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1.  Current knowledge about the hydrophilic and nanostructured SLActive surface 
This review summarizes the present documentation for the SLActive surface, a hydrophilic and nanostructured surface produced by Straumann Company in Switzerland, and covers the results from 15 in vitro, 17 in vivo, and 16 clinical studies. The SLActive surface is a development of the large grit-blasted and acid-etched SLA surface, and is further processed to a high degree of hydrophilicity. In general, the in vitro and in vivo studies of the SLActive surface demonstrate a stronger cell and bone tissue response than for the predecessor, the SLA surface, produced by the same company. However, in most studies, this difference disappears after 6–8 weeks. In the clinical studies, a stronger bone response was reported for the SLActive surface during the early healing phase when compared with the SLA surface. However, the later biological response was quite similar for the two surfaces and both demonstrated very good clinical results.
doi:10.2147/CCIDEN.S15949
PMCID: PMC3652359  PMID: 23674916
SLActive; surface; in vitro; in vivo; clinical results
2.  Requirement for Both Micron and Submicron Scale Structure for Synergistic Responses of Osteoblasts to Substrate Surface Energy and Topography 
Biomaterials  2007;28(18):2821-2829.
Objective
Surface roughness and surface free energy are two important factors that regulate cell responses to biomaterials. Previous studies established that titanium substrates with micron-scale and submicron scale topographies promote osteoblast differentiation and osteogenic local factor production and that there is a synergistic response to microrough Ti surfaces that have retained their high surface energy via processing that limits hydrocarbon contamination. This study tested the hypothesis that the synergistic response of osteoblasts to these modified surfaces depends on both surface microstructure and surface energy.
Methods
Ti disks were manufactured to present three different surface structures: smooth pretreatment surfaces (PT) with Ra of 0.2 µm; acid-etched surfaces (A) with a submicron roughness Ra of 0.83 µm; and sandblasted/acid-etched surfaces (SLA) with Ra of 3–4 µm. Modified acid-etched (modA) and modified sandblasted/acid-etched (modSLA) titanium substrates, which have low contamination and present a hydroxylated/hydrated surface layer to retain high surface energy, were compared with regular low surface energy A and SLA surfaces. Human osteoblast-like MG63 cells were cultured on these substrates and their responses, including cell shape, growth, differentiation (alkaline phosphatase, osteocalcin), and local factor production (TGF-β1, PGE2, osteoprotegerin [OPG]) were analyzed (N=6 per variable). Data were normalized to cell number.
Results
There were no significant differences between smooth PT and A surfaces except for a small increase in OPG. Compared to A surfaces, MG63 cells produced 30% more osteocalcin on modA, and 70% more on SLA. However, growth on modSLA increased osteocalcin by more than 250%, which exceeded the sum of independent effects of surface energy and topography. Similar effects were noted when levels of latent TGF-β1, PGE2 and OPG were measured in the conditioned media.
Conclusions
The results demonstrate a synergistic effect between high surface energy and topography of Ti substrates and show that both micron scale and submicron scale structural features are necessary.
doi:10.1016/j.biomaterials.2007.02.024
PMCID: PMC2754822  PMID: 17368532
Titanium; Surface energy; Microstructure; Submicron roughness; Osteoblast differentiation
3.  Blue-Violet Laser Modification of Titania Treated Titanium: Antibacterial and Osteo-Inductive Effects 
PLoS ONE  2013;8(12):e84327.
Background
Many studies on surface modifications of titanium have been performed in an attempt to accelerate osseointegration. Recently, anatase titanium dioxide has been found to act as a photocatalyst that expresses antibiotic properties and exhibits hydrophilicity after ultraviolet exposure. A blue-violet semiconductor laser (BV-LD) has been developed as near-ultraviolet light. The purpose of this study was to investigate the effects of exposure to this BV-LD on surface modifications of titanium with the goal of enhancing osteoconductive and antibacterial properties.
Methods
The surfaces of pure commercial titanium were polished with #800 waterproof polishing papers and were treated with anatase titania solution. Specimens were exposed using BV-LD (λ = 405 nm) or an ultraviolet light-emitting diode (UV-LED, λ = 365 nm) at 6 mW/cm2 for 3 h. The surface modification was evaluated physically and biologically using the following parameters or tests: surface roughness, surface temperature during exposure, X-ray diffraction (XRD) analysis, contact angle, methylene blue degradation tests, adherence of Porphyromonas gingivalis, osteoblast and fibroblast proliferation, and histological examination after implantation in rats.
Results
No significant changes were found in the surface roughness or XRD profiles after exposure. BV-LD exposure did not raise the surface temperature of titanium. The contact angle was significantly decreased, and methylene blue was significantly degraded. The number of attached P. gingivalis organisms was significantly reduced after BV-LD exposure compared to that in the no exposure group. New bone was observed around exposed specimens in the histological evaluation, and both the bone-to-specimen contact ratio and the new bone area increased significantly in exposed groups.
Conclusions
This study suggested that exposure of titanium to BV-LD can enhance the osteoconductivity of the titanium surface and induce antibacterial properties, similar to the properties observed following exposure to UV-LED.
doi:10.1371/journal.pone.0084327
PMCID: PMC3866166  PMID: 24358355
4.  The Effect of Carbon Dioxide (CO2) Laser on Sandblasting with Large Grit and Acid Etching (SLA) Surface  
Introduction: The purpose of this study was to investigate the effect of 6W power Carbon Dioxide Laser (CO2) on the biologic compatibility of the Sandblasting with large grit and acid etching (SLA) titanium discs through studying of the Sarcoma Osteogenic (SaOS-2) human osteoblast-like cells viability.
Methods: Sterilized titanium discs were used together with SaOS-2 human osteoblast-like cells. 6 sterilized SLA titanium discs of the experimental group were exposed to irradiation by CO2 laser with a power of 6W and 10.600nm wavelength, at fixed frequency of 80Hz during 45 seconds in both pulse and non-contact settings. SaOS-2 human osteoblast-like cells were incubated under 37°C in humid atmosphere (95% weather, 5% CO2) for 72 hours. MTT test was performed to measure the ratio level of cellular proliferation.
Results: The results indicated that at 570nm wavelength, the 6W CO2 laser power have not affected the cellular viability.
Conclusion: CO2 laser in 6w power has had no effect on the biologic compatibility of the SLA titanium surface
PMCID: PMC4281976  PMID: 25606313
CO2 laser ; titanium; SLA
5.  Effect of cleaning and sterilization on titanium implant surface properties and cellular response 
Acta biomaterialia  2011;8(5):1966-1975.
Titanium (Ti) has been widely used as an implant material due to the excellent biocompatibility and corrosion resistance of its oxide surface. Biomaterials must be sterile before implantation, but the effects of sterilization on their surface properties have been less well studied. The effects of cleaning and sterilization on surface characteristics were bio-determined using contaminated and pure Ti substrata first manufactured to present two different surface structures: pretreated titanium (PT, Ra = 0.4 μm) (i.e. surfaces that were not modified by sandblasting and/or acid etching); (SLA, Ra = 3.4 μm). Previously cultured cells and associated extracellular matrix were removed from all bio-contaminated specimens by cleaning in a sonicator bath with a sequential acetone–isopropanol–ethanol–distilled water protocol. Cleaned specimens were sterilized with autoclave, gamma irradiation, oxygen plasma, or ultraviolet light. X-ray photoelectron spectroscopy (XPS), contact angle measurements, profilometry, and scanning electron microscopy were used to examine surface chemical components, hydrophilicity, roughness, and morphology, respectively. Small organic molecules present on contaminated Ti surfaces were removed with cleaning. XPS analysis confirmed that surface chemistry was altered by both cleaning and sterilization. Cleaning and sterilization affected hydrophobicity and roughness. These modified surface properties affected osteogenic differentiation of human MG63 osteoblast-like cells. Specifically, autoclaved SLA surfaces lost the characteristic increase in osteoblast differentiation seen on starting SLA surfaces, which was correlated with altered surface wettability and roughness. These data indicated that recleaned and resterilized Ti implant surfaces cannot be considered the same as the first surfaces in terms of surface properties and cell responses. Therefore, the reuse of Ti implants after resterilization may not result in the same tissue responses as found with never-before-implanted specimens.
doi:10.1016/j.actbio.2011.11.026
PMCID: PMC3618465  PMID: 22154860
Titanium; Sterilization; Roughness; Hydrophilicity; MG63 cells
6.  The roles of Wnt signaling modulators Dickkopf-1 (Dkk1) and Dickkopf-2 (Dkk2) and cell maturation state in osteogenesis on microstructured titanium surfaces 
Biomaterials  2009;31(8):2015-2024.
Osteoblast differentiation on tissue culture polystyrene (TCPS) requires Wnt/beta-catenin signaling, regulating modulators of the Wnt pathway like Dickkopf-1 (Dkk1) and Dkk2. Osteoblast differentiation is increased on microstructured titanium (Ti) surfaces compared to TCPS; therefore, we hypothesized that surface topography and hydrophilicity affect Dkk1 and Dkk2 expression and that their roles in osteoblast differentiation on Ti differs depending on cell maturation state. Human osteoblast-like MG63 cells, normal human osteoblasts (HOBs), and human mesenchymal stem cells (MSCs), as well as MG63 cells stably silenced for Dkk1 or Dkk2 were grown for 6 days on TCPS and Ti surfaces (PT [Ra<0.2 μm], SLA [Ra = 4 μm], modSLA [hydrophilic-SLA]). Dkk1 and Dkk2 mRNA and protein increased on SLA and modSLA for all cell types, but exogenous rhDkk1 and rhDkk2 affected MSCs differently than MG63 cells and HOBs. Silencing Dkk1 reduced MG63 cell number on TCPS and PT, but increased differentiation on these substrates. Silencing Dkk2 reduced stimulatory effects of SLA and modSLA on osteoblast differentiation; Dkk2 but not Dkk1 restored these effects. Antibodies to Dkk1 or Dkk2 specifically blocked substrate-dependent changes caused by the proteins, demonstrating their autocrine action. This indicates major roles for Dkk1 and the canonical Wnt pathway in early-stage differentiation, and for Dkk2 and Wnt/Ca2+-dependent signaling in late-stage differentiation on microstructured and hydrophilic surfaces, during osseointegration.
doi:10.1016/j.biomaterials.2009.11.071
PMCID: PMC3618462  PMID: 20004015
Osseointegration; Titanium; Osteoblast; Mesenchymal stem cell; Surface roughness; Cell signaling
7.  The responses to surface wettability gradients induced by chitosan nanofilms on microtextured titanium mediated by specific integrin receptors 
Biomaterials  2012;33(30):7386-7393.
Microtexture and chemistry of implant surfaces are important variables for modulating cellular responses. Surface chemistry and wettability are connected directly. While each of these surface properties can influence cell response, it is difficult to decouple their specific contributions. To address this problem, the aims of this study were to develop a surface wettability gradient with a specific chemistry without altering micron scale roughness and to investigate the role of surface wettability on osteoblast response. Microtextured sandblasted/acid-etched (SLA, Sa = 3.1 μm) titanium disks were treated with oxygen plasma to increase reactive oxygen density on the surface. At 0, 2, 6, 10, and 24 h after removing them from the plasma, the surfaces were coated with chitosan for 30 min, rinsed and dried. Modified SLA surfaces are denoted as SLA/h in air prior to coating. Surface characterization demonstrated that this process yielded differing wettability (SLA0 < SLA2 < SLA10 < SLA24) without modifying the micron scale features of the surface. Cell number was reduced in a wettability-dependent manner, except for the most water-wettable surface, SLA24. There was no difference in alkaline phosphatase activity with differing wettability. Increased wettability yielded increased osteocalcin and osteoprotegerin production, except on the SLA24 surfaces. mRNA for integrins α1, α2, α5, β1, and β3 was sensitive to surface wettability. However, surface wettability did not affect mRNA levels for integrin α3. Silencing β1 increased cell number with reduced osteocalcin and osteoprotegerin in a wettability-dependent manner. Surface wettability as a primary regulator enhanced osteoblast differentiation, but integrin expression and silencing β1 results indicate that surface wettability regulates osteoblast through differential integrin expression profiles than microtexture does. The results may indicate that both microtexture and wettability with a specific chemistry have important regulatory effects on osseointegration. Each property had different effects, which were mediated by different integrin receptors.
doi:10.1016/j.biomaterials.2012.06.066
PMCID: PMC3781581  PMID: 22835642
Wettability; Oxygen plasma; Chitosan; Titanium; Osteoblast; Integrin
8.  Use of polyelectrolyte thin films to modulate Osteoblast response to microstructured titanium surfaces 
Biomaterials  2012;33(21):5267-5277.
The microstructure and wettability of titanium (Ti) surfaces directly impact osteoblast differentiation in vitro and in vivo. These surface properties are important variables that control initial interactions of an implant with the physiological environment, potentially affecting osseointegration. The objective of this study was to use polyelectrolyte thin films to investigate how surface chemistry modulates response of human MG63 osteoblast-like cells to surface microstructure. Three polyelectrolytes, chitosan, poly(l-glutamic acid), and poly(l-lysine), were used to coat Ti substrates with two different microtopographies (PT, Sa = 0.37 µm and SLA, Sa = 2.54 µm). The polyelectrolyte coatings significantly increased wettability of PT and SLA without altering micron-scale roughness or morphology of the surface. Enhanced wettability of all coated PT surfaces was correlated with increased cell numbers whereas cell number was reduced on coated SLA surfaces. Alkaline phosphatase specific activity was increased on coated SLA surfaces than on uncoated SLA whereas no differences in enzyme activity were seen on coated PT compared to uncoated PT. Culture on chitosan-coated SLA enhanced osteocalcin and osteoprotegerin production. Integrin expression on smooth surfaces was sensitive to surface chemistry, but microtexture was the dominant variable in modulating integrin expression on SLA. These results suggest that surface wettability achieved using different thin films has a major role in regulating osteoblast response to Ti, but this is dependent on the microtexture of the substrate.
doi:10.1016/j.biomaterials.2012.03.074
PMCID: PMC3618464  PMID: 22541354
Wettability; Titanium; Surface roughness; Osteoblast
9.  Response of osteoblast-like cells cultured on zirconia to bone morphogenetic protein-2 
Purpose
The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2.
Methods
MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days.
Results
At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7.
Conclusions
The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.
doi:10.5051/jpis.2011.41.5.227
PMCID: PMC3213233  PMID: 22087413
Bone morphogenetic protein-2; Cell differentiation; Cell proliferation; Zirconium oxide
10.  MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization 
The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells.
doi:10.1155/2012/743465
PMCID: PMC3364553  PMID: 22675359
11.  Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro 
Clinical oral implants research  2011;23(3):301-307.
Objectives
The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation.
Materials and methods
Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [Ra<0.2 μm]), acid-etched (A [Ra<0.8 μm]), A modified to be hydrophilic (modA), sand-blasted/acid-etched (SLA [Ra = 4 μm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA–DNA hybridization. Results: Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva.
Conclusions
The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used.
doi:10.1111/j.1600-0501.2011.02184.x
PMCID: PMC4287405  PMID: 21492236
biofilm; hydrophilicity; microstructure; titanium
12.  Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol 
BMC Biotechnology  2009;9:9.
Background
Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.
Results
The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.
Conclusion
The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.
doi:10.1186/1472-6750-9-9
PMCID: PMC2654889  PMID: 19220883
13.  Microstructured Titanium Regulates Interleukin Production by Osteoblasts, an Effect Modulated by Exogenous BMP-2 
Acta biomaterialia  2012;9(3):5821-5829.
Microtextured implant surfaces increase osteoblast differentiation in vitro and enhance bone-to-implant contact in vivo and clinically. These implants may be used in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2) to enhance peri-implant bone formation. However, the effect of surface modifications alone or in combination with rhBMP-2 on osteoblast-produced inflammatory microenvironment is unknown. MG63 cells were cultured on tissue culture polystyrene or titanium substrates: smooth pretreated (PT, Ra=0.2μm), sandblasted/acid-etched (SLA, Ra=3.2μm), or hydrophilic-SLA (modSLA). Expression and protein production of pro-inflammatory interleukins (IL1b, IL6, IL8, IL17) and anti-inflammatory interleukins (IL10) were measured in cells with or without rhBMP-2. To determine which BMP signaling pathways were involved, cultures were incubated with BMP pathway inhibitors to blocking Smad (dorsomorphin), TAB/TAK1 ((5Z)-7-oxozeaenol), or PKA (H-8) signaling. Culture on rough SLA and modSLA surfaces decreased pro-inflammatory interleukins and increased anti-inflammatory IL10. This effect was negated in cells treated with rhBMP-2, which caused an increase in pro-inflammatory interleukins and a decrease in anti-inflammatory interleukins through TAB/TAK signaling. The results suggest that surface microtexture modulates the inflammatory process during osseointegration, an effect that may enhance healing. However, rhBMP-2 in combination with microtextured titanium implants can influence the effect of cells on these surfaces, and may adversely affect cells involved in osseointegration.
doi:10.1016/j.actbio.2012.10.030
PMCID: PMC3618455  PMID: 23123301
Microstructure; Inflammation; BMP (bone morphogenetic protein); Titanium
14.  Mechanisms Regulating Increased Production of Osteoprotegerin by Osteoblasts Cultured on Microstructured Titanium Surfaces 
Biomaterials  2009;30(20):3390-3396.
Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2μm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4μm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4μm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-β1, PKC, and α2β1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-β1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-β1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-β type II receptor or by reducing TGF-β1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of α2 or a double knockdown of α2β1 also reduced OPG, as well as production of TGF-β1. These results indicate that substrate dependent OPG production is regulated by TGF-β1, PKC, and α2β1 and suggest a mechanism by which α2β1-signaling increases PKC, resulting in TGF-β1 production and TGF-β1 then acts on its receptor to increase transcription of OPG.
doi:10.1016/j.biomaterials.2009.03.047
PMCID: PMC2700751  PMID: 19395022
Osteoblast; TGF-β1; Osteoprotegerin; Titanium; Microtopography
15.  Behavior of osteoblastic cells cultured on titanium and structured zirconia surfaces 
Head & Face Medicine  2008;4:29.
Background
Osseointegration is crucial for the long-term success of dental implants and depends on the tissue reaction at the tissue-implant interface. Mechanical properties and biocompatibility make zirconia a suitable material for dental implants, although surface processings are still problematic. The aim of the present study was to compare osteoblast behavior on structured zirconia and titanium surfaces under standardized conditions.
Methods
The surface characteristics were determined by scanning electron microscopy (SEM). In primary bovine osteoblasts attachment kinetics, proliferation rate and synthesis of bone-associated proteins were tested on different surfaces.
Results
The results demonstrated that the proliferation rate of cells was significantly higher on zirconia surfaces than on titanium surfaces (p < 0.05; Student's t-test). In contrast, attachment and adhesion strength of the primary cells was significant higher on titanium surfaces (p < 0.05; U test). No significant differences were found in the synthesis of bone-specific proteins. Ultrastructural analysis revealed phenotypic features of osteoblast-like cells on both zirconia and titanium surfaces.
Conclusion
The study demonstrates distinct effects of the surface composition on osteoblasts in culture. Zirconia improves cell proliferation significantly during the first days of culture, but it does not improve attachment and adhesion strength. Both materials do not differ with respect to protein synthesis or ultrastructural appearance of osteoblasts. Zirconium oxide may therefore be a suitable material for dental implants.
doi:10.1186/1746-160X-4-29
PMCID: PMC2614982  PMID: 19063728
16.  Role of non-canonical Wnt signaling in osteoblast maturation on microstructured titanium surfaces 
Acta biomaterialia  2011;7(6):2740-2750.
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra = 0.2 μm), sand-blasted and acid-etched SLA (Ra = 3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.
doi:10.1016/j.actbio.2011.02.030
PMCID: PMC4287412  PMID: 21352958
Cell signaling; Titanium surface roughness; Osteoblast differentiation; Gene expression; Regulatory factors
17.  In vitro preliminary study of osteoblast response to surface roughness of titanium discs and topical application of melatonin 
Objectives: To observe human osteoblast behavior cultured in vitro on titanium discs (Ti) in relation to surface roughness and melatonin application. Study Design: Human osteoblasts (MG-63) were cultured on 60 Ti6Al4V discs divided into three groups: Group I: discs treated with dual acid etching; Group II dual acid etching and blasting with calcium phosphate particles; Group III (control) machined discs. Surface roughness and topography of the discs were examined with scanning electron microscope (SEM) and confocal laser scanning electron microscope( CLSM). Osteoblast adhesion, proliferation and cell morphology were determined by means of fluorescence microscopy with Image-Pro Plus software and SEM. Results: Group II presented the roughest discs, while the least rough were Group III. Cell adhesion was greatest in Group II. The addition of melatonin improved cell proliferation. Conclusions: 1. Surface treatments (dual acid etching, calcium phosphate impaction) increase surface roughness in comparison with machined titanium. 2. Greater surface roughness tends to favor cell adhesion after 24-hour cell culture. 3. The addition of melatonin tends to favor osteoblast proliferation.
Key words:Osteoblasts, titanium, roughness, melatonin, dental implants, osseointegration.
doi:10.4317/medoral.19953
PMCID: PMC4320427  PMID: 25129252
18.  The role of phospholipase D in osteoblast response to titanium surface microstructure 
Biomaterial surface properties such as microtopography and energy can change cellular responses at the cell-implant interface. Phospholipase D (PLD) is required for the differentiation of osteoblast-like MG63 cells on machined and grit-blasted titanium surfaces. Here, we determined if PLD is also required on microstructured/high-energy substrates and the mechanism involved. shRNAs for human PLD1 and PLD2 were used to silence MG63 cells. Wild-type and PLD1 or PLD1/2 silenced cells were cultured on smooth-pretreatment surfaces (PT); grit-blasted, acid-etched surfaces (SLA); and SLA surfaces modified to have higher surface energy (modSLA). PLD was inhibited with ethanol or activated with 24,25-dihydroxyvitamin-D3 [24R,25(OH)2D3]. As surface roughness/energy increased, PLD mRNA and activity increased, cell number decreased, osteocalcin and osteoprotegerin increased, and protein kinase C (PKC) and alkaline phosphatase specific activities increased. Ethanol inhibited PLD and reduced surface effects on these parameters. There was no effect on these parameters after knockdown of PLD1, but PLD1/2 double knockdown had effects comparable to PLD inhibition. 24R,25(OH)2D3 increased PLD activity and the production of osteocalcin and osteoprotegerin, but decreased cell number on the rough/high-energy surfaces. These results confirm that surface roughness/energy-induced PLD activity is required for osteoblast differentiation and that PLD2 is the main isoform involved in this pathway. PLD is activated by 24R,25(OH)2D3 in a surface-dependent manner and inhibition of PLD reduces the effects of surface microstructure/energy on PKC, suggesting that PLD mediates the stimulatory effect of microstructured/high-energy surfaces via PKC-dependent signaling.
doi:10.1002/jbm.a.32596
PMCID: PMC4287411  PMID: 19705469
phospholipase D; osteoblast differentiation; titanium surface microstructure and surface energy; vitamin D metabolites; mechanism of cell surface interaction
19.  EFFECTS OF PHOSPHATED TITANIUM AND ENAMEL MATRIX DERIVATIVES ON OSTEOBLAST BEHAVIOR IN VITRO 
Purpose
The purpose of this study was to evaluate the effects of phosphated titanium and EMD on osteoblast function.
Materials and Methods
Primary rat osteoblasts were cultured on discs of either phosphated or non-phosphated titanium and in half of the samples 180μg of EMD was immediately added. Media was changed every 2 days for 28 days, and then analyzed by TGF-β1 and IL-1β ELISAs. Scanning electron microscopy (SEM) and light microscopy (LM) was used to evaluate nodule formation and mineralization.
Results
Microscopic evaluation revealed no differences in osteoblast attachment on all discs, regardless of treatment. Osteoblast nodule formation was observed in all groups. In the absence of mineralizing media, nodules on the non-phosphated titanium samples showed no evidence of mineralization. All nodules on the phosphated titanium had evidence of mineralization. ELISA analysis revealed no significant differences in IL-1β production between any of the groups. The EMD treated osteoblasts produced significantly more TGF-β1 than non-EMD treated cells for up to 8 days, and osteoblasts on phosphated titanium produced significantly more Tgf-ß1 at 8 days.
Discussion and Conclusion
Osteoblast attachment appeared unaffected by surface treatment. EMD initiated early TGF-β1 production, but production decreased to control levels within 10 days. Phosphated titanium increased Tgf-ß1 production at 8 days, and induced nodule mineralization even in the absence of mineralizing medium.
PMCID: PMC3215085  PMID: 17974103
phosphate; titanium; osteoblasts; enamel matrix derivatives; TGF-β1; IL-1β
20.  Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics 
Clinical oral implants research  2011;22(8):865-872.
Objective
Functionalizing surfaces with specific peptides may aid osteointegration of orthopedic implants by favoring attachment of osteoprogenitor cells and promoting osteoblastic differentiation. This study addressed the hypothesis that implant surfaces functionalized with peptides targeting multiple ligands will enhance osteoblast attachment and/or differentiation. To test this hypothesis, we used titanium (Ti) surfaces coated with poly-l-lysine-grafted polyethylene glycol (PLL-g-PEG) and functionalized with two peptides found in extracellular matrix proteins, arginine–glycine–aspartic acid (RGD) and lysine–arginine–serine–arginine (KRSR), which have been shown to increase osteoblast attachment. KSSR, which does not promote osteoblast attachment, was used as a control.
Materials and methods
Sandblasted acid-etched titanium surfaces were coated with PLL-g-PEG functionalized with varying combinations of RGD and KRSR, as well as KSSR. Effects of these surfaces on osteoblasts were assessed by measuring cell number, alkaline phosphatase-specific activity, and levels of osteocalcin, transforming growth factor beta-1 (TGF-β1), and PGE2.
Results
RGD increased cell number, but decreased markers for osteoblast differentiation. KRSR alone had no effect on cell number, but decreased levels of TGF-β1 and PGE2. KRSR and RGD/KRSR coatings inhibited osteoblast differentiation vs. PLL-g-PEG. KSSR decreased cell number and increased osteoblast differentiation, indicated by increased levels of osteocalcin and PGE2.
Conclusions
The RGD and KRSR functionalized surfaces supported attachment but did not enhance osteoblast differentiation, whereas KSSR increased differentiation. RGD decreased this effect, suggesting that multifunctional peptide surfaces can be designed that improve peri-implant healing by optimizing attachment and proliferation as well as differentiation of osteoblasts, but peptide combination, dose and presentation are critical variables.
doi:10.1111/j.1600-0501.2010.02074.x
PMCID: PMC4287399  PMID: 21244501
KRSR; KSSR; microstructure; non-fouling; osteoblast differentiation; PLL-g-PEG; RGD; surface modification; surface roughness; titanium
21.  The Effects of Different Wavelength UV Photofunctionalization on Micro-Arc Oxidized Titanium 
PLoS ONE  2013;8(7):e68086.
Many challenges exist in improving early osseointegration, one of the most critical factors in the long-term clinical success of dental implants. Recently, ultraviolet (UV) light-mediated photofunctionalization of titanium as a new potential surface treatment has aroused great interest. This study examines the bioactivity of titanium surfaces treated with UV light of different wavelengths and the underlying associated mechanism. Micro-arc oxidation (MAO) titanium samples were pretreated with UVA light (peak wavelength of 360 nm) or UVC light (peak wavelength of 250 nm) for up to 24 h. UVC treatment promoted the attachment, spread, proliferation and differentiation of MG-63 osteoblast-like cells on the titanium surface, as well as the capacity for apatite formation in simulated body fluid (SBF). These biological influences were not observed after UVA treatment, apart from a weaker effect on apatite formation. The enhanced bioactivity was substantially correlated with the amount of Ti-OH groups, which play an important role in improving the hydrophilicity, along with the removal of hydrocarbons on the titanium surface. Our results showed that both UVA and UVC irradiation altered the chemical properties of the titanium surface without sacrificing its excellent physical characteristics, suggesting that this technology has extensive potential applications and merits further investigation.
doi:10.1371/journal.pone.0068086
PMCID: PMC3702557  PMID: 23861853
22.  Effect of heat treatment on H2O2/HCl etched pure titanium dental implant: An in vitro study 
Summary
Background
Surface chemistry of dental implant plays an important role in osseointegration. Heat treatment might alter surface chemistry and result in different biological response. The aim of this study was to investigate the roles of heat treatment of H2O2/HCl-treated Ti implants in cell attachment, proliferation and osteoblastic differentiation.
Material/Methods
Sandblasted, dual acid-etched and H2O2/HCl heat-treated discs were set as the control group and sandblasted, dual acid-etched H2O2/HCl-treated discs were the test group. Both groups’ discs were sent for surface characterization. MC3T3-E1 cells were seeded on these 2 groups’ discs for 3 hours to 14 days, and then cell attachment, cell proliferation and cell differentiation were evaluated.
Results
Scanning electron microscope analysis revealed that the titanium discs in the 2 groups shared the same surface topography, while x-ray diffraction examination showed an anatase layer in the control group and titanium hydride diffractions in the test group. The cell attachment of the test group was equivalent to that of the control group. Cell proliferation was slightly stimulated at all time points in the control group, but the alkaline phosphatase (ALP) activity and osteocalcin (OC) production increased significantly in the test group compared with those in the control group at every time point investigated (p<0.05 or p<0.01). Moreover, the osteoblastic differentiation-related genes AKP-2, osteopontin (OPN) and OC were greatly up-regulated in the test group (p<0.05 or p<0.01).
Conclusions
The results implied that surface chemistry played an important role in cell response, and H2O2/HCl etched titanium surface without subsequent heat treatment might improve osseointegration response.
doi:10.12659/MSM.883204
PMCID: PMC3560775  PMID: 22739726
titanium implant; heat treatment; anatase; titanium hydride
23.  Regulation of Angiogenesis during Osseointegration by Titanium Surface Microstructure and Energy 
Biomaterials  2010;31(18):4909-4917.
Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces. Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA. Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.
doi:10.1016/j.biomaterials.2010.02.071
PMCID: PMC2896824  PMID: 20356623
Titanium; microstructure; surface energy; osteoblast; angiogenesis; VEGF
24.  Sex dependent regulation of osteoblast response to implant surface properties by systemic hormones 
Background
Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)2D3 and 17β-oestradiol (E2)] and microstructured surfaces on osteoblasts are sex dependent.
Methods
Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)2D3, E2, or E2 conjugated to bovine serum albumin (E2-BSA).
Results
Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-β1, osteoprotegerin and prostaglandin E2 in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E2 and E2-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)2D3 increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells.
Conclusions
Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)2D3 had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones.
doi:10.1186/2042-6410-1-4
PMCID: PMC3010104  PMID: 21208469
25.  A Prospective Study of Early Loaded Single Implant-Retained Mandibular Overdentures: Preliminary One-Year Results 
Purpose. To investigate the predictability of simplifying mandibular overdenture treatment using one-stage surgery and early prosthetic loading of a single implant. Materials and Methods. Twenty edentulous patients with problematic existing mandibular dentures were treated. A single implant with a chemically modified surface (SLActive, Straumann AG, Basel, Switzerland) was placed into the mandibular midline. The patients were recalled at 3, 6 and 12 months. Clinical assessments and marginal bone loss using standardized radiographs were recorded. All complications, failures and maintenance were noted. Visual analog-scale questionnaires were used to record patient satisfaction in five categories. ANOVA was used to determine differences between means of marginal bone loss and different categories of patient staisfaction (P = 0.05). Results. The 20 early loaded implants were all surviving at the 12-month recall. All implants showed less than 1 mm of marginal bone loss by the end of the 1-year with a significant increase during the follow-up period. Few prosthetic problems were reported. Patient satisfaction was high with a significant increase in all comfort and functional parameters. Conclusions. These preliminary 1-year results indicate that early loading of a single chemically modified surface implant used to retain a mucosa-borne mandibular overdenture is a safe, reliable, and cost-effective treatment.
doi:10.1155/2012/236409
PMCID: PMC3312253  PMID: 22505895

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