Insights into the interactions between porcine collagen and a Zr–Al–Ti metal complex

Abstract

Porcine acelluar dermal matrix (pADM), known as pure collagen with three dimensional structure, was used to explore the interactions between porcine collagen and a metal complex in this study. The metal complex mainly consists of elements Zr, Al and Ti (patented product named DMT-II). Tests of Fourier transform infrared spectrometry (FTIR), the crosslinking degree, Diffraction Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD) were carried out to further probe the microscopic changes between collagen and the metal complex. Results have revealed that the DMT-II could react with collagen between fibers and at functional groups in the collagen molecules. The unique structure of collagen has been retained with only small changes of intensity of the characteristic absorption peaks appeared. The highest thermal denaturation temperature of pADM after reaction with DMT-II (50%) was 86.6 °C, which has been improved by 18.9 °C. The crystal structure analyzed by XRD showed that DMT-II affected the triple helical structure of collagen to some degree, proving that the reaction took place at the collagen molecules. Through morphology observation, it was clear that DMT-II changed the fiber distribution, and the fibers in pADM assembled together to form a tight layer. By a series of tests, results showed that the reaction between collagen and DMT-II could take place both in fibers and in collagen molecules, which paved a new way for collagen modification.

---

1 Introduction

Collagen, the most abundant protein in vertebrates, is generally extracted from tendons, skin, ligaments, corneas and other tissues. For a long time, collagen has been known to have a unique biocompatibility, safety, low antigenicity and high hydrophilicity, which has led to it being used in biomaterial fields. In particular, it was found that collagen has the ability to enhance cellular activities, including cell attachment and proliferation.¹ For these reasons, collagen has been widely applied to wound healing dressings,^2,3 hemostatic sponge,⁴ bone substitution,^5–7 skin replacement⁸ and drug delivery systems.⁹ Notably, porcine acellular dermal matrix (pADM) is another way of making full use of collagen. As is known, pADM has frequently been used as a dermal substitute,^10,11 in defect repairing,¹² in tissue reconstruction,¹³ in biomaterial fields and as a filling material¹⁴ in plastic surgery. The three dimensional structure of pure collagen fibers endows pADM with unique functions. However, the tough issue of modification has hindered the further development of collagen based materials. Thus, a lot of research of collagen modification has been carried out, including blending with other natural polymers,^15–17 and crosslinked by glutaraldehyde,^18,19 carbodiimide,²⁰ epoxy compound,²¹ nano materials,^22,23 genipin²⁴ and other chemicals.²⁵ But still, the potential possibility of introducing viruses or other infectious agents from animals remains controversial, making synthetic materials another option for researchers. In a word, more efforts are still needed to avoid these risks to broaden the utilization of natural collagen.

Apart from being used as a biomaterial, collagen has also been studied for hundreds of years in the leather industry. As the main constitution of skin, collagen has to undergo complicated processes before being turned into leather. In those complex processes, tanning has been considered as the most important one and is the key process to turn raw hide into leather. In the tanning process, the tanning agent acts as the vital factor affecting the properties of the final products, including the flexibility, mechanical strength, thermal stability and stiffness. Among a number of different tanning agents, chrome tanning agents and vegetable tanning agents are most commonly used to produce light leather and heavy leather respectively. However, considering that the chrome tanning agent could inevitably bring harm to the environment and human health to some extent, researchers turned towards seeking other environmentally friendly substitutions. Therefore, other metal compounds like aluminum, zirconium and titanium have gained extensive attention. Especially in recent years, in an attempt to find a suitable substitute to reduce or replace the use of chromium, our group has been devoted to the development of the Zr–Al–Ti complex. Compared with the most commonly used metal chromium, zirconium, aluminum and titanium have potential to be used as substitutes because of their low toxicity. Efforts have been made to study the effects and mechanism of the metal complex on skin collagen.^26–28

It is vital to have more knowledge about the basic structure of the metal complex before more research is carried out, especially about the coordination bond types. The electronic structure of Zr⁴⁺ is 4d⁰5s⁰5p⁰, the same as that of Ti⁴⁺; this allows them to make different hybrid orbits, including sp³, d²sp³, d³sp³, d⁴sp⁴, etc. These hybrid orbits make it possible to form various configurations. The electronic structure of Al³⁺ is 2s²2p⁶3s⁰3p⁰3d⁰,²⁹ allowing easy formation of outer orbital coordination compounds. As for Zr⁴⁺, the electron pairs tend to enter the inner empty d orbit, which is comparatively stable because of its low energy. The coordination compound formed is relatively stable with a strong bonding energy. But for Al³⁺, the electron pairs only get the chance to enter the outer d orbit, which is unstable because of its high energy. The bonding energy between the central metal ion and ligand is relatively low. Ti⁴⁺ generally exists as TiO²⁺ in solution; the complex formed is comparatively unstable. Though the states of the different complexes in solution are acknowledged, it is still easy for them to form other more complicated coordination compounds when three of them added together. The early research³⁰ about DMT-II showed that the composition of the Zr–Al–Ti complex solution included Zr–Al, Zr–Ti, Ti–Al and Zr–Al–Ti with various ratios, which makes it difficult to reveal the interaction mechanism of DMT-II with other chemicals. However, efforts have been made for a long period of time to unveil this mystery.

Though the interaction mechanisms between collagen and the single metal complex have been revealed to some degree,³¹ the interactions between the Zr–Al–Ti complex and pADM has not been explored yet. Since the state in solution of the metal complex affects the reaction directly, it is necessary to go further in probing the molecular interactions between the Zr–Al–Ti complex and collagen.

2 Materials and methods

2.1 Materials

pADM and DMT-II were prepared strictly according to our patents ZL200410022506.9 and ZL200710048440.4, respectively. Briefly, porcine hide was used as the raw material. It has to undergo fleshing, washing, dehairing, alkaline swelling, shaving and enzymatic digestion processes before being lyophilized. In order to simplify the manufacturing process, pADM was directly provided by the Jiangyin Benshine Biological Technology CO. Ltd. with the authorization of our corresponding patent. DMT-II was firstly prepared by mixing Zr(SO₄)₂, Ti(SO₄)₂, Al₂(SO₄)₃ and citric acid in a certain given ratio, then the pH was adjusted to ∼1.5 by adding sodium hydroxide (1 mol L⁻¹). The final solution has to be stored for 24 h before further use. Other related chemicals were all of analytical grade.

2.2 pADM treatment with DMT-II

pADM was cut to 4 cm × 6 cm. 6 groups of 1 g pADM were firstly added to conical flasks respectively and 20 mL of pickling liquor (pH = 2.5, °Be′ ≈ 6.5–7.0) was added to each group to reach equilibrium. After shaking for 30 min at room temperature, DMT-II of different dosages of 10%, 30%, 50%, 100%, 200% and 300% (m_DMT-II/m_collagen) was added to each group and shaken for 2 h. Subsequently, pH of each group was adjusted to 4.0–4.2 by using sodium bicarbonate slowly, and finally deionized water was used to keep each flask at the same volume of solution. The reaction proceeded for another 2 h after elevating the temperature to 40 °C, and then the samples were stored overnight. Each sample was washed thoroughly and lyophilized before testing.

2.3 Crosslinking degree

The crosslinking degree was characterized by free amino content analysis. A ninhydrin reaction was performed to determine the free amino content.³² Briefly, 0.5 g ninhydrin was dissolved in 100 mL 95% ethanol and stored at 4 °C. 1 g KIO₃ was firstly dissolved in 300 mL deionized water, and then 200 mL 95% ethanol was added to achieve a total volume of 500 mL. Afterwards, 1 mg of the sample, 4 mL PBS and 1 mL ninhydrin solution were placed into a colorimetric tube and heated in a boiling water bath for 30 min. After that, the tubes were cooled to room temperature in a cold water bath. Finally, the optical absorbance of the solution was detected at 568 nm. Before the test, glycine with different concentrations (0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16 mmol L⁻¹) was used to make the standard curve. The linear equation was achieved by computer linear fitting. Subsequently, the free amino content was calculated according to the linear equation, and the crosslinking degree was defined as:

Crosslinking degree (%) = (NH₀ − NH_t)/NH₀ × 100

In this equation, NH_t and NH₀ represent the free amino content after treatment with DMT-II and the blank group of pADM respectively; 5 groups of each sample were measured to get the average value and standard deviation.

2.4 Differential scanning calorimetry analysis

Certain weights (3–5 mg) of different samples of pADM treated with DMT-II were applied to thermal property analysis in a temperature range of 20–150 °C using a differential scanning calorimeter (DSC-200PC PHOX, Netzsch CO., Japan). Each sample was sealed in an aluminum cell and heated at a rate of 5 K min⁻¹. Before the test, samples were directly stored in a controlled dry container at room temperature after being lyophilized.

2.5 Fourier transform-infrared spectroscopy analysis

Functional group analysis was carried out by scanning the compressed blend film of KBr and samples (m_KBr/m_s ≈ 100–150) using an FT-IR instrument (Nicolet iS10, Thermo Scientific CO., America). All spectra were recorded 32 times at 4000–400 cm⁻¹ under room temperature and with humidity around 65%.

2.6 X-ray diffraction analysis

Crystalline structure analysis was conducted using an X’Pert X-ray diffractometer (Philips, Holland) with Cu Kα-radiation.

2.7 Morphology

The morphology of pADM treated with DMT-II was recorded by SEM (Phenom ProSuite, Phenom-World CO., Netherlands) at an accelerating voltage of 10 kV.

3 Results and discussions

DMT-II was the subject of a great deal of research in our laboratory for a long period of time before a patent was applied for it. As we previously concluded,³³ the composition and molecular structure of DMT-II are shown in Fig. 1 and 2 respectively. The crystal structure of DMT-II was simulated by Materials Studio 5.5, and the obtained polyhedron model was showed from three different angles. The Zr–Al–Ti complex is not simply piled up with Zr, Al and Ti, but is formed by the coexisting covalent bond, ionic bond and hydrogen bond. In particular, S and O play a significant role in bridging Zr, Al and Ti together.

Fig. 1 Basic composition of DMT-II.

Fig. 2 Polyhedron model of the crystal structure of DMT-II.

As mentioned above, it is significant to acknowledge the state of Zr, Al and Ti in solution before a further study of the interactions between DMT-II and pADM is performed. As for the Zr complex, the coordination polymer in solution mainly exists as a tetramer. It has been proved that the Zr complex can combine with collagen at pH 1.5–2.5. At this condition, pH deviates from pI of porcine collagen (pI ≈ 6.5) and the amino groups act as the main reaction sites. According to other research,^26,27,34,35 it has been commonly deemed that the Zr complex reacts with amino groups in collagen, but there is still a chance for it to react with carboxyl groups and oxygen atoms on undissociated carboxyl groups. As for the Al complex, there has been a long history of more than 100 years to get to know the states of the Al complex in solution, and it differs from the composition of ligands. The ligancy of Al³⁺ is 6 and there is often a dynamic equilibrium where the ligancy increases to 6 as [Al(H₂O)₆]³⁺ in an acidic condition, while it decreases to 4 as [Al(OH)₄]⁻ in an alkaline condition. The existing state of the Al complex is usually coordination with carboxyl groups and hydroxyl groups, and the reactivity and bonding interaction are weaker compared with that of the Zr complex. While for the Ti complex, it often exists as the most stable +4 valence state. The electronic structure of the Ti complex is similar to that of the Zr complex. Therefore, researchers have speculated that there is high possibility for the Ti complex to share a similar reaction mode with the Zr complex.²⁹ Even though the structure of DMT-II has been speculated and simulated (Fig. 2), the interaction mode with collagen is not simply a matter of adding it together with the single element.

3.1 Crosslinking degree

The free amino content in pADM can be effectively used to characterize the reacting degree at the site of the amino group, which was marked as the crosslinking degree. Before the experiment, we have speculated that the crosslinking degree would increase with the increase of the dosage of DMT-II. But the results turned out differently, as displayed in Fig. 3; the 50% dosage of DMT-II ranked first, while the 10% group was the lowest one. According to the tanning chemistry in leather manufacturing, the reaction process with hide collagen consists of two procedures: the metal complex with a smaller molecule at a lower pH value firstly penetrates into the collagen fibers and disperses into the three dimensional structured collagen. Then with the increase of the pH value, the smaller molecule of the metal complex enlarges through the coordinating process and begins to combine with the functional groups in collagen, like the carboxyl groups, amino groups, hydroxyl groups, etc.²⁹ During the first stage (entering into the collagen fibers), the concentration of DMT-II would affect the amount of molecules penetrated. Under the same conditions, a lower concentration is better for penetrating because of the osmotic pressure, while the groups with a higher concentration of DMT-II would undoubtedly have a lower speed of penetration. Besides, the pH of the DMT-II solution differed from the concentration, namely, the pH of the higher concentration group is also higher than that of the group with a lower concentration. As is known, pH plays a significant role in the processes of both penetration and combination. At lower pH, DMT-II penetration is the main process, while on the contrary, once the pH value increases, DMT-II combines with collagen in various ways. In the reaction process, we have observed that a small proportion of collagen fibers in pADM hydrolyzed, which, we speculated, may be due to the low pH value. This behavior has jeopardized the molecular structure of collagen to some extent and also decreased the crosslinking degree, which might explain the phenomenon of the lower crosslinking degree of groups with a higher dosage of DMT-II. After increasing the pH, the molecule of DMT-II began enlarging and combining with collagen. The specific number of active groups in collagen determined the amount of combined DMT-II, therefore the dosage of 50% might have reached the limit of combination, a higher dosage of DMT-II would have no more effect in increasing the crosslinking degree. Because of the complex changes of DMT-II in solution, it is still tough to control the size and structure of the metal complex, which is far more significant in modifying collagen.

Fig. 3 Crosslinking degree of DMT-II treated collagen with various concentrations.

3.2 Differential scanning calorimetry analysis

Collagen is the main component of pADM. As reported,³⁶ the thermal denaturation of collagen depends on its water content, the pH of the environmental medium and the degree of crosslinking. Moreover, the moisture content was considered to have a different influence on the hydrothermal stability of collagen, peculiarly, when the moisture is less than the given value, the hydrothermal stability has a small relation with it.³⁷ Thus, under similar conditions of water content and pH value, DSC was applied as an effective way to characterize the thermal properties of collagen and to observe the effect of DMT-II crosslinked with collagen. As exhibited in Fig. 4, all DSC curves showed the same trend. Two groups of endothermic peaks were recorded; the first endothermic peaks, appearing at around 26–30 °C, are the turning points of the loosening triple helical structure of collagen, which has often been reported as the breaking of hydrogen bonds among collagen molecules. While the second major endothermic peaks are the transition of the collagen triple helix to a random coil, often marked as the denaturation temperature of collagen T_d.³⁸ In this work, besides the influence of the water content, the difference in T_d is due to the new chemical bonds formed between inter- and intra-collagen molecules by different dosages of DMT-II. Compared with the blank group T_d of 76.7 °C, the highest T_d of 86.6 °C appeared in the 50% DMT-II group, which is useful for improving the stability of collagens when being used as either biomaterials or leather products. Combined with early research,³⁰ the new chemical bonds formed in the tertiary structure of collagen may include electrostatic, coordination and covalent interactions. Notably, the rank of T_d in each group is T_d50% > T_d100% > T_d200% > T_d30% > T_d0% > T_d300% > T_d10%, showing the same trend with that of the crosslinking degree, which has reasonably confirmed that the enhanced T_d was related to the crosslinks in collagen and DMT-II. But the specific bonding types at specific sites of collagen and DMT-II still remain unrevealed.

Fig. 4 DSC curves of pADM treated with different dosages of DMT-II.

3.3 Fourier transform-infrared spectroscopy analysis

The characteristic absorption bands at 1647 cm⁻¹, 1542 cm⁻¹ and 1238 cm⁻¹ represent the amide I, amide II and amide III bands of collagen, and are caused by the stretching vibration of C=O in the peptide chain, bending vibration of N–H and stretching vibration of C–N respectively. Amide A and amide B at 3387 cm⁻¹ and 2927 cm⁻¹ were caused by the N–H group stretching vibration. Generally, amide I, amide II and amide III were used to investigate the integrity of the structure of collagen. In particular amide I (1647 cm⁻¹), directly related to the stretching vibrations on carboxyl groups along the polypeptide backbone, is a useful marker of the polypeptide secondary structure.³⁹ As seen in Fig. 5, all characteristic absorption bands in all groups were recorded, with only small changes in the intensity. By comparison, it is clear that the structure of collagen in each group has not been destroyed, but the lowest intensity of the 50% group indicated that the increase in T_d and the crosslinking degree might have been at the cost of affecting the molecular structure of collagen to some extent. In addition, another absorption peak appeared at 2850 cm⁻¹ in the experimental groups, which was the stretching vibration of C–H in –CH₃ or –CH₂. This small change might be caused by the hydrolysis of collagen. More information on the microstructure changes needs to be gathered to further determine the final structure and interactions between collagen and DMT-II.

Fig. 5 FTIR spectra of pADM treated with different dosages of DMT-II.

3.4 X-ray diffraction analysis

XRD has often been used to study the crystal structure of materials. As for collagen, it is commonly considered that the tiny sharp peak around ∼8° represents the distance (d) between molecular chains; the broad band around 15–25° demonstrates the amorphous area of the collagen fibers and another small peak around ∼32° represents the distance between the two adjacent amino acid residues in the collagen triple helical structure, respectively. These three characteristic peaks are often used to reveal the molecular structure of collagen. Fig. 6 displays the XRD patterns of each group. It is obvious that with the addition of DMT-II, small changes appeared. Compared with the blank group, the peak at 7–8° in each experimental group shifted to 9–10°. According to the Bragg equation 2d sin θ = nλ (λ = 0.154 nm), the value of d is in inverse proportion to that of θ. It has been reported that the axial repeat per residue is around 0.286 nm, when viewed at the left handed individual helices.⁴⁰ Thus, in an attempt to observe the changes in the three dimensional structure of collagen, the distances calculated from 2θ at around 8° (d₁) and 32° (d₂) of each sample measured by XRD are listed in Table 1. From Fig. 6 and Table 1, d₁ calculated by the value of 2θ at around 8° showed an obvious decreasing trend after treated by DMT-II, from the original 1.213 nm to 1.00–1.19 nm. The slight changes in d₁ indicate that the distance between the collagen molecular chains has been shortened, which is due to the interactions of inter-collagen molecules introduced by DMT-II. Note that when the dosage of DMT-II was 50%, d₁ decreased to the minimum 1.088 nm, showing a consistent agreement with the results of DSC and the crosslinking degree. However, the peak at 32° disappeared slowly with the increasing dosage of DMT-II, especially in the 100%, 200% and 300% groups where the characteristic peak can hardly be observed. But it is still clear from the 10%, 30% and 50% groups that there is a minor decrease compared with the blank group; the d₂ value of 0.285 nm decreased to around 0.280 nm. d₂ is useful for reflecting the interactions of the intra-collagen molecules. But in contrast to the result of d₁, no obvious law could be drawn from d₂. By comparison, the intensity of the variation of d₁ is higher than that of d₂ at a lower concentration of DMT-II, suggesting that there is a high possibility that the interactions may take place in inter-collagen molecules, while a high concentration of DMT-II may be needed to achieve the goal of interactions in intra-collagen molecules. In general, from the shift of 2θ observed from Fig. 6, the triple helix of collagen has been affected to some extent in each experimental group, and the changes were due to the reactions between DMT-II and the –COOH, –NH₂ and –OH functional groups on amino acids in the collagen chains.

Fig. 6 XRD diagrams of pADM treated with different dosages of DMT-II.

Table 1 The distance between collagen chains and the axial repeat per residue

Group number	d1/(nm)	d2/(nm)
Blank	1.213	0.285
1 (10%)	1.121	0.278
2 (30%)	1.156	0.282
3 (50%)	1.088	0.281
4 (100%)	1.096	—
5 (200%)	1.096	—
6 (300%)	1.194	—

---

3.5 Morphology

The interactions between collagen and DMT-II have been confirmed by the crosslinking degree, DSC, FTIR and XRD, however, the influence on the morphology also matters before further use. The inserts of Fig. 7 exhibit pADM treated with DMT-II with different concentrations, both observed from the longitudinal section and flat section. Before any treatment (Fig. 7A), collagen fibers linked together to form bundles, and the size of the pore was uniform. It is clear in Fig. 7B (DMT-II 10%) that the shape of the pores in pADM was destroyed to a certain degree, and collagen fiber bundles turned into a tight layer upon layer structure. The collapsed three dimensional structure of collagen in the flat section is further proof of the hydrolysis of pADM treated with 10% DMT-II, which is consistent with the results of the crosslinking degree and DSC analysis. Interestingly, in other groups treated with DMT-II, the collagen fiber bundles also changed into a regular layer by layer structure, and the pore size became much more uniform. In the present research, 50% DMT-II is the best dosage to crosslink collagen in pADM based on the results of the crosslinking degree, DSC analysis, FTIR and XRD. Moreover, it can be seen from the SEM insert (Fig. 7D) that the collagen fiber bundles loosened up to a certain extent to further form a network structure with a relatively enlarged pore, the three dimensional structure of which remained. However, when the dosage of DMT-II increased to 100%, 200% and 300%, the flat section exhibited a more compact structure and the dense layer by layer structure decreased the flexibility of pADM distinctly. The morphology verified that the interactions between collagen and DMI-II have obviously changed the microstructure of collagen in pADM.

Fig. 7 SEM images of pADM treated with different dosages of DMT-II. (A) Blank, (B) 10% DMT-II, (C) 30% DMT-II, (D) 50% DMT-II, (E) 100% DMT-II, (F) 200% DMT-II, and (G) 300% DMT-II.

4 Conclusions

Metal complexes, being used as tanning agents, have a long history in leather manufacturing. Since the main component of leather is collagen fiber, this has led us to further study the interactions between the metal complex and collagen for the future modification of collagen.

The results revealed that DMT-II could react well with –NH₂, –COOH and –OH in collagen. There was a saturation point for the crosslinking; with the dosage increase of DMT-II, the crosslinking degree increased to some extent and reached the highest when the amount of DMT-II was 50%. Meanwhile, DSC curves showed that the highest T_d (DMT-II 50%) was 86.6 °C, which increased 18.9 °C compared with that of pADM without treatment, which is beneficial for the stability and strength of collagen based materials. This change in T_d is the result of interactions between collagen and DMT-II, including the electrostatic, coordination and covalent bonds. Furthermore, these speculations have been confirmed by FTIR and XRD analysis. FTIR showed that the characterized absorption peaks have all been retained, demonstrating that the structure of collagen after crosslinking by DMT-II has not been destroyed severely. But in the XRD results, it was revealed that with a larger addition of DMT-II, the crystal structure of collagen is relatively affected. Due to the interactions, the distance between the collagen molecular chains has been shortened, obviously, and the triple helical structure has been stretched to some degree. Finally, the morphology observation showed that the collagen fiber bundles have been changed into a tight layer by layer structure, and the pore size and distribution became much more uniform.

By a series of tests, the interactions between collagen and DMT-II have been preliminarily observed. In the present study, pADM was used to simulate collagen, and the Zr–Al–Ti metal complex, DMT-II, was applied to probe its interactions with collagen for the first time, which paved a new way for collagen modification. But still, more deep research is needed to confirm the specific interaction sites on collagen and the Zr–Al–Ti complex.

Acknowledgements

The authors would like to give their sincere thanks to the National Natural Science Foundation of China (contract grant number 21276164) and Ph.D. Programs Foundation of Ministry of Education of China (20130181110092).

References

1. S. H. Ahn, Y. B. Kim, H. J. Lee and G. H. Kim, J. Mater. Chem., 2012, 22, 15901–15909.
2. R. N. Chen, G. M. Wang, C. H. Chen, H. O. Ho and M. T. Sheu, Biomacromolecules, 2006, 7, 1058–1064.
3. C. Helary, A. Abed, G. Mosser, L. Louedec, D. Letourneur, T. Coradin, M. M. G. Guille and A. M. Pelle, Biomater. Sci., 2015, 3, 373–382.
4. K. M. Lewis, A. Schiviz, H. C. Hedrich, J. Regenbongen and A. Goppelt, Int. J. Surg., 2014, 12, 940–944.
5. L. M. Wang and J. P. Stegemann, Acta Biomater., 2011, 7, 2410–2417.
6. Z. Xia, M. M. Villa and M. Wei, J. Mater. Chem. B, 2014, 2, 1998–2007.
7. S. T. Bendtsen and M. Wei, J. Mater. Chem. B, 2015, 3, 3081–3090.
8. G. H. Kim, S. Ahn, Y. Y. Kim, Y. Cho and W. Chun, J. Mater. Chem., 2011, 21, 6165–6172.
9. T. Takezawa, T. Takeuchi, A. Nitani, Y. Takayama, M. Kino-oka, M. Taya and S. Enosawa, J. Biotechnol., 2007, 131, 76–83.
10. J. K. Chai, L. M. Liang, H. M. Yang, R. Feng, H. N. Yin, F. Y. Li and Z. Y. Sheng, Burns, 2007, 33, 719–725.
11. T. M. MacLeod, P. Sarathchandra, G. Williams, R. Sanders and C. J. Green, Burns, 2004, 30, 431–437.
12. C. E. Butler, N. K. Burns, K. T. Campbell, A. B. Mathur, M. V. Jaffari and C. N. Rios, Journal of the American College of Surgeons, 2010, 211, 368–376.
13. K. M. Patel, M. Y. Nahabedian, F. Albino and P. Bhanot, Am. J. Surg., 2013, 205, 209–212.
14. H. Lin, W. H. Dan and N. H. Dan, West China Med. J., 2010, 25, 1213–1215.
15. R. Tsaryk, A. Gloria, T. Russo, L. Anspach, R. D. Santis, S. Ghanaati, R. E. Unger, L. Ambrosio and C. J. Kirkpatrick, Acta Biomater., 2015, 20, 10–21.
16. Z. G. Chen, X. M. Mo, C. L. He and H. S. Wang, Carbohydr. Polym., 2008, 72, 410–418.
17. P. Ramasamy and A. Shanmugam, Int. J. Biol. Macromol., 2015, 74, 93–102.
18. Z. H. Tian, K. Wu, W. T. Liu, L. R. Shen and G. Y. Li, Spectrochim. Acta, Part A, 2015, 140, 356–363.
19. H. R. Kayed, K. H. Sizeland, N. Kirby, A. Hawley, S. T. Mudie and R. G. Haverkamp, RSC Adv., 2015, 5, 3611–3618.
20. H. Yang, L. Liu, W. H. Dan, N. H. Dan, Z. P. Gu and X. X. Yu, Int. J. Biol. Macromol., 2013, 55, 221–230.
21. N. H. Dan, W. H. Dan, L. B. Guan, H. Lin and K. J. Wang, Funct. Mater., 2009, 40, 1352–1358.
22. K. V. Srivatsan, N. Duraipandy, S. Begum, R. Lakra, U. Ramamurthy, P. S. Korrapati and M. S. Kiran, Int. J. Biol. Macromol., 2015, 75, 306–315.
23. C. Alliraja, J. R. Rao and P. Thanikaivelan, RSC Adv., 2015, 5, 20939–20944.
24. G. Gorczyca, R. Tylingo, P. Szweda, E. Augustin, M. Sadowska and S. Milewski, Carbohydr. Polym., 2014, 102, 901–911.
25. S. Liu, R. J. Xie, J. Cai, L. Wang, X. T. Shi, L. Ren and Y. J. Wang, RSC Adv., 2015, 5, 46088–46094.
26. L. R. He, S. M. Cai, B. Wu, C. D. Mu, G. Z. Zhang and W. Lin, J. Inorg. Biochem., 2012, 117, 124–130.
27. N. N. Fathima, M. Balaraman, J. R. Rao and B. U. Nair, J. Inorg. Biochem., 2003, 95, 47–54.
28. N. N. Fathima, M. C. Bose, J. R. Rao and B. U. Nair, J. Inorg. Biochem., 2006, 100, 1774–1780.
29. W. Y. Chen and G. Y. Li, Tanning chemistry [M], Chinese Light Industry Press, BeiJing, 2011, vol. 21, ch. 22, pp. 32–36.
30. K. J. Wang, S. P. Jia, N. H. Dan, M. Liu, S. W. Xiao and W. H. Dan, J. Soc. Leather Technol. Chem., 2013, 2, 80–83.
31. M. A. Haroun, P. K. Khirstova, G. A. Gasmelseed and A. D. Covington, Thermochim. Acta, 2009, 484, 4–10.
32. M. K. Yeh, Y. M. Liang, K. M. Cheng, N. T. Dai, C. C. Liu and J. J. Young, Polymer, 2011, 52, 996–1003.
33. S. W. Xiao, Q. He, W. H. Dan and N. H. Dan, 2015 Asia-Pacific Biological Engineering and Biomedical Conference, Shanghai, China, 2015, pp. 109–115.
34. B. Madhan, N. Nishiad Fathima and J. Raghahava Rao, et al., J. Am. Leather Chem. Assoc., 2003, 98, 445–450.
35. K. J. Sreeram, R. Aravindhan and J. Raghava, et al., J. Am. Leather Chem. Assoc., 2004, 99, 110–118.
36. R. Usha and T. Ramasami, Thermochim. Acta, 2004, 409, 201–206.
37. Y. J. Wang, J. Guo, H. Chen and Z. H. Shan, J. Therm. Anal. Calorim., 2010, 99, 295–300.
38. C. D. Mu, D. F. Li, W. Lin, Y. W. Ding and G. Z. Zhang, Biopolymers, 2007, 86, 282–287.
39. J. T. Zang, Q. Xin, S. Q. Li, D. S. Li, Y. B. Gong, C. Yang, F. Xu and J. G. Liu, Chem. Res. Chin. Univ., 2010, 26, 662–666.
40. J. Engel and H. P. Bachinger, Top. Curr. Chem., 2005, 247, 7–33.

---