Patrizia Monti

Raman spectroscopic studies of silk fibroin from Bombyx mori

This study was focused on the conformational characterization of Bombyx mori silk fibroin in film, fiber and powder form by means of Fourier transform Raman spectroscopy. Native and regenerated silk fibroin films prepared by casting dilute silk fibroin solutions (<1%, w/v) display characteristic conformationally sensitive bands at 1660 cm-1 (amide I), in the range 1276–1244 cm-1 (a complex amide III region with multiple detectable maxima) and at 1107 and 938 cm-1. This spectral pattern can be related to a prevalently random coil conformation, with traces of α-helix. Liquid silk, prepared by casting the silk gland content (fibroin concentration 20–25%, w/v), shows almost the same wavenumbers in the amide I and III ranges, while differences appear below 1000 cm-1, where three bands at 952, 930 and 867 cm-1 increase in intensity. The spectral differences between films and liquid silk are discussed with a view to identifying possible markers for silk I structure, a crystalline modification of silk fibroin. The treatment of both native and regenerated films with 50% (v/v) methanol solution induces the conformational transition to a β-sheet structure, as demonstrated by the shift of amide I to 1665 cm-1 and the appearance of new maxima at 1262 and 1236 cm-1 (amide III) and at 1084 cm-1. When liquid silk is cast at above 50°C, the prevailing conformation taken by silk fibroin is β-sheet, whatever the rate of drying. By comparing the Raman spectra of silk fibroin fiber and powder, both having a β-sheet structure, a difference in the tyrosine doublet bands and in the amide I band can be observed. The value of the I853/I830 (Rtyr) intensity ratio increases in the powder while amide I shifts to lower wavenumbers, suggesting that the hydrogen bonds involving the tyrosil residues are weaker in the powder than in the fiber.

Structure and molecular conformation of tussah silk fibroin films: Effect of heat treatment

Structural changes of tussah (Antheraea pernyi) silk fibroin films induced by heat treatment were studied as a function of the treatment temperature in the range 200–250°C. The DSC curve of tussah films with α-helix molecular conformation displayed characteristic endo and exo peaks at 216 and 226°C, respectively. These peaks first weakened and then completely disappeared after heating at 230°C. Accordingly, the TMA thermal shrinkage at 206°C disappeared when the films were heated at 230°C. The onset of weight loss was monitored at 210°C by means of TG measurements. X-ray diffraction profiles gradually changed from α-helix to β-sheet crystalline structure as the treatment temperature increased from 200 to 250°C. On raising the heating temperature above 200°C, the intensity of IR and Raman bands characteristic of β-sheet conformation increased in the whole ranges of amide and skeletal modes. The sample treated at 200°C showed a spectral pattern intermediate between α-helix and β-sheet molecular conformation. The IR marker band for random coil structure, still detectable at 200°C, disappeared at higher treatment temperatures. Spectral changes attributable to the onset of thermal degradation appeared at 230°C.

Binding of Co(II) and Cu(II) cations to chemically modified wool fibres: an IR investigation

Wool fibres were modified by treatment with tannic acid (TA) solution or by acylation with ethylenediaminetetraacetic (EDTA) dianhydride. The unmodified and modified fibres were subsequently treated with Cu 2þ and Co 2þ solutions, at alkaline pH, and analysed by Attenuated Total Reflectance, ATR/IR spectroscopy to evaluate the changes induced in the structure of the fibre by metal binding. The spectral changes were correlated to metal adsorption results obtained by Inductive Coupled PlasmaAtomic Emission Spectrometry (ICP-AES). The IR results were discussed in relation to our previous findings on the metal binding mode of Bombyx mori and Tussah silk fibres; the changes observed in the spectra were explained by considering the different affinity of the fibres for the modifying reagent and the amount of the metal absorbed. More relevant spectral changes were observed upon Cu 2þ complexation rather than Co 2þ complexation, according to the metal absorption results. The most relevant changes were observed for the EDTAmodified wool sample treated with Cu 2þ , according to the higher affinity of wool for EDTA. The IR spectra were quantitatively evaluated by the intensity ratio between the Amide I and Amide II bands (IAmideI/IAmideII) and its trend as a function of metal absorption was reported. The present investigation demonstrated that the interaction between fibre and metal and the subsequent fibre modification depend on the chemical nature of the fibre, the metal cation and the modifying reagent. q 2003 Elsevier Science B.V. All rights reserved.

Vibrational and thermal study on the in vitro and in vivo degradation of a poly(lactic acid)-based bioabsorbable periodontal membrane

Fourier transform Raman (FT-Raman), attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectra and differential scanning calorimetry (DSC) measurements were performed on a poly(lactic acid)-based biodegradable periodontal membrane in order to study its in vitro and in vivo degradation mechanism and kinetics. For this purpose, the hydrolitic in vitro degradation of the membrane was investigated in two aqueous media: saline phosphate buffer (SPB, pH=7.4) and 0.01 M NaOH solution. Moreover, a membrane implanted in vivo for four weeks for treatment of contiguous vertical bony defects, was examined. Vibrational and thermal measurements show that the membrane has a prevalently amorphous structure and is composed of low molecular weight polymeric chains. The degradation is faster in NaOH solution than in SPB and occurs heterogeneously without any significative increase in crystallinity. The DSC and spectroscopic measurements are discussed in comparison with the trend of % weight loss and show a progressive decrease in molecular weight. Regarding the Raman analysis, the I875/I1452 intensity ratio was identified as a marker of the degree of degradation. Regarding the in vivo degradation, the presence, spectroscopically revealed, of a biological component entrapped in the membrane proves the good integration of the membrane with the surrounding tissues. The membrane seems to degrade faster in vivo than in vitro. A comparison with the degradation mechanism and kinetics of a periodontal membrane previously studied, Vicryl® periodontal mesh, is made.

IR study on the binding mode of metal cations to chemically modified Bombyx mori and Tussah silk fibres

Abstract Bombyx mori ( B. mori ) and Tussah ( Antheraea pernyi ) silk fibres were modified by treatment with tannic acid (TA) or ethylenediaminetetraacetic (EDTA) dianhydride, subsequently treated with Cu 2+ and Co 2+ solutions, at alkaline pH, and analysed by attenuated total reflectance/infrared spectroscopy to evaluate the changes induced in their structure by metal binding. The spectral changes were correlated to metal binding results obtained by inductive coupled plasma-atomic emission spectrometry. Upon Co 2+ complexation, the spectra of all the B. mori and Tussah silk samples showed a decrease in intensity of the Amide I band with a trend related to the metal uptake. The most relevant changes in the whole spectra were observed for the EDTA- and TA-modified samples in the case of B. mori and Tussah silks, respectively. Upon Cu 2+ complexation, the decrease in intensity of the Amide I band follows the trend of the metal uptake only in the case of Tussah silk. Moreover, the most relevant changes with respect to the untreated sample were observed for the TA-modified B. mori silk sample. The spectral changes were explained by considering the different affinities of the fibres for the modifying reagent, the amount of the metal bound and the relative stability of the complexes formed.

Structure and molecular conformation of tussah silk fibroin films treated with water–methanol solutions: Dynamic mechanical and thermomechanical behavior

The thermal response of tussah (Antheraea pernyi) silk fibroin films treated with different water–methanol solutions at 20°C was studied by means of dynamic mechanical (DMA) and thermomechanical (TMA) analyses as a function of methanol concentration and treatment time. The DMA curves of α-helix films (treated with ≥80% v/v methanol for 2 min and 100% methanol for 30 min) showed the sharp fall of storage modulus at about 190°C, and the loss peak in the range 207–213°C. The TMA curves were characterized by a thermal shrinkage at 209–211°C, immediately followed by an abrupt extension leading to film failure. Both storage and loss modulus curves significantly shifted upwards for β-sheet films, obtained by treatment with ≤60% methanol for 30 min. The loss peak exhibited a maximum at 236°C. Accordingly, the TMA shrinkage at above 200°C disappeared. The films broke beyond 330°C, failure being preceded by a broad contraction step. Intermediate DMA and TMA patterns were observed for the other solvent-treated films. The loss peak shifted to higher temperature (219–220°C), and a minor loss modulus component appeared at about 230°C. This coincided with the onset of a plateau region in the storage modulus curve. The TMA extension–contraction events in the range 200–300°C weakened, and the samples displayed a final broad contraction (peak temperature 326–338°C) before breaking. The DMA and TMA response of these films was attributed to partial annealing by solvent treatment, which resulted in the formation of nuclei of β-sheet crystallization within the film matrix. The increased thermal stability was probably due to the small β-sheet crystals formed, which acted as high-strength junctions between adjacent random coil and α-helix domains.

Applications of Raman spectroscopy to the ophthalmological field : Raman spectra of soft contact lenses made of poly-2-hydroxyethylmethacrylate (PHEMA).

Abstract Raman and i.r. spectra of soft contact lenses made of PHEMA are discussed in relation with their structural properties and clinical applications. Complementary TG,DSC and TMA measurements are also presented. The results of this study show that such lenses present the requested characteristics of biocompatibility.

Vibrational and thermal study on the in vitro and in vivo degradation of a bioabsorbable periodontal membrane: Vicryl® Periodontal Mesh (Polyglactin 910)

Fourier transform Raman (FT-Raman), attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectra and differential scanning calorimetry (DSC) measurements were performed on a biodegradable periodontal membrane, the Vicryl® periodontal mesh, in order to study its in vitro and in vivo degradation mechanism and kinetics. The hydrolitic in vitro degradation was investigated in two aqueous media: a saline phosphate buffer (SPB, pH=7.4) and a 0.01 M NaOH solution. Moreover, a membrane implanted in vivo for 4 weeks for treatment of contiguous vertical bony defects, was examined. Vibrational and thermal measurements show that the Vicryl® membrane presents a semicrystalline structure. It degrades faster in the NaOH solution than in the SPB and degradation occurs heterogeneously with a progressive increase in the percentage of crystallinity and shortening of the polymeric chains both in vitro and in vivo. The trends of % weight loss and IR I627/I1415 intensity ratio (identified as a marker of crystallinity) are discussed in comparison with the DSC results. The IR I627/I1415 intensity ratio and Xc% allow to determine the % weight loss undergone by the membrane degraded in vivo. The result obtained shows that the Vicryl® membrane degrades faster in vivo than in vitro with the formation of oligomers which are more easily absorbed by the surrounding tissues than they are soluble in the degradation media examined.

Hydrogen bonds in calcium acid phosphates by infrared and Raman spectra

Abstract Infrared and Raman spectra of Ca(H2PO4)2, Ca(H2PO4)2·H2O and deuterated analogues are presented and discussed. The behaviour of the asymmetric stretching vibration νOH and of the in-plane δOH and out-of-plane γOH deformations shows that hydrogen bonds of different strengths are present in both compounds, in agreement with X-ray diffraction data. The perturbation brought about by the water molecule on the strength of the hydrogen bond is also considered.

In vivo bioactivity of titanium and fluorinated apatite coatings for orthopaedic implants: a vibrational study

Abstract The bone integration of implants is a complex process which depends on chemical composition and surface morphology. To accelerate osteointegration, metal implants are coated with porous metal or apatites which have been reported to increase mineralisation, improving prosthesis fixation. To study the influence of composition and morphology on the in vivo bioactivity, titanium screws coated by Plasma Flame Spraying (PFS) with titanium or fluorinated apatite (K690) were implanted in sheep tibia and femur for 10 weeks and studied by micro-Raman and IR spectroscopy. The same techniques, together with thermogravimetry, were used for characterising the pre-coating K690 powder. Contrary to the manufacturer report, the K690 pre-coating revealed to be composed of a partially fluorinated apatite containing impurities of Ca(OH) 2 and CaCO 3 . By effect of PFS, the impurities were decomposed and the crystallinity degree of the coating was found to decrease. The vibrational spectra recorded on the implanted screws revealed the presence of newly formed bone; for the K690-coated screws at least, a high level of osteointegration was evidenced.