Materials 2015, 8, 6018-6028; doi:10.3390/ma8095288
OPEN ACCESS
materials ISSN 1996-1944 www.mdpi.com/journal/materials Article
Characterization of Human Gingival Fibroblasts on Zirconia Surfaces Containing Niobium Oxide Young-Dan Cho 1,: , Ji-Cheol Shin 2,: , Hyung-In Yoon 2 , Young Ku 1 , Hyun-Mo Ryoo 3 , Dae-Joon Kim 4 , Do-Gyoon Kim 5 and Jung-Suk Han 2, * 1
Department of Periodontology, School of Dentistry, Seoul National University, Seoul 110-749, Korea; E-Mails:
[email protected] (Y.-D.C.);
[email protected] (Y.K.) 2 Department of Prosthodontics, School of Dentistry, Seoul National University, Seoul 110-749, Korea; E-Mails:
[email protected] (J.-C.S.);
[email protected] (H.-I.Y.) 3 Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, Seoul National University, Seoul 110-749, Korea; E-Mail:
[email protected] 4 Department of Advanced Materials Engineering, Sejong University, Seoul 143-747, Korea; E-Mail:
[email protected] 5 Division of Orthodontics, College of Dentistry, Ohio State University, Columbus, OH 43210, USA; E-Mail:
[email protected] :
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +82-2-2072-3711; Fax: +82-2-2072-3860. Academic Editor: Dusan Losic Received: 11 May 2015 / Accepted: 2 September 2015 / Published: 10 September 2015
Abstract: It was indicated that tetragonal zirconia polycrystal (TZP) containing yttria (Y2 O3 ) and niobium oxide (Nb2 O5 ) ((Y,Nb)-TZP) could be an adequate dental material to be used at esthetically important sites. The (Y,Nb)-TZP was also proved to possess its osteogenic potential comparable with those conventional dental implant material, titanium (Ti). The objective of the current study was to characterize cellular response of human gingival fibroblasts (HGFs) to smooth and rough surfaces of the (Y,Nb)-TZP disc, which were obtained by polishing and sandblasting, respectively. Various microscopic, biochemical, and molecular techniques were used to investigate the disc surfaces and cellular responses for the experimental (Y,Nb)-TZP and the comparing Ti groups. Sandblasted rough (Y,Nb)-TZP (Zir-R) discs had the highest surface roughness. HGFs cultured on polished (Y,Nb)-TZP (Zir) showed a rounded cell morphology and light spreading at 6 h
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after seeding and its proliferation rate significantly increased during seven days of culture compared to other surfaces. The mRNA expressions of type I collagen, integrin α2 and β1 were significantly stimulated for the Zir group at 24 h after seeding. The current findings, combined with the previous results, indicate that (Y,Nb)-TZP provides appropriate surface condition for osseointegration at the fixture level and for peri-implant mucosal sealing at the abutment level producing a suitable candidate for dental implantation with an expected favorable clinical outcome. Keywords: dental implant; zirconia; niobium; human gingival fibroblasts (HGFs); mucosal sealing
1. Introduction Implantology, a highly specialized field in dentistry, has rapidly developed the rehabilitation of dentition. Titanium (Ti) is a widely used dental implant material because of its excellent biocompatibility and strength [1]. However, its drawback of a visible metallic and grayish color in the esthetically important zone such as the anterior part of the oral cavity has necessitated developing new implant materials that have a similar color of tooth [2]. Zirconia has been introduced as an alternative material that satisfies the criteria of biocompatibility and esthetics, while the associated phenomenon of low strength by low temperature degradation (LTD) limited its clinical applications [3,4]. In a previous study, the LTD phenomenon in zirconia was substantially reduced by addition of niobium oxide (Nb2 O5 ) [5,6] or tantalum oxide (Ta2 O5 ) [7]. Our previous reports also proved that LTD reinforced tetragonal zirconia polycrystal (TZP) discs containing yttria (Y2 O3 ) and niobium oxide (Nb2 O5 ) ((Y,Nb)-TZP) have osteogenic potential similar to that of Ti [8], and that the new aerosol deposition technique for hydroxyapatite (HA) coating is significantly effective in enhancing the osteogenic potential [9]. Biocompatibility with soft tissue at the implant interface is also crucial for obtaining a successful implant system [10]. A sufficient gingival growth and mucosal sealing around the dental implant are essential to maintain the stable connection avoiding the risk of bacterial infections [11,12]. Periodontal soft tissue is mainly composed of periodontal ligament and gingival fibroblasts [13]. Gingival fibroblasts are involved in the maintenance and production of the gingival connective tissue [14]. Poorly formed gingival connective tissue around the implant allows ease of bacterial invasion that causes inflammation resulting in marginal bone loss [15]. Hence, the formation of a tight and firm mucosal sealing between the implant and the soft tissue interface is important for a long term clinical success of the implant system [16]. It has been reported that the property of collagen fibers around the implant neck area is similar in vivo regardless of the type of materials (Ti and Zir) [17]. In vitro observation showed the presence of integrin subunits in human gingival fibroblasts (HGFs) and the morphological alteration of HGFs due to the surface roughness of Ti [18]. Surface topography is an important factor in cell attachment, adhesion, proliferation, and differentiation [19], which also affects cell orientation and migration [20]. However, the association between functional cellular activity and the surface roughness of materials is
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still In particular, particular, opposite opposite results results have have been still controversial. controversial. In been reported reported about about whether whether increasing increasing cellular cellular proliferation dependent on a smooth surface of biomaterials [21,22] or or not [23,24]. [23,24]. Thus, Thus, the objective proliferation is dependent biomaterials [21,22] of this study was to characterize cellular response of HGFs to smooth and rough surfaces of the (Y,Nb)-TZP (Y,Nb)-TZP disc, disc,which whichwere wereobtained obtainedby bypolishing polishingand andsandblasting, sandblasting,respectively. respectively. 2. 2. Results Results and and Discussion Discussion 2.1. 2.1. Surface Surface Characterization Characterization of of Titanium Titanium and and Zirconia Zirconia Discs Discs Proper Proper adhesion adhesion of of gingival gingival fibroblasts fibroblasts to to the the implant implant surface surface is is of of importance importance in in obtaining obtaining successful dental implantation and osseointegration [25]. Hence, surface topography is an important successful dental implantation and osseointegration [25]. Hence, surface topography is an important modulator surface topography impacts cell cell adhesion, proliferation, and modulator of of fibroblast fibroblastadhesion adhesion[26]. [26].AsAs surface topography impacts adhesion, proliferation, differentiation [22], the current study examined the different characters Ti-machined and differentiation [22], the current study examined thesurface different surfacebetween characters between (Ti-M), Ti-anodizing (Y,Nb)-TZP (Zir)(Y,Nb)-TZP and sandblasted (Zir-R)(Y,Nb)-TZP groups The average Ti-machined (Ti-M),(Ti-R), Ti-anodizing (Ti-R), (Zir)(Y,Nb)-TZP and sandblasted (Zir-R) roughness (Raroughness ) and surface topography of the specimens analyzed by three-dimensional groups Thevalues average values (Ra) and surface topographywere of the specimens were analyzed by confocal laser microscopy as shown in Figureas1A. TheinRFigure ofThe Ti-M Ti-R were three-dimensional confocal(3D-CLM) laser microscopy (3D-CLM) shown 1A. Ra and values of Ti-M a values 0.281 ˘ 0.03 and ± 0.689 0.04 respectively, andrespectively, those of Zir and and those Zir-R of were ˘ 0.01were µm and Ti-R wereµm 0.281 0.03˘μm andµm, 0.689 ± 0.04 μm, Zir0.092 and Zir-R and ˘ μm 0.05and µm, respectively. Ra value The of Zir was lowest due lowest to applied fineapplied polishing 0.0920.739 ± 0.01 0.739 ± 0.05 μm,The respectively. Ra value of Zir was due to fine (p < 0.05). In order to increase the roughness to a similar level as that of titanium, sandblasting was polishing (p < 0.05). In order to increase the roughness to a similar level as that of titanium, performed with alumina particles. conditions usedThe for conditions sandblasting were µm aluminawere (Al250 O3 )μm at sandblasting was performed with The alumina particles. used for50 sandblasting 2alumina bar pressure (Al2O3for ) at12min. bar pressure for 1 min.
Figure 1. (A) Three-dimensional confocal laser microscopy (3D-CLM) of the examined Figure 1. (A) Three-dimensional confocal laser microscopy (3D-CLM) of the examined substrate surfaces showing the roughness, Ra . (B) Scanning electron microscopy (SEM) substrate surfaces showing the roughness, Ra. (B) Scanning electron microscopy (SEM) images at 1500ˆ magnification, and (C) atomic force microscopy (AFM) images. images at 1500× magnification, and (C) atomic force microscopy (AFM) images. Surface Surface morphology morphology was was observed observed by by using using scanning scanning electron electron microscopy microscopy (SEM) (SEM) (Figure (Figure 1B). 1B). Machined Machined Ti Ti (Ti-M) (Ti-M) showed showed regular regular scratches scratches caused caused by by mechanical mechanical machining machining operation, operation, while while the the
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surface of of anodized anodized Ti Ti (Ti-R) crater like like patterns owing to to the of surface (Ti-R) was was porous porous and and displayed displayed crater patterns owing the presence presence of crystalline structures structuresininthethe form of rutile anatase. Polished mirror-like (Zir) showed crystalline form of rutile andand anatase. Polished mirror-like zirconiazirconia (Zir) showed a smootha smooth fine dotted resulting the process sintering. However,after aftersandblasting sandblasting with with and fineand dotted patternpattern resulting from from the process of of sintering. However, alumina, Zir-R Zir-R exhibited in alumina, exhibited uneven uneven rough rough patterns. patterns. The The atomic atomic force force microscopy microscopy (AFM) (AFM) data data showed showed in Figure 1C 1C were were in in good good agreement agreement with with the the RRaa values values (Figure (Figure 1A). 1A).InIn respect respect of of surface surface chemistry, chemistry, Figure blasting with with Al Al22O O33 powder powder may may change change the the surface surface characteristics characteristics as as aa result result of of broken broken bonds bonds of of ZrO ZrO22 blasting comprising the the surface, Thus, the the adhesion adhesion is is comprising surface, leading leading to to surface surface energy energy higher higher than than as-sintered as-sintered one. one. Thus, prompted to to lower lower the Nevertheless, the the change change in in composition composition of of the the surface surface prompted the energy energy of of the the system. system. Nevertheless, due to to the the blasting blasting is is likely likely to to be be negligible negligible since since the the ceramic ceramic processing processing to to produce produce the the specimens specimens was was due ˝ including the C and including the ball ball milling, milling, homogeneous homogeneous heat heat treatment treatment atat1250 1250 °C and the the following following attrition attrition milling, milling, is known to provide uniform distribution of components so that the composition in bulk and surface is is known to provide uniform distribution of components so that the composition in bulk and surface is reasonably identical. identical. reasonably 2.2. Cell Cell Attachment Attachment and and Morphology Morphology 2.2. It is well established that smooth smooth Ti Ti surfaces surfaces favor favor fibroblast attachment, whereas rough Ti surfaces In order order to to investigate investigate this this phenomenon phenomenon between between (Y,Nb)-TZP (Y,Nb)-TZP and promote osseointegration [27,28]. In and CLM CLM observation observation was followed Ti, HGFs were seeded onto the discs and harvested at 6 and 24 h, and At the early cell culture stage (6 h), cells to examine cellular attachment and morphology (Figure 2). At were observed to be well attached and stretched on the Ti-M and Ti-R compared to that on Zir or Zir-R ofof cell morphology waswas somewhat similar between the (Figure 2A). After After 24 24hhofofcell cellseeding, seeding,the thestate state cell morphology somewhat similar between Ti and Zir, regardless of their surface roughness (Figure 2B). Cells theon Zirthe were to be widely the Ti and Zir, regardless of their surface roughness (Figure 2B). on Cells Zirobserved were observed to be spread and elongated with good attachment. However, those on those Zir-R on were narrower and less stretched widely spread and elongated with good attachment. However, Zir-R were narrower and less than Zir, probably weaker binding the surface. stretched than Zir, indicating probably indicating weakerwith binding with the surface.
Figure 2. Microscopic observation after human gingival fibroblast (HGF) cells were seeded Figure 2. Microscopic observation after human gingival fibroblast (HGF) cells were onto the discs at (A) 6 h and (B) 24 h. Original magnification is 300ˆ and scale bar = 50 µm. seeded onto the discs at (A) 6 h and (B) 24 h. Original magnification is 300× and scale bar = 50 μm.
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2.3. Cell Proliferation Cell proliferation was evaluated by the picogreen assay. For this purpose, the HGFs were seeded onto discs andproliferation cultured forwas one,evaluated four, and by seven (Figure 3). HGFs were observed proliferate in Cell the days picogreen assay. For this purpose, thetoHGFs were well seeded cell on cultured all types for of surfaces. on Zir significantly increased at Day 4 ontocultures discs and one, four,The andproliferation seven days rate (Figure 3). was HGFs were observed to proliferate (p < 0.05) proliferation wasofpromoted Day 7, indicating positive effect of surface roughness. well in celland cultures on all types surfaces.until The proliferation rate ona Zir was significantly increased at Day Generally, surfaces Ti-M or Zir) induced higher acell proliferation to the 4 (p < 0.05)the andsmooth proliferation was(i.e., promoted until Day 7, indicating positive effect of comparison surface roughness. rough surfaces (i.e., Ti-R or Zir-R). These results correlate those observed in case cell Generally, the smooth surfaces (i.e., Ti-M or Zir) induced higherwell cell with proliferation comparison to theofrough attachment and morphology, which results showedcorrelate a more well aligned spreaded pattern in of thecell Zir attachment and Ti-M surfaces (i.e., Ti-R or Zir-R). These withand those observed in case (Figure 2). and morphology, which showed a more aligned and spreaded pattern in the Zir and Ti-M (Figure 2).
Figure 3. Cell proliferation assay (picogreen assay) of MC3T3-E1 cells at Day 1, 4 and Figure 3. Cell proliferation assay (picogreen assay) of MC3T3-E1 cells at Day 1, 4 and 7 7 seeded on Ti- or Zr-discs. Data are expressed as the mean ˘standard deviation (SD) of seeded on Ti- or Zr-discs. Data are expressed as the mean ±standard deviation (SD) of three independent experiments. Significance was tested by one-way analysis of variance three independent experiments. Significance was tested by one-way analysis of variance (ANOVA) test (* p < 0.05). (ANOVA) test (* p < 0.05). 2.4. 2.4. Cell Cell Differentiation Differentiation Quantitative Quantitative real real time time polymerase polymerase chain chain reaction reaction (RT-PCR) (RT-PCR)was wasperformed performed to to evaluate evaluate the the mRNA mRNA expression level of collagen and integrin subunits. HGFs were seeded onto discs and cultured expression level of collagen and integrin subunits. HGFs were seeded onto discs and cultured for for 24, 24, 48 48 and and 72 72 hh to to analyze analyze mRNA mRNA expression expression levels levels of of Type TypeIIcollagen, collagen,Integrin Integrinα2, α2, Integrin Integrin β1 β1 (Figure (Figure 4). 4). Collagen I is mainly produced in the gingival fibroblasts, osteoblasts and periodontal ligament [29,30]. Collagen I is mainly produced in the gingival fibroblasts, osteoblasts and periodontal ligament [29,30]. It It is is an an important important factor factor of of gingival gingival connective connective tissue tissue and and contributes contributes to to rapid rapid periodontal periodontal tissue tissue regeneration In this this experiment, experiment, the the mRNA mRNA levels regeneration and and maintenance maintenance of of tissue tissue architecture architecture [31,32]. [31,32]. In levels of of Type Type II collagen collagen were were significantly significantly high high on on Zir Zir after after 24 24 and and 72 72 hh of of cell cell culture culture (p (p
