Hans-Ludwig Graf

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.

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.

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.

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.

Biocompatibility of the titanium-based implant surfaces: Effect of the calcium dihydrogen phosphate on osteoblast cells

Investigated was the influence of the presence of calcium dihydrogen phosphate in acidic media on titanium-based implant surfaces (Ticer), used in clinical practice, and its white form (Ticer white), on osteoblast cells. Novel surfaces, denoted M1 and M2 , were obtained by immersing Ticer and Ticer white surfaces in calcium dihydrogen phosphate solution at pH 3.5. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) investigations were performed to characterize the surfaces. Comparative results of interaction between investigated surfaces and human osteoblast cells from indirect biocompatibility (MTT and SRB assays), proliferation (DAPI assay) and mode of cell death (acridine orange/ethidium bromide (AO/EB) double staining) were found to be in good agreement, as well as findings from osteocalcin (OC) and bone sialoprotein (BSP) expression. Surfaces obtained by employing anodic plasma-electrochemical oxidation with spark discharges, without subsequent surface modifications were found to be more compatible. Soaking Ticer and Ticer white in phosphate solution resulted in toxic materials ( M1 and M2 ) which induced apoptosis and secondary necrosis in osteoblast cells.

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-β (TGF-β) 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-β (Cercon different) was found.

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.