Marcus Oliver Klein

Bisphosphonates: restrictions for vasculogenesis and angiogenesis: inhibition of cell function of endothelial progenitor cells and mature endothelial cells in vitro.

Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is one of the main side effects in patients treated with bisphosphonates for metastasis to the bone or osteoporosis. BP-ONJ usually occurs in patients treated with highly potent nitrogen-containing bisphosphonates. The exact mechanism of action and etiopathology is still unknown. In addition to inhibition of bone remodelling, an anti-angiogenetic effect has become the focus of research. The aim of these study was to investigate the effect of different bisphosphonates on human umbilicord vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), which play an important role in angiogenesis. Using varying concentrations, the impact of one non-nitrogen-containing bisphosphonate (clodronate) and three nitrogen-containing bisphosphonates (ibandronate, pamidronate and zoledronate) on HUVEC and EPC was analysed. The biologic behaviour of HUVEC after incubation with different bisphosphonates was measured in a Boyden migration assay as well as in a 3D angiogenesis assay. The number of apoptotic cells was measured by Tunnel assay. To underline the importance of neoangiogenesis in the context of BP-ONJ, we measured the EPC number after incubation with different bisphosphonates in vitro. HUVEC and EPC were significantly influenced by bisphosphonates at different concentrations compared with the non-treated control groups. The nitrogen-containing bisphosphonates pamidronate and zoledronate had the greatest impact on the cells, whereas clodronate followed by ibandronate was less distinct on cell function. These results underline the hypothesis that inhibited angiogenesis induced by bisphosphonates might be of relevance in the development and maintenance of BP-ONJ. The increased impact by highly potent bisphosphonates on HUVEC and EPC may explain the high prevalence of BP-ONJ in patients undergoing this treatment.

Submicron Scale-Structured Hydrophilic Titanium Surfaces Promote Early Osteogenic Gene Response for Cell Adhesion and Cell Differentiation

BACKGROUND AND PURPOSE Titanium (Ti) surface roughness and surface hydrophilicity are key factors to regulate osteogenic cell responses during dental implant healing. In detail, specific integrin-mediated interactions with the extracellular environment trigger relevant osteogenic cell responses like differentiation and matrix synthesis via transcriptions factors. Aim of this study was to monitor surface-dependent osteogenic cell adhesion dynamics, proliferation, and specific osteogenic cell differentiation over a period of 7 days. MATERIALS AND METHODS Ti disks were manufactured to present smooth pretreatment (PT) surfaces and rough sandblasted/acid-etched (SLA) surfaces. Further processing to isolate the uncontaminated TiO(2) surface from contact with atmosphere provided a highly hydrophilic surface without alteration of the surface topography (modSLA). Tissue culture polystyrene (TCPS) served as control. Human osteogenic cells were cultivated on the respective substrates. After 24 hours, 48 hours, 72 hours, and 7 days, cell morphology on the Ti substrates was visualized by scanning transmission electron microscopy. As a marker of cellular proliferation, cell count was assessed. For the analysis of cell adhesion and differentiation, specific gene expression levels of the integrin subunits β1 and αv, runx-2, collagen type Iα (COL), alkaline phosphatase (AP), and osteocalcin (OC) were obtained by real-time RT-PCR for the respective time points. Data were normalized to internal controls. RESULTS TCPS and PT surfaces preserved a rather immature, dividing osteogenic phenotype (high proliferation rates, low integrin levels, and low specific osteogenic cell differentiation). SLA and especially modSLA surfaces promoted both cell adhesion as well as the maturation of osteogenic precursors into post-mitotic osteoblasts. In detail, during the first 48 hours, modSLA resulted in lowest cell proliferation rates but exhibited highest levels of the investigated integrins, runx-2, COL, AP, and OC. CONCLUSION Our results revealed a strong synergistic effect between submicron-scale roughness and surface hydrophilicity on early osteogenic cell adhesion and maturation.

Use of a new cross-linked collagen membrane for the treatment of dehiscence-type defects at titanium implants: a prospective, randomized-controlled double-blinded clinical multicenter study.

OBJECTIVES The aim of the present randomized-controlled double-blinded clinical multicenter study was to assess the use of either a new cross-linked (VN) or a native collagen membrane (BG) for the treatment of dehiscence-type defects at titanium implants. MATERIAL AND METHODS A total of n=54 patients were recruited in four German university clinics. According to a parallel-groups design, dehiscence-type defects at titanium implants were filled with a natural bone mineral and randomly assigned to either VN or BG. Submerged sites were allowed to heal for 4 months. Primary (e.g., changes in defect length - DeltaDL, quality of newly formed tissue [0-4] - TQ) and secondary parameters (e.g., membrane exposure, tissue conditions at dehisced sites) were consecutively recorded. RESULTS Four patients were excluded due to an early wound infection (VN:3; BG:1), and one patient was lost during follow-up (VN). The mean DeltaDL was 3.0 +/- 2.5 mm in the VN, and 1.94 +/- 2.13 mm in the BG group. The assessment of TQ revealed comparable mean values in both groups (VN: 3.05 +/- 1.66, BG: 3.46 +/- 1.48). A significant correlation between membrane exposure and inflammation of the adjacent soft tissue was observed in the VN group. In both groups, the mean DL and TQ values were not significantly different at either non-exposed or exposed implant sites. CONCLUSION The results of the present study have indicated that VN supported bone regeneration on a level non-inferior to BG. However, in case of a premature membrane exposure, cross-linking might impair soft-tissue healing or may even cause wound infections.

Long‐term response of osteogenic cells on micron and submicron‐scale‐structured hydrophilic titanium surfaces: sequence of cell proliferation and cell differentiation

OBJECTIVE Modifications of surface topography and surface chemistry are key factors for guiding target cells during dental implant healing. Recent in vitro studies confirmed promotion of early osteogenic cell differentiation on submicron scaled surfaces in particular when hydrophilized. However, no long-term observations on both osteogenic cell proliferation as well as on cell maturation have been reported for respectively modified surfaces. Aim of this study was to monitor osteogenic cell proliferation and expression of specific osteogenic cell differentiation markers on a protein level over an extended period of 3 weeks with respect to surface modifications. MATERIAL AND METHODS Modified titanium (Ti) disks were obtained from Institute Straumann, representing the following surfaces: smooth pretreatment (PT), sandblasted/acid etched (SLA), and hydrophilized (modSLA). Surface topography was analyzed by scanning electron microscopy, surface elemental composition was assessed by X-Ray Photoelectronic Spectroscopy (XPS). Tissue culture polystyrene (TCPS) served as a control substrate. Human osteogenic cells (HOB-c) were cultivated on the respective substrates. After 24 hrs, 48 hrs, 72 hrs, 7 d, 14 d and 21 d, cell count was assessed as well as osteogenic cell differentiation utilizing cellular Quantitative Immuno-Cytochemistry (QIC) assay for collagen type I (COL), alkaline phosphatase (AP), osteopontin (OPN) and osteocalcin (OC). Data were normalized with respect to internal controls. RESULTS In contrast to the other modified Ti disks, modSLA stands out due to low surface carbon contamination. TCPS and PT surfaces preserved a rather immature, mitotic active osteogenic phenotype (high proliferation rates, no increase of OC production), SLA and especially modSLA surfaces promoted the maturation of osteogenic precursors into post-mitotic osteoblasts. In detail, modSLA resulted in lowest cell proliferation rates, but exhibited highest expression rates of OC. DISCUSSION Our results, which confirm previous studies, reveal long-term promotion of osteogenic cell maturation by topography (micron and submicron scale roughness) and surface hydrophilicity.

Primary Stability of a Hybrid Self-Tapping Implant Compared to a Cylindrical Non-Self-Tapping Implant with Respect to Drilling Protocols in an Ex Vivo Model

BACKGROUND Modifications of implant design have been intending to improve primary stability. However, little is known about investigation of a hybrid self-tapping implant on primary stability. PURPOSES The aims of this study were to evaluate the primary stability of two hybrid self-tapping implants compared to one cylindrical non-self-tapping implant, and to elucidate the relevance of drilling protocols on primary stability in an ex vivo model. MATERIALS AND METHODS Two types of hybrid self-tapping implants (Straumann® Bone Level implant [BL], Straumann® Tapered Effect implant [TE]) and one type of cylindrical non-self-tapping implant (Straumann® Standard Plus implant [SP]) were investigated in the study. In porcine iliac cancellous bones, 10 implants each were inserted either using standard drilling or under-dimensioned drilling protocol. The evaluation of implant-bone interface stability was carried out by records of maximum insertion torque, the Periotest® (Siemens, Bensheim, Germany), the resonance frequency analysis (RFA), and the push-out test. RESULTS In each drilling group, the maximum insertion torque values of BL and TE were significantly higher than SP (p=.014 and p=.047, respectively). In each group, the Periotest values of TE were significantly lower than SP (p=.036 and p=.033, respectively). The Periotest values of BL and TE were significantly lower in the group of under-dimensioned drilling than standard drilling (p=.002 and p=.02, respectively). In the RFA, no statistical significances were found in implants between two groups and between implants in each group. In each group, the push-out values of BL and TE were significantly higher than SP (p=.006 and p=.049, respectively). CONCLUSION Hybrid self-tapping implants could achieve a high primary stability which predicts them for use in low-density bone. However, there is still a debate to clarify the influence of under-dimensioned drilling on primary stability.

Pore characteristics of bone substitute materials assessed by microcomputed tomography

OBJECTIVES Pore configurations of alloplastic biomaterial scaffolds play a major role for new bone formation in vivo. Current studies on characteristics of pores in bone substitute materials focus on individual particles or single blocks. Thus, three-dimensional (3-D) architecture of particle aggregates, representing the clinical relevant in vivo situation is not adequately taken into account. The aim of this study was the visualization and quantification of pore properties, both of the scaffold structure of single particles as well as of the micro-morphology of complex 3-D aggregated particle-conglomerates. MATERIAL AND METHODS In model experiments, standardized plexiglass cylinders were stuffed with commercial bone substitute material particles with diverse chemical composition (HA, beta-TCP, HA-SiO(2), HA-beta-TCP, bioactive glass), origin (phycogenic, bovine, synthetic) and granulation (50 mum-2000 mum). Analogue to establish procedures for native (human) bone samples, non-fixed bone substitute materials were scanned by high-resolution microcomputed tomography. In addition to computer animated two-dimensional and 3-D reconstruction of the samples, median pore thickness and pore size distribution were determined. Materials representative for their chemical constitution were documented by SEM imaging. RESULTS Investigated specimens significantly were different in micro-morophology and pore properties, ranging from highly porous to rather solid. The most voluminous pores were localized interparticularly. Within one product line, the determined pore properties showed a significant correlation with single particle grain sizes. CONCLUSION The generation and interpretation of micro-CT based 3-D pore models can provide further insight into the expected osteoconduction dynamics and therefore might serve as a basis for further modifications of scaffold size and geometry as well as for further invasive studies on the biological behaviour of the scaffolds.

Adsorption and Conformation Behavior of Biotinylated Fibronectin on Streptavidin-Modified TiOX Surfaces Studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.

Promotion of Osteogenic Cell Response Using Quasicovalent Immobilized Fibronectin on Titanium Surfaces: Introduction of a Novel Biomimetic Layer System

PURPOSE Despite the undeniable potential of cell adhesion molecules such as fibronectin to support osteogenic cell responses and consecutive dental implant healing, the most beneficial mode of application onto titanium implant surfaces still requires investigation. Unspecific fibronectin adsorption on titanium dioxide (TiO(2)) surfaces can result in low-loading, high-desorption rates and protein-metal interactions with impaired biologic activity. The aim of the present study was to monitor the osteogenic cell responses (cell adhesion, proliferation, and differentiation) specifically to fibronectin biofunctionalized TiO(2). MATERIALS AND METHODS An innovative biomimetic streptavidin-biotin layer system allows flexible, but stable, specific binding of biotinylated biomolecules such as fibronectin on TiO(2) surfaces. Transparent glass disks were sputtered with TiO(2). The biomimetic layer system was immobilized by self-assembly and consisted of silane, biotin-derivate, streptavidin, and biotinylated fibronectin (bFN). For the control group, unbiotinylated fibronectin was directly coated onto TiO(2). Early cell adhesion dynamics were quantified using automated processing of light microscopy images within the first 24 hours. Relative mRNA expression of integrin-β1, cyclin D1, runt-related gene 2, alkaline phosphatase, and osteocalcin was obtained using quantitative real-time polymerase chain reactions 3 and 7 days after incubation. RESULTS Although untreated TiO(2) preserved a rather immature osteogenic phenotype, both unbiotinylated fibronectin and bFN promoted osteogenic cell adhesion and cell differentiation. In particular, runt-related gene 2 expression was significantly promoted by bFN after 3 days. In contrast, cyclin D1 expression was decreased for unbiotinylated fibronectin and bFN after 7 days. CONCLUSIONS The introduced biomimetic layer system contributes a coherent immobilization approach of adhesion molecules with promotion of osteogenic cell response in vitro.

Analysis of implant-failure predictors in the posterior maxilla: A retrospective study of 1395 implants

The aim of this study was to analyze predictors for dental implant failure in the posterior maxilla. A database was created to include patients being treated with dental implants posterior to the maxillary cuspids. Independent variables thought to be predictive of potential implant failure included (1) sinus elevation, (2) implant length, (3) implant diameter, (4) indication, (5) implant region, (6) timepoint of implant placement, (7) one-vs. two-stage augmentation, and (8) healing mode. Cox regression analysis was used to evaluate the influence of predictors 1-3 on implant failure as dependent variable. The predictors 4-9 were analyzed strictly descriptively. The final database included 592 patients with 1395 implants. The overall 1- and 5-year implant survival rates were 94.8% and 88.6%, respectively. The survival rates for sinus elevation vs. placement into native bone were 94.4% and 95.4%, respectively (p = 0.33). The survival rates for the short (<10 mm), the middle (10-13 mm) and the long implants (>13 mm) were 100%, 89% and 76.8%, respectively (middle-vs. long implants p = 0.62). The implant survival rates for the small- (<3.6 mm), the middle- (3.6-4.5 mm) and the wide diameter implants (>4.5 mm) were 92.5%, 87.9% and 89.6%, respectively (p = 0.0425). None of the parameters evaluated were identified as predictor of implant failure in the posterior maxilla.

Streptavidin-coated TiO2 surfaces are biologically inert: Protein adsorption and osteoblast adhesion studies †

Non-fouling TiO2 surfaces are attractive for a wide range of applications such as biosensors and medical devices, where biologically inert surfaces are needed. Typically, this is achieved by controlled surface modifications which prevent protein adsorption. For example, polyethylene glycol (PEG) or PEG-derived polymers have been widely applied to render TiO2 surfaces biologically inert. These surfaces have been further modified in order to achieve specific bio-activation. Therefore, there have been efforts to specifically functionalize TiO2 surfaces with polymers with embedded biotin motives, which can be used to couple streptavidin for further functionalization. As an alternative, here a streptavidin layer was immobilized by self-assembly directly on a biotinylated TiO2 surface, thus forming an anti-adhesive matrix, which can be selectively bio-activated. The anti-adhesive properties of these substrates were analyzed by studying the interaction of the surface coating with fibronectin, lysozym, and osteoblast cells using surface plasmon resonance spectroscopy, atomic force microscopy, and light microscopy. In contrast to non-modified TiO2 surfaces, streptavidin-coated TiO2 surfaces led to a very biologically inert substrate, making this type of surface coating a promising alternative to polymer coatings of TiO2 surfaces.