Effect of UV irradiation on stabilized collagen: role of chromium(III)

Research on the effect of UV radiation on stabilized collagen is an area of potential interest owing to the fact that collagen is an important biomaterial finding immense use in various fields. In this present study, effect of UV irradiation on collagen stabilized using chromium(III) has been studied. The physical and optical properties affected by UV irradiation have been detailed. Viscosity measurements have shown that chromium(III) treated collagen has better stability against UV radiation than native collagen. Circular dichroic studies indicate that increase in concentration of chromium(III) does not affect the conformation of collagen however, the duration of irradiation has profound impact on the conformation of collagen. The fluorescence intensity of native collagen has been found to decrease more than that of chromium(III) treated collagen. The difference absorption spectra also shows that chromium(III) treatment brings about more stability to collagen against UV irradiation.     

THE HAIRLESS MOUSE MODEL OF PHOTOAGING: EVALUATION OF THE RELATIONSHIP BETWEEN DERMAL ELASTIN, COLLAGEN, SKIN THICKNESS AND WRINKLES

Quantitative and qualitative changes in dermal collagen and elastin occur in response to chronic ultraviolet (UV) irradiation. These changes have been implicated in the genesis of the wrinkling seen in chronically irradiated, or photoaged skin. We examined the relationship between wrinkle formation and changes in dermal structural protein content and type. Skh-1 hairless mice were irradiated with suberythemal doses of UV-B three times a week for up to 20 wk. Visible wrinkling was present after 6-7 wk of irradiation. Dermal elastic fiber content was quantified by color image analysis of paraffin-embedded tissue. There was no significant difference in dermal elastic fiber content between irradiated and age-matched control mice after either 10 or 20 wk of irradiation. The effect of UV-B irradiation on total dermal collagen content, ratio of collagen type III-type I, and extent of glycosylation and crosslinking of collagen was no different in irradiated and age-matched control mice after 10 wk of irradiation. Increased epidermal thickness was evident in frozen sections after 6 wk of irradiation, and the thickness increased with continued irradiation. Dermal thickening was evident after 10 wk of irradiation. Sufficient UV-B irradiation will eventually cause changes in dermal elastin and collagen content; however, wrinkle formation precedes such changes. A causal relationship between wrinkle formation and dermal structural protein content changes in Skh-1 hairless mice could not be established in this study.     

Unique size-change behavior of photo-crosslinked cinnamic acid derivative nanoparticles during hydrolytic degradation

A unique size change of photo-crosslinkable poly[(3,4-dihydroxycinnamic acid)-co-(4-hydroxycinnamic acid)] nanoparticles was observed during hydrolytic degradation depending on the crosslinking degree. The diameter of uncrosslinked nanoparticles decreased from 850 to 300 nm during hydrolysis, whereas that of 75% crosslinked nanoparticles increased from 700 to 950 nm. The diameter changes of crosslinked nanoparticles during hydrolysis might be induced by swelling of the crosslinked networks depending on the crosslinking degree. Moreover, the diameter of the uncrosslinked nanoparticle recovered by additional UV irradiation during hydrolysis. These results suggested that the diameter of the nanoparticles could be controlled even during hydrolysis by UV irradiation.     

Thermal and mechanical properties of UV irradiated collagen/chitosan thin films

The thermal and mechanical properties of collagen/chitosan blends before and after UV irradiation have been investigated using thermal analysis and mechanical (Instron) techniques. Comparisons were made with the thermal and mechanical properties of both collagen and chitosan films. Air-dried collagen, chitosan and collagen/chitosan films were exposed to UV irradiation (wavelength 254 nm) for different time intervals. Thermal properties of collagen/chitosan blends depend on the composition of the blend and are not significantly altered by UV irradiation.Mechanical properties such as ultimate tensile strength and ultimate percentage of elongation were much better for collagen films than for collagen/chitosan films. The results have shown that the mechanical properties of the blends were greatly affected by the duration of UV irradiation. Ultimate tensile strength and ultimate percentage elongation decreased after UV irradiation of the blend. Increasing UV irradiation leads to an increase in Young's modulus of the collagen/chitosan blend.     

FORMATION OF PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IN COLLAGEN TYPES I AND IV EXPOSED TO ULTRAVIOLET LIGHT

Abstract— Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.     

The influence of UV light on collagen/poly(ethylene glycol) blends

The photodegradation behaviour of the collagen and poly(ethylene glycol) PEG blends has been studied by Fourier transform infrared spectroscopy (FTIR), UV-Vis spectroscopy and viscometry. Surface properties before and after UV irradiation were observed using optical microscope.Collagen and PEG were immiscible and the films obtained from the mixture were fragile with poor mechanical properties. The photochemical stability of the collagen and PEG blend was different from that of the single components. In general collagen/PEG blends are less stable under UV irradiation than pure collagen. The influence of PEG on the photochemical stability of collagen depends on its concentration in the blend. Microscope photographs showed that the surface characteristics of collagen and collagen/PEG blends in film form are not drastically altered after UV irradiation.     

Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea

The thermal stability of UVB irradiated collagen in bovine lens capsules and in bovine cornea has been investigated by differential scanning calorimetry (DSC). During UVB irradiation the lens capsules and cornea were immersed in water to keep the collagen in a fully hydrated condition at all times. UV irradiation induced changes in collagen which caused both stabilization and destabilization of the collagen structure. The helix-coil transition for non-irradiated collagen in cornea occurred near 66 degrees C, instead for the irradiated one for 3h it occurred at 69 degrees C. After irradiating for longer times (20-96h) the helix-coil transition peak occurred at much lower temperatures. The peak was very broad and suggested that collagen was reduced by UV to different polypeptides of different molecular weight and different lower thermal stabilities. The irradiation of lens capsules with UVB light in vitro resulted in changes in the thermal properties of type-IV collagen consistent with increased cross-linking. DSC of lens capsules showed two major peaks at melting temperatures at 54 degrees C Tm1 and 78 degrees C Tm2, which can be attributed to the denaturation of the triple helix and 7S domains, respectively. UVB irradiation of lens capsules in vitro for 6 h caused an increase in Tm1 from 54 to 57 degrees C. The higher temperature required to denature the type-IV collagen after irradiation in vitro suggested an increase of intermolecular cross-linking.     

Photochemical stability of collagen/poly(ethylene oxide) blends

The photochemical stability of the blends of collagen and poly(ethylene oxide) PEO has been studied by Fourier transform infrared spectroscopy (FTIR), UV–vis spectroscopy and viscosimetry. Surface properties before and after UV irradiation were observed using an optical microscope.Collagen and PEO were immiscible in diluted solution and only small interactions between the two components in the solid state were observed. New materials based on the blending of collagen and PEO that we obtained have a different photochemical stability than those of single components. In general, collagen/PEO blends are less stable under UV irradiation than pure collagen. The influence of PEO on the photochemical stability of collagen depends on the concentration of this polymer in the blend. Microscopic photographs show that the surface characteristics of thin films of collagen/PEO blends are not drastically altered after UV irradiation.     

Identification of free radicals induced by UV irradiation in collagen water solutions

Electron paramagnetic resonance (EPR) method has shown that hydrogen atoms and acetic acid free radicals appear in surrounding acetic acid-water solution of collagen under ultraviolet (UV) irradiation. These free radicals interact with the collagen molecule; consequently, seven superfine components of EPR spectrum with the split of aH = 11.3G and g-factor 2.001 appear. It is assumed that this spectrum is related to the free radical occurred on the proline residue in collagen molecule. In order to discover .OH hydroxyl radicals even in minor concentration, spin trap 5.5-dimethyl-1-pyrroline N-oxide (DMPO) has been applied. During the irradiation of collagen water solution in the presence of spin trap, EPR spectrum of the DMPO/.OH adduct has not been identified, while the above mentioned spectrum has been observed once the hydrogen peroxide H2O2 and FeSO4 were added to the sample. That means that water photolysis does not take place in collagen water-solution due to UV irradiation. It was suggested that occurrence of hydrogen radical is connected with the electron transmission to the hydrogen ion. The possible source of free electrons can be aromatic residues, photo ionization of which takes place in collagen molecule due to UV irradiation.     

EFFECTS OF CHRONIC EXPOSURE ULTRAVIOLET-A INCLUDING 2% ULTRAVIOLET-B ON FREE RADICAL REDUCTION SYSTEMS IN HAIRLESS MICE

Chronic ultraviolet (UV) irradiation is known to cause a variety of changes in the skin, including wrinkles, pigmented spots and carcinogenesis. To explore time dependent changes in several parameters with chronic UV irradiation, we examined the molecular changes in connective tissue, intracellular defence enzymes and free radical antioxidant substances in hairless mice skin caused by chronic exposure to UV-A including 2% UV-B. Connective tissue changes were estimated using hydroxyproline and isodesmosine assays as a measure of collagen and elastin concentrations, respectively. After 6 weeks irradiation, the insoluble collagen and elastin were both substantially elevated, as were the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Continued UV irradiation resulted in a steady decline in SOD and lipid soluble antioxidants, while the GSH-Px remained elevated, suggesting that SOD and lipid soluble antioxidants in the skin may be involved in protecting it from UV damage and deteriorate with chronic irradiation.     

PHOTOCHEMICAL CROSSLINKING OF ATP TO HISTONE H4

Abstract— A covalent crosslink occurs between histone H4 and the adenine moiety of ATP, when the complex they form is irradiated with UV light of Λ > 290 nm in the presence of acetone. Within 1 h of irradiation a 48% yield of crosslinked product is thus obtained. It is also shown that in the photosensitized reaction, 80% of the crosslinked product is monomeric, whereas protein precipitation and aggregation occur as a result of direct irradiation with 254 nm light.     

Changes induced by ultraviolet light in fluorescence of collagen in the presence of β-carotene

The influence of UV radiation (253.7 nm) on collagen fluorescence in the absence, and presence, of β-carotene was investigated. It was found that UV radiation of 253.7 nm causes irreversible destruction of tyrosyl and phenylalanyl residues. The fluorescence of collagen (excitation at 275 nm, emission at 305 nm) decreased rapidly during irradiation and a new fluorescence large band at 400–500 nm formed under UV radiation. Smaller changes in the fluorescence of collagen in the presence of β-carotene suggest that it makes collagen less sensitive to the action of UV radiation.     

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice.

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.     

Living cationic polymerization of a coumarin‐substituted vinyl ether and reversible photoinduced crosslinking of the resulting homopolymers and amphiphilic block copolymers

Living cationic polymerization of 4‐methyl‐7‐(2‐vinyloxyethoxy)coumarin (CMVE) was achieved using SnCl₄ in the presence of nBu₄NBr as an added salt at 0 °C. The number‐average molecular weight of the resulting polymers increased in direct proportion to the monomer conversion while retaining relatively low polydispersity. Structural analysis revealed that the resulting polymers carried pendant coumarinyl moieties. These coumarinyl moieties were crosslinked by irradiation with UV light at λ_max = 366 nm, and the crosslinked sites were then cleaved by irradiation with UV light at λ_max = 254 nm. The crosslinking behaviors of the polymers were studied by UV and FTIR spectroscopic measurement. PolyCMVE was soluble in dichloromethane but was found to be insoluble upon UV light irradiation. We also synthesized amphiphilic block polymers bearing coumarinyl moieties by living cationic copolymerization with an amphiphilic vinyl ether. The resulting block polymers were soluble in an aqueous medium and also formed micelle‐like aggregates. Upon UV irradiation of aqueous solutions above the critical micelle concentration, an efficient crosslinking reaction occurred. Photoinduced structural changes of these polymer aggregates in the solution state were further investigated. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Spectroscopic studies into the influence of UV radiation on elastin in the presence of collagen

An investigation into the influence of UV irradiation on elastin hydrolysates in the presence of collagen was carried out using UV-Vis spectroscopy and spectrofluorometry. It was found that the absorbance of elastin hydrolysates in solution increased during irradiation more than the absorbance of the elastin/collagen blend. The fluorescence of elastin hydrolysates was observed at 305nm and at 380nm after excitation at 270nm. For the elastin/collagen mixture in solution, fluorescence spectrum shows only one maximum at 305nm. UV irradiation caused fluorescence fading at 305nm. For irradiated elastin the fluorescence at 305nm decreased faster than for the irradiated elastin/collagen mixture. The maximum of the fluorescence peak was shifted for elastin by 4nm, whereas for the elastin/collagen blends the shift was only 1-2nm. All the obtained results point out the ability of mixing elastin and collagen, and suggest that the elastin/collagen mixture in solution is less sensitive to UV irradiation than elastin hydrolysates alone.     

Photochemical stability of poly(vinyl pyrrolidone) in the presence of collagen

The photochemical stability of poly(vinyl pyrrolidone) (PVP) in the presence of 1%, 3% and 5% of collagen has been studied by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetry analysis (TG) and derivative thermogravimetry (DTG). Surface properties have been studied by contact angle measurements. PVP samples and samples containing 1%, 3% and 5% of collagen were irradiated with UV light of wavelength λ = 254 nm in air for up to 24 h. The amount of gel created during UV irradiation was estimated.PVP in the presence of 1%, 3% and 5% of collagen is less stable both thermally and photochemically. Collagen enhances photochemical processes leading to crosslinking of PVP. The contact angle measurements and values of surface free energy showed that the wettability of PVP films was changed by the addition of collagen and by UV irradiation. The increase of polarity of samples indicates an efficient photooxidation on the surface upon UV irradiation.     

Dynamics of cyclodimerization and viscoelasticity of photo‐crosslinkable PVA

We investigated the interfacial properties of poly(vinyl alcohol) carrying UV‐crosslinkable pendant quaternized stilbazole (styrylpyridinium), PVA‐SbQ. The extent and dynamics of PVA‐SbQ cyclodimerization reactions and crosslinking induced by UV irradiation were monitored in situ and in real time by quartz crystal microgravimetry (QCM). Sensograms reflecting time‐dependent changes in density and viscoelasticity of crosslinking films followed a Boltzmann sigmoidal model, depending on precursor film composition and irradiation power. The shifts in QCM frequency and energy dissipation upon PVA‐SbQ cyclodimerization correlated with three photo‐crosslinking phases involving soft‐to‐rigid transitions, namely, induction (initiation), main crosslinking (interaction), and termination. PVA‐SbQ films crosslinked to different degree were used as protein carriers and a slower release profile was determined for the films that underwent more extensive crosslinking. Overall, this study demonstrates for the first time the dynamics of PVA‐SbQ crosslinking and its impact in system viscoelasticity and protein release. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 345–355     

Effects of ultraviolet light on free and peptide-bound pyridinoline and deoxypyridinoline cross-links. Protective effect of acid pH against photolytic degradation

Little is known about the photodegradation of pyridinoline (Pyd) and deoxypyridinoline (Dpd), which are two mature cross-links stabilizing collagen within extracellular matrix. In this study, highly purified free Pyd and Dpd cross-links have been degraded by irradiation with ultraviolet light and we have shown that photolysis varies with the pH value. Assessment of photolysis in basic (pH 9) and neutral (pH 7) solutions by high-performance liquid chromatography as well as by UV absorbance measurement indicates that both cross-links are degraded after a 24 h UV exposure, while in acidic solution (pH 3) only Dpd is photolysed, suggesting that acid pH provides major protection against Pyd photolysis. Photodegradation products have been studied by amino-acid and mass spectral analysis. Both methods confirm the lack of Pyd degradation in acid pH. Furthermore, amino-acid analysis allows us to identify hydroxylysine and lysine as a result of Pyd and Dpd photolysis, respectively, indicating that the mechanism of photodegradation involves the cleavage of the pyridinium ring on each side of the quaternary nitrogen. Finally, we have also studied the photolysis of different molecular species of type I collagen peptides, obtained by digestion with collagenase of demineralized turkey bone. Our results indicate that even when they are part of the structure of collagen peptide, Pyd and Dpd can be photolysed. However, we have shown that the larger the peptide is, the smaller are the effects of UV irradiation.     

紫外光照射对皮胶原热降解活化能的影响

将皮胶原经不同时间的紫外光照射,采用热重法(TG)和微分热重分析(DTG)研究了照射前后皮胶原的热降解行为,用Horowitz-Metzger法和Coats-Redfern法计算了其热降解活化能,得到了未照射皮胶原和经过不同紫外线照射时间处理后的皮胶原的热降解活化能。研究发现,在较短时间(0~4h)的照射后,皮胶原的热降解活化能略有增大;在较长时间(8~64h)的照射后,皮胶原的热降解活化能大幅降低。这可能是由于在紫外光照射的过程中,皮胶原分子链发生了以交联为主和以断链为主的复杂光化学反应。