Step down Vascular Calcification Analysis using State-of-the-Art Nanoanalysis Techniques.

New insights into the architecture and formation mechanisms of calcific lesions down to the nanoscale open a better understanding of atherosclerosis and its pathogenesis. Scanning electron - and atomic force microscope based nano-analytical characterization techniques were adapted to the assessment of an ex-vivo calcified coronary artery. Human atherosclerotic tissue and bone tissue reside a typical chemistry of Magnesium and Sodium rich Calcium phosphates, identified as whitlockite and Calcium apatite, respectively. Despite the obvious similarities in both chemistry and crystallography, there are also clear differences between calcified vascular tissue and bone such as the highly oriented growth in bone, revealing meso-crystal character, as opposed to the anisotropic character of calcified vascular lesions. While the grain size in vascular calcified plaques is in the range of nanometers, the grain size in bone appears larger. Spherical calcific particles present in both the coronary artery wall and embedded in plaques reveal concentric layers with variations in both organic content and degree of hydration.

Development and evaluation of a device for simultaneous uniaxial compression and optical imaging of cartilage samples in vitro.

In this paper, we present a system that allows imaging of cartilage tissue via optical coherence tomography (OCT) during controlled uniaxial unconfined compression of cylindrical osteochondral cores in vitro. We describe the system design and conduct a static and dynamic performance analysis. While reference measurements yield a full scale maximum deviation of 0.14% in displacement, force can be measured with a full scale standard deviation of 1.4%. The dynamic performance evaluation indicates a high accuracy in force controlled mode up to 25 Hz, but it also reveals a strong effect of variance of sample mechanical properties on the tracking performance under displacement control. In order to counterbalance these disturbances, an adaptive feed forward approach was applied which finally resulted in an improved displacement tracking accuracy up to 3 Hz. A built-in imaging probe allows on-line monitoring of the sample via OCT while being loaded in the cultivation chamber. We show that cartilage topology and defects in the tissue can be observed and demonstrate the visualization of the compression process during static mechanical loading.

Ex vivo human trabecular bone model for biocompatibility evaluation of calcium phosphate composites modified with spray dried biodegradable microspheres.

Our aim was to study the suitability of the ex-vivo human trabecular bone bioreactor ZetOS to test the biocompatibility of calcium phosphate bone cement composites modified with spray dried, drug loaded microspheres. We hypothesized, that this bone bioreactor could be a promising alternative to in vivo assessment of biocompatibility in living human bone over a defined time period. Composites consisting of tetracycline loaded poly(lactic-co-glycolic acid) microspheres and calcium phosphate bone cement, were inserted into in vitro cultured human femora head trabecular bone and incubated over 30 days at 37°C in the incubation system. Different biocompatibility parameters, such as lactate dehydrogenase activity, alkaline phosphatase release and the expression of relevant cytokines, IL-1ß, IL-6, and TNF-α, were measured in the incubation medium. No significant differences in alkaline phosphatase, osteocalcin, and lactate dehydrogenase activity were measured compared to control samples. Tetracycline was released from the microspheres, delivered and incorporated into newly formed bone. In this study we demonstrated that ex vivo biocompatibility testing using human trabecular bone in a bioreactor is a potential alternative to animal experiments since bone metabolism is still maintained in a physiological environment ex vivo.

Effect of bone morphogenetic protein-2 on tendon-bone integration in an in vitro cell culture.

The goal of this study was to evaluate the influence of bone morphogenetic protein-2 (BMP-2) on tendon-bone integration in a bovine in vitro cell culture. Seventy-two bovine tendons were cultivated over 3 months. The effects of BMP-2 were evaluated by generation in 4 subgroups. The groups differed in 2 parameters: the application of BMP-2 and the application of primary bovine osteoblasts. Results were analyzed biochemically by determining alkaline phosphatase activity and histologic tendon calcification, both markers for graft incorporation. Histological analysis demonstrated a positive effect of BMP-2 on the production of extracellular matrix and therefore the induction of osteogenesis. In addition, the results showed a superior cell ingrowth on the tendon in the BMP-2-stimulated groups. Calcium carbonate-like structures and organized ossification zones could only be detected in the BMP-2-stimulated tendons. The histological results matched those of the biochemical alkaline phosphatase analysis. The highest alkaline phosphatase activity was detected using BMP-2 stimulation in the first month (P<.001). High alkaline phosphatase values suggest high osteoblast activity and a high potential for mineralization. Furthermore, a positive effect of BMP-2 on fibroblasts existed with regard to the overall integration process. These results confirm the positive influence and triggering effect of BMP-2 on the mineralization process. Bone morphogenetic protein-2 seems to accelerate and optimize tendon-bone integration in the early process of graft incorporation. Besides the influence of BMP-2 on bovine osteoblasts, an additional positive effect of BMP-2 on bovine fibroblasts was detected; therefore, graft incorporation may be carried out by osteoblasts and fibroblasts.

Cement interdigitation and bone-cement interface after augmenting fractured vertebrae: A cadaveric study.

BACKGROUND: The treatment of painful osteoporotic vertebral compression fractures with transpedicular cement augmentation has grown significantly over the last 20 years. There is still uncertainty about long-term and midterm effects of polymethyl methacrylate in trabecular bone. Preservation of the trabecular structures, as well as interdigitation of the cement with the surrounding bone, therefore has been gaining increasing attention. Interdigitation of cement is likely relevant for biological healing and the biomechanical augmentation process. In this study a cutting and grinding technique was used to evaluate the interdigitation for 4 augmentation techniques. METHODS: By use of a standardized protocol, wedge fractures were created in vertebrae taken from a fresh-frozen spine. Thereafter the vertebrae were assigned to 1 of 4 similar groups with regard to the vertebral size and force required to produce the fracture. The 4 groups were randomized to the following augmentation techniques: balloon kyphoplasty, radiofrequency (RF) kyphoplasty, shield kyphoplasty, and vertebral stenting. Histologic analysis was designed to examine the bone structure and interdigitation after the augmentation. RESULTS: For the void-creating procedures, the distance between bone and cement was 341.4 ± 173.7 µm and 413.6 ± 167.6 µm for vertebral stenting and balloon kyphoplasty, respectively. Specifically, the trabecular bone was condensed around the cement, forming a shield of condensed bone. The procedures without a balloon resulted in shorter distances of 151.2 ± 111.4 µm and 228.1 ± 183.6 µm for RF and shield kyphoplasty, respectively. The difference among the groups was highly significant (P < .0001). The percentage of interdigitation was higher for the procedures that did not use a balloon: 16.7% ± 9.7% for balloon kyphoplasty, 20.5% ± 12.9% for vertebral stenting, 66.45% ± 12.35% for RF kyphoplasty, and 48.61% ± 20.56% for shield kyphoplasty. The difference among the groups was highly significant (P < .00001). CONCLUSIONS: Cavity-creating procedures reduce the cement interdigitation significantly and may accordingly reduce the effectiveness of the augmentation procedures.

Comparison of optical coherence tomography, microcomputed tomography, and histology at a three-dimensionally imaged trabecular bone sample.

We investigate optical coherence tomography (OCT) as a method for imaging bone. The OCT images are compared directly to those of the standard methods of bone histology and microcomputed tomography (microCT) on a single, fixed human femoral trabecular bone sample. An advantage of OCT over bone histology is its noninvasive nature. OCT also images the lamellar structure of trabeculae at slightly higher contrast than normal bone histology. While microCT visualizes the trabecular framework of the whole sample, OCT can image additionally cells with a penetration depth limited approximately to 1 mm. The most significant advantage of OCT, however, is the absence of toxic effects (no ionizing radiation), i.e., continuous images may be made and individual cell tracking may be performed. The penetration depth of OCT, however, limits its use to small animal models and small bone organ cultures.

Polyethylene and cobalt-chromium molybdenium particles elicit a different immune response in vitro.

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Particles of prosthetic material stimulate immune competent cells to release cytokines, which may cause bone loss and loosening of the prosthesis. This study examined the following hypothesis. Polyethylene and titanium particles elicit a different immune response in vitro. To test these hypotheses, we used the human bone marrow cell culture model that we have established and previously used to examine particle associated cytokine release. Ultra high molecular weight polyethylene (UHMW-PE) induced a proliferation of CD14 positive cells (monocytes/macrophages) whereas cobalt chromium molybdenium (CoCrMb) particles demonstrated an increased proliferation of CD66b positive cells (granulocytes). Light and scanning microscopic evaluation revealed that the UHMW-PE particles, which have built large clusters of particles (Ø7, 5 microm), were mainly surrounded by the cells and less phagocytosed. On the other hand the smaller particles from CoCrMb have been phagocytosed by the cells. These results provide strong support for our hypothesis: that wear particles derived from prosthetic materials of different material can elicit significantly different biologic responses. In summary the results suggest that the "in vitro" response to wear particles of different biomaterials should be investigated by culture systems of various lineages of cells.

Correlation of in vitro and in vivo results of vacuum plasma sprayed titanium implants with different surface topography.

Research has proven that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The purpose of this study was to evaluate the correlation between the expression of bone cell-associated proteins to Vacuum Plasma-Sprayed Titanium implants (VPS-Ti) with different surface textures in vitro and the bone integration in vivo. The biological performances of the surfaces were evaluated over a period of 8 weeks using human bone marrow cell cultures and Göttinger mini pigs. Cells were cultured on VPS-Ti with two respectively different surface-roughnesses (Ra). The level of Osteoprotegerin (OPG), Osteocalcin (OC) and alkaline phosphatase activity (ALP) were evaluated. The bone integration in vivo was evaluated by histomorphological analyses. A cancellous structured titanium (CS-Ti) construct was used as reference material in both study designs. Comparison of data was conducted using the Scheffé tests and the paired t-test with Bonferroni's correction. A comparative analysis was done to measure the degree of association between the in vitro and in vivo data. A total amount of OC was significantly increased for VPS-Ti for cells cultured on both VPS-Ti and CS-Ti, while OPG was only detectable after 8 weeks without any significant differences. The ALP activity on all surfaces was not statistically increased. For VPS-Ti with Ra ranging from 0.025 mm up to 0.059 mm, bone integration response was increased, but there was no statistical difference between the VPS-Ti. Expression of OPG, OC and ALP correlated with the histomorphological data over the 8-week period. The in vitro data suggest the superiority of VPS-Ti over CS-Ti, but more importantly, the biocompatibility of testing an in vitro model to predict the outcome and possible integration of implants in vivo.