that after 1 day of culture, the osteoblast cells spread well and covered more surface areas on the higher surface energy substrates than on the lower surface energy substrates of the MgO –PSZ [7]. Initial events during cell-biomaterial interactions (such as cell adhesion and concomitant morphology) affect functions, such as cell population motility, proliferation, and synthesis of extracellular matrix proteins of anchoragedependent cells [25 – 27]. Increased osteoblast adhesion coupled with enhanced proliferation and synthesis of alkaline phosphatase has been observed on surfaces (for example, borosilicate glass and titanium) modified with immobilized peptide sequences (such as arginine– glycine– aspartic acid –serine (RGDS) and lysine –arginine– serine– arginine (KRSR) [28,29] contained in extracellular matrix proteins such as vitronectin and collagen. The enhanced cell adhesion [30] on nanophase ceramics brought about the enhanced cell functions of these materials [31]. The results of the present in vitro study provided the first evidence of enhanced functions (specifically, cell proliferation, synthesis of alkaline phosphatase, and osteocalcin in the extracellular matrix) of osteoblasts on CO2 laser treated MgO – PSZ. These improved functions would certainly promote and sustain osteointegration of surrounding bone that are needed to improve implant efficacy.
30 0
5. Conclusions
0.12 0.08 0.04 0.00 0.1
0.2
0.3
0.4
0.5
0.6
Wettability, cos θ (Glycerol)
0.7
0.8
Fig. 6. The relationship between optical density (cell proliferation), osteocalcin score, LAPA score, and wettability of the MgO – PSZ.
The results of the present in vitro study provided the first evidence of enhanced functions (specifically, cell proliferation, synthesis of alkaline phosphatase, and osteocalcin in the extracellular matrix) of osteoblasts on CO2 laser treated MgO – PSZ. The CO2 laser treatment of the MgO – PSZ brought about changes in surface energy, surface roughness, surface oxygen content, and microstructure that resulted in higher wettability of the MgO – PSZ compared with
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untreated specimen. Wettability, which could influence protein adsorption and initial cell response, could be a predominant mechanism active in improvement of the cell functions. These improved functions would certainly promote and sustain osteointegration of surrounding bone that is needed to improve implant efficacy.
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