Biomineralization of calcium phosphate on human hair protein film and formation of a novel...

Biomineralization of Calcium Phosphate on Human Hair ProteinFilm and Formation of a Novel Hydroxyapatite–ProteinComposite Material

Toshihiro Fujii,1 Teppei Tanaka,2 Kousaku Ohkawa3

1 Bioengineering Course, Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University,Ueda, Nagano 386-8567, Japan

2Department of Kansei Engineering, Faculty of Textile Science and Technology, Shinshu University,Ueda, Nagano 386-8567, Japan

3 Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University,Ueda, Nagano 386-8567, Japan

Received 9 August 2008; revised 10 March 2009; accepted 2 April 2009Published online 25 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31426

Abstract: Human hair protein can be used not only as a totally biodegradable material but

also as a ‘‘self-originated’’ material, which may avoid an undesirable immune reaction, if it

has been prepared from a certain individual and implanted into the same person. In this study,

a novel organic–inorganic composite, which contains human hair proteins and hydroxyapatite,

was investigated as biomineral-scaffolding materials. The human hair protein was extracted

by our original ‘‘Shindai method’’ (Nakamura et al., Biol Pharm Bull 2002;25:569–572; Fujii

et al., Biol Pharm Bull 2004;27:89–93). The extracts were exposed to CaCl2 solution for

fabrication into flat films, which mainly consisted of a-keratin. After washing with distilled

water, �3 Ca21 ions per 1 keratin molecule bound to the film. The Ca21-binding was slightly

sensitive to the ionic strengths, and only Mg21 inhibited binding of Ca21. A composite of the

human hair protein and calcium phosphate was prepared via alternate soaking processes using

CaCl2 and Na2HPO4 solutions. As the soaking cycle proceeded, the film weight increased and

its color became white, indicating successful deposition of calcium phosphate. The diameters of

deposited calcium phosphate particles were about 2–4 lm. The proteins were not solubilized

and degraded during the soaking processes. FTIR and WAXD analyses indicated that calcium

phosphate was first deposited as amorphous, then transformed into crystalline monohydrogen

calcium phosphate during the earlier soaking cycle, and, via octacalcium phosphate, finally

converted into hydroxyapatite after 20 cycles. The present human hair protein/hydroxyapatite

composite film is a ‘‘self-originated’’ and also an intact proteinaceous material without

chemical modification, and thus, a promising material for hard tissue engineering. ' 2009 Wiley

Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 91B: 528–536, 2009

Keywords: human hair protein; composite film; calcium phosphate; keratin; biomineraliza-

tion; hydroxyapatite

INTRODUCTION

Human hair is a typical renewable biomaterial consisting of

proteins, lipids, colored matter, several minerals, and water.

The protein comprises �80% of the total mass of the

human hair and mainly consists of a-keratins, keratin-

associated proteins, and high molecular weight components.

The human hair material can be collected in a large

quantity at an individual level. Thus, a variety of protein

materials will be produced by the obtained hair from a cer-

tain specific individual, which are applicable as a donor

material with a high biocompatibility.

The hair and skin keratins constitute one of the most

extensive protein families among the intermediate filaments

with a diameter of around 10 nm and provide resistance

against mechanical stress in the epidermal cells and kerati-

nized tissues. Some different keratin polypeptides have been

described, which can be classified into two different types,

that is, I and II, based on their molecular weights and iso-

electric points.1,2 After solubilization of the intermediate

Correspondence to: K. Ohkawa (e-mail: [email protected])Contract grant sponsor: Ministry of Education, Culture, Sports, Science and

Technology of Japan.

' 2009 Wiley Periodicals, Inc.

528

filaments using denaturants, the monomers can retain the

ability of self-polymerization depending on the ionic

strengths and form a filamentous structure.3,4 The convenient

methods, such as precast, postcast, and soft postcast meth-

ods, have been already developed by us for the preparation

of keratin films from human hair, and the time required for

the fabrication is within 24 h.5,6 The keratin films, which are

prepared by the soft postcast method using divalent or

monovalent cations, were more flexible than those prepared

by other methods. The films have been subjected to the patch

test to examine their tissue compatibility, and the result

clearly indicated that the human hair protein film has no tis-

sue toxicity after continuous skin contact over 40 h (8 h 3 5

days).5 Both the keratin solutions and films had little effect

on rat mast cells that are widely distributed in various tissues

containing epithelia and responsible for type 1 allergies.7

These observed properties of the human hair protein film

suggest their promising use as medical materials.

To expand the material application of the human hair pro-

tein film, the use of the human hair protein film as a scaffold

material can be considered, especially for hard tissue regen-

eration or repair. The hard tissues, such as bone or teeth, can

be recognized as an organic–inorganic composite, in which

the organic components are usually proteins including colla-

gens and phosphorylated extracellular matrix proteins, and

the inorganic components are calcium phosphate, especially

present as hydroxyapatite.8 A number of material chemistry

studies associated with hard tissue engineering have already

been reported, and a significant part of these studies is the

material design of the bone scaffolds, which are prepared

from the possible combinations of organic materials of natu-

ral origin, inorganic materials of natural origin, synthetic or-

ganic materials, and synthetic inorganic materials.9 The

bone scaffold have been fabricated as film,10 hydrogels,11,12

textiles,13 and also native tissue.14 Among them, as for the

proteinaceous fabrics, the hydrogel,11 sponge,12 and films15

of collagen or gelatin are the most focused materials,

because the extracellular matrix mainly consisted of collagen

microfibrils, which act as the initial deposition and nuclea-

tion sites of the biogenic calcium phosphate.

The common requirements of a scaffold material for the

biogenic calcium phosphate are as follows: (i) efficient and

selective binding ability of the divalent calcium cations

(Ca21) and phosphate anions (PO422), (ii) restoration of dis-

solution-precipitation equilibrium of calcium phosphate

around the insoluble or moderately swollen polymer matrix

to form the amorphous calcium phosphate,16 and (iii) induc-

tion of the nucleation of calcium phosphate and the deposi-

tion of crystalline products as brushite (CaHPO4 � H2O) and

octacalcium phosphate,17 the latter of which is a metastable

precursor of the biogenic hydroxyapatite as the most stable

crystalline phase. In most cases, the proteinaceous materials,

especially for the collagen, have been investigated to exam-

ine the three subjects described earlier.

Keratins are also involved in the tissue calcification, in

some cases, as tooth enamel matrix or horn development, and

the physiological comparison and relationship between the

tissue keratinization and the calcification (biomineralization)

have been discussed for a long time.18,19 The physiological

function of the keratinization associated with the tissue calci-

fication, even to date, can inspire ideas for the novel applica-

tion of keratin as a biomedical material. Thus, this study

has been conducted to evaluate the human hair protein film

as a hard tissue scaffold. The experimental objectives were

(i) selective binding ability of Ca21, (ii) stability of the human

hair protein and morphology of the calcium phosphate de-

position, and (iii) phase transition of calcium phosphate depo-

sition onto the human hair protein film surface. Finally,

the possibility using the resulting human hair protein–hydroxy-

apatite composite films for medical applications was discussed.

MATERIALS AND METHODS

Preparation of Human Hair Protein Solution andProtein Films

Human hair protein solutions were prepared according to

the "Shindai method," which has been previously described

by us.20 When the human hair protein extract, which were

prepared by the Shindai method, were exposed to various

solutions containing CaCl2, MgCl2, NaCl, KCl, and acetate

buffer, the human hair proteins were aggregated onto the

dish surfaces to form wet films.6 Depending on the reagents

for protein aggregation in the preparation of the human

hair protein films, the fabrication methods are designated as

the postcast (for use of 100 mM acetate buffer at pH 4.0,

casting the human hair protein extract first, then overlaying

the salt solution) and soft postcast (CaCl2, MgCl2, KCl,

and NaCl), since the latter method yielded mechanically

more flexible films.5 Among these inorganic salts, CaCl2and acetate buffer were mainly used in this study.

Briefly, ethanol-treated volunteer hair without any chem-

ical treatments, such as bleach and a permanent, was

extracted using a solution containing 20 mM Tris-HCl, pH

8.5, 2.6M thiourea, 5M urea, and 150 mM dithiothreitol

(DTT) at 508C for 1 day. After filtration and centrifugation

of the extracted solution, the extracted protein solution was

further used for the preparation of the films.

The human hair protein films were prepared using the

soft postcast method.5 The hair protein solution was poured

into tissue culture dishes, then a salt solution containing 50

mM CaCl2 was gently overlayed. A membrane-like aggre-

gate was formed that spread out on the bottom of the dish.

The films were washed five times for 1 h by rinsing with

distilled water. The human hair protein films were recov-

ered after drying at room temperature.

Assays of Human Hair Proteins

The films were incubated with Shindai solution containing

5% 2-mercaptoethanol (2-ME) instead of 150 mM DTT at

508C for 3 h. The dissolved protein solution was used for

the measurement of the protein and calcium ion contents.

529BIOMINERALIZATION OF CALCIUM PHOSPHATE ON HUMAN HAIR PROTEIN FILM

Journal of Biomedical Materials Research Part B: Applied Biomaterials

The protein concentrations were determined according to

Bradford using bovine serum albumin as the standard.21 The

concentrations of Ca21 and Mg21 were spectrophotometri-

cally measured using a specific dye (BioAssay Systems),

according to the manufacturer’s protocol (n 5 3–4). The

protein components were analyzed by sodium dodecyl sul-

fate-polyacrylamide gel electrophoresis (SDS-PAGE).22 The

molecular weight of the a-keratin was calculated to be

50,000 Da. In our previous study, the SDS-PAGE analysis

of the human hair protein films indicated that the recovered

proteins in the films are mainly composed of a-keratins.6

Deposition of Calcium Phosphate onto Human HairProtein Films

The deposition of calcium phosphate on human hair pro-

teins films was performed according to the so-called alter-

nate soaking procedure.11–13 The calcium-bound human

hair protein films, which were prepared in a culture dish,

were immersed in a 120 mM Na2HPO4 solution for 30–60

s and then fully rinsed twice with distilled water. After

draining off the water, the films were immersed in a 200

mM CaCl2 solution for 30–60 s and rinsed again with dis-

tilled water. The series of operations was assumed to be

one cycle, and this cycle was repeated up to 20 times.

Characterization of Calcium Phosphate

The human hair protein films before and after the deposi-

tion of calcium phosphate were sputtered with platinum,

then the morphology of the deposited calcium phosphate

was observed using a scanning electron microscope

(Hitachi S-2380N) at the accelerating voltage of 20 kV.

For the qualitative comparisons of the amounts of the

deposited calcium phosphate on the human hair protein

films, a Fourier-transform infrared (FTIR) spectrum was

obtained after 0, 10, and 20 cycles of the alternate soaking

procedure. The samples were prepared by the usual proce-

dure using a KBr pellet, and the spectrum was recorded in

the transmission mode (HORIBA FT720). The intensities

of the infrared absorption bands for the P¼¼O stretching

vibration (1036 cm21) were compared for each sample, as

normalized for the intensities of the amide II (1541 cm21).

This calibration provides a relative comparison of the phos-

phate salt amounts deposited onto the films.

The crystalline phase analysis of calcium phosphate on

the human hair protein film was performed by wide angle

X-ray diffraction measurements (WAXD) using a RIGAKU

Geiger Flex apparatus at 40 kV and 150 mA.

RESULTS

Interaction between Ca21 Ion and Human HairProtein Films

After forming film-like aggregates, the proteins remained

insoluble even after washing with distilled water for 1–2

days. The yields of the human hair proteins in the resulting

films were 60–80% based on the observed protein concen-

trations in the human hair protein extract and the actual

weights of the recovered films.

To examine the ionic interaction between the human

hair proteins and inorganic ions, the stoichiometry between

the proteins and ions was first determined. The films pre-

pared in a 50 mM CaCl2 solution were washed from one to

seven times every hour with fresh distilled water to remove

any unbound Ca21. The films were then dissolved again in

the Shindai solution containing 2-ME, followed by mea-

surement of the remaining Ca21 amount. As a-keratins

(types I and II) are the major components of the human

hair protein films, the remaining Ca21 in the film are

considered to have electrostatically interacted with the

a-keratin molecules, and thus, the amount of bound Ca21

was represented as molar ratios toward the keratin mole-

cules (Figure 1). The bound Ca21 decreased with the

increasing washing times, and the molar ratio almost

became constant after the fourth or fifth washings. This

result clearly indicates that the a-keratin molecules in the

human hair protein films can significantly entrap Ca21 ions

in the molar ratio of approximately three Ca21 ions per

one a-keratin molecule.

Second, the CaCl2 concentrations in the protein aggrega-

tion step during the preparation of the human hair protein

film were varied from 10 to 100 mM. When the CaCl2 con-

centration was lower than 10 mM, protein aggregation did

not occur, therefore, the human hair protein cannot be

recovered.6 Thus, the human hair protein films were pre-

pared with 10–100 mM CaCl2 for the protein aggregation

steps, and the resulting film was washed five times every

Figure 1. Effects of washing time on the Ca21-binding ratio of thehair protein films. The human hair protein extract was treated with a

50 mM CaCl2 solution, and the obtained films were washed 1–7

times for 1 h using distilled water, followed by drying at room tem-perature. The washed films were dissolved in the Shindai solution to

extract the residual Ca21 ions, and then the amounts of the solubi-

lized Ca21 and protein were measured.

530 FUJII, TANAKA, AND OHKAWA


hour with fresh portions of distilled water. After recovery

of the human hair protein film, the remaining Ca21 was

quantified to estimate the molar ratio of the bound Ca21

and a-keratin molecules. Figure 2 shows the result. The

bound Ca21 increased with the increasing concentrations of

the CaCl2 solution and reached a plateau at 3.0–3.2 mole

Ca21/mole a-keratin, the values of which are similar to

that observed in Figure 1, supporting the accuracy of the

estimated Ca21 binding ability of one a-keratin molecule.

The half-maximum Ca21 bound ratio was estimated to be

about 1.5 mole Ca21/mole a-keratin by the extrapolation of

the saturation curve in Figure 2, which corresponds to the

CaCl2 concentration of �5 mM. This value further indi-

cates that during the protein aggregation step, Ca21 ions

will be irreversibly bound to the a-keratin molecules.

Divalent Cation-Binding Properties of Human HairProtein Films

As the aggregation of the human hair protein, especially

for the a-keratin, was also observed in the cases of inor-

ganic salts other than CaCl2, such as KCl, NaCl, and

MgCl2, a competitive adsorption experiment was conducted

in the coexistence of KCl or MgCl2. Figure 3(A) represents

the Ca21-binding ratio per one a-keratin molecule in the

presence of 50 mM CaCl2 and the predetermined concen-

trations of KCl at 0, 100, 300, and 500 mM. In the pres-

ence of a low concentration of KCl at 100 mM, the Ca21-

binding ratio was 3.1 mole Ca21/mole a-keratin, and this

value was similar to that in the absence of KCl (3.0–3.2

mole Ca21/mole a-keratin). At the higher concentration of

KCl (500 mM), the Ca21-binding ratio was 2.7 mole Ca21/

mole a-keratin, in which only a slight decrease can be

observed as compared to that in the absence of KCl. The

competitive inhibitory effect of KCl on the Ca21-binding

to a-keratin was calculated to be �10% at 500 mM KCl.

For the MgCl2, however, the Ca21-binding ratio signifi-

cantly decreased to 1.5 mole Ca21/mole a-keratin in the

presence of 50 mM MgCl2, and the amount of Mg21 bound

to the film was 1.0 mole Mg21/mole a-keratin [Figure

3(B)]. Because CaCl2 and MgCl2 coexist at the same con-

centrations (50 mM), this result clearly indicated that the

Mg21 ion can compete with Ca21 ions to occupy the diva-

lent cation-binding sites in the a-keratin molecules with

less ion selectivity. At the MgCl2 concentrations above 100

mM, the Mg21-binding ratio saturated at about 2.0 mole

Mg21/mole a-keratin, while the Ca21-binding ratio

decreased to 1.0–0.5 mole Ca21/mole a-keratin. These

results suggest that the selectivity in binding metal ions for

Ca21 is only slightly higher than that for Mg21.Figure 2. Relationship between the CaCl2 concentration during the

protein-aggregation step for the preparation of the human hair pro-

tein film and the residual amount of Ca21 ions bound to the film. Af-ter preparation of the human hair proteins using various

concentrations of CaCl2 (10–100 mM) solutions, the films were

washed five times with distilled water, and then the amount of Ca21

ions bound to the film was determined.

Figure 3. Effects of KCl (A) and MgCl2 (B) on the Ca21-binding ratio

of the human hair protein films. (A) The CaCl2 concentration wasconstant at 50 mM in the presence of 0, 100, 300, 500 mM KCl. (B)

The CaCl2 concentration was constant at 50 mM in the presence of

25, 50, 75, 100, 150, 200, 250 mM MgCl2, and then the amounts

bound to the human hair protein film were separately measured forCa21 and Mg21 ions.



Deposition of Calcium Phosphate onto Human HairProtein Film

The alternate soaking procedure is a convenient method for

testing the deposition of inorganic salts. In the case of cal-

cium phosphate, Taguchi et al.12 first reported the combina-

tion of 200 mM calcium chloride and 120 mM disodium

hydrogen phosphate, in which the ratio of the concentra-

tions between the calcium and phosphate ions are 1.67:1.0,

corresponding to those formulated in the crystalline

hydroxyapatite (Ca5(PO4)3OH). The calcium chloride and

disodium hydrogen phosphate solutions were alternately

poured into and removed from the dish, and the dish round

wall was also repeatedly contacted to the both solutions.

The calcium phosphate crystals, however, were not depos-

ited on the dish wall, indicating that calcium phosphate

deposition is accelerated by the presence of the human hair

protein film. The increase in the weight was first tested on

the deposition of calcium phosphate during the alternate

soaking process.

Figure 4 represents the increase in the weight of the

human hair protein films, which are prepared by two kinds

of casting methods: one is the soft postcast method involv-

ing a 50 mM CaCl2 solution for the protein aggregation

step, and the other is the postcast method using the usual

acetate buffer solution. The weight of the film prepared by

the soft postcast method (50 mM CaCl2) increased in pro-

portion to the soaking cycle. On the other hand, the weight

of the film prepared by the postcast method (acetate buffer)

was unchanged. These results suggest that a-keratin mole-

cules in the as-prepared human hair protein film via the

soft postcast method already have bound Ca21 ions, which

can act as a seed stimulating the conjugation of Ca21 and

PO422 complexes from the CaCl2 and Na2HPO4 solutions.

The average amount of deposited calcium phosphate was

140 lg/cm2 per one cycle under the present experimental

conditions.

The inorganic reaction between CaCl2 and Na2HPO4

usually yields multiple compositions of calcium phosphate,

including the amorphous state and potentially five kinds of

crystalline products, such as brushite (CaHPO4 � H2O),

monocalcium dihydrogen phosphate (Ca(H2PO4)2), octacal-

cium phosphate (Ca8H2(PO4)6), and hydroxyapatite

(Ca5(PO4)3OH). An in situ byproduct, HCl, is also gener-

ated in stoichiometric amounts depending on the experi-

mental conditions. The proteins would be possibly

damaged by the acid-mediated hydrolysis and cleaved into

lower molecular weight polypeptides.

To exclude the undesired possibility described earlier,

the SDS-PAGE analysis was next performed to determine

the molecular weight of the human hair proteins in the film

matrix in parallel with the alternate soaking process. The

human hair proteins were redissolved in the Shindai solu-

tion containing 2-ME at 0, 10, 20, and 30 cycles of the

alternate soaking process, and the protein composition

of the soluble fraction was analyzed by SDS-PAGE

(Figure 5). As previously reported by us,5,6 almost all of

the proteins in the soluble extract at cycle 0 are the a-kera-

tin types I and II having molecular masses of 40–50 kDa

and 55–60 kDa, respectively, while the coexisting keratin-

associated proteins were rather minor. As the alternate

soaking process proceeded up to 30 cycles, no significant

Figure 4. Increase in the weight of the human hair protein film dur-

ing the alternate soaking procedure for the deposition of the cal-

cium phosphate. The soft postcast method involves the proteinaggregation step using 50 mM CaCl2, while in the postcast method,

the acetate buffer was used.

Figure 5. SDS-polyacrylamide gel electrophoresis of the a-keratinin the human hair protein films during the alternate soaking proce-

dure for the deposition of calcium phosphate. Numbers on the topof the lanes denote the repetitive cycle number of the alternate

soaking procedure, and the numbers with arrows on the right side

indicate the mobilities of the molecular weight markers. The bands

of the a-keratins (types I and II) are marked on the left side, and nodetectable degradation of the a-keratins was observed.



degradation of the a-keratins was observed. In addition,

a-keratins in the human hair protein films were not

degraded after storage for more than 10 months at room

temperature (data not shown), indicating that the human

hair protein films are stable not only at the morphological

level but also at the molecular level.

The appearance of the as-prepared human hair protein

film via the soft postcast method (50 mM CaCl2) was trans-

lucent and slightly light brown, as the three-letter object

‘‘HAP’’ beyond the film can be clearly seen through the

film (Figure 6, 0 cycle, upper left panel). The fine structure

of the film surface was observed under SEM, and the as-

prepared human hair protein film has a porous structure, in

which the apparent size of each pore is frequently less than

1 lm (Figure 6, 0 cycle, lower left panel). After repeating

the alternate soaking processes five times or more, the

translucent film gradually turned opaque and white, and at

20 cycles, the surface of the human hair protein film was

completely covered with deposited calcium phosphate par-

ticles (Figure 6, 20 cycles, upper right panel). After repeat-

ing the alternate soaking processes 15 times or more, the

size distribution of the calcium phosphate particles at 20

cycles was estimated to be 0.4–2.0 lm (Figure 6, 20

cycles, lower right panel). The opposite side of the film

surface, which contacts the tissue culture dish used as the

substrate, remained unchanged (data not shown) without

any observable deposition of calcium phosphate, indicating

that the CaCl2 and Na2HPO4 solutions did not fully diffuse

through the film. As a result, the binding of CaCl2 and

Na2HPO4 to the a-keratin molecules will occur only on one

side of the human hair protein film.

Characterization of Calcium Phosphate Deposited ontoHuman Hair Protein Film

The relative amounts of the deposited calcium phosphate

after 0, 10, and 20 cycles were determined by FTIR, in

which the absorbance at the P¼¼O stretching vibration

(1036 cm21) was compared, after normalization of the ab-

sorbance at the amide II band (1541 cm21) as 0.1 for all

the spectra (Figure 7). The absorbance at the P¼¼O stretch-

ing vibration increased with the increasing cycle of the

alternate soaking, supporting the results of the weight

increase as seen in Figure 4.

Figure 8 represents the wide-angle X-ray diffraction

(WAXD) patterns of the human hair protein film at the

alternate soaking cycle numbers of 0, 4, 10, 14, and 20.

For the diffraction pattern before the altanate soaking, no

clear diffraction peak was observed, indicating that the ker-

atin molecules in the human hair protein film are not in an

oriented package having a periodic structure. At cycle num-

ber 4, two clear diffraction peaks were detected at 2y 5

4.98 and 2y 5 11.98. The peak at 2y 5 4.98 can be

assigned to the (100) plane in the crystal lattice of the octa-

calcium phosphate and the peak at 2y 5 11.98 can be con-

sidered to be the (020) plane of brushite (CaHPO4 � H2O).

At cycle numbers 10, a diffraction peak at 2y 5 31.98developed as the altanate soaking proceeded. This diffrac-

tion peak corresponded to the (211) plane of hydroxyapa-

tite. The diffraction pattern at cycle number 14 clearly

indicated that the deposited calcium phosphate on the

human hair protein film involved at least four kinds of

crystalline polymorphs such as brushite, octacalcium phos-

phate, and hydroxyapatite. Among them, brushite can stim-

Figure 6. Changes in appearances of the human hair protein films (upper photographs) and surface

morphology (lower SEM images) during the alternate soaking procedure for calcium phosphate deposi-tion. Numbers on the top of the panels indicate the cycle number of the alternate soaking. The human

hair protein film before soaking is transparent as the three-letter object ‘‘HAP’’ can be clearly seen

through the film, and after 20 cycles of soaking, the surface was fully covered with calcium phosphate.

Scale bars: 10 mm for upper photographs; 50 lm for lower SEM images.



ulate the deposition of octacalcium phosphate as the direct

precursor of hydroxyapatite.17 At cycle number 20, the

peak at 2y 5 31.98 became the predominant component of

the entire diffraction pattern, indicating that hydroxyapatite

is the major polymorph of the calcium phosphate deposited

onto the human hair protein film.

DISCUSSION

This study proposed the utilization of the human hair pro-

tein film as a biomineral scaffold. The properties of the

human hair protein film were examined on the basis of the

common characteristics as the biomineral-scaffolding mate-

rials, that is, (i) calcium ion-binding ability, (ii) chemical

stability during deposition of calcium phosphate, and (iii)

reproduction of in vivo process of hydroxyapatite formation.

Ca21-Binding Ability

The Ca21 ion binding ability of the a-keratin in the human

hair protein films have been confirmed (Figures 1 and 2).

However, Ca21 ion selectivity of the human hair protein

film was not obviously shown from the experimental data

(Figure 3). On the other hand, assuming the practical use

of the human hair protein for implantation, for example,

during surgery, Ca21 ions can be adsorbed on the human

hair protein film before the surgical operations, and the pre-

adsorbed Ca21 is expected to act as a seed for the precipi-

tation of amorphous calcium phosphate from the body

fluid.

The metal cation uptake properties of wool and related

materials have been concisely summarized by Masri and

Friedman.23 According to their description, native wool is

capable of binding 0.13 mole Mg21/gram wool. This value

roughly corresponds to 2.8–3.1 mole Mg21/mole a-keratin,

assuming that microfibrils occupy 50% of the wool fiber

structure.24 This calculation is quite coincident with the

present results (Figure 3) in the case of the human hair a-

keratins. Together with a high homology between the

amino acid sequences of wool keratin and human hair a-

keratins (types I and II),2,25 the divalent metal ion-binding

ability of human hair a-keratins can be explained by the

same mechanism proposed by Masri and Friedman.23 The

human hair protein film can actually interact with Ca21

ions in a relatively higher ratio than Mg21; therefore, this

material was next subjected to the deposition of calcium

phosphate to examine the human hair protein film as a

scaffold for hard tissue development.

Chemical Stability during Deposition of CalciumPhosphate

The chemical stability of the a-keratin molecules as the

major component of the human hair protein film has been

Figure 7. Fourier-transform infrared spectra of the human hair pro-

tein films before (solid line) and after 10 (broken line) and 20 (dottedline) cycles of the alternative soaking procedure. All spectra were

recorded using the KBr pellet preparations of the samples. The ab-

sorbance were normalized as 0.1 at the peak frequency of the am-

ide II bands (1541 cm21) to compare the relative amounts ofphosphate ion as seen at 1036 cm21, which were deposited onto

human hair protein film as calcium phosphate.

Figure 8. Transition of wide-angle X-ray diffraction patterns of the

human hair protein films during the alternative soaking procedure

for the deposition of the calcium phosphate. The numbers on theright side indicate the cycle numbers of the alternate soaking. The

‘‘HAP’’ on right side denotes the authentic hydroxyapatite sample.

The (hkl) indexes were indicated on the top. OCP, octacalcium

phosphate; DCPD, brushite (calcium hydrogenphosphate dihydrate);HAP, hydroxylapatite.



confirmed by no detectable hydrolysis or undesired degra-

dation during the calcium phosphate deposition (Figure 5).

Furthermore, as for the polymer chemical stability of a-

keratin molecules, the polypeptide chain conformation of

a-keratin can be discussed with the FTIR spectra. At the

protein-specific absorption region, the amide I and II bands

of the human hair protein film, the peak of amide I was

observed at 1649 cm21 before and after alternate soaking

(Figure 7).

According to Bendit, the amide I bands of the nonhy-

drated a-keratin from horse hair appeared at 1642 and 1648

cm21, respectively, for those parallel and perpendicular to

the fiber axis, and the peak of the amide I band shifts to

1655–1656 cm21 because of the swelling of the horse hair

fiber.26 Therefore, the amide I peak of the human hair pro-

tein film indicates that the film did not absorb water after

the calcium phosphate deposition. The amide II peak of the

human hair protein film was observed at 1541 cm21, and

there was no observable peak shift before and after the cal-

cium phosphate deposition. The relationships between the

peak frequencies of the amide I and II bands and the chain

conformation have been thoroughly investigated, and

according to Miyazawa and Blout, the observed and calcu-

lated frequencies of the a-helical polypeptides are well

matched at 1650–1652 cm21 for the amide I band and at

1540–1546 cm21 for the amide II band (polarized IR, per-

pendicular to the helical axis).27 The observed peak fre-

quencies of the amide I and II bands of the human hair

protein film suggest that the a-keratin molecules are partly

in an a-helical chain conformation and also that a disor-

dered chain conformation coexists.

Reproduction of In Vivo Process of HydroxyapatiteFormation

The WAXD spectra with increasing number of the alternate

soaking cycle (Figure 8) clearly indicated that hydroxyapa-

tite crystals were formed from the precursor polymorph,

octacalcium phosphate, with the coexisting brushite. The

first appeared crystalline phase, brushite, can promote the

deposition of the second crystalline phase, octacalcium

phosphate,17 which is the precursor polymorph of the bio-

logical hydroxyapatite.18 Therefore, the observed crystalline

phase transition of the calcium phosphate deposited onto

the human hair protein film is almost same as the in vivoprocess of hydroxyapatite formation.

CONCLUSION

The present human hair protein film satisfied above three

characteristics (i)–(iii), which are required for the utiliza-

tion as the biomineral-scaffolding material. Additional mer-

its of the human hair protein film are as follows: (iv) intact

proteinaceous material without any types of chemical modi-

fication, (v) use as a ‘‘self-originated’’ material, which may

avoid an undesirable immune reaction, if the human hair

protein film has been prepared from a certain individual

and implanted into the same person, and (vi) totally biode-

gradable material. In particular, the expected property (v) is

achieved only by use of the human hair protein and distin-

guished from other sources of a-keratin, such as wools.28

In the practical use of the human hair protein film in the

surgical operation, the alternate soaking process to deposit

the calcium phosphate crystal cannot be employed. Alterna-

tively, one possible design of the working materials is the

immobilization of the osteoinductive factors, such as bone

morphogenic protein (BMP)-2, onto the porous and water-

insoluble human hair protein film surface, together with b-

tricalcium phosphate (TCP). Abarrategi et al. reported that

the BMP-2-b-TCP-coated chitosan film stimulates in vivonew bone formation.29 By means of this approach, the pres-

ent human hair protein film can be modified as a practical

material for implant-use in hard tissue engineering. The

synthetic phosphorylated polypeptides30 are also expected

as the candidates for immobilized osteoinductive factors,

which can be applied as a composite of the human hair

protein film.

As for another possible therapeutic procedure, Akashi

and coworkers reported on osteoconductive and hemostatic

properties of a HAP/agarose gel composite, which under-

went alternate soaking before the surgical operation.31,32

Implantation of the HAP/agarose gel into the surgically

created infrabony periodontal defects on dog jawbones pro-

moted the regeneration of bone.31 The HAP/agarose gel is

also effective for the regeneration of bony defects in mon-

key jawbones.32 These facts inspire us to consider that a

pretreatment of the human hair protein film by means of

alternate soaking and subsequent implantation into bone

defect is one of the most acceptable therapeutic procedures

in clinical use. In conclusion, the human hair protein

film is rather promising for utilization as a biomineral-

scaffolding material.

The authors are indebted to the Division of Gene

Research Center, Research Center for Human and Environ-

mental Science, Shinshu University, for allowing the use of

their facilities.

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