Synthesis and Redox Properties of Superbenzene Porphyrin Conjugates.

Superbenzene porphyrin conjugates find wide range of applications from nonlinear optical materials to semiconductors. Herein, we report the synthesis and characterization of 5,15-bis(3,5-di-tert-butylphenyl)-10,20-bis(pentaphenylphenyl)phenylporphyrin and its Zinc-metallated complex. Oxidative planarization of 5,15-bis(3,5-di-tert-butylphenyl)-10,20-bis(pentaphenylphenyl)phenylporphyrin and its metallated complex was carried out by using NOBF4 as an oxidizing agent. The formation of superbenzene porphyrin conjugates validates its Scholl type reactions. The laboratory-synthesized porphyrin conjugates were characterized experimentally using spectroscopic techniques such as 1H NMR, 13C NMR, electron spin resonance, and ultraviolet-visible spectroscopy for structural conformation. In addition, density functional theory calculations were carried out to validate the experimental results. The theoretical and experimental results show that the 4-(pentaphenylphenyl)phenyl ligand increases the stability, optical properties, and rate of planarization of synthesized porphyrins. The conjugates exhibited intense and distant electronic communication between two hexabenzocoronene sites, taking advantage of porphyrin as a π-spacer. The π-radical cation has also been found to be an intermediate in oxidative C-C bond formation. NICS calculations support such a conclusion.

Quantitative 3D MRI reveals limited intra-lesional bony overgrowth at 1 year after microfracture-based cartilage repair.

OBJECTIVE: Intra-lesional bony overgrowth (BO) identified during or following cartilage repair treatment is being frequently described through subjective reports focusing primarily on incidence. Our objective was to quantify the exact volume of intra-lesional BO at 12 months post-cartilage repair treatment, to determine if a correlation exists between the extent of BO and clinical outcomes, and to visualize and characterize the BO. DESIGN: MRI scans were systematically obtained during a randomized clinical trial for cartilage repair (Stanish et al., 2013) that compared two microfracture-based treatments in 78 patients. Semi-automated morphological segmentation of pre-treatment, 1 and 12 months post-treatment scans utilizing a programmed anatomical atlas for all knee bone and cartilage structures permitted three-dimensional reconstruction, quantitative analysis, as well as qualitative characterization and artistic visualization of BO. RESULTS: Limited intra-lesional BO representing only 5.8 ± 5.7% of the original debrided cartilage lesion volume was found in 78 patients with available MRIs at 12 months. The majority (80%) of patients had very little BO (<10%). Most occurrences of BO carried either spotty (56.4%) or planar (6.4%) morphological features, and the remaining balance (37.2%) was qualitatively unobservable by eye. Pre-existing BO recurred at 12 months in the same intra-lesional location in 36% of patients. No statistical correlations were found between BO and clinical outcomes. CONCLUSIONS: Intra-lesional BO following microfracture-based treatments may not be as severe as previously believed, its incidence is partly explained by pre-existing conditions, and no relationship to clinical outcomes exists at 12 months. Morphologically, observable BO was categorized as comprising either spotty or planar bone.

A polarized light microscopy method for accurate and reliable grading of collagen organization in cartilage repair.

OBJECTIVES: Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. DESIGN: This PLM score uses an ordinal scale of 0-5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. RESULTS: The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. CONCLUSION: This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Structural characteristics of the collagen network in human normal, degraded and repair articular cartilages observed in polarized light and scanning electron microscopies.

OBJECTIVE: This study characterizes collagen organization (CO) in human normal (n = 6), degraded (n = 6) and repair (n = 22) cartilages, using polarized light (PLM) and scanning electron (SEM) microscopies. DESIGN: CO was assessed using a recently developed PLM-CO score (Changoor et al. Osteoarthritis Cartilage 2011;19:126-35), and zonal proportions measured. SEM images were captured from locations matched to PLM. Fibre orientations were assessed in SEM and compared to those observed in PLM. CO was also assessed in individual SEM images and combined to generate a SEM-CO score for overall CO analogous to PLM-CO. Fibre diameters were measured in SEM. RESULTS: PLM-CO and SEM-CO scores were correlated, r = 0.786 (P < 0.00001, n = 32), after excluding two outliers. Orientation observed in PLM was validated by SEM since PLM/SEM correspondence occurred in 91.6% of samples. Proportions of the deep (DZ), transitional (TZ) and superficial (SZ) zones averaged 74.0 ± 9.1%, 18.6 ± 7.0%, and 7.3 ± 1.2% in normal, and 45.6 ± 10.7%, 47.2 ± 10.1% and 9.5 ± 3.4% in degraded cartilage, respectively. Fibre diameters in normal cartilage increased with depth from the articular surface [55.8 ± 9.4 nm (SZ), 87.5 ± 1.8 nm (TZ) and 108.2 ± 1.8 nm (DZ)]. Fibre diameters were smaller in repair biopsies [60.4 ± 0.7 nm (SZ), 63.2 ± 0.6 nm (TZ) and 67.2 ± 0.8 nm (DZ)]. Degraded cartilage had wider fibre diameter ranges and bimodal distributions, possibly reflecting new collagen synthesis and remodelling or collagen fibre unravelling. Repair tissues revealed the potential of microfracture-based repair procedures to produce zonal CO resembling native articular cartilage structure. Values are reported as mean ± 95% confidence interval. CONCLUSION: This detailed assessment of collagen architecture could benefit the development of cartilage repair strategies intended to recreate functional collagen architecture.

Ultrastructure of hybrid chitosan-glycerol phosphate blood clots by environmental scanning electron microscopy.

Chitosan-based polymers have been extensively studied for biomedical applications. Recently, liquid solutions of chitosan in a glycerol phosphate buffer (chitosan-GP) with physiological pH and osmolality were mixed with autologous blood to form hybrid chitosan-GP/blood implants that improved the repair of articular cartilage lesions in a large animal model. The mixture of chitosan-GP and blood forms a viscous liquid, which solidifies in minutes via normal blood coagulation as well as chitosan-mediated mechanisms. Here we have examined the ultrastructure of these chitosan-GP/blood clots as well as regular blood clots and chitosan-GP gels, the latter produced by heating. Both unfixed and fixed samples of chitosan-GP/blood clots, regular blood clots, and chitosan-GP gels were investigated by environmental scanning electron microscopy (ESEM) in conjunction with energy dispersive X-ray spectrometry (EDS), the former permitting direct observation of the ultrastructure in hydrated conditions simulating the natural state. By examination of unfixed specimens using ESEM we found that chitosan formed a network structure in both chitosan-GP gels and chitosan-GP/blood clots; however this structure was altered by aldehyde fixation to produce artifactual aggregates of chitosan microparticles. We were also able to identify chitosan in chitosan-GP/blood clots by washing samples in low concentration NaCl solutions followed by local EDS analyses to identify excess chloride versus sodium, and thus presence of cationic chitosan in analyzed features. Additional results indicated that the majority of glycerol phosphate diffuses freely from chitosan-GP gels (by EDS of phosphorus) and that hyperosmotic paraformaldehyde-based fixatives (i.e. 4% w/v) significantly disturb erythrocyte morphology in fixed whole blood clots.

Efficient cobalt(II) phthalocyanine-catalyzed reduction of flavones with sodium borohydride.

Efficient and facile reductions of flavones with sodium borohydride (NaBH4) catalyzed by cobalt(II) phthalocyanines afford cis-flavan-4-ols in 89-97% yields.

Shear stress-induced apoptosis of adherent neutrophils: a mechanism for persistence of cardiovascular device infections.

The mechanisms underlying problematic cardiovascular device-associated infections are not understood. Because the outcome of the acute response to infection is largely dependent on the function of neutrophils, the persistence of these infections suggests that neutrophil function may be compromised because of cellular responses to shear stress. A rotating disk system was used to generate physiologically relevant shear stress levels (0-18 dynes/cm(2); 1 dyne = 10 microN) at the surface of a polyetherurethane urea film. We demonstrate that shear stress diminishes phagocytic ability in neutrophils adherent to a cardiovascular device material, and causes morphological and biochemical alterations that are consistent with those described for apoptosis. Complete neutrophil apoptosis occurred at shear stress levels above 6 dynes/cm(2) after only 1 h. Morphologically, these cells displayed irreversible cytoplasmic and nuclear condensation while maintaining intact membranes. Analysis of neutrophil area and filamentous actin content demonstrated concomitant decreases in both cell area and actin content with increasing levels of shear stress. Neutrophil phagocytosis of adherent bacteria decreased with increasing shear stress. Biochemical alterations included membrane phosphatidylserine exposure and DNA fragmentation, as evaluated by in situ annexin V and terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) assays, respectively. The potency of the shear-stress effect was emphasized by comparative inductive studies with adherent neutrophils under static conditions. The combination of tumor necrosis factor-alpha and cycloheximide was ineffective in inducing >21% apoptosis after 3 h. These findings suggest a mechanism through which shear stress plays an important role in the development of bacterial infections at the sites of cardiovascular device implantation.

Shear stress effects on bacterial adhesion, leukocyte adhesion, and leukocyte oxidative capacity on a polyetherurethane.

Infection of implanted cardiovascular biomaterials still occurs despite inherent host defense mechanisms. Using a rotating disk system, we investigated Staphylococcus epidermidis and polymorphonuclear leukocyte (PMN) adhesion to a polyetherurethane urea (PEUU-A') under shear stress (0-17.5 dynes/cm2) for time periods up to 6 h. In addition, the superoxide (SO) release capacity of PMNs after transient exposure to PEUU-A' under shear stress was determined. Bacterial adhesion in phosphate-buffered saline (PBS) showed a linear shear dependence, decreasing with increasing shear stress. Overall adhesion in PBS decreased with time. However, bacterial adhesion in 25% human serum was similar for all time points up to 360 min. Adhesion was observed at all shear levels, displaying no shear dependence. In contrast, PMN adhesion demonstrated a strong shear dependence similarly for times up to 240 min, decreasing sharply with increasing shear stress. Although PMNs preexposed to shear stress showed a slightly diminished SO release response compared to fresh cells for all stimuli, it was not statistically significant regardless of the stimulus. We conclude that circulating leukocytes are unable to adhere in regions of high shear which may contain adherent bacteria. In addition, exposure to PEUU-A' and shear stress (in the range 0-18 dynes/cm2) is insufficient to cause a depression in the oxidative response of PMNs.

Influence of biomaterial surface chemistry on the apoptosis of adherent cells.

A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.

Effects of photochemically immobilized polymer coatings on protein adsorption, cell adhesion, and the foreign body reaction to silicone rubber.

Photochemical immobilization technology was utilized to covalently couple polymers to silicone rubber either at multiple points along a polymer backbone or at the endpoint of an amphiphilic chain. The coating variants then were tested in vitro and in vivo for improvement of desired responses compared to uncoated silicone rubber. All coating variants suppressed the adsorption of fibrinogen and immunoglobulin G, and most also inhibited fibroblast growth by 90-99%. None of the coating variants inhibited monocyte or neutrophil adhesion in vitro. However, the surfaces that supported the highest levels of monocyte adhesion also elicited the lowest secretion of pro-inflammatory cytokines. None of the materials elicited a strong inflammatory response or significantly (p< 0.05) reduced the thickness of the fibrous capsule when implanted subcutaneously in rats. Overall, the most passivating coating variant was an endpoint immobilized polypeptide that reduced protein adsorption, inhibited fibroblast growth by 90%, elicited low cytokine secretion from monocytes, and reduced fibrous encapsulation by 33%. In general, although some coating variants modified the adsorption of proteins and the behavior of leukocytes or fibroblasts in vitro, none abolished the development of a fibrous capsule in vivo.

In vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) hydrogels.

The in vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were assessed in order to investigate the influence of poly(ethylene glycol) molecular weight and copolymer composition. These materials have application as injectable cardiovascular implants; cytotoxicity due to leachable products, as well as inflammation caused by the biomaterial itself, may ultimately affect the biocompatibility of the implant. We utilized a 7-day in vitro cytotoxicity assay to quantify cell density and cellular proliferation in the presence of copolymer films. The copolymer films exhibited slight to moderate cytotoxicity toward cultured endothelial cells, showing 20-86% viability relative to controls. Cell viability increased with an increasing weight percent of PEG or, to a lesser extent, the molecular weight of PEG. In vivo biocompatibility was assessed using a cage implantation model over a 21-day time period. This system was used to characterize the local cellular and humoral inflammatory response in the surrounding exudate, as well as the size and density of macrophages adherent to the material itself. All copolymer formulations exhibited excellent biocompatibility relative to controls with no significant differences in total leukocyte count among the different formulations. The in vivo inflammatory reaction displayed normal wound healing over 21 days as shown by a progressive decrease in both leukocyte concentration and enzymatic activity. The surface coverage of the copolymer films remained relatively constant from 7 to 21 days. There were no cells larger than 0.003 mm2, which was previously shown to be the threshold value for foreign-body giant cells. These data suggest that P(PF-co-EG) hydrogels have potential for use as injectable biomaterials.