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1.  Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics 
Clinical oral implants research  2011;22(8):865-872.
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.
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.
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.
PMCID: PMC4287399  PMID: 21244501
KRSR; KSSR; microstructure; non-fouling; osteoblast differentiation; PLL-g-PEG; RGD; surface modification; surface roughness; titanium
2.  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.
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.
The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used.
PMCID: PMC4287405  PMID: 21492236
biofilm; hydrophilicity; microstructure; titanium
3.  Electrical Implications of Corrosion for Osseointegration of Titanium Implants 
Journal of Dental Research  2011;90(12):1389-1397.
The success rate of titanium implants for dental and orthopedic applications depends on the ability of surrounding bone tissue to integrate with the surface of the device, and it remains far from ideal in patients with bone compromised by physiological factors. The electrical properties and electrical stimulation of bone have been shown to control its growth and healing and can enhance osseointegration. Bone cells are also sensitive to the chemical products generated during corrosion events, but less is known about how the electrical signals associated with corrosion might affect osseointegration. The metallic nature of the materials used for implant applications and the corrosive environments found in the human body, in combination with the continuous and cyclic loads to which these implants are exposed, may lead to corrosion and its corresponding electrochemical products. The abnormal electrical currents produced during corrosion can convert any metallic implant into an electrode, and the negative impact on the surrounding tissue due to these extreme signals could be an additional cause of poor performance and rejection of implants. Here, we review basic aspects of the electrical properties and electrical stimulation of bone, as well as fundamental concepts of aqueous corrosion and its electrical and clinical implications.
PMCID: PMC3215755  PMID: 21555775
biopotentials; electrical stimulation; corrosion; titanium; bone; osseointegration of dental and orthopedic implants
4.  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.
PMCID: PMC2700751  PMID: 19395022
Osteoblast; TGF-β1; Osteoprotegerin; Titanium; Microtopography
5.  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.
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.
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.
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.
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.
PMCID: PMC2754822  PMID: 17368532
Titanium; Surface energy; Microstructure; Submicron roughness; Osteoblast differentiation
6.  1,25-Dihydroxy Vitamin D3 Is an Autocrine Regulator of Extracellular Matrix Turnover and Growth Factor Release via ERp60-Activated Matrix Vesicle Matrix Metalloproteinases 
Cells, Tissues, Organs  2008;189(1-4):70-74.
As growth plate chondrocytes mature and hypertrophy, they reorganize their proteoglycan-rich type II collagen extracellular matrix (ECM), involving 1,25(OH)2D3-dependent regulation of matrix metalloproteinases (MMPs). Stromelysin-1 (MMP-3) and 72-kD gelatinase (MMP-2) are found in extracellular matrix vesicles (MVs) and release and activate ECM-bound latent TGF-β1 and TGF-β2, respectively. 1,25(OH)2D3 regulates incorporation of MMP-2 and MMP-3 into MVs and release of these enzymes in the ECM. Plasma membranes (PMs) and MVs contain the 1α,25(OH)2D3 membrane receptor ERp60 (protein disulfide isomerase A3), phospholipase A2 (PLA2), PLA2-activating protein, the nuclear vitamin D receptor and caveolin-1. 1,25(OH)2D3 secreted by chondrocytes binds MV ERp60, activating PLA2. Resulting lysophospholipids destabilize MV membranes, releasing active MMPs. We examined 1,25(OH)2D3-dependent activation of latent TGF-β1 stored in cartilage ECM. Interestingly, TGF-β1 regulates 1,25(OH)2D3 production. 1α,25(OH)2D3 activates PM protein kinase C (PKC)-α via ERp60-dependent PLA2-signaling, lysophospholipid production and phospholipase C-γ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching MVs in PKC-ζ. Direct activation of MV MMP-3 requires ERp60 based on blocking antibodies and PKC based on inhibitor studies. However, treatment of MVs with 1,25(OH)2D3 decreases MV PKC-ζ activity, suggesting more complex feedback mechanisms, potentially involving MV lipid signaling. Our observations indicate that one role of MVs is to provide MMPs at sites distant from the cells. Chondrocytes secrete 1,25(OH)2D3, which acts directly on MV-membranes via ERp60, releasing MMPs. MMP-specific ECM components are hydrolyzed, resulting in release and activation of growth factors that can act back on the cells.
PMCID: PMC2824185  PMID: 18765931
1α,25(OH)2D3; Matrix vesicles; Extracellular matrix; TGF-β1, latent activation; Matrix metalloproteinases; MMP-3

Results 1-6 (6)