Rafael Arcesio Delgado-Ruız Gerardo Gomez Moreno Antonio Aguilar-Salvatierra Aleksa Markovic nchez Jose Eduardo Mate-Sa Jose Luis Calvo-Guirado

Human fetal osteoblast behavior on zirconia dental implants and zirconia disks with microstructured surfaces. An experimental in vitro study

Authors’ affiliations: Rafael Arcesio Delgado-Ruız, Department of Prosthodontics, School of Dental Medicine, Stony Brook University, Stony Brook, NY, USA Gerardo Gomez Moreno, Antonio AguilarSalvatierra, Faculty of Medicine and Dentistry, Granada University, Granada, Spain Aleksa Markovic, School of Medicine and Dentistry, Belgrade University, Belgrade, Serbia Jose Eduardo Mate-S anchez, Jose Luis CalvoGuirado, Faculty of Medicine and Dentistry, Murcia University, Murcia, Spain

Key words: Alizarin, alkaline phosphatase, cell density, dental implants, femtosecond laser,

Corresponding author: Rafael Arcesio Delgado-Ruız Department of Prosthodontics School of Dental Medicine 1103 Westchester Hall School of Dental Medicine at Stony Brook University Stony Brook, NY 11794-8712, USA Tel.: +631 632 6913 Fax: +631 632 9443 e-mail: Rafael.Delgado-Ruiz@stonybrookmedicine. edu

human fetal osteoblast, microgrooved surfaces, Zirconia Abstract Objectives: To measure the lateral surface area of microgrooved zirconia implants, to evaluate the cell geometry and cell density of human fetal osteoblasts seeded on zirconia microgrooved implants, to describe the surface roughness and chemistry, and to evaluate the activity of human fetal osteoblasts seeded on zirconia microgrooved disks. Materials and methods: This experimental in vitro study used 62 zirconia implants and 130 zirconia disks. Two experimental groups were created for the implants: 31 non-microgrooved implants (Control) and 31 microgrooved implants (Test); two experimental groups were created for the disks: 65 non-microgrooved disks (Control) and 65 microgrooved disks (Test). The following evaluations of the implants were made: lateral surface area (LSA), cell morphology, and density of human fetal osteoblasts seeded on implant surfaces. On the disks, surface parameters (roughness and chemistry) and cell activity (alkaline phosphatase – ALP and alizarin red – ALZ) were evaluated at 7 and 15 days. Results: LSA was lower for control implants (62.8 mm) compared with test implants (128.74 mm) (P < 0.05). Cell bodies on control surfaces were flattened and disorganized, while in the test group, they were aligned inside the microgrooves. Control group cells showed few lamellipodia, which were attached mainly inside topographical accidents (surface cracks, valleys, and pits). Test group implants presented cells rich in lamellipodia prolongations, attached to the inner walls or to the borders of the microgrooves and in the flat areas between the microgrooves. Cell density was higher in the test group compared with controls (P < 0.05) Surface roughness and oxygen content increased in test disks samples compared with controls (P < 0.05). Carbon and aluminum were reduced in disks test samples compared with controls (P < 0.05), and ALP and ALZ levels were significantly increased on test surfaces (P < 0.05) at both study times. Conclusions: Within the limitations of this experimental study, it may be concluded that (i) Roughness is increased and chemical composition enhanced on the surface of zirconia implants with microgrooves. (ii) The LSA of microgrooved zirconia implants is greater and provides more available surface compared with implants of the same dimensions without microgrooves. (iii) Microgrooves on zirconia implants modify the morphology and guide the size and alignment of human fetal osteoblasts. (iv) Zirconia surfaces with microgrooves of 30 lm width and 70 lm separation between grooves enhance ALP and ALZ expression by human fetal osteoblasts.

Date: Accepted 8 February 2015 To cite this article: Delgado-Ruız RA, Gomez Moreno G, Aguilar-Salvatierra A, Markovic A, Mate-Sanchez JE, Calvo-Guirado JL. Human fetal osteoblast behavior on zirconia dental implants and zirconia disks with microstructured surfaces. An experimental in vitro study. Clin. Oral Impl. Res. 00, 2015, 1–10 doi: 10.1111/clr.12585

Compared with other dental ceramics with implant applications, yttria partially stabilized tetragonal zirconia offers several advantages due to zirconia’s higher resistance to fracture and flexural strength (Piconi & Maccauro 1999; Albrektsson et al. 2008). Based on histological examination, zirconia is well tolerated by both connective tissue and bone (Christel 1989; Ichikawa et al. 1992; Covacci et al. 1999; Senn-

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

erby et al. 2005), and animal studies have observed that the osseointegration of zirconia implants is similar to that of titanium (Albrektsson et al. 1986, 2008; Akagawa et al. 1993; Scarano et al. 2003; Langhoff et al. 2008; Kohal et al. 2009). To improve the biocompatibility and mechanical performance of zirconia implants, different surface treatments have been assayed

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Delgado-Ruız et al  Cell behavior on zirconia implants

that include the following: magnesium (Mg) ion implantation (Liang et al. 2007); the aggregation of hydroxyapatite (Rocchietta et al. 2009) or calcium phosphate (CaP) nanolayers (Lee et al. 2009); ultra violet (UV) radiation to increase the hydrophilic properties of the zirconia surfaces (Att et al. 2009); sandblasting with aluminum oxide particles (Gahlert et al. 2007; Gahlert et al. 2008); and simple etching with hydrochloric or hydrofluoric acids (Schliephake et al. 2010). Previous studies have demonstrated that the increased surface roughness of titanium disks created by sandblasting with different sized aluminum oxide particles (Al2O3) influences the growth and metabolic activity of human osteoblasts, inducing more pronounced cell proliferation and differentiation compared with smooth surfaces. (Guizzardi et al. 2004). However, on zirconia surfaces, osteoblast response to surface roughness modifications is contradictory. Singh et al. (2012) found that by increasing nanoroughness, MG-63 cell attachment decreased, while the division and proliferation of attached cells were faster. On the other hand, Ito et al. (2013) demonstrated that increased micro- and nanotopographies in TZP surface treatments enhanced adhesion, proliferation, and differentiation of MC3T3-E1 cells. In general, when smooth implant surfaces are modified and roughened, the total surface area available increases, facilitating cellular phenomena that may enhance cell–material interactions and cellular activity (Ellingsen et al. 2006). In addition to roughness, isotropic (organized) or anisotropic (disorganized) surface organization stimulates cell response as demonstrated by Rajnicek et al. (1997), who found that substrate topography of microgrooved quartz (a series of repeating parallel microgrooves created by electron beam lithography) was a potent morphogenetic factor for developing CNS neurons and regulating neuronal polarity. Cells react to substrate topography by changing cytoskeletal organization, modifying cell adhesion, and cell-tocell interactions (Boyan et al. 1996). Recently, our research team has developed a technique for microstructuring zirconia dental implants by femtosecond laser ablation, which creates an isotropic pattern of microgrooves on the implant surface (Delgado Ruiz et al. 2010). Tests of these surfaces have found increased surface roughness, decreased contaminants, and decreased monoclinic phase on the femtosecond laser microgrooved surfaces. Animal studies have found that zirconia implants with microgrooved surfaces inserted

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in dog mandibles and immediately loaded preserve the thickness of soft tissues and achieve higher bone-to-implant contact (BIC) and bone density (Calvo-Guirado et al. 2013; Delgado-Ruiz et al. 2014a). Compared with titanium implants under immediate loading, the same implants were found to achieve similar BIC and crestal bone levels (CBL) (Calvo-Guirado et al. 2013). Furthermore, several studies have demonstrated that collagen fiber orientation surrounding zirconia implants with microgrooved surfaces is mainly perpendicular to the implant surface (Delgado-Ruiz et al. 2014b); it has also been shown that microgrooves on zirconia implants will host bioactive molecules, enhancing the initial stages of bone formation in rabbits (CalvoGuirado et al. 2014). The creation of microgrooves on zirconia implant surfaces changes the microstructure of the implant surface; surface area (or lateral area) is a factor that must be determined to evaluate bone formation objectively (Schicho et al. 2007). For this reason, mathematical formulae for calculating lateral surface area or computerized microtomography (micro CT) may provide valuable information for implant surface evaluation (Schicho et al. 2007). There is a lack of information on just how the presence of microgrooves on zirconia implants affects lateral surface area and cell behavior and in particular, the response of human fetal osteoblasts (HFOB) seeded on zirconia implants with microstructured surfaces that could explain the enhanced clinical behavior of these surfaces obtained in experimental animal studies. Therefore, the aims of this study were as follows:

tion (CPA) were used for microstructuring, using the technique reported by Delgado Ruiz et al. (2010). Laser pulses travel through air to a focusing system, which consists of an achromatic doublet lens and an off-axis imaging system (lens, beam splitters and CCD Philips Lucaâ cameras) for the visualization of the processing area, which facilitates implant positioning and beam focusing. Implants or disks were placed on a motorized platform with three-axis motion, X, Y, and Z, controlled by Micos ES100â software (Nanotec Electronic GMBH & Co., Munich, Germany), so that laser pulses impinged perpendicularly onto the object. For implant processing, an OWISâ (Nanotec Electronic GMBH & Co. Munich, Germany) rotating motorized base turned the base at speeds varying between 0° and 30°/s, combining rotation and movement in the y-axis, allowing the whole periphery of the implant to be modified without altering focusing conditions. To process the disks, the rotation movement was stopped maintaining a translation movement in the x-axis alone for the creation of microgrooves, and displacements in the y-axis to ensure the creation of micro grooves of 30 lm width and 70 lm pitch without altering the focusing conditions. In this way, the same texture was reproduced all over the implant surface. Two parallel in vitro experiments were performed as follows: Experiment 1 (zirconia implants)

Materials and methods

62 White SKY (Bredent medicalâ GMBH & Co. KG, Senden, Germany) implants of 4 mm diameter and 10 mm length were used. The implants were manufactured using high-pressure sintering of tetragonal zirconium oxide polycrystals stabilized with 3% molar ratio yttrium oxide at temperatures in the range of 1173–2370 °C; the implant surfaces were modified by sandblasting with aluminum oxide particles. The implants were divided into two groups: 31 sandblasted zirconia implants (Control samples); 31 sandblasted and microgrooved zirconia implants (Test samples). The following variables were evaluated: lateral surface area and cell morphology and cell density of human fetal osteoblasts seeded on the implant surfaces at 7 and 15 days.

Laser processing

Lateral surface area

A commercial Ti:Sapphire laser (Tsunami; Spectra Physics, Berlin, Germany) and a regenerative amplifier system (Spitfire; Spectra Physics) based on chirped pulse amplifica-

To calculate the lateral surface area, 10 implants (5 per group) were embedded in epoxy resin and sectioned through the longitudinal axis of the implant to obtain two

To describe the lateral surface area of microgrooved zirconia implants. To evaluate the cell morphology and cell density of human fetal osteoblasts seeded on zirconia microgrooved implants. To analyze the surface roughness and chemistry of zirconia microgrooved disks. To evaluate the cell activity of human fetal osteoblasts seeded on zirconia microgrooved disks.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Delgado-Ruız et al  Cell behavior on zirconia implants

halves using a microtome under continuous water-cooling. The specimens obtained were ground using SiC paper with up to 1200 grit size, polished with 3 lm diamond paste and ultrasonically cleaned in distilled water. Then the samples were degreased in ethanol solutions and desiccated with acetone and finally sputtered with gold palladium in the SCD 040 (Balzers Union, Wallruf, Germany). The observation parameters used were as follows: a focal distance of 32 mm, 20 Kv, and 150 9 magnification. Image analysis was performed on a Macintosh computer using Image J software (NIH Image Program U.S. National Institutes of Health); the linear profile of four implants per group was measured, using the following mathematical formula: Lateral Surface Area ¼ LSA : p  r  g

p = 3.1416; r = Radius of the cylinder base in mm; g = Length in mm of the implant lateral wall profile, measured from implant shoulder to apex in the control group (the profile of the walls including threads and valleys represented by the letter a); in the test group this was equivalent to the sum of the letters a + b1 + b2 + c measured from the implant shoulder to the apex (Fig. 1). Cell culture and seeding process

The protocol for this experimental in vitro study was approved by the ethics committee of Murcia University (September/2013) following local and European directives of November 24, 1986 (86/609). Human Fetal Osteoblasts hFOB 1.19 (ATCC; American Culture Collection, Manassas, VA, USA) were used.

Culture medium

The cells were grown in a basic culture medium composed of 1:1 DMEM (Dulbecco’s modified Eagle’s medium) without phenol red, plus Ham’s F12 nutrient mixture (Sigma Chemical Company, St Louis, MO, USA), supplemented with 10% (v/v) calf fetal serum, 100 IU/ml penicillin, 100 lg/ml streptomycin, 1.5% (v/v) HEPES buffer, and 1% (v/v) L-glutamine (all from BioWhittakerâ; Lonza, Walkersville, MD, USA), and maintained at 35 °C in a humidified atmosphere containing 5% CO2. The medium was changed twice a week. Cell seeding

Fifty-two zirconia implants (26 per group) were cleaned and degreased with ethanol at different concentrations and sterilized with dry heat at 134 °C for 15 min. The implants were inserted horizontally into a layer of 0.8% agarose with 2 mm thickness (to stabilize them) on the bottom of 24-well culture flasks (BD Falcon, North Ryde, NSW, Australia). The upper surface of the implants opposite to the agarose layer was identified with a permanent marker. The cells were seeded at a density of 3 9 104 cells per implant using a micropipette. Cell morphology and cell density

Cell morphology and density were evaluated on the implant surfaces. Fifty-two implants were used for the analysis. Thirteen implants per group were removed after 7 (26 implants) and 15 days (26 implants) incubation and washed three times with PBS to remove nonadherent cells and fixed using 4% glutaraldehyde in DPBS for at least 48 h. Afterward, the samples were immersed for 24 h in water–ethanol solution with increasing ethanol content (until absolute ethanol), desiccated with ace-

Fig. 1. Lateral surface area evaluation. Control group profile analyzed along the entire surface, from the implant platform to a point at the center of the implant apex. Test group received the same evaluation, including measurement of microgroove dimensions.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

tone, and finally sputtered with gold palladium in an SCD 040 (Balzers Union) for cell morphology and cell density scanning electron microscope (SEM) observation. The observation parameters were a focal distance of 32 mm, 20 Kv and 150 9 , 450 9 , and 1500 9 magnification. Cell morphology analysis was performed by description of the cell bodies and cell alignment on the implant surfaces at both study times. Cell surface density (Cells per unit area/ 300 lm2) was calculated for each implant in ten micro areas of 300 9 300 lm inside a rectangle (2 mm width 9 8 mm length) located at the top of the implant surface, using a Macintosh computer equipped with image analysis software (Image J, NIH Image Program U.S. National Institutes of Health). After performing threshold contrast with respect to the substrate, cells were identified and counted digitally inside each micro area; surface density values per implant were calculated. The total surface density was evaluated for each group at 7 and 15 days and expressed as percentages, means, and standard deviations. Experiment 2 (zirconia disks)

130 zirconia disks of 6 mm diameter and 2 mm height were divided into two groups: Control: 65 disks treated by sandblasting only; Test: 65 disks sandblasted and treated with femtosecond laser pulses to create micro grooves of 30 lm width, 70 lm pitch. The following variables were evaluated: surface roughness, chemical elements, and the cell activity markers, alkaline phosphatase (ALP) and alizarin red (AL). Surface roughness evaluation

The surface roughness of 10 zirconia disks was evaluated by optical interferometric profilometry with a Veeko NT 1100VR interferometric microscope (Wyco Systems, New York, USA). Ten sampling micro areas of 227.2 lm 9 298.7 lm were defined; a Gaussian filter of 100 lm 9 100 lm was used; and measures were performed with a lateral resolution of 0.7 mm, vertical resolution of 0.5 lm, and 20.7 9 magnification in VSI mode. The arithmetic mean of the absolute values of the surface height within the sampling area (Sa) and the developed surface area ratio (Sdr) were calculated for each sample. Afterward, mean values and standard deviations were calculated for both groups. SEM: quantification of elements

Element analysis was carried out by energy dispersive X-ray spectroscopy (EDX) analysis

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Delgado-Ruız et al  Cell behavior on zirconia implants

(a)

(c)

(b)

(d)

Fig. 2. The different profiles under evaluation. (a) profile of implant thread; (b) profile of inter-thread area; (c) measurement of microgroove pitch area; (d) microgroove profile and walls.

Table 1. Lateral surface area expressed as mm, from the measures of the line profile by implant group (Mean average  SD). The LSA was clearly higher for the test group compared to controls. p = 3.1416; r = radius of the cylinder; g = Length. Mann–Whitney U-test showed that the twotailed P value is 0.0079, considered very significant

Sample

LSAmm (Lateral surface area) LSA= p *r * g Mean  SD

Median

P-value

Control (n = 5) Test (n = 5)

63.06  0.88 mm 128.752  0.29 mm

62.900 128.74

NS 0.01

using an OXFORD INCA 300 system (Oxford Instruments; Abingdon, Oxfordshire, UK). The same zirconia disks used for the analysis of the roughness were analyzed (five specimens per group) for chemical characterization of elements present at the surface. Specimens were degreased in ethanol solutions and desiccated with acetone; they were then were coated with conductive carbon in a sputter-coating unit (SCD 004 Sputter-Coater with OCD 30 attachment, Bal-Tec, Vaduz, Liechtenstein). Elemental analysis was carried out in ten micro areas of 30 lm 9 30 lm per disk.

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The observation parameters used were a focal distance of 32 mm, 20 kV, and 450 9 magnification. Values were expressed as percentages. Using the same culture medium as in Experiment 1, human fetal osteoblasts were cultured and seeded on the zirconia disks for the evaluation of ALP and AL activity as follows: Cell activity markers

The 120 remaining zirconia disks were cleaned, degreased, and sterilized using the same procedure as previously described. The

zirconia disks were placed directly at the bottom of 24-well culture flasks, and human fetal osteoblasts were seeded at a density of 3 9 104 cells/disks. After 7 and 15 days, cell activity was evaluated by ALP and ALZ assays. ALP activity

ALP activity was evaluated on 60 zirconia disks, 15 disks per group (test and control) at 7 and 15 days. The disks were removed from the plates and the cells were washed twice with ice-cold PBS and scraped in a 0.2% aqueous solution of surfactant (NonidetP40; Roche Diagnostics, Meylan, France). The cell suspension was sonicated on ice for 30 s (XL 2000; Misonix Inc., Farmingdale, NY, USA) and centrifuged for 5 min at 4 °C. ALP activity was measured by colorimetry at pH 10.3 in 0.1 M 2-amino-2-methyl-1-propanol containing 1 mM MgCl2 and an equal amount of para-nitrophenyl phosphate (10 mM). Optical density was read at 405 nm using an lQuant spectrophotometer. Measurements

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Delgado-Ruız et al  Cell behavior on zirconia implants

(a)

(b)

(c)

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(e)

(f)

Fig. 3. SEM evaluation of cell morphology in control and test groups at 7 days. (a) a cell in the base of an implant thread in the control group; (b) close-up view of a couple of cell bodies close to the base of the thread; (c) a cell body with very short cell lamellipodia located in a crack of a control group surface (shown at high magnification); (d) cell at the border of a microgroove; (e) lamellipodia extend inside microgrooves, bridging microgroove borders; (f) cell body aligned in the direction of the microgroove.

were compared with p-nitrophenol standards and normalized using the total protein amounts (Protein assay; Bio-Rad, Tokyo, Japan). ALZ activity

(a)

(c)

(b)

(d)

Fig. 4. Cells on test surface at 7 days (high magnification). (a) lateral view of multiple cells firmly adhered to the inner surface of the microgrooves; (b) cell body alignment at the base of the microgrooves; (c) lamellipodia network at walls and base of the microgrooves; (d) inner wall of a microgroove showing filopodia connected to the nanorough texture of the microgroove walls.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

ALZ activity was evaluated on the remaining 60 zirconia disks, measured by the mineralization staining method. After 7 and 15 days of culture, the culture medium was removed, cells were washed twice with PBS and fixed in 4% paraformaldehyde in 0.1 M PBS for 20 min at room temperature and then rinsed once with deionized water. After fixation, the cultures were stained with 2% solution of Alizarin Red S in 0.1M PBS for 5 min at room temperature. Excess dye was washed off with deionized water. Samples were visualized under a microscope (Nikon SMZ1000; Nikon, Tokyo, Japan). The area covered with a red stain, representing mineralized bone-like nodules, was then measured with image analysis software (ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ ij/). The total surface areas covered by stained nodules were measured and expressed as percentages.

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Delgado-Ruız et al  Cell behavior on zirconia implants

(a)

(b)

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(f)

Fig. 5. Comparison of control and test groups at 15 days. (a, b, and c) cells on control group surface. The cells established intercellular contacts and formed layers; contact between the cell and the surface occur mainly in topographic accidents (high magnification). (d–f) cells fill the microgroove completely; cells also form in the pitch areas.

Table 2. Cell density. Data expressed as mean percentage of cells  SD at 7 and 15 days Cell density

1 week cells %  SD

Control Test

23  2.41 37  1.78*

Median

P-value 1 week

2 week cells %  SD

Median

P-value 2 weeks

22.900 37.206

0.062 0.002

35  1.61 64  1.92*

35.204 64.948

NS 0.01

The test group showed significant more cell density compared to controls *P < 0.05.

Statistical analysis

The t-test was applied for the comparison of the surface roughness and chemical composition of both groups. Mann–Whitney U-test for independent groups was performed to confirm the normal distribution of the samples. For the analysis of the cell activity (ALP and ALZ) regarding the effects of two different surfaces at two periods of time, a two-way ANOVA test with Kruskal–Wallis post-test was performed. The level of significance was set at P < 0.05.

Results Experiment 1 Implant lateral surface area (LSA)

A single microgroove profile had a total measurement of 105  7 lm, this multiplied by the number of grooves resulted in increased g values for microgrooved surfaces,

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showing increased LSA values for test samples. LSA was higher in the test group with a mean difference of 65.94  0.20 mm, increasing LSA twofold in comparison with control samples (Fig. 2 & Table 1). Cell morphology

with lamellipodia directed toward the inner surface and connected to the base and walls of the microgrooves. Typically, in the flat surfaces between the micro grooves, the cells grew with the same pattern as on control surfaces (Fig. 3d–f). Detailed observation at high magnification showed cell bodies deposited inside the microgrooves (Fig. 4a and b), a network of lamellipodia was observed on the inner surfaces of the microgrooves (Fig. 4c); cell adhesion was supported by filopodia extensions retained in the nanometric structures of the microgroove walls (Fig. 4d). Second week

First week In the control group, cell bodies were flattened, with a slight tendency to growth in areas of surface topographical changes such as fissures or surface cracks and in the base of the threads (Fig. 3a and b). Lamellipodia were observed extending from the cell bodies in all directions (Fig. 3c). In the test group, cells were observed inside the microgrooves, aligned along the groove axis; lamellipodia extensions were observed directed from the cell body toward the microgroove walls. In addition, cells were observed at the borders of the microgrooves

In the control group, cells established intercellular contacts (Fig. 5a–c) and formed monolayers. In the test group, cells completely filled the microgrooves (Fig. 5d); cell geometry was polygonal with shortened lamellipodia (Fig. 5e), and multiple cell contacts were observed. Fixation was established with the walls and base of the microgrooves; cells were also observed in the areas between the microgrooves (Fig. 5f). Cell density

After 7 days, cell density was significantly higher in the test group compared with the

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Delgado-Ruız et al  Cell behavior on zirconia implants

(a)

significantly higher compared with the control group (P < 0.05) (Table 5 & Fig. 8). ALZ percentages at 15 days revealed that the test group increased the activity threefold compared with the control group (P < 0.05) (Table 6 & Fig. 9).

(c)

Discussion

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(d)

Fig. 6. Images under interferometric and reflection microscope. (a) control group surface under reflection microscope; (b) control surface under optical interferometric microscope; (c) test group surface under reflection microscope, displaying microgrooves microfeatures; (d) test group surface under interferometric microscope

Table 3. Surface roughness parameters. Sa and Sdr expressed as lm (Mean  SD). Control (sandblasted zirconia). Test (sandblasted + laser micro grooved zirconia). Roughness values were higher for test group Sample

Sa (lm)

Sdr (lm)

P-value

Control (n = 5) Test (n = 5)

2.73  0.16 9.1  0.52

2.81  0.13 9.3  0.47

NS 0.01

Table 4. Elements present in the surface expressed as percentages (Mean  SD). Traces of other elements usually present in zirconia samples as Hf were not detected. C and Al were predominant in Controls; meanwhile, O and Zr were present in test groups EDX surface analysis

C%

Al %

O%

Zr %

Control (n = 5) Test (n = 5)

19.7  0.8* 0.3  0.12

4.3  0.9* 0.18  0.1

12.6  0.5 23.1  0.12*

60.2  0.7 76.3  0.2*

*Higher values for the evaluated chemical elements. Significance was set as P < 0.05.

control group (P < 0.05). After 15 days, this relation was maintained with greater cell density in the test group (P < 0.05). Cell density increased between the 7-day and 15-day study times in both groups but this change was greater for test samples (33.6%) compared with control samples (12.6%) Table 2.

SEM: quantification of elements

Experiment 2

Table 4 shows the relative abundance of the main components of zirconia together with the percentages of contaminant elements (carbon and aluminum) for both groups. Test surfaces exhibited higher proportions of zirconium and oxygen than control surfaces; control surfaces showed increased carbon and aluminum content.

Surface roughness

Cell activity markers

All statistical roughness parameters indicated a noticeable increase in surface roughness as a result of laser modification. Surface roughness increased significantly in the test group compared with the control group (Fig. 6 & Table 3).

Although both groups showed evidence of ALP and ALZ activity, greater ALP and ALZ activity was detected at day 15 compared with day 7 (Fig. 7). Data analysis revealed that ALP values in the test group at both study times were

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

The purpose of the present study was to evaluate how modification of zirconia implant surfaces with microgrooves altered lateral surface area (LSA), cell geometry, and cell density compared with non-microgrooved zirconia implants. The study also evaluated how this modification influenced surface roughness, chemical composition, and the cell activity of human fetal osteoblasts cultured on zirconia disks with microgrooves. For the evaluation of LSA (circumference x height), implants were sectioned axially, to allow a linear evaluation along the entire implant profile. Those implants with microgrooves presented higher values, which resulted in higher LSA values compared with control implants. This line represents implant height, but the creation of microgrooves produces a longer linear contour making more surface available for cell and bone interactions. As far as the authors are aware, this is the first study to describe the LSA of zirconia implants with microgrooves in relation to cell density. LSA in the test group was found to be twice that of control samples. In this way, the LSA of any implant treated to create microgrooves (of the same dimensions used in this experiment) could be doubled. The surfaces of short implants could be modified by the creation of microgrooves (30 lm width and 70 lm pitch), thus potentially increasing the LSA available for cell adhesion, and increased cell density, which in turn could produce greater bone-to-implant contact (BIC). This could be potentially useful in areas with reduced height availability or with low bone density. The results of the present study demonstrated that osteoblast adhesion occurred first in the microgrooves and later on flat surfaces. Moreover, cells migrated inside the microgrooves and acquired the geometry and alignment of the microgrooves after 15 days, while on control surfaces, cells had a tendency to adhere to accidental topographic surface features such as pits, cracks, or the base of the threads.

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Delgado-Ruız et al  Cell behavior on zirconia implants

(a)

(c)

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Fig. 7. Optical microscope images of cells under ALP and ALZ observation. Image (a) and (b) green stain indicates ALP (at 7 days); pink indicates ALZ (at 7 days). (c) and (d) increased ALP and ALZ activity at 15 days.

Table 5. Cell activity for ALP in lg. Variations recorded for both groups at 7 and 15 days Group

ALP 7 days control

ALP 15 days control

ALP 7 days test

ALP 15 days test

Mean Standard deviation (SD) Sample size Standard error of mean (SEM) Lower 95% conf. limit Upper 95% conf. limit Median P-value

8.7 lg 0.2500 15 0.064550 8.5615 8.8385 8.612 NS

27.7 lg 0.7600 15 0.19623 27.797 28.121 27.954 NS

14.32*lg 0.1800 15 0.0464 14.220 14.420 14.395 0.023

63.6*lg 0.8100 15 0.20914 63.151 64.049 63.325 0.001

The test group showed significant more cell activity compared to controls in both periods of time * P < 0.05.

Fig. 8. ALP activity at 7 and 15 days. Increased ALP activity at 15 days, greater in test group.

This is in agreement with previous studies in which 30 lm microgrooves guided osteoblast cellular growth optimally, and served as

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reservoirs for the cell bodies (Marotti et al. 1975; Zambonin Zallone 1977; Pucket et al. 2008). It was decided to study cell morphology and density on implant surfaces because cells cultured in 2D models do not provide an accurate representation of cells in situ, given that native three-dimensional conditions act as cues for cell behavior (Chang et al. 1999; Li & Liu 1999; Lumbikanonda & Sammons 2001). This approach allowed the researchers to observe more accurately the effects of the microgrooved surface on the cell morphology. Cell morphology differed between control surfaces (flat, irregular, and with short lamellipodia) and test surfaces (elongated, organized, and with multiple adhesion foci). Various factors in the cellular microenvironment give signals to cells, including interaction with neighboring cells (Owen &

Schoicet 2010) and surface roughness. When micro- and nanoroughness were increased (test group), osteoblast behavior was enhanced in terms of both morphology and adhesion. Increased cell density was observed in the test group compared with the control group. It may be hypothesized that the cells were attracted to the interior of the microgrooves by the presence of nanoroughness, which is easily recognized by the filopodia. However, it could be that the microgrooves served as deposits for the proteins present in the culture medium, which adsorbed to the implant surface enhancing the establishment of cell contact and cell adhesion (Nune et al. 2014). Another explanation may be that the additional space created for cells on the walls and the base of the microgrooves ensured more area for adhesion. These results are in agreement with results obtained by Lee et al. (2010) in a study using titanium surfaces; the authors found that combined submicron- and microtopography with relevant microdimensions and structures enhanced surface hydrophilicity, which acted as the essential factor influencing osteoblast maturation on microgrooved titanium substrata. The roughness values and chemical composition reported in the present work are in agreement with previous data obtained by Delgado Ruiz et al. (2010), who analyzed zirconia implant surfaces modified by femtosecond laser. The technique applied to flat surfaces (disks) or to curved surfaces (implants) was shown to be reproducible with the resulting surfaces containing minimal amounts of surface contaminants. This is in agreement with the results of Massaro et al. (2002), who demonstrated that laser treatment of surfaces reduced the presence of contaminants, which could affect osseointegration levels whenever these elements remain on the surfaces of implant materials. As for cell activity, the ALP values obtained in the present study were seen to increase in the test group, a finding that agrees with Marinuci et al. (2006), who found that microroughness values of over 3 lm increased osteoblast metabolic activity. Given that the present test surfaces presented roughness values of over 8 lm, increased osteoblast metabolic activity was to be expected. Ito et al. (2013) found that ALP activity increased on zirconia implants with acidetched and sandblasted surfaces, because these modifications created micro- and

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Delgado-Ruız et al  Cell behavior on zirconia implants

Table 6. Cell activity for ALZ. Variations recorded for both groups at 7 and 15 days Group

ALZ 7 days control

ALZ 15 days control

ALZ 7 days test

ALZ 15 days test

Mean Standard deviation (SD) Sample size Standard error of mean (SEM) Lower 95% conf. limit Upper 95% conf. limit Median P-value

17.65% 1.6400 15 0.42345 16.742 18.558 17.242 NS

23.61% 1.5200 15 0.39246 22.768 24.452 23.932 NS

24.83 2.1300 15 0.54996 23.650 26.010 25.126 NS

78.22* 2.3500 15 0.60677 76.918 79.522 78.353 0.033

The test group showed significant more cell activity compared to controls at 15 days *P < 0.05.

Fig. 9. ALZ activity at 7 and 15 days. At 7 days, no differences were observed between groups; an increase in ALZ activity was observed in the test group at 15 days.

nanotopographies that enhanced cell proliferation and differentiation. Apparently, integrins a2b1 are responsible for focal adhesions, which occur during cell adhesion. If cell adhesion is fast, cell metabolism increases resulting in higher ALP expression (Wang et al. 2014). To explain this, we can refer to the study by Anselme et al. (2002), who compared different surface treatments on titanium implants, analysing the orientation and pro-

liferation of human osteoblasts on surfaces of varying microroughness. It was found that cultured human osteoblasts preferred surfaces with relatively high microroughness, which generated increased cell activity. Nadeem et al. 2013 investigated the effects of micropatterned surfaces on cell responses. These authors created different sized microgrooves (100 lm/50 lm, and 10 lm/10 lm groove/pitch) on ceramic disks, which were then seeded with human mesenchymal stem cells. More osteoid matrix nodules shown by osteopontin and osteocalcin were observed on the surfaces with larger grooves after 21 days cell culture, indicating a higher level of osteogenicity. Another outcome resulting from micropatterned surfaces is an increase in extracellular matrix (ECM). Matsuzaka et al. (2003) demonstrated that microgrooved surfaces increased osteoblast activity by inducing greater extracellular matrix formation, responsible for increased mineralization. Recently, Hayashi et al. (2014) have shown that hydrocarbons present on titanium surfaces attenuated osteoblastic activity measured by ALP and ALZ. These authors used titanium surfaces modified by

the presence of different carbon/titanium (C/ Ti) ratios (0.3, 0.7, and 1.0). Subsequently, MC3T3-E1 cells were seeded onto the surfaces, with the 0.3 C/Ti ratio producing the best response. This is in agreement with the present results, whereby the test group showed greater ALP and ALZ activity. The presence of carbon (C) was reduced after laser treatment in the test group, so the presence of higher levels of C in the control group may explain the lower cell density values and cell activity observed. The increased roughness, the isotropy of the surface, and the reduction of carbon content might explain the increased cell density and activity of human fetal osteoblasts cultured on microgrooved zirconia surfaces.

Conclusions Within the limitations of this experimental study, it may be concluded that: The LSA of microgrooved zirconia implants is greater and provides more available surface compared with implants of the same dimensions without microgrooves. Microgrooves on zirconia implants modify the morphology and guide the size and alignment of human fetal osteoblasts. Roughness is increased and chemical composition enhanced on the surface of zirconia disks with microgrooves. Zirconia disks surfaces with microgrooves of 30 lm width and 70 lm separation between grooves enhance ALP and ALZ expression by human fetal osteoblasts.

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Human fetal osteoblast behavior on zirconia dental implants and zirconia disks with microstructured surfaces. An experimental in vitro study.

To measure the lateral surface area of microgrooved zirconia implants, to evaluate the cell geometry and cell density of human fetal osteoblasts seede...
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