Attachment of Human Primary Osteoblast Cells to Modified

Mar 10, 2009 - E-mail: [email protected] (R.H.B.); [email protected] (A.H.C.P.)., † ... The attachment and function of human primary osteoblast-...
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Attachment of Human Primary Osteoblast Cells to Modified Polyethylene Surfaces Alexandra H. C. Poulsson,*,†,‡ Stephen A. Mitchell,† Marcus R. Davidson,† Alan J. Johnstone,† Neil Emmison,§ and Robert H. Bradley*,† Materials & Biomaterials Research Centre, The Robert Gordon UniVersity, Aberdeen AB10 1FR, and School of Life Sciences, The Robert Gordon UniVersity, Aberdeen AB25 1HG, United Kingdom ReceiVed June 11, 2008. ReVised Manuscript ReceiVed NoVember 16, 2008 Ultra-high-molecular-weight polyethylene (UHMWPE) has a long history of use in medical devices, primarily for articulating surfaces due to its inherent low surface energy which limits tissue integration. To widen the applications of UHMWPE, the surface energy can be increased. The increase in surface energy would improve the adsorption of proteins and attachment of cells to allow tissue integration, thereby allowing UHMWPE to potentially be used for a wider range of implants. The attachment and function of human primary osteoblast-like (HOB) cells to surfaces of UHMWPE with various levels of incorporated surface oxygen have been investigated. The surface modification of the UHMWPE was produced by exposure to a UV/ozone treatment. The resulting surface chemistry was studied using X-ray photoelectron spectroscopy (XPS), and the topography and surface structure were probed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which showed an increase in surface oxygen from 11 to 26 atom % with no significant change to the surface topography. The absolute root mean square roughness of both untreated and UV/ozone-treated surfaces was within 350-450 nm, and the water contact angles decreased with increasing oxygen incorporation, i.e., showing an increase in surface hydrophilicity. Cell attachment and functionality were assessed over a 21 day period for each cell–surface combination studied; these were performed using SEM and the alamarBlue assay to study cell attachment and proliferation and energy-dispersive X-ray (EDX) analysis to confirm extracellular mineral deposits, and total protein assay to examine the intra- and extracellular protein expressed by the cells. HOB cells cultured for 21 days on the modified UHMWPE surfaces with 19 and 26 atom % oxygen incorporated showed significantly higher cell densities compared to cells cultured on tissue culture polystyrene (TCPS) from day 3 onward. This indicated that the cells attached and proliferated more readily on the UV/ozone-treated UHMWPE surfaces than on untreated UHMWPE and TCPS surfaces. Contact guidance of the cells was observed on the UHMWPE surfaces by both SEM and AFM. Scanning electron micrographs showed that the cells were confluent on the modified UHMWPE surfaces by day 10, which led to visible layering of the cells by day 21, an indicator of nodule formation. In Vitro mineralization of the extracellular matrix expressed by the HOB cells on the modified UHMWPE surfaces was confirmed by SEM and EDX analysis; spherulite structures were observed near cell protrusions by day 21. EDX analysis confirmed the spherulites to contain calcium and phosphorus, the major constituents in calcium phosphate apatite, the mineral phase of bone. Overall cell attachment, functionality, and mineralization were found to be enhanced on the UV/ozone-modified UHMWPE surfaces, demonstrating the importance of optimizing the surface chemistry for primary HOB cells.

Introduction Understanding and controlling cell-surface interactions is of critical importance in biomedical technologies. In particular, achieving satisfactory cell attachment and long-term viability is essential in most areas of tissue culture and implant development. Polymers such as polystyrene (PS) and ultrahigh molecular weight polyethylene (UHMWPE) are particularly attractive as biomaterials: the former is extensively used in tissue culture vessels, while the latter has high strength and wear properties and is of significant importance in the manufacture of components of the larger artificial joints such as hips and knees and for the onepiece fabrication of smaller components, for example, finger joints. Through surface modification the utilization of polymers such as UHMWPE can be widened to applications where tissue integration is important, e.g., craniomaxillofacial implants. Improving cellular attachment by increasing the polar surface * To whom correspondence should be addressed. E-mail: r.bradley@ rgu.ac.uk (R.H.B.); [email protected] (A.H.C.P.). † Materials & Biomaterials Research Centre. ‡ Current address: AO Research Institute, AO Foundation, Davos Platz CH-7270, Switzerland. § School of Life Sciences.

free energy is well established.1 The surface can be modified by the incorporation of functional groups, particularly those containing oxygen. A variety of direct modification techniques1-4 and deposition techniques5-8 are now routinely used for this purpose. The incorporated surface groups mediate the resulting interactions which occur with physiological fluids; in particular these surface groups strongly influence hydrogen bonding interactions with water and other polar species present in physiological fluids. The subsequent adsorption interactions of the hydrated surfaces with serum proteins, such as albumin, and (1) Amstein, C. F.; Hartman, P. A. J. Clin. Microbiol. 1975, 2(1), 46–54. (2) Mathieson, I.; Bradley, R. H. Effects of Ultra Violet/Ozone on the Surface Chemistry of Polymer Films. AdVances in Engineering Materials; Trans Tech Publications: Stafa–Zürich, Switzerland, 1994; pp 185-192. (3) Callen, B. W.; Ridge, M. L.; Lahooti, S.; Neumann, A. W.; Sodhi, R. N. S. J. Vac. Sci. Technol. 1995, 13(4), 2023–2029. (4) Welle, A.; Gottwald, E. Biomed. MicrodeVices 2002, 4(1), 33–41. (5) Mitchell, S. A.; Emmison, N.; Shard, A. G. Surf. Interface Anal. 2002, 33, 742–747. (6) Chu, P. K.; Chen, J. Y.; Wang, L. P.; Huang, N. Mater. Sci. Eng., R 2002, 36, 143–206. (7) Falconnet, D.; G., C.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063. (8) France, R. M.; Short, R. D. J. Chem. Soc., Faraday Trans. 1997, 93(17), 3173–3178.

10.1021/la801820s CCC: $40.75  2009 American Chemical Society Published on Web 03/10/2009

Attachment of HOB Cells to Polyethylene Surfaces

cell attachment proteins, such as fibronectin and vitronectin,9-12 are influenced by the way in which the initial molecules have adsorbed. Overall, the type and physical distribution of polar surface groups effectively determines the structure of the hydrated surface layer, directing the interaction between the adsorbed proteins and cellular integrins and therefore subsequent cellattachment characteristics of the polymer surfaces.13-16 Many studies have attempted to identify which specific oxygen functional groups lead to increased cellular adhesion on hydrophobic polymers, though the findings have often been contradictory.17,18 However, more recent work indicates that individual types of functionality may play different and specific roles in the attachment, proliferation, differentiation, and longer term stability of some cell types.19 This has led to the study of monofunctional surfaces, e.g., produced using self-assembled monolayer systems (SAMs), and surfaces of tightly defined functional group composition.19,20 This approach promises much in providing a detailed understanding of the precise roles of individual surface species in the overall cell-surface interactions. However, the chemospecific functionalization methods which lead to monofunctional surfaces, for example, SAMs, are multistep and are therefore likely to be expensive if applied to the routine manufacture of biomedical devices. Consequently, less expensive, generic methods for the functionalization of biomaterials and devices on a larger scale are also currently of great interest. This is especially true for the fabrication of bone replacement and regeneration materials for orthopedic applications, which is highly topical as a result of complications with metal implant materials and the surrounding biological tissues. The rise in nickel allergies has led to the move away from the use of stainless steel implants,21 and assessment by MRI or X-ray of implant integration can be obscured by the presence of metal devices.22 The use of polymeric materials has therefore become more attractive for low loadbearing applications due to their good biocompatibility and radiolucent properties and the use of surface modification techniques which can increase the surface energy to allow increased cellular and tissue integration. Of great importance when implant surfaces are evaluated is the choice of cell type. While many biomaterials investigations have employed cell lines to probe surface cytocompatibility, there is always the concern that, by their intrinsic nature, these transformed cell types will behave atypically in comparison to the human primary sourced cell types. In addition, primary animalderived cultures can also behave very differently. Many cell lines have certain specific phenotypic characteristics upregulated abnormally high or not expressed at all, but fundamentally the limitation is that these cell lines do not experience contact inhibition, which thereby leads to a totally different cellular response to a biomaterial than would be observed in vivo.23-26 (9) Altanov, G.; Groth, T. J. Mater. Sci.: Mater. Med. 1994, 5, 732–737. (10) Couchourel, D.; Escoffier, C.; Rohanizadeh, R.; Bohic, S.; Daculsi, G.; Fortun, Y.; Padrines, M. J. Inorg. Biochem. 1999, 73, 129–136. (11) Garcia, A. J.; Reyes, C. D. J. Dent. Res. 2004, 84(5), 407–413. (12) Steele, J. G.; Dalton, B. A.; Johnson, G.; Underwood, P. A. Biomaterials 1995, 16, 1057–1067. (13) Browne, M. M.; Lubarsky, G. V.; Davidson, M. R.; Bradley, R. H. Surf. Sci. 2004, 553, 155–167. (14) Hubbell, J. A. Trends Polym. Sci. 1994, 2(1), 20–25. (15) Brighton, C. T.; Albelda, S. M. J. Orthop. Res. 1992, 10(6), 766–773. (16) Kasemo, B. Curr. Opin. Solid State Mater. Sci. 1998, 3, 451–459. (17) Curtis, A. S. G.; Forrester, J. V.; Clark, P. J. Cell Sci. 1986, 86, 9–24. (18) Keselowsky, B. G.; Collard, D. M.; Garcia, A. J. Biomaterials 2004, 25, 5947–5954. (19) Curran, J. M.; Chen, R.; Hunt, J. A. Biomaterials 2006, 27, 4783–4793. (20) Tidwell, C. D.; Ertel, S. I.; Ratner, B. D. Langmuir 1997, 13, 3404–3413. (21) Hallab, N.; Merritt, K.; Jacobs, J. Bone Joint Surg. Am. 2001, 83-A, 428–436. (22) Cizek, G. R.; Boyd, L. M. Spine 2000, 25, 2633–2636. (23) Lian, J. B.; Stein, G. S. Crit. ReV. Oral Biol. Med. 1992, 3, 269–305.

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Thus, it is very important, where feasible, to probe biomaterial surfaces using cells of direct relevance to the ultimate application for which the surface is to be used. The work presented here is based on a study of the attachment, proliferation, and viability of human primary osteoblast-like (HOB) cells as a function of the surface oxygen chemistry on UHMWPE surfaces, of a grade similar to that used in artificial joints. Oxygen levels were controlled using exposure in a UV/ ozone system which is suitable for commercial batch or semicontinuous treatment of biomedical and tissue culture materials. The surface compositions and functional group chemistry of the surfaces were characterized by X-ray photoelectron spectroscopy (XPS), and the topography and that of cells adhered to the surfaces were imaged using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The study shows that UV/ozone surface oxidation treatment leads to enhanced osteoblast attachment and that the attached cells retain functionality in that de noVo bone formation was confirmed by the presence of nodular calcium phosphate formations within the extracellular matrix produced by the cells.

Experimental Section Materials. Sheets of 1 mm thick UHMWPE were supplied by Goodfellow (United Kingdom). Samples were machined into disks to fit inside 20 cm2 Petri dishes or punched out to fit inside circular 24-well plates (Nunclon, Denmark) with a diameter of 14 mm. The samples were cleaned first by sonication in methanol for 20 min to remove any residues from the machining process and then twice in doubly distilled water (resistivity 18 MΩ) for 20 min. Once dry, the samples were placed into the bacteriological grade PS dishes or 24-well plates. The samples were secured by the addition of a few drops of acetone, which slightly dissolved the PS surface of the dish or well to form a sufficiently secure bond with the UHMWPE. This technique was developed to keep the UHMWPE disks from floating during cell culture experiments. The samples were used for cell experiments in either as-received form, which had 11 atom % surface oxygen, or after surface modification by UV/ozone treatment. Surface Modification. The UHMWPE-surfaced dishes were treated in a UV/ozone reactor (Jelight Co. Inc.) of the type used in our previous studies.2,27,28 A high-intensity, low-pressure mercury vapor grid lamp irradiated samples at approximately 1120 µW/cm2 at a distance of 3 cm under atmospheric conditions. The lamp was warmed for 1 h prior to surface treatments to ensure optimal transmission of the UV and UV radiation emitted includes wavelengths of 184.9 and 253.7 nm.31 Once irradiated, the dishes with the inset UHMWPE disks were washed on a rotating platform using Millipore water (resistivity 18 MΩ) twice for 20 min and then dried in a class I laminar flow hood before being surface analyzed or plated with cells. The experimental surfaces with 19 and 26 atom % oxygen were produced using this method. X-ray Photoelectron Spectroscopy. Surfaces were analyzed both before and after surface treatment by XPS using a Kratos Axis HSi 5 channel instrument with monochromated Al KR radiation (energy 1486.6 eV) operated at 150 W in a residual vacuum of approximately 5 × 10-9 mbar with the analyzer in fixed analyzer transmission (FAT) mode. Charge neutralization was utilized on all samples and had a -2.3 V bias voltage, -1.0 V electron filament voltage, and (24) Clover, J.; Gowan, M. Bone 1994, 15, 585–591. (25) Anselme, K.; Linez, P.; Bigerelle, M.; Le Maguer, D.; Le Maguer, A.; Hardouin, P.; Hildebrand, H. F.; Iost, A.; Leroy, J. M. Biomaterials 2000, 21, 1567–1577. (26) Scheven, B. A. A.; Marshall, D.; Aspden, R. M. Cell Biol. Int. 2002, 26(4), 337–346. (27) Teare, D. O. H.; Emmison, N.; Ton-That, C.; Bradley, R. H. Langmuir 2000, 16, 2818–2824. (28) Mitchell, S. A.; Poulsson, A. H. C.; Davidson, M. R.; Emmison, N.; Shard, A. G.; Bradley, R. H. Biomaterials 2004, 25, 4079–4086. (29) Briggs, D. Applications of XPS in Polymer Technology. In Practical Surface Analysis; Briggs, D., Seah, M. P., Eds.; Wiley: New York, 1990; Chapter 9, pp 437-483.

3720 Langmuir, Vol. 25, No. 6, 2009 +1.9 A filament current. All spectra were acquired in hybrid mode, using both the electrostatic and magnetic lenses. The sample area was approximately 1 mm2, and spectra were taken from three replicate samples. Survey spectra were measured, with a pass energy of 80 eV, in the binding energy range between 0-1100 eV for UHMWPE. Narrow scans were obtained with a pass energy of 20 eV, between 282 and 295 eV for carbon 1s and between 526 and 536 eV for O 1s photoelectrons. The surface elemental composition was calculated from peak areas gained from narrow scan spectra after subtraction of the linear background using Kratos Vision software, as described previously.26 Surface charging was corrected for by shifting the full width at half-maximum (fwhm) of the C 1s (hydrocarbon) peak to 285.0 eV. Sample surface compositions were determined from an average of a minimum of three measurements from a minimum of three different samples. Atomic Force Microscopy. The topography of polymer surfaces and the surfaces with adhered cells were studied using a Digital Instruments Nanoscope IIIa (Veeco Instruments, United Kingdom) under ambient conditions. Cells were fixed in 5% gluteraldehyde/ PBS solution following the SEM fixation procedure described below. The atomic force microscope (AFM) was calibrated in the x, y, and z directions using a DI calibration standard (Veeco). The polymer surfaces were investigated in contact mode, while the adhered cells were analyzed in tapping mode, with silicon nitride tips (Veeco). Digital Instruments version 4.23r6 software was used to analyze the data. The root mean square (rms) roughness (Rq) was determined using standard methods; the same size sample areas (20 × 20 µm) were analyzed from three areas on three replicate samples (i.e., n ) 9). The same size area was examined on all the polymer surfaces since roughness is known to increase with increasing area. The areas examined with attached cells were approximately 150 × 150 µm to allow individual cells to be visualized. Contact Angles. Water contact angles for all UHMWPE surfaces were measured using an FTÅ125 dynamic contact angle analyzer (First Ten Ångstroms). A sessile drop of ∼20 µL of water was advanced toward the surface at a set rate. As the drop was released from the needle, a sequence of digital images were taken automatically by the camera and then analyzed to give an advancing contact angle. A minimum of three replicate readings were taken from each surface type and repeated three times. Scanning Electron Microscopy and Energy-Dispersive X-ray Analysis. The samples were analyzed with a LEO SEM S430 with LEO-32 V02.04 software. Scanning electron micrographs were obtained at accelerating voltages between 10 and 25 kV. Lower voltages were used for high-magnification micrographs of cells and extracellular matrix mineralization to minimize sample damage. The surface topography of the UHMWPE surfaces was also examined before and after UV/ozone treatment to identify whether the treatment caused morphological changes. Energy-dispersive X-ray (EDX) analysis was used to analyze regions in which extracellular matrix deposits had been identified. The EDX instrument eXL2 (Oxford Instruments, United Kingdom) was attached to the scanning electron microscope, and Oxford Microanalysis Group XAN.70 software was utilized for this analysis. The cells were fixed in ice cold 5% gluteraldehyde/PBS solution after 3 washes with PBS on ice. The dishes were stored at 4 °C in gluteraldehyde/PBS solution for a minimum of 1 h and subsequently prepared for critical point drying for SEM/EDX analysis. Immediately before critical point drying, the samples were washed with increasing concentrations of ethanol, 50-100% at 5 min intervals. The final 100% ethanol wash was substituted by 70-100% washes of acetone. Once the samples had reached equilibrium in the acetone, they were placed inside the critical point drier, and fluid was removed using standard procedures. After dehydration by critical point drying, the UHMWPE disks with and without adhered cells were sputter-coated with gold/palladium to a thickness of approximately 5-10 nm to prevent surface charging during SEM/EDX analysis. Cell Culture. Human primary osteoblast-like (HOB) cells were isolated from femoral heads which had been removed during total joint replacement (TJR) operations. The femoral heads were removed due to osteoporotic (OP) complications. The individual femoral heads

Poulsson et al. were not specifically tested to identify OP disease, and as a consequence, the cells sourced may not themselves have been affected by OP, although the disease was the underlying reason for the TJR operation. The femoral heads were mechanically fragmented into pieces, and the cancellous bone was then removed from the surrounding cartilage or trabecular bone. The cancellous bone chips were washed with PBS to remove blood and fat. The chips were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 80 µg/mL L-ascorbic acid, 1% penicillin/ streptomyocin, and 1% nonessential amino acids at 37 °C with 5% CO2 at 100% humidity to allow the osteoblast cells to migrate out of the chips. The cells used in the experiments were at passage 3 and were grown to approximately 70% confluence. HOB cells were plated onto the experimental UHMWPE surfaces and Nunclon tissue culture polystyrene (TCPS) dishes (Nunc, Denmark), which were used as control surfaces at 5000 cells/cm2. To aid mineralization, R-MEM supplemented with 10% FCS, 1% penicillin/streptomyocin, 10 µM dexamethasone, and 10 mM β-glycerophosphate was used. Cell attachment and functionality were assessed over 21 days at time points of 1, 2, 3, 10, and 21days. Cell and Protein Assays. Cell density was measured using standard alamarBlue (Serotec, United Kingdom). The cell density (cells/cm2) was determined by plating standard cell densities varying from 0 to 50 000 cells/cm2 in dishes to produce a standard curve. After a minimum of 4 h postplating, the medium was removed from the dishes and fresh medium with 10% (v/v) alamarBlue added. The dishes were then incubated under the standard culture conditions as described previously for 4 h. The reduction of alamarBlue was measured with a microtiter plate reader (Dyntech, United Kingdom) at absorbances of 570 nm (oxidized) and 600 nm (reduced), and the degree of reduction can be estimated as described by Serotec. The degree of reduction was then correlated with the cell density. For all measurements described here a standard curve was created for each set of long-term experiments individually. The total protein assay used was a bicinchoninic acid, BCA, kit provided by Pierce Perbio (United Kingdom), which is a simpler variation of the Lowry assay and is less sensitive to interfering substances. The assay was performed according to the manufacturer’s protocol. Statistical Analysis. Statistical analysis was performed on the data from the biological experiments. The statistical analysis was designed to take into account variability due to the femoral head source and compare the data from each surface to those of a control, TCPS. An ANOVA, general linear model, using Dunnett’s simultaneous test was used to compare each surface to the control surface. This statistical analysis was performed using Minitab software.

Results and Discussion Surface Characterization. XPS survey spectra of the asreceived UHMWPE contained the characteristic polymeric C1s peak at 285.0 eV which is usually observed for UHMWPE.29 In addition, for this particular material, a significant O1s peak at 534 eV is also present, which equates to a surface oxygen level of approximately 11 atom %. This was not removed or decreased by washing and sonication in methanol and water. Analysis of freshly cleaved surfaces of the UHMWPE revealed no surface oxygen. The 11 atom % oxygen surface contamination was interpreted as arising from manufacture and processing, where the striations observed in the UHMWPE surfaces were plate lines, a result of the machining process, and have been previously observed for UHMWPE.30,32-34 Survey scans can be found in (30) Buncick, M. C.; Thomas, D. E.; McKinny, K. S.; Jahan, M. S. Appl. Surf. Sci. 2000, 156, 97–109. (31) Davidson, M. R.; Mitchell, S. A.; Bradley, R. H. Surf. Sci. 2005, 581, 169–177. (32) Mao, C.; Yuan, J.; Mei, H.; Zhu, A.; Shen, J.; Lin, S. Mater. Sci. Eng., C 2004, 24, 479–485. (33) Vaisman, L.; Gonzalez, M. F.; Marom, G. Polymer 2003, 44, 1229–1235.

Attachment of HOB Cells to Polyethylene Surfaces

Figure 1. Surface oxygen concentration of washed samples produced after UV/ozone treatment of UHMWPE compared to TCPS, determined by XP narrow scan spectra. There was an increase in atomic oxygen content with increasing treatment time to 900 s (standard deviations >5%, individual standard deviations were calculated, n ) 9).

the Supporting Information, as the only significant feature of the spectra from the materials produced in this study is the variance of the C1s to O1s peak intensities of the samples. The C1s narrow scan envelope from the untreated UHMWPE, shown in the Supporting Information, shows the main hydrocarbon peak at 285 eV, the fwhm of which is 0.9 eV. This is similar to that reported from other studies of UHMWPE.32 The surface oxygen gives rise to a chemically shifted peak at 286.5 eV which indicates that the native oxygen is present as primarily CO-R (alcohol/ether) groups. Since the UHMWPE surfaces already contained surface oxygen, the UV/ozone treatment only led to a further increase with a maximum level of 26.1 atom %. Figure 1 shows the relationship between UV/ozone exposure time and incorporated oxygen after washing for UHMWPE and also shows the concentration in TCPS for comparison. The commercial TCPS control used in this study had a surface oxygen content of approximately 14.5 atom %. Samples of UHMWPE with surface oxygen levels of 11 (as received), 19, and 26 atom % oxygen were used for HOB experiments. As the UHMWPE contained some surface oxygen, the modification of the existing alcohol/ether groups was also likely to occur during the UV/ozone treatment. The saturation level, determined from the unwashed samples, was achieved at treatment times >600 s, which corresponded to a steady state between oxygen incorporation by photooxidation and the loss of volatile photolysis products from the UHMWPE surface.27,28,31,36,37 This process is accompanied by the formation of low-molecular-weight oxidized material, which as previously reported may have an adverse effect on the mechanical properties of the surface and would have a detrimental effect upon the cells.31 All samples used for cell culture were washed to remove this loosely bound material, leaving a stable oxidized surface. (34) Wilson, D. J. Plasma Treatment of Polymers for Modifying Haemocompatibillity. Scientific Thesis, University of Liverpool, Liverpool, U.K., 2000. (35) Karlsson, M.; Palsgard, E.; Wilshaw, P. R.; Di Silvio, L. Biomaterials 2003, 24, 3039–3046. (36) Teare, D. O. H.; Ton-That, C.; Bradley, R. H. Surf. Interface Anal. 2000, 29.

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Figure 2. High-resolution carbon 1s peak envelope from an UHMWPE surface after 180 s of UV/ozone treatment showing fitted peaks corresponding to residual functional groups remaining after washing.

C1s narrow scan spectra from a UV/ozone-treated washed UHMWPE surface, in this instance the 180 s treated UHMWPE, had 19 atom % oxygen. Four peaks were fitted within the C1s peak envelope of the treated materials (Figure 2): the main C-C/ C-H hydrocarbon peak at 285 eV, which is a composite of two peaks, separated by ∼0.4 eV and associated with the vibrational fine structure of the untreated UHMWPE, plus three additional chemically shifted peaks due to the UV/ozone modification of the UHMWPE surface. These are attributable to alcohols/ethers, CO-R, at ∼286.5 eV, carbonyl/diether groups, CdO, at ∼288 eV, and carboxyl/ester groups, OsCdO, at ∼289.5. The photooxidation of UHMWPE is known to be less complex than, for example, that of PS, as it is an aliphatic polymer and therefore the oxidation occurs through the polymer backbone.38 Oosterom et al. investigated UV/ozone-treated UHMWPE and found it to have detrimental effects on the bulk properties; however, their surface treatment was for 30 min,39 which was much longer than those examined in this study. No surface crazing or embrittlement was observed as a result of the UV/ozone treatment in the present study. Figure 3 shows representative topographical AFM images of untreated and UV/ozone-treated UHMWPE surfaces. Striations in the surface are clearly visible running vertically through all of the AFM images. Figure 3a shows the as-received UHMWPE material (11 atom % oxygen) where a peak-trough structure with an approximate height difference of 3 µm and a distance from peak to peak of approximately 5 µm is apparent. Figure 3b shows a representative image from the 19 atom % surface (180 s treatment), where the microtopography appeared not to be a result of the surface treatment as the features were very similar to those of the untreated surfaces. In the images from UHMWPE surfaces which have 26 atom % oxygen (UV/ozonetreated for 600 s) the striations appear a little more prominent; the peak-trough distance is ∼4-5 µm, while the peak to peak distance is still 5 µm, indicating that any oxidation-induced microroughening of these surfaces was very limited, as shown in Figure 3b. (37) Ton-That, C.; Teare, D. O. H.; Campbell, P. A.; Bradley, R. H. Surf. Sci. 1999, 433-435, 278–282. (38) Costa, L.; Luda, M. P.; Trossarelli, L. Polym. Degrad. Stab. 1997, 58, 41–54.

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Figure 4. Root mean square Rq surface roughness of washed UV/ozonetreated UHMWPE surfaces as a function of the treatment time. The surface roughness of TCPS is shown for comparison with an average roughness of 1.63 nm (n ) 9 for all surfaces).

Figure 5. Advancing water contact angles for washed UV/ozone-treated and untreated UHMWPE as a function of the UV/ozone treatment time, compared with that of TCPS.

Figure 3. AFM contact mode images of (a) untreated, (b)150 s UV/ ozone-treated (19 atom % oxygen), and (c) 600 s UV/ozone-treated (26 atom % oxygen) UHMWPE depicting topographical height variations by color, with lighter colors indicating higher topography, as shown in the legend, showing the variations from peak to trough (scan size 20 µm and z range 5 µm).

The average roughness measured as rms (Rq) for the untreated and treated UHMWPE showed that the microroughening effect of the surface treatment was very limited; the average roughness was between 350-450 nm range for all the washed UHMWPE surfaces, as shown in Figure 4. However, the roughness was very high when compared to that of TCPS, which has a surface roughness of ∼1.6 nm. The UHMWPE sheets contained surface features, mainly striations, on both the micrometer and submicrometer scales of a type which has previously been reported to affect protein adsorption, conformation, and subsequent cell attachment.25,40-46 The water contact angles for the untreated UHMWPE surfaces were measured to approximately 88°, which is consistent with the 11 atom % oxygen and lower than the value of 98° previously reported for pure, non-oxygen-containing UHMWPE.47 As shown in Figure 5, the contact angle decreases with increasing UV/ ozone treatment time to approximately 73° after 600 s of treatment, and the contact angle was observed to stabilize thereafter. This is consistent with the XPS data where no appreciable change in

Attachment of HOB Cells to Polyethylene Surfaces

Figure 6. Cell density of the HOB cells determined by the alamarBlue assay on the untreated UHMWPE, UV/ozone-treated UHMWPE, and TCPS surfaces, as shown in the legend. The cell densities of the untreated and treated UHMWPE surfaces were compared to those of TCPS, and there was an initial plating density of 5000 cells/cm2 (the asterisk indicates p < 0.05 by ANOVA, general linear model, using Dunnett’s simultaneous test to compare to the control surface TCPS, n ) 9).

chemistry was shown after 600 s of treatment, which equates to the residual chemistry resulting from the steady-state process mentioned previously. Cell Attachment and Proliferation. Cell attachment and proliferation were determined by alamarBlue assay, and cell attachment was imaged by SEM; both demonstrated that the surface modification of UHMWPE led to a surface which was cytocompatible. Measuring the cell density on the surface of a material, and thereby the proliferation, allows a better understanding of the effect of the surfaces on the cells.26,35,48,49 Initial cell adhesion has been previously reported to be a useful tool to gauge surface biocompatibility.50 The cell densities on the UHMWPE untreated and UV/ozone-treated surfaces determined by alamarBlue were compared to those measured on TCPS, and the cell densities were determined at 1, 2, 3, 10, and 21 days postplating and are shown in Figure 6. The cell densities were significantly higher on the UHMWPE surfaces with 19% surface oxygen in comparison to TCPS throughout the 21 day culture period. On day 1, all of the UHMWPE surfaces had a higher cell density, though by day 2 only the UHMWPE with 19 atom % oxygen had a significantly higher cell density. The cell densities of the UHMWPE untreated surface and UHMWPE surface with 26 atom % oxygen were only just increased compared to that of TCPS. By day 3 all of the UHMWPE surfaces had significantly higher cell densities. There was a greater increase in cell density from day 3 to day 10 than from day 10 to day 21, which indicated that the cells had reached confluence on UV/ozone-treated UHMWPE surfaces (39) Oosterom, R.; Ahmed, T. J.; Poulis, J. A.; Bersee, H. E. N. Med. Eng. Phys. 2006, 28, 323–330. (40) Callister, W. D. In Characteristics, Applications and Processing of Polymers; Callister, W. D., Ed.; Wiley: New York, 2003; pp 480-526. (41) Curtis, A. S. G.; Wilkinson, C. D. W. Biomaterials 1997, 18, 1573–1583. (42) Manwaring, M. E.; Walsh, J. F.; Tresco, P. A. Biomaterials 2004, 25, 3631–3638. (43) Dalby, M. J.; Childs, S.; Riehle, M. O.; Johnstone, H. J. H.; Affrossman, S.; Curtis, A. S. G. Biomaterials 2003, 24, 927–935. (44) Kasemo, B.; Gold, J. AdV. Dent. Res. 1999, 13, 8–20. (45) Meyer, U.; Buchter, A.; Weismann, H. P.; Joos, U.; Jones, D. B. Eur. Cells Mater. 2005, 9, 39–49. (46) Singhvi, R.; Stephanopoulos, G.; Wang, D. I. Biotechnol. Bioeng. 1994, 43, 764–771. (47) Sheu, M.-S.; Hoffman, A. S.; Ratner, B. D.; Feijen, J.; Harris, J. M. J. Adhes. Sci. Technol. 1993, 7(10), 1065–1076. (48) Anderson, S. I.; Downes, S.; Perry, C. C.; Caballero, A. M. J. Mater. Sci.: Mater. Med. 1998, 9, 731–735. (49) Rizzi, S. C.; Heath, D. J.; Coombes, A. G. A.; Bock, N.; Textor, M.; Downes, S. J. Biomed. Mater. Res. 2001, 55, 475–486.

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Figure 7. Total protein produced by the HOB cells on untreated UHMWPE, UV/ozone-treated UHMWPE, and TCPS over the 21 days, as shown in the legend. The total protein on the treated and untreated UHMWPE surfaces was compared to that on TCPS, and the initial plating density was 5000 cells/cm2 (a single asterisk indicates p < 0.05, two asterisks indicate p < 0.01, and three asterisks indicate p < 0.001 by ANOVA, general linear model, using Dunnett’s simultaneous test to compare to the control surface TCPS, n ) 9, mean ( standard deviation).

by day 10. By day 10 the cell densities of the UHMWPE surfaces with both 19 and 26 atom % oxygen were significantly increased (nearly doubled) compared to that of TCPS, and the cell densities of these surfaces continued to be significantly increased to day 21. The untreated UHMWPE and TCPS surfaces may have reached cell confluence after day 10, but the clear trend of cell confluence shown on the treated UHMWPE surfaces was not as apparent. Total protein levels, measured by the BCA assay, for the corresponding surfaces are given in Figure 7 and reflect the general increase in cell density shown in Figure 6, but also show the protein expression on the UHMWPE surfaces was significantly higher compared to that on TCPS throughout the 21 days. The significantly higher level of total protein expression indicates that the UHMWPE surfaces lead to up-regulation of protein expression as a result of cell confluence reached between 3 and 10 days. Cell Imaging. Since UHMWPE surfaces are opaque, cells could not be easily imaged by using optical microscopy, so SEM was therefore used to study cellular attachment, morphology, and confluence. Due to the striations on the UHMWPE substrates it was also possible to examine the cellular orientation on the surfaces by both SEM and AFM. Figure 8 shows representative SEM images of the cells on the UHMWPE and TCPS surfaces, the latter imaged by optical microscopy, over the 21 days. As a result of the use of acetone during the fixation process to dehydrate the samples further before critical point drying, the TCPS samples could not be fixed by this method, as TCPS is soluble in acetone. The scanning electron micrographs clearly show the higher cell densities observed on the UV/ozone-treated UHMWPE surfaces and the untreated UHMWPE surfaces, confirming the alamarBlue data given in Figure 6. The optical images show that the cells on the TCPS had not actually reached cell confluence by day 21, whereas the scanning electron micrographs show that the UHMWPE surfaces clearly had. It is also evident that the cells on the UHMWPE surfaces exhibited contact guidance and were seen to have oriented themselves to the microstriations, which is consistent with other reported findings.41,42,51-53 As (50) Scotchford, C. A.; Ball, M.; Winkelmann, M.; Voros, J.; Csucs, C.; Brunette, D. M.; Danuser, G.; Textor, M. Biomaterials 2003, 24, 1147–1158.

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Figure 8. Scanning electron micrographs of cell growth on the UHMWPE surfaces and optical microscope images of the cell growth on TCPS, taken at 3, 10, and 21 day time points (individual scale bars are shown on the SEM images of 10 and 20 µm, and for the optical microscope images the scale bar indicates 100 µm).

Figure 9. AFM tapping mode images of cell attachment on TCPS, 2 days postplating. (a) shows a 2-D topographical image depicting variations in height by color, as described in the legend. The black arrows show examples of stress fibers. (b) shows a 3-D image of the surface topography, showing that the cell was randomly oriented, as the surface topography was relatively smooth.

Figure 10. AFM tapping mode images of cell attachment on the untreated UHMWPE surface, 2 days postplating. (a) shows a 2-D topographical image depicting variations in height by color, as shown in the legend. (b) shows a 3-D image of the cell and surface topography, showing contact guidance more clearly.

described previously, the surface striations were approximately 5 µm apart and the height difference from trough to peak was

∼3-5 µm depending on the treatment time. The HOB cells were 10-20 µm wide and could be more than 80 µm long. The cells were therefore wider than the distance between the striations, although it is clear that the long axes of the cells were aligned with the striations. In addition, by day 21 cellular multilayers were observed on the UHMWPE surfaces, and some of the upper

(51) von Recum, A. F.; van Kooten, T. G. J. Biomater. Sci., Polym. Ed. 1995, 7(2), 181–198. (52) Curtis, A. S. G.; Wilkinson, C. D. W. J. Biomater. Sci., Polym. Ed. 1998, 9(12), 1313–1329.

Attachment of HOB Cells to Polyethylene Surfaces

Figure 11. AFM tapping mode images of cell attachment on UV/ozonetreated UHMWPE with 19% surface oxygen, 2 days postplating. (a) shows a 2-D topographical image depicting variations in height by color, as shown in the legend. (b) shows a 3-D image of the cell and surface topography, to show contact guidance more clearly.

layers contained cells which were randomly aligned, i.e., where the influence of the striations had been lost, while other cells were still experiencing guidance effects due to either the influence of cells already aligned or other, longer range, effects of the striations. The change in morphology was attributed to contact guidance as a result of the striations. AFM was also used to image the cells on all the UHMWPE surfaces and to directly compare cell behavior to that on the TCPS surfaces. This technique is generally utilized to examine the topography of surfaces, but here it was utilized to visualize the orientation of the cells relative to the surface striations. The scan area in tapping mode is limited, with the areas examined being a maximum of 160 µm wide; it was therefore important that the cells were not confluent, as this would have led to the surface striations being completely obscured. The TCPS surfaces did not contain striations, and the surface roughness was ∼1.6 nm, i.e., much less rough than the UHMWPE surfaces. Consequently, the fine-cellular structures were clearly defined on these surfaces, with the 2-D image in Figure 9a showing that the cell had spread in all directions with stress fibers, indicated by black arrows. Figure 9b shows the topography in 3-D: the cell is shown in relief from the surface, and again the random spread fibers were clear and the edges of the cell were readily defined against the smooth surface. In comparison, Figure 10 shows representative AFM images of cell attachment to untreated UHMWPE 2 days postplating. Although the detail of the cellular attachment was not as clear as that shown for TCPS, the cell alignment along the striations in the surface was just visible with the cells and striations being identified by the variations in height (the darker colors signify

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Figure 12. AFM tapping mode images of cell attachment on UV/ozonetreated UHMWPE with 26% surface oxygen, 2 days postplating. (a) shows a 2-D topographical image depicting variations in height by color, as shown on the legend. (b) shows a 3-D image of the surface topography. (c) shows a 2-D image showing more detail of the height differences of the cell and depicting the stress fibers used for anchoring the cell attachment to the UHMWPE surface.

lower topography; as the colors become lighter the height of the topography increases). To visualize a whole cell on the relatively rough UHMWPE topography, the area examined was necessarily at the upper limit for the area that can be examined using AFM, which resulted in some distortions due to the height fluctuation from cell peak to surface trough. The z range for the tip was set to 6 µm, and consequently, the cells were clearly visible, but the striations in the surface were less visible as the variation in surface topography was small relative to the cells. The highest point of the cells was greater than 6 µm (cell peak to surface trough), and the cells appear flattened in some of the images because the tip is scanning at its maximum height. Figure 10a shows that the cells resemble mountain-like ranges with their long axis orientated parallel to the striations, confirming the contact guidance, though the cells were actually spread across several striations. The 3-D topographical image given in Figure 10b shows the surface striations more clearly with the edges of the cell filopodia visible in the troughs. Figure 11 shows the cell attachment to UHMWPE surfaces with 19 atom % surface oxygen 2 days postplating. The images shown are representative and again confirm that there was a higher cell density at this time point. Figure 11a shows the cells as the lighter regions due to their height, with the darker colors signifying flatter topography. The height of the cells was more than 6 µm in places, so the images become unclear from where the cantilever reached its maximum z range. The images do however clearly show that all the cells were aligned to the direction of the striations. Figure 11b shows that the cell extensions or

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Figure 13. Representative EDX spectrum and the region where this was taken from showing mineralization on the (a) untreated UHMWPE, (b) UHMWPE surfaces with 19 atom % oxygen, and (c) UHMWPE surfaces with 26 atom % oxygen 21 days postplating. Elemental analysis with EDX showed P and Ca peaks in the regions where spherulite formations were present on the scanning electron micrographs of the surfaces.

filopodia and lamellipodia were extended along the troughs of the striations. Unfortunately, due to the large z range, the topographical variations of the surface were less obvious. The 3-D image displays “mountain” ridges similar to those observed on the untreated UHMWPE, and there are more regions where the cells were too high for the AFM cantilever to follow, leading to abrupt flat regions. Figure 12 shows representative images of the cell attachment to the UV/ozone-treated UHMWPE surface with 26 atom % oxygen. The region imaged here is smaller than in Figures 10 and 11 and shows the surface topography more clearly as the z range could thereby be decreased. Again the long axis of the cell, shown in Figure 12, was oriented to the striations, and the cell had spread across several of them. There are also clear filopodia spreading across the striations. Figure 12b shows the topography of the area, and the cell appeared to have spread across large amounts of the surface visualized. A closer examination of the changes in height seen at the base of the cell is shown in Figure 12c. The cell appears to have stress fibers fanning out from the main body of the cell. These fibers were ∼1.5 µm higher than the surface and appear to be involved in cell anchorage. It must also be noted that the topography on all the AFM images which contain cells is not that of the surface itself, but that of the adsorbed protein layer, and the variation in the total protein expression by the HOB cells on all surfaces was therefore investigated, as shown in Figure 7. HOB Cell Mineralization. SEM was also used to locate extracellular mineral deposits which were subsequently analyzed by EDX analysis to obtain constituent elemental information, compared to that of non-mineralized areas. In the final stages of osteogenic differentiation, the osteoidlike extracellular matrix (ECM) is mineralized, so to confirm the longer term cytocompatibility of the modified surface chemistry, it was important to prove that in Vitro mineralization was occurring. Initial stages of mineralization, both in Vitro and in ViVo, are known to be based on the formation of mineral spherulites.54-59 These are thought to be present at the initiation sites of mineral formation60 and consist primarily of calcium and phosphate, which has been confirmed by elemental analyses of mineralizing regions.57,61,62 (53) Rajnicek, A. M.; Britland, S.; McCaig, C. D. J. Cell Sci. 1997, 110, 2905–2913. (54) Coelho, M. J.; Cabral, A. T.; Fernandes, M. H. Biomaterials 2000, 21, 1087–1094. (55) Livingstone, T.; Ducheyne, P.; Garino, J. J. Biomed. Mater. Res. 2002, 62, 1–13. (56) Lu, H. H.; Tang, L.; Oh, S. C.; Spalazzi, J. P.; Dionisio, K. Biomaterials 2005, 26, 6323–6334. (57) Morais, S.; Carvalho, G. S.; Faria, J. L.; Gomes, H. T.; Sousa, J. P. Biomaterials 1998, 19, 23–29. (58) Pederson, A. W.; Ruberti, J. W.; Messersmith, P. B. Biomaterials 2003, 24, 4881–4890. (59) Wang, Y.; Cui, F. Z.; Zhai, Y.; Wang, X.; Kong, X.; Fan, D. Mater. Sci. Eng., C 2006, 26, 635–638.

Examination of the cell growth on the surfaces by SEM clearly revealed that at the later time points mineralization was occurring on all UHMWPE surfaces. Mineral deposits were obvious in the representative SE micrographs, shown in Figure 8, as white spherical formations at the edge of the cells. This is shown more clearly in the high-resolution images given in Figure 13, where 21 day images show the presence of mineral-like formations surrounding the cells. The layering of cells on top of those cells aligned to the direction of the surface striations of the UHMWPE surfaces was also more clearly visible. The more random orientation of these upper layer cells confirms previous observations that contact guidance effects are reduced when cells form layers.42 EDX spectra from 21 day samples, untreated UHMWPE surfaces with 11 atom % and treated UHMWPE surfaces with 19 atom % oxygen, respectively, after 21 days of culture, given in parts a and b of Figure 13 contain peaks due to Ca and P. The additional peaks from Au and Pd are due to the sputter coating used to aid sample conductivity for the SEM analysis. The peaks seen for calcium and phosphorus are typical of nodule formation, indicative of mineralization,54,55,61,62 and correlate well with contemporaneous Alizarin Red staining data,63 which further confirms the mineralization on these surfaces. The calcium phosphate spherulites surrounding the cells are shown alongside each image and are where the spectra were taken from. EDX is a semiquantitative technique, so the levels indicated purely confirm the presence of the elements and cannot therefore be used to assess whether there was a higher density of mineralization on one surface compared to another. Figure 13c shows that the UHMWPE surfaces with 26 atom % oxygen have a similar elemental analysis, confirming the presence of calcium and phosphorus. The micrographs show individual calcium phosphate spherulite formation in contact with cell protrusions, and larger conglomerates of mineralization are also present. The presence of a mineralized inorganic phase containing calcium phosphate was confirmed on the UHMWPE surfaces after 21 days of culture. These formations were not observed at the earlier time points, confirming their production by the cells, and less of these formations were observed on the untreated surfaces.

Conclusions The attachment and growth of HOB cells on UHMWPE surfaces has been studied. The as-supplied surfaces were found to have micro- and macrotopography in the form of surface striations which had peak to trough heights of approximately 3 µm and peak to peak separations of 5 µm. An intrinsic surface (60) Mann, S. In Biomineralisation: Principles and Concepts in Bioinorganic Materials Chemistry, 1st ed.; Compton, R. G., Davies, S. G., Evans, J., Eds.; Oxford Chemistry Masters, Vol. 5; Oxford Press: Oxford, U.K., 2001; p 198. (61) Xynos, I. D.; Hukkanen, M. V. H.; Batten, J. J.; Buttery, L. D.; Hench, L. L.; Polak, J. M. Calcif. Tissue Int. 2000, 67, 321–329. (62) Yamamoto, M.; Kato, K.; Ikada, Y. J. Biomed. Mater. Res. 1997, 37, 29–36.

Attachment of HOB Cells to Polyethylene Surfaces

oxygen level on UHMWPE of 11 atom %, resulting from the manufacturing process, was shown by XPS to be mainly in the form of hydroxyl/ether groups. UV/ozone treatment was used to produce higher levels of oxidation on UHMWPE, of up to 26 atom %, which resulted in the incorporation of carbonyl and carboxyl/ester groups. The surface striations were not altered significantly by the oxidation treatment. The as-supplied UHMWPE and those with the two levels of UV/ozone treatment chosen for these experiments, resulting in 19 and 26 atom % oxygen, all had absolute rms Rq roughness within the 350-450 nm range, i.e., 2 orders of magnitude greater than that of the TCPS. Water contact angles of the UHMWPE surfaces decreased from 88° to approximately 73° with increasing oxygen incorporation. HOB cells were cultured on each polymer surface and characterized at 1, 2, 3, 10, and 21 days postplating. HOB cells cultured on the treated UHMWPE surfaces with 19 atom % oxygen showed significantly higher densities compared to cells cultured on TCPS throughout the 21 day period, and the treated UHMWPE surfaces with 26 atom % oxygen had significantly higher cell densities from 3 days onward. This shows that the cells attached and proliferated more readily on the UV/ozonetreated UHMWPE surfaces than on conventional culture plastics (TCPS). Contact guidance was observed by both SEM and AFM and showed that the cells on the UHMWPE surfaces had aligned to the striations. Scanning electron micrographs showed the UHMWPE surfaces were confluent by day 10, which led to visible layering of the cells by day 21. Cell layering is an indicator of nodule formation.64,65 Where cell layering occurred, cell orientation in the subsequent layers was less influenced by the (63) Poulsson, A. H. C. Modification of Polymer Surfaces to Aid the Attachment of Cells Derived from Bone. Thesis, Robert Gordon University, Aberdeen, U.K., 2007. (64) Di Silvio, L.; Gurav, N. Osteoblasts. In Human Cell Culture: Primary Mesenchymal Cells; Koller, M. R., Palsson, B. O., Masters, J. R. W., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001; pp 221-241. (65) Stanford, C. M.; Jacobson, P. A.; Eanes, E. D.; Lembke, L. A.; Midura, R. L. J. Biol. Chem. 1995, 270(16), 9420–9428.

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direction of the surface striations, as expected. Mineralization was confirmed by SEM and EDX analysis, which showed that there was no mineralization at the early time points, but by day 10 initial spherulite formations were observed near cell protrusions. By day 21, there were higher concentrations of these on all of the UHMWPE surfaces, and more spherulites were observed on the UV/ozone-treated surfaces, though this was only a qualitative observation. EDX analysis confirmed that the spherulites contained calcium and phosphorus. Calcium and phosphorus are the major constituents in the mineral phase of bone. Overall, UV/ozone surface treatment leads to an enhancement of the HOB cell attachment and functionality which is significantly better than that observed under the same conditions for TCPS. The cells which attached to the UHMWPE retain their functionality and go on to mineralize by the production of calcium and phosphorus as demonstrated by SEM/EDX analysis of the spherulitic structures. The cell functionality on the untreated UHMWPE surfaces proved that the effect of the UV/ozone treatment enhanced cell functionality, independent of the topography. These results are promising for both the enhanced in Vitro culture of human primary osteoblast cells and the use of UV/ozone treatment to improve the adhesion and manipulation on implant surfaces. Acknowledgment. We acknowledge financial aid from the EPSRC and SHEFC. We thank the Department of Orthopaedic Surgery, IMS, Aberdeen University, and the surgical team at Woodend Hospital for kindly providing advice, tissues, and patient consent. We also thank Mr. Iain Tough for advice and assistance with the SEM and EDX. Supporting Information Available: A list of abbreviations and representative XPS survey and narrow scans of all the surface treatments described. This material is available free of charge via the Internet at http://pubs.acs.org. LA801820S