Porous keratin scaffold–promising biomaterial for tissue engineering and drug delivery

Porous Keratin Scaffold–Promising Biomaterial for TissueEngineering and Drug Delivery

Balaji Srinivasan,1 Ramadhar Kumar,1 Kirubanandan Shanmugam,1 Uma Tiruchirapalli Sivagnam,1

Neelakanta Puily Reddy,2 Praveen Kumar Sehgal1

1 Bioproduct Laboratory, Biomaterial Division, Central Leather Research Institute (Council of Scientific & Industrial Research),Adyar, Chennai 600020, India

2 Bioorganic Laboratory & Neurochemistry Department, Central Leather Research Institute (Council of Scientific & IndustrialResearch), Adyar, Chennai 600020, India

Received 11 February 2009; revised 29 April 2009; accepted 12 June 2009Published online 27 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31483

Abstract: A porous keratin scaffold, prepared from the reduced keratin solution, has shown

good cell viability which makes it a potential candidate for cell seeding. An aqueous solution of

reduced keratin was extracted from horn meal using a mixture of urea, sodium dodecyl

sulfate, mercaptoethanol, and water at 608C. The molecular mass of the extracted keratin is

found to be ranging between 225 and 150 KDa. The CD spectrum of aqueous solution of

keratin shows the presence of ‘-helical structure with b-turns as negative absorption band at

225 nm and as positive absorption band at 195 nm. The FTIR spectrum of the same confirms

‘-helical structure with b-turns. Its characteristic absorption bands are assigned mainly to the

peptide bonds for amide I, II, and III respectively. DSC and TGA data of the reduced keratin

peaks fall in region 2008C–2508C and 2008C–4008C temperatures, respectively. They

correspond to the ‘-helix denaturation of the material. ' 2009 Wiley Periodicals, Inc. J Biomed

Mater Res Part B: Appl Biomater 92B: 5–12, 2010

Keywords: keratin; porous scaffold; horn meal; tissue engineering

INTRODUCTION

The regeneration of tissues at the site of injury is really a

challenging task in the field of biomedical science.1 The

need of suitable biomaterials, which influence the formation

of in vivo extra cellular matrix and support cellular prolifera-

tion for tissue regeneration, have motivated the researchers/

scientists toward tissue engineering which led to the develop-

ment of implantable porous scaffolds to be used at the dam-

aged site. These scaffolds temporarily act as the supporting

materials for growth of the cells and they degrade along with

in vivo production of extra cellular matrix leading to vascula-

rization and tissue regeneration.1 Biomaterials made from

fibrous protein mimic the extra cellular matrix at the site of

injury for regeneration of tissues.2,3 Collagen is one such bio-

material and is already prominently placed in the develop-

ment of tissue engineering constructs but with a few

limitations. Because of some environmental reasons, colla-

gen denatures into gelatin and it looses its triple helix confor-

mation. Also collagen needs cross linking to improve its

mechanical properties, which normally causes cytotoxity.4 In

place of collagen, keratin, a less thermolabile protein is an

alternative for such applications.5 This fibrous protein is

found in the extracellular matrix and present in the outer cov-

ering of wool, hair feather, and nail.

At molecular level, the most distinctive feature of kera-

tin is the high concentration of half-cystine residues (7%–

20% of the total amino acid residues). Most of these half-

cystine residues are localized at the terminal regions of the

proteins.5,6 Keratin biomaterials in the form of sponge, film

were already developed from wool and human hair for var-

ious biomedical applications such as wound dressings and

neural tissue engineering applications.7

Keratin extracted from wool, silk, and human hair con-

tained cell adhesion sequence, RGD (Arg-Gly-Asp), and

LDV (Leu-Asp-Val) which are found in the extra cellular

matrix proteins such as fibronectin.8,9 Keratin contains

cellular-binding motifs which mimic the sites of cellular

attachment found in the native extra cellular matrix because

of which, keratin could be used for the development of

tissue engineering constructs.

The protein based porous scaffolds have several

advantages to enhance the growth of cells at the site of

damage. The microstructures of the scaffold, its high

porosity (greater than 90%) and interconnected pore

network are requisite for its development. The preferred

pore size of the scaffold for growth of cells, vasculariza-

Correspondence to: Dr. P. K. Sehgal (e-mail: [email protected])

' 2009 Wiley Periodicals, Inc.

5

tion and regeneration of tissues is in the range of

50–500 microns.9–15

In this study, we have attempted to prepare porous scaf-

fold by extracting keratin in the reduced form from horn

meal by means of chemical methods and characterized its

physiochemical properties. We have also fabricated and

characterized the keratin porous scaffold for assessment of

cell viability.

MATERIALS AND METHODS

Preparation of Horn Meal

Raw horns of slaughtered cattle and buffaloes collected

from the local slaughterhouse at Perambur Chennai were

washed and subjected to high steam pressure (40 psi) in a

wet rendering plant (FMC, Australia) for 3 hours (raw horn

water ratio 100:30 w/v). The resulting material (solid) was

dried in a dryer (BHL, Ahmadabad) and pulverized using a

pulverizer (FMC, Australia) to get horn meal. Horn meal

thus prepared in the pilot plant of Central Leather Research

Institute is used as a starting material for further

experiment.

Extraction of Reduced Keratin from Horn Meal

This horn meal was washed, dried, and defatted by soxhlet

extraction using 1:1v/v mixture of hexane and dichlorome-

thane. The defatted horn meal (10 g) was mixed with 7 M

urea (180 mL), SDS (6 g), and 2-mercaptoethanol (15 mL)

in a 300 mL round-bottom flask and shaken at 608C for 12

hours to extract reduced keratin at pH 6–8. The resulting

mixture was centrifuged for 10 minutes at 10,000 rpm and

the filtrate was dialyzed against degassed water for 5 days.

Preparation of Porous Keratin Scaffold

10 mL of reduced keratin solution (containing 420 mg of

protein) after degassing was poured into a 3 cm dia Petri

dish (glass) frozen at 2808C for 2 days and lyophilized to

form a sponge (5 cm diameter, 1.00 mm thickness). The

sponge, thus, obtained was washed with Phosphate buffered

saline solution (pH 7.4, 0.07 M) at room temperature to

remove excess SDS, Mercaptoethanol, and Urea.

SDS PAGE of Keratin

The aqueous solution of reduced keratin was subjected to

one-dimensional slab SDS-PAGE at 158C using a 10%–

15% gradient gel at 250 V and 10 mA. The gel was stained

using Coomassie brilliant blue R-250.

Quantification of Proteins and Amino Acid Residues

The protein-content was determined by the Lowry’s

method.16 The amino acid content in the reduced keratin

and sponge were quantified by the High Performance

Liquid chromatography (HPLC Water model) with column

SUPELCOSILTM LC-DABS HPLC. About 3 lm Column,

particle size, L X I.D. 15 cm 3 4.6 mm (Sigma) by detect-

ing the elute at 254 nm on UV spectrophotometer. The

amino acid composition was expressed as mol % for each

amino acid, determined against external standard calibra-

tion kit (Sigma).

Circular Dichroism Spectra of Reduced Keratin

Circular Dichroism (CD) spectrum of reduced keratin was

taken on a JASCO J-20 spectropolarimeter in a thermostati-

cally controlled 1 mm jacketed cell. Liquid nitrogen was

circulated through the instrument for 30 minutes to main-

tain a constant temperature and protect the nativity of

protein during the period of experiment. A cell with 1 mm

path length was employed. Baseline adjustment was carried

out with 0.025 M acetic acid solution. The CD measure-

ment of each of the samples was made from 195 to

250 nm. The CD data were expressed in terms of the mean

residue ellipticity, y in deg cm2/dmol.

Fourier Transform Infra Red Spectra of Reduced Keratin

Fourier transform infra red (FTIR) spectra of lyophilized

reduced keratin was obtained by using the ATR technique

(Attenuated Total Reflection). Keratin pellet was prepared

by mixing it with potassium bromide. An infrared spectrum

of keratin was recorded from 400 to 2000 cm21 using a

Nicolet 20 DXB FT-IR spectrophotometer.

Differential Scanning Calorimeter Analysis ofReduced Keratin

Differential scanning calorimeter (DSC) analysis of reduced

keratin after conditioning the samples at 248C, 65% R.H

was performed from 308C to 4008C, at 108C/min. using

universal V4.4A TA instruments. The instrument was cali-

brated by an indium standard and the calorimeter cell was

flushed with 100 mL/min liquid nitrogen.

Thermogravimetric Analysis of Reduced Keratin

Thermogravimetric analysis (TGA) analysis of reduced ker-

atin was performed using universal V4.4A TA instruments.

About 3 mg of the sample was heated at 108C/min at a

temperature range of 308C–6008C using Al2O3 crucibles.

Scanning Electron Microscopy for PorousKeratin Scaffold

The porous keratin scaffold was sputter coated with gold

and observed under a SEM (JSM-T330, JEOL Co., CLRI,

Chennai) at 5 kV. The surface, cross, and transverse section

of the scaffold were scanned.

6 SRINIVASAN ET AL.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

Water Uptake and Swelling Studies of Keratin Scaffolds

The rectangular keratin sponge measuring 1.7 3 1.2 3

0.3 cm was dried under vacuum at 708C for 4 hours and

cooled in a desiccator at ambient temperature (25 6 38C).

Subsequently the weight and length of the longer side of

the dried sponge was recorded. The preconditioned sponge

was immersed in PBS, pH 7.4 for 24 hours to achieve com-

plete saturation. The sponge was removed from PBS, im-

mediately weighed and its length (Longer side) was again

measured. Swelling ratio was measured and an average of

four values was taken as per the given equation.

Swelling ratio ¼ ðLw � LdÞLW 3 100

Where Lw and Ld represents the length of wet and dry

sponge, respectively.

The swelling ratios of the sponge was also measured in

acetate (pH 4), (pH 7), and Tris-HCl (pH 10) buffer

solution.

Water uptake was determined according to the following

equation and an average of four values was taken to find

the percentage water uptake.

Water uptake ¼ ðWw �Wd=WwÞW 3 100

Where Ww and Wd represents the weight of wet and dry

sponge, respectively.

Measurement of Tensile Strength

The keratin scaffold (1.7 3 1.2 cm, thickness 0.3 cm) was

tested for its mechanical strength. The samples were condi-

tioned (Dried and weighed) as described earlier. Stress–strain

measurement was carried out on an autograph (recorder) at a

crosshead speed of 20 mm/min, 258C temperature and RH of

65% and 85% respectively. Percentage of elongation at break

(%) was measured using a universal testing machine (INS-

TRON model 1405) according to Vogel at an extension rate

of 5 mm/min.

Statistical Analysis

The statistical analysis was performed using SPSS (version

4.01). Data was expressed as mean 6 SD of three inde-

pendent experiments.

MTT Assay for Cell Viability

Monolayer of fibroblast cell line NIH 3T3 [National centre

for Cell Science (NCCS), Pune, India] were grown on keratin

coated 24 well culture plate (Corning, NY) and maintained

in Dulbecco’s modified Eagles medium (sigma) with 10%

fetal calf serum (sigma) supplemented with antibiotics

(sigma), penicillin (120 units/mL), streptomycin (75 mg/mL),

gentamycin (160 mg/mL), and amphotericin B (3 mg/mL) at

378C humidified with 5% CO2. After 24 and 48 hours, the

MTT (sigma) assays were performed to see the percentage of

cell viability.18,19

RESULTS

Reduced Keratin

100 kg of raw horns yielded 60 kg of horn meal. An aque-

ous solution of the reduced keratin was obtained from horn

meal using mixture of urea, sodium dodecyl sulfate, and

mercaptoethanol at 608C. This solution was stable for 1

year when stored at ambient temperature (208C–248C). The

reduced keratin solution was extracted at neutral pH range

of 6–8 because keratin could not be extracted at acidic pH

(\5) and underwent decomposition at alkaline pH ([9).

SDS PAGE of Keratin

The electrophoretical analysis of reduced keratin is shown

in Figure 1. Two high molecular mass bands corresponding

to 225 and 150 Kda and three lower molecular mass bands

between 25 and 15 Kda were observed. Bands at 225 and

150 Kda represent more than 75% of reduced keratin.

Amino Acid Residues

Figure 2 reveals the amino acid composition of reduced kera-

tin and keratin sponge. Difference in the composition of two

preparations is because of the oxidation and reduction of cys-

tine and half cystine during sponge preparation from reduced

keratin. The value of the each amino acid has been presented

as mole percent. The cystine content was found to be 56%

and 44% in reduced keratin and keratin sponge, respectively,

but the half cystine was found to be 41% and 58% in reduced

keratin and sponge because of increased number of S��S

bond because of partial removal of reductants.

CD Spectra of Reduced Keratin

The CD spectrum of aqueous solution of keratin confirms

anti parallel beta sheet structure with negative minimum

absorption band at 225 nm and a weak positive maximum

absorption band at 195 nm shown in Figure 3.

FTIR Spectra of Reduced Keratin

The FTIR spectrum of reduced keratin is shown in Figure

4. Characteristic absorption band mainly to the peptide

bonds (��CONH��) is seen. The vibrations in the peptide

bonds can be attributed to amide A, amide I, II, and III.

The amide A band, which falls at 3292.12 cm21 is because

of the stretching vibration of N��H bonds. The amide I

band caused by C��O stretching vibration occurs in the

range of 1700–1600 cm21. Sharp peak was observed at

1650.8 cm21.

The amide II, which falls at 1541.47 cm21 is related to

N��H bending and C��H stretching vibration. The amide

III band (1220–1300 cm21) was observed at 1231.18

7POROUS KERATIN SCAFFOLD–PROMISING BIOMATERIAL


cm21as a sharp peak. It results from in phase combination

of C��N stretching and N��H in plane bending, with some

contribution from C��C stretching and C¼¼O bending

vibrations.

Differential Scanning Calorimeter Analysis ofReduced Keratin

The DSC thermograms of reduced keratin are shown in

Figure 5. The first endothermic peaks seen as a sharp peak

at 93.618C is because of water evaporation. The second

peak falls in range of 2008C–2508C is related to the alpha

helix denaturation.

TGA Analysis of Reduced Keratin

The thermogravimetric curve for reduced keratin sample

(7.511 mg) is shown in Figure 6. The initial weight loss at

1008C–1508C is because of moisture loss. The second

weight losses took place in the temperature range 2008C–

4008C. About 54% weight loss was observed between

225.248C and 387.878C.

Preparation of Porous Keratin Sponge

The porous keratin Scaffold prepared by chemical method

was frozen (2808C) and lyophilized. The SEM observation

of the keratin scaffold at 50 x reveals heterogeneous porous

Figure 3. Circular Dichroism (CD) Spectroscopy of aqueouskeratin.

Figure 4. Fourier Transform Infra Red (FTIR) Spectra of Reduced

Keratin obtained from Horn Meal. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

Figure 1. SDS-PAGE; Standard (Lane 1), Reduced Keratin fromHorn Meal (Lane 2).

Figure 2. Amino acid composition (mol %) of reduced keratin in

solution and keratin Sponge. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

8 SRINIVASAN ET AL.


microstructures of transverse section [Figure 7(A)]. Surface

view of sponge at 20 x shows heterogeneous porous micro-

structure with pore size of 40–200 lm [Figure 7(B)]. The

cross section of keratin sponge also reveals heterogeneous

pores as shown in Figures 7(C–E).

Water Uptake and Swelling Studies of Keratin Sponge

The water uptake of the sponge was found to be 85 6 3%.

The sponge swells 5% at pH 7.4 and 8.5% at pH 9.1.

Measurement of Tensile Strength

The keratin sponge was found to have a tensile strength of

0.05 Mpa (Table I). The sponge was prepared without

cross-linking agent it stretched on applying load. Results

are presented as mean 6 SD (n 5 3).

Cell Viability

MTT assay showed more than 90% viability (NIH 3T3)

after 24 and 48 hours of culture on keratin coated wells

when compared with noncoated wells.

DISCUSSION

Biomaterials based on fibrous protein strongly influences

formation of the extracellular matrix at the site of injury.

Polymeric biomaterials are biodegradable and used for tis-

sue engineering construct, but they are very expensive and

improper degradation of polymeric biomaterials under

physiological conditions cause inflammatory response in

the host tissue. Biomaterials made from proteinous biopoly-

mers such as collagen or other extra cellular matrix compo-

nents mimic the native cellular environment to facilitate

normal development of tissues and organs under in-vitroconditions but the thermal instability of these proteinous

biomolecules limits their utilization. Collagen denatures

and in turn looses its functional triple helical conformation

which is a very important property for the biomaterial

development for both tissue engineering and dermal drug

delivery in relation to wound dressing and healing. The dis-

covery of new classes of biomaterials may provide another

opportunity to address clinical needs. The development of

keratin biomaterials from biowastes such as horns and

human hair20–22 is an alternative to collagen, other biopoly-

meric biomaterial and synthetic polymeric biomaterial and

has been used as the scaffolds for the regeneration of sev-

eral tissue types. Keratins are a large family of structural

proteins found in the cytoskeleton and in the protective tis-

sues of vertebrates. The hard keratins, as the name implies,

form the more resilient structures such as hair, horn, and

hooves. Hair and wool which form soft keratins have been

investigated as a source of biomaterials since the early

1900s. Early applications included wound healing and drug

delivery.

Keratin provides better attachment for the cells as it has

cell adhesion sequence in its structure, which is very cru-

cial for cell signaling. Keratin biomaterials also contain

intrinsic sites of cellular recognition that mimic the ECM.

It has been shown that in addition to the widely known

RGD motif, the ‘‘X’’-Aspartic Acid-‘‘Y’’ motif on fibronec-

tin (where X equals glycine, leucine, or glutamic acid and

Y equals serine or valine) is also recognized by the integrin

a4b1. Keratin biomaterials derived from human hair con-

tain these same binding motifs. A recent search of the

NCBI protein database revealed sequences for 71 discrete,

unique human hair keratin proteins. Of these, 78% contain

Figure 5. Differential scanning calorimeter analysis (DSC) of

Reduced Keratin obtained from Horn meal.

Figure 6. Thermogravimetric analysis (TGA) of Reduced Keratinobtained from Horn Meal.



at least one fibronectin-like integrin receptor-binding motif

and 25% contain at lease two or more.8,9

We have reported development of porous keratin-based

biomaterials that demonstrate cell instructive capabilities.

Some keratin biomaterials from biowastes such as wool

and hair have been shown to be mitogenic and chemotactic

for a variety of cell types, and to mediate changes in gene

expression consistent with the promotion of wound healing,

the development of keratin-based biomaterials from various

sources such as wool and human hair23 have already done

and they are effective biomaterial for osteoblast differentia-

tion and fibroblast cultivation and also for wound repair.

Horns are biowaste, collected from slaughter house, which

have rich content of keratin in condensed form. We have

extracted keratin in reduced form from this material.24,25

The electrophoretical analysis of reduced keratin shows

two high molecular mass bands 225 and 150 Kda and the

lower molecular mass bands (25–15 Kda). Whereas

Figure 7. (A–E) Scanning electron microscopic observation of porous Keratin Scaffold prepared by

freezing for 2 days at 2808C and subsequent lyophilization. Transverse, Surface and Cross Section of

keratin sponge figures heterogeneous porous microstructures were observed at differentmagnification.

TABLE I. Mechanical Property of Keratin Sponge

S.No

Maximum

Load (N)

Maximum

Extension (mm)

Elongation

Break (%)

Tensile

Strength (Mpa)

1 0.50 2.92 19.44 0.05

2 0.57 2.67 17.78 0.06

3 0.59 2.63 17.68 0.06

Mean 6 SD 0.53 6 0.55* 2.74 6 2.76* 18.03 6 18.05* 0.05 6 0.07*

Results are presented as mean 6 SD (n 5 3).

* p\ 0.01 was considered statistically significant.

10 SRINIVASAN ET AL.


reduced keratin from wool showed bands at 52 and 62–69

Kda.26 Wool Keratin regenerated from the formic acid

showed bands at 60 and 45–28 Kda.27 More amount of half

cysteine in case of keratin sponge reflects the existence of

more number of S��S bonds.

The FTIR spectrum also confirms alpha helical structure

with ß turns with characteristic absorption bands assigned

mainly to the peptide bonds (��CONH��) for amide I, II,

and III at 1650.8, 1541.47, and 1231.18 cm21, respectively.

The amide I mode was resolved in Gauss shaped bands

corresponding to alpha helix (1650 cm21) beta-turn (1620

cm21). On the basis of literature data, the absorption at

1650 cm21 suggests the presence of alpha helix structure,

whereas the bands related to beta turn structure fall in the

1631–1515 cm21 range. The reduced keratin from horn

meal might contain combination of alpha helix and beta

turn combination in the reduced conditions.28

The CD spectrum of aqueous solution of keratin also

confirms alpha helical structure with ß turn negative mini-

mum absorption band at 225 nm and a weak positive maxi-

mum absorption band at 195 nm. The CD spectra shows

beta turn presents in the keratin of reduced conditions.17

The thermoanalytical (DSC) investigations show an

endothermic peak in the temperature range of 2308C–

2408C which has been indexed as the helix peak and the

area under the peak represents a measure of the relative

helix content of the sample.29 This corresponds to our cur-

rent findings for horn meal keratin in which the peak that

fall in the 2008C–2508C temperature range, related to the

alpha helix denaturation. In TGA analysis, the initial

weight loss at 1008C–1508C is because of moisture loss

and the weight loss of horn meal keratin started at about

2008C and continued up to 4008C which correlates with

helix denaturation.30 The reported value of endothermic

peak of wool and hair is around 2308C–2458C and can be

attributed to helical denaturation superimposed by various

decomposition reactions.

We have purified keratin from horn meal which demon-

strated several remarkable characteristics. Keratins from the

horn meal are highly biocompatible because purified sam-

ples contain no cellular material so they might not be elic-

iting an immune response to the hosts. Certain keratins

have an incredible ability for molecular self-assembly that

results in the spontaneous formation of network structures.

Keratin contains cellular-binding motifs that mimic the

sites of cell attachment found in the native extra cellular

matrix components which facilitates better growth via

providing proliferation signals to the cells and minimizes

apoptotic cell death.

Various biopolymers have been used for dermal drug

delivery systems especially for wound dressing and healing

but keratin based system can be a better choice because of

its physio-biochemical characteristics. The thermal analysis

of keratin biomaterial shows that material can withstand

change in temperature during preparation of biomaterials.

The porous keratin sponge contains lot of heterogenious

pores and the water uptake is also high which shows that it

may have capacity to absorb the wound exudes at the

wound site and maintains moist environment when used as

wound dressings.31

CONCLUSIONS

Keratin biomaterial can be advocated as a promising candi-

date in the field of biomedical science to be used for tissue

engineering and dermal drug delivery systems. The prob-

lem of environmental pollution could also be addressed

through the fruitful utilization of solid biowastes.32

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Porous keratin scaffold–promising biomaterial for tissue engineering and drug delivery