Comparing and visualizing titanium implant integration in rat bone using 2D and 3D techniques.

The aim was to compare the osseointegration of grit-blasted implants with and without a hydrogen fluoride treatment in rat tibia and femur, and to visualize bone formation using state-of-the-art 3D visualization techniques. Grit-blasted implants were inserted in femur and tibia of 10 Sprague-Dawley rats (4 implants/rat). Four weeks after insertion, bone implant samples were retrieved. Selected samples were imaged in 3D using Synchrotron Radiation-based µCT (SRµCT). The 3D data was quantified and visualized using two novel visualization techniques, thread fly-through and 2D unfolding. All samples were processed to cut and ground sections and 2D histomorphometrical comparisons of bone implant contact (BIC), bone area (BA), and mirror image area (MI) were performed. BA values were statistically significantly higher for test implants than controls (p < 0.05), but BIC and MI data did not differ significantly. Thus, the results partly indicate improved bone formation at blasted and hydrogen fluoride treated implants, compared to blasted implants. The 3D analysis was a valuable complement to 2D analysis, facilitating improved visualization. However, further studies are required to evaluate aspects of 3D quantitative techniques, with relation to light microscopy that traditionally is used for osseointegration studies.

The role of mechanical force and ROS in integrin-dependent signals.

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5ß1 or αvß3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.

Introducing a novel analysis technique for osseointegrated dental implants retrieved 29 years postsurgery.

PURPOSE: To investigate osseointegration of oral implants, which were retrieved from a patient after 29 years in situ, we use novel three-dimensional analysis methods and visualization techniques that supplement conventional two-dimensional analysis. MATERIALS AND METHODS: The sample processing involved nondecalcification and embedment in resin. Conventional two-dimensional histomorphometrical methods were conducted. Additionally, the quantification was extended to three-dimensional by using synchrotron radiation micro-computed tomography (SRµCT) technique and two relevant visualization methods for the three-dimensional data were introduced. RESULTS: The three-dimensional results involved three-dimensional quantification and visualization of two implant samples with methods beyond state-of-the-art. Traditional two-dimensional histomorphometrical results revealed a mean bone-implant contact (BIC) of about 50%. In most samples, bone area (BA) was lower inside the treads compared with out-folded mirror images, which were confirmed by the three-dimensional quantification. The BIC along four selected regions showed highest percentages in the bottom/valley region and lowest in the thread-peak region. Qualitative observations revealed ongoing bone remodeling areas in all samples. The apical hole demonstrated high osseointegration. CONCLUSION: The novel techniques including an animation and an out-folding of BIC and BA enabled a simultaneous visualization of the three-dimensional material obtained from SRµCT data. However, the two-dimensional histological sections were needed for qualitative and quantitative evaluation of osseointegration and, thus, both methods are considered equally important.

Extracting 3D information on bone remodeling in the proximity of titanium implants in SRµCT image volumes.

Bone-implant integration is measured in several ways. Traditionally and routinely, 2D histological sections of samples, containing bone and the biomaterial, are stained and analyzed using a light microscope. Such histological section provides detailed cellular information about the bone regeneration in the proximity of the implant. However, this information reflects the integration in only a very small fraction, a 10 µm thick slice, of the sample. In this study, we show that feature values quantified on 2D sections are highly dependent on the orientation and the placement of the section, suggesting that a 3D analysis of the whole sample is of importance for a more complete judgment of the bone structure in the proximity of the implant. We propose features describing the 3D data by extending the features traditionally used for 2D-analysis. We present a method for extracting these features from 3D image data and we measure them on five 3D SRµCT image volumes. We also simulate cuts through the image volume positioned at all possible section positions. These simulations show that the measurement variations due to the orientation of the section around the center line of the implant are about 30%.