R.P. Franke

Biostimulatory Windows in Low-Intensity Laser Activation: Lasers, Scanners, and NASA's Light-Emitting Diode Array System

OBJECTIVE The purpose of this study was to assess and to formulate physically an irreducible set of irradiation parameters that could be relevant in the achieving reproducible light-induced effects in biological systems, both in vitro and in vivo. BACKGROUND DATA Light-tissue interaction studies focusing on the evaluation of irradiation thresholds are basic for the extensively growing applications for medical lasers and related light-emitting systems. These thresholds are of central interest in the rejuvenation of collagens, photorefractive keratectomy, and wound healing. METHODS There is ample evidence that the action of light in biological systems depends at least on two threshold parameters: the energy density and the intensity. Depending on the particular light delivery system coupled to an irradiation source, the mean energy density and the local intensity have to be determined separately using adequate experimental methods. RESULTS From the observations of different research groups and our own observations, we conclude that the threshold parameters energy density and intensity are biologically independent from each other. CONCLUSIONS This independence is of practical importance, at least for the medical application of photobiological effects achieved at low-energy density levels, accounting for the success and the failure in most of the cold laser uses since Mesters pioneering work.

Influence of rheological parameters on the velocity of erythrocytes passing nailfold capillaries in humans

One thousand two hundred and fifty-six subjects (apparently healthy subjects and patients with cardiovascular diseases) were registered in a prospective study including demographical and clinical data, rheological parameters (hematocrit, plasma viscosity, erythrocyte aggregation, erythrocyte deformability) as well as the erythrocyte velocity in human nailfold capillaries under resting and postischemic conditions. A multivariate regression analysis showed that under resting conditions there was no correlation between rheological parameters and erythrocyte velocity in capillaries. The blood flow regulation seemed to be so effective, that pathological changes of the blood fluidity showed no effect on the velocity of an erythrocyte passing the capillaries. During vessel paralysis in the early phase of the postischemic hyperemia following a stasis of three minutes in the vasculature distal to a pressure cuff at the upper arm a very clear correlation between the plasma viscosity and the maximum postischemic erythrocyte velocity in ipsilateral cutaneous capillaries could be observed (p < 0.0001) while none of the other rheological parameters seemed to play a role. In a subgroup of diabetic patients the erythrocyte aggregation (measured during stasis) also correlated with the erythrocyte velocity (p = 0.0175) besides the plasma viscosity. This shows that a correlation of rheological parameters with the capillary perfusion could only be found during vessel paralysis. In of diabetic patients besides the plasma viscosity also the erythrocyte aggregation correlated with the mean capillary erythrocyte velocity. Theses results are in agreement with the hypothesis from Barras that plasma viscosity determines the perfusion of microvessels. Under certain conditions e.g. diabetic disorder, also the erythrocyte aggregation plays a role.

Biocompatibility testing of novel multifunctional polymeric biomaterials for tissue engineering applications in head and neck surgery: an overview.

Biomaterial research and tissue engineering are rapidly growing scientific fields that need an interdisciplinary approach where clinicians should be included from the onset. Biocompatibility testing in vitro and in vivo comprise the agarose-overlay test, the MTT test, direct cell seeding tests and the chorioallantoic membrane test for angiogenic effects, among others. Molecular biology techniques such as real-time polymerase chain reaction and microarray technology facilitate the investigation of tissue integration into biomaterials on a cellular and molecular level. The physicochemical characterization of biomaterials is conducted using such methods as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Excellent biocompatibility and biofunctionality were demonstrated for a series of recently developed multifunctional biodegradable, polymeric biomaterials both in vitro and in vivo. Novel, multifunctional polymeric biomaterials offer a highly specific adjustment to the physiological, anatomical and surgical requirements and can thereby facilitate new therapeutic options in head and neck surgery.

Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

Stimulation of monocytes and macrophages: possible influence of surface roughness.

The aim of this study was to understand the mechanisms of interaction of monocytes/macrophages and foreign body giant cell (FBGC) with implant materials, with respect to the roughness and the solubility of calcium phosphate based coatings. Anderson et al. (Bone Engineering, J.E. Davies, ed., Toronto, 2000, pp. 81-93) showed that the presence of FBGCs and monocytes/macrophages influenced the strength of the implant-tissue integration and that more monocytes/macrophages rested on smooth surfaces compared to rough surfaces. We seeded human bone marrow cells on uncoated ultrasmooth polished TiAl6V4 samples as well as on coated TiAl6V4 discs of the same diameter with two different calcium phosphates coatings, monetite (DCP) and hydroxyapatite (OHAp), both with rougher surfaces. On uncoated ultrasmooth polished TiAl6V4 discs (UUTi, diameter 16 mm, thickness 2 mm) and on TiAl6V4 discs of same diameter coated with OHAP or DCPA, human bone marrow cells (HMBC) were seeded and cultivated under standard culture conditions for 90 days without addition of inducing substances like ascorbic acid, Na-beta-glycerophosphate or dexamethasone. The roughnesses of the virgin samples were assessed with atomic force microscopy and light profilometry. After 90 days of cultivation a fraction of the samples, with cells and extracellular matrix, were stained with hematoxylin eosin (HE) and examined in light microscopy. R(a) roughness values of virgin uncoated TiAl6V4 samples were 0.001 microm, of DCP coated discs 4 microm and of OHAp coated discs 3 microm. The examination of HE stained samples showed a high number of FBGC and monocytes/macrophages on the UUTi samples. On the DCP coated samples there were less FBGC and monocytes/macrophages and on the OHAp coated samples we could not find any FBGC and monocytes/macrophages. The extracellular matrix (ECM) we found on the UUTi samples was finer and thinner than on the coated samples. The ECM was vastly spread and not dense on the UUTi samples in contrast to the calcium phosphate coated samples, where the ECM was much thicker and stronger. The ultrasmooth surface of the uncoated TiAl6V4 samples, a material which is accepted to be biocompatible, evidently induced the differentiation of cells of the monocytic lineage and the formation of FBGC out of the cell populations present in the human bone marrow.

Relevance of the sterilization-induced effects on the properties of different hydroxyapatite nanoparticles and assessment of the osteoblastic cell response

Hydroxyapatite (Hap) is a calcium phosphate with a chemical formula that closely resembles that of the mineral constituents found in hard tissues, thereby explaining its natural biocompatibility and wide biomedical use. Nanostructured Hap materials appear to present a good performance in bone tissue applications because of their ability to mimic the dimensions of bone components. However, bone cell response to individual nanoparticles and/or nanoparticle aggregates lost from these materials is largely unknown and shows great variability. This work addresses the preparation and characterization of two different Hap nanoparticles and their interaction with osteoblastic cells. Hap particles were produced by a wet chemical synthesis (WCS) at 37°C and by hydrothermal synthesis (HS) at 180°C. As the ultimate in vivo applications require a sterilization step, the synthesized particles were characterized ‘as prepared’ and after sterilization (autoclaving, 120°C, 20 min). WCS and HS particles differ in their morphological (size and shape) and physicochemical properties. The sterilization modified markedly the shape, size and aggregation state of WCS nanoparticles. Both particles were readily internalized by osteoblastic cells by endocytosis, and showed a low intracellular dissolution rate. Concentrations of WCS and HS particles less than 500 μg ml−1 did not affect cell proliferation, F-actin cytoskeleton organization and apoptosis rate and increased the gene expression of alkaline phosphatase and BMP-2. The two particles presented some differences in the elicited cell response. In conclusion, WCS and HS particles might exhibit an interesting profile for bone tissue applications. Results suggest the relevance of a proper particle characterization, and the interest of an individual nanoparticle targeted research.

Shear resistance of human umbilical endothelial cells on different materials covered with or without extracellular matrix: Controlled in-vitro study

A variety of medical grade polymeric materials are used in tissue engineering and biomedical technology. Dense non-porous polymeric foils were used as substrates for endothelial cell layers. Half a the test samples (polymers and control materials) were seeded with bovine corneal endothelial cells (BCEC) which more or less covered the substrates with an extracellular matrix (ECM) in the consecutive culturing period. Afterwards the ECM covered as well as the uncovered materials were seeded with human umbilical venous endothelial cells (HUVEC). HUVEC seeded samples were cultured either under static or under dynamical conditions in a cone/plate rheometer with a mean low arterial shear stress of 8.2 dyn/cm2 to simulate the flow conditions in a coronary vein graft. With the exemption of polyvinyl chloride all other materials could be coated with ECM at least partially. Under static conditions the best results with respect to complete coverage with ECM and HUVEC were seen on polyester and polyurethane. Under shear load, however, the complete HUVEC layer together with the ECM detached from the polymer surface within a short time. ECM and HUVEC remained no longer than 43 minutes on anyone of the materials tested. The materials as supplied and tested were clearly not appropriate as implants in contact to the flowing blood.

Reversibility of echinocyte formation after contact of erythrocytes with various radiographic contrast media

Various radiographic contrast media (RCM) significantly influence the morphology of erythrocytes, especially the formation of echinocytes [Scand. J. Clin. Lab. Invest. 35 (1975), 1-43; Microvasc. Res. 60 (2000), 193-200; Herz 23 (2003), 35-41]. Microscopic studies, however, have shown that these changes of erythrocyte morphology are possibly reversible [Acta Radiol. 37 (1996), 214-217]. The aim of this study was to proof if the RCM-induced echinocyte formation can be reversed by a resuspension in autologous plasma. In this study four RCMs were tested (Iodixanol, Iohexol, Iomeprol and Iopromide). These RCM induced echinocyte formation (after suspension of erythrocytes in plasma/RCM mixtures for 10 min at 37 degrees C), which was reversible after resuspension in autologous RCM-free plasma (resuspension time 5 min at 37 degrees C). Especially for Iomeprol and Iopromide - the RCMs which induced the strongest echinocyte formation - an echinocyte reduction from 94.2% to 44.5% and for Iopromide from 80.6% to 50.4% occurred. The echinocyte formation was influenced by the type of RCM as well as by the RCM concentration. The same was true for the reversibility of echinocyte formation due to resuspension in autologous plasma (type of RCM: p</=0.0001; concentration of RCM: p=0.0847). Iodixanol was associated with the least numbers echinocytes formed (after suspension in the plasma/RCM-mixture as well as after the resuspension in autologous plasma). A 100% reversibility back to discocytes was observed in none of the RCMs after resuspension in autologous RCM-free plasma.In conclusion, a significant reversibility of RCM-induced echinocyte formation in autologous plasma could be observed.

Effect of radiographic contrast media on the spectrin/band3-network of the membrane skeleton of erythrocytes.

The membrane of red blood cells consists of a phospholipid bilayer with embedded membrane proteins and is associated on the cytoplasmatic side with a network of proteins, the membrane skeleton. Band3 has an important role as centre of the functional complexes e.g. gas exchange complex and as element of attachment for the membrane skeleton maintaining membrane stability and flexibility. Up to now it is unclear if band3 is involved in the morphology change of red blood cells after contact with radiographic contrast media. The study revealed for the first time that Iopromide induced markedly more severe alterations of the membrane skeleton compared to Iodixanol whose effects were similar to erythrocytes suspended in autologous plasma. A remarkable clustering of band3 was found associated with an accumulation of band3 in spicules and also a sequestration of band3 to the extracellular space. This was evidently accompanied by a gross reduction of functional band3 complexes combined with a dissociation of spectrin from band3 leading to a loss of homogeneity of the spectrin network. It could be demonstrated for the first time that RCM not only induced echinocyte formation but also exocytosis of particles at least coated with band3.

Effect of cytochrome P450-dependent epoxyeicosanoids on Ristocetin-induced thrombocyte aggregation

Epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 (CYP)-dependent epoxidation of arachidonic acid (AA) inhibit thrombocyte adhesion to the vascular wall. Upon dietary omega-3 fatty acid supplementation, EETs are partially replaced by eicosapentaenoic acid (EPA)-derived epoxyeicosatetraenoic acids (EEQs) and docosahexaenoic acid (DHA)-derived epoxydocosapentaenoic acids (EDPs). We hypothesized that the omega-3 epoxy-metabolites may exhibit superior anti-thrombogenic properties compared to their AA-derived counterparts. To test this hypothesis, we analyzed the effects of 11,12-EET, 17,18-EEQ and 19,20-EDP on Ristocetin-induced thrombocyte aggregation (RITA), a process that mimics thrombocyte adhesion to the vascular wall. The eicosanoids were added for 5, 30, or 60 minutes to thrombocyte-rich plasma freshly prepared immediately after blood collection from stringently selected apparently healthy subjects. Thrombocyte aggregation was then induced by Ristocetin (0.75 mg/mL) and assessed by turbidimetric measurements. After 60 minutes of preincubation, all three epoxy-metabolites significantly decreased the rate of RITA. 17,18-EEQ and 19,20-EDP were effective already at 1 μM, whereas 5-fold higher concentrations were required with 11,12-EET. Addition of AUDA, an inhibitor of the soluble epoxide hydrolase, potentiated the effect of 17,18-EEQ resulting in a significant further decrease of the velocity as well as amplitude of the aggregation process. In contrast to their profound effects on RITA, none of the epoxy-metabolites was effective in reducing collagen- or ADP-induced thrombocyte aggregation. These results indicate a highly specific role of CYP-eicosanoids in preventing thromboembolic events and suggest that the formation of 17,18-EEQ and 19,20-EDP may contribute to the anti-thrombotic effects of omega-3 fatty acids.