Guoying Li

Preparation and characterization of collagen/hydroxypropyl methylcellulose (HPMC) blend film.

This study aimed to prepare and characterize the collagen/HPMC blend film (1/1). Thermogravimetric analysis and differential scanning calorimetry were used to investigate the thermal properties of the film. Both thermal decomposition temperature and denaturation temperature of the blend film were higher than those of the collagen film due to the intermolecular hydrogen bonding interaction between collagen and HPMC, which was demonstrated by Fourier transform infrared spectroscopy. Additionally, the morphologies, mechanical properties and hydrophilicity of films were examined. The blend film exhibited a more homogeneous and compact structure compared with that of the collagen film, as observed from scanning electron microscopy and atomic force microscopy. The tensile strength, ultimate elongation and hydrophilicity of the blend film were superior to those of the pure collagen film. Furthermore, the introduction of polyethylene glycol 1500 had almost no influence on the thermal properties of the blend film but obviously improved its stretch-ability and smoothness.

Effects of NaCl on the rheological behavior of collagen solution

The effects of NaCl on the rheological properties of collagen solutions were studied by steady shear test and thixotropic loop test. The results showed the rheological properties of the collagen solutions with different NaCl concentrations were quite different. With low NaCl concentrations (0 to 0.1 mol/L), the collagen solutions exhibited salting in effect. The pseudoplastic behavior of the solutions became weak, while the thixotropy became strong. The collagen solutions with high NaCl concentrations (0.1 to 0.3 mol/L) exhibited salting out effect. The pseudoplastic behavior of the solutions became strong and the thixotropy had no obvious variation. In addition, Ostwald de-waele model, Carreau model and Herschel-Bulkley model were used to fit the experimental data. The superimposed experimental data and the model curves indicated the suitability of the models used, except for the up curves in thixotropic loop test.

Two-dimensional infrared spectroscopic study on the thermally induced structural changes of glutaraldehyde-crosslinked collagen.

The thermal stability of collagen solution (5 mg/mL) crosslinked by glutaraldehyde (GTA) [GTA/collagen (w/w)=0.5] was measured by differential scanning calorimetry and Fourier transform infrared spectroscopy (FTIR), and the thermally induced structural changes were analyzed using two-dimensional (2D) correlation spectra. The denaturation temperature (Td) and enthalpy change (ΔH) of crosslinked collagen were respectively about 27°C and 88 J/g higher than those of native collagen, illuminating the thermal stability increased. With the increase of temperature, the red-shift of absorption bands and the decreased AIII/A1455 value obtained from FTIR spectra indicated that hydrogen bonds were weakened and the unwinding of triple helix occurred for both native and crosslinked collagens; whereas the less changes in red-shifting and AIII/A1455 values for crosslinked collagen also confirmed the increase in thermal stability. Additionally, the 2D correlation analysis provided information about the thermally induced structural changes. In the 2D synchronous spectra, the intensities of auto-peaks at 1655 and 1555 cm(-1), respectively assigned to amide I band (CO stretching vibration) and amide II band (combination of NH bending and CN stretching vibrations) in helical conformation were weaker for crosslinked collagen than those for native collagen, indicating that the helical structure of crosslinked collagen was less sensitive to temperature. Moreover, the sequence of the band intensity variations showed that the band at 1555 cm(-1) moved backwards owing to the addition of GTA, demonstrating that the response of helical structure of crosslinked collagen to the increased temperature lagged. It was speculated that the stabilization of collagen by GTA was due to the reinforcement of triple helical structure.

The influence of chondroitin 4-sulfate on the reconstitution of collagen fibrils in vitro

Collagen fibrils were in vitro reconstituted from the aggregated collagen solution in the presence of a wide range of collagen/chondroitin 4-sulfate (Col/C4S) ratios. As revealed by turbidimetric measurement, the collagen fibril formation is significantly accelerated by C4S. The turbidity values of both the solution after 30 min preincubation at 4°C and the gels after 60 min preincubation at 37°C become larger with the increase of C4S amount. According to the results obtained from turbidimetric measurement and atomic force microscopy observation of solutions, it is predicted that the preincubation of Col/C4S blends at 4°C is necessary to initiate the Col/C4S binding and then promote the further lateral fusion of collagen aggregates in solution. The interactions between collagen and C4S are also vital in the growth phase of collagen self-assembly. Collagen quantitation data show that the amounts of collagen incorporated into self-assembled cofibrils increase a lot as a result of the presence of C4S. Differential scanning calorimetry measurement shows that the thermal stability of cofibrils keeps increasing with the ascending amount of incorporated C4S. It is suggested that the bound C4S might be captured inside the cofibrils acting as promoter and stabilizer. Atomic force microscopy and scanning electron microscopy observations of self-assembled fibrils indicate that the size increase of the self-assembled cofibrils depends on the lateral accretion of small collagen fibrils, while the self-assembly mode of collagen is not affected.

Physicochemical properties of collagen solutions cross-linked by glutaraldehyde

Abstract The physicochemical properties of collagen solutions (5 mg/ml) cross-linked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w) = 0–0.5] under acidic condition (pH 4.00) were examined. Based on the results of the determination of residual amino group content, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, dynamic rheological measurements, differential scanning calorimetry and atomic force microscopy (AFM), it was proved that the collagen solutions possessed strikingly different physicochemical properties depending on the amount of GTA. At low GTA amounts [GTA/collagen (w/w) ≤ 0.1], the residual amino group contents of the cross-linked collagens decreased largely from 100% to 32.76%, accompanied by an increase in the molecular weight. Additionally, increases of the fiber diameter and the values of G′, G″ and η* were measured, while the thermal denaturation temperature (Td) did not change visibly and the fluidity of collagen samples was still retained with increasing the GTA amount. When the ratio of GTA to collagen exceeded 0.1, although the residual amino group content only decreased by ∼8.2%, the cross-linked collagen solution [GTA/collagen (w/w) = 0.3] displayed a clear loss of flow and a sudden rise (∼2.0 °C) of the Td value compared to the uncross-linked collagen solution, probably illustrating that the collagen solution was converted into a gel with mature network structure-containing nuclei observed in AFM image. It was conjectured that the physicochemical properties of the collagen solutions might be in connection with the cross-linking between collagen molecules from the same aggregate or different aggregates.

Effect of hydroxypropyl methylcellulose on collagen fibril formation in vitro.

Collagen and hydroxypropyl methylcellulose (HPMC) were mixed to obtain blends and the effect of HPMC on collagen self-assembly was studied. As deduced from atomic force microscopy (AFM), the amount of nuclei in collagen-HPMC solutions was changed with the addition of HPMC. Under physiological conditions, the kinetics curves of fibril formation showed that the turbidity of blends at 313 nm was higher than that of native collagen. More HPMC was involved in the hydrogel network for blends with higher HPMC/collagen. However, both the thermal stability and the storage moduli of hydrogels, which was evaluated by UV and rheological measurements respectively, reached the maximum just when HPMC/collagen=0.25. Furthermore, it was showed by AFM that denser fibrils with smaller diameter would be obtained as HPMC/collagen<0.25, while more addition of HPMC (HPMC/collagen>0.25) would bring about fibrils with larger diameter. However, HPMC did not significantly affect the characteristic D-periods of the fibrils for all blends.

Characterization of acylated pepsin-solubilized collagen with better surface activity.

A new kind of acylated collagen with water solubility and better surface activity was prepared via reaction of pepsin-solubilized calf skin collagen with lauroyl chloride and succinic anhydride in this paper. The equilibrium surface tension and the isoelectric point were 55.92 mN/m and 4.93 respectively, suggesting that acylated collagen had surface activity as well as water solubility. Meanwhile, the results of Fourier transform infrared spectroscopy analyses and electrophoresis patterns demonstrated that the triple helix conformation of collagen was not destroyed, but the subunits of acylated collagen shifted to higher molecular weight than those of native collagen. Scanning electron microscope and differential scanning calorimeter measurements revealed that lyophilized acylated collagen exhibited relatively well-distributed pore structure and its denaturation temperature was about 9.0 °C higher than that of native collagen. Additionally, the increase of the diameter of the fibrils was observed by atomic force microscopy. Acylated collagen with water solubility and better surface activity might broaden the application of collagen-based materials to cosmetics, drug delivery and pharmacotherapy.

Rheological properties of glutaraldehyde-crosslinked collagen solutions analyzed quantitatively using mechanical models

Understanding the rheological behavior of collagen solutions crosslinked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w)=0-0.1] is fundamental either to design optimized products or to ensure stable flow. Under steady shear, all the samples exhibited pseudoplasticity with shear-thinning behavior, and the flow curves were well described by Ostwald-de Waele model and Carreau model. With increased amounts of GTA, the viscosity increased from 6.15 to 168.54 Pa·s at 0.1s(-1), and the pseudoplasticity strengthened (the flow index decreased from 0.549 to 0.117). Additionally, hysteresis loops were evaluated to analyze the thixotropy of the native and crosslinked collagen solutions, and indicated that stronger thixotropic behavior was associated with higher amount of GTA. Furthermore, the values of apparent yield stress were negative, and a flow index <1 for all the systems obtained via Herschel-Bulkley model confirmed that the native and crosslinked collagen solutions belonged to pseudoplastic fluid without apparent yield stress. However, the increment of dynamic denaturation temperature determined by dynamic temperature sweep was not obvious. The viscoelastic properties were examined based on creep-recovery measurements and then simulated using Burger model and a semi-empirical model. The increase in the proportion of recoverable compliance (instantaneous and retardant compliance) reflected that the crosslinked collagen solutions were more resistant to the deformation and exhibited more elastic behavior than the native collagen solution, accompanied by the fact that the compliance value decreased from 39.317 to 0.152 Pa(-1) and the recovery percentage increased from 1.128% to 87.604%. These data indicated that adjusting the amount of GTA could be a suitable mean for manipulating mechanical properties of collagen-based biomaterials.

Changes in aggregation behavior of collagen molecules in solution with varying concentrations of acetic acid

A critical aggregation concentration of 0.30-0.50mg/mL was previously obtained for type I collagen at 0.1M acetic acid (AA). In the present study, the aggregation behavior of collagen in solution (0.5mg/mL) in the presence of 0.1-2.0M AA was investigated. Circular dichroism showed that the three helix structure was maintained across the whole AA concentration range. However, the ratio of positive peak intensity over negative peak intensity varied depending on the conformational state of collagen aggregates. Ultra-sensitive differential scanning calorimetry revealed that transition temperatures Tm1 and Tm2 decreased by 8.35°C and 7.80°C, respectively, between 0.1M and 2.0M, indicating a possible relationship between the aggregation state and the thermal effect. The surrounding polarity of collagen molecules in solution containing pyrene was investigated by fluorescence spectroscopy, which demonstrated that disaggregation of collagen aggregates was enhanced with increasing AA concentration. This observation was correlated with changes in collagen fiber size observed by atomic force microscopy. Furthermore, collagen tyrosine residues were blue-shifted in an intrinsic fluorescence spectra, further indicating changes in aggregation behavior with increasing AA concentration. Finally, the dynamic response of collagen molecules to AA was analyzed by two-dimensional correlation fluorescence spectra.

Fluorescence study on the aggregation of collagen molecules in acid solution influenced by hydroxypropyl methylcellulose.

The effect of hydroxypropyl methylcellulose (HPMC) on the aggregation of collagen molecules with collagen concentrations of 0.25, 0.5 and 1.0mg/mL was studied by fluorescence techniques. On one hand, both the synchronous fluorescence spectra and fluorescence emission spectra showed that there was no change in the fluorescence intensity of collagen intrinsic fluorescence when 30% HPMC was added, while it decreased obviously when HPMC content ≥ 50%. From the two-dimensional fluorescence correlation analysis, it was indicated that collagen molecules in 0.25 and 0.5mg/mL collagen solutions were more sensitive to HPMC than those in 1.0mg/mL collagen solution. On the other hand, the pyrene fluorescence and the fluorescence anisotropy measurements indicated that HPMC inhibited the collagen aggregation for 0.25 and 0.5mg/mL collagen, but promoted it for 1.0mg/mL collagen. The atomic force microscopy images further confirmed the effect of HPMC on collagen with different initial states.