Physical vapour deposition of zirconia on titanium: fabrication, characterization and interaction with human osteoblast cells.

The physical vapor deposition of zirconia was used to prepare two new titanium-based surfaces M1 and M2 with a different layer thickness. These novel surfaces were characterized for chemistry, topography and morphology by surface and solid state techniques. Primary osteoblast cells were used for in vitro studies. DAPI assay was applied for cell proliferation, while for bone sialoprotein (BSP), osteonectin and transforming growth factor-ß (TGF-ß) expression immunohistochemical analyses were employed. Materials M1 and M2 affected cell proliferation accordingly to their surface roughness with their impact on cell number being between the impact of two rough (Ticer, SS) and two smooth surfaces (Ti cp and Cercon). Different influence of the investigated materials on the osteoblastic production of BSP (all materials similar impact), ON (Cercon-higher; SS-lower for others) and TGF-ß (Cercon different) was found.

A Key Role of Autophagy in Osteoblast Differentiation on Titanium-Based Dental Implants.

Autophagy plays an important role in embryogenesis, for the maintenance of tissue homeostasis and the elimination of damaged subcellular structures. Furthermore, autophagy could be a mode of physiological cell death and also be implicated in cell differentiation. Thus, we hypothesized that autophagy may have an impact on the differentiation of osteoblast cells influenced by various titanium-based surfaces. Interactions between smooth, commercially available pure titanium (Ti cp), rough Ticer, acid-etched Ti cp (SS) and M1-M3 (comprised of the monoclinic phase of sodium-titanium oxides and rutile; M2 contains amorphous calcium phosphates) and human osteoblast cells were investigated. Immunofluorescent staining was used for detecting autophagy, cell cluster formation and collagen type I (Col-1) expression. Flow cytometry was employed to identify autophagy, the production of endogenous nitric oxide (NO) and the size and granularity of the cells. Rough surfaces caused osteoblast differentiation via the autophagic-dependent PI3/Akt signalling pathway. These surfaces induced the formation of discrete populations of large, granular cells, i.e. mature osteoblasts. In addition, M1-M3 provoked the development of a third population of small, granular cells, responsible for cell cluster formation, which are important for the formation of bone noduli and mineralisation. The same surfaces induced faster osteoblast maturation and enhanced NO production, a hallmark of the already mentioned processes. Neither the mature osteoblasts nor the small cells appeared after the inhibition of autophagy. Inhibition of autophagy also prevented cell cluster formation. We demonstrate that autophagy plays an essential role in the osteoblast differentiation on titanium-based surfaces with rough topography.

Proliferation, apoptosis and expression of non-collagenous proteins: differences between the upper and the lower jaw bone in vitro.

One of the effects observed during several screening studies for osteocompatibility in vitro was that cells derived from the upper and lower jaw exhibited distinct differences regarding proliferation. Therefore, the aim of this study was to examine systematically whether a single osteoblast possesses abilities which are specific to the upper or lower jaw. Both human maxillary and mandibular bone samples without any clinical or radiographic evidence of pathology were obtained from 4 male donors aged between 40 and 45 years. Cells were cultured for up to 25 days to investigate in vitro development. Total and apoptotic cell numbers were estimated by image analysis. Cells were identified as bone-like cells using immunocytochemical determination of bone sialoprotein (BSP) and osteocalcin expression. The number of healthy cells was significantly higher for cells of the lower jaw compared to those of the upper jaw. The number of apoptotic cells showed an inverse pattern. The expression pattern of osseo-inductive BSP correlated with the proliferation rate of the cells. The pattern of osteocalcin expression was related to the number of apoptotic cells. Our findings are new but were anticipated regarding the well-known differences in the healing process around implants in the lower jaw versus the upper jaw. Additionally, a relationship between our results and some diseases of the lower/upper jaw seems obvious. Future work on cell responses to biomaterials should define the origin of the cells more precisely.

Type IV secretion of Helicobacter pylori CagA into oral epithelial cells is prevented by the absence of CEACAM receptor expression.

BACKGROUND: Helicobacter pylori typically colonizes the human stomach, but it can occasionally be detected in the oral cavity of infected persons. Clinical outcome as a result of gastric colonization depends on presence of the pathogenicity island cagPAI that encodes a type-IV secretion system (T4SS) for translocation of the effector protein CagA and ADP-heptose. Upon injection into target cells, CagA is phosphorylated, which can be demonstrated by in vitro infection of the gastric epithelial cell line AGS, resulting in cell elongation. Here we investigated whether H. pylori can exert these responses during interaction with cells from the oral epithelium. To this purpose, three oral epithelial cell lines, HN, CAL-27 and BHY, were infected with various virulent wild-type H. pylori strains, and CagA delivery and ADP-heptose-mediated pro-inflammatory responses were monitored. RESULTS: All three oral cell lines were resistant to elongation upon infection, despite similar bacterial binding capabilities. Moreover, T4SS-dependent CagA injection was absent. Resistance to CagA delivery was shown to be due to absence of CEACAM expression in these cell lines, while these surface molecules have recently been recognized as H. pylori T4SS receptors. Lack of CEACAM expression in HN, CAL-27 and BHY cells was overcome by genetic introduction of either CEACAM1, CEACAM5, or CEACAM6, which in each of the cell lines was proven sufficient to facilitate CagA delivery and phosphorylation upon H. pylori infection to levels similar to those observed with the gastric AGS cells. Pro-inflammatory responses, as measured by interleukin-8 ELISA, were induced to high levels in each cell line and CEACAM-independent. CONCLUSIONS: These results show that lack of CEACAM receptors on the surface of the oral epithelial cells was responsible for resistance to H. pylori CagA-dependent pathogenic activities, and confirms the important role for the T4SS-dependent interaction of these receptors with H. pylori in the gastric epithelium.

Titanium dental implant surfaces obtained by anodic spark deposition - From the past to the future.

Commercial titanium-based dental implants are obtained applying various methods such as machining, acid etching, anodization, plasma spraying, grit blasting or combination techniques yielding materials with smooth or micro-roughened surfaces. Those techniques are used to optimize the surface properties and to maximize biocompatibility and bioactivity with bone tissue. Present review is focused on the material surfaces obtained by anodic spark deposition (ASD). From the early 1980s till present, the results of numerous studies have shown that anodically oxidized surfaces with different dopants express a positive effect on osteoblasts behavior in vitro and osseointegration in vivo. Those surfaces demonstrated a high biocompatibility and rapid osseointegration in clinical application. This paper provides an overview of the preparation of implant surfaces by employing ASD process. Moreover, reviewed are clinically used ASD implant surfaces (Ticer, TiUnite, Osstem, etc.). The electrolyte variations in ASD process and their influence on surface properties are given herein. Using different electrolytes, anode voltages and temperatures, the above fabrication process can yield various surface morphologies from smooth to rough, porous surfaces. Furthermore, ASD enables thickening of oxide layers and enrichment with different dopands from used electrolyte, which hinder release of potentially toxic titanium ions in surrounding tissue. Particularly exciting results were achieved by calcium and phosphorus doping of the oxide layer (Ticer, ZL Microdent; TiUnite, Nobel Biocare Holding AB) which significantly increased the osteocompatibility. Ticer, a dental implant with anodically oxidized surface and the first among similar materials employed in clinical practice, was found to promote fast osteoblast cell differentiation and mineralization processes. Moreover, Ticer accelerate the integration with the bone, increase the bone/implant contact and improve primary and secondary stability of the implants. Additionally, potential innovations in this field such as fabrication of nanotubes on the implant surfaces as well as novel approaches (e.g. coating with proteins, nanostructured topography; combining implant body and surface derived from titanium and zirconia) are elaborated in this review. Besides, biochemical aspects on implant surface cell/tissue interaction are summarized. From the clinical point of view implant surfaces fabricated by ASD technology possess fast and improved osseointegration, high healing rates and long term prognosis.

Plasma-electrochemical deposition of porous zirconia on titanium-based dental material and in vitro interactions with primary osteoblasts cells.

Three new porous zirconia-coated titanium materials using anodic plasma-electrochemical oxidation have been fabricated and characterized by scanning electron microscopy, electron probe microanalysis and X-ray diffraction. These ZrO2/TiO2 surfaces contained up to 43 wt% of ZrO2, 49 wt% TiO2 ( M1: - M3: ) and 8 wt% P2O5 ( M2: , M3: ). Zirconium titanate was detected as dominant microcrystalline phase. Primary human osteoblast cells were used for in vitro investigations. Cell proliferation and immunohistochemical analyses of morphology and expression of bone sialoprotein and osteocalcin were performed. Novel coatings M2: and M3: were shown to induce proliferation and expression of osteocalcin and bone sialoprotein to the extent comparable to that of Ticer, a material already employed in clinical practice.

First titanium dental implants with white surfaces: preparation and in vitro tests.

OBJECTIVES: To evaluate first white titanium surfaces developed for improvement of existing clinically used titanium-based implants Ticer. METHODS: The anodic plasma-electrochemical oxidation in aqueous solutions of sodium hydroxide and calcium dihydrogen phospate was used to prepare three novel anodic conversion layers with white titanium oxide surfaces. The surfaces have been characterized by the means of scanning electron microscopy, surface microanalysis and X-ray diffraction. In vitro studies were conducted on primary human osteoblast cells using novel surfaces (M1-M3) as well as commercially pure titanium (Ti cp), Ticer and SS (subtracted surface). An indirect toxicity test using MTT and SRB assays has been carried out. Furthermore, immunohistochemical analysis of cell proliferation, morphology, and expression of non-collagenous bone matrix proteins (sialoprotein, BSP, and osteocalcin, OC) were performed. RESULTS: The basic morphology of the surfaces shows clusters in a size of 100 µm of knob-like structures. The coatings are composed of rutile and monoclinic sodium titanates. Novel white surfaces (M1-M3) induced proliferation rates, morphological changes and influenced the expression of OC and BSP similarly to Ticer. On the other hand, Ti cp and SS exhibited different in vitro behavior. SIGNIFICANCE: The novel surfaces expressed similar in vitro behavior as Ticer, successfully used in clinical practice. Furthermore, due to their white color they are also promising from the esthetic point of view. The results described herein open the door toward a new generation of white titanium dental implants.

Zirconia coated titanium for implants and their interactions with osteoblast cells.

The anodic plasma-electrochemical oxidation in aqueous electrolytes of Zr(SO4)2 was used to prepare new zirconia/titania-based surfaces M1 (Ti, Zr and O: 7-10, 22-27 and 65-69 at.%) and M2 (Ti, Zr and O: 11-13, 20-23 and 64-69 at.%). The chemical composition and the microstructure of these coatings were characterized by surface and solid state techniques such as scanning electron microscopy, electron probe microanalysis, Raman spectroscopy and X-ray diffraction. These mixed oxides of ZrO2/TiO2 surfaces consist up to 84% (m/m) of ZrO2 and 16% (m/m) of TiO2. Monoclinic zirconia was detected as the dominant microcrystalline phase. In vitro studies were conducted on primary human osteoblast cells. MTT and DAPI assays were used for assessment on cell proliferation. Immunohistochemical analyses of morphology, cell cluster formation and expression of bone sialoprotein (BSP) and osteocalcin (OC) were performed. Novel surfaces M1 and M2 induced proliferation and expression of OC and BSP similarly to Ticer, used in clinical practice. Furthermore, the presence of zirconia on titanium surface has a higher beneficial effect on the osteoblast morphological changes and cell cluster formation.

Lateral alveolar ridge augmentation using a synthetic nano-crystalline hydroxyapatite bone substitution material (Ostim): preliminary clinical and histological results.

OBJECTIVES: The purpose of this preliminary two-center clinical prospective study was to evaluate the tissue composition of augmented sites after the use of a nano-crystalline hydroxyapatite (ncHA) bone substitution material by clinical and histological examinations. MATERIAL AND METHODS: A synthetic ncHA augmentation material was used without any additives in 14 patients requiring lateral ridge augmentation 6-7 months before (10 patients) or at implant placement (four patients). The ncHA material was covered by a titanium mesh for space maintenance. Clinical and radiographic parameters were evaluated and bone biopsy cores, obtained 6-7 months following augmentation, were assessed histologically and histomorphometrically. RESULTS: One patient showed gingival swelling, redness and pain at the augmentation site requiring removal of the titanium mesh 6 weeks postoperatively. In seven patients, a premature exposure of the titanium mesh without any inflammatory symptoms was noted. The width of the fixed gingival and the alveolar ridge height did not change significantly at least 6 months following augmentation (P>0.5), whereas a significant gain in alveolar ridge width (P=0.01) was noted. After a median period of prosthetic loading of 24 months, no implant was considered to be a failure. Histology revealed ncHA remnants in peripheral and central parts of biopsy cores obtained from seven patients after at least 6 months without histological symptoms of inflammation, whereas histomorphometry of bone cores revealed no significant differences of the mean percentage area of ncHA in peripheral (23.4%) and central (15.1%) parts of biopsy cores (P=0.262). The mean percentage area of bone colonizing the defect was 52.3%. CONCLUSIONS: Small amounts of ncHA were found after at least 6 months in bone biopsies. The former defect space was filled with bone. The alveolar ridge width gain was found to be significant after lateral augmentation utilizing ncHA, providing a quantitatively and qualitatively sufficient site for primary stable implant placement.

Effects of dental implant surfaces on the expression of bone sialoprotein in cells derived from human mandibular bone.

BACKGROUND: Protein adsorption is believed to be the first event that takes place after contact of natural tissue with an artificial surface. Bone sialoprotein (BSP) is one of the major noncollagenous proteins in the extracellular matrix of bone. The expression of BSP coincides with initial bone mineralization and is believed to be a center of crystallization for hydroxyapatite formation. MATERIAL/METHODS: We used a variety of four differently designed dental implant surfaces (SLA, CPT, ANOX, TICER) to investigate the effects on the development of adult human mandibular bone at days 5, 10, 15, 20, and 25 in vitro. The time course of the expression of BSP and labeling of fibroblasts was visualized immunohistochemically. The distribution patterns of cells were determined semiquantitatively on both the surface and the tissue-implant borderline. RESULTS: BSP immunoresponse increased from day 5 before decreasing after day 15 in vitro. The distribution of BSP-expressing cells changed during that time. Cell counts revealed that the time course of the settlement of cells depended on the design of the surface of the implant. The design of the border of the implant affected both the cell distribution patterns and the survivals of cells to a higher degree than did the design of the implant surface. CONCLUSIONS: Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue-engineering strategies. The hydroxyapatite-based implant material TICER could be a good scaffold to guide and promote the regeneration of bone tissue.