Differentiation between normal and tumor vasculature of animal and human glioma by FTIR imaging

Malignant gliomas are very aggressive tumors, highly angiogenic and invading heterogeneously the surrounding brain parenchyma, making their resection very difficult. To overcome the limits of current diagnostic imaging techniques used for gliomas, we proposed using FTIR imaging, with a spatial resolution from 6 to 10 μm, to provide molecular information for their histological examination, based on discrimination between normal and tumor vasculature. Differentiation between normal and tumor blood vessel spectra by hierarchical cluster analysis was performed on tissue sections obtained from xenografted brain tumors of Rag-gamma mice 28 days after intracranial implantation of glioma cells, as well as for human brain tumors obtained in clinics. Classical pathological examination and immunohistochemistry were performed in parallel to the FTIR spectral imaging of brain tissues. First on the animal model, classification of FTIR spectra of blood vessels could be performed using spectral intervals based on fatty acyl (3050-2800 cm(-1)) and carbohydrate (1180-950 cm(-1)) absorptions, with the formation of two clusters corresponding to healthy and tumor parts of the tissue sections. Further data treatments on these two spectral intervals provided interpretable information about the molecular contents involved in the differentiation between normal and tumor blood vessels, the latter presenting a higher level of fatty acyl chain unsaturation and an unexpected loss of absorption from osidic residues. This classification method was further successfully tested on human glioma tissue sections. These findings demonstrate that FTIR imaging could highlight discriminant molecular markers to distinguish between normal and tumor vasculature, and help to delimitate areas of corresponding tissue.     

Functional histology of glioma vasculature by FTIR imaging

Fourier-transform infrared (FTIR) imaging has been used to investigate brain tumor angiogenesis using a mice solid tumor model and bare-gold (∅ 25 nm) or BaSO₄ (∅ 500 nm) nanoparticles (NP) injected into blood vasculature. FTIR images of 20-μm-thick tissue sections were used for chemical histology of healthy and tumor areas. Distribution of BaSO₄-NP (using the 1,218–1,159 cm⁻¹ spectral interval) revealed clearly all details of blood vasculature with morphological abnormalities of tumor capillaries, while Au-NP (using the 1,046–1,002 cm⁻¹ spectral interval) revealed also diffusion properties of leaky blood vessels. Diffusion of Au-NP out of vascular space reached 64 ± 29 μm, showing the fenestration of “leaky” tumor blood vessels, which should allow small NP (<100 nm, as for Au-NP) to diffuse almost freely, while large NP should not (as for BaSO₄-NP in this study). Therefore, we propose to develop FTIR imaging as a convenient tool for functional molecular histology imaging of brain tumor vasculature, both for identifying blood capillaries and for determining the extravascular diffusion space offered by vessel fenestration.     

Controlled cell proliferation on an electrochemically engineered collagen scaffold

Therapies for corneal disease and injury often rely on artificial implants, but integrating cells into synthetic corneal materials remains a significant challenge. The electrochemically formed collagen-based matrix presented here is non-toxic to cells and controls the proliferation in the corneal fibroblasts seeded onto it. Histology and biomolecular studies show a behavior similar to corneal stromal cells in a native corneal environment. Not only is this result an important first step toward developing a more realistic, multi-component artificial cornea, but it also opens possibilities for using this matrix to control and contain the growth of cells in engineered tissues.     

Characterization of keratin–collagen 3D scaffold for biomedical applications

Fabrication of keratin–collagen (KC) 3D scaffold with improved thermal denaturation rate is reported. In vitro application of (KC) scaffold stimulates basic extra cellular matrix constituents. KC Scaffold considerably reduced undesirable properties of both collagen and keratin while collagen incorporation reduces the fragility with increases of strength and flexibility in the scaffold. In addition to this, the scaffold showed homogenous well‐interconnected pores in the range of 10–100 µm when observed in scanning electron microscope. Usage of keratin in KC scaffold offers increased biodegradation rate and higher denaturation rate in addition to its rapid cell growth with normal morphology ultimately reaching cell population of 3.9–9.7 million per cm³ after 48 hr in KC scaffold. Circular dichroism (CD) and Fourier transform spectroscopy (FT‐IR) of KC showed presence of helical structure of collagen and ß‐turns of keratin confirming retention of native structures of both the proteins KC scaffold showed good swelling behavior and water uptake. Our study strongly supports the superidity of KC scaffold over the collagen or keratin when they are independently used for tissue engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.     

The function of p27KIP1 during tumor development

Timely cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves cyclin dependent kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different phases of the cell cycle. One CKI of importance is p27^KIP1. The regulation and cellular localization of p27^KIP1 can result in biologically contradicting roles when found in the nucleus or cytoplasm of both normal and tumor cells. The p27^KIP1 protein is mainly regulated by proteasomal degradation and its downregulation is often correlated with poor prognosis in several types of human cancers. The protein can also be functionally inactivated by cytoplasmic localization or by phosphorylation. The p27^KIP1 protein is an unconventional tumor suppressor because mutation of its gene is extremely rare in tumors, implying the normal function of the protein is deranged during tumor development. While the tumor suppressor function is mediated by p27^KIP1''s inhibitory interactions with the cyclin/CDK complexes, its oncogenic function is cyclin/CDK independent, and in many cases correlates with cytoplasmic localization. Here we review the basic features and novel aspects of the p27^KIP1 protein, which displays genetically separable tumor suppressing and oncogenic functions.     

Zirconia nanoparticles enhanced grafted collagen tri-helix scaffold for unmediated biosensing of hydrogen peroxide

A novel, biocompatible, thermally steady, and nontoxic zirconia enhanced grafted collagen tri-helix scaffold was prepared on a graphite electrode. This scaffold provided a microenvironment for loading biomolecules and helped to retain their natural structure. UV-vis spectroscopy and scanning electron microscopy were used to characterize the scaffold and the structure of immobilized biomolecules. Using horseradish peroxidase (HRP) as an example, this scaffold accelerated its electron transfer and led to its direct electrochemical behavior with a good thermal stability up to 80 degrees C. The surface electron-transfer rate constant of the immobilized HRP was (5.55 +/- 0.43) s(-)(1) in 0.1 M pH 7.0 PBS at 18 degrees C. The immobilized HRP showed an electrocatalytic activity to the reduction of hydrogen peroxide (H(2)O(2)) without aid of an electron mediator. The linear response range of the biosensor for H(2)O(2) was from 1.0 to 73.0 microM with a correlation coefficient of 0.999 (n = 14), a limit of detection down to 0.25 microM and an apparent Michaelis-Menten constant of (0.28 +/- 0.02) mM. The biosensor exhibited high sensitivity, acceptable stability, and reproducibility. The ZrO(2) grafted collagen provided an excellent matrix for protein immobilization and biosensor preparation.     

Stage-specific alterations of cyclin expression during UVB-induced murine skin tumor development

We have evaluated the in vivo correlation between the expression of cell cycle markers and skin tumor development in SKH-1 hairless mice in a complete photocarcinogenesis protocol. Irradiated mice developed an average of 16 tumors per animal by week 23 with the average number of carcinomas per mouse being 2.1. The expression of p53 and cyclins A and D1 was confined initially to sporadic single cells and gradually developed into foci of patchy intense staining in the basal and granular layers of UVB-exposed epidermis. p53 was expressed in all the papilloma sections examined, whereas cyclins D1 and A were expressed in 68 and 71% of these lesions, respectively. In UVB-induced squamous cell carcinomas (SCC), p53 was expressed in >90% of the tumors, whereas cyclin D1 was detected in 55% of the lesions, and cyclin A staining was limited to 27%. These immunohistochemical observations were confirmed by Western blotting and protein kinase assays. We observed an early wave of cyclin A overexpression and cyclin A protein kinase activity preceding the appearance of detectable tumors. Cyclin D1 and p53 overexpression were coupled with the development of tumors, and these changes are likely to be relevant to the pathogenesis of these lesions.     

葡萄劣变过程中挥发性物质的FTIR光谱分析

葡萄在运输和贮藏过程中极易发生变质,对葡萄劣变进行预警可有效降低大规模腐败的风险.研究了葡萄在劣变过程中所产生挥发性物质的FTIR光谱特性.实验证明了葡萄劣变中挥发性物质的主要成分为乙酸乙酯、乙醇、二氧化碳和水汽.通过光谱定量化分析研究挥发性物质在劣变中的变化规律,发现葡萄在劣变开始发生时的气体释放速率会发生阶跃性变化.论文采用主成分分析法(PCA)对挥发性物质的红外光谱进行了分类,可以准确地区分未变质、轻度变质和重度变质的葡萄.论文的结论说明挥发性物质的FTIR光谱分析可以有效鉴别贮藏中葡萄的劣变程度.而且由于挥发性气体在葡萄劣变中的阶跃变化性质,使这种鉴别方法具有不易受葡萄数量、存放方式影响的优点.论文的研究为葡萄劣变监测设备研制提供了理论和技术基础.     

Fabrication and characterization of low-cost freeze-gelated chitosan/collagen/hydroxyapatite hydrogel nanocomposite scaffold

In the present research, chitosan/collagen and chitosan/collagen/nano-hydroxyapatite (nHAP) hydrogel nanocomposites were prepared using naturally extracted chitosan from Persian Gulf shrimp wastes and rat tail-tendon collagen. Freeze-gelation method was used to prepare highly porous scaffolds. The morphology, chemical structure, water retainability, and thermal properties were characterized using SEM, FTIR, water content experiment, simultaneous thermal analysis (STA), respectively. Atomic force microscopy (AFM) nanoindentation and unconfined compression test were used to assess different feature of the mechanical properties of the hydrogels. The obtained results were so promising that the prepared nanocomposites can be considered as a potential candidate for cartilage tissue engineering.     

Cyclotriveratrylene (CTV) as a new chiral triacid scaffold capable of inducing triple helix formation of collagen peptides containing either a native sequence or Pro-Hyp-Gly repeats

A new triacid scaffold is described based on the cone-shaped cyclotriveratrylene (CTV) molecule that facilitates the triple helical folding of peptides containing either a unique blood platelet binding collagen sequence or collagen peptides composed of Pro-Hyp-Gly repeats. The latter were synthesized by segment condensation using Fmoc-Pro-Hyp-Gly-OH. Peptides were coupled to this CTV scaffold and also coupled to the Kemp's triacid (KTA) scaffold. After assembly of peptide H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu(OAll)-Arg-Gly-Val-Glu (OAll)-Gly-[Pro-Hyp-Gly]2-NH2 (13) by an orthogonal synthesis strategy to both triacid scaffolds, followed by deprotection of the allyl groups, the molecular constructs spontaneously folded into a triple helical structure. In contrast, the non-assembled peptides did not. The melting temperature (Tm) of (+/-) CTV[CH2C(O)N(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (14) is 19 degrees C, whereas KTA[Gly-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (15) has a Tm of 20 degrees C. Thus, it was shown for the first time that scaffolds were also effective in stabilizing the triple helix of native collagen sequences. The different stabilizing properties of the two CTV enantiomers could be measured after coupling of racemic CTV triacid to the collagen peptide, and subsequent chromatographic separation of the diastereomers. After assembly of the two chiral CTV scaffolds to the model peptide H-Gly-Gly-(Pro-Hyp-Gly)5-NH2 (24), the (+)-enantiomer of CTV 28b was found to serve as a better triple helix-inducing scaffold than the (-)-enantiomer 28a. In addition to an effect of the chirality of the CTV scaffold, a certain degree of flexibility between the CTV cone and the folded peptide was also shown to be of importance. Restricting the flexibility from two to one glycine residues resulted in a significant difference between the two collagen mimics 20a and 20b, whereas the difference was only slight when two glycine residues were present between the CTV scaffold and the peptide sequence in collagen mimics 30a and 30b.     

Interactions between collagen chains and fiber formation.

The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6-1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radioactively labeled collagen into fibers was measured in 0-0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39 degrees C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30 degrees C. We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.     

FTIR studies of collagen model peptides: complementary experimental and simulation approaches to conformation and unfolding

X-ray crystallography of collagen model peptides has provided high-resolution structures of the basic triple-helical conformation and its water-mediated hydration network. Vibrational spectroscopy provides a useful bridge for transferring the structural information from X-ray diffraction to collagen in its native environment. The vibrational mode most useful for this purpose is the amide I mode (mostly peptide bond C=O stretch) near 1650 cm-1. The current study refines and extends the range of utility of a novel simulation method that accurately predicts the infrared (IR) amide I spectral contour from the three-dimensional structure of a protein or peptide. The approach is demonstrated through accurate simulation of the experimental amide I contour in solution for both a standard triple helix, (Pro-Pro-Gly)10, and a second peptide with a Gly --> Ala substitution in the middle of the chain that models the effect of a mutation in the native collagen sequence. Monitoring the major amide I peak as a function of temperature gives sharp thermal transitions for both peptides, similar to those obtained by circular dichroism spectroscopy, and the Fourier transform infrared (FTIR) spectra of the unfolded states were compared with polyproline II. The simulation studies were extended to model early stages of thermal denaturation of (Pro-Pro-Gly)10. Dihedral angle changes suggested by molecular dynamics simulations were made in a stepwise fashion to generate peptide unwinding from each end, which emulates the effect of increasing temperature. Simulated bands from these new structures were then compared to the experimental bands obtained as temperature was increased. The similarity between the simulated and experimental IR spectra lends credence to the simulation method and paves the way for a variety of applications.     

FTIR study on the formation of TiO2 nanostructures in supercritical CO2

TiO(2) nanospherical and fibered structures were obtained via a one-step sol-gel method in supercritical carbon dioxide (scCO(2)) involving polycondensation of the alkoxide monomers titanium isopropoxide (TIP) and titanium butoxide (TBO) with acetic acid (HAc). The resulting materials were characterized by means of electron microscopy (SEM and TEM), X-ray diffraction (XRD), thermal analysis (TGA), and attenuated total reflection Fourier transmission infrared (ATR-FTIR) analysis. Depending on the experimental conditions, TiO(2) anatase nanospheres with a diameter of 20 nm or TiO(2) anatase/rutile nanofibers with a diameter of 10-100 nm were obtained. Fiber formation was enhanced by a higher HAc/Ti ratio and the use of the titanium isopropoxide (TIP) monomer. The mechanism of the microstructure formation was studied using in situ FTIR analysis in scCO(2). The FTIR results indicated that the formation of nanofibers was favored by a titanium hexamer that leads to one-dimensional condensation, while nanospheres were favored by a hexamer that permits three-dimensional condensation.     

Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus

Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.     

L-Glutamic acid loaded collagen chitosan composite scaffold as regenerative medicine for the accelerated healing of diabetic wounds

Diabetic wounds (DWs) are characterized by prolonged inflammation, which poses a significant challenge for clinicians and researchers to promote healing. In this study, we fabricate L-Glutamic acid (LGA) loaded collagen/chitosan (COL-CS) composite scaffold for the accelerated healing of DW. The characterization outcomes of the composite scaffold revealed that a crosslinked scaffold holds optimum porosity, low matrix degradation, and sustained drug release in contrast to a non-crosslinked scaffold. In vitro, LGA composite scaffolds have not exhibited any toxicity on 3T3L1 cell lines. In vivo, the LGA composite scaffold has shown significantly (p < 0.001), higher rates of wound contraction than those in control and COL-CS scaffold treated groups. In addition, MMP-9 levels were also significantly reduced in LGA composite scaffold-treated group compared with those in the control and COL-CS scaffold treated group. Thus, the LGA composite scaffold may serve as a promising therapy in DW due to its unique modulatory effect on inflammatory biomarker MMP-9.     

Thermodynamic modeling and investigation of the formation of electrospun collagen fibers

Electrospun type I collagen fibers are very promising materials for tissue scaffold applications, but are typically fabricated from toxic solvents. Recently, electrospinning of type I collagen fibers by using environmentally friendly phosphate buffer saline (PBS)/ethanol solution has been explored. PBS/ethanol solvent systems offer better cell compatibility, but the high surface tension and high boiling point of the solvent system make the collagen difficult to electrospin and can cause inferior fiber morphology. In this study, the influence of solvent surface tension on the morphology of electrospun collagen fibers has been experimentally investigated and analyzed from a thermodynamics perspective. The analytical results indicate that solvents with high surface tension drive the formation of beads along the smaller, thinner fibers. In addition, beads with relatively small angular eccentricity were thermodynamically favorable. The experimental results presented herein corroborate the theoretical analysis and conclusions drawn from this study. The surface tension of the solvent has significant influence on the bead formation, especially in an aqueous system. The environmental humidity for the electrospinning process and the collagen concentration were also investigated. These parameters may result in variations of the evaporation-solidification rates, which consequently impact the formation and morphologies of electrospun collagen fibers. According to the thermodynamic analysis, uniform electrospun collagen fibers without beads can be obtained by manipulating solvent surface tension during the electrospinning process.     

The use of FTIR microscopy for evaluation of herpes viruses infection development kinetics

The kinetics of Herpes simplex infection development was studied using an FTIR microscopy (FTIR-M) method. The family of herpes viruses includes several members like H. simplex types I and II (HSV I, II), Varicella zoster (VZV) viruses which are involved in various human and animal infections of different parts of the body. In our previous study, we found significant spectral differences between normal uninfected cells in cultures and cells infected with herpes viruses at early stages of the infection. In the present study, cells in cultures were infected with either HSV-I or VZV and at various times post-infection they were examined either by optical microscopy or by advanced FTIR-M. Spectroscopic measurements show a consistent decrease in the intensity of the carbohydrate peak in correlation with the viral infection development, observed by optical microscopy. This decrease in cellular carbohydrate level was used as indicator for herpes viruses infection kinetics. This parameter could be used as a basis for applying a spectroscopic method for the evaluation of herpes virus infection development. Our results show also that the development kinetics of viral infection has an exponential character for these viruses.     

A collagen network formation effector from leaves of Premna subscandens.

As a part of the search for biologically active plant products, M cells, which form a collagen fiber network in vitro after a prolonged culture period, were used. The n-BuOH-soluble fraction of a methanol extract of leaves of Premna subscandens exhibited promotion of collagen network formation by M cells. Extensive isolation work guided by a bioassay afforded a phenylethanoid, acteoside, as an active compound.     

Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage

A MHDS has been employed to fabricate 3D scaffolds from PLGA with acetyl endgroups to achieve in vivo regeneration of cartilage tissue. The fabricated acetylated-PLGA scaffold showed open pores and interconnected structures. Rabbit chondrocytes were seeded on the PLGA scaffolds and transplanted immediately into subcutaneous sites of athymic mice. Chondrocytes transplantation with untreated PLGA scaffolds served as a control. Histological analysis of the implants at 4 weeks with H&E staining and alcian blue staining revealed higher extracellular matrix and GAG expression at the neocartilage in the PLGA-6Ac scaffolds than that of the PLGA-6OH scaffold group. This endgroup-modified scaffold may be useful for successful cartilage tissue engineering in orthopedic applications.