Calcium phosphate bone cements, functionalized with VEGF: release kinetics and biological activity.

Calcium phosphate bone cements are of great interest for bone replacement since the nanocrystalline structure allows their remodelling into native bone tissue. A strategy to accelerate vascularization of the implant region is the functionalization with vascular endothelial growth factor (VEGF), which is known to mediate angiogenesis in vivo. In this study, the release of recombinant human VEGF (rhVEGF(165)) following physical adsorption to Biocement D (BioD) and several modifications were investigated. Our data demonstrate a high VEGF binding capacity of BioD and a sustained release with a moderate initial burst. A proliferation assay using endothelial cells revealed maintenance of biological activity of VEGF after release from BioD. Release behavior of BioD was not improved by modification with mineralized collagen type I, as well as with a combination of mineralized collagen with O-phospho-L-serine and sodium citrate, respectively. In contrast, a positive impact of these modifications on the activity of released VEGF was observed; in case of the phosphoserine- and sodium citrate-modified cements, the biological efficacy of released VEGF was even higher than that of nonreleased control VEGF. We conclude that the bone implant material BioD and, especially, the phosphoserine modification may support activation of angiogenesis by delivery of VEGF in a local and sustained manner.

Electron holography of non-stained bacterial surface layer proteins.

We report transmission electron microscopy (TEM) investigations on bacterial surface layers (S-layers) which belong to the simplest biomembranes existing in nature. S-layers are regular 2D protein crystals composed of single protein or glycoprotein species. In their native form, S-layers are weak phase objects giving only poor contrast in conventional TEM. Therefore, they are usually examined negatively stained. However, staining with heavy metal compounds may cause the formation of structural artefacts. In this work, electron microscopy studies of non-stained S-layers of Bacillus sphaericus NCTC 9602 were performed. Compared to other proteins, these S-layers are found relatively stable against radiation damage. Electron holography was applied where information about phase and amplitude of the diffracted electron wave is simultaneously obtained. In spite of small phase shifts observed, the phase image reconstructed from the hologram of the non-stained S-layer is found to be sensitive to rather slight structure and thickness variations. The lateral resolution, obtained so far, is less than that of conventional electron microscopy of negatively stained S-layers. It corresponds to the main lattice planes of 12.4 nm observed in the reconstructed electron phase image. In addition, as a unique feature of electron holography the phase image provides thickness information. Thus, the existence of double layers of the protein crystals could be easily visualized by the height profile of the specimen.

Induction of apoptosis in human cancer cells by targeting mitochondria with gold nanoparticles.

A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.

Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D.

In order to generate a calcium-phosphate bone cement as a transient replacement for bone defects, we modified Biocement D (Merck Biomaterial GmbH) containing mineralised collagen with osteocalcin, the most abundant non-collageneous protein of bone. Osteocalcin was added to the cement paste during setting in order to control the crystallisation kinetics of hydroxyapatite (HAP) as well as to stimulate the interaction of osteoblasts and osteoclasts with the bone replacement material. Analysis by SEM and AFM shows, that the addition of osteocalcin causes a nanosize microstructure of the calcium cement, which can be explained by inhibited growth of HAP crystals. The fracture strength of the material decreased by incorporation of osteocalcin, pointing onto a higher defect concentration of the crystalline structure. The impact of osteocalcin onto the interaction of bone cells with HAP-Collagen I-cements was studied in a cell culture system using the human osteosarcoma cell line SAOS-2. Results suggest, that osteocalcin might possibly improve the initial adherence of osteoblast-like cells, whereas proliferation of the cells is not effected.

Investigation of the thermal stability of Ni/C multilayers by X-ray methods.

Microstructural properties of Ni/C multilayers prepared by PLD (pulsed laser deposition) have been investigated after heat treatment in vacuum at temperatures in the range of 50 degrees C to 500 degrees C. X-ray diffractometry, X-ray reflectometry, fluorescence EXAFS (extended X-ray absorption fine structure) and HREM (high resolution transmission electron microscopy) have been applied to characterize samples in the initial state and after annealing. The multilayer reflectivity remained unchanged or increased at temperatures below 400 degrees C due to sharpening of the interfaces caused by the formation of alpha-nickel and nickel carbide. The reflectivity decreased at temperatures above 400 degrees C because of the fragmentation of the nickel layers. It can be shown, that both chemical and mechanical driving forces are responsible for the observed modifications of the initial specimen state.

125Iodinated fibrinogen uptake in inflammatory joint disease as a complicating factor in the detection of deep vein thrombosis.

An enhanced uptake of 125I-fibrinogen observed in the arthritic knee joint of a patient suspected of deep vein thrombosis was confirmed in a number of patients with inflammatory joint disease in the absence of thrombosis. Whilst this is a potentially serious source of false positive diagnoses, the possibility also exists that this phenomenon could be used to evaluate the degree to which active disease is present in the joints of patients suffering from inflammatory disease.

Technetium-99m-labelled diethyl-acetanilido-iminodiacetate: a new hepatobiliary agent. A preliminary report.

A new hepatobiliary reagent 99Tcm diethyl-acetanilido-iminodiacetate has been examined in a small group of patients to evaluate its potential as a clinical reagent. The radiopharmaceutical accumulated readily in the hepatocytes and was rapidly excreted via the biliary system. It would appear possible to develop a dynamic test of hepatocyte function coupled with an evaluation of the patency of the biliary system. No adverse reactions were observed.

Thallium 201 chloride uptake in tumours, a possible complication in heart scintigraphy.

An abnormal uptake pattern of 201Tl thallous chloride observed during myocardial scintigraphy of a patient with known thoracic tumour localization suggested the possibility that thallous chloride may also accumulate in tumours. This was confirmed by observing 201thallous chloride accumulation in rhabdomyosarcoma implants in rats. Thallium 201 accumulation in tumour cells should be borne in mind as a possible complicating factor when using this reagent for the evaluation of cardiac conditions in oncological patients.

Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid.

In this study, the proliferation and differentiation of rat calvarial osteoblasts cultured on either (1) calcium-phosphate bone cement Biocement D, (2) Biocement D with 2.5% (w/w) mineralized collagen type I, or (3) Biocement D with 2.5% (w/w) mineralized collagen type I and 3% (w/w) citric acid were investigated. Incubation of the composites in cell-culture medium resulted in a fast decrease of pH and calcium concentration as well as in an increase of phosphate concentration. Although these effects occurred with all investigated materials, the lowest extent could be observed for the citric-acid-containing composites. As shown by scanning-electron microscopy, osteoblasts adhered to the composite surfaces. Proliferation and differentiation of the cells grown on the composites were found to be reduced compared to cells grown on tissue-culture polystyrene. Cells cultured in the vicinity of the composites but without direct contact also exhibited a reduced rate of proliferation, reduced alkaline phosphatase activity, and reduced mineralization. Simulating the changes in calcium and phosphate concentration occasioned by the composites through exposing cells to EGTA and phosphate gives rise to the same effects of reducing proliferation, ALP activity, and mineralization. No indication for apoptosis in cells exposed to low calcium and high phosphate concentrations was found. The number of necrotic cells, however, increased after incubation with EGTA and phosphate. For assessment of cell-composite interactions and the success of the composites in vivo, as well as for more effective material development, it seems to be important to know how changes in microenvironmental pH and ion composition of the material affect cellular proliferation and differentiation.

Lymphatic drainage in patients after replantation of extremities.

Lymph drainage was studied by means of lymph scintigraphy in eight patients in whom successful replantation of a totally or subtotally amputated extremity had been performed. Scintigrams were made after subcutaneous injection of technetium-99m in the replanted part of the patient and the contralateral, normal extremity. In all scintigrams, axillary or inguinal lymph node activity is seen, implying drainage of lymph by means of the lymph vessels. Retention of colloid in the replanted part (79 to 94 percent) shows no significant difference with the contralateral, normal side (86 to 94 percent). Unquestionable evidence of regeneration of lymphatics in humans is delivered in the three patients, in whom lymph node activity and normal retention percentages are seen on the scintigrams after total amputation of an extremity followed by replantation without anastomosing of interrupted lymph vessels.

Effects of different titanium alloys and nanosize surface patterning on adhesion, differentiation, and orientation of osteoblast-like cells.

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced patterns of parallel Ti oxide lines with different widths (0.2-10 microm) and distances (2-20 and 1,000 microm), but a common height of only 12 nm. These structures did not influence ALP activity (days 5-9), but had a positive effect on cell alignment. Two days after plating, the majority of the focal contacts were placed on the oxide lines. The portion of larger focal adhesions bridging two lines was inversely related to the line distance (2-20 microm). In contrast, the portion of aligned cells did not depend on the line distance. On average, 43% of the cells orientated parallel towards the lines, whereas 34% orientated vertically. In the control pattern (1,000 microm line distance), cell distribution was completely at random. Because a significant surplus of the cells preferred a parallel alignment, the nanosize difference in height between Ti surface and oxide lines may be sufficient to orientate the cells by contact guiding. However, gradients in electrostatic potential and surface charge density at the Ti/Ti oxide interface may additionally influence focal contact formation and cell guidance.

Scanning Force Microscopy of the Fine Structure of Cartilage Irradiated with a CO2 Laser.

Alterations of the cartilage matrix structure under non-destructive laser irradiation have been investigated by scanning force microscopy. Porcine nasal septum cartilage was irradiated with a CO2 laser with a power density of 50 W/cm(2) under two different time regimes: for 3 s and for 30 s. Short-time irradiation had little effect on the structure of the cartilage matrix. In comparison with non-irradiated cartilage, small channels of 100-400 nm in cross-section appeared. This observation gives evidence that the underlying mechanism of laser-induced stress relaxation of cartilage is based on short-time depolymerisation and subsequent re-formation of proteoglycan units. The 30 s laser treatment results in melting and denaturation of the matrix. For the first time, small crystals, 100-800 nm, were found on cut sections of the laser treated cartilage. The crystals mainly consist of resolvable sodium carbonate. Thus, they cannot be responsible for the formation of a stable cartilage configuration after laser treatment.

Initial nucleation of platinum clusters after reduction of K(2)PtCl(4) in aqueous solution: a first principles study.

The initial nucleation of platinum clusters after the reduction of K(2)PtCl(4) in aqueous solution is studied by means of first principles molecular dynamics simulations. A reaction mechanism leading to a Pt dimer is revealed both by gas-phase simulations and by simulations which model the solution environment. The key step of the observed reaction process is the formation of a Pt-Pt bond between a Pt(I) complex and an unreduced Pt(II) complex. In light of this result, we discuss the reduction process leading to the formation of platinum nanoparticles. In the generally accepted model, the nucleation of Pt particles starts only when a critical concentration of Pt(0) atoms is reached. Here, we discuss a complementary mechanism where metal-metal bonds form between Pt complexes in higher oxidation states. This is consistent with a number of experimental results which show that a high concentration of zerovalent atoms is not necessary to start the nucleation.

Use of a mineralized collagen membrane to enhance repair of calvarial defects in rats.

The aim of the present study was to evaluate mineralized collagen membranes for enhancement of bone regeneration in calvarial defects. Forty adult female Sprague-Dawley rats received calvarial full thickness defects with a diameter of 8 mm. In 20 animals, the defects were covered with a mineralized collagen membrane, and 20 animals served as controls. After 6, 13, 26 and 52 weeks, bone regeneration was evaluated using undecalcified thick-section histometry. There was no clear enhancement of bone regeneration during the first 26 weeks after the operation. Bone regeneration underneath the membrane produced consistently thicker bone, albeit without statistical significance. Accumulation of membrane material occurred in the center of the defects surrounded by multinuclear giant cells during early stages of healing. After complete resorption of the membrane, significantly increased bone formation was seen after 52 weeks in the defects that had received membrane coverage. It was concluded that mineralization in the present form did not increase mechanical strength of the membrane to prevent interference of the membrane with bone regeneration in the defect. The reason for the increase in bone formation after resorption of the membrane after 26 weeks remains to be clarified.

Phosphoserine--a convenient compound for modification of calcium phosphate bone cement collagen composites.

Temporary bone replacement materials on the basis of calcium phosphates and hydroxyapatite (HAP) are used in surgery for filling bone defects. Components which are able to control the nucleation and crystal growth of HAP through their functional groups and which can additionally activate bone cells may be helpful in the development of materials with enhanced remodelling in vivo. In this study, the influence of O-phospho-L-serine (PS) on the materials properties of calcium phosphate bone cement composites was investigated. For up to an addition of 25 mg/g PS a strong increase in the stability of the cements under load was determined. The material was studied by scanning electron microscopy and transmission electron microscopy. A more dense microstructure and a plate-like morphology of the HAP-crystals were detected in the modified composites compared with the non-modified samples. By X-ray powder diffraction an inhibition of the dissolution of alpha-tricalcium phosphate (alpha-TCP) and dicalciumphosphate anhydrous (DCPA) particles was found. alpha-TCP and DCPA are the main constituents of the cement precursor. The results of cell culture studies using rat calvaria osteoblasts demonstrate a good viability of the cells on the PS-modified material. Furthermore, the proliferation and differentiation were found to be enhanced on the PS-modified material.