Materials Science and Engineering C 47 (2015) 123134

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Materials Science and Engineering C

j ourna l homepage: www.e lsev ie r .com/ locate /msecMechanical and biological properties of oxidized horn keratinQuanbin Zhang a, Guanghua Shan b, Ping Cao c, Jia He c, Zhongshi Lin c, Yaoxiong Huang a, Ningjian Ao a,a Department of Biomedical Engineering, Jinan University, Guangzhou 510632, Chinab Cardiology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, Chinac Shenzhen Testing Center of Medical Devices, Shenzhen 518057, China Corresponding author.E-mail address: taonj@jnu.edu.cn (N. Ao).

http://dx.doi.org/10.1016/j.msec.2014.11.0510928-4931/ 2014 Elsevier B.V. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:Received 21 September 2014Accepted 12 November 2014Available online 14 November 2014

Keywords:Horn keratinProtein oxidationMechanical propertiesBiocompatibilityBiomaterialThe goal of this study was to investigate the mechanical and biological properties of oxidized keratin materials,which were obtained by using buffalo horns to oxidize. It could provide a way to evaluate their potential for clin-ical translatability. The characterization on their composition, mechanical properties, and biological responseswas performed. It is found that the oxidation process could lead the disulfide bond to break down and then toform sulfonic acid, or even make partial peptide chain to be fragment for the new modification of amino acid.Hence the oxidized horn keratins have lower thermal stability and hydrolytic stability in comparison withhorn keratin, but the degradation products of oxidized horn keratins have no significant difference. In addition,the mechanical properties of oxidized horn keratins are poorer than that of horn keratin, but the oxidized hornkeratins still have disulfide bonds to form a three-dimensional structure, which benefits for their mechanicalproperties. The fracture toughness of oxidized horn keratins increases with the increase in the degree of oxida-tion. After oxidation, the oxidized horn keratins have lower cytotoxicity and lower hemolysis ratio. Moreover,when the oxidized horn keratins, aswell as different concentration of degradation products of oxidized horn ker-atins, are directly in contact with platelet-rich plasma, platelets are not activated. It suggests that the oxidizedhorn keratins have good hemocompatibility, without triggering blood thrombosis. The implantation experimentin vivo also demonstrates that the oxidized horn keratins are compatible with the tissue, because there are min-imal fibrous capsule and less of infiltration of host cells, without causing serious inflammation. In summary, theoxidized horn keratins can act as implanted biomaterial devices that are directly in contact with blood and tissue.

2014 Elsevier B.V. All rights reserved.1. Introduction

Keratin is one of the most abundant proteins [1]. The first study ofkeratin used as biomaterial was published by Noishiki and his col-leagues. They coated a heparinized keratin derivative onto a vasculargraft, which was implanted into a dog, without thrombosis for morethan 200 days [2]. In fact, keratin is the major component of any tissueof a living organism, such as hair, wool, feathers and horns, which isoften associated with various biological functions. For example, theycan serve as a barrier for environmental stress, regulate moisture andcommunicate with others. Keratin can be distinguished as soft andhard keratin [3,4]. Soft keratin is often found in epidermis and callusesand it has lower sulfur content. However, hard keratin has higher sulfurcontent. Hard keratin can be classified into two groups. The one is hard-keratin,which is found inmammalian epidermal appendages, such ashorns, hairs and nails, and the other one is -keratin, which is found inavian and reptilian tissues. The-keratin has an-helical coil structure,but the -keratin has a twisted -sheet structure. In recent years, thekeratinousmaterials have attracted increasing attention, such as equinehoof [5], bovid hoof [6], wool [7], and especially the sheep horn [8].

Horn appears on animals coming from the bovid family, which in-cludes cattle, sheep, and waterbuck, and it is composed of a keratinoussheath overlying a bony core [9]. The horn keratin is a hard -keratin,and has crystalline fiber phase and amorphous matrix phase [10]. Thecrystalline phase contains microfibrils with -helical structure, but theamorphous phase is made up of microfibrils with non-helical structureand other morphological components. In a horn, the keratin fibers aresubstantially parallel to the growth direction and are stacked witheach other to form lamellar structure [11]. Besides, keratin fibers areembedded in an amorphous and non-fibrous protein matrix throughmany bonds, such as disulfide bonds, hydrogen bonds, van der Waalsforces and ionic interaction [10]. Thus, the horn keratin forms an excel-lent biological model with a hierarchical structure from nanometer tomicrometer scale [12]. As a result, horn keratin has good performances,such as high toughness, stiffness and strength. However, the stablethree-dimensional structure of horn, which is formed by disulfide brid-ges and other crosslinks, makes keratin have high chemical stability inphysiological environment, resulting in its non-degradability, which ishindering its application [13]. Many extraction methods for the solublekeratin have been studied extensively, such as oxidation and reduction

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124 Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134[1417]. The soluble keratin would have advantages in several fields,such as wound care, tissue reconstruction, cell seeding and diffusion,and drug delivery [18]. But it doesn't have three-dimensional structure,whichmakes it have poormechanical property and processability. Thus,the practical applications of soluble keratin are restricted [19]. There-fore, it is desired to prepare a keratin with both good mechanical prop-erty and degradation.

Therefore, in order to design a sort of degradable keratin with three-dimensional structure, we prepared an oxidized horn keratin biomaterialby using oxidative conditions and investigated its general chemical, me-chanical, and biological properties. In addition, the hemocompatibility ofthe oxidized horn keratin was studied by, hemolysis ratio, platelet adhe-sion tests, partial thromboplastin time (PTT), activated partial thrombo-plastin time (APTT) and fibrinogen (Fib) assay.

2. Materials and methods

2.1. Sample preparation

The buffalo horns from subadult and healthy bovine were obtainedwithin 24 h after slaughter from a local slaughterhouse (butchered fordietary reasons; Baiyun District, Guangzhou, China). The horn sheathwas isolated naturally from the bony core without destroying the natu-ral structure after 30 day storage at ambient conditions. Distal segmentswere cut 30mm. And then the distal part of hornwas cut alongwith thegrowth direction of the horn, and the thickness was perpendicular tothe radial direction, as shown in Fig. 1. About 2 mm external surfaceand internal surface in the horns were cut away from each sample.Samples were cut into rectangular prisms of dimensions 40 mm 5mm2mm(lengthwidth thickness)with a handsaw and varietyof fine rasps. In samples' milling process, therewas no overheating phe-nomenon in the horns.

2.2. The oxidation of horn keratin

The horn samples were washed with distilled water and 0.1 N NaClsolution, and then immersed into ethyl ether to remove fat. After that,the samples were washed three times with distilled water and air-dried at 25 C at a relative humidity of 60 2%. Samples were treatedin an aqueous bath with a liquid-to-horn ratio of 30:1 (v/v). The hydro-gen peroxide concentration was set at 20% and 30% respectively. Thebath temperature was controlled at 25 C, and the treatment time was12 h and 48 h respectively. After the reaction, samples were immersedin distilled water under ultrasonic vibration for 48 h, during which thewater was changed every 12 h, and then dried at 80 C under vacuumto remove the residual hydrogen peroxide. The removal of hydrogenperoxide was confirmed by the reduction method of potassium iodide[20]. The oxidized horn keratins, soaked in 20% H2O2 for 12 h, 30%Fig. 1. (A) Schematic drawing of the horn samples, showing the orientation and positionwhere the samples were cut. (B) The inset shows the sample orientation for tests(not to scale).H2O2 for 12 h and 30% H2O2 for 48 h respectively, were denoted asOH1220, OH1230 and OH4830 respectively. In addition, the hornkeratin was denoted as OH.

2.3. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra were obtained by using the attenuated total reflectance(ATR) technique of FTIR (EQUINOX55, BRUKER). An average of 32 scanswas taken from 4000 to 600 cm1 with a resolution of 4 cm1.

2.4. Thermal analysis

The thermal degradation behavior of the samples was investigatedby a thermogravimetry instrument (TG, 209F3-ASC, NETZSCH). A fewmilligrams of samples were heated from 30 to 400 C at a heating rateof 10 Cmin1, under nitrogen and air atmospherewith flowing atmo-sphere (10 mlmin1) respectively.

The phase transition temperature of the samples was examined by adifferential scanning calorimetric instrument (DSC, 204F1, NETZSCH). Afew milligrams of samples were heated from 50 to 320 C at the heatingrate of 10 Cmin1, flushing the crucible with 100 mlmin1 nitrogen.

2.5. Tensile tests

Quasi-static tensile tests were conducted at room temperatureusing a computer-controlled universal testing machine (DL-D series,Xinzhengwei Corporation, Jiangdu, China). In order to prevent the sampledamage and slip, both the ends of the samples were pasted with squarealuminum grips (10 mm 6 mm 1 mm) by an epoxy resin adhesive,which could transfer the load smoothly and uniformly to the two endsof the samples. Tests were performed at a constant crosshead speed of10 mmmin1 with a load cell of 2000 N. The force and displacementdata were automatically recorded by the built-in measurement software.Three samples (40mm5mm2mm)were taken from each set of thesamples for measurement, and the water content of samples was con-trolled at about 9%. The results were expressed as mean values stan-dard deviation.

2.6. Scanning electron microscopy

After tensile tests, the fracture surfaces of samples were coated withgold and observed by a scanning electron microscope (SEM, PhilipsXL-30, Netherland).

2.7. In vitro degradation and SDS-PAGE analysis

Each sample (40mm 5mm 2mm)was placed in a test tube con-taining 10ml of phosphate-buffered saline (PBS, pH 7.4) and incubated at37 C. The buffer solutions were replaced by fresh ones every two weeks.After incubation, the samples were washed and dried in vacuum to con-stant weight. Results were expressed as percentage of weight loss (W%)and calculated according to the equation: W = [(W0 Wt) / W0] 100%, where W0 was the weight of the dry sample at time 0 and Wtwas the weight of the dry sample at time t.

In addition, the molecular weight of the degradation products wasmeasured by the method of sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) [21]. At first, in order to remove un-dissolved debris, the degradation solutions of samples were centrifugedat 2500 rpm for 5 min using a microcentrifuge (SC-02, ZONKIA, Anhui,China). Protein content in the supernatant of all samples was collectedand concentrated to be about 1 ml degradation solution respectively.100 l of each solution was mixed with 5 l of 5 SDS loading buffer(Bio Rad) containing 0.6 M b-mercaptoethanol (Bio-Rad). Sampleswere denatured by boiling in SDS/b-mercaptoethanol solution for5 min and then immediately placed into ice water. 30 l of these cold,denatured solutions was loaded onto lanes of precast TrisHCl gels

125Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134(5% stacking gel and 12% separating gel) (Bio-Rad). Separation was per-formed at 80 V for approximately 3 h. After separation, gels were rinsedwith ultrapure water for 5 min before staining with Bio-Safe Coomassiestain (G250, Bio Rad) for 10 min under boiling water bath. Destainingwas done overnight in ultrapure water with gentle rotation. Sampleswere compared to a standard ladder (Benchmark Prestained ProteinLadder, Invitrogen, Carlsbad, CA) and the gels were imaged in DIAmode with an Image Scanner (GE, USA).

2.8. In vitro cell viability

Cell viability in the presence of horn keratin and oxidized horn ker-atin was assessed by anMTT assay. All keratin samples (4mm 2mm),which had been soaked in PBS solution (pH 7.4) for 24 h and thendrained, were sterilized in a steam autoclave at 120 C for 30 minprior to NIH-3T3 and human umbilical vein endothelial cell (HUVEC)(Medical Laboratory, Jinan University, China) culture experiments. Sub-sequently, all the sterilized samples were placed in a 48-well cultureplate (Corning Life Sciences), and each sample had 5 duplications. TheNIH-3T3 and HUVECs were seeded at a density of 5000 cells/cm2 andallowed to grow at 37 C atmosphere of 5% CO2. The negative controlconsisted of cells without samples. After incubation of certain time,such as 24 h, 48 h and 72 h respectively, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/ml,Sigma) was added and incubated for further 4 h. Mitochondrial dehy-drogenases of viable cells cleaved the tetrazolium ring, yielding purpleformazan crystals. Formazan crystals were then dissolved in DMSO so-lution (Sigma). Afterwards, 200 l of the blue solutions was transferredto a 96-well plate. The absorbancewasmeasured at 490 nmby amicro-plate reader (Bio-Rad).

2.9. In vitro hemocompatibility

The fresh rabbit's blood used in our experiments was obtained legal-ly from the Shenzhen Testing Center of Medical Devices, China. Theanalysis was performed within 12 h after blood donation. The amountsof the samples used for statistical count were not less than three.

2.9.1. Hemolysis ratio4 ml blood was diluted by 5 ml of 0.9% (w/v) sodium chloride solu-

tion. Each testing sample (40 mm 5 mm 2 mm) was added 10 ml0.9% (w/v) sodium chloride solution. Additionally, 10ml of 0.9% (w/v) so-dium chloride solution and 10 ml double distilled water were preparedrespectively for antitheses. All the samples were kept at 37 C for 72 h,and then immediately added 0.2 ml of the diluted blood. Incubationwas performed at 37 Cwith test tubes. After 60min incubation, the sam-ples were centrifuged at 750 g for 5 min. Then the supernatant was mea-sured at 545 nm by 722 s spectrophotometer (Shanghai Sunny HengpingScientific Instrument Co., Ltd.). Hemolysis ratio was calculated as follows:

hemolysis ODtestODneg

= ODposODneg h i

100%;

where ODtest, ODneg, and ODpos were the absorbance values of the testsample, negative control (saline), and positive control (water), respec-tively. All the hemolysis experiments were done in triplicate.

2.9.2. Platelet adhesion and activationPlatelet adhesion experimentwas carried out to evaluate the surface

thrombogenicity of the samples and to examine the interaction be-tween blood and thematerials in vitro [30]. The size of all of the sampleswas 5 mm 5 mm. The rabbit whole blood was treated with anti-coagulant (EDTAK2, Hunan Liuyang Medical Instrument Factory,China). After centrifuging at 1000 rpm for 15min, a platelet-rich plasma(PRP) was obtained. The samples were immersed in PRP and incubatedat 37 C for 30 min. The samples were subsequently rinsed with a PBSsolution (pH 7.4) to remove weakly adherent platelets. The adheredplatelets were fixed in 2.5% glutaraldehyde solutions at room tempera-ture for 2 h, and then dehydrated and dried at room temperature. Thesamples were then coated with gold and observed by SEM (PhilipsXL-30, Netherland).

2.9.3. PTT, APTT and fibrinogen (Fib) assayFor the partial thromboplastin time (PTT) measurement, samples

were added to each plastic test tube. The rabbitwhole blood,which con-tains anticoagulant (sodium citrate 1:9, Guangzhou Improve MedicalLtd., China), was centrifuged at 2000 rpm for 15 min to obtainplatelet-poor plasma (PPP). Thereafter, 1 ml PPP was added onto thesamples (5 mm 5 mm 2 mm), which were completely immersedand incubated at 37 C for 15min. 100 l incubated PPP was transferredto a test tube and 100 l PTT reagent was added to the same test tube,followed by the addition of 0.025MCaCl2 solution (100 l). The suspen-sionwas stirred by amagnetic stick and the coagulation timewas deter-mined at 37 C using a coagulation instrument (ACL 8000, BeckmanCoulter, Inc.).

For the activated partial thromboplastin time (APTT) test, 400 l PPPwas mixed with PBS (as a control) or the degradation solution of thesamples (200 l) and then incubated at 37 C for 15 min. After mixing,100 l plasmamixture, 100 l APTT reagent and 100 l 0.025MCaCl2 so-lution were added to the same test tube. Subsequently, the suspensionwas stirred by a magnetic stick and the coagulation time was deter-mined at 37 C using the same coagulation instrument. The Fib mea-surements were carried out with the same procedure of APTT, exceptthat 100 l Fib reagent was added.

2.10. In vivo implantation experiment

All animal procedures were performed under a protocol approvedby the Institutional Animal Care and Use Committee. To assess thevivo biocompatibility of oxidized horn keratin biomaterials, smallautoclaved samples (0.1 g, about 8 mm 5 mm sections) were im-planted subcutaneously into mice. Before implantation, the sampleswere incubated in sterile PBS (pH 7.4) for 1 h. Adult female BALB/cmice (Experimental Animal Laboratories, Guangdong, China), approxi-mately 2025 g in weight, were implemented under general anesthesiaby intraperitoneal injections of chloral hydrate (AR, Sigma). The opera-tive site on the back was shaved and cleansed with Betadine (Guang-dong Hengjian Pharmaceutical Co., Ltd.). A 2 cm lateral skin incisionwas made on the mid-portion of the back and tissue pockets were cre-ated by gross dissection laterally using blunt scissors. Sterilized sampleswere implanted into the subcutaneous pocket of each mouse. Skinclosures were performed with nonabsorbable nylon sutures (ShanghaiMedical Suture Needle Co., Ltd.). At 1, 3, 6, and 10 weeks, animalswere euthanized and an incision was made on the back of each mouse(n = 2 mice/time point, each sample). Digital photographs of sampleswere taken in situ and observed grossly for inflammation and capsuleformation. The entire implant site was excised, but the sample was re-moved because of its hard texture.

Tissue explants were fixed in 10% neutral buffered formalin (NBF,Fisher Scientific) for 48 h, and then dehydrated in increasing concentra-tions of ethanol (Fisher Scientific) and embedded in paraffin (Fisher Sci-entific). Cross-sections at 5 mm thickness were cut on a microtome(Ultramicrotome RM2235, Leica Microsystems Inc.), and mounted onCITOGLASglass slides, and air dried. Sectionswere stainedwith hema-toxylin & eosin (H&E, Fisher Scientific) to assess the presence and thick-ness of fibrous tissue, cellular response, and vascularization using anoptical microscope (Axio Scope A1, Zeiss).

2.11. Statistical analyses

Descriptive statistics were performed on all of the experimentaldata to obtain the means and the standard errors with Origin Pro7.5.

126 Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134One-way analysis of variance (ANOVA) was applied to the measureddata for each experiment described above. The difference is consideredsignificant when p b 0.05.3. Results and discussion

3.1. Analysis of FTIR

During the oxidizing reaction of hydrogen peroxide, many perhy-droxyl species are formed from hydrogen peroxide, such as HO2, whichcan attack much substance, including the keratins [22,23]. The attackingof disulfide bonds by perhydroxyl species would produce disulfide oxida-tion products from ruptured \S\S\ bonds, which are cysteic acid andintermediate sulfoxides.

The FTIR spectra of horn keratin and oxidized horn keratins areshown in Fig. 2. According to the curves of samples, there are threemajor band regions, which should be assigned to amides I, II, IIIFig. 2. FTIR spectra of OH, OH1220, OH1230 and OH4830, as shown in A; the fragmentof FTIR spectra resolved into components (over range 17101590 cm1, R2 N 0.999), asshown in B.respectively. The band in the range of 16001700 cm1 is assigned tothe amide I, which is related to the C_O stretching. While the amideII,which is observed in 14001500 cm1, is caused by theN\Hbendingand C\H stretching vibration. But the amide III, occurring in 12201300 cm1, results from the combination of C\N stretching and N\Hin plane bending, with some distribution from C\C stretching andC_O bending [24,25].

In fact, the amide I is very sensitive to the secondary structure ofthe proteins [26]. In order to eliminate the water absorption band(16001650 cm1), which could interferewith the amide I, the sampleswere dried in vacuum at 80 C for 2 days before the acquisition of spec-tra. And then, the spectra in 17101590 cm1 were baseline correctedand smoothed with the SavitskyGolay method (9 points) [27]. Atlast, the manipulated spectra were resolved by second order derivativeusing the method of Marquardt, as shown in Fig. 2. The peak in 16501658 cm1 indicates an -helix structure, while the bands in 16401610 cm1 have been assigned to -sheet [25,28]. Both -sheet and-helix are found in the horn keratin and oxidized samples. Moreover,the contents of two structures are different in all samples, as shown inTable 1. With the increase in the degree of oxidation, the content of-sheet structure increases while the content of -helical structure de-creases, when the oxidized samples are compared with the horn kera-tin. On the other hand, the region of 12401220 cm1 corresponds tothe random coin and -sheet structure, which increases graduallywith the increase in the degree of oxidation [29,30]. These band shiftsare caused by the distinct hydrogen bonding states, which are producedby the different protein conformations. It can indicate that the oxidizedhorn keratin would change its secondary structure, for example, the ox-idized horn keratin lost its ordered -helical structure to form a disor-dered structure, such as -sheet [31,32]. This may be due to hydrogenperoxide which forms strong interaction with the polar side chaingroups of keratin [32]. The result is that the molecular chains becomecloser. In this way, this organization can promote crystallization in -sheet structure embedded in an amorphous matrix [25]. Therefore,the amorphous matrix in oxidized samples increases gradually withthe increase in the degree of oxidation. The horn possesses a moresolid and compacted structure, but the structure of the oxidized samplesis different. Because their hydrogen bonds and disulfide bonds, evenpeptide bonds, are split apart, and some crystals and amorphous regionsin keratin are destroyed. In addition, band at about 1390 cm1 is relatedwith the C\H and O\H bending vibration, which decreases graduallywith the increase in the degree of oxidation. The disulfide bond in cys-tine of the horn keratin is broken down to form sulfate oxides in thepro-cess of oxidation. For all the oxidized samples, a group of small peakswith different intensities, laying in 11801022 cm1, is related to thecontent of different sulfate oxides, such as cysteine acid, \SO3H [24,33]. The intensity increases with the increase in the degree of oxidation.It is due to the changes in some sulfur-containing groups during the ox-idation. The peaks in the range of 630650 cm1 can be attributed tothe C\S band stretching vibrations [24,30]. It can be shown that the di-sulfide bond is not completely broken down during the oxidation.

3.2. TG-DSC measurement

From the TG-DTG curves, all samples show two evident mass lossstages, as shown in Fig. 3A. The first stage in the temperature range ofTable 1Characteristic of the amide bands of horn keratin and oxidized horn keratins.

Materials -Helix -Sheet

Band position (cm1) Area (%) Band position (cm1) Area (%)

OH 1650.8 48.74 1617.0 and 1631.2 31.69OH1220 1652.3 45.80 1617.1 and 1632.3 35.35OH1230 1653.9 42.42 1619.9 and 1634.0 38.15OH4830 1656.2 37.66 1620.3 and 1634.3 42.10

image of Fig.2

Fig. 3. TG-DTG and DSC curves of the samples: TG-DTG (A), DSC (B). In addition, a, b, c and d for OH, OH1220, OH1230 and OH4830 respectively.

127Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 12313430150 C generally corresponds to the evaporation of moisture, andthe second stage is assigned to the thermal degradation of samples,which occurs in the temperature range of 220400 C.

With the increase in the degree of oxidation, the mass loss of oxi-dized samples in the temperature range of 30150 C decreases, namelyabout 7.93%, 7.61% and 6.58% for OH1220, OH1230 and OH4830 re-spectively. But the mass loss of horn keratin is about 6.82% in the tem-perature range of 30150 C. From the DTG curves, the temperature inthermal degradation of horn keratin is higher than that of oxidized sam-ples and the temperature peak is broader. However, as for oxidizedsamples, the order of temperature in thermal degradation is OH4830 N OH1230 N OH1220. This suggests that the oxidized sampleshave lower thermal stability compared to the horn keratin; in addition,the thermal stability of oxidized samples would increase with the in-crease in degree of oxidation.

The DSC curves of samples are shown in Fig. 3B. There are three en-dothermic peaks in the DSC curves, and they roughly correspond to twoevident mass losses. For horn keratin, the well-known endothermicpeak at about 92 C resulted from water evaporation and glass transi-tion. Due to the complex structure of horn keratin, the glass transitionoften occurs in a temperature range rather than at a fixed temperature[34,35]. Moreover, this temperature range is usually overlapped withthe peak of water evaporation in a DSC curve [36]. The endothermicpeak at 236 C is ascribed to the denaturation of the -keratin crystal-lites and the area under the curve can be used to measure the -helixcontent; and the endothermic peak at 312 C corresponds to the

image of Fig.3

Table 2The values of mechanical characteristics in horn keratin and oxidized horn keratins.

Samples Tensile strength (MPa) Young's modulus (GPa) Fracture strain (%)

OH 117.581 3.129 1.553 0.031 24.602 3.599OH1220 100.011 2.341 1.137 0.029 37.566 4.113OH1230 85.758 5.386 0.869 0.021 56.180 4.252OH4830 72.680 2.063 0.732 0.022 70.529 6.371

n = 3 for each set of samples. Values are means s.e.m. and p b 0.05 by comparisons atall samples.

128 Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134destruction of crosslinks, such as disulfide bonds, hydrogen bonds, andsalt links [37,38]. However, the peaks of oxidized samples are differentfrom those of horn keratin, suggesting that themicrostructure of the ox-idized samples changed during the process of oxidation.

It is well known that horn keratin is made up of amorphous ma-trix and crystalline regions [10]. The former provides weakly boundwater sites, but the latter provides strongly bound structural water sites[3]. From FTIR, the amount of hydrophilic groups, such as \COOHand\SO3H, and the amorphous matrix in oxidized samples increasesduring the process of oxidation, which can present more weakly boundwater sites aswell as increase the affinity towater in the oxidized sampleswhen compared with the horn keratin. Therefore, more hydrophilicgroups absorb water and more moisture-bonded structures are formedin oxidized samples. This corresponds to the first mass loss of samplesin TG curves.Moreover, the temperature ofwater evaporation in oxidizedsamples increases from 84 C to 107 C. But the temperature at about 92C in horn keratin is caused by the presence of strongly bound water,mainly provided by crystalline phase. It indicates that more amounts ofhydrophilic groups provide more strongly bound water than that incrystalline phase.

The results fromDSC curves also show that as the increase in the de-gree of oxidation, the denaturation temperature (246 C, 245 C, 242 Crespectively) for -keratin crystallites is higher than that in horn kera-tin; however, the temperature (295 C, 296 C, 298 C respectively) fordestruction of crosslinks is lower than that in horn keratin. The lowerdenaturation temperature of horn keratin may be resulted from thelower amount of crystalline -sheet structure. In -sheet structure, in-termolecular interaction between the protein chains is stronger thanthat in the keratin fibers [39]. However, with increase in the degree ofoxidation, -helical structure decreases evidently, which makes thechange of crystal and the reduction of the crystallinity, resulting in thedecrease of the denaturation temperature. On the other hand, the crys-talline denaturation peak is broader in horn keratin, which can reflect adistribution of crystal sizes in horn keratin [40]. In addition, the betterthermal-stability of horn keratin is a result of the cross-linking betweenthemacromolecules bymore disulfide bonds and hydrogen bonds. Afteroxidation, the decrease of disulfide bonds weakens the crosslinks, oreven the crosslinks are broken down, diminishing the stability to heatand resulting in the decrease of the destruction temperatures of thecrosslinks. However, with the increase in the degree of oxidation, onlythe crosslinks that are not easily to be destroyed are left. Furthermore,the increase of amorphous matrix will produce more hydrogen bonds[34]. Therefore, the destruction temperature of crosslinks in oxidizedhorn keratin increases gradually with the increase in the degree of oxi-dation, which is in accordance with the performance of the samples inTG-DTG curves.

3.3. The effects of oxidation on the mechanical properties of horn keratin

During the oxidizing reaction of hydrogen peroxide, the disulfidebond and hydrogen bond, or even peptide bond, are attacked and partlybroken down, which has significant effects on the mechanical proper-ties of horn keratin [41]. In previous studies, the tensile strength,Young's modulus and fracture strain of untreated samples with watercontent of 9% were found to be 117.58 3.13 (MPa), 1.553 0.031(GPa) and 24.60 3.60 (%) respectively [13]. In comparison, the tensilestrength and Young's modulus of the oxidized horn keratin are moder-ately degraded, and the failure strain increases with the increase in thedegree of oxidation (p b 0.05), as shown in Table 2. Despite that, the ox-idized samples could be fully recovered to its original shape and dimen-sions after the tensile test.

The cross-linkages in keratin are formed by disulfide bonds, togetherwith the van der Waals forces, hydrogen bonds and ionic interaction.The cross-linkages contribute to mechanical properties as well as struc-tural stability, making the horn keratin have better stiffness andstrength [13]. After oxidation, the disulfide bonds, van derWaals forces,hydrogen bonds and ionic interaction, or even peptide bonds, are bro-ken somewhat, resulting in less coherent interactionswithin the proteinstructure [42]. On the other hand, the crystalline regions with-helicalstructure are responsible for the strength of horn keratin, but the amor-phous regions, which have relatively fewer bonds between the polymerchains and random distribution of the chains, provide the horn keratinwith elasticity and flexibility [43]. From the results of FTIR, the-helical structure decreases and amorphousmatrix increases after ox-idation. As a result, this would cause larger matrix region with freedomof movement and reduce the stability of the matrix, resulting in thedecrease of the tensile strength and Young's modulus and the improve-ment of failure strain of oxidized horn keratin. However, high concen-tration of hydrogen peroxide might contribute to the production ofexcessive amounts of perhydroxyl species, which can react with moresubstances. If the time of oxidation is longer, it would let perhydroxylspecies have more time to attack the proteins [41]. This can cause fur-ther breakage of bonds, even polypeptide chains. Therefore, as the de-gree of oxidation increases, there is a significant reduction in tensilestrength and Young's modulus in oxidized horn keratin, but significantincrease in toughness. It indicates that the mechanical properties ofhorn keratin are largely affected by the concentration of hydrogen per-oxide and the time of oxidation. However, the tensile strength and frac-ture strain of oxidized horn keratin are stronger than those of othersynthetic materials, such as polycarbonate (67 MPa and 15%) [44] andpolylactide (65 MPa and 9%) [45]; even the tensile strength of OH1220 can rival that of fiberglass (110MPa) [46]. Therefore, with appropri-ate oxidation treatment, the oxidized horn keratin can maintain itsmechanical property not to change too much.

3.4. Microstructure of fracture surfaces

The horn keratin is a hierarchical material and has laminate struc-ture [8]. Related to the fracture surfaces of oxidized samples, the SEMimages of samples reveal different failure phenomena, as seen inFig. 4. It is clearly shown that the fracture surface in buffalo horn is rel-atively smooth, neat and wavy, indicating that the buffalo horn has adense laminate structure. But the fibers are pulled out and the lamellasare partially torn in OH1220. Moreover, the fracture surfaces of OH1230 and OH4830 show an extremely ductile fracture mode, evidencedby a very deep, convoluted cup-and-cone type fracture. At the sametime, the samples had fully recovered to their original shape and dimen-sion after a certain time. The larger failure strain also indicates that ox-idized horn keratin is more resilient than horn keratin. It may be due tothe more compliant matrix that can yield and flow more readily withthe increase in the degree of oxidation, which is corresponding to theresults of the mechanical properties of the samples.

3.5. In vitro hydrolytic stability and SDS-PAGE analysis

Numerous disulfide bonds permanently bind the peptide chains,which contributes to the insolubility or low insolubility of keratin inwater. The degradation behavior of horn keratin after oxidation was in-vestigated using an in vitro degradation experiment, which would pro-vide a good understanding in the hydrolytic stability of oxidized hornkeratin. From theweight loss ratiotime curve in Fig. 5, the degradationrate of OH is extremely slow, nearly no degradation during 10 weeks.

Fig. 4. The fracture surfaces of the samples under tensile test. A for OH, B for OH1220, C for OH1230 and D for OH4830.

129Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134However, with the disulfide bonds being oxidized to break down, thehydrolytic stability of oxidized horn keratin decreases gradually.Compared with the horn keratin, there is a significant difference in thedegradation rate of oxidized samples (p b 0.05). The degradation rateof oxidized horn keratins gradually accelerates with the increase indegree of oxidation. For example, after 16 weeks, the degradation rateof OH4830 is up to about 90%, nearly completely degraded; but forOH1220, the weight loss ratiotime curve is substantially linear,which indicates that the degradation behavior of OH1220 is relativelystable. However, there is a sudden sharp rise in weight loss ratiotimecurve ofOH1230andOH4830. As expected,withmorebrokendisulfidebonds, the degradation behavior of the oxidized horn keratin is signifi-cantly enhanced, resulting in less hydrolytic stability.

In SDS-PAGEmethod, themobility of protein depends on its relativemolecular mass, regardless of the electric charge and molecular shapes.Therefore, after the qualitative analysis of protein fractions in degradationproducts by SDS-PAGE, the protein bands from 3 separate degradationproducts of oxidized horn keratins reveal similar patterns, includinghigh molecular mass bands (N40 kDa) and low molecular mass bands(b25 kDa), as shown in Fig. 6. The protein bands at about 4060 kDaFig. 5. The weight loss ratiotime curves of the samples. Each value is expressed asmean standard deviation.are attributed to monomeric keratin subunits that are mainly low-sulfurcontent of -helical keratins; however, the lesser low molecular massbands at about 1520 kDa are attributed to high-sulfur content of matrix[47]. In addition, the protein bands at about 60160 kDamay be assignedto obligate keratin heterodimers. Due to the proteinprotein interactionor cross-linkings, the higher molecular mass bands (N160 kDa) may cor-respond to stable tetramers or larger multimers of keratins [48]. The mo-lecular mass of degradation products in three oxidized horn keratins ismainly more than 20 kDa, with less of low molecular mass; moreover,for OH1220 and OH1230, protein bands are mainly focused on the re-gion of high molecular mass, even higher protein bands (N160 kDa).However, compared with OH1220 and OH1230, the molecular massof degradation product of OH4830 is decreased to some extent. Thismaybedependedon the extent of damage to the structure of horn keratinby oxidation. For example, the destruction of disulfide bonds or evenpeptide bonds would make the protein chains interrupt randomly to beprotein fragments, which decreases the molecular mass of protein. Withthe increase in degree of oxidation, single protein will be cut into moreFig. 6. SDS-PAGE of degradation solution of oxidized horn keratins.

image of Fig.4image of Fig.5image of Fig.6

Table 3Hemolysis ratio and PTT of horn keratin and oxidized horn keratins. The hemolysis ratio ofthe positive control (water) and negative control (saline) was 1 and 0, respectively.

Samples Hemolysis ratio (%) PTT (s)

Original plasma 63.23 1.17Positive control 30.57 0.93OH 1.80 0.35 63.55 1.21OH1220 1.54 0.23 60.73 1.86OH1230 1.99 0.16 61.13 3.49OH4830 1.49 0.77 59.33 1.76

Fig. 8.APTT and Fib of the original plasmaand theplasma contactedwith different concen-trations of degradation product of the oxidized keratins.

130 Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134protein fragments bymore perhydroxyl species, resulting that the proteinfragments have lower molecular mass. As a result, the protein fragmentsare released during the degradation, reflected in the SDS-PAGE. However,the mechanism of degradation in the oxidized horn keratin needs to beproved by more experiments.

3.6. In vitro hemocompatibility

3.6.1. Hemolysis ratioHemolysis ratio is an important factor to evaluate the blood compat-

ibility of a biomaterial. When the red blood cells swell to the criticalbulk, which would make the cell membranes break up, hemolysis isformed. In this way, the adenosine diphosphate is released from thebroken red blood cells, which can intensify the assembly of platelets.As a result, the formation of clot and thrombus is accelerated [49]. Ifthe hemolysis ratio is lower, the blood compatibility is better with lessbroken red blood cells. The hemolysis ratios of all samples in Table 3are all well within 5%. As a novel material contacting with the blood, ifits hemolysis ratio is less than the accepted threshold value of 5%, ithas a good hemocompatibility [50]. It directly demonstrates that the ox-idized horn keratin can be used as biomaterials without causing the redblood cell to have change in deformability and fracture to form anyhemolysis.

3.6.2. Platelet adhesion and activationPlatelet adhesion and activation are known as the main intuitive in-

dicators and often used to assess the hemocompatability of materials[51]. If platelets are spreading and aggregating on the surfaces of mate-rials, the platelets are activated. It is a major mechanism for the forma-tion of thrombosis. On the surface of the tested samples, some adherentplatelets are in a moderate degree aggregation, but most adherentFig. 7. Morphology of adherent platelets on the surfaces of the samplplatelets still remain individual and spherical, separated without pseu-dopodium, as shown in Fig. 7. In addition, the adherent platelets arenot found to further induce a large number of platelet to aggregate.The process of blood coagulation is initiated when platelets are aggre-gating with the formation of a fibrin network. After that, a thrombus issubsequently formed [52]. However, if the surface of material is passiv-ated by a thin layer of platelets, without activation, it will have betterhemocompatibility [53]. As there are no platelet aggregation and activa-tion on the surfaces of oxidized horn keratins, it directly demonstratesthat the oxidized horn keratinswould not activate blood clotting systemto form thrombus.3.6.3. Fibrinogen activationFibrinogen is a serum protein and plays a dominant role in the for-

mation of thrombus [54]. The Fib levels of samples fall within normallevel, without showing significant difference with each other (p N0.05), but have a significant difference to positive control (p b 0.05),as shown in Fig. 8. Generally, the increase of Fib would enhance theblood coagulation to increase the thrombus formation. This may bedue to the conformational changes of fibrinogen, which would makees. A for OH, B for OH1220, C for OH1230 and D for OH4830.

image of Fig.7image of Fig.8

Fig. 9. The results of MTT assay of the samples, A for 3T3 cell and B for HUVECs. Each value is expressed as mean standard deviation.

131Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134fibrinogen to combine with the GPIIb/IIIa integrin receptor on plateletmembrane and further trigger the platelets to aggregate [55]. Thenormal Fib levels show that the degradation products of oxidized hornkeratin would not activate the platelets to trigger thrombus.

3.6.4. PTT and APTTCoagulation cascade system has intrinsic and extrinsic pathways,

which both converge at a common point. When the factor X is activatedFig. 10. The peri-implant tissue was obtained at 1 week to 10 weeks. Gross examination of theelapsing. AD for OH, EH for OH1220, IL for OH1230 and MP for OH4830 respectively.to Xa, prothrombin is sequentially activated to convert to thrombin,which would trigger and accelerate the formation of fibrin from fibrin-ogen [56]. The intrinsic pathway is initiated when the material iscontacted with the blood, which would sequentially activate theclotting process to lead to thrombosis. In addition, it is well knownthat PTT and APTT are used to detect the abnormalities of factors inintrinsic pathway, such as factors I, II, V, VIII, IX, X, XI, and XII, and fibrin-ogen [57]. As shown in Table 3, the PTTs of samples do not showimplant area did not show any observable inflammation or capsule formation with time

image of Fig.9image of Fig.10

Fig. 11. The H&E staining of the peri-implant tissue. AD for OH; EH for OH1220; IL for OH1230 and MP for OH4830 respectively.

132 Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134significant difference with each other (p N 0.05), but have a significantdifference to positive control (p b 0.05). PTT, without adding activators,can revealwhether there is activation betweenplasma and thematerial.Shortening of the PTT will increase the risk of thromboembolism. FromFig. 12. The high-magnification micrograph of H&E staining. AD for OH; Ethe results of PTT falling within the normal level, the horn keratin andthe oxidized horn keratin would not activate the platelets. On theother hand, comparedwith the positive control and the original plasma,the APTT variation falls within the normal level (p N 0.05) for all theH for OH1220; IL for OH1230 and MP for OH4830 respectively.

image of Fig.11image of Fig.12

133Q. Zhang et al. / Materials Science and Engineering C 47 (2015) 123134tested concentrations of degradation solution, as seen in Fig. 8. It indi-cates that the degradation solution (01 mg/ml) does not effectivelybenefit to the activation of intrinsic blood coagulation system.

However, the keratin biomaterials, whichwere extracted fromwool,hair, etc., have been demonstrated to be as an efficient hemostatic agentin several animal models [58]. In this way, the extracted keratin doesnot have three-dimensional structure, and the main component of itsdegradation is a low molecular weight of polypeptide or protein. But itis different from the oxidized horn keratin of this article, as can beseen from the results of FTIR, DSC, mechanical properties and SDS-PAGE, which may cause the oxidized horn keratin not to play the roleof procoagulant. From the results on hemocompatibility analysis, itcan obviously show that horn keratin and oxidized horn keratinswould not interfere with the normal functioning of platelets, withoutsignificant influence on the coagulation system. It directly demonstratesthat the oxidized horn keratins have better security because they meetwith the basic requirements of hemocompatibility of biomaterials.

3.7. Cell viability

The cells observed by optical microscopy growwell with normal cellmorphology both in control group and the test group; moreover, thereare discrete particles within the cytoplasm, without cytolysis. The MTTassay is usually used to evaluate the cytotoxicity of material by quanti-fying relative cell numbers [19]. As shown in Fig. 9, the horn keratin andoxidized horn keratin have no significant effects on the viability of 3T3and HUVECs when exposed to cells for 24 h, 48 h and 72 h respectively.Compared to serum-containing media, there is no significant evidenceof cytotoxicity and cell viability of samples is statistically equivalent. Itis demonstrated that the horn keratin and oxidized horn keratin havenon-cytotoxicity in vitro and compliance with the requirements ofbiomaterials.

3.8. In vivo tissue response

After implantation, thematerial would be seen as a foreign body andattacked by the immune system of the recipient. In the absence of otherfactors, if thematerial is toxic, it causes death of the surrounding tissue;if thematerial is nontoxic, the reaction between the tissue and implantsis primarily aseptic inflammation and fibrous capsule [59]. Early tissueresponse is mild or moderate acute aseptic inflammation, such asedema, hyperemia, and neutrophil infiltration, which is caused by im-plant irritation. After two weeks, the acute inflammatory would changeto chronic inflammation, including macrophages, lymphocytes and fi-broblast proliferation. Organism eliminates foreign body throughphagocytosis and enzymatic digestion, or by fibrous capsule wrappingto insulate implants [60,61].

During the experiments, the activities and diet of experimentalmicewere normal, without accidental death. In early stage of implantation,the dissected inner side of the skin shows acute inflammation, such ashyperemia around the implants; and then the inflammatory responsechanges to chronic inflammation, as shown in Fig. 10. At later timepoints, hyperemia in the subcutaneous tissue decreases, particularlyaround the implants, which can reflect that the inflammation is dimin-ished. In addition, the implants are isolated by fibrous capsule, whichcould make the implants not be eliminated by the cellular immune sys-tem. The thickness of fibrous capsule may reflect the histocompatibilitybetween implants and tissue [48,62]. If the capsule is thicker, the foreignbody reactionwould beheavier, resultingworse histocompatibility; andvice versa. However, the results do not exhibit thicker fibrous capsule atall-time points. So it can demonstrate that there are no adverse reac-tions between the implants and tissue, without obvious inflammationor fibrosis at late time points.

From the results of histological section in the vicinity of samples, itcan confirm that host cell migrates, such as inflammatory cells, endo-thelial cells, macrophages and fibroblasts, as shown in Figs. 11 and 12.In early stage of the implantation, the peri-implant tissue responsedoes not appear toxic reaction, such as cell lysis and destruction, and ap-pears to be predominantly neutrophils and a small amount of lympho-cytes. Neutrophils are the first responders for the acute inflammation.After that, macrophages, endothelial cells and fibroblasts are coming[63]. After 3 weeks, neutrophils have decreased, but there is an increasein the number of lymphocytes, macrophages and activated fibroblasts.However, the total number of inflammatory cells on the periphery ofimplants significantly reduces, revealing the chronic inflammation.Later, the peri-implant tissue response is dominated by mature fibro-blasts with a small number of lymphocytes. Inflammatory cell accumu-lation reaches the maximum number within 13 weeks and thengradually goes down,whichmay be due to the short life-time of neutro-phils and macrophages. It indicates that the inflammation decreasesduring the time. On the other hand, foreign body giant cells also existin all time points, which can reveal that the oxidized horn keratins arehighly compatible with tissue.

From the above results of assessments, the insertions of horn keratinand oxidized horn keratins subcutaneously implanted into mice do notcause a substantial inflammatory reaction. All responses around the im-plants are limited to mild foreign body reactions. Thus it demonstratesthat horn keratin and oxidized horn keratins have good biocompatibil-ity and can act as an implanted biomaterial for clinical applications.

4. Conclusions

Overall, our study can demonstrate some general properties of oxi-dized horn keratin and support its use as a biomaterial. The FTIR revealsthat the disulfide bonds, or even partial peptide chain of horn keratin,are broken down during oxidation. With the change in microstructure,the oxidized horn keratin has lower thermal stability and hydrolytic sta-bility in comparison with horn keratin. The mechanical properties ofoxidized horn keratins are poorer than those of horn keratin, but theoxidized horn keratins still have disulfide bonds to form a three-dimensional structure, which benefits for their mechanical properties.However, the fracture toughness of oxidized horn keratin increaseswith the increase in the degree of oxidation. After oxidation, the oxi-dized horn keratins have lower cytotoxicity and lower hemolysis ratio.Moreover, when the oxidized horn keratins, aswell as different concen-trations of degradation products of oxidized horn keratins, are directlyin contact with platelet-rich plasma, platelets are not activated. It sug-gests that the oxidized horn keratins have good hemocompatibility,without triggering blood thrombosis. The implantation experimentin vivo also demonstrates that the oxidized horn keratins are com-patible with the tissue, because there are minimal fibrous capsuleand less of infiltration of host cells, without causing serious inflam-mation. Therefore, the oxidized horn keratin may offer a potentialapplication of implanted biomaterial that is directly in contactwith blood and tissue.

Acknowledgments

This work has been financially supported by the National NaturalScience Foundation of China (Grant No. 20976068/B060805) andthe Science and Technology Program of Guangdong Province (No.268017). In addition, the authors would like to thank ShenzhenTesting Center of Medical Devices, China. They would also like tothank Yuan Tian, coming from Biomedical Engineering of JinanUniversity.

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Mechanical and biological properties of oxidized horn keratin1. Introduction2. Materials and methods2.1. Sample preparation2.2. The oxidation of horn keratin2.3. Fourier transform infrared spectroscopy (FTIR)2.4. Thermal analysis2.5. Tensile tests2.6. Scanning electron microscopy2.7. In vitro degradation and SDS-PAGE analysis2.8. In vitro cell viability2.9. In vitro hemocompatibility2.9.1. Hemolysis ratio2.9.2. Platelet adhesion and activation2.9.3. PTT, APTT and fibrinogen (Fib) assay

2.10. In vivo implantation experiment2.11. Statistical analyses

3. Results and discussion3.1. Analysis of FTIR3.2. TG-DSC measurement3.3. The effects of oxidation on the mechanical properties of horn keratin3.4. Microstructure of fracture surfaces3.5. In vitro hydrolytic stability and SDS-PAGE analysis3.6. In vitro hemocompatibility3.6.1. Hemolysis ratio3.6.2. Platelet adhesion and activation3.6.3. Fibrinogen activation3.6.4. PTT and APTT

3.7. Cell viability3.8. In vivo tissue response

4. ConclusionsAcknowledgmentsReferences