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Carbohydrate Polymers 157 (2017) 1650–1656

Contents lists available at ScienceDirect

Carbohydrate Polymers

j ourna l ho me pa g e: www.elsev ier .com/ locate /carbpol

reparation of oxidized sodium alginate with different moleculareights and its application for crosslinking collagen fiber

ei Dinga, Jianfei Zhoua, Yunhang Zenga, Ya-nan Wanga,∗, Bi Shia,b

National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, PR ChinaKey Laboratory of Leather Chemistry and Engineering (Sichuan University), Ministry of Education, Chengdu 610065, PR China

r t i c l e i n f o

rticle history:eceived 15 July 2016eceived in revised form1 November 2016ccepted 15 November 2016vailable online 17 November 2016

hemical compounds:odium alginate (PubChem CID: 5102882)odium periodate (PubChem CID:3667635)thylene glycol (PubChem CID: 174)thanol (PubChem CID: 702)cetone (PubChem CID: 180)ydroxylamine hydrochloride (PubChemID: 443297)

a b s t r a c t

A series of periodate oxidized sodium alginate (OSA) were prepared as green polysaccharide-basedcrosslinkers. The molecular weight of OSA decreased, while their aldehyde group content increased withincreasing dosage of sodium periodate. A typical OSA was further fractionated to four fractions by ethanolwith a narrower molecular weight distribution. Then the crosslinking performances of OSAs/fractions oncollagen fiber (CF) were investigated. DSC and SEM analyses showed that the thermal stability and dis-persion degree of crosslinked CF was considerably enhanced with decreasing molecular weight of OSA.The effect of aldehyde group content of OSA on its crosslinking performance was less obvious than thatof molecular weight, probably because the aldehyde group content in each OSA sample was higher thanthe amino group content of CF involved in the crosslinking reaction. In general, molecular weight of OSAplays a decisive role in improving properties of the crosslinked CF.

© 2016 Elsevier Ltd. All rights reserved.

eywords:odium alginatexidationolecular weight

ldehyde groupollagen fiber

rosslinking performance

. Introduction

Sodium alginate (SA) is a polysaccharide consisting of linearhains of (1-4)-�-d-mannuronic acid (M) units, �-l-guluronic acidG) units and their sodium salts (Zia, Zia, Zuber, Rehman, & Ahmad,015). SA is commonly used in food, cosmetics, and pharma-eutical industries as additive and auxiliaries due to its gellingbility, stabilizing property and high viscosity in aqueous solu-ions (Ci et al., 1999; Drury & Mooney, 2003; Kong, Smith, &

ooney, 2003). In addition, SA is applied for preparing biomedical

aterials owing to its low toxicity and superior biocompatibility

Venkatesan, Bhatnagar, Manivasagan, Kang, & Kim, 2015). In par-icular, it can be further oxidized partially using sodium periodate

∗ Corresponding author.E-mail address: wangyanan@scu.edu.cn (Y.-n. Wang).

ttp://dx.doi.org/10.1016/j.carbpol.2016.11.045144-8617/© 2016 Elsevier Ltd. All rights reserved.

to decrease its molecular weight and improve its biodegradabilityfor better application as hydrogels in drug delivery systems and tis-sue engineering (Kristiansen, Potthast, & Christensen, 2010; Lee &Mooney, 2012). Multiple functional aldehyde groups are formed onthe backbone of oxidized sodium alginate (OSA), which enhancesthe interactions of OSA with the amino groups of active proteinor polypeptides (Balakrishnan & Jayakrishnan, 2005; Bouhadir,Hausman, & Mooney, 1999). This fact suggests the possibility ofOSA as an effective and green crosslinker in the fixation of protein(Xu, Li, Yu, Gu, & Zhang, 2012).

Collagen fiber, one of the most abundant and renewable biopoly-mers, has many favorable characteristics as for preparation offunctional materials, such as affinity to metal ions, hydrophilic-ity, flexibility and hierarchical fibrous structure (Sizeland et al.,

2013). However, the specific structure of raw collagen fiber is easyto be damaged by heating, acid, alkali and proteases (Charulatha& Rajaram, 2003). Chemical modifications are undertaken to fix

W. Ding et al. / Carbohydrate Polymers 157 (2017) 1650–1656 1651

and r

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Scheme 1. Crosslinking process

he structure of collagen fiber by multivalent metal ions, polyphe-ols or aldehydes (Covington, 1997; Jayakumar, Kanth, Rao, & Nair,015). Compared to these crosslinkers, OSA is supposed to haveetter biocompatibility and biodegradability, and has been useds a crosslinker of soluble collagen (Hu et al., 2014; Kanth, Rao,

Nair, 2008). This means that OSA is capable of forming cross-inks between collagen fibers. Scheme 1 illustrates that Schiff’sase structure could be formed between aldehyde group of OSAnd amino group in the hierarchical structure of collagen fiber.owever, the penetration and crosslinking reaction of OSA in theierarchical structure of collagen fiber is more complicated thanhat in collagen solution. The molecular size of OSA could have a

ore important influence on the crosslinking performance. Theelationship between the molecular weight and structure of OSAnd its crosslinking performance on collagen fiber has not beenlear yet. Therefore, further understanding the structure-propertyelations of OSA is meaningful for developing green polysaccharide-ased crosslinkers and expanding their applications in preparingollagen-fiber-based materials.

In the present work, SA was oxidized to OSA with increasingosage of sodium periodate. Then a typical OSA sample was fur-her fractionated by graded ethanol precipitation to obtain four OSAactions with a narrower range of molecular weight. The structurend properties of OSA samples and fractions, including moleculareight and aldehyde group content, were investigated by gel per-eation chromatography (GPC), atomic force microscope (AFM),

iscosity measurement, Fourier transform infrared spectroscopyFT-IR) and potentiometric titration. The crosslinking performancesf OSA samples and fractions on collagen fiber were evaluatedsing differential scanning calorimetry (DSC) and scanning elec-ron microscopy (SEM). The structure-property relations of OSA as

crosslinker are expected to be clarified.

. Materials and methods

.1. Materials

Sodium alginate (Mw is about 3 × 105 g/mol, 10 g/L; viscosity

s about 1600 mPa·s, 20 g/L), sodium periodate, ethylene glycol,thanol and acetone were of analytical grade and purchased fromhengdu Kelong Chemical Co., Ltd (China). All the other reagentssed for analysis were of analytical grade. Collagen fiber was pre-

eaction of OSA in collagen fiber.

pared from cattle hide according to our previous work (Liao et al.,2004). Briefly, cattle hide pelt was first prepared by removing thenon-collagen components of cattle hide through soaking, degreas-ing, liming, splitting and deliming processes. Then the pelt waswashed by 150% aqueous solution of acetic acid (concentration16 g/L) three times to remove mineral substances. The pH of peltwas adjusted to 4.8-5.0 using acetic acid-sodium acetate buffersolution and then dehydrated by absolute ethyl alcohol. The dehy-drated pelt was dried in a vacuum to moisture content ≤10%,ground and sieved. 10–20 mesh hide powder (collagen fiber) withmoisture ≤12%, ash content ≤0.3%, and pH = 5.0–5.5 was obtained.

2.2. Preparation of OSA

Oxidized sodium alginate (OSA) was prepared according to apreviously reported method (Balakrishnan & Jayakrishnan, 2005).10.0 g sodium alginate (SA) was solubilized in 500 mL distilledwater, and then sodium periodate was added under stirring awayfrom light at room temperature. The mol ratios of sodium perio-date to monomeric unit of SA were 0.2:1, 0.4:1, 0.6:1, 0.8:1 and 1:1,respectively. After reaction for 24 h, the oxidation was quenched byadding equimolar ethylene glycol to sodium periodate under stir-ring for 0.5 h. The resultant solution was filtered and the filtratecontaining OSA was collected.

2.3. Preparation of OSA fractions

OSA solution prepared with a 0.8:1 mol ratio of sodium perio-date to SA monomeric unit (OSA-0.8) was fractionally precipitatedin ethanol-water solutions with different ethanol concentrations(35%, 50%, 75%, v/v) (Brillouet, Joseleau, Utille, & Lelisvre, 1982).Firstly, OSA-0.8 was mixed with a certain amount of ethanol toobtain 35% ethanol-water solution, and then the solution was pre-cipitated at 4 ◦C for 24 h. The precipitate was filtered out, rinsed byacetone, and then lyophilized to obtain the fraction named P-35.The filtrate was subsequently precipitated by 50% ethanol-water

solution to prepare the fraction P-50 in the same way as above. Thenthe filtrate was further precipitated by 75% ethanol-water solutionto prepare the fraction P-75. The residual filtrate was freeze-driedto obtain the fraction named P-S.

1 Polymers 157 (2017) 1650–1656

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Table 1Molecular weights of SA and OSA.

Samples Mw/(g/mol) Mn/(g/mol) Mw/Mn

SA 275,057 188,204 1.461OSA−0.2a 54,683 20,702 2.641OSA-0.4 39,806 6204 6.416OSA-0.6 27,249 2639 10.323OSA-0.8 24,723 2074 11.920OSA-1.0 24,614 1946 12.647

652 W. Ding et al. / Carbohydrate

.4. Determination of molecular weight

Gel Permeation Chromatography (Malvern 270 max GPC,alvern Instruments, UK) equipped with a TSKgel GMPWXL

7.8 mm × 300 mm, Tosoh Bioscience, Japan) chromatographic col-mn was used to determine the molecular weight of SA, OSA andractions of OSA-0.8. 10 mL aqueous sample solution (10 mg/mL)as filtered through a 0.22 �m pore membrane to eliminate dustarticles. The injection volume of sample was 100 �L. The elu-nt was 0.1 mol/L NaNO3 at a flow rate of 0.8 mL/min under0 ◦C elution temperature. The specific refractive index incrementdn/dc) adopted is 0.155 (Rioux, Turgeon, & Beaulieu, 2007). Before

easurements, the apparatus was calibrated using pullulan P-50tandard (Sigma-Aldrich, USA). Weight-average molecular weightMw), number average molecular weight (Mn) and polydispersityMw/Mn) of samples were calculated by software (OmniSEC, ver-ion 4.7).

.5. Atomic force microscope (AFM) observations

The SA/OSA solutions (100 mg/L) were annealed at 40 ◦C for0 min and ten times diluted. Then 3 �L of the diluted solutionas spread onto a new mica using a micropipette and dried in air

or 48 h. The aggregation structure of SA/OSA in aqueous solutionas observed using AFM (SPM-9600, SHIMADZU, Japan) in tappingode.

.6. Viscosity measurements

Viscosity measurements of SA (20 g/L) and OSA (20 g/L) wereonducted using a rotational rheometer (MARS III, HAKKE,ermany) equipped with a cone-and-plate geometry (35 and0 mm diameter, cone angle 1◦). Samples were sheared continu-usly at a rate of 40 s−1 under 30 ◦C. Measurements were made inriplicate, and the results were presented as the means ± standardeviation.

.7. Fourier transform infrared spectroscopy (FT-IR)eterminations

The FT-IR spectra of SA and OSA were recorded using a FTIR spec-rophotometer (Thermo Scientific Nicolet IS10, USA). Samples wereressed as KBr pellet and measured in the range of 1000–4000 cm−1

t room temperature, using 32 scans and a resolution of 4 cm−1.

.8. Determination of aldehyde group content and formaldehydeontent

Potentiometric titration by hydroxylamine hydrochlo-ide/sodium hydroxide was used to determine the aldehyderoup content of OSA and fractions of OSA-0.8 (Zhao & Heindel,991). The amino group in hydroxylamine hydrochloride reactsith the aldehyde group in OSA, releasing an equimolar amount

f hydrogen chloride to aldehyde group. Thus, aldehyde groupontent (mmol/g) can be quantified by titrating the amount ofeleased hydrogen chloride using 0.1 M NaOH standard solution.he analytical reactions are as follows:

OSA-(CHO)n + n(H2N-OH·HCl) → OSA-(CH = N-OH)n

+ nH2O + nHCl (1)

Cl + NaOH → NaCl + H2O (2)

Aldehyde group content measurements were conducted in trip-icate. Data were presented as means ± standard deviation.

a means the mol ratio of sodium periodate to monomeric unit of SA was 0.2:1,similarly hereinafter.

2.9. Crosslinking of collagen fiber with OSA

1.00 g collagen fiber (hide powder) was suspended in 20 mLdistilled water, and then 20 mL crosslinker (OSA or fractions of OSA-0.8, 20 g/L) and 2.40 g NaCl were added. The mixture was shaken at120 rpm in a water bath oscillator under 30 ◦C for 2 h. Then 0.48 gNaHCO3 was added into the mixture to adjust pH to 8.0 for enhanc-ing the crosslinking reaction. After shaking for 4 h, the mixture wasstood under 30 ◦C for 18 h. The crosslinked collagen fiber was col-lected by suction filtration, rinsed with 100 mL distilled water forthree times and lyophilized.

2.10. Differential scanning calorimetry (DSC) determinations

Collagen fiber and crosslinked collagen fiber were conditioned at20 ◦C, 65% RH for 48 h. Then 3–5 mg sample was put into a standardaluminum pan and sealed. An empty aluminum pan was used asreference. The thermal stability of the sample was measured usingDSC (Netzsch DSC 204 F1, Germany) in dynamic mode from 20 to150 ◦C at a heating rate of 40 K/min under N2 atmosphere.

2.11. Scanning electron microscope (SEM) observations

The morphologies of collagen fiber and crosslinked collagenfiber were observed using SEM (JEOL, JSM-7500F, Japan). Sampleswere fixed on the conductive adhesive, sputter coated with gold,and then observed with an accelerating voltage of 5 kV.

3. Results and discussion

3.1. Molecular weight

The molecular weights (Mw and Mn) and polydispersities(Mw/Mn) of SA and OSA oxidized with different dosages of sodiumperiodate are shown in Table 1. The molecular weight decreaseddrastically after oxidation due to the extensive rupture of polysac-charide chains. Moreover, a gradual reduction in the molecularweight was observed with increasing dosage of sodium perio-date, which indicates that the degree of oxidative degradation wasincreased. This can be supported by the AFM images (Fig. 1), whereSA with dense network structure was dramatically destroyed toparticles after oxidation, and the particle size of OSA was reducedas the dosage of sodium periodate increased. When the mol ratio ofsodium periodate to monomeric unit of SA exceeded 0.8, the changeof molecular weight was no longer remarkable. Additionally, thepolydispersity (Mw/Mn) was increased gradually with increasingdosage of sodium periodate, suggesting that the components of OSAwere increasingly diversified.

The diversified components with different molecular weightsand reactive group contents may perform different crosslinking

effects on collagen fiber. Accordingly, it is interesting to fur-ther grade OSA into fractions so as to better understand thestructure-activity relationship of OSA. As we know, the addition ofethanol promotes the intermolecular associations of water-soluble

W. Ding et al. / Carbohydrate Polymers 157 (2017) 1650–1656 1653

Fig. 1. AFM images of the aggregation structures of SA and OSA in aqueous solutions (10 mg/L).

Table 2Molecular weight of the fractions of OSA-0.8.

Fractions Mw/(g/mol) Mn/(g/mol) Mw/Mn Yield/%

P-35 40,459 21,135 1.914 28.64P-50 18,223 9260 1.968 51.36

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Table 3Aldehyde group content of OSA and the fractions of OSA-0.8.

Samples Aldehyde group content/(mmol/g)

OSA-0.2 3.15 ± 0.17OSA-0.4 4.55 ± 0.30OSA-0.6 6.73 ± 0.23OSA-0.8 7.81 ± 0.26OSA-1.0 8.37 ± 0.18P-35 6.83 ± 0.09P-50 7.43 ± 0.13

P-75 10,496 3558 2.950 10.26P-S 1517 962 1.577 2.64

olysaccharides, thus allowing polysaccharides to aggregate andrecipitate. The molecules with higher molecular weight tend torecipitate in lower concentration of ethanol (Huggins, 1942; Jiant al., 2014). OSA-0.8 was fractionally precipitated using gradedthanol concentrations (35%, 50%, 75%, v/v). As presented in Table 2,he molecular weight of OSA-0.8 fractions was significantly reducedith increasing ethanol concentration. The polydispersity of each

raction was lower than 3.0, indicating relatively narrow moleculareight distribution. The total yield of all fractions was higher than

0%. These results demonstrated that the components in OSA-0.8ere effectively fractionated.

.2. Viscosity

In consideration of its intrinsic high viscosity, SA would be dif-cult to penetrate into the interfibrillar spaces of collagen fibernd form crosslinking between fibrils. Fortunately, the viscosity ofSA was remarkably reduced (Fig. S1) due to the drastic decreasef molecular weight (as shown in Table 1) after oxidation byodium periodate. The apparent viscosity of OSA-0.2 (9.81 mPa·s)as considerably lower than that of SA (1608 mPa·s) at the same

oncentration (20 g/L). In addition, the viscosity of OSA decreasedlightly with further increasing the extent of oxidation. It is deducedhat lower molecular weight as well as lower viscosity of OSA isavorable for intra-crosslinking of collagen fiber. To confirm thisypothesis, more investigations were carried out in the followingections.

.3. FT-IR

Fig. S2 shows the FT-IR spectra of SA and typical OSA. Com-ared to the spectrum of SA, OSA had a new characteristic peak at734 cm−1, which was assigned to the stretching vibration of –CHOBouhadir, Hausman, & Mooney, 1999; Fan et al., 2011). Moreover,he peak became stronger with increasing dosage of sodium peri-date, suggesting that more aldehyde groups were formed due tohe oxidation of hydroxyl groups at C2 and C3 positions of uronicnits and the cleavage of C2 C3 bond. The ratio of OH stretching

−1

ibration peak (3440 cm ) increased after oxidation due to thecission of glycosidic bonds and the formation of more hydroxylroups (Wasikiewicz, Yoshii, Nagasawa, Wach, & Mitomo, 2005).his result also certified that SA was significantly degraded dur-

P-75 8.44 ± 0.26P-S 4.27 ± 0.22

ing oxidation, which is in accordance with the results of molecularweight and viscosity.

3.4. Aldehyde group content

Hydroxyl groups on C2 and C3 of the repetitive unit of SA canbe oxidized by sodium periodate to create aldehyde groups, whichmakes it possible for OSA to crosslink with amino groups of collagenfiber (Kanth, Rao, & Nair, 2008). Table 3 shows that the aldehydegroup content of OSA increased with increasing dosage of sodiumperiodate. But the increased degree of aldehyde group content wasrelatively smaller when higher molar ratio of sodium periodate tomonomeric unit of SA (0.8:1, 1:1) was used. This should be dueto the formation of hemiacetal at high concentration of aldehydegroups in OSA. A hemiacetal was formed between an aldehydegroup and a hydroxyl group in the monomeric units, resulting inthe protection of the hydroxyl group from further oxidation. Thus,it led to a lower aldehyde group content compared to theoreticalvalues (Painter, 1982).

The aldehyde group contents of the fractions of OSA-0.8 werealso determined. We have known from Table 2 that the molecularweight of the fractions decreased with increasing ethanol concen-tration. Table 3 indicates that the fractions with lower molecularweight had higher aldehyde group contents. This means both thedegrees of oxidation and degradation of SA are positively corre-lated. The only exception was the residual fraction (P-S) whosealdehyde group content was the lowest (4.27 mmol/L), probablybecause of the presence of impurities, such as iodates, in the frac-tion.

3.5. Crosslinking performance of OSA in collagen fiber

Raw collagen fiber exhibits poor thermal stability, mechani-cal strength and chemical resistance, which largely hinders itsapplication as a high-performance biomaterial. Generally, the mod-

ifications by crosslinkers (namely tanning in leather industry) canimprove physicochemical properties of collagen fiber. It is widelyacknowledged that thermal stability of crosslinked collagen fibercan be characterized by thermal denaturation temperature (Td). A

1654 W. Ding et al. / Carbohydrate Polymers 157 (2017) 1650–1656

Fig. 2. DSC thermograms of collagen fiber crosslinked with OSA (A) and fractions of OSA-0.8 (B) obtained at a heating rate of 40 K/min under dynamic N2 atmosphere.

F er (Ac n fibe

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ig. 3. SEM images (×1000) of raw collagen fiber and OSA crosslinked collagen fibollagen fiber; D: OSA-0.6 crosslinked collagen fiber; E: OSA-0.8 crosslinked collage

igher crosslinking degree usually results in higher Td, represent-ng higher thermal stability of crosslinked collagen fiber (Pietrucha,005). The DSC thermograms of collagen fibers crosslinked withSA samples and fractions are shown in Fig. 2. Within the tem-erature range of 30–120 ◦C, only one broad endothermal peakppeared for each sample, which was attributed to the helix-coilransition induced by thermal disruption of hydrogen bonds sta-ilizing the triple helical structure of collagen (Safandowska &ietrucha, 2013).

Fig. 2A shows that the Td for raw collagen fiber is 72.6 ◦C, whichs close to literature data (Td = 67.0 ◦C) (Usha & Ramasami, 2000).he thermal stability of collagen fiber was enhanced after crosslink-ng. Td of crosslinked collagen fiber increased from 76.9 to 87.9 ◦C

ith increasing oxidation degree of OSA, which is likely to relateo the decrease of molecular weight and the increase of aldehyderoup content of OSA. Fig. 2B also shows that Td of crosslinked colla-en fiber was higher when using the fraction with lower moleculareight. Surprisingly, the highest Td was present on the sample

rosslinked by P-S whose aldehyde group content was the lowest.urthermore, it was found that the Td of collagen fiber crosslinkedy OSA-0.2 and P-35 were the same (76.9 ◦C). These two crosslink-rs almost had equal Mn (2 × 104 g/mol), while the aldehyde group

ontent of P-35 was twice higher than that of OSA-0.2. All theseesults demonstrate that the thermal stability of crosslinked colla-en fiber mainly depends on the molecular weight of OSA. Collagenber is a hierarchical structured material assembled through triple-

: raw collagen fiber; B: OSA-0.2 crosslinked collagen fiber; C: OSA-0.4 crosslinkedr; F: OSA-1.0 crosslinked collagen fiber).

helical collagen molecules, microfibrils, fibrils, elemental fibers andeven larger fiber bundles (Deng, Wu, Liao, & Shi, 2008). The OSAwith lower molecular weight would penetrate into more primarystructures and crosslink between microfibril or even the peptidechains of collagen molecules to endow crosslinked collagen fiberwith higher thermal stability. On the other hand, the existence ofaldehyde group in OSA is necessary as a crosslinker, but the con-tent of aldehyde group in OSA is a less important factor comparedto molecular weight. The number of amino group on collagen fiberis limited. This implies that the aldehyde groups in all of the OSAsamples were excessive for crosslinking reaction.

The morphologies of crosslinked collagen fiber were observedby SEM. Compared with the morphology of raw collagen fiber, theOSA crosslinked collagen fiber appeared to be less compact andsticking. The dispersion degree of fibers increased with reducingmolecular weight of OSA (Fig. 3). Similar results were found inFig. 4, where the collagen fiber crosslinked by the fractions withsmaller molecular size displayed looser weave and larger porosity.According to the theory of tanning chemistry, better crosslinkingperformance is in favor of higher opening-up and fixing degrees ofcollagen fibers, leading to an increase in porosity of the fiber matrix(Fathima, Baias, Blumich, & Ramasami, 2010; Li, Wang, Li, & Shi,

2016). Therefore, the SEM further demonstrated that the OSA withlower molecular weight exhibit better crosslinking performance oncollagen fiber, which was in agreement with the results of thermalstability. These results provide a useful suggestion for OSA appli-

W. Ding et al. / Carbohydrate Polymers 157 (2017) 1650–1656 1655

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ig. 4. SEM images (×500) of collagen fiber crosslinked by fractions of OSA-0.8: (Aollagen fiber; D: P-S crosslinked collagen fiber).

ation in cleaner leather tanning technology (Kanth et al., 2007)s well as preparation of collagen-fiber-based functional materi-ls, such as porous adsorbents, catalysts and microwave absorptionaterials (Guo et al., 2012; Liao, Ding, Wang, & Shi, 2006; Liu, Tang,e, Liao, & Shi, 2009).

. Conclusions

Oxidized sodium alginate (OSA) crosslinkers with differentolecular weights and aldehyde group contents can be prepared

y oxidation using different dosages of sodium periodate andractional precipitation. Compared to aldehyde group content,

olecular weight of OSA plays a more dominant role in crosslink-ng of collagen fiber. Lower molecular weight of OSA favors itsenetration and reaction in the hierarchical structure of collagenber, and therefore leads to higher thermal stability and bet-er dispersion degree of collagen fiber. The understanding of thetructure-property relations of OSA in this investigation may pro-ide a guide in developing green polysaccharide-based crosslinkersith favorable performance for collagen-fiber-based materials andromoting the applications of polysaccharides in fabrication of bio-aterials.

cknowledgments

This work was financially supported by the National Naturalcience Foundation of China (21476149, 21506129) and South Wis-om Valley Innovative Research Team Program. The authors would

ike to thank Dr. Wang Hui (Analytical & Testing Center, Sichuanniversity) for the SEM analysis.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.carbpol.2016.11.45.

crosslinked collagen fiber; B: P-50 crosslinked collagen fiber; C: P-75 crosslinked

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