a r t i c l e
i n f o
Article history:
Received 21 September 2014
Accepted 12 November 2014
Available online 14 November 2014
Keywords:
Horn keratin
Protein oxidation
Mechanical properties
Biocompatibility
Biomaterial
a b s t r a c t
The 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 clinical translatability. The characterization on their composition, mechanical properties, and biological responses
was performed. It is found that the oxidation process could lead the disulde bond to break down and then to
form sulfonic acid, or even make partial peptide chain to be fragment for the new modication of amino acid.
Hence the oxidized horn keratins have lower thermal stability and hydrolytic stability in comparison with
horn keratin, but the degradation products of oxidized horn keratins have no signicant difference. In addition,
the mechanical properties of oxidized horn keratins are poorer than that of horn keratin, but the oxidized horn
keratins still have disulde bonds to form a three-dimensional structure, which benets for their mechanical
properties. The fracture toughness of oxidized horn keratins increases with the increase in the degree of oxidation. After oxidation, the oxidized horn keratins have lower cytotoxicity and lower hemolysis ratio. Moreover,
when the oxidized horn keratins, as well as different concentration of degradation products of oxidized horn keratins, are directly in contact with platelet-rich plasma, platelets are not activated. It suggests that the oxidized
horn keratins have good hemocompatibility, without triggering blood thrombosis. The implantation experiment
in vivo also demonstrates that the oxidized horn keratins are compatible with the tissue, because there are minimal brous capsule and less of inltration of host cells, without causing serious inammation. In summary, the
oxidized 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 rst study of
keratin used as biomaterial was published by Noishiki and his colleagues. They coated a heparinized keratin derivative onto a vascular
graft, which was implanted into a dog, without thrombosis for more
than 200 days [2]. In fact, keratin is the major component of any tissue
of a living organism, such as hair, wool, feathers and horns, which is
often associated with various biological functions. For example, they
can serve as a barrier for environmental stress, regulate moisture and
communicate with others. Keratin can be distinguished as soft and
hard keratin [3,4]. Soft keratin is often found in epidermis and calluses
and it has lower sulfur content. However, hard keratin has higher sulfur
content. Hard keratin can be classied into two groups. The one is hard
-keratin, which is found in mammalian epidermal appendages, such as
horns, hairs and nails, and the other one is -keratin, which is found in
avian and reptilian tissues. The -keratin has an -helical coil structure,
but the -keratin has a twisted -sheet structure. In recent years, the
Corresponding author.
E-mail address: taonj@jnu.edu.cn (N. Ao).
http://dx.doi.org/10.1016/j.msec.2014.11.051
0928-4931/ 2014 Elsevier B.V. All rights reserved.
124
Fig. 1. (A) Schematic drawing of the horn samples, showing the orientation and position
where the samples were cut. (B) The inset shows the sample orientation for tests
(not to scale).
(5% stacking gel and 12% separating gel) (Bio-Rad). Separation was performed at 80 V for approximately 3 h. After separation, gels were rinsed
with ultrapure water for 5 min before staining with Bio-Safe Coomassie
stain (G250, Bio Rad) for 10 min under boiling water bath. Destaining
was done overnight in ultrapure water with gentle rotation. Samples
were compared to a standard ladder (Benchmark Prestained Protein
Ladder, Invitrogen, Carlsbad, CA) and the gels were imaged in DIA
mode with an Image Scanner (GE, USA).
2.8. In vitro cell viability
Cell viability in the presence of horn keratin and oxidized horn keratin was assessed by an MTT assay. All keratin samples (4 mm 2 mm),
which had been soaked in PBS solution (pH 7.4) for 24 h and then
drained, were sterilized in a steam autoclave at 120 C for 30 min
prior to NIH-3T3 and human umbilical vein endothelial cell (HUVEC)
(Medical Laboratory, Jinan University, China) culture experiments. Subsequently, all the sterilized samples were placed in a 48-well culture
plate (Corning Life Sciences), and each sample had 5 duplications. The
NIH-3T3 and HUVECs were seeded at a density of 5000 cells/cm2 and
allowed to grow at 37 C atmosphere of 5% CO2. The negative control
consisted of cells without samples. After incubation of certain time,
such as 24 h, 48 h and 72 h respectively, a 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/ml,
Sigma) was added and incubated for further 4 h. Mitochondrial dehydrogenases of viable cells cleaved the tetrazolium ring, yielding purple
formazan crystals. Formazan crystals were then dissolved in DMSO solution (Sigma). Afterwards, 200 l of the blue solutions was transferred
to a 96-well plate. The absorbance was measured at 490 nm by a microplate reader (Bio-Rad).
2.9. In vitro hemocompatibility
The fresh rabbit's blood used in our experiments was obtained legally from the Shenzhen Testing Center of Medical Devices, China. The
analysis was performed within 12 h after blood donation. The amounts
of the samples used for statistical count were not less than three.
2.9.1. Hemolysis ratio
4 ml blood was diluted by 5 ml of 0.9% (w/v) sodium chloride solution. Each testing sample (40 mm 5 mm 2 mm) was added 10 ml
0.9% (w/v) sodium chloride solution. Additionally, 10 ml of 0.9% (w/v) sodium chloride solution and 10 ml double distilled water were prepared
respectively for antitheses. All the samples were kept at 37 C for 72 h,
and then immediately added 0.2 ml of the diluted blood. Incubation
was performed at 37 C with test tubes. After 60 min incubation, the samples were centrifuged at 750 g for 5 min. Then the supernatant was measured at 545 nm by 722 s spectrophotometer (Shanghai Sunny Hengping
Scientic Instrument Co., Ltd.). Hemolysis ratio was calculated as follows:
hemolysis
h
i
ODtest ODneg = ODpos ODneg 100%;
where ODtest, ODneg, and ODpos were the absorbance values of the test
sample, negative control (saline), and positive control (water), respectively. All the hemolysis experiments were done in triplicate.
2.9.2. Platelet adhesion and activation
Platelet adhesion experiment was carried out to evaluate the surface
thrombogenicity of the samples and to examine the interaction between blood and the materials in vitro [30]. The size of all of the samples
was 5 mm 5 mm. The rabbit whole blood was treated with anticoagulant (EDTAK2, Hunan Liuyang Medical Instrument Factory,
China). After centrifuging at 1000 rpm for 15 min, a platelet-rich plasma
(PRP) was obtained. The samples were immersed in PRP and incubated
at 37 C for 30 min. The samples were subsequently rinsed with a PBS
125
126
-Helix
Band position (cm
Fig. 2. FTIR spectra of OH, OH1220, OH1230 and OH4830, as shown in A; the fragment
of FTIR spectra resolved into components (over range 17101590 cm1, R2 N 0.999), as
shown in B.
OH
OH1220
OH1230
OH4830
1650.8
1652.3
1653.9
1656.2
-Sheet
1
Area (%)
Area (%)
48.74
45.80
42.42
37.66
31.69
35.35
38.15
42.10
127
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.
the thermal stability of oxidized samples would increase with the increase in degree of oxidation.
The DSC curves of samples are shown in Fig. 3B. There are three endothermic peaks in the DSC curves, and they roughly correspond to two
evident mass losses. For horn keratin, the well-known endothermic
peak at about 92 C resulted from water evaporation and glass transition. Due to the complex structure of horn keratin, the glass transition
often occurs in a temperature range rather than at a xed temperature
[34,35]. Moreover, this temperature range is usually overlapped with
the peak of water evaporation in a DSC curve [36]. The endothermic
peak at 236 C is ascribed to the denaturation of the -keratin crystallites and the area under the curve can be used to measure the -helix
content; and the endothermic peak at 312 C corresponds to the
128
Table 2
The values of mechanical characteristics in horn keratin and oxidized horn keratins.
Samples
OH
OH1220
OH1230
OH4830
117.581
100.011
85.758
72.680
1.553
1.137
0.869
0.732
24.602
37.566
56.180
70.529
3.129
2.341
5.386
2.063
0.031
0.029
0.021
0.022
3.599
4.113
4.252
6.371
n = 3 for each set of samples. Values are means s.e.m. and p b 0.05 by comparisons at
all samples.
hydrogen bonds and ionic interaction, or even peptide bonds, are broken somewhat, resulting in less coherent interactions within the protein
structure [42]. On the other hand, the crystalline regions with -helical
structure are responsible for the strength of horn keratin, but the amorphous regions, which have relatively fewer bonds between the polymer
chains and random distribution of the chains, provide the horn keratin
with elasticity and exibility [43]. From the results of FTIR, the
-helical structure decreases and amorphous matrix increases after oxidation. As a result, this would cause larger matrix region with freedom
of movement and reduce the stability of the matrix, resulting in the
decrease of the tensile strength and Young's modulus and the improvement of failure strain of oxidized horn keratin. However, high concentration of hydrogen peroxide might contribute to the production of
excessive amounts of perhydroxyl species, which can react with more
substances. If the time of oxidation is longer, it would let perhydroxyl
species have more time to attack the proteins [41]. This can cause further breakage of bonds, even polypeptide chains. Therefore, as the degree of oxidation increases, there is a signicant reduction in tensile
strength and Young's modulus in oxidized horn keratin, but signicant
increase in toughness. It indicates that the mechanical properties of
horn keratin are largely affected by the concentration of hydrogen peroxide and the time of oxidation. However, the tensile strength and fracture strain of oxidized horn keratin are stronger than those of other
synthetic materials, such as polycarbonate (67 MPa and 15%) [44] and
polylactide (65 MPa and 9%) [45]; even the tensile strength of OH12
20 can rival that of berglass (110 MPa) [46]. Therefore, with appropriate oxidation treatment, the oxidized horn keratin can maintain its
mechanical property not to change too much.
3.4. Microstructure of fracture surfaces
The horn keratin is a hierarchical material and has laminate structure [8]. Related to the fracture surfaces of oxidized samples, the SEM
images of samples reveal different failure phenomena, as seen in
Fig. 4. It is clearly shown that the fracture surface in buffalo horn is relatively smooth, neat and wavy, indicating that the buffalo horn has a
dense laminate structure. But the bers are pulled out and the lamellas
are partially torn in OH1220. Moreover, the fracture surfaces of OH12
30 and OH4830 show an extremely ductile fracture mode, evidenced
by a very deep, convoluted cup-and-cone type fracture. At the same
time, the samples had fully recovered to their original shape and dimension after a certain time. The larger failure strain also indicates that oxidized horn keratin is more resilient than horn keratin. It may be due to
the more compliant matrix that can yield and ow more readily with
the increase in the degree of oxidation, which is corresponding to the
results of the mechanical properties of the samples.
3.5. In vitro hydrolytic stability and SDS-PAGE analysis
Numerous disulde bonds permanently bind the peptide chains,
which contributes to the insolubility or low insolubility of keratin in
water. The degradation behavior of horn keratin after oxidation was investigated using an in vitro degradation experiment, which would provide a good understanding in the hydrolytic stability of oxidized horn
keratin. From the weight loss ratiotime curve in Fig. 5, the degradation
rate of OH is extremely slow, nearly no degradation during 10 weeks.
129
Fig. 4. The fracture surfaces of the samples under tensile test. A for OH, B for OH1220, C for OH1230 and D for OH4830.
However, with the disulde bonds being oxidized to break down, the
hydrolytic stability of oxidized horn keratin decreases gradually.
Compared with the horn keratin, there is a signicant difference in the
degradation rate of oxidized samples (p b 0.05). The degradation rate
of oxidized horn keratins gradually accelerates with the increase in
degree of oxidation. For example, after 16 weeks, the degradation rate
of OH4830 is up to about 90%, nearly completely degraded; but for
OH1220, the weight loss ratiotime curve is substantially linear,
which indicates that the degradation behavior of OH1220 is relatively
stable. However, there is a sudden sharp rise in weight loss ratiotime
curve of OH1230 and OH4830. As expected, with more broken disulde
bonds, the degradation behavior of the oxidized horn keratin is signicantly enhanced, resulting in less hydrolytic stability.
In SDS-PAGE method, the mobility of protein depends on its relative
molecular mass, regardless of the electric charge and molecular shapes.
Therefore, after the qualitative analysis of protein fractions in degradation
products by SDS-PAGE, the protein bands from 3 separate degradation
products of oxidized horn keratins reveal similar patterns, including
high molecular mass bands (N40 kDa) and low molecular mass bands
(b 25 kDa), as shown in Fig. 6. The protein bands at about 4060 kDa
Fig. 5. The weight loss ratiotime curves of the samples. Each value is expressed as
mean standard deviation.
130
Table 3
Hemolysis ratio and PTT of horn keratin and oxidized horn keratins. The hemolysis ratio of
the positive control (water) and negative control (saline) was 1 and 0, respectively.
Samples
PTT (s)
Original plasma
Positive control
OH
OH1220
OH1230
OH4830
1.80
1.54
1.99
1.49
63.23
30.57
63.55
60.73
61.13
59.33
0.35
0.23
0.16
0.77
1.17
0.93
1.21
1.86
3.49
1.76
Fig. 8. APTT and Fib of the original plasma and the plasma contacted with different concentrations of degradation product of the oxidized keratins.
platelets still remain individual and spherical, separated without pseudopodium, as shown in Fig. 7. In addition, the adherent platelets are
not found to further induce a large number of platelet to aggregate.
The process of blood coagulation is initiated when platelets are aggregating with the formation of a brin network. After that, a thrombus is
subsequently formed [52]. However, if the surface of material is passivated by a thin layer of platelets, without activation, it will have better
hemocompatibility [53]. As there are no platelet aggregation and activation on the surfaces of oxidized horn keratins, it directly demonstrates
that the oxidized horn keratins would not activate blood clotting system
to form thrombus.
3.6.3. Fibrinogen activation
Fibrinogen is a serum protein and plays a dominant role in the formation of thrombus [54]. The Fib levels of samples fall within normal
level, without showing signicant difference with each other (p N
0.05), but have a signicant difference to positive control (p b 0.05),
as shown in Fig. 8. Generally, the increase of Fib would enhance the
blood coagulation to increase the thrombus formation. This may be
due to the conformational changes of brinogen, which would make
Fig. 7. Morphology of adherent platelets on the surfaces of the samples. A for OH, B for OH1220, C for OH1230 and D for OH4830.
131
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.
Fig. 10. The peri-implant tissue was obtained at 1 week to 10 weeks. Gross examination of the implant area did not show any observable inammation or capsule formation with time
elapsing. AD for OH, EH for OH1220, IL for OH1230 and MP for OH4830 respectively.
132
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.
the results of PTT falling within the normal level, the horn keratin and
the oxidized horn keratin would not activate the platelets. On the
other hand, compared with the positive control and the original plasma,
the APTT variation falls within the normal level (p N 0.05) for all the
Fig. 12. The high-magnication micrograph of H&E staining. AD for OH; EH for OH1220; IL for OH1230 and MP for OH4830 respectively.
tested concentrations of degradation solution, as seen in Fig. 8. It indicates that the degradation solution (01 mg/ml) does not effectively
benet to the activation of intrinsic blood coagulation system.
However, the keratin biomaterials, which were extracted from wool,
hair, etc., have been demonstrated to be as an efcient hemostatic agent
in several animal models [58]. In this way, the extracted keratin does
not have three-dimensional structure, and the main component of its
degradation is a low molecular weight of polypeptide or protein. But it
is different from the oxidized horn keratin of this article, as can be
seen from the results of FTIR, DSC, mechanical properties and SDSPAGE, which may cause the oxidized horn keratin not to play the role
of procoagulant. From the results on hemocompatibility analysis, it
can obviously show that horn keratin and oxidized horn keratins
would not interfere with the normal functioning of platelets, without
signicant inuence on the coagulation system. It directly demonstrates
that the oxidized horn keratins have better security because they meet
with the basic requirements of hemocompatibility of biomaterials.
3.7. Cell viability
The cells observed by optical microscopy grow well with normal cell
morphology both in control group and the test group; moreover, there
are discrete particles within the cytoplasm, without cytolysis. The MTT
assay is usually used to evaluate the cytotoxicity of material by quantifying relative cell numbers [19]. As shown in Fig. 9, the horn keratin and
oxidized horn keratin have no signicant effects on the viability of 3T3
and HUVECs when exposed to cells for 24 h, 48 h and 72 h respectively.
Compared to serum-containing media, there is no signicant evidence
of cytotoxicity and cell viability of samples is statistically equivalent. It
is demonstrated that the horn keratin and oxidized horn keratin have
non-cytotoxicity in vitro and compliance with the requirements of
biomaterials.
3.8. In vivo tissue response
After implantation, the material would be seen as a foreign body and
attacked by the immune system of the recipient. In the absence of other
factors, if the material is toxic, it causes death of the surrounding tissue;
if the material is nontoxic, the reaction between the tissue and implants
is primarily aseptic inammation and brous capsule [59]. Early tissue
response is mild or moderate acute aseptic inammation, such as
edema, hyperemia, and neutrophil inltration, which is caused by implant irritation. After two weeks, the acute inammatory would change
to chronic inammation, including macrophages, lymphocytes and broblast proliferation. Organism eliminates foreign body through
phagocytosis and enzymatic digestion, or by brous capsule wrapping
to insulate implants [60,61].
During the experiments, the activities and diet of experimental mice
were normal, without accidental death. In early stage of implantation,
the dissected inner side of the skin shows acute inammation, such as
hyperemia around the implants; and then the inammatory response
changes to chronic inammation, as shown in Fig. 10. At later time
points, hyperemia in the subcutaneous tissue decreases, particularly
around the implants, which can reect that the inammation is diminished. In addition, the implants are isolated by brous capsule, which
could make the implants not be eliminated by the cellular immune system. The thickness of brous capsule may reect the histocompatibility
between implants and tissue [48,62]. If the capsule is thicker, the foreign
body reaction would be heavier, resulting worse histocompatibility; and
vice versa. However, the results do not exhibit thicker brous capsule at
all-time points. So it can demonstrate that there are no adverse reactions between the implants and tissue, without obvious inammation
or brosis at late time points.
From the results of histological section in the vicinity of samples, it
can conrm that host cell migrates, such as inammatory cells, endothelial cells, macrophages and broblasts, as shown in Figs. 11 and 12.
133
134
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