Keratin saçın en önemli yapı taşıdır. Saçın iç lifleri ve dış kılıfı keratin yapısındadır. Keratin vücutta sentezlendiği gibi ağız yoluyla da alınmaktadır. Uygun dozda alınan keratin saç kalitesini artırır, saçın uzamasını hızlandırır, saç kırıklarını engeller, saçta solma ve çökme gibi durumlara engel olur. Saç dökülmesini engelleme etkisi olmasa da saçın uzama dönemi olan anageni uzatır.
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Bilimsel yayın 1
A Review of Keratin-Based Biomaterials for Biomedical Applications
Jillian G. Rouse and Mark E. Van Dyke *
Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston-Salem, North
Carolina, USA; E-Mail: jrouse@wfubmc.edu (J.G.R.)
* Author to whom correspondence should be addressed; E-Mail: mavandyk@wfubmc.edu;
Tel.: +1-336-713-7266; Fax: +1-336-713-7290.
Received: 6 January 2009; in revised form: 24 January 2010 / Accepted: 26 January 2010 /
Published: 3 February 2010
Abstract: Advances in the extraction, purification, and characterization of keratin proteins
from hair and wool fibers over the past century have led to the development of a keratinbased
biomaterials platform. Like many naturally-derived biomolecules, keratins have
intrinsic biological activity and biocompatibility. In addition, extracted keratins are capable
of forming self-assembled structures that regulate cellular recognition and behavior. These
qualities have led to the development of keratin biomaterials with applications in wound
healing, drug delivery, tissue engineering, trauma and medical devices. This review
discusses the history of keratin research and the advancement of keratin biomaterials for
biomedical applications.
Keywords: keratin; human hair protein; natural biomaterial; protein film; scaffold
1. Introduction
One of the primary goals of biomaterials research is the development of a matrix or scaffolding
system that mimics the structure and function of native tissue. For this purpose, many researchers have
explored the use of natural macromolecules due to their intrinsic ability to perform very specific
biochemical, mechanical and structural roles. In particular, protein-based biomaterials have emerged as
potential candidates for many biomedical and biotechnological applications due their ability to
function as a synthetic extracellular matrix that facilitates cell-cell and cell-matrix interactions. Such
substrates contain a defined, three-dimensional microstructure that supports cellular proliferation and
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cell-guided tissue formation, both of which are important characteristics for biomaterial scaffolds. In
addition, the strong bioactivities and diverse physiochemical properties of proteinaceous
macromolecules are attractive for other biomedical applications for which biocompatibility is essential,
such as medical devices, bioactive surfaces, hygiene products, etc.
Several proteins have been investigated in the development of naturally-derived biomaterials,
including collagen, albumin, gelatin, fibroin and keratin. Of these, keratin-based materials have shown
promise for revolutionizing the biomaterial world due to their intrinsic biocompatibility,
biodegradability, mechanical durability, and natural abundance. This review focuses on the history of
keratin research and the development of keratin-based biomaterials for biomedical applications. A
brief review of keratin biology is also discussed with an emphasis on how the proteins are developed
within the hair fiber.
2. Keratin Biology
The term “keratin” originally referred to the broad category of insoluble proteins that associate as
intermediate filaments (IFs) and form the bulk of cytoplasmic epithelia and epidermal appendageal
structures (i.e., hair, wool, horns, hooves and nails). Subsequent research of these structural proteins
led to the classification of mammalian keratins into two distinct groups based on their structure,
function and regulation. “Hard” keratins form ordered arrays of IFs embedded in a matrix of cystinerich
proteins and contribute to the tough structure of epidermal appendages. “Soft” keratins
preferentially form loosely-packed bundles of cytoplasmic IFs and endow mechanical resilience to
epithelial cells [1−3]. In 2006, Schweizer et al. [4] developed a new consensus nomenclature for hard
and soft keratins to accommodate the functional genes and pseudogenes for the full complement of
human keratins. This system classifies the 54 functional keratin genes as either epithelial or hair
keratins. The structural subunits of both epithelial and hair keratins are two chains of differing
molecular weight and composition (designated types I and II) that each contain non-helical endterminal
domains and a highly-conserved, central alpha-helical domain. The type I (acidic) and type II
(neutral-basic) keratin chains interact to form heterodimers, which in turn further polymerize to form
10-nm intermediate filaments. Although hard and soft keratins have closely related secondary
structures, distinct differences in amino acid sequences contribute to measurable differences between
the filamentous structures. Most notably, hair keratins contain a much higher content of cysteine
residues in their non-helical domains and thus form tougher and more durable structures via
intermolecular disulfide bond formation [2,5,6].
2.1. Hair Keratins
Hair fibers are elongated keratinized structures that are composed of heavily crosslinked hard
keratins. Each fiber is divided into three principle compartments: the cuticle, cortex, and medulla. The
thin outer surface of the fiber, the cuticle, is a scaly tubular layer that consists of over-lapping flattened
cells. The cuticle primarily contains beta-keratins that function to protect the hair fiber from physical
and chemical damage. The major body of the hair fiber is referred to as the cortex, which is composed
of many spindle-shaped cells that contain keratin filaments. Occasionally, in the very center of the hair
fiber is a region called the medulla that consists of a column of loosely connected keratinized cells [7].
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Within the cortex of the hair fiber are two main groups of proteins: (1) low-sulfur, “alpha” keratins
(MW 40−60 kDa) and (2) high-sulfur, matrix proteins (MW 10−25 kDa). Collectively, the hair fiber
consists of 50−60% alpha keratins and 20−30% matrix proteins [7]. The alpha keratins assemble
together to form microfibrous structures known as keratin intermediate filaments (KIFs) that impart
toughness to the hair fiber. The matrix proteins function primarily as a disulfide crosslinker or glue that
holds the cortical superstructure together and are also termed keratin associated proteins or KAPs [4].
In total, there are 17 human hair keratin genes (11 type I; 6 type II) [4] and more than 85 KAP genes
[8] that potentially contribute to the hair structure in humans.
2.2. Development of Hair Keratins
Hair morphogenesis begins in a proliferative compartment at the base of the hair follicle called the
bulb. Within this region, cells divide and differentiate to form the various compartments of the hair
follicle. The hair follicle is a cyclic regeneration system comprised of actively migrating and
differentiating stem cells responsible for the formation and growth of hair fibers. The follicle
undergoes a continuous cycle of proliferation (anagen), regression (catagen), and quiescence (telogen)
that is regulated by over thirty growth factors, cytokines and signaling molecules [9,10]. The mature
anagen hair follicle contains a concentric series of cell sheaths, the outermost of which is called the
outer root sheath (ORS), followed by a single cell layer called the companion sheath. The inner root
sheath (IRS) lies adjacent to the companion layer and consists of three compartments: the Henle layer,
the Huxley layer, and the IRS cuticle. The hair fiber fills the center of this multilayered cylinder, which
is itself divided into cuticle, cortex and medulla [8−10]. As cells within the hair shaft terminally
differentiate, they extrude their organelles and become tightly packed with keratin filaments. The
cysteine-rich keratins become physically crosslinked upon exposure to oxygen and give strength and
flexibility to the hair shaft [10].
Keratin genes have complex, differential, and in many cases sequential expression patterns within
the cuticle and cortex of the hair follicle [5,11−14]. For example, only a few keratins are expressed in
the hair-forming matrix of the cortex and cuticle, whereas others are sequentially switched on upon
differentiation in the lower cortex. The bulk of keratins are expressed in the middle cortex
(“keratinizing zone”) of the ascending hair fiber. Other keratin expressions are restricted to the hair
cuticle and are sequentially expressed during hair morphogenesis [5,13]. The highly regulated
expression pattern of keratins during hair morphogenesis is indicative of the functional differences
between acidic and basic keratins, although this relationship is not yet fully understood [11,12].
3. History of Keratin Biomaterials
3.1. Early Uses of Keratins
The earliest documented use of keratins for medicinal applications comes from a Chinese herbalist
named Li Shi-Zhen in the 16th century. Over a 38-year period, Shi-Zhen wrote a collection of 800
books known as the Ben Cao Gang Mu that describe more than 11,000 therapeutic prescriptions.
Among them is a substance made of ground ash from pyrolized human hair that was used to accelerate
wound healing and blood clotting called Xue Yu Tan, also known as Crinis Carbonisatus. Although
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the details about the discovery of the biological activity of human hair are not reported in great detail,
its uses for medicinal purposes are clearly documented [15].
The word “keratin” first appears in the literature around 1850 to describe the material that made up
hard tissues such as animal horns and hooves (keratin comes from the Greek “kera” meaning horn). At
the time, keratins intrigued scientists because they did not behave like other proteins. In particular,
normal methods for dissolving proteins were ineffective for solubilizing keratin. Although methods
such as burning and grinding had been known for some time, many scientists and inventors were more
interested in dissolving hair and horns in order to make better products. The resolution to the
insolubility problem came in 1905 with the issue of a United States patent to John Hoffmeier that
described a process for extracting keratins from animal horns using lime. The extracted keratins were
used to make keratin-based gels that could be strengthened by adding formaldehyde [16].
During the years from 1905 to 1935, many methods were developed to extract keratins using
oxidative and reductive chemistries [17−22]. These technologies were initially applied to animal horns
and hooves, but were also eventually used to extract keratins from wool and human hair. The
biological properties of the extracts led to increased interest in the development of keratins for medical
applications, and among the first inventions were keratin powders for cosmetics, composites, and
coatings for drugs [23−25].
During the 1920s, keratin research changed its focus from products made from keratin to the
structure and function of keratin proteins. Several key papers were published that analyzed oxidatively
and reductively extracted keratins [21,22]. These scientists soon concluded that many different forms
of keratin were present in these extracts, and that the hair fiber must be a complex structure, not simply
a strand of protein. In 1934, a key research paper was published that described different types of
keratins, distinguished primarily by having different molecular weights [22]. This seminal paper
demonstrated that there were many different keratin homologs, and that each played a different role in
the structure and function of the hair follicle.
3.2. Keratin Research from 1940−1970
It was during the years of World War II and immediately after that one of the most comprehensive
research projects on the structure and chemistry of hair fibers was undertaken. Driven by the
commercialization of synthetic fibers such as Nylon and polyester, Australian scientists were charged
with protecting the country’s huge wool industry. Synthetic fibers were seen as a threat to Australia’s
dominance in wool production, and the Council for Scientific and Industrial Research (later the
Commonwealth Scientific and Industrial Research Organisation or CSIRO) established the Division of
Protein Chemistry in 1940. The goal of this fundamental research was to better understand the
structure and chemistry of fibers so that the potential applications of wool and keratins could be
expanded. Earlier work at the University of Leeds and the Wool Industries Research Association in the
UK had shown that wool and other fibers were made up of an outer cuticle and a central cortex.
Building on this information, scientists at CSIRO conducted many of the most fundamental studies on
the structure and composition of wool. Using X-ray diffraction and electron microscopy, combined
with oxidative and reductive chemical methods, CSIRO produced the first complete diagram of a hair
fiber [26].
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CSIRO scientists also conducted extensive studies on the wool proteins themselves. Many methods
for the extraction, separation, and identification of these keratins were developed. Other fundamental
studies included wool surface chemistry, processing of products, fellmongering (harvesting of wool
from sheep), felting, carbonising, surface treatments, flammability, denaturation, chemical
modification, dyeing, photochemical degradation, and application of polymers to wool. This
monumental effort was conducted over a period of more than 30 years and resulted in over 660
publications, 20 patents, and three books. In the meantime, the use of oxidative and reductive
chemistry to extract keratins from hair fibers was being applied by other scientists across the world. In
The Netherlands, researchers patented a method for making films and textile fibers from reductively
extracted keratins from ground up hooves [27].
Probably nowhere in the world was keratin research more active than in Japan. Between the years
of 1940 and 1970, applications for keratin-based inventions submitted to the Japanese patent office
numbered more than 700. This was a renaissance in keratin research that was trending toward the
fundamentals of materials science and biomaterials. Driven by the development of reliable methods to
solubilize keratins, researchers were beginning to understand the many sub-classes of keratins, and
their different properties [28−32]. In 1965, CSIRO scientist W. Gordon Crewther and his colleagues
published the definitive text on the chemistry of keratins [7]. This chapter in Advances in Protein
Chemistry contained references to more than 640 published studies on keratins.
3.3. Keratin Research from 1970-Present
Advances in the extraction, purification and characterization of keratins, led to the exponential
growth of keratin materials and their derivatives. In the 1970s, methods to form extracted keratins into
powders, films, gels, coatings, fibers, and foams were developed and published by several research
groups [33−35]. All of these methods made use of the oxidative and reductive chemistries developed
decades earlier, or variations thereof.
The prospect of using keratin as a biomaterial in medical applications was obvious. During the
1980s, collagen became a commonly used biomolecule in many medical applications. Other naturally
derived molecules soon followed such as alginates from seaweed, chitosan from shrimp shells, and
hyaluronic acid from animal tissues. The potential uses of keratins in similar applications began to be
explored by a number of scientists. In 1982, Japanese scientist published the first study describing the
use of a keratin coating on vascular grafts as a way to eliminate blood clotting [36], as well as
experiments on the biocompatibility of keratins [37]. Soon thereafter in 1985, two researchers from the
UK published a review article speculating on the prospect of using keratin as the building block for
new biomaterials development [38]. In 1993, a Japanese scientists published a commentary on the
prominent position keratins could take at the forefront of biomaterials development [39].
4. Keratin Biomaterials
The solid foundation for keratin research led to the development of many keratin-based biomaterials
for use in biomedical applications. This foundation is based on several key properties of keratins that
contribute to the overall physical, chemical and biological behavior of these biomaterials. First,
extracted keratin proteins have an intrinsic ability to self-assemble and polymerize into porous, fibrous
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scaffolds. The spontaneous self-assembly of keratin solutions has been studied extensively at both the
microscale [40−42] and macroscale levels [43]. This phenomenon of self-assembly is evident in the
highly conserved superstructure of the hair fiber and, when processed correctly, is responsible for the
reproducible architecture, dimensionality and porosity of keratin-based materials. In addition, keratin
biomaterials derived from wool and human hair have been shown to possess cell binding motifs, such
as leucine-aspartic acid-valine (LDV) and glutamic acid-aspartic acid-serine (EDS) binding residues,
which are capable of supporting cellular attachment [44,45]. Together, these properties create a
favorable three dimensional matrix that allows for cellular infiltration, attachment and proliferation.
Like other intermediate filaments, keratins are also believed to participate in some regulatory functions
that mediate cellular behavior [46,47]. Thus, the conservation of biological activity within regenerated
keratin biomaterials could prove advantageous for the control of specific biological functions in a
variety of tissue engineering applications.
The enhanced physical, chemical and biological properties of keratins as well as the desire to
exploit wool and human hair fibers as a renewable natural resource have fueled keratin biomaterials
research over the past three decades. Much has been done to both fabricate and characterize new
keratin-based products such as films, sponges, scaffolds and fibers. In many cases, these novel keratin
materials have been shown to possess excellent biocompatibility. In addition, many researchers have
discovered methods for modulating the physical and mechanical properties of keratins in order to
create biomaterials that have appropriate characteristics for their application of interest.
4.1. Keratin Films
The preparation of protein films from keratin extracted from wool and human hair has been used for
a number of years to explore the structural and biological properties of self-assembled keratins.
Yamauchi et al. [48] were among the first to begin to investigate the properties of products made from
extracted wool keratins and in doing so described the physiochemical and biodegradational properties
of solvent-cast keratin films. Although pure keratin films were too fragile for practical use, the addition
of glycerol resulted in a transparent, relatively strong, flexible, and biodegradable film [48]. In an
additional publication, Yamauchi et al. [49] described the cell compatibility of this film by cultivation
of mouse fibroblasts on the surface. When compared to the growth of cells on collagen and glass, the
keratin substrate proved to be more adhesive to the cells and more supportive of cellular proliferation
[49]. Fujii et al. [50] also demonstrated that hair keratins were useful for preparing protein films and
described a rapid casting method. This research also revealed the feasibility of incorporating such
bioactive molecules as alkaline phosphatase into the keratin films for controlled-release applications.
The films, however, had poor strength and flexibility [50]. Together these early studies demonstrated
the feasibility of preparing keratin films and demonstrated their potential for use as biomaterials in
medical applications.
Like many natural-derived biomaterials, however, the practical use of keratin-based products was
ultimately limited by their poor mechanical characteristics. Thus, keratin film research shifted to focus
on the optimization of the physical strength and flexibility of films while maintaining their excellent
biological activity. Several approaches for controlling the physical and biological properties have been
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considered, including the addition of natural [51−56] and synthetic [57,58] polymers to keratinblended
systems and new preparation techniques for pure keratin films [59,60].
In 2002, Yamauchi’s group enhanced the mechanical properties of their glycerol containing keratin
films by the addition of chitosan. Chitosan is a well investigated biomolecule for biomaterial
applications, and is known to possess high biocompatibility and biological function for wound healing
and antibacterial activity. Addition of chitosan into the keratin films resulted in improved mechanical
strength. Furthermore, the chitosan-keratin films also demonstrated antibacterial properties and were
shown to be good substrates for cell culture [51]. The biological activity of keratin films was also
increased by incorporating a cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), at the free cysteine
residues of reduced keratin extracts. RGDS-carrying keratin films proved to be excellent substrates for
mammalian cell growth, and this work again demonstrated the potential and versatility of keratin
biomaterials [52].
Silk fibroin (SF) is another natural polymer that has received much attention as a biomaterial due to
its intrinsic biocompatibility and biodegradability. Keratin-SF films have been studied extensively in
order to understand the interactions that occur between the two biomolecules and how they relate to
the overall mechanical and biological characteristics of the biomaterial. Lee et al. [53] studied the
secondary structure of keratin-SF films and observed a transition from random coil to β-sheet structure
for fibroin due to the presence of the polar amino acids present in keratin. These blended films were
shown to have enhanced antithrombogenicity properties and increased biocompatibility in comparison
to SF or keratin only films [54], most likely due to the enhanced surface polarity of the blends
generated by the conformational transformation of the proteins [55]. Vasconcelos et al. [56] further
explored the mechanical and degradation properties of keratin-SF blended films and concluded that SF
and keratin interactions are not simply additive but rather the two proteins are capable of unique
intermolecular interactions that directly affect the bulk properties of the films. Ultimately, the nature
and strength of these interactions and knowledge of the degradation rates will allow for the design of
matrices for release of active compounds that are suitable for future biomedical applications [56].
In addition to natural biopolymers, the interaction between keratin and synthetic polymers has also
been studied [57,58]. Tonin et al. [57] explored the relationship between poly(ethylene oxide) (PEO)
and keratin blended films in order to develop a keratin-based material with improved structural
properties. Morphological, structural and thermal analyses of the keratin/PEO films revealed that at
appropriate levels, keratin inhibits PEO crystallization and PEO interferes with the keratin selfassembly,
inducing a more thermally-stable, β-sheet secondary protein structure. The improved
structural properties of keratin/PEO blends enables the development of keratin materials for use as
scaffolds for cell growth, wound dressings and drug delivery membranes [57]. The intermolecular
interactions between keratin and polyamide 6 (PA6) have also been studied with the goal of creating
keratin-based materials that have practical use for a wide variety of applications ranging from
biomedical devices to active water filtration and textile fibers [58].
In addition to creating blended keratin systems with natural or synthetic polymers, researchers have
also investigated alternative fabrication techniques for creating keratin films with more suitable
mechanical properties. Katoh et al. [60] reported an alternative method for processing keratin films to
overcome the limited versatility associated with solution-cast methods. Compression molding of Ssulfo
keratin powder proved to be an effective technique for producing pure keratin films of distinct
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shape. Control of the mechanical properties of the films was obtained by controlling the molding
temperature and water content of the film, and the biocompatibility of the S-sulfo films was also
demonstrated by fibroblast attachment and proliferation on the keratin substrates [60]. In a separate
study, an improved procedure for preparing pure keratin films with translucent and flexible properties
was reported, and the practical application and compatibility of the films were demonstrated by testing
their compatibility with human skin [59].
Recently, Reichl et al. [61] characterized two different approaches for substrate coatings and
demonstrated the growth behavior of twelve different cell lines cultured on the keratin films. Results
showed that growth substrates formed by casting of a keratin nanosuspension supported cell adherence
and improved cell growth as compared to uncoated polystyrene or keratin coatings formed by
trichloroacetic acid precipitation. The new approach is believed to be a low cost, standardized
alternative to commonly used coatings such as collagen and fibronectin [61].
4.2. Keratin Sponges and Scaffolds
The ability of extracted keratin proteins to self-assemble and polymerize into complex three
dimensional structures has led to their development as scaffolds for tissue engineering. Fabrication of
wool keratin scaffolds for long term cell cultivation was first reported by Tachibana et al. [44] in 2001.
The matrices were created by lyophilization of aqueous wool keratin solutions after controlled
freezing, which resulted in a rigid and heat-stable structure with a homogenously porous microarchitecture.
The keratins, which were shown to contain RGD and LDV cell adhesion sequences,
exhibited good cell compatibility and supported the attachment and proliferation of fibroblasts over a
long-term cultivation period of 23−43 days. In addition, the free cysteine residues present within the
scaffold were shown to be potential modification sites for the immobilization of bioactive substances
[44]. In later work, lysozyme was used as a model compound and linked to the keratin sponge via
disulfide and thioether bonds. Disulfide-linked lysozyme was gradually released over a 21-day period
whereas lysozyme linked via thioether bonds was stably maintained for up to two months. This work
demonstrated that the selection of a chemical crosslinker can uniquely determine the stability of an
immobilized bioactive substance on keratin sponges [62].
Functionalization of active free thiol in the keratin sponges using various chemical treatments has
also been demonstrated using iodoacetic acid, 2-bromoethylamine, and iodoacetamide to produce
carboxyl-, amino-, and amido-sponges, respectively. These chemically-modified keratin sponges have
been shown to mimic extracellular matrix proteins, and the large presence of active groups within the
sponges has allowed for further hybridization with bioactive molecules. This technique was
demonstrated by Tachibana et al. [63] in 2005 with the hybridization of keratin sponges with calcium
phosphate. Two types of calcium phosphate composite sponges were fabricated by either chemically
binding calcium and phosphate ions or trapping hydroxyapatite particles within the keratin carboxysponges.
Both hybridized materials supported osteoblast cultivation and altered their differentiation
pattern based on the expression pattern of alkaline phosphatase [63]. Keratin carboxy-sponges have
also been functionalized with bone morphogenetic protein-2 (BMP-2), which was shown to associate
tightly within the keratin sponge and to localize the differentiation of preosteoblasts grown with the
construct. Cells outside of the BMP-2-loaded construct did not differentiate, suggesting that no
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significant amount of BMP-2 leaked out and that the effects were confined inside the modified keratin
sponge. These findings are significant for in vivo applications because it is expected that the use of
these scaffolds will promote internal osteogenesis while avoiding external heterotopic
ossification [64].
Regulation of pore size and porosity of keratin scaffolds was achieved by Katoh et al. [65] using a
compression molding/particulate leaching (CM/PL) technique. The ability to regulate the pore
diameter and interconnectivity of scaffolds for tissue engineering applications is desired for allowing
adequate cellular infiltration and nutrient delivery. In addition to having regulated pore size, scaffolds
created using the CM/PL method were water tolerable, which presents significant superiority over
collagen materials that are soluble in water without the use of UV irradiation or cytotoxic chemical
crosslinkers [65].
The in vivo biodegradation of keratin bars was explored by Peplow et al. [66] in order to establish a
relationship between mass and physical strength. Rectangular bars of reconstituted keratins were
subcutaneously implanted into adult rats, and dry weight and elastic modulus of the explanted bars
were monitored over an 18-week time period. The dry weight of the bars decreased gradually with a
maximum weight degradation of 22% at 18 weeks. The elastic modulus of the keratin bars decreased
abruptly between 3 and 6 weeks accompanied by an increase in the number of fissures and cavitations
at the surface of the bars. This gradual degradation and quick loss of mechanical integrity are
indications that this form of keratin is more suited as a resorbable implant material to provide
scaffolding for non-load bearing applications [66].
The construction, characterization and cytocompatibility of human hair protein scaffolds for in vitro
tissue engineering applications has recently been reported by Verma et al. [45]. Keratin proteins
extracted from hair were fabricated into porous sponges via lyophilization of frozen protein
suspensions. Characterization of the sponges was performed using swelling experiments and
morphological assessments made by scanning electron microscopy (SEM), which showed that the
sponges were capable of swelling 48% within a period of 60 minutes and that the sponge surface had
an average pore diameter of 150 μm. The interconnectivity and pore diameters supported cell
attachment and survival. The authors suggest that these scaffolds are prospective materials for tissue
engineering applications due to their human origin, biodegradability and cytocompatibility [45].
4.3. Keratin Fibers
In recent years, research on the electrospinning of biocompatible polymeric materials has greatly
increased due to the abundance of potential biomedical applications for nanofibrous materials.
Electrospinning is a technique that utilizes a high voltage to create an electrically charged jet of
polymer that is drawn toward a grounded collection plate or mandrel. The resulting fibers have
diameters in the nano- to micro-scale range and are randomly arranged to form a non-woven fibrous
mat. The enhanced physical configuration (i.e., small pore size, high porosity, three-dimensional
features, and high surface area-to-volume ratio) of nanostructured nonwovens promotes cell adhesion
and growth, which has led to the development of electrospun membranes for such uses as bandages for
wound healing and scaffolds for tissue engineering. Recently, the electrospinning process has also
been extended to include regenerated keratin extracted from hair and wool fibers. Due to the
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intrinsically poor mechanical characteristics of pure keratin, however, many researchers have resorted
to the addition of synthetic or natural polymers in order to increase the processability of keratin for
fiber formation. Much work has been done to characterize the intermolecular interactions between the
keratin and “additive” macromolecule in order to correlate the properties of the blend solution to the
properties of the electrospun fibers.
Aluigi et al. [67,68] created keratin/PEO materials by combining aqueous keratin solutions and
PEO powder. In the first of two studies, the investigators identified the electrospinning parameters to
create defect-free fibrous materials. Blended solutions with a keratin/PEO weight ratio of 50:50 and
7% and 10% total polymer concentrations were shown to have sufficient viscosities to electrospin with
few defects. Spectroscopic and thermal analyses indicated that the electrospinning process destabilized
the natural self-assembly of keratin and promoted a less complex protein conformation [67]. In further
work, keratin and PEO were combined in different proportions in order to correlate the chemical,
physical, and rheological properties of the blend solutions with the morphological, structural, thermal
and mechanical properties of the electrospun mats. The keratin/PEO solutions were shown to have
increased viscosities in comparison to both pure PEO and keratin, and the blends exhibited a non-
Newtonian flow behavior with strong shear-thinning properties that were dependent on PEO
concentration. The low viscosity of blends with higher keratin content greatly hindered their ability to
form fibers; however, solutions with a lower composition of keratin were successfully electrospun
without defects. Comparisons between actual and theoretical rheological properties using Graessley’s
theory showed that the broadening of molecular weight distribution and possible bonding between
PEO and keratin macromolecules at certain keratin/PEO ratios are responsible for the shear viscosity
behavior of the blends, which ultimately correlate with the morphology of the electrospun fibers [69].
The practical uses of the keratin/PEO nanofibrous mats, however, were ultimately limited by their
water instability and poor mechanical properties [68].
Fibroin regenerated from silk has also been used to improve the processability of keratin for
electrospinning applications [70]. Characterizations of the rheological behavior of keratin/fibroin
solutions revealed macromolecule interactions that promoted the formation of network structures with
maximum synergy at a 50/50 (w/w) blend ratio. At this ratio, the synergistic effects on the protein
interactions resulted in the formation of smaller-diameter, finer nanofibers as compared to fibers
formed using solutions of unequal ratios of keratin/fibroin. Conformational analyses confirmed the
prevalence of β-sheet secondary structure in keratin/fibroin films except at the 50/50 blend in which
the proteins showed a propensity to assemble in the α-helix-coiled structure. On the contrary, the
electrospinning process was shown to induce changes in secondary structure at all blend ratios by
preventing β-sheet formation and promoting a random coil or α-form structure. In addition, the α-
crystallites formed by electrospinning were shown to be less thermally stable, most likely due to the
high rate of fiber formation that limits the molecular rearrangement and crystallization of the keratin
chains [70].
Wet-spinning is another fiber-forming technique that has traditionally been used for manufacturing
synthetic fibers for the textile industry, but has recently been employed to create single fiber
biomaterials. This method involves extrusion of a dope solution through a spinneret into a coagulation
bath and subsequent drawing/stretching to promote polymer chain alignment and fiber formation. The
physical limitations of keratin materials have hindered the production of pure keratin fibers, yet
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researchers have overcome these challenges using blends of synthetic and natural polymers with
improved material properties.
Katoh et al. [71] improved upon the fiber-forming capabilities of aqueous keratin solutions using
poly-(vinyl alcohol) (PVA). PVA acted to increase the viscosity of the spinning dope, which allowed
fibers with a keratin content ranging from 13−46% to be spun. Due to the fragility of fibers with high
amounts of keratin, the maximum keratin content for sufficient fiber formation was determined to be
30%. This combination of keratin and PVA proved to be advantageous in terms of mechanical
strength, waterproof characteristics, and the adsorption of toxic substances. According to the authors,
keratin-PVA fibers are expected to have wide-spread industrial applications as absorbents for toxic
substances such as heavy metals ions and formaldehyde gas [71].
Wrzesniewska-Toski et al. [72] also employed wet-spinning techniques to create novel fibrous
keratin-based materials that have potential application as hygienic fabrics. Keratin extracted from
chicken feathers and bio-modified cellulose were combined and used to create fibers that were
characterized as having better sorption properties, higher hygroscopicity, and a smaller wetting angle
than cellulose-only fibers. Although introduction of keratin into cellulose fibers decreased the
mechanical properties, a level was achieved that still enabled their application for manufacturing
composite fibrous materials. In addition, the cellulose-keratin fibers had better biodegradation than
cellulose fibers.
5. Keratin Biomaterials in Tissue Engineering and Regenerative Medicine
Much work has been done to fabricate and characterize keratin-based materials and to demonstrate
their cytocompatibility and biodegradation. Until recently, however, few of these biomaterial
developments had been applied in models of tissue regeneration.
Sierpinski et al. [73] and Apel et al. [74] demonstrated that keratin-based hydrogels were
neuroinductive and capable of facilitating regeneration in a peripheral nerve injury model in mice.
Human hair keratins enhanced the in vitro activity of Schwann cells by inducing cellular proliferation
and migration, and by upregulating expression of specific genes required for important neuronal
functions. When translated into a mouse tibial nerve injury model, keratin gel-filled conduits served as
a neuroinductive provisional matrix that mediated axon regeneration and improved functional recovery
compared to sensory nerve autografts [73]. In another study, the time course of peripheral nerve
regeneration was evaluated with respect to neuromuscular recovery and nerve histomorphometry.
Keratin-filled hydrogels were shown to accelerate nerve regeneration as evidenced by improved
electrophysiological recovery and increased axon density at early time points. This early development
of neuromuscular contacts resulted in more functional connections with the target muscle that in turn
promoted increased axon myelination at six months. The authors concluded that these results showed
that keratin-based scaffolds made from human hair can facilitate peripheral nerve regeneration and
promote neuromuscular recovery that is equivalent to the gold standard, sensory nerve autografts [74].
Keratin hydrogels derived from human hair have also been shown to act effectively as a hemostatic
agent in a rabbit model of lethal liver injury. In comparison to other commonly used hemostats
(QuickClot® and HemCon® bandage), the keratin hemostatic gel improved 24 hr survival and
performed consistently as well, if not better than, conventional hemostats in terms of total blood loss
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and shock index. The keratin gel used in these experiments acted on the injury site by instigating
thrombus formation and by forming a physical seal of the wound site that acted as a porous scaffold to
allow for cellular infiltration and granulose tissue formation [75]. The ability for keratin-based
biomaterials to be translated into the human clinical setting is dependent on further research to
elucidate the mechanisms by which these materials regulate hemostasis and nerve regeneration.
6. Conclusions
It would appear that keratin biomaterials have been in the collective conscience of materials
researchers for many decades, yet there are no keratin biomaterials currently in clinical use. This
comprehensive review has shown an impressive level of activity, diversity, and ingenuity, albeit at a
relatively low level compared to other mainstream biomaterials. Keratin biomaterials possess many
distinct advantages over conventional biomolecules, including a unique chemistry afforded by their
high sulfur content, remarkable biocompatibility, propensity for self-assembly, and intrinsic cellular
recognition. As these properties become better understood, controlled and exploited, many biomedical
applications of keratin biomaterials will make their way into clinical trials.
References
1. Moll, R.; Franke, W.W.; Schiller, D.L.; Geiger, B.; Krepler, R. The catalog of human
cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982, 31,
11−24.
2. Fraser, R.D.; MacRae, T.P.; Parry, D.A.; Suzuki, E. Intermediate filaments in alpha-keratins.
Proc. Natl. Acad. Sci. USA 1986, 83, 1179−1183.
3. Coulombe, P.A.; Bousquet, O.; Ma, L.; Yamada, S.; Wirtz, D. The ‘ins’ and ‘outs’ of intermediate
filament organization. Tr. Cell Biol. 2000, 10, 420−428.
4. Schweizer, J.; Bowden, P.E.; Coulombe, P.A.; Langbein, L.; Lane, E.B.; Magin, T.M.; Maltais,
L.; Omary, M.B.; Parry, D.A.; Rogers, M.A.; Wright, M.W. New consensus nomenclature for
mammalian keratins. J. Cell Biol. 2006, 174, 169−174.
5. Moll, R.; Divo, M.; Langbein, L. The human keratins: Biology and pathology. Histochem. Cell
Biol. 2008, 129, 705−733.
6. Yu, J.; Yu, D.; Checkla, D.M.; Freedberg, I.M.; Bertolino, A.P. Human Hair Keratins. J. Invest.
Dermatol. 1993, 101, 56S−59S.
7. Crewther, W.G.; Fraser, R.D.B.; Lennox, F.G.; Lindley, H. The Chemistry of Keratins. In
Advances in Protein Chemistry; Anfinsen, C.B., Anson, M.L., Edsall, J.T., Richards, F.M., Eds.;
Academic Press: New York, USA, 1965; Volume 20, pp. 191−347.
8. Rogers, M.A.; Langbein, L.; Praetzel-Wunder, S.; Winter, H.; Schweizer, J. Human hair keratinassociated
proteins (KAPs). Int. Rev. Cytol. 2006, 251, 209−263.
9. Stenn, K.S.; Paus, R. What controls hair follicle cycling? Exp. Dermatol. 1999, 8, 229−233.
10. Alonso, L.; Fuchs, E. The hair cycle. J. Cell Sci. 2006, 119, 391−393.
11. Langbein, L.; Rogers, M.A.; Winter, H.; Praetzel, S.; Beckhaus, U.; Rackwitz, H.R.; Schweizer, J.
The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle. J.
Biol. Chem. 1999, 274, 19874−19884.
Materials 2010, 3
1011
12. Langbein, L.; Rogers, M.A.; Winter, H.; Praetzel, S.; Schweizer, J. The catalog of human hair
keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of
human type I and II keratins. J. Biol. Chem. 2001, 276, 35123−35132.
13. Langbein, L.; Schweizer, J. Keratins of the human hair follicle. Int. Rev. Cytol. 2005, 243, 1−78.
14. Schweizer, J.; Langbein, L.; Rogers, M.A.; Winter, H. Hair follicle-specific keratins and their
diseases. Exp. Cell Res. 2007, 313, 2010−2020.
15. Zhen, L.S. Ben Cao Gang Mu; The Time Literature & Art Press: Changchun, Jilin, China, 2005 .
16. Hofmeier, J. Horn-lime plastic masses from keratin substances. German Pat. DE184915, 18
December 1905.
17. Breinl, F.; Baudisch, O. The oxidative breaking up of keratin through treatment with hydrogen
peroxide. Z Physiol. Chem. 1907, 52, 158−169.
18. Neuberg, C. Process of producing digestable substances from keratin. US Pat. 926,999, 6 July
1909.
19. Lissizin, T. Behavior of keratin sulfur and cystin sulfur in the oxidation of these proteins by
potassium permanganate I. Biochem. Bull. 1915, 4, 18−23.
20. Zdenko, S. Solubility and digestibility of the degradation products of albumoids I. Z Physiol.
Chem. 1924, 136, 160−172.
21. Lissizin, T. The oxidation products of keratin by oxidation with permanganate II. Z Physiol.
Chem. 1928, 173, 309−311.
22. Goddard, D.R.; Michaelis, L. Derivatives of Keratin. J. Biol. Chem. 1935, 112, 361−371.
23. Beyer, C. The keratin or horny substance of the hair. German Pat. DE22643, 14 October 1907.
24. Goldsmith, B.B. Thermoplastic composition containing keratin. US Pat. 922,692, 25 May 1909.
25. Dale, H.N. Keratin and other coatings for pills. Pharm. J. 1932, 129, 494−495.
26. Rivett, D.E.; Ward, S.W.; Belkin, L.M.; Ramshaw, J.A.M.; Wilshire, J.F.K. Keratin and Wool
Research. In The Lennox Legacy; CSIRO Publishing: Collingwood, VIC, Australia, 1996.
27. van den Bergh, J.; Milo, G.J.; van Dijk, H.E.P. Keratin-resin threads, films, etc. Netherlands Pat.
NL51000577, 15 December 1941.
28. Orwin, D.F.G; Baumann, H.; Asquith, R.S.; Parry, D.A.D. In Fibrous Proteins: Scientific,
Industrial and Medical Aspects; Parry, D.A.D., Creamer, L.K., Eds.; Academic Press: New York,
NY, USA 1979; pp. 271–427.
29. Earland, C.; Knight, C.S. Structure of keratin II: Amino acid content of fractions isolated from
oxidized wool. Biochem. Biophys. Acta 1956, 22, 405−411.
30. Kikkawa, M.; Chonan, Y.; Toyoda, H. Solubilization of keratin 6: Solubilization of feather keratin
by oxidation with performic acid. Hikaku Kagaku 1974, 20, 151−162.
31. Buchanan, J.H. A cystine-rich protein fraction from oxidized alpha-keratin Biochem. J. 1977, 167,
489−491.
32. Matsunga, A.; Chonan, Y.; Toyoda, H. Studies on the chemical properties of human hair keratin,
Part 1: Fractionation and amino acid composition of human hair solubilized by performic acid
oxidation. Hikaku Kagaku 1981, 27, 21−29.
33. Anker, C.A. Method of preparing keratin-containing films and coatings. US Pat. 3,642,498, 15
February 1972.
34. Kawano, Y.; Okamoto, S. Film and gels of keratin. Kagaku Seibutsu 1975, 13, 291−292.
Materials 2010, 3
1012
35. Okamoto, S. Formation of films from some proteins. Nippon Shokuhin Kogyo Gakkaishi 1977,
24, 40−50.
36. Noishiki, Y.; Ito, H.; Miyamoto, T.; Inagaki, H. Application of Denatured Wool Keratin
Derivatives to an Antithrombogenic Biomaterial-Vascular Graft Coated with a Heparinized
Keratin Derivative. Kobunshi Ronbunshu 1982, 39, 221−227.
37. Ito, H.; Miyamoto, T.; Inagaki, H.; Noishiki, Y. Biocompatibility of Denatured Keratins from
Wool. Kobunshi Ronbunshu 1982, 39, 249−256.
38. Jarman, T.; Light, J. Prospects for novel biomaterials development. In World Biotech Report;
Pinner: Middlesex, UK, 1985; Volume 1, pp. 505−512.
39. Various Authors Biomaterial forefront: Keratin which can be extracted by simple chemical
technique. Kogyo Zairyo 1993, 41, 106−109.
40. van de Locht, M. Reconstitution of microfibrils from wool and filaments from epidermis proteins.
Melliand Textilberichte 1987, 10, 780−786.
41. Steinert, P.M.; Gullino, M.I. Bovine epidermal keratin filament assembly in vitor. Biochem.
Biophys. Res. Commun. 1976, 70, 221−227.
42. Thomas, H.; Conrads, A.; Phan, P.H.; van de Locht, M.; Zahn, H. In vitor reconstitution of wool
intermediate filaments. Int. J. Biol. Macromol. 1986, 8, 258−264.
43. Ikkai, F.; Naito, S. Dynamic light scattering and circular dichroism studies on heat-induced
gelation of hard-keratin protein aqueous solutions. Biomacromolecules 2002, 3, 482−487.
44. Tachibana, A.; Furuta, Y.; Takeshima, H.; Tanabe, T.; Yamauchi, K. Fabrication of wool keratin
sponge scaffolds for long-term cell cultivation. J. Biotechnol. 2002, 93, 165−170.
45. Verma, V.; Verma, P.; Ray, P.; Ray, A.R. Preparation of scaffolds from human hair proteins for
tissue-engineering applications. Biomed. Mater. 2008, 3, 25007.
46. Magin, T.M.; Vijayaraj, P.; Leube, R.E. Structural and regulatory functions of keratins. Exp. Cell
Res. 2007, 313, 2021−2032.
47. Izawa, I.; Inagaki, M. Regulatory mechanisms and functions of intermediate filaments: A study
using site- and phosphorylation state-specific antibodies. Cancer Sci. 2006, 97, 167−174.
48. Yamauchi, K.; Yamauchi, A.; Kusunoki, T.; Kohda, A.; Konishi, Y. Preparation of stable aqueous
solution of keratins, and physiochemical and biodegradational properties of films. J. Biomed.
Mater. Res. 1996, 31, 439−444.
49. Yamauchi, K.; Maniwa, M.; Mori, T. Cultivation of fibroblast cells on keratin-coated substrata. J.
Biomat. Sci.-Polym. E. 1998, 9, 259−270.
50. Fujii, T.; Ogiwara, D.; Arimoto, M. Convenient procedures for human hair protein films and
properties of alkaline phosphatase incorporated in the film. Biol. Pharm. Bull. 2004, 27, 89−93.
51. Tanabe, T.; Okitsu, N.; Tachibana, A.; Yamauchi, K. Preparation and characterization of keratinchitosan
composite film. Biomaterials 2002, 23, 817−825.
52. Yamauchi, K.; Hojo, H.; Yamamoto, Y.; Tanabe, T. Enhanced cell adhesion on RGDS-carrying
keratin film. Mat. Sci. Eng. C-Bio. S. 2003, 23, 467−472.
53. Lee, K.Y.; Ha, W.S. DSC studies on bound water in silk fibroin/S-carboxymethyl kerateine blend
films. Polymer 1999, 40, 4131−4134.
Materials 2010, 3
1013
54. Lee, K.Y.; Kong, S.J.; Park, W.H.; Ha, W.S.; Kwon, I.C. Effect of surface properties on the
antithrombogenicity of silk fibroin/S-carboxymethyl kerateine blend films. J. Biomater. Sci.
Polym. Ed. 1998, 9, 905−914.
55. Lee, K.Y. Characterization of Silk/Fibroin/S-carboxymethyl Kerateine Surfaces: Evaluation of
Biocompatibility by Contact Angle Measurements. Fibers Polym. 2001, 2, 71−74.
56. Vasconcelos, A.; Freddi, G.; Cavaco-Paulo, A. Biodegradable materials based on silk fibroin and
keratin. Biomacromolecules 2008, 9, 1299−1305.
57. Tonin, C.; Aluigi, A.; Vineis, C.; Varesano, A.; Montarsolo, A.; Ferrero, F. Thermal and
structural characterization of poly(ethylene-oxide)/keratin blend films. J. Therm. Anal. Calorim.
2007, 89, 601−608.
58. Zoccola, M.; Montarsolo, A.; Aluigi, A.; Varesano, A.; Vineis, C.; Tonin, C. Electrospinning of
polyamide 6/modified-keratin blends. E-Polym. 2007, no. 105.
59. Fujii, T.; Ide, Y. Preparation of translucent and flexible human hair protein films and their
properties. Biol. Pharm. Bull. 2004, 27, 1433−1436.
60. Katoh, K.; Shibayama, M.; Tanabe, T.; Yamauchi, K. Preparation and physicochemical properties
of compression-molded keratin films. Biomaterials 2004, 25, 2265−2272.
61. Reichl, S. Films based on human hair keratin as substrates for cell culture and tissue engineering.
Biomaterials 2009, 30, 6854−6866.
62. Kurimoto, A.; Tanabe, T.; Tachibana, A.; Yamauchi, K. Keratin sponge: Immobilization of
lysozyme. J. Biosci. Bioeng. 2003, 96, 307−309.
63. Tachibana, A.; Kaneko, S.; Tanabe, T.; Yamauchi, K. Rapid fabrication of keratin-hydroxyapatite
hybrid sponges toward osteoblast cultivation and differentiation. Biomaterials 2005, 26, 297−302.
64. Tachibana, A.; Nishikawa, Y.; Nishino, M.; Kaneko, S.; Tanabe, T.; Yamauchi, K. Modified
keratin sponge: Binding of bone morphogenetic protein-2 and osteoblast differentiation. J. Biosci.
Bioeng. 2006, 102, 425−429.
65. Katoh, K.; Tanabe, T.; Yamauchi, K. Novel approach to fabricate keratin sponge scaffolds with
controlled pore size and porosity. Biomaterials 2004, 25, 4255−4262.
66. Peplow, P.V.; Dias, G.J. A study of the relationship between mass and physical strength of keratin
bars in vivo. J. Mater. Sci. Mater. Med. 2004, 15, 1217−1220.
67. Aluigi, A.; Varesano, A.; Montarsolo, A.; Vineis, C.; Ferrero, F.; Mazzuchetti, G.; Tonin, C.
Electrospinning of keratin/poly(ethylene oxide) blend nanofibers. J. Appl. Polym. Sci. 2007, 104,
863−870.
68. Aluigi, A.; Vineis, C.; Varesano, A.; Mazzuchetti, G.; Ferrero, F.; Tonin, C. Structure and
properties of keratin/PEO blend nanofibres. Eur. Polym. J. 2008, 44, 2465−2475.
69. Varesano, A.; Aluigi, A.; Vineis, C.; Tonin, C. Study on the shear viscosity behavior of
keratin/PEO blends for nanofibre electrospinning. J. Polym. Sci. Poly. Phys. 2008, 46,
1193−1201.
70. Zoccola, M.; Aluigi, A.; Vineis, C.; Tonin, C.; Ferrero, F.; Piacentino, M.G. Study on cast
membranes and electrospun nanofibers made from keratin/fibroin blends. Biomacromolecules
2008, 9, 2819−2825.
71. Katoh, K.; Shibayama, M.; Tanabe, T.; Yamauchi, K. Preparation and properties of keratinpoly(
vinyl alcohol) blend fiber. J. Appl. Polym. Sci. 2004, 91, 756−762.
Materials 2010, 3
1014
72. Wrześniewska-Tosik, K.; Wawro, D.; Ratajska, M.; Stęplewski, W. Novel composites with
feather keratin. Fibres Text. East. Eur. 2007, 15, 157−162.
73. Sierpinski, P.; Garrett, J.; Ma, J.; Apel, P.; Klorig, D.; Smith, T.; Koman, L.A.; Atala, A.; Van
Dyke, M. The use of keratin biomaterials derived from human hair for the promotion of rapid
regeneration of peripheral nerves. Biomaterials 2008, 29, 118−128.
74. Apel, P.J.; Garrett, J.P.; Sierpinski, P.; Ma, J.; Atala, A.; Smith, T.L.; Koman, L.A.; Van Dyke,
M.E. Peripheral nerve regeneration using a keratin-based scaffold: Long-term functional and
histological outcomes in a mouse model. J. Hand Surg. Am. 2008, 33, 1541−1547.
75. Aboushwareb, T.; Eberli, D.; Ward, C.; Broda, C.; Holcomb, J.; Atala, A.; Van Dyke, M. A
Keratin biomaterial gel hemostat derived from human hair: Evaluation in a rabbit model of lethal
liver injury. J. Biomed. Mater. Res. B 2009, 90, 45−54.
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license.
Bilimsel yayın 2
Ageing processes influence keratin and KAP expression in human hair follicles
Melanie Giesen1,
Sabine Gruedl1,
Olaf Holtkoetter1,
Guido Fuhrmann1,
Andrea Koerner2,
Dirk Petersohn1
Article first published online: 16 MAY 2011
DOI: 10.1111/j.1600-0625.2011.01301.x
Get PDF (340K)
Abstract
Abstract: In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.
Background
Since ancient times, the human hair is an attribute for health, youth and attractiveness and plays an important role in people’s self-perception (1). Therefore, alterations in the appearance of hair, such as premature greying or changes in hair structure, often stress the well-being and self-confidence of the affected persons.
The hair follicle is a complex mini organ that shows cyclic activity during postnatal life with periods of active growth, involution and resting (2). In each anagen phase, it produces the visible hair shaft; thus, synthesis of hair keratin is an essential prerequisite for the growth of strong and healthy hair. But like all biological systems, the hair follicle, the biological active part of the hair, also undergoes an ageing process, which is not only characterized by loss of pigmentation (3–6). But even if there are some general features besides greying indicating follicular ageing, such as hair loss, reduction in hair diameter as well as anecdotal evidence that hair becomes more fragile at an older age, little is known about further alterations and the molecular reasons underlying the known macroscopic changes.
Questions addressed
The objective of this study was to characterize the age-related changes in the gene expression of structural proteins in human hair follicles.
Experimental design
For the evaluation of differential gene expression in groups of different ages, 10 scalp hair follicles for every 20 healthy volunteers (10 women/10 men) below 25 years and above 50 years of age were plucked and total RNA was isolated.
Following standard reverse transcription of each RNA sample, quantitative polymerase chain reaction was performed with gene-specific primer sets for different hair keratins (Table S1). The resulting relative expression values of both groups were compared, whereby statistical significant differences were proven by a student t-test.
For extended gene expression analysis, the samples were pooled and the experiments were performed at Miltenyi Biotec (Bergisch Gladbach, Germany) using an Agilent whole-human genome microarray according to the manufacturer’s instructions. All methods are described in detail in Appendix S1 in the Supporting information.
Results
Our data show that the ageing process in follicles is associated among other things with a decline of structural proteins such as certain hair keratins and keratin-associated proteins (KAPs). Molecular analysis of keratin gene expression in hair follicles of donors above 50 years and below 25 years of age revealed striking differences between these two groups. While the expression of KRT31, KRT32, KRT36, KRT85 and KRT86 seemed to remain unaffected by ageing, we registered a statistically significant decline in gene activity of KRT33A and KRT34 in the older group of above 50 years of age compared with the younger group (Fig. 1). Remarkably, the regulated hair-specific keratins KRT33A and KRT34 belong to a group of keratins which are expressed in the upper part of the follicular cortex, representing late differentiation products within the hair-forming compartment (7).
Figure 1. Gene expression analysis of different hair keratins using a quantitative PCR method. Data show significant down-regulation of KRT33A and KRT34 in the older follicles compared to the younger ones. Statistics are given as SEM, * P < 0.05.
Furthermore, whole-human genome microarray analysis demonstrated a clear diminution of gene expression regarding certain KAPs, which represent the cross-linking network between the keratin intermediate filaments (8), in the hair follicles of older volunteers. From 39 analysed KAP genes, seven members of the family are downregulated significantly in aged hair follicles, which have been proven byANOVA/t-test. Thereby, the group of KAP4 genes seems to undergo the most vigorous age-related changes. Additionally, two members of two further KAP families are underrepresented in RNA samples of mature hair follicles (Fig. 2).
Figure 2. Gene expression analysis using a cDNA microarray. Data show significant down-regulation of different keratin associated proteins in the older follicles compared to the younger ones. Statistics are given as SEM, * P < 0.05.
Conclusion
The present studies enabled us to get a deeper insight into molecular events accompanying hair follicle ageing, especially the parameters related to hair fibre composition. Keratins are the most abundant structural proteins in hair (9) and the microfibrils or intermediate filaments they create are primarily responsible for hair’s mechanical properties (10). Thus, their adequate synthesis might be a prerequisite to maintain hair’s juvenescent characteristics. Here, we showed that the expression of two members of the acidic keratin family, keratin KRT33A and keratin KRT34, decrease with age. As they represent the later differentiation of the hair follicle, we speculate that this might be aetiological for the previously reported changes in hair structure during ageing (11). Besides the hair keratins, KAPs represent the other main components of the hair fibre, forming the protein matrix between the keratin microfibrils (12) and playing a crucial role in forming a strong hair shaft (8). As the KAP4 family not only represents the largest KAP family but also has been shown to be expressed predominantly in the highly differentiated portions of the middle and upper cortex and is suggested to be heavily involved in the terminal keratinization of the hair fibre cortex (13), the strong decline in gene expression therefore might influence hair shaft stability and flexibility. To evaluate whether the age-related changes in the gene expression pattern also become manifest in the ultrastructure of the hair follicle and fibre, further analysis using transmission electron microscopy as it has been proposed by Morioka (14) might be of interest. Already today, we have first evidence that the described age-related changes in the hair follicle influence the later hair fibre, as protein extracts of the hair shaft analysed by mass spectroscopy following separation by gel electrophoresis showed certain age-dependent differences in the peptide pattern (data not shown). Further studies will show whether those alterations can explain the macroscopic changes of mature hair and how they influence the mechanical behaviour.
Acknowledgements
We thank Ms. Sabine Gruedl for her technical assistance and the analysis of parts of the data sets. Mr. Olaf Holtkoetter contributes to the analysis of the microarray data sets. Mr. Guido Fuhrmann supported the sample collection and preparation of the samples. Mrs. Andrea Koerner was responsible for the analysis of hair fibres using mass spectroscopy techniques and Mr. Dirk Petersohn revised and approved the manuscript. Mrs. Melanie Giesen designed the study, analysed parts of the data sets and wrote the paper.
Conflict of interest
The authors state no conflict of interest.
References
Trüeb R M. J Cosmet Dermatol 2005: 4: 60–72.
Direct Link:
Abstract
Full Article (HTML)
PDF(344K)
References
2
Botchkarev V A, Kishimoto J. J Invest Dermatol Symp Proc 2003: 8: 46–55.
CrossRef,
PubMed,
CAS,
Web of Science® Times Cited: 58
3
Birch M P, Messenger J F, Messenger A G. Br J Dermatol 2001: 144: 297–304.
Direct Link:
Abstract
Full Article (HTML)
PDF(713K)
References
Courtois M, Loussouarn G, Hourseau C et al. Br J Dermatol 1995: 132: 86–93.
Van Neste D. Thickness, medullation and growth rate of female scalp hair are subject to significant variation according to pigmentation and scalp location during ageing. Eur J Dermatol 2004: 14: 28–32.
Tobin D J. Gerontobiology of the Hair Follicle. In: TrüebR M, TobinD J, eds. Aging Hair. Berlin: Springer-Verlag 2010: 1–8.
CrossRef
Langbein L, Schweizer J. Int Rev Cytol 2005: 243: 1–78.
CrossRef,
PubMed,
CAS,
Web of Science® Times Cited: 72
Shimomura Y, Ito M. Human hair keratin-associated proteins. J Invest Dermatol Symp Proc 2005: 10: 230–233.
CrossRef,
PubMed,
CAS,
Web of Science® Times Cited: 5
Lee Y J, Rice R H, Lee Y M. Mol Cell Proteomics 2006: 5: 789–800.
CrossRef,
PubMed,
Web of Science® Times Cited: 20
Zahn H. Int J Cosmet Sci 2002: 24: 163–169.
Direct Link:
Abstract
Full Article (HTML)
PDF(630K)
References
11
Mandt N, Blume-Peytavi U. Hautarzt 2005: 56: 340–346.
CrossRef,
PubMed,
CAS,
Web of Science® Times Cited: 1
Powell B C, Rogers G E. The role of keratin proteins and their genes in the growth, structure and properties of hair. In: JollésP,ZahnH, HöckerH, eds. Formation and Structure of Human Hair. Basel: Birkhäuser Verlag 1997: 59–148.
Kariya N, Shimomura Y, Ito M. J Invest Dermatol 2005: 124: 1111–1118.
CrossRef,
PubMed,
CAS,
Web of Science® Times Cited: 9 Morioka K. Exp Dermatol 2009: 18: 577–582.
Bilimsel yayın 3
Nicolas R. Barthélemya, Audrey Bednarczyka, Christine Schaeffer-Reissa, Dominique Jullienb, Alain Van Dorsselaera, Nükhet Cavusoglu Proteomic tools for the investigation of human hair structural proteins and evidence of weakness sites on hair keratin coil segments Analytical Biochemistry Volume 421, Issue 1, 1 February 2012, Pages 43–55
Human hair is principally composed of hair keratins and keratin-associated proteins (KAPs) that form a complex network giving the hair its rigidity and mechanical properties. However, during their growth, hairs are subject to various treatments that can induce irreversible damage. For a better understanding of the human hair protein structures, proteomic mass spectrometry (MS)-based strategies could assist in characterizing numerous isoforms and posttranslational modifications of human hair fiber proteins. However, due to their physicochemical properties, characterization of human hair proteins using classical proteomic approaches is still a challenge. To address this issue, we have used two complementary approaches to analyze proteins from the human hair cortex. The multidimensional protein identification technology (MudPit) approach allowed identifying all keratins and the major KAPs present in the hair as well as posttranslational modifications in keratins such as cysteine trioxidation, lysine, and histidine methylation. Then two-dimensional gel electrophoresis coupled with MS (2-DE gel MS) allowed us to obtain the most complete 2-DE gel pattern of human hair proteins, revealing an unexpected heterogeneity of keratin structures. Analyses of these structures by differential peptide mapping have brought evidence of cleaved species in hair keratins and suggest a preferential breaking zone in α-helical segments.
