Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach

AbstractThe effects of alkaline pH on the initial adhesion of osteoblasts to titanium surfaces was analyzed by single cell force microscopy (SCFM). In the SCFM measurements, the same cells were used to compare their unspecific adhesion to uncoated titanium with their specific adhesion to collagen coated titanium. When the maximum detachment forces (MDFs) were compared at pH 7.4 and 8.0, only slight differences were found on pure titanium, while the MDFs were significantly increased at collagen coated surfaces at pH 8.0. Effects on the subsequent proliferation and gene expression were investigated in an in vitro model system consisting of an alkalizing polyvinyl alcohol (PVA) matrix and a perforated titanium disc. The sodium hydroxide releasing matrix maintained the medium pH between pH 7.6 and pH 8.4 during the entire experiment. Under these conditions, cell counts were significantly increased with respect to the control system after 7 days in culture. These results were supported by gene expression analyses, which showed an upregulation of proliferation-controlling genes of the EGFR1 and PI3K/AKT pathways after 14 days in culture. The SCFM data were complemented by findings of an intensive regulation of genes known to be associated with focal adhesion such as Itga8 and Tnn.

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Effect of Multi-Phosphonate Coating of Titanium Surfaces on Osteogenic Potential

Materials ◽

10.3390/ma13245777 ◽

2020 ◽

Vol 13 (24) ◽

pp. 5777

Author(s):

Christian Wehner ◽

Christian Behm ◽

Selma Husejnagic ◽

Andreas Moritz ◽

Xiaohui Rausch-Fan ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Tissue Culture ◽

Bone Turnover ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Tissue Culture Plastic ◽

Titanium Surfaces ◽

Coated Surfaces

The aim of this study was to evaluate the impact of a novel multi-phosphonate (MP) coating strategy of dental implant surfaces on the expression of osteogenesis-related factors in vitro. MG-63 human osteoblast-like cells, bone marrow mesenchymal stem cells (BM-MSCs), and human periodontal ligament stem cells (hPDLSCs) were cultured separately on titanium disks with and without MP coating. Cell attachment was visualized by focal adhesion and actin cytoskeleton staining. The proliferation and gene expression of the markers related to osteogenesis and bone turnover were measured after 48 and 120 h of cell culture. Actin cytoskeleton assembly and focal adhesion were similar between test surfaces within each cell type but differed from those on tissue culture plastic (TCP). The proliferation of MG-63 cells and PDLSCs was comparable on all surfaces, while BM-MSCs showed an increase on tissue culture plastic (TCP) versus titanium. The gene expression of osteoprotegerin and receptor activator of nuclear factor-kappa B ligand was higher in MG-63 cells grown on MP-coated surfaces. At the same time, osteocalcin was decreased compared to the other surfaces. Collagen type I gene expression after 120 h was significantly lower in hPDLSCs cultivated on MP-coated surfaces. Within the limitations of this study, MP coating on titanium surfaces might have a slight beneficial effect on bone turnover in vitro.

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Adhesion Behaviour of Primary Human Osteoblasts and Fibroblasts on Polyether Ether Ketone Compared with Titanium under In Vitro Lipopolysaccharide Incubation

Materials ◽

10.3390/ma12172739 ◽

2019 ◽

Vol 12 (17) ◽

pp. 2739 ◽

Cited By ~ 1

Author(s):

Korbinian Benz ◽

Andreas Schöbel ◽

Marisa Dietz ◽

Peter Maurer ◽

Jochen Jackowski

Keyword(s):

Gene Expression ◽

Protein Level ◽

Binding Protein ◽

Sem Images ◽

Human Osteoblasts ◽

Polyether Ether Ketone ◽

Primary Human Osteoblasts ◽

Titanium Surfaces ◽

Ether Ketone

The aim of this in vitro pilot study was to analyse the adhesion behaviour of human osteoblasts and fibroblasts on polyether ether ketone (PEEK) when compared with titanium surfaces in an inflammatory environment under lipopolysaccharide (LPS) incubation. Scanning electron microscopy (SEM) images of primary human osteoblasts/fibroblasts on titanium/PEEK samples were created. The gene expression of the LPS-binding protein (LBP) and the LPS receptor (toll-like receptor 4; TLR4) was measured by real-time polymerase chain reaction (PCR). Immunocytochemistry was used to obtain evidence for the distribution of LBP/TLR4 at the protein level of the extra-cellular-matrix-binding protein vinculin and the actin cytoskeleton. SEM images revealed that the osteoblasts and fibroblasts on the PEEK surfaces had adhesion characteristics comparable to those of titanium. The osteoblasts contracted under LPS incubation and a significantly increased LBP gene expression were detected. This was discernible at the protein level on all the materials. Whereas no increase of TLR4 was detected with regard to mRNA concentrations, a considerable increase in the antibody reaction was detected on all the materials. As is the case with titanium, the colonisation of human osteoblasts and fibroblasts on PEEK samples is possible under pro-inflammatory environmental conditions and the cellular inflammation behaviour towards PEEK is lower than that of titanium.

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In vitro study of monocyte viability during the initial adhesion to albumin- and fibrinogen-coated surfaces

Biomaterials ◽

10.1016/s0142-9612(00)00246-5 ◽

2001 ◽

Vol 22 (8) ◽

pp. 827-832 ◽

Cited By ~ 23

Author(s):

M. Werthén ◽

A. Sellborn ◽

M. Källtorp ◽

H. Elwing ◽

P. Thomsen

Keyword(s):

In Vitro Study ◽

Vitro Study ◽

Initial Adhesion ◽

Coated Surfaces

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The influence of titanium surfaces treated by alkalis on macrophage and osteoblast-like cell adhesion and gene expression in vitro

RSC Advances ◽

10.1039/c5ra10701f ◽

2015 ◽

Vol 5 (99) ◽

pp. 81378-81387 ◽

Cited By ~ 6

Author(s):

Ting Ma ◽

Xi-Yuan Ge ◽

Sheng-Nan Jia ◽

Xi Jiang ◽

Yu Zhang ◽

...

Keyword(s):

Gene Expression ◽

Alkaline Phosphatase ◽

Cell Adhesion ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Related Gene ◽

Titanium Surfaces

The effect of alkali-treated titanium surfaces on inflammation-related gene expression of macrophages and alkaline phosphatase activity of osteoblast-like cells.

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Osteosphere Model to Evaluate Cell–Surface Interactions of Implantable Biomaterials

Materials ◽

10.3390/ma14195858 ◽

2021 ◽

Vol 14 (19) ◽

pp. 5858

Author(s):

Ana Carolina Batista Brochado ◽

Victor Hugo de Souza ◽

Joice Correa ◽

Suzana Azevedo dos Anjos ◽

Carlos Fernando de Almeida Barros Mourão ◽

...

Keyword(s):

Cell Surface ◽

3D Model ◽

Bone Cells ◽

Three Dimensional ◽

Surface Interactions ◽

Force Microscopy ◽

Tissue Therapy ◽

Cell Surface Interactions ◽

Titanium Surfaces

Successful biomaterials for bone tissue therapy must present different biocompatible properties, such as the ability to stimulate the migration and proliferation of osteogenic cells on the implantable surface, to increase attachment and avoid the risks of implant movement after surgery. The present work investigates the applicability of a three-dimensional (3D) model of bone cells (osteospheres) in the evaluation of osteoconductive properties of different implant surfaces. Three different titanium surface treatments were tested: machined (MA), sandblasting and acid etching (BE), and Hydroxyapatite coating by plasma spray (PSHA). The surfaces were characterized by Scanning Electron Microscopy (SEM) and atomic force microscopy (AFM), confirming that they present very distinct roughness. After seeding the osteospheres, cell–surface interactions were studied in relation to cell proliferation, migration, and spreading. The results show that BE surfaces present higher densities of cells, leaving the aggregates towards than titanium surfaces, providing more evidence of migration. The PSHA surface presented the lowest performance in all analyses. The results indicate that the 3D model allows the focal analysis of an in vitro cell/surfaces interaction of cells and surfaces. Moreover, by demonstrating the agreement with the clinical data observed in the literature, they suggest a potential use as a predictive preclinical tool for investigating osteoconductive properties of novel biomaterials for bone therapy.

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Platelet Adhesion on Commercially Pure Titanium Plates in Vitro II. Immunofluorescence Visualization of PDGF-B, TGFβ1, and PPARγ Released from Activated Adherent Platelets

Dentistry Journal ◽

10.3390/dj7040109 ◽

2019 ◽

Vol 7 (4) ◽

pp. 109 ◽

Cited By ~ 2

Author(s):

Tetsuhiro Tsujino ◽

Akira Takahashi ◽

Taisuke Watanabe ◽

Kazushige Isobe ◽

Yutaka Kitamura ◽

...

Keyword(s):

Industrial Development ◽

Platelet Adhesion ◽

Alveolar Bone ◽

Platelet Rich Plasma ◽

Pure Titanium ◽

Commercially Pure Titanium ◽

Commercially Pure ◽

Titanium Surfaces ◽

Cp Ti

Recent progress in the industrial development of dental implants has improved their surface bio-affinity, while clinical implantologists attempt to improve it through coating with various compounds, including platelet-rich plasma (PRP) in clinical settings. However, it is poorly understood how PRP acts on titanium surfaces. To validate this surface modification method and demonstrate how platelet-derived soluble biomolecules released from the activated adherent platelets act on plain, commercially pure-titanium (cp-Ti) plates, we evaluated the distribution of biomolecules by immunofluorescence. PPARγ, PDGF-B, and TGFβ1 were similarly released at immunofluorescence levels from activated adherent platelets, retained in the surrounding extra-platelet spaces for a while, and did not immediately diffuse away to distant spaces. Exogenously added CaCl2 augmented release and retention of those biomolecules along with activation and aggregation. Taken together with our previous data regarding platelet adhesion, these findings suggest that especially when treated with CaCl2, platelets immediately adhere on cp-Ti plates to release their stored biomolecules in the absence of plasma proteins and that these biomolecules do not diffuse away, but stay longer in extra-platelet spaces around the platelets by newly formed, immature fibrin fiber fragments. Consequently, these retained biomolecules are anticipated to cooperatively stabilize implants by stimulating alveolar bone regeneration and integration.

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Different Effects of Cisplatin and Transplatin on the Higher-Order Structure of DNA and Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms21010034 ◽

2019 ◽

Vol 21 (1) ◽

pp. 34 ◽

Cited By ~ 2

Author(s):

Toshifumi Kishimoto ◽

Yuko Yoshikawa ◽

Kenichi Yoshikawa ◽

Seiji Komeda

Keyword(s):

Gene Expression ◽

Nuclear Dna ◽

Biological Effects ◽

Inhibitory Effect ◽

Higher Order ◽

Luciferase Assay ◽

Order Structure ◽

Force Microscopy ◽

Order Of Magnitude

Despite the effectiveness of cisplatin as an anticancer agent, its trans-isomer, transplatin, is clinically ineffective. Although both isomers target nuclear DNA, there is a large difference in the magnitude of their biological effects. Here, we compared their effects on gene expression in an in vitro luciferase assay and quantified their effects on the higher-order structure of DNA using fluorescence microscopy (FM) and atomic force microscopy (AFM). The inhibitory effect of cisplatin on gene expression was about 7 times that of transplatin. Analysis of the fluctuation autocorrelation function of the intrachain Brownian motion of individual DNA molecules showed that cisplatin increases the spring and damping constants of DNA by one order of magnitude and these visco-elastic characteristics tend to increase gradually over several hours. Transplatin had a weaker effect, which tended to decrease with time. These results agree with a stronger inhibitory effect of cisplatin on gene expression. We discussed the characteristic effects of the two compounds on the higher-order DNA structure and gene expression in terms of the differences in their binding to DNA.

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Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy

Blood ◽

10.1182/blood.v101.3.929 ◽

2003 ◽

Vol 101 (3) ◽

pp. 929-936 ◽

Cited By ~ 38

Author(s):

Dipti Patel ◽

Heikki Väänänen ◽

Markéta Jiroušková ◽

Thomas Hoffmann ◽

Carol Bodian ◽

...

Keyword(s):

Platelet Adhesion ◽

Thrombus Formation ◽

Wild Type Mouse ◽

Poisson Model ◽

Human Platelets ◽

Wild Type ◽

Initial Adhesion ◽

Platelet Deposition ◽

Coated Surfaces

Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

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Preparation of Bioactive Titanium Surfaces via Fluoride and Fibronectin Retention

International Journal of Biomaterials ◽

10.1155/2012/290179 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Carlos Nelson Elias ◽

Patricia Abdo Gravina ◽

Costa e Silva Filho ◽

Pedro Augusto de Paula Nascente

Keyword(s):

Dental Implants ◽

Dental Implant ◽

Osteoblastic Cells ◽

Acid Etching ◽

Implant Surface ◽

Force Microscopy ◽

Dental Implant Surface ◽

Before And After ◽

Titanium Surfaces

Statement of Problem. The chemical or topographic modification of the dental implant surface can affect bone healing, promote accelerated osteogenesis, and increase bone-implant contact and bonding strength.Objective. In this work, the effects of dental implant surface treatment and fibronectin adsorption on the adhesion of osteoblasts were analyzed.Materials and Methods. Two titanium dental implants (Porous-acid etching and PorousNano-acid etching followed by fluoride ion modification) were characterized by high-resolution scanning electron microscopy, atomic force microscopy, and X-ray diffraction before and after the incorporation of human plasma fibronectin (FN). The objective was to investigate the biofunctionalization of these surfaces and examine their effects on the interaction with osteoblastic cells.Results. The evaluation techniques used showed that the Porous and PorousNano implants have similar microstructural characteristics. Spectrophotometry demonstrated similar levels of fibronectin adsorption on both surfaces (80%). The association indexes of osteoblastic cells in FN-treated samples were significantly higher than those in samples without FN. The radioactivity values associated with the same samples, expressed as counts per minute (cpm), suggested that FN incorporation is an important determinant of thein vitrocytocompatibility of the surfaces.Conclusion. The preparation of bioactive titanium surfaces via fluoride and FN retention proved to be a useful treatment to optimize and to accelerate the osseointegration process for dental implants.

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The Influence of the Operating Parameters of Titanium Electropolishing to Obtain Nanostructured Titanium Surfaces

Materials Science Forum ◽

10.4028/www.scientific.net/msf.727-728.1638 ◽

2012 ◽

Vol 727-728 ◽

pp. 1638-1642

Author(s):

Leonardo Marasca Antonini ◽

Rafael Gomes Mielczarski ◽

Caroline Pigatto ◽

Iduvirges Lourdes Müller ◽

Célia de Fraga Malfatti

Keyword(s):

Operating Parameters ◽

Nanostructured Titanium ◽

Nanostructured Surfaces ◽

Force Microscopy ◽

Angle Measurements ◽

Atomic Force ◽

Different Temperatures ◽

Titanium Surfaces

Titanium and Ti alloys have been widely used as biomaterial due to their mechanical properties and high in vitro and in vivo cytocompatibility. Studies have showed that the acceleration of the osseointegration process is associated to the modification of the surface morphology. The aim of this work is to study the influence of the operating parameters of titanium electropolishing to obtain nanostructured titanium surfaces. The titanium electropolishing was carried out with different temperatures (7°C, 18°C and 25°C), current density of 0.19 A/cm2 and electropolishing time of 8 minutes. After the electropolishing process the titanium samples were characterized by Atomic Force Microscopy, profilometry (mechanical profilometer) and contact angle measurements. Preliminary results showed that the Ti nanostructured surfaces formation, strongly depends on the control of operating parameters.

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