Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition

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Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition J. Lincks a'b B D. B o y a n b'c'd'* C.R. Blanchard e C.H. L o h m a n n c Y. Liu c, D.L. C o c h r a n b, D . D . D e a n c, Z. Schwartz b'~'f 9

~

~

a Wilford Hall Medical Center, Lackland AFB b Department of Periodontics, University of Texas Health Science Centre, San Antonio, TX, USA c Department of Orthopaedics, University of Texas Health Science Centre, San Antonio, TX, USA dDepartment of Biochemistry, University of Texas Health Science Centre, San Antonio, TX, USA eSouthwest Research Institute, San Antonio, Texas, USA f Department of Periodontics, Hebrew University Hadassah Faculty of Dental Medicine, Jerusalem, Israel

Abstract The success of an implant is determined by its integration into the tissue surrounding the biomaterial. Surface roughness and composition are considered to influence the properties of adherent cells. The aim of this study was to determine the effect of chemical composition and surface roughness of commercially pure titanium (Ti) and Ti-6A1-4V alloy (Ti-A) on MG63 osteoblast-like cells. Unalloyed and alloyed Ti disks were machined and either fine-polished or wet-ground, resulting in smooth (S) and rough (R) finishes, respectively. Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by cold field emission scanning electron microscopy and profilometry, while chemical composition was determined using Auger electron spectroscopy and Fourier transform infrared spectroscopy. The effect on the cells was evaluated 24 h postconfluence by measuring cell number, [3H]-thymidine incorporation into DNA, cell and cell layer alkaline phosphatase specific activity (ALPase), osteocalcin and collagen production, [-35S]-sulfate incorporation into proteoglycan, and prostaglandin E 2 (PGE2) and transforming growth factor-/~ (TGF-/~) production. When compared to plastic, the number of cells was reduced on the pure Ti surfaces, while it was equivalent on the Ti-A surfaces; [3H]-thymidine incorporation was reduced on all surfaces. The stimulatory effect of surface roughness on ALPase in isolated cells and the cell layer was more pronounced on the rougher surfaces, with enzyme activity on Ti-R being greater than on Ti-A-R. Osteocalcin production was increased only on the Ti-R surface. Collagen production was decreased on Ti surfaces except Ti-R; [35S]-sulfate incorporation was reduced on all surfaces. Surface roughness affected local factor production (TGF-/~, PGE2). The stimulatory effect of the rougher surfaces on PGE2 and TGF-/~ was greater on Ti than Ti-A. In summary, cell proliferation, differentiation, protein synthesis and local factor production were affected by surface roughness and composition. Enhanced differentiation of cells grown on rough vs. smooth surfaces for both Ti and Ti-A surfaces was indicated by decreased proliferation and increased ALPase and osteocalcin production. Local factor production was also enhanced on rough surfaces, supporting the contention that these cells are more differentiated. Surface composition also played a role in cell differentiation, since cells cultured on Ti-R surfaces produced more ALPase than those cultured on Ti-A-R. While it is still unknown which material properties induce which cellular responses, this study suggests that surface roughness and composition may play a major role and that the best design for an orthopaedic implant is a pure titanium surface with a rough microtopography. 9 1998 Published by Elsevier Science Ltd. All rights reserved Keywords: Osteoblasts; Titanium; Titanium alloy; Surface roughness; PGE2; TGF-/~; In vitro

1. Introduction The m o r p h o l o g y of an implant surface, including m i c r o t o p o g r a p h y and roughness, has been shown to be *Corresponding author. Tel.: (210) 567-6326; fax: (210) 567-6295; internet: [email protected]

related to successful bone fixation [1, 2]. In addition, the m a n u f a c t u r i n g process used to achieve the surface texture, either chemical [3] or mechanical [4], also influences clinical success. At present, titanium implants in clinical use vary with respect to surface roughness and composition, with consensus being limited to the fact that bone forms m o r e readily on a rough surface whereas

0142-9612/98/$--See front matter ~ 1998 Published by Elsevier Science Ltd. All rights reserved. PII SO 1 42-96 1 2(98)00 1 44-6

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fibrous connective tissue is found more frequently on a smooth surface [5]. In vitro studies have provided some insight into the response of specific cell types to surface properties. It is clear that surface roughness affects cell response. In particular, osteoblast-like cells exhibit roughness-dependent phenotypic characteristics. They tend to attach more readily to surfaces with a rougher microtopography [6, 7]. Moreover, they appear to be more differentiated on rougher surfaces with respect to morphology, extracellular matrix synthesis, alkaline phosphatase specific activity and osteocalcin production, and response to systemic hormones such as 1,25-(OH)zD3 [8, 9]. The degree of roughness also affects production of local factors such as transforming growth factor beta (TGF-fl) and prostaglandin E2 (PGE2) 1-10], both of which can act on the osteoblastic cells as autocrine regulators [-11, 12], and can modulate the activity of bone resorbing cells via paracrine mechanisms [13, 14]. The morphology of the surface also plays a role. A variety of cells can orient themselves in the grooves of micromachined surfaces [15-17]. Depending on the degree of roughness, these cells may actually see the groove as smooth. On a randomly rough surface as is created by grit blasting or chemical etching, cells may form different focal attachments which result in a phenotype that is distinct from that seen on the grooved surface with the same degree of roughness. Titanium implants which are currently in clinical use in dentistry and orthopaedics, vary with respect to surface roughness and composition. In dentistry, commercially pure titanium (Ti) has become one of the most commonly used implant materials whereas in orthopaedics Ti alloys have virtually replaced Ti because of strength requirements [18, 19]. Both Ti and Ti-6A1-4V (Ti alloy) develop a surface oxide layer due to the natural passivation of Ti [20,21]. However, differences in the crystallinity of the underlying metal as well as the segregation of alloy components, may cause the oxide that forms on Ti to be quite different from the oxide that forms on Ti alloys. Several studies have shown that even subtle differences in surface composition, including Ti oxide crystallinity, can modify cell response, even when surface roughness is held constant [-6, 22-28]. We previously showed that when MG63 osteoblastlike osteosarcoma cells are cultured on Ti discs with average surface roughness values (Ra) varying from <0.1 lum (smooth) to 3-4 gm (rough) to > 6 gm (very rough), there are distinct differences in phenotypic expression [8, 10]. For these studies, the smooth surfaces were obtained by electropolishing following chemical etching; rough surfaces were obtained by coarse grit blasting; and very rough surfaces were achieved via Ti plasma spray. The results showed that as surface roughness increased, expression of a differentiated osteoblastic phenotype increased, including reduced cell number and

DNA synthesis (proliferation), and increased alkaline phosphatase specific activity (ALPase), osteocalcin production, collagen synthesis, proteoglycan sulfation, and production of latent TGF-/~ and PGE2. The optimal surface appeared to be those with R a values around 4 lam; cell proliferation was reduced but not blocked and phenotypic differentiation was enhanced. In contrast, cells on the smooth surface had high proliferation rates but ALPase and osteocalcin production were low, indicative of a loss of a differentiated osteoblastic phenotype. To determine whether the composition of the surface or microtopography are more important variables in determining osteoblastic phenotype, we examined the response of MG63 cells to machined surfaces with smooth R a values as well as with rough R a values that were prepared from Ti and Ti alloy. The results of the present study using machined surfaces were compared to those of our previous work using grit-blasting to obtain similar R a values.

2. Materials and methods

2.1. Titanium disk preparation and characterization 2.1.1. Disk preparation Titanium disks (14.75 mm diameter; 0.8 mm thick) were fabricated from sheets of either commercially pure titanium (Ti: medical grade 2, ASTM F67, 'unalloyed Ti for medical applications') or titanium-6 wt% aluminum4 wt% vanadium alloy (Ti-6A1-4V; Ti-A) obtained from Timet, Inc. (O'Fallon, MO). Chemical composition was provided by the supplier and was not verified prior to surface preparation. Each sheet was sectioned into one foot by one foot plates for ease of handling and to ensure a consistent finish. The disks were either polished or ground to acquire the desired surface finishes. Polishing to create the smooth surface was performed by lapping with 18T grit (oil based 500-600 grit aluminum oxide) followed by polishing with 4.0 paper (1200 grit aluminum oxide) by French Grinding Service, Inc. (Houston, TX). The rough surface was prepared by wet sanding using a carborundum brand zirconium oxide/aluminum oxide resin bonded to a cloth belt by Metal Samples, Inc. (Mumford, AL). Disks were stamped using an automated metal punch and cleaned in an acetone bath using an ultrasonic cleaner for one hour. The disks were then washed in Jet-A fuel (grade AL-24487-F; Diamond Shamrock, San Antonio, TX) in an ultrasonic cleaner for one hour and was followed by four washes with Versa Clean (Fisher Scientific, Pittsburgh, PA). Between each wash with Versa Clean the disks were rinsed twice with deionized, distilled water. After the final wash, the disks were rinsed with 70% ethanol and then dried in vacuo. Prior to use each disk was washed again three times with ethanol and rinsed three times with deionized, distilled water. The

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disks were individually wrapped in gauze to prevent damage and then sterilized by autoclaving.

2.1.2. Surface characterization Representative disks from each group were subjected to surface analysis. The surface microtopography of the disks was examined using an Amray 1645 cold field emission scanning electron microscope (Amray, Bedford, MA) with a nonthermally assisted tip and secondary and backscattered electron capability. Two samples from each group were examined at 100 to 500 x. Surface roughness was measured by profilometry using a Taylor-Hobson Surtronic 3 profilometer (Leicester, UK). Average surface roughness (Ra) measurements were taken at ten different locations on each one foot x one foot sheet to obtain an accurate assessment. For the smooth surfaces, measurements were made in all directions, whereas on the rough surfaces, measurements were taken perpendicular to the machine markings. Following the punching operation, four disks from each sheet were randomly sampled to confirm the R a values obtained earlier. Auger electron spectroscopy was performed to analyze the Ti oxide layer using a Perkin-Elmer Model 595 scanning Auger microprobe (Perkin Elmer, Physical Electronics Division, Eden Prairie, MN). Spectra were obtained from two representative disks from the two groups with a smooth surface (Ti and Ti-A) to determine the chemical profile of the subsurface layer. Rough disks were not examined to avoid artifacts associated with rough morphologies; further, the thickness and composition of the surface oxides on the rough and smooth disks for each material would be expected to be identical since all disks were machined and cleaned using the same protocol. The spectra were obtained at regular sputtering intervals at a sputtering rate of 400 A min- ~. Comparing spectra and relative peak heights at given surface depths provided information about the chemistry of the oxide layer. Fourier transform infrared spectroscopy (FTIR) was performed to determine if an organic residue remained on the disk surfaces after cleaning. Spectra were obtained from four disks (two from the smooth Ti group and two from the smooth Ti-A group) using a Nicolet Magna FTIR in reflection mode. Spectra were collected using 32 scan summations at a resolution of 16 cm- 1. FTIR spectroscopy was not performed on the rough surfaces, because artifactual measurements are obtained on rough samples.

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a relatively immature osteoblast, including the stimulation of alkaline phosphatase activity and osteocalcin synthesis and inhibition of proliferation in response to treatment with l~,25-(OH)zD3 [29,30]. As a result they are a good model for examining the early stages of osteoblast differentiation. However, the culture conditions under which MG63 cells will mineralize their matrix have not been defined, so terminal differentiation cannot be studied using these cells. Despite this limitation, we selected this model in preference to fetal rat calvarial cells since the latter are derived from embryonic rat bone which may differ significantly from adult human bone. We recognize that MG63 cells are not normal osteoblasts and data interpretation must take this into consideration. MG63 cells were obtained from the American Type Culture Collection (Rockville, MD). For all experiments, cells were cultured on disks placed in 24 well plates (Corning, Corning, IL). Controls consisted of cells cultured directly on the polystyrene surface of the 24 well plate. Cells were plated at 9300 cells cm-2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.5% antibiotics (diluted from a stock solution containing 5000 U m l - 1 penicillin, 5000 U ml-1 streptomycin; GIBCO, Grand Island, NY) and cultured at 37~ in an atmosphere of 100% humidity and 5% CO2. Media were changed every 48 h until the cells reached confluence. Because of the opacity of the Ti disks, there was no practical way to assess confluency of the cultures. As a result, when cells reached visual confluence on plastic, cultures on all other surfaces were treated exactly as those grown on plastic.

2.3. Cell morphology To determine whether cell morphology varied as a function of surface roughness, the cultures were examined by scanning electron microscopy. At harvest, the culture media were removed and the samples rinsed three times with phosphate-buffered saline (PBS) and fixed with 1% OsO4 in 0.1 M PBS for 15-30 min. After fixation, the disks were rinsed with PBS, sequentially incubated for 30-45rain each in 50, 75, 90 and 100% ter-butyl alcohol, and vacuum dried. A thin layer of goldpalladium was sputter-coated onto the samples prior to examination in a JEOL 6400 FEC cold field emission scanning microscope (JEOL USA, Inc. Peabody, MA).

2.4. Cell proliferation 2.2. Cell culture MG63 osteoblast-like cells were used for these experiments because they were obtained from a human osteosarcoma [29] and have been well-characterized. They display numerous osteoblastic traits that are typical of

2.4.1. Cell number At harvest, cells were released from the culture surface by addition of 0.25% trypsin in Hank's balanced salt solution (HBSS) containing 1 mM ethylenediamine tetraacetic acid (EDTA) for ten minutes at 37~ and this

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was followed by addition of DMEM containing 10% FBS to stop the reaction. Previous studies demonstrated that two trypsinizations are necessary to quantitatively harvest MG63 cells from rough Ti surfaces [8]. Accordingly, a second trypsinization was performed to ensure that any remaining cells had been removed from the surface. Cell suspensions from both trypsinizations were combined and centrifuged at 500 x 9 for 10 min. Cell pellets were washed with PBS and resuspended in PBS. Cell number was determined by use of a Coulter Counter (Coulter Electronics, Hialeah, FL). Cells harvested in this manner exhibit > 95 % viability based on trypan blue dye exclusion.

2.4.2. [~H]-thymidine incorporation DNA synthesis was estimated by measuring [3H]thymidine incorporation into trichloroacetic acid (TCA) insoluble cell precipitates as previously described by Schwartz et al. [31]. MG63 cells were cultured on the plastic surface or Ti disks until the cells on plastic reached visual confluence. Media were changed and the incubation continued for an additional 24 h. Four hours prior to harvest, 50 ~1 [3H]-thymidine (from a 1 ~Ci ml- 1 stock solution) was added to the cultures. At harvest, the cell layers were washed twice with cold PBS, twice with 5% TCA, and then treated with ice-cold saturated TCA for 30 min. TCA-precipitable material was dissolved in 0.25 ml 1% sodium dodecyl sulfate (SDS) at 20~ and radioactivity measured by liquid scintillation spectroscopy.

2.5. Cell differentiation 2.5.1. Alkaline phosphatase specific activity At harvest, either cell layers, as described below, or isolated cells, as described above, were prepared and their protein content determined by use of commercially available kits (Micro/Macro BCA, Pierce Chemical Co., Rockford, IL). Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, alkaline; E.C. 3.1.3.1] activity was assayed as the release of p-nitrophenol from p-nitrophenylphosphate at pH 10.2 as previously described [32] and specific activity determined. Cell layers were prepared following the method of Hale et al. [33]. At harvest, culture media were decanted, cell layers washed twice with PBS, and then removed with a cell scraper. After centrifugation, the cell layer pellets were washed once more with PBS and resuspended by vortexing in 0.5 ml deionized water plus 25 l.tl 1% Triton-X-100. Pellets were further disrupted by freeze/thawing three times. Isolated cells were harvested as described above for the determination of cell number, except that after the cell pellets had been washed twice with PBS, the cells were resuspended by vortexing in 0.5 ml of deionized water with 25 t.tl of 1% Triton-X-100.

Enzyme assays were performed on both cell and cell layer lysates.

2.5.2. Osteocalcin production The production of osteocalcin by the cultures was measured using a commercially available radioimmunoassay kit (Human Osteocalcin RIA Kit, Biomedical Technologies, Stoughton, MA). Culture media were concentrated five-fold by lyophilization and reconstituted in 100 ~1 normal rabbit serum, 10 btl rabbit anti-human osteocalcin antibody, 100 ~1 [125I]-human osteocalcin, and 200 ~1 Tris-saline buffer and placed overnight on an orbital platform shaker (approximately 80 rpm) at room temperature. Goat anti-rabbit antibody and polyethylene glycol (100 l.tl each) were added to each tube the following morning. After vortexing, the samples were placed on an orbital shaker for 2 h at room temperature. One ml of Tris-saline buffer was added to each sample. The solution was then vortexed and centrifuged at 500 x 9 for 20 min at 4~ The supernatant was decanted and the pellet placed in scintillation cocktail and counted. Osteocalcin concentrations were determined by correlating the percentage bound over unbound counts to a standard curve.

2.6. Matrix production 2.6.1. Collagen production Matrix protein synthesis was assessed by measuring the incorporation of [3H]-proline into collagenase digestible (CDP) and noncollagenase digestible (NCP) protein [34]. When the cells reached confluence on plastic, the media in all cultures were replaced with 500 gl DMEM containing 10% FBS, antibiotics, and 50 ~gm1-1 /%amino proprionitrile (Sigma, St. Louis, MO), and 10 gCim1-1 of L[GaH]-proline (New England Nuclear, Boston, MA). After 24 h, media were discarded. Cell layers (cells and matrix) were obtained by scraping and resuspending in two 0.2 ml portions of 0.2 N NaOH. Proteins were precipitated with 0.1 ml 100% TCA containing 1% tannic acid, washed three times with 0.5 ml 10% TCA + 1% tannic acid, and then twice with icecold acetone. The final pellets from the cell layers were dissolved in 500 l.tl 0.05 N NaOH. Digestion of the cell layer pellet was performed using highly purified clostridial collagenase (Calbiochem, San Diego, CA; 138 U mg-1 protein) as described previously [8]. NCP synthesis was calculated after multiplying the labeled proline in NCP by 5.4 to correct for its relative abundance in collagen [34]. Percent collagen production was calculated by comparing CDP production with total CDP + NCP production (i.e.: [CDP/(CDP + NCP)] x 100). The protein content of each fraction was determined by miniaturization of the method of Lowry et al. [35]. This assay does not take into account any

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degradation that may have occurred during the incubation or during sample preparation.

2.6.2. Proteoglycan sulfation Proteoglycan synthesis was assessed by [-35S]-sulfate incorporation according to the method of O'Keefe et al. [36]. Previously, we found that the amount of radiolabeled proteoglycan secreted into the media by MG63 cells was less than 15 % of the total radiolabeled proteoglycan produced. Because more than 85% of the radiolabeled proteoglycan was in the cell layer, we examined the incorporation of [35S]-sulfate only in the cell layer. At confluence, 50 ~tl D M E M containing 90 ~tCiml-1 [35S]-sulfate were added to the media to make a final concentration of 9 ~tCiml-1. Four hours later, the media were discarded and the wells washed one time with 500 ~tl PBS. The cell layer was collected in two 0.25 ml portions of 0.25 M NaOH. The protein content was determined by the method of Lowry et al. [35]. To measure [-3SS]sulfate incorporation into the cell layers, the total volume was adjusted to 0.7 ml by the addition of 0.15 M NaC1 and the sample dialyzed in a 12000-14000 molecular weight cut-off membrane against buffer containing 0.15 M NaC1, 20 mM NazSO4, and 20 mM NazHPO4 at pH 7.4 and 4~ The dialysis solution was changed until the radioactivity in the dialysate reached background levels. The amount of [35S]-sulfate incorporated was determined by liquid scintillation spectrometry and was calculated as dpm mg-1 cell layer protein.

2. 7. Local factor production 2. 7.1. Prostaglandin E: The amount of PGE2 produced by the cells and released into the media was assessed using a commercially available competitive binding radioimmunoassay kit (NEN Research Products, Boston, MA). In this assay, unlabeled PGE2 in the sample was incubated overnight with radiolabeled PGE2 and unlabeled PGE2 antibody. Antigen-antibody complexes were separated from free antigen by precipitation with polyethylene glycol. Sample PGE2 concentrations were determined by correlating the percentage bound over unbound counts to a standard curve.

2. 7.2. Transforming growth factor-beta (TGF-fi) In order to measure the level of total TGF-~ production by the cells, a commercially available enzyme-linked immunoassay (ELISA) kit (Promega Corp., Madison, WI) specific for human TGF-~I was used. Immediately prior to assay, conditioned media were diluted 1:10 in D M E M and the 1:10 dilution further diluted by adding four volumes of PBS. The media were then acidified by the addition of 1 M HC1 for 15 min to activate latent TGF-fi (LTGF-fl), followed by neutralization with 1 M

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NaOH. The assay was performed according to the manufacturer's directions. Intensity measurements were conducted at 450nm using a BioRad Model 2550 EIA Reader (Hercules, CA). Sample concentrations were determined by comparing the absorbance value to a known standard curve. The amount of TGF-fil in the cell layer was not examined because of difficulties associated with quantitatively extracting this cytokine from the matrix.

2.8. Statistical analysis Experiments were conducted at least twice and the data shown are from one representative experiment. For any given experiment, each data point represents mean + SEM of six individual cultures. Data were first analyzed by analysis of variance; when statistical differences were detected, the Student's t-test for multiple comparisons using Bonferroni's modification was used. P-values <0.05 were considered to be significant. m

3. Results

3.1. Disk characteristics 3.1.1. Morphology When the Ti-S and Ti-A-S disks were examined by scanning electron microscopy, the surfaces were found to be very similar (Fig. 1A and C). Morphologically, the disks had small pits (2 jam in diameter) and randomly oriented scratches from the polishing operation, which were only evident at high magnification (data not shown). The Ti-R and Ti-A-R disks also had a similar appearance (Fig. 1B and D) and contained parallel, longitudinal grooves with both sharp and serrated edges, resulting from the grinding operation. Parallel grooves of varying heights were prominent; in addition, the distance between the grooves varied. On both rough surfaces, curved sheets of material were observed occasionally at the apex of the grooves. Additionally, the Ti-A-R surface contained areas with pits that were 10-20 jam in diameter.

3.1.2. Surface roughness Based on profilometry (Table 1) the smooth surfaces, Ti-S and Ti-A-S, had similar R, values of 0.22 and 0.23 ~tm, respectively. The Ti-R surface was the roughest and had a n R a of 4.24 ~tm, while the Ti-A-R surface had a n Ra of 3.20 ~tm. Both rough surfaces were significantly rougher than both smooth surfaces.

3.1.3. Auger electron spectroscopy Both smooth surfaces (Ti-S and Ti-A-S) were found to contain Ti, O, and C by Auger electron spectroscopy before sputtering. In the alloyed surface, A1 was also found. After 10 s of sputtering, the C signal was virtually gone at a depth of 67 A in both Ti and Ti-A disks. In

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Fig. 1. Scanning electron micrographs of the different disk surfaces used in this study. Panel A: Ti-S; Panel B: Ti-R; Panel C: Ti-A-S; Panel D: Ti-A-R. Bar = 200 ~tm. Original magnification: 100x.

Table 1 Average surface roughness values for the Ti and Ti-alloy disks used in this study

the O signal virtually disappeared. N o v a n a d i u m was found in the disks.

Surface

Ra value

Ti-S Ti-R Ti-A-S Ti-A-R

0.22 + 0.00a 4.24 + 0.13 b 0.23 + 0.00a 3.20 + 0.12

3.1.4. Fourier transform infrared spectroscopy F T I R analysis of the disks confirmed that no organic residue was left on the surface of either the Ti-S or Ti-A-S disks.

Note: Ti and Ti alloy (Ti-A) disks were prepared with either a smooth (S) or rough (R) surface as described in the Materials and Methods. The Ra value for each disk type was determined by profilometry. Data shown in the table represent the mean +_SEM for four disks in each group; each disk was measured in four areas. a p < 0.05, smooth vs. rough surface. b p < 0.05, T i - R vs. T i - A - R .

addition to Ti and O, A1 was also present in the alloy. Twenty seconds of sputtering to a depth of 134 ,~ produced a continuously decreasing O signal while sputtering t h r o u g h the oxide layer, and an increasing Ti signal. After one minute, the Ti signal became very strong, and

evidence of

3.2. Cell morphology The a p p e a r a n c e of the cells varied with surface roughness and chemical composition of the disks. Cells grown on the Ti-S surface were spread out across the surface and grew as a monolayer, but this m o n o l a y e r was not continuous (Fig. 2C and D). The cells had a dendritic appearance, with extensions that were up to 10 ~tm in length and had ruffled m e m b r a n e s on their surfaces. Cells cultured on Ti-R (Fig. 2A and B) and Ti-A-S (Fig. 3C and D) disks grew as a continuous, thin m o n o l a y e r across the surface. O n the Ti-R surface, all cracks and fissures were covered by a m o n o l a y e r of cells (Fig. 2A and B). Cultures on the Ti-A-R surface induced the cells to grow as a multilayer (Fig. 3A and B), with m a n y cells p r o d u c i n g

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Fig. 2. Scanning electron micrographs of MG63 osteoblast-like cells cultured on smooth and rough Ti surfaces. Panel A: Ti-R, magnification: 100• bar = 10 ~tm; Panel B: Ti-R, magnification: 500x, bar = 1 ~tm; Panel C: Ti-S, magnification: 100• bar = 10 ~tm; Panel D: Ti-S, magnification: 500x, bar = 1 ~tm.

extensions that covered distances of up to 10 lam. In addition, the cells were oriented along the parallel cracks and grooves and grew over the sharp edges, forming a multilayer.

3.3. Cell proliferation 3.3.1. Cell number Cell number was affected by both chemical composition and surface roughness (Fig. 4). Compared to plastic, cell number was reduced by 36% on Ti-R. Although not statistically significant, cell number was also reduced by 20% on Ti-S. Fewer cells were present on the Ti-R surfaces than on Ti-A-R as well. The numbers of the cells grown on the Ti-A-S and Ti-A-R surfaces were similar to that seen on the plastic.

3.3.2. [3H]-thymidine incorporation [3H]-thymidine incorporation was reduced on all metal surfaces when compared to plastic (Fig. 5). The effect was comparable on the alloyed Ti surfaces (49%) and the Ti-R surface (48%). However, the decrease seen on the Ti-S surface was significantly less than on the other surfaces (19%).

3.4. Cell differentiation 3.4.1. Alkaline phosphatase specific activity Enzyme activity varied with surface roughness and composition (Fig. 6). Cell layers from cells cultured on all different surfaces contained significantly more alkaline phosphatase specific activity than on the plastic control (1.6 fold to 2.2 fold). Activity on Ti-R was 20% greater than on Ti-A-R. Activity on the rough surfaces was consistently greater than on smooth surfaces. Alkaline phosphatase on Ti-R was 1.5-fold greater than on Ti-S; on Ti-A-R, alkaline phosphatase was 1.3-fold greater than on Ti-A-S. When enzyme activity of isolated cells was measured, similar observations were made (Fig. 7). Cells grown on Ti-R surfaces exhibited a 1.8-fold increase in enzyme activity over that seen on plastic. On Ti-R, the increase was 1.4-fold, and on the smooth surface disks, there was a 1.3-fold increase. Activity was greater on Ti-R in comparison to Ti-S and in comparison to Ti-A-R. These results also showed that the effects of surface roughness and composition on alkaline phosphatase specific activity were primarily due to enzyme present in the matrix. Specific activity of the cell layer was consistently

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Fig. 3. Scanning electron micrographs of MG63 osteoblast-like cells cultured on smooth and rough Ti-A-surfaces. Panel A: Ti-A-R, magnification: 100• bar = 10~m; Panel B: Ti-A-R, magnification: 500• bar = 1 ~m; Panel C: Ti-A-S, magnification: 100• bar = 10 ~tm; Panel D: Ti-A-S, magnification: 500• bar = 1 ~tm.

Fig. 4. Number of MG63 osteoblast-like cells released by two trypsinizations of the Ti disks 24 h after they had reached confluence on the plastic. Values are the mean ___ SEM of six cultures. *P < 0.05, Ti disk vs. plastic; # P < 0.05, Ti-A-R vs. Ti-R. Data are from one of two replicate experiments. t w o t i m e s t h a t of t h e i s o l a t e d cells, d e s p i t e t h e l a r g e r d e n o m i n a t i o n d u e to t h e p r e s e n c e of m a t r i x p r o t e i n . T h e f o l d - i n c r e a s e s n o t e d as a f u n c t i o n of e i t h e r r o u g h n e s s or c o m p o s i t i o n w e r e g r e a t e r w h e n a s s a y i n g cell layers, res u l t i n g in s i g n i f i c a n t l y g r e a t e r real e n z y m e a c t i v i t y t h a n w a s seen in t h e i s o l a t e d cells. T h i s w a s p a r t i c u l a r l y evid e n t for cell l a y e r s c u l t u r e d o n Ti-R.

Fig. 5. [3H]-Thymidine incorporation by MG63 osteoblast-like cells during culture on plastic or Ti disks. When the cells reached confluence on plastic, the media were changed and culture continued for another 24 h. Four hours prior to harvest, [3H]-thymidine was added and incorporation into TCA insoluble cell precipitates measured. Values are the mean _+ SEM of six cultures. *P < 0.05, Ti disk vs. plastic: # P < 0.05, Ti-S vs. Ti-R. Data are from one of two replicate experiments.

3.4.2. Osteocalcin production Cell c u l t u r e s g r o w n o n t h e T i - R surface s h o w e d a significant i n c r e a s e (1.9 fold) in o s t e o c a l c i n p r o d u c t i o n c o m p a r e d to p l a s t i c (Fig. 8). T h e o s t e o c a l c i n p r o d u c t i o n by

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Fig. 6. Alkaline phosphatase specific activity of cell layers produced by MG63 osteoblast-like cells during culture on Ti disks. After cells had reached confluence on plastic, cultures were continued for an additional 24 h and then harvested by scraping. Enzyme activity was measured in the cell layer lysate. Values are the mean + SEM of six cultures. * P <0.05, titanium vs. plastic; # P <0.05, Ti-A-R vs. Ti-R; 9 P < 0.05, smooth vs. rough surface of same material. Data are from one of two replicate experiments.

Fig. 8. Osteocalcin production by MG63 osteoblast-like cells during culture on Ti disks. After cells reached confluence on plastic, the media were changed and the culture continued for an additional 24 h. At harvest, the media were collected, and osteocalcin content measured by RIA. Values are the mean • SEM of six cultures. * P < 0.05, titanium vs. plastic. Data are from one of two replicate experiments.

Fig. 7. Alkaline phosphatase specific activity of trypsinized MG63 osteoblast-like cells after culture on Ti disks. After cells had reached confluence on plastic, cultures were continued for an additional 24 h and then harvested by trypsinization. Enzyme activity was measured in lysates of the cells. Values are the mean _+ SEM of six cultures. * P <0.05, titanium vs. plastic; # P <0.05, Ti-A-R vs. Ti-R; 9 P < 0.05, smooth vs. rough surface of same material. Data are from one of two replicate experiments.

Fig. 9. Percent collagen production by MG63 osteoblast-like cells during culture on Ti disks. Values were derived from CDP and NCP production and are the mean _+ SEM of six cultures. * P < 0.05, titanium vs. plastic; # P < 0.05, Ti-A-R vs. Ti-R; 9 P < 0.05, smooth vs. rough surface of same material. Data are from one of two replicate experiments.

cells g r o w n plastic.

o n all t h e o t h e r

s u r f a c e s w a s s i m i l a r to

p r o d u c t i o n b y t h e cells w a s s i g n i f i c a n t l y d e c r e a s e d ( 1 5 % ) o n r o u g h T i - A - R s u r f a c e s c o m p a r e d to T i - R surfaces. M o r e o v e r , cells o n T i - S p r o d u c e d 3 1 % less c o l l a g e n t h a n

3.5. Matrix production

o n T i - R , a n d cells o n T i - A - S p r o d u c e d 1 7 % less c o l l a g e n than on Ti-A-R.

3.5.1. Collagen production

3.5.2. Proteoglycan sulfation

C o l l a g e n s y n t h e s i s w a s a l s o affected b y s u r f a c e c o m -

Compared

to

plastic,

[-35S]-sulfate

p o s i t i o n a n d r o u g h n e s s (Fig. 9). W h i l e c o l l a g e n s y n t h e s i s

by M G 6 3

w a s u n a f f e c t e d in cells c u l t u r e d o n T i - R , cells g r o w n o n

surfaces e x a m i n e d ( 3 5 - 4 8 % )

Ti-S, T i - A - R a n d T i - A - S s u r f a c e s s y n t h e s i z e d 1 4 - 3 0 %

least p r o n o u n c e d

less c o l l a g e n c o m p a r e d

surface. N o

t o plastic. T h e p e r c e n t c o l l a g e n

incorporation

cells w a s s i g n i f i c a n t l y r e d u c e d

o n all d i s k

(Fig. 10). T h i s effect w a s

in cells g r o w n o n t h e s m o o t h T i - A - R

significant

difference in t h e

[35S]-sulfate

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Effect of Disk Surface on [3ss]-sulfate Incorporation

Fig. 10. [35S]-Sulfate incorporation by MG63 osteoblast-like cells during culture on Ti disks. When the cells reached confluence on plastic, the media were changed and culture continued for another 24 h. Four hours prior to harvest, [35S]-sulfate was added and incorporation into the cell layer measured. Values are the mean + SEM of six cultures. * P < 0.05, titanium vs. plastic. Data are from one of two replicate experiments.

Fig. 12. Latent transforming growth factor fl (LTGFfl) production by MG63 osteoblast-like cells during culture on Ti disks. After the cells reached confluence on plastic, the media were changed and the culture continued for an additional 24 h. At harvest, the media were collected, and LTGFfi content measured by ELISA. Values are the mean _+ SEM of six cultures. * P < 0.05, titanium vs. plastic; # P < 0.05, Ti-A-R vs. Ti-R; 9 P < 0.05, smooth vs. rough surface. Data are from one of two replicate experiments.

those on the Ti-A-S surface. T h e levels on b o t h s m o o t h surface p r e p a r a t i o n s were n o t significantly different f r o m plastic.

3.6.2. Transforming growth factor-~

Fig. 11. Prostaglandin E2 (PGE2) production by MG63 osteoblast-like cells during culture on Ti disks. After cells reached confluence on plastic, the media were changed and the culture continued for an additional 24 h. At harvest, the media were collected, and PGE2 content measured by RIA. Values are the mean _ SEM of six cultures. * P <0.05, titanium vs. plastic; # P <0.05, Ti-A-R vs Ti-R; 9 P < 0.05, smooth vs. rough surface. Data are from one of two replicate experiments.

i n c o r p o r a t i o n a m o n g the different surface r o u g h n e s s e s a n d c o m p o s i t i o n s was observed.

3.6. Local factor production 3.6.1. Prostaglandin E2 T h e level of P G E 2 p r o d u c t i o n by the cells was affected by the different surface t r e a t m e n t s (Fig. 11). Significantly m o r e P G E 2 was p r o d u c e d by cells c u l t u r e d on Ti-R w h e n c o m p a r e d to plastic (3.9-fold) a n d to Ti-S surface (2.0 fold). Cells on the Ti-A-R surface synthesized 2.9-fold m o r e P G E 2 t h a n those on plastic a n d 3.3-fold m o r e t h a n

T h e level of latent T G F - / / i n the c o n d i t i o n e d m e d i a was also influenced by culture on the different surfaces (Fig. 12). L a t e n t TGF-/~ levels were increased by 1.7-fold on the Ti-A-R a n d 2.7-fold on the Ti-R surfaces. L a t e n t TGF-/~ p r o d u c t i o n was greater on the Ti-R surface comp a r e d to cultures g r o w n on the Ti-A-R surface (1.6-fold) a n d 2.1-fold greater w h e n c o m p a r e d to Ti-S. T h e r e was a slight, b u t insignificant, increase in L T G F / ~ levels prod u c e d by cells g r o w n on b o t h s m o o t h surfaces c o m p a r e d to plastic.

4. Discussion This study confirms previous o b s e r v a t i o n s t h a t osteoblast-like cells r e s p o n d in a differential m a n n e r to b o t h surface r o u g h n e s s [7, 37-39] a n d m a t e r i a l comp o s i t i o n [ 2 5 , 4 0 - 4 2 ] . As n o t e d previously [ 8 - 1 0 ] , M G 6 3 cells g r o w n on Ti-R surfaces exhibited a m o r e differentiated p h e n o t y p e as evidenced by r e d u c e d cell proliferation a n d increased alkaline p h o s p h a t a s e specific activity a n d osteocalcin p r o d u c t i o n . Cells g r o w n on Ti-S surfaces also exhibited r e d u c e d cell proliferation, a n d they h a d elevated alkaline p h o s p h a t a s e in c o m p a r i s o n with cultures g r o w n on plastic, b u t the effects were less r o b u s t t h a n those seen on Ti-R. M o r e o v e r , osteocalcin p r o d u c t i o n was u n a l t e r e d in these M G 6 3 cells, indicating t h a t they were n o t as differentiated as those cells g r o w n on Ti-R.

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Although [3H]-thymidine incorporation was reduced in cells cultured on Ti-A-R, total cell number was unaffected. The latter value is a cumulative measure of the viable cells in the culture, whereas the former value is an indication of the rate of DNA synthesis, and therefore, cell replication during the radiolabeling period, in our case, the last four hours of culture. This indicates that the cells grown on Ti-A-R ceased to proliferate and initiated expression of the mature osteoblastic phenotype at a slower rate than cells cultured on Ti-R, since proliferation is negatively correlated with phenotypic expression [43]. This hypothesis is supported by the fact that alkaline phosphatase activity on Ti-A-R was elevated, but to a lesser degree than seen on Ti-R, and the MG63 cells on Ti-A-R did not exhibit elevated osteocalcin production. Even for the alloy disks, however, the cells cultured on the rougher surfaces were more differentiated than the cells cultured on the smoother surfaces. Other aspects of osteoblast function were sensitive to the substrate, either with respect to roughness or to the bulk composition of the material. Production of extracellular matrix vesicles was affected by the nature of the substrate based on differences in cell layer alkaline phosphatase, where matrix vesicles are present, when compared to enzyme activity in isolated cells. Alkaline phosphatase is an early marker of osteogenic differentiation. While this enzyme activity is present in all cell membranes, it is found in higher levels in cells which mineralize their matrix such as osteoblasts [44]. As osteoblasts mature, they produce extracellular matrix vesicles which are enriched in alkaline phosphatase specific activity; because of this specific enrichment, alkaline phosphatase is the marker enzyme for this extracellular organelle [45]. Matrix vesicles are associated with the onset of calcification and they contain enzymes necessary for matrix modification necessary for crystal deposition and growth [46, 47]. The results of the present study show clearly that the effects of surface roughness were targeted to the matrix vesicles, whether the cells were cultured on Ti-R or Ti-A-R, since the fold increases in enzyme activity in the cell layer were significantly greater than the fold increases observed in the isolated cells. In addition, the effects of material composition were also found predominately in the matrix vesicle compartment, supporting previous in vivo and in vitro observations. Studies examining endosteal healing adjacent to various implant materials demonstrate that matrix vesicle production and function are sensitive to the type of material used [-41, 48, 49]. Similarly, when cells were cultured on thin films of various implant materials which had been sputtered onto tissue culture plastic, the effects of material composition were targeted to the matrix vesicles [26]. In comparison to plastic, proteoglycan sulfation was reduced in all of the cultures to a comparable extent. In

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contrast, collagen production was differentially affected by the nature of the surface topography and the material used. In general, synthesis on rougher surfaces was greater than seen on smoother surfaces, correlating with the production of latent TGF-/~. The expression of this growth factor is associated with the collagen deposition in the extracellular matrix of osteoblasts [50]. Similarly, production of PGE2 was greater on the rougher surfaces, supporting our previous observation that there is a positive correlation of latent TGF-/~ and PGE2 production with increasing surface roughness [10]. Both latent TGF-/3 and PGE2 are produced by osteoblasts as paracrine and autocrine regulators of cell function and differentiation. Their release by the MG63 cells cultured on Ti-S and Ti-A-S was essentially identical to the basal levels seen on plastic, another smooth surface. However, on the rougher Ti-R and Ti-A-R surfaces, their production was markedly enhanced, although in a material-specific manner, with the greatest production being observed in cells grown on Ti-R. This supports the contention that these cells exhibit a more differentiated osteoblastic phenotype. Whether more differentiated cells produce higher levels of these local factors, or whether the cells are more differentiated because they produce and respond to higher levels of these factors, is not known. The amounts of PGE2 produced per culture are well within the limits of prostaglandin known to be osteogenic and not inflammatory [ 11]. Since all of the TGF-/~ released into the media was in latent form, it is difficult to comment on its contribution to the differentiation of the MG63 cells. However, recent studies in our lab [51] and others [52] indicate that the latent TGF-/3 which is incorporated into the matrix may be activated locally via the action of matrix vesicles and may regulate the phenotypic expression of the cells. There is some indication that this is the case in the present study. In cells cultured on Ti-R surfaces, both ALPase and osteocalcin production were increased, whereas on Ti-A-R surfaces, ALPase was stimulated and osteocalcin production was not. When osteoblasts are treated with TGF-/3, alkaline phosphatase, an early marker of osteoblastic differentiation, is stimulated [12], whereas production of osteocalcin, a marker of terminal differentiation, is inhibited [12]. Whether TGF-/~ is modulating the differential expression of osteoblastic phenotypic markers in the MG63 cells is certainly not established by this study but the potential for regulation of this type is evidenced by the fact that production of local regulatory factors is sensitive to the material used. The results presented here also support our previous observation that roughness may play a more important role in determining cell response than the type of topography, as long as the R a values can be sensed by the cells. For practical purposes, the distance between peaks

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should not exceed the ability of the cell to form focal attachments on two or more peaks; otherwise, the cell would sense a rough surface as smooth. In the present study, surface roughness was achieved by machining, resulting in parallel grooves, whereas our previous studies used commercially pure Ti disks that were roughened by grit-blasting and acid-etching, resulting in random peaks and valleys. In general, the MG63 cells responded to smooth surfaces in a manner similar to their behavior on tissue culture plastic and to machined Ti-R surfaces in a manner similar to grit-blasted Ti surfaces with comparable R a values. The morphology of the cells on the Ti-R and Ti-A-R surfaces demonstrates that they have assumed a more cuboidal shape with dendritic extensions, similar to the morphology noted on rough cpTi surfaces achieved by grit-blasting, and typical of a more differentiated osteoblast. Similar observations have been noted with chick embryonic osteoblasts [37]. In contrast, cells on the smoother surfaces appear more flattened and fibroblastic. Our data also show that MG63 cells are sensitive to the bulk composition of the material, whether the surface is smooth or rough. Even though a titanium oxide layer formed on both the Ti and Ti-alloy surfaces, it is unlikely that the oxides were identical. Certainly mosaicism of the alloy components would result in a more complex surface chemistry. This would have a direct effect on the nature of the conditioning film that forms as the material surface interacts with the culture medium [53-55]. In addition, ions released from the alloy could also modulate cellular response. Recently, studies using fibroblast cultures demonstrated that locally released vanadium ions from Ti-6A1-4V alloy surfaces negatively impacted cell adhesion [56]. Thompson and Puleo [57] have also shown that Ti-6A1-4V ion solutions can inhibit expression of the osteogenic phenotype by bone marrow stromal cells, suggesting that ions released from implants could also impair normal bone formation. Despite the differences in cellular response due to material composition, roughness remains the overriding variable in promoting osteogenic differentiation. As strength requirements of orthopaedic implants necessitate the need for alloyed titanium preparations, it is essential that the optimal surface characteristics be determined, potentially mitigating any negative effects of the bulk material on bone formation and function.

Acknowledgements The authors gratefully acknowledge the expert assistance of Sandra Messier, Monica Luna, Kimberly Rhame, and Roland Campos in the preparation of the manuscript. Jack Lincks is a fellow in the Air Force Institute of Technology. This work does not necessarily reflect the views of the United States Air Force. Funding

for this research was provided by the Center for the Enhancement of the Biology/Biomaterials Interface at the University of Texas Health Science Center at San Antonio. Support for Dr. Lohmann was provided by a grant from the B. Braun Foundation. Melsungen, Germany.

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