Observation of liquid crystalline collagen with atomic force microscopy ( AFM )

The effects of concentration and sonication on the liquid crystalline phases of collagen were investigated by several methods, especially by the atomic force microscopy (AFM). The X-ray diffraction (XRD) results revealed that the triple-helical structure of the collagen was nearly unchanged after sonication. Moreover, the differential scanning calorimetry (DSC) examinations indicated that the thermal stability of the sonicated collagen was close to that of native collagen. The AFM observations showed that collagen with a concentration of 60 mg/mL had more ordered arrays compared to that of 30 mg/mL when both samples were treated by sonication. Furthermore, the 60 mg/mL collagen solution without sonication could still form pre-cholesteric patterns, while the liquid phase could not be observed for the 30 mg/mL collagen solution under the same conditions. Generally, AFM was an effective tool for the study of the liquid crystalline phases of collagen.


INTRODUCTION
Collagen is the major structural component in connective tissues, which accounts for 20-30% of the total amount of protein in the mammalian body. 1 As an important biomass, collagen has been a hot research topic, as it is biocompatible, biodegradable, and shows low immunogenicity and favors cell adhesion and proliferation. 2,3In connective tissues such as bone, cornea, dermis and tendon, collagen can be assembled into extracellular domains in specific and ordered arrays, which is classically described in the literature 4 .It is of great interest to investigate if the collagen solutions are able to mimic the supramolecular assemblies described in these connective tissues, which means that collagen could be biomaterial substitutes for living tissues.
Giraud-Guille has reported that collagen at high concentrations in acid solution could be gathered spontaneously in liquid crystalline phases. 52][13] Furthermore, the concentrated collagen solution has been used successfully as a liquid crystal biological template for silica structuration from Nano to microscopic scales. 11he aforementioned studies investigated the liquid crystalline model both in vivo and in vitro commonly using the polarized light microscopy (PLM) and the transmission electron microscopy (TEM).As widely known, the atomic force microscopy (AFM) is useful tool to characterize microstructures since minimal specimen preparation is required and very high vertical resolution (< 0.1 nm) is achieved. 146][17][18][19] One of the main advantages of AFM is the possibility to visualize the nonconductive materials in a non-vacuous environment directly, such as air or liquids, without any staining.This technique is a very good supplement for the other ultra-structural methods (such as TEM), as it requires collagen gels without any additional preparation steps (like fixation or coating) to minimize artefacts.In addition, this technique appears to be cost-and time-effective.
The aim of our present study is therefore to test the possible application of the AFM to the liquid crystalline phases of collagen.This study may provide useful information on the suitability of AFM as a tool to identify the liquid crystalline orders in condensed collagen.In this paper, the influence of both concentration and sonication were considered as the critical factors for the formation of liquid crystals, and the liquid crystalline assemblies of collagen was investigated via AFM.

Preparation of collagen
Collagen was extracted from calf skin by following the method described by Zhang et al. 20 The delimed bovine split pieces was neutralized with 0.5 mol/L acetic acid containing 3% pepsin (EC 3.4.23.1, 1: 10,000, Sigma Chemical Co.).The mixture was centrifuged at 9000 G, the supernatant was salted out, and the precipitate was redissolved in 0.5 mol/L acetic acid.After dialysis in 0.01 mol/L acetic acid, the collagen solution was stored at 4 ℃ prior to the following experiments.

Production of collagen liquid crystalline phases
Aliquots of 10 mL collagen stock solutions were subjected to ultrasonic treatment at 20 kHz and 50 W at 4 ℃ for 10 min, and then concentrated to 30 mg/mL or 60 mg/mL by evaporating the solvent at reduced pressure.The obtained concentrated collagen samples were then examined by atomic force microscopy (AFM).Part of the concentrated solutions was gelated by placing the samples in ammonia vapor for 1 h and then overnight at room temperature, and then lyophilized prior to the testing of Xray diffraction field (XRD) and differential scanning calorimetry (DSC).The transition from a sol state at acid pH to a gel state at neutral pH without any dilution of the samples retained the initial three-dimensional order observed in the liquid crystalline phases, according to Besseau and Giraud-Guille. 23or simplicity's sake, the sonicated collagen sample (30 mg/mL), un-sonicated collagen sample (30 mg/mL), sonicated collagen sample (60 mg/mL) and un-sonicated collagen sample (60 mg/mL) were referred to as S-col (30), U-col (30), S-col (60) and U-col (60) in the following text.

X-ray diffraction (XRD)
X-ray powder diffraction measurements of the collagen samples were performed using a diffractometer (Panalytical X'pert Pro MPD, Netherlands) at a scanning rate of 1°/min in the 2θ range from 5 to 80°, with a 0.2-mm Ni filter plate and Cu Kα radiation (λ=0.154056nm).

Differential scanning calorimetry (DSC)
The thermal transition of collagen samples was determined by a differential scanning calorimetry (DSC) (Netzsch DSC 200PC, Germany).The lyophilized collagen samples were dissolved with 0.05 mol/L acetic acid to reach a concentration of 5 mg/mL.The dissolved samples (~10 mg) were weighed accurately in a DSC aluminum pan, sealed, and scanned over the range from 22.5 to 50.0 ℃ at a heating rate of 3 ℃ /min in a nitrogen atmosphere.Liquid nitrogen was used for cooling.The reference pan contained only 10 mg 0.05 mol/L acetic acid.

Atomic force microscopy (AFM)
The collagen solution was transfered slowly to a silicon wafer, and then exposed on acetic acid steam for 48 h.After rapid drying, AFM images were obtained by an atomic force microscopy (MFP-3D-origin, Asylum Research of Oxford Instruments) using square pyramidal silicon nitride tips with 0.58 N/m nominal spring constant.Scanning rate was set at 2 Hz, with all samples imaged in the height mode.Images presented were neither processed nor filtered and represented direct output from AFM.

Physicochemical properties of collagen
The supramolecular structure of the collagen films was examined by XRD (Fig. 1).X-ray diffraction diagrams of collagen yielded the first sharp peak at around 1.2 nm, which indicated the distance between molecular chains, while the second broad peak was caused by diffuse scattering.The third peak at around 0.29 nm was from the unit height of a typical triple helical structure, which was considered to be important to the conformational integrity of collagen.
According to the Bragg diffraction equation 21 as follows, 2dsinθ=0.154056nm (1) The distances between molecular chains of all collagen samples were calculated to be 1.197, 1.190, 1.187 and 1.180 nm for S-col (30), U-col (30), S-col (60) and U-col (60), respectively.These data demonstrated that the distances between molecular chains of collagen slightly decreased when collagen concentration increased from 30 mg/mL to 60 mg/mL.The corresponding distance decreased slightly due to the action of ultrasonic waves.
The third peak was calculated to be 0.285, 0.281, 0.287 and 0.292 nm for these four samples.Therefore, no significant variations were observed at the position of the third peak (0.281-0.292 nm), suggesting that the triplehelical structure of collagen was nearly unchanged after sonication.
Fig. 1.X-ray diffraction diagrams of collagen films derived from collagen fibrillogenesis, induced by exposure to ammonia vapors Fig. 2 displays the differential scanning calorimetry thermos-grams of collagen solutions.All the collagen samples possessed the main denaturation peak at 39.0 ℃, derived from the collapse of the collagen triple helix to a random coil, that is, the breakage of bonds that stabilized the secondary structure of collagen. 22The peak locating at 33.0 to 33.5 ℃ for the sonicated collagen solutions of different concentrations indicate different thermal stabilities for the two collagen samples of different molecular populations.In general, the DSC results show that a small part of the collagen molecules was degraded in the sonication treatment, and most of them retained intact structure.

Texture observation of condensed phases of collagen by AFM
Fig. 3 showed the textures of sonicated collagen solutions of 30 and 60 mg/mL concentration, respectively.It was interesting to find that the two textures were analogous to precholesteric phase (Fig. 3 a1, a2 and b1, b2).However, compared to the S-col (30), the arrangement of the collagen fibrils was much more uniform for S-col (60).At higher magnifications (Fig. 3 a3, a4 and b3, b4), The images clearly show the laminas of arced patterns constituted by microfibrillar aggregates of 10-20 nm in average diameter.In addition, it seemed that the diameter of S-col (60) was larger than that of S-col (30).periodicity appeared every 180ºrotation of the molecular directions, which was defined as the half cholesteric pitch (P/2). 23The P/2 for the collagen of 60 mg/mL concentration was ~3μm, while it could not be estimated for the collagen of 30 mg/mL concentration, as the orientation was not clear.In conclusion, the AFM images clearly show the textures of collagen, and the detail structures of the microfibrillar aggregates of collagen.
The overall appearance of the collagen matrices was firstly apprehended by AFM on transverse semi-thin sections in gels.It was thus possible to confirm the positive impact of ultrasonic waves on the homogeneity of collagen solutions.Fig. 4 showed the two samples of the same concentration of 60 mg/mL: a sonicated gel (Fig. 4 a1-a3) and an un-sonicated gel (Fig. 4 b1-b3).Classical precholesteric textures were observed in both gels.In other words, the collagen fibrils were actually arranged in an undulated pattern.
Fig. 5 shows the AFM images for the two collagen samples prepared with the collagen solution of the same concentration (30 mg/mL): a sonicated gel (Fig. 5a1-a3) and an un-sonicated gel (Fig. 5b1-b3).In the first gel, precholesteric textures could still be observed, however, the orientation was weaker compared to the one prepared with 60 mg/mL solution.Nevertheless, there was no fingerprint pattern characteristic of a cholesteric liquid crystal phase observed from the un-sonicated collagen prepared with the 30 mg/mL solution.
Fig. 3 AFM images of sonicated collagen solutions with concentrations of 30 mg/mL (a1, a2, a3 and a4) and 60 mg/mL (b1, b2, b3 and b4) with different sizes Fig. 6 shows the AFM images for S-col (30) (Fig. 6 a), Ucol (30) (Fig. 6 b), S-col (60) (Fig. 6 c) and U-col (60) (Fig. 6d) at a higher magnification.Compared to S-col (30) and U-col (30), a new collagen texture was observed for Scol (60) and U-col (60).It appeared as dots (with dimensions similar to the half pitch of the cholesteric phase) that regularly interrupted cholesteric banding (Fig. 6 c and d for S-col (60) and U-col (60), respectively).In some regions, the dots from adjacent cholesteric stripes were aligned and showing staggered periodic structures (see arrows in Fig. 6  d).In other regions, the dots fused together.In addition, it seemed that this new texture was more clearly shown for the U-col (60) than for the S-col (60).
The transversal striations completely overtook the cholesteric pattern with increasing concentrations, suggesting a phase transition.Peixoto et al. 7 observed this kind of collagen texture at higher concentration other than the cholesteric phase, and believed that this optical response revealed a periodic discontinuity either in the cholesteric director orientation or in collagen organization.Fig. 4. AFM images of sonicated (a1, a2 and a3) and un-sonicated (b1, b2 and b3) collagen solutions with concentrations of 60 mg/mL While the formation of ordered arrays of collagen may be influenced by many factors, the present work focused on the effect of collagen concentration and sonication on the liquid crystalline structure of collagen.Firstly, we found that the concentration of collagen was crucial to the formation of liquid crystal.Compared to the collagen prepared with 30 mg/mL solution, the texture observed by AFM was more obvious orientation for collagen prepared with 60 mg/mL solution.This phenomenon suggested that the higher concentration would be beneficial to the longrange assemblies via liquid crystalline processes.It has been reported that the hydrophilic interaction would be reduced, while the hydrophobic effect would be enhanced when collagen concentration was increased.On the one hand, collagen molecules tend to attract each other due to the enhanced hydrophobic effect.On the other hand, they also tend to repel each other by the electrostatic force, as collagen molecules bear positive charges in acidic conditions.Therefore, equilibrium existed between the two forces in concentrated collagen solutions, and the collagen molecules adopted different alignments layer-by-layer to obtain the liquid phases 6 .Fig. 5. AFM images of sonicated (a1, a2 and a3) and un-sonicated (b1, b2 and b3) collagen solutions with concentrations of 30 mg/mL Fig. 6.AFM images (2×2 μm 2 ) of sonicated (a) and un-sonicated (b) collagen with concentration of 30 mg/mL; sonicated (c) and un-sonicated (d) collagen with concentration of 60 mg/mL Secondly, the formation of liquid crystalline phase could be promoted by proper sonication.Compared to the collagen treated by sonication, it was easier to form the precholesteric patterns for the un-sonicated collagen.Giraud-Guille compared the assembly properties of concentrated solutions of type I collagen before and after 5min sonication, and found that 300-nm triple helices could be broken into short segments of about 20 nm with a strong polydispersity.Giraud-Guille 5 also suggested that the breakage of collagen chains would be helpful for the short collagen triples helices to organize to cholesteric phases.Nevertheless, it could be deduced from the XRD and DSC results that only a small part of collagen molecules were degraded, which means that the native triple helices were maintained for most of the collagen molecules.
In the aqueous solution, collagen can be self-associated with enlarged concentration, which was mainly ascribed to the reduction in the repelling action between collagen molecular chains and thus the enhancement in intra-and inter-molecular hydrogen bonds. 24,25 hi et al. 26 reported the aggregation behavior of collagen in 0.1 mol/L acetic solution and proved that the aggregation behavior occurred in collagen solution when collagen concentration was increased to 0.5 mg/mL or above.Therefore, aggregates were formed in collagen solutions with initial concentration of 5 mg/mL employed in the present work.The mobility of collagen chains would be restrained by the entanglement effect amongst the very elongated collagen molecules, resulting in the difficulty in the long-range alignment.When treated by sonication, collagen molecules in the aggregates would be partly dispersed, thus the alignment of collagen molecules would be promoted during the concentrating process, and finally the liquid crystal phase would be easier to obtain.
Furthermore, it should be noted that 60 mg/mL collagen solution without sonication could still form precholesteric patterns, whereas 30 mg/mL collagen solution without sonication formed disorganized morphology revealed by AFM.It seemed that both the hydrophobic effect and electrostatic repelling force between collagen molecules could be enhanced as collagen concentration reached 60 mg/mL, leading to the formation of liquid crystals, even if the treatment of sonication was absent.At low collagen concentration (e.g. 30 mg/mL), the hydrophobic and electrostatic effects alone were not sufficient to cause formation of liquid crystals.In other words, the sonication was not essential for the collagen solution of higher concentration (60 mg/mL) to form precholesteric patterns, while it played an critical role in the formation of liquid crystalline phase for the collagen solution of lower concentration (30 mg/mL).
AFM turned out to be a useful tool to investigate the influence of sonication and concentration on the alignment behavior of collagen molecules.The high resolution AFM images clearly show the fine structures of cholesteric stripes formed by a microfibrils mass, as well as the new collagen texture dots which could not be observed with PLM.The sample preparation for AFM observation was much simpler than for TEM.Moreover, the dehydration and embedding procedures for TEM may affect the texture of collagen.

CONCLUSIONS
This work was attempted to study the liquid crystal of collagen using atomic force microscopy (AFM).It was found that sonication treatment was essential for collagen solution of 30 mg/mL concentration to form the texture of liquid crystal, while it was unnecessary when the concentration of collagen solution reached 60 mg/mL.It seemed that there was no significant degradation of collagen caused by the treatment of sonication.The reason for the positive effect of sonication on the formation of precholesteric patterns of collagen might be that the aggregated collagen molecules were partly dispersed by the ultrasonic treatment, promoting the alignment of the collagen molecules during the concentrating process.
A new texture of collagen was discovered by the AFM analyses, which was shown as dots aligned from adjacent cholesteric stripes with staggered periodic structures.In conclusion, AFM was shown to be a useful tool for the study of collagen in condense phase, and the findings in this study could provide useful information for the synthesis of collagen-based tissue-like organized matrices for medical and pharmaceutical applications.