The influence of silkworm species on cellular interactions with novel pva/silk sericin hydrogels

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The Influence of Silkworm Species on CellularInteractions with Novel PVA/SilkSericin Hydrogels

Khoon S. Lim, Joydip Kundu, April Reeves, Laura A. Poole-Warren,Subhas C. Kundu, Penny J. Martens*

Sericin peptides and PVA are chemically modified with methacrylate groups to produce acovalent PVA/sericin hydrogel. Preservation of the sericin bioactivity following methacryl-ation is confirmed, and PVA/sericin hydrogels are fabricated for both B. mori and A. mylittasericin. Cell adhesion studies confirm the preservationof sericin bioactivity post incorporation in PVA gels.PVA/A. mylitta gels are observed to facilitate celladhesion to a significantly greater degree thanPVA/B. mori gels. Overall, the incorporation of sericindoes not alter the physical properties of the PVA hydro-gels but does result in significantly improved cellularinteraction, particularly from A. mylitta gels.

1. Introduction

Hydrogels are soft-crosslinked polymers with high water

content and good macromolecular diffusivity. Although

these characteristics are similar to the extracellular matrix

(ECM), our group and others have recognized the critical

need for incorporation of biological molecules into syn-

thetic hydrogels to augment biofunctionality. Of particular

importance for tissue engineering applications are

improvements in cell adhesive and cell signaling proper-

ties.[1–3] ECM proteins such as collagen[4,5] and its derivative

gelatin,[6,7] laminin[8,9] and glycosaminoglycans such as

K. S. Lim, A. Reeves, Prof. L. A. Poole-Warren, Dr. P. J. MartensGraduate School of Biomedical Engineering, University of NewSouth Wales, Sydney 2052, AustraliaE-mail: [email protected]. Kundu, Prof. S. C. KunduDepartment of Biotechnology, Indian Institute of Technology,Kharagpur 721302, India

K.S.Lim and J. Kundu contributed equally to this paper.

Macromol. Biosci. 2012, 12, 322–332

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonline

chondroitin sulfate[1] and heparin,[3] are the most com-

monly studied biological modifiers for synthetic hydrogels.

More recently, the silk protein sericin has shown promise

for enhancing cell interactions with hydrogels that have

otherwise low interactivity.[2,10,11] Sericin is a protein

secreted from the middle silk gland of a mature silkworm

larva and acts as the glue for adhesion of fibroin based fibers

during cocoon formation.[12,13] The glue-like protein is

composed of random coil and b-sheet secondary structures

with a high abundance of hydrophilic amino acids that

confers water solubility.[10,12,14–16] It has been previously

reported that sericin from the domesticated silkworm

Bombyxmorihas antioxidant properties as well as being UV

protective, cryoprotective and chemoprotective.[13,17–22] In

terms of cellular interactions, there is some evidence that

B. Mori sericin promotes cell adhesion and cell survival

of fibroblasts,[20,23] epithelial cells[24] and osteoblasts,[11]

although the mechanism for this is unknown. As there are

no mammalian proteins or peptides possessing multi-

functional characteristics such as those of sericin, it is of

interest to copolymerize sericin with synthetic polymers to

library.com DOI: 10.1002/mabi.201100292

The Influence of Silkworm Species on Cellular Interactions with . . .

www.mbs-journal.de

create biosynthetic hydrogels, and to study whether the

bioactivity of sericin is transmitted to the gel. Other

advantages are that the source of sericin is sustainable and

the likelihood of viral and prion transfer tends to be lower

for insect proteins compared with mammalian proteins.

While the majority of research on silk proteins has

centered on proteins derived from B. mori, there is a

growing interest in other silkworm species. Sericin

extracted from the wild-type silkworm Antheraea mylitta,

appears to be significantly different to that of its

domesticated cousin. B. mori sericin consists of proteins

with major molecular weight fractions at 150, 250 and

400 kDa while A. mylitta sericin has fraction at 70, 200 and

>200 kDa.[12,15,16] Moreover, antibody cross-reactivity

between A. mylitta sericin and B. mori sericin is variable

suggesting that the two sericins are biochemically differ-

ent.[12] A. mylitta sericin has also been reported to have

lower tyrosine and serine contents than B. mori sericin.[25]

However there has been little research directly comparing

the bio-functionality of the two species which will be

addressed in this study.

The key challenges for incorporating proteins such as

sericin in hydrogels are in the formation of stable, covalent

linkage of the protein to the synthetic polymer and in

maintenance of the function of the biological molecule after

integration. Another key issue revolves around using the

minimum biological information required to achieve the

desired cellular functions. By reducing the amount of

protein used and achieving efficient incorporation, the

physical and mechanical properties of the base polymer are

minimally perturbed and likely to remain stable following

fabrication and use in specific applications.

Previous studies have produced sericin-based gels via

simply blending the sericin into gels,[20] by ethanol

treatment to induce gelation, or crystallinity of seri-

cin[10,26,27] or have integrated sericin with synthetic

hydrogels using chemical crosslinkers such as glutaralde-

hyde.[2,17,28] The problems with such approaches relate to

the stability of gels in the former instances and the

likelihood for chemical toxicity and regulatory concerns in

the latter.

The present study proposes an approach to produce a

copolymerized network of sericin and poly(vinyl alcohol)

(PVA) to form a dimensionally stable hydrogel with

improved biological performance. Several studies have

shown that PVA can be functionalized with acrylate based

side groups,[1,3,29] and photopolymerized to form cross-

linked hydrogels in the presence of a non-toxic, water

soluble initiator.[30] This research will study the feasibility

of chemically modifying sericin proteins using equivalent

approaches to those used for functionalizing PVA. This will

be extended to examine the impact of this modification on

selected biological functions of sericin. Finally, the bioac-

tivity of functionalized sericin after incorporation into PVA

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Macromol. Biosci. 20

� 2012 WILEY-VCH Verlag Gmb

hydrogels will be examined based on cell responses to this

co-hydrogel.

2. Experimental Section

2.1. Macromer Synthesis

2.1.1. Isolation and Characterization of Sericin from

Silkworm Cocoons

A. mylitta and B. mori silk cocoons were collected from Jhargram

Tropical Tasar Farms and Debra Sericulture Farm, West Midnapore,

West Bengal, India, respectively. Silk sericin peptides were

extracted from the cocoons according to a method modified from

Sofia et al.[31] In brief, A. mylitta and B. mori cocoons were cut into

small pieces then boiled in excess sodium carbonate (Na2CO3)

solution (0.02 M) for 1 and 4 h, respectively. The supernatant was

centrifuged at 1000 rpm for 10 min to remove fibers and impurities,

then dialyzed against deionized water for 48 h in a 10 kDa

molecular weight cut-off tubing. The solution was then lyophilized

to obtain dried sericin. The extracted sericin was characterized by

sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-

PAGE) to confirm the presence and molecular weights of the

peptides. The peptides were separated by using an 8% SDS-PAGE gel

at a constant voltage of 80 V for approximately 3 h. Peptide bands

were visualized using Coomassie Brilliant Blue R-250 (Sigma) and a

methanol/water 1:1 destaining solution that contained 20% acetic

acid.

2.1.2. Synthesis of PVA Methacrylate (PVA-MA)

PVA-MA was prepared by reacting PVA (Sigma-Aldrich, 13–23 kDa,

98% hydrolyzed) with 2-isocyanatoethyl methacrylate (ICEMA)

(Sigma-Aldrich, 98% purity) in dimethyl sulfoxide (DMSO) accord-

ing to a method described by Bryant et al.[1] In a typical experiment,

a 10 wt% solution of PVA (10 g) in DMSO (100 mL) was heated to

60 8C under nitrogen atmosphere until the PVA was completely

dissolved. ICEMA (0.405 cm3) was then added with vigorous stirring

and the solution was left to react for 4 h. To stop the reaction, the

solution was precipitated in toluene. The precipitated polymer was

redissolved in water and then purified by ultrafiltration through a

10 kDa molecular weight cut-off membrane. Lastly, the purified

solution was sterilized by filtration through a 0.22mm sterile filter,

and then lyophilized to obtain sterile dried PVA-MA.

2.1.3. Synthesis of Sericin Methacrylate (Sericin-MA)

Sericin was methacrylated using a similar method to PVA but with

slight modifications.[1] In brief, sericin (1 g) was dissolved in lithium

chloride (LiCl) in DMSO solution (100 cm3, 1 M) to form 1 wt%

solution. The solution was then purged with N2 gas for 30 min,

followed by the addition of ICEMA (0.0283 cm3), then left to react for

5 h under N2 atmosphere. The reaction was stopped by precipitat-

ing the solution in ethanol, and then centrifuged at 1000 rpm for

10 min to collect the precipitate. Purification was done by re-

dissolving the sericin-MA in water then dialyzed against deionized

water for 48 h in a 10 kDa molecular weight cut-off tubing. The

purified sericin-MA solution was filtered through a 0.22mm sterile

filter, and then lyophilized to obtain sterile dried sericin-MA.

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K. S. Lim, J. Kundu, A. Reeves, L. A. Poole-Warren, S. C. Kundu, P. J. Martens

2.2. Macromer Characterization

2.2.1. Macromer Characterization Using NMR

Both PVA-MA and sericin-MA were dissolved in deuterium oxide

(D2O) then analyzed using 1H NMR (300 MHz Bruker Advance DPX-

300 spectrometer) to quantify the amount of methacrylate groups

attached. To calculate percent methacrylation for PVA, the area of

methacrylate vinyl proton peaks, d¼6.1 (s, H1) and 5.8 (s, H2) was

compared to the area of the protons in the PVA backbone, d¼ 4.0

(s, H3).[3,32] Similarly, percent methacrylation of sericin is calculated

by comparing the area of the methacrylate vinyl proton peaks to

the area of the protons associated with the serine and glycine

groups, d¼3.6–4.0 (m, Ser bCH2 and Gly aCH2), in the sericin

backbone.[33] The number of crosslinkers per chain was then

calculated from

Crossl

% Cel

inker per chain ¼ % methacrylation �MWpolymer

MWRU(1)

where, MW is molecular weight and RU is the repeating unit of the

polymer.

2.2.2. L929 Cell Growth Inhibition Study

Samples (PVA, PVA-MA, sericin and sericin-MA) were prepared at

concentrations of 4 mg �mL�1 (in 0.9% saline) and diluted to

1 mg �mL�1 with Eagle’s minimum essential medium (EMEM)

containing 10% fetal bovine serum (FBS) and 1% penicillin/

streptomycin (PS). Murine dermal fibroblasts (L929) at a concen-

tration of 5�104 cells �mL�1 were seeded onto 35-mm diameter

tissue culture dishes. After 24 h of incubation at 37 8C in a 5% CO2

humidified atmosphere, the media was discarded and 1 mL of

sample solution was added to the cells. Following an additional 48 h

of incubation, the cells were trypsinized, and counted with a cell

viability analyzer (Vi-cell XR, Beckman Coulter). The percentage cell

growth inhibition was calculated according to

l growth inhibition ¼ 1 � Number of cells in sample dish

Number of cells in media control dish

(2)

2.2.3. Antioxidant Properties of Sericin

The antioxidant properties of sericin-MA compared to the non-

methacrylated sericin isolated from both B. mori and A. mylitta

cocoons was determined following a previously described proto-

col.[15] In brief, human keratinocytes (HaCaT cells) were seeded in

tissue culture dishes (35 mm diameter) at a density of

1�106 cells �mL�1 and allowed to attach and grow for 24 h. The

cells were pre-treated with sericin and sericin-MA (50 and

100 ng �mL�1) for 24 h and then subjected to hydrogen peroxide

(H2O2, 0.8�10�3M) induced oxidative stress. The pre-treated and

stressed cells were then washed and harvested by trypsinization.

The cells were resuspended in sterile PBS and homogenized then

centrifuged for 10 min at 5 000 rpm to collect the supernatant of

each sample for biochemical assays. HaCaT cell damage caused

by H2O2 was quantitatively assessed from the ratio of catalase,

lactate dehydrogenase (LDH) and malondialdehyde (MDA) released



from damaged cells to the undamaged cells after the induction of

oxidative stress. Catalase is an enzyme secreted when cells are

oxidative damaged, and was determined by measuring the

reduction in absorbance of NADH at 240 nm for 5 min following

the breakdown of hydrogen peroxide. LDH activity corresponding

to the cell membrane integrity was expressed as change in

absorbance of the reaction mixture at 340 nm per min following the

breakdown of pyruvate by the enzyme on addition of NADH. MDA

concentration in the medium, which reflects the degree of lipid

peroxidation, was quantified by measuring the absorbance at

535 nm following reaction with thiobarbituric acid reagent

(0.5 cm3, 1:1 v/v, mixture of 0.67% thiobarbituric acid and acetic

acid).

2.2.4. L929 Cell Adhesion and Proliferation Studies on

Sericin Coatings

24 well polystyrene plates (well area of 2 cm2), were coated with

sericin and sericin-MA. In brief, sterile filtered sericin and sericin-

MA solutions were added to the wells then allowed to dry on a hot

plate kept at 37 8C in a sterile laminar hood for 2 h. All the coatings

were at a final concentration of 25mg � cm�2. Tissue culture plastics

(TCP), i.e. non-coated wells, were used as controls in this

experiment. L929 fibroblasts suspended in EMEM containing

10% FBS and 1% PS were seeded on the coatings and controls at

a concentration of 6000 cells �mL�1, which corresponds to a density

of 30 cells �mm�2. At the time points of interest (4 h, 1 d and 4 d), the

media and non-adherent cells were removed from the well, and

1 mL of fresh media were re-applied to each well. Each well was

then photographed (3 pictures per well) using a phase contrast

microscope at 10� magnification. ImageJ was used to count the

attached cells.

2.3. Fabrication and Characterization of PVA/Sericin

Hydrogels

2.3.1. Fabrication of PVA/Sericin Hydrogels

Dried PVA-MA was dissolved in water at 80 8C.[3] Upon complete

dissolution, the solution was cooled to room temperature and the

photoinitiator, 2-hydroxy-1-[4-hydroxyethoxy(phenyl)]-2-methyl-

1-propanone (Irgacure 2959, Ciba Specialty Chemicals), was added

to the polymer solution (0.05 wt%). The samples were UV-irradiated

(Greenspot, peaks 310–365 nm, 30 mW � cm�2) for 3 min.[3,32,34] To

prepare PVA/sericin hydrogels, both PVA-MA and sericin-MA were

dissolved separately then mixed at room temperature prior to

addition of initiator. The total content of macromer in the hydrogels

was 20 wt%.

2.3.2. Swelling Study and Mass Loss Analysis

Hydrogel discs (10 mm diameter, 1 mm thick) were fabricated for

the swelling and mass loss studies.[3,32,34,35] The initial wet mass of

each sample (minitial;t0) was measured, and three samples were

immediately lyophilized to obtain their dry weights (mdry;t0). This

enabled the calculation of the actual starting% macromer for all

samples,[3]

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% macromer ¼mdry;t0

minitial;t0

� 100 (3)

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The initial dry mass of the remaining samples (mi,dry) was then

calculated using the actual % macromer (Equation 3) and each

disc’s individual starting wet weight (minitial).

www.M

mi;dry ¼ minitial � % macromer (4)

se samples were then submerged in a sink of phosphate-
The
buffered saline solution (pH¼7.4), and incubated in an orbital

shaker incubator set at 37 8C. Samples were removed from the

incubator after 1 d, blotted dry and weighed (mswollen). The swollen

samples were then freeze-dried and weighed again (mdry). The mass

swelling ratio (q) and mass loss/sol fraction were calculated as

follows:[3,32]

q ¼ mswollen

mdry(5)

Mass loss% ¼mi;dry �mdry

mi;dry

� �� 100% (6)

The sol fraction of the hydrogels is equal to the mass loss at 1 d, as

it has been shown that any polymer chains not attached to the

network will diffuse out of the sample in this timeframe.[34,35]

2.3.3. Sericin Release Studies

PVA/sericin hydrogels were prepared (10 mm diameter, 1 mm

thick), immersed in 2 mL phosphate buffer saline (PBS) and

incubated at 37 8C. 0.5 mL of supernatant was withdrawn after

1 d of incubation and stored frozen. The released sericin was

quantified using the microbicinchoninic acid (microBCA) assay

(BCA kit for protein determination, Pierce). In brief, the supernatant

containing sericin was mixed with the BCA reagents, then

measured for absorbance at 562 nm. The mass of sericin in the

supernatant was determined by using sericin standard curves

constructed for the two different species, respectively. The sericin

mass loss percentage was calculated as follows:

Sericin mass loss% ¼ msericin in supernatant

msericin in hydrogel

� �� 100% (7)

where msericin in hydrogel is the amount of sericin originally

incorporated into the hydrogel.


PVA/Sericin Hydrogels

Hydrogels (7.5 mm diameter, 1 mm thick) were prepared under

sterile conditions, and then soaked in media to extract the sol

fraction in the network. After 1 d, the media was discarded and

hollow metal fences were placed on top of the hydrogels to prevent

the hydrogels from floating and to ensure the cells were seeded only

on the hydrogel surface. Cells were seeded onto the hydrogels

through the metal fences (12.57 mm2) at a density of

80 cells �mm�2. Media was added through the side path of the

metal fences to surround the hydrogels. All samples were prepared

in triplicate. Live-dead assay was done to determine the amount of

the adhered cells after 4 h, 1 d and 3 d of incubation. In brief, the gels

were washed with Dulbecco’s phosphate buffered saline (DPBS)

then stained with propidium iodide (PI) and Calcein-AM (Cal-AM)

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both at 1mg �mL�1. After 10 min incubation with the stains, the gels

were again washed with DPBS then images were captured using

the fluorescent microscope. ImageJ was used to count the attached

live cells. In a separate experiment, cells were seeded onto TCP

through the metal fences, and visualization suggested that

adhesion and morphology of cells was similar to those on PVA/

sericin hydrogels.

2.4. Statistical Analysis

All results were analyzed using an ANOVA model with replication

and combination of fixed and random variables. The models were

constructed using Minitab statistical software (Minitab Inc.,

version 15). Samples in a study (n¼1) were all prepared in

triplicates, and all studies were repeated 3 times (n¼ 3). A p<0.05

was considered as statistically significant.

3. Results and Discussion

3.1. Macromer Synthesis

3.1.1. Isolation of Sericin from Silkworm Cocoons

The molecular weights of the isolated silk sericin peptides

from both species were measured by 8% SDS-PAGE analysis,

and were observed to range from 20 to 200 kDa (results not

shown), and is consistent with the literature.[12,14,16,19,36]

Current processes used to extract sericin from silkworm

cocoons involve a range of treatments including heat

(121 8C), acid (citric acid), alkali (Na2CO3) and urea. Recently,

it was shown that these extraction processes induce sericin

degradation or hydroxylation, which can further influence

the molecular weight of the sericin products.[21,37] How-

ever, numerous studies showed that these degraded sericin

peptides retain their beneficial biological properties, such

as moisture absorption, antioxidation, tyrosinase activity

inhibition, anticancer activity and protecting cells from

death caused by serum deprivation.[19,38] It was also

reported that alkali degraded B. mori sericin are suitable

for cell culture and promoted collagen production from

fibroblasts.[20] Hence, it has been well established that the

degraded sericin remained bioactive, and it is expected that

incorporating these degraded sericin peptides into PVA

hydrogels will improve the gel’s bioactivity.

3.2. Macromer Characterization

3.2.1. PVA and Sericin Methacrylation

PVA and both species of sericin peptides were all

successfully functionalized with methacrylate groups as

shown by 1H NMR (Figure 1). The methacrylate groups will

allow the polymers to be photopolymerized into cross-

linked hydrogels. For PVA-MA, ICEMA reacted with the

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Figure 1. (a) 1H NMR (D2O) of sericin peptide isolated from thecocoons of B. mori. (b) 1H NMR (D2O) of methacrylated B. morisericin. Inset: Schematic numbering of the vinyl hydrogen atomsfor methacrylate group attached to the sericin molecule.

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hydroxyl groups on the PVA backbone where as for sericin-

MA, ICEMA was hypothesized to react with the hydroxyl,

amino and carboxyl groups.[1,39] The percent functionaliza-

tion was determined by 1H NMR. Sample NMR spectra of

rawB.mori sericin and the functionalizedB.mori sericin are

shown in Figure 1 for reference. By comparing the spectra of

the two species of silkworms (data not shown), it was

observed that the area under the curve for peak composed of

serine and glycine (Ser b-CH2þGly a-CH2) for B. mori is

larger than that for A. mylitta. This agrees with previous

Table 1. Number of crosslinkers/chain of the functionalized macrom

Component Methacrylationa)

[%]

PVA-MA 1.1

B. mori sericin-MA 2.0

A. mylitta sericin-MA 2.0

a)As determined by means of 1H NMR.



studies that the total amount of serine and glycine inB.mori

cocoon sericin (45.5 mol%) is higher than that found in

A. mylitta cocoon sericin (35.9 mol%).[16,18] As reported in

the literature, the molecular weight of the alkali degraded

B. mori and A. mylitta sericins are between 20 and

200 kDa.[12,14,16,19,36] Thus, using 1H NMR and the area

under the respective peaks, it was quantified that the 2%

methacrylated sericins had anywhere from 4 to 40 cross-

linkers per chain, depending on the length of the sericin

chain (Table 1).

3.2.2. L929 Cell Growth Inhibition Studies

The first test to characterize the biological functionality of

sericin and sericin-MA is to ensure that the macromers do

not cause cell toxicity. Cell growth inhibition tests are

commonly used as a measure of toxicity, where previous

studies have shown that ester acrylate modified PVA

displayed minimal growth inhibition of L929 cells, and that

the modification resulted in only slight increase in the

percentage growth inhibition over unmodified PVA.[32] It

was also reported that the levels of inhibition increased

with increasing amounts of functional groups.[40,41] How-

ever, it was observed in this study that both PVA and PVA-

MA showed relatively low inhibitory effect on cell growth,

and there was no difference between PVA and PVA-MA

(Figure 2). Raw silk sericin samples also resulted in small

amounts of cell growth inhibition (<10%). B. mori sericin

has been reported to enhance cell proliferation at a

concentration of 1 mg �mL�1,[42,43] however these results

were not reflected in this study as there was still a small

percentage of inhibition. Furthermore, sericin-MA for both

A.mylitta and B.mori showed higher cell growth inhibition

as compared to native sericin (Figure 2), although again this

difference was not statistically different. The slight increase

in cell growth inhibition with the methacrylated sericin

indicates that the methacrylate groups themselves are

interacting with and influencing the cells. These studies

reflect a worst case scenario, as the cells are exposed to the

unreacted functional groups for 48 h. In the final hydrogel

system, cells would only be exposed to the methacrylates

for less than 5 min if they were being encapsulated, or not at

all if the cells were only exposed to the scaffold material

ers.

Molecular weight

[kDa]

Crosslinkers

per chain

13–23 3–6

20–200 4–40

20–200 4–40

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Figure 3. Percentage release of (a) catalase, (b) LDH, (c) malonal-dehyde (MDA) in normal cells (control), cells exposed to H2O2,cells preincubated with sericin and sericin-MA followed byexposure to H2O2. Error bars represent the standard deviationof n¼ 3. Sericin and sericin-MA of both species and concentrationwere significantly different (p<0.05) to H2O2 for catalase, LDHand MDA release.

Figure 2. Inhibitory effect of PVA-MA and sericin-MA used forPVA/sericin hydrogels fabrication on L929 cells growth. Error barsrepresent standard deviation for n¼ 3.


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after polymerization. Therefore, the slight increase in cell

growth inhibition for the methacrylated sericins does

indicate their possible detrimental effects on cells, however

due to the relatively low inhibition (less than 16%) this is

not a concern when considering the final application of the

materials.

3.2.3. Antioxidant Properties

Methacrylate groups were attached by substituting hydro-

xyl, amino and carboxyl groups on the sericin chain.

Therefore, biological functionality of the resulting metha-

crylated sericin might be compromised depending on

where and how many of those substitutions occurred.

Sericins have been shown to possess photo-protective effect

against UVB-induced acute damage,[15] and protect a skin

fibroblast cell line against H2O2 induced oxidative stress.[14]

Figure 3 shows the percentage of catalase, LDH and MDA

activities released in the medium by normal keratinocytes

cells (control), cells treated with 0.8� 10�3M H2O2 for 24 h

and cells pre-incubated with 50 and 100 ng �mL�1 of sericin

and sericin-MA before treatment with H2O2. The catalase

activity (Figure 3a) was found to be significantly lower in

cells pre-treated with sericin and sericin-MA of both B.mori

and A. mylitta respectively, when compared to H2O2-

treated cells. As catalase is an enzyme known to be elevated

in oxidative damaged cells,[15] this result indicates that

cells treated with the sericins are less damaged, and

further suggests that the sericins are protecting the cells

against H2O2 induced oxidative stress. Moreover, the sericin

pre-treated cells also showed lower LDH release when

compared to cells treated with H2O2 (Figure 3b). LDH

activity is a measure of cell membrane integrity,[15] and this

result shows that the membranes of sericin pre-treated cells

are less damaged. This result is further confirmed by the

lower levels of MDA examined in sericin pre-treated cells

(Figure 3c). MDA is a product of lipid peroxidation and

serves as a measure of the damage level caused to the cell

membrane. Sericin had been previously reported to be




antioxidative and was believed to be due to the higher

content of hydrophobic amino acids.[15,16,44,45] The anti-

oxidant properties of the sericin and sericin-MA of

A. mylitta do appear to be slightly better than the

B. mori, however this is not significantly different. While

sericin in general is shown to have anti-oxidative proper-

ties, it is important to compare the methacrylated to non-

methacrylated sericin. As can be seen (Figure 3), there are no

significant differences when methacrylates groups are

added as both B. mori and A. mylitta sericin-MA showed

similar protective effect to that of the non-functionalized

sericins on keratinocytes. As only a small portion of the

residues on the sericins were functionalized (2%), it was

anticipated that the overall antioxidant property of the

sericin would not be affected.

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Figure 4. Number of attached L929 cells on sericin and sericin-MAcoatings at t¼4 h, 1 d and 4d. Error bars represent the standarderror mean (SEM) for n¼ 3. Sericin and sericin-MA of both speciesare statistically different (p<0.001) to TCP at t¼4 d.

Table 2. Averagemass swelling ratio (q) andmass loss of PVA andPVA/sericin hydrogels 1 d post-polymerization.

PVA-MA

[wt%]

Sericin-MA

[wt%]

Mass

swelling ratio

[q]

Mass

loss

[%]

20 – 6.4� 0.3 21.8� 3.1

19 1 (B. mori) 6.6� 0.2 21.0� 3.7

19 1 (A. mylitta) 6.6� 0.3 21.1� 2.8

18 2 (B. mori) 6.9� 0.4 22.1� 2.8

18 2 (A. mylitta) 6.8� 0.5 23.4� 5.4

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Sericin Coatings

The ability of sericin to encourage cell attachment is one of

the key biological properties that are focused on this paper.

L929 fibroblasts are well known to adhere and proliferate on

TCP, and therefore TCP was used as a ‘‘gold standard’’

reference in comparison to the sericin coatings. Figure 4

shows the attachment of L929s on sericin coatings at

various time points. At the initial attachment period (4 h

and 1 d), the number of cells attached on the TCP and sericin

coatings were similar. These results confirmed that sericin

of both species does facilitate cell attachment, even though

there were no cell adhesion sequences such as RGD and

YIGSR present in the protein. After 4 d of incubation, there

were significantly more cells attached on the sericin

coatings as compared to TCP. Previous studies that were

focusing on B. mori sericin reported that it supports

fibroblasts adhesion and proliferation.[23,46] As the cell

attachment properties of A. mylitta had never been

characterized, this study highlights that sericins of both

species are similar in terms of facilitating cell attachment

and proliferation. It has been reported that the serine-rich

repetitive sequences found inB.mori sericin are responsible

for the cell attachment activity.[23] Therefore, as B. mori

sericin has more serine (39 mol%) as compared toA.mylitta

(18 mol%),[25,28] it was expected that more cells would

adhere and proliferate on B. mori sericin coatings but

statistical analysis showed no significant difference

between the two species. Next, the grafting of sericin with

methacrylate groups did not result in a significant decrease

in cell proliferation activity, and sericin-MA coatings from

both species showed a higher amount of attached cells

compared to the TCP control (Figure 4). Although it was

hypothesized that the functionalization process might

denature or degrade the sericin peptides, hence poses a

negative effect on the bioactivity, it was not reflected in this



study. Furthermore, sericin-MA from both B. mori and

A. mylitta were similar in terms of the cell adhesion and

proliferation activity. Hence, it can be concluded from this

study that sericins extracted from both species possess

similar biofunctionality in terms of fibroblast adhesion and

proliferation, and that the bioactivity is retained after

methacrylation. While the methacrylation process did

decrease the cellular activity, as compared to unmodified

sericin, it was still enhanced over the TCP control.

3.3. Fabrication of PVA/Sericin Hydrogels

PVA/sericin hydrogels were successfully fabricated from

both silkworm species at ratios of 19/1 and 18/2 PVA/

sericin-MA with 0.05 wt% initiator. However, it should be

noted that while a gel formed for the 18/2 PVA/A. mylitta

combination, the gels were observed to be of lower quality

with excess liquid present after polymerization was

complete. The reason this only occurred in the higher

percentage A. mylitta as opposed to the B. mori sericin was

hypothesized to be due to the structural difference between

A. mylitta and B. mori sericin. Sericin is a protein composed

of random coil and b-sheet molecular structures, and

A. mylitta cocoon sericin has been reported to have less b-

sheet inducing amino acids such as tyrosine and serine as

compared to B. mori.[12] The A. mylitta has more random

coils, and thus may be harder for the methacrylate groups to

be available for hydrogel formation, or cause radical

entrapment.[34,47,48] The antioxidative property of seri-

cin,[14] might also cause radical scavenging where the

radicals are neutralized by receiving electrons from

sericin.[49] It was reported that A. mylitta is more

antioxidative (Figure 3),[14] hence can act as a greater

radical scavenger.

3.3.1. Swelling Study and Mass Loss Analysis

The mass swelling ratio (q) and the percent mass loss were

measured for the pure PVA and the PVA/sericin hydrogels at

1 and 7 d after polymerization and incubation in PBS. The

results at 1 d are given in Table 2, and similar results were

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obtained at 7 d (data not shown). It has been previously

shown that any polymer chains not incorporated in the PVA

gels will be released with the first 24 h,[34,35] and no further

changes in swelling or mass loss occur, and these results

demonstrated similar findings. Statistical analysis showed

that there was no significant difference between the mass

swelling ratio (q) of the pure PVA gels and either of the 19/1

PVA/sericin gels. However, the values of q for 18/2 PVA/

sericin gels from both species were statistically higher than

that of the pure PVA gels. Previous studies have reported

that incorporation of ionic biological polymers such as

heparin and chondroitin sulfate into PVA hydrogels has

increased the swelling capacity,[1,3,50] and this study also

demonstrates a trend towards increased swelling with the

incorporation of sericin. Sericin proteins have been reported

to be negatively charged at neutral pH,[26,50] and therefore it

would be expected that the more negatively charged chains

that are incorporated the more the swelling will increase.

All the mass loss values obtained (Table 2) were

comparable to each other, and to previous studies done

on photopolymerized methacrylate hydrogels.[3,30,34,35]

Statistical analysis showed that there were no significant

differences in the mass loss for PVA and PVA/sericin

hydrogels, which indicates that the sericin peptides did not

disrupt the polymerization process. Although the photo-

polymerization process with this PVA macromer is not

100% efficient, it is known that the polymerization kinetics

can be controlled through a variety of techniques. Previous

studies done in our lab had shown that using other

functional groups that are more reactive (ester acrylates) or

increasing the amount of methacrylates grafted on the PVA

backbone can increase the polymerization efficiency.[35]

3.3.2. Sericin Release Studies

Another indication of the quality of the gel formation is the

measurement of the amount of sericin that is lost from

Figure 5. Sericin release (%) from fabricated PVA/sericin hydrogelsafter 1 d of incubation. Three gels were prepared for each exper-iment (n¼ 1) and a total of three repeats of each experiment wereperformed (n¼ 3). As sericin release varied throughout the threeexperiments, all nine data points were shown.




the gel. As shown in Figure 5, there is a large amount of

sericin that was lost from all of the gels in the first 24 h,

although in all cases a significant portion of the sericin still

remained within the gel. This is consistent with previous

findings that all unincorporated molecules will diffuse out

of the hydrogel during this time frame.[34,35] The amount of

sericin released from the gels ranged between 20 and 60% of

the original amount of sericin incorporated. The release and

the quantification of the amount of sericin was variable

throughout the three experiments, therefore all of the data

is shown in Figure 5 for completeness. The 18/2 PVA/

A. mylitta gels had the greatest variability in the results,

although were within the same range as the other gels. It

was hypothesized that increasing the concentration of

sericin may perturb the crosslinking process and thus result

in an increase in ‘‘unbound’’ sericin able to be released. The

mechanism underlying this perturbation is likely to relate

to the antioxidative radical scavenging properties of sericin.

This is supported by the observation that the higher

variability of release in A. mylitta gels correlated with the

A. mylitta sericin being more anti-oxidative. The two

silkworm species are biochemically different, and therefore

incomparable biological behaviors can be expected. This

increase in variability, along with the visual differences in

these gels, supports the hypothesis that the sericin is acting

in some way to decrease the effectiveness of the

polymerization, whether through radical scavenging or

another method. Increasing the percent methacrylation of

sericin would support formation of more highly crosslinked

networks of PVA and sericin, which could reduce the

variation in sericin release. The disadvantages associated

Figure 6. Number of attached L929 cells on PVA and PVA/sericinhydrogels at t¼4 h, 1 d, and 3 d. Error bars represent the standarderrormean (SEM) for n¼ 3. The numbers of cells attached on PVA/sericin hydrogels are significantly different to 20% PVA hydrogels(p<0.001). PVA/sericin hydrogels of A. mylitta has significantlyhigher cell attachment compared to B. mori (p¼0.001). In termsof sericin concentration, cell attachment on 18/2 PVA/sericinhydrogels is significantly higher to 19/1 PVA/sericin for B. mori(p<0.05) but not for A. mylitta (p>0.05). For noting, in aseparate experiment, cells were grown on TCP by the samemethods and the number of attached L929 cells were quantifiedto be 68� 3 (t¼ 4h), 98� 4 (t¼ 1 d) and 436� 20 (t¼ 3 d).

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with increasing the sericin functional group density relate

to the potential for disruption of biological function with

higher methacrylate substitution. This would need to be

studied in future research.

3.3.3. Cell Adhesion Studies

The cell adhesion study was used to evaluate the biological

functionality of the PVA/sericin hydrogels. The aim was to

determine if the sericin still had bio-functionality after

chemical modification and incorporation into the gels, and

also if the sericins were accessible to cells exposed to the

surface of the gels. It is known that cells do not adhere to

pure PVA hydrogels due to its highly hydrophilic nat-

ure.[51,52] Therefore, pure PVA hydrogels were used as a

control, and as expected, L929 cells did not adhere and

proliferate on them (Figure 6). Previous studies have

reported that by incorporating biological polymers such

Figure 7. L929 cells attached on PVA/sericin hydrogels at t¼4 h, 1 d and 3 d respectively:(a) 20% PVA; (b) 19/1 PVA/B. mori; (c) 18/2 PVA/B. mori; (d) 19/1 PVA/A. mylitta; (e) 18/2PVA/A. mylitta. Green¼ live cells; Red¼dead cells. Scale bar¼ 200mm.

as chitosan into PVA hydrogels, the

attachment of fibroblasts on the hydrogel

surfaces increased greatly due to electro-

static interactions between the cells and

chitosan amino acid groups,[53] hence it

was expected that PVA/sericin hydrogels

would have better cell adhesion proper-

ties as compared to pure PVA hydrogels. A

marked difference was observed in cell

numbers for both types of sericin

incorporation, even at the low amounts

of sericin addition (Figure 6). The 19/1

PVA/B. mori gels showed similar low

numbers of cells up to the 1 d time point,

however by 3 d a significant number of

cells had attached to the surface. In

addition, it was observed that 18/2

PVA/B. mori gels showed a higher cell

attachment at 1 and 3 d, when compared

to 19/1 PVA/B. mori gels. A more sig-

nificant difference was seen through the

incorporation of the A. mylitta sericin, as

cells adhered to the gels within 4 h, and

the cells continued to thrive and prolifer-

ate for the entire length of the study (3 d).

However, a comparison done between

19/1 PVA/A. mylitta and 18/2 PVA/

A. mylitta gels showed that there were

no distinct differences in terms of the gels’

ability to facilitate cell adhesion. Both the

19/1 PVA/A. mylitta and 18/2 PVA/

A. mylitta showed comparable cell adhe-

sive properties to TCP (see caption of

Figure 6). For both species, it is clear that

the incorporation of sericin was success-

ful, and the biological activity of the

peptides was maintained. In addition,



this study confirmed that biological activity was imparted

into the PVA hydrogels.

Figure 7 illustrates the different morphologies of the

attached cells at the 4 h, 1 d and 3 d time point. It can be

clearly noted that the cells were attached but rounded on

the PVA, 19/1 and 18/2 PVA/B. mori gels, whereas the cells

take on their more natural spread morphology on the 19/1

and 18/2 PVA/A. mylitta gels. Moreover, it can be observed

that the amount of cells attached on the PVA/sericin

hydrogels after 1 and 3 d of incubation exceeded the initial

amount of cells seeded (80 cells �mm�2). Therefore, this

study demonstrated that the incorporated silk sericin

peptides enhanced not only the attachment, but also the

proliferation and spreading of the L929 cells, and A. mylitta

provided better cellular interactions in comparison to

B.mori. It was initially hypothesized that A.mylitta being a

wild type silkworm, is more capable of withstanding the

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harsh conditions of the extraction and functionalization

processes used to synthesize sericin-MA.[14,15] In this case,

theA.mylitta sericin would therefore be less denatured and

degraded than the B. mori sericin, and retained more of its

structure, biological and chemical characteristics of the

natural sericin. However, the cell adhesion and prolifera-

tion study done on sericin coatings showed no difference

between both species, before and after methacrylation

(Figure 4), this finding implies that the resistive nature ofA.

mylitta may not be the answer to the vast difference

observed. As such, it may be due to the way sericin is bound

in the hydrogel network and presented to cells. The

different amino acid composition and structural conforma-

tion of both sericins may affect the radicals pathway during

the crosslinking process, and cause dissimilarity in sericin

distribution in the hydrogels. The interaction between

sericin-MA and PVA-MA during the hydrogel fabrication is

yet to be explored and is beyond the scope of this paper. As

the exact nature of the biofunctionality of the sericin

proteins or peptides is not known, the precise reason for the

differences between the two types of sericins is also difficult

to determine. While none of the known cell adhesion

sequences are found in the sericin (e.g., RGD, YIGSR), they

remain to be fully sequenced. It is hypothesized that

the sericin may contain other, lesser known adhesion

sequences such as the serine-rich repetitive sequence found

in B. mori,[23] or that the sericins are inducing attached

fibroblasts to secrete type 1 collagen that is known to

enhance cell proliferation. The characterization of the

structure, chemistry and denaturation of the sericin

proteins or peptides, is outside the scope of this paper;

however, it is clear that sericin retained its bioactivity, i.e.

antioxidative and cell adhesion properties, after modifica-

tion, and these attributes were successfully transmitted

into PVA hydrogels. Moreover, there are very distinct

differences between the two species and the exact nature of

those differences needs to be explored.

4. Conclusion

In conclusion, isolated cocoon sericin peptides from B. mori

and A. mylitta were chemically modified and successfully

copolymerized with PVA to form PVA/sericin hydrogels.

Both the modified and unmodified sericins were character-

ized to be similar in terms of bio-functionality. The

incorporation of sericin did not significantly affect the

quality of the gels as compared to the pure PVA gels. In

addition, while not all incorporated sericin-MA molecules

were crosslinked into the hydrogel network, a significant

portion of the peptides remained after polymerization and

incubation. There were distinct differences between the

properties of sericin obtained from the two silkworm

species. These results demonstrated that the A. mylitta




sericin appeared to have a higher radical scavenging

capability, and was also more biologically active and

available after incorporation into the hydrogels as com-

pared to B. mori sericin. This work demonstrates that

PVA/sericin hydrogels have the potential to be used in

biomedical device applications, especially as tissue engi-

neering scaffolds, and that the addition of sericin might be

an easy method of changing the bio-functionality of

pure synthetic hydrogels without interfering with the

base network structure. This novel PVA/sericin hydrogel

may be applied in encapsulation systems for various cell

types where cell survival and differentiated cell function

combined with immunoprotection of encapsulated cells

are required. Specifically, such encapsulation systems may

be used for insulin producing islet cells for treatment of

diabetes, where the incorporated sericin provides antiox-

idative capacity and UV protection to cells during the photo-

encapsulation process. Furthermore, the presence of sericin

in the gel may also protect encapsulated cells in vivo by

scavenging harmful reactive oxygen species.

Acknowledgements: The authors kindly thank Ms. Cathy Liu forher work on the mass loss and swelling studies, and Dr. Ross Odellfor his help with statistical analysis. This work was supported bythe Australian Government under the Australia-India StrategicResearch Fund (BF010049) and Indo Australia Biotechnology Fund,DBT (BT/PR9552/ICD/16/755/2006), Government of India, NewDelhi, and is a joint effort from the laboratories of Prof. S. C. Kunduat the Indian Institute of Technology, Kharagpur and Prof. Poole-Warren and Dr. Martens at the University of New South Wales.

Received: July 22, 2011; Revised: September 28, 2011; Publishedonline: January 16, 2012; DOI: 10.1002/mabi.201100292

Keywords: biomaterials; hydrogels; photopolymerization; poly-(vinyl alcohol); silk sericin

[1] S. J. Bryant, K. A. Davis-Arehart, N. Luo, R. K. Shoemaker, J. A.Arthur, K. S. Anseth, Macromolecules 2004, 37, 6726.

[2] B. B. Mandal, B. Ghosh, S. C. Kundu, Int. J. Biol. Macromol.2011, 49, 125.

[3] A. Nilasaroya, L. A. Poole-Warren, J. M. Whitelock,P. Jo Martens, Biomaterials 2008, 29, 4658.

[4] E. Farrell, F. J. O’Brien, P. Doyle, J. Fischer, I. Yannas, B. A.Harley, B. O’Connell, P. J. Prendergast, V. A. Campbell, TissueEng. 2006, 12, 459.

[5] H. J. Lee, J. S. Lee, T. Chansakul, C. Yu, J. H. Elisseeff, S. M. Yu,Biomaterials 2006, 27, 5268.

[6] J. W. Nichol, S. T. Koshy, H. Bae, C. M. Hwang, S. Yamanlar,A. Khademhosseini, Biomaterials 2010, 31, 5536.

[7] J. Zimmermann, K. Bittner, B. Stark, R. Mulhaupt, Biomaterials2002, 23, 2127.

[8] S. P. Massia, S. S. Rao, J. A. Hubbell, J. Biol. Chem. 1993, 268,8053.

12, 12, 322–332


332

www.mbs-journal.de


[9] S. Hou, Q. Xu, W. Tian, F. Cui, Q. Cai, J. Ma, I.-S. Lee, J. Neurosci.Methods 2005, 148, 60.

[10] H. Teramoto, K. Nakajima, C. Takabayashi, Biosci. Biotechnol.Biochem. 2005, 69, 845.

[11] A. Motta, B. Barbato, C. Foss, P. Torricelli, C. Migliaresi,J. Bioact. Compat. Polym. 2011, 26, 130.

[12] R. Dash, S. K. Ghosh, D. L. Kaplan, S. C. Kundu, Comp. Biochem.Physiol. Part B: Biochem. Mol. Biol. 2007, 147, 129.

[13] T. Khire, J. Kundu, S. C. Kundu, V. K. Yadavalli, Soft Matter2010, 6, 2066.

[14] R. Dash, C. Acharya, P. Bindu, S. Kundu, BMB Rep. 2008, 41,236.

[15] R. Dash, M. Mandal, S. Ghosh, S. Kundu, Mol. Cell Biochem.2008, 311, 111.

[16] R. Dash, S. Mukherjee, S. C. Kundu, Int. J. Biol. Macromol. 2006,38, 255.

[17] Y. Q. Zhang, Biotechnol. Adv. 2002, 20, 91.[18] M. Mondal, K. Trivedy, S. Nirmal Kumar, Caspian J. Env. Sci.

2007, 5, 63.[19] P. Aramwit, S. Damrongsakkul, S. Kanokpanont, T. Srichana,

Biotechnol. Appl. Biochem. 2010, 55, 91.[20] P. Aramwit, S. Kanokpanont, T. Nakpheng, T. Srichana, Int. J.

Mol. Sci. 2010, 11, 2200.[21] Y. Q. Zhang, M. L. Tao, W. D. Shen, Y. Z. Zhou, Y. Ding, Y. Ma,

W. L. Zhou, Biomaterials 2004, 25, 3751.[22] K. Tsujimoto, H. Takagi, M. Takahashi, H. Yamada,

S. Nakamori, J. Biochem. 2001, 129, 979.[23] K. Tsubouchi, Y. Igarashi, Y. Takasu, H. Yamada, Biosci. Bio-

technol. Biochem. 2005, 69, 403.[24] N. Nagai, T. Murao, Y. Ito, N. Okamoto, M. Sasaki, Biol. Pharm.

Bull. 2009, 32, 933.[25] S. C. Kundu, B. C. Dash, R. Dash, D. L. Kaplan, Prog. Polym. Sci.

2008, 33, 998.[26] H. Oh, J. Lee, A. Kim, C. Ki, J. Kim, Y. Park, K. Lee, Fibers Polym.

2007, 8, 470.[27] B. C. Dash, B. B. Mandal, S. C. Kundu, J. Biotechnol. 2009, 144,

321.[28] B. B. Mandal, A. S. Priya, S. C. Kundu, Acta Biomater. 2009, 5,

3007.[29] A. Muhlebach, B. Muller, C. Pharisa, M. Hofmann, B. Seiferling,

D. Guerry, J. Polym. Sci. Part A: Polym. Chem. 1997, 35, 3603.[30] F. Cavalieri, F. Miano, P. D’Antona, G. Paradossi, Biomacro-

molecules 2004, 5, 2439.



[31] S. Sofia, M. B. McCarthy, G. Gronowicz, D. L. Kaplan, J. Biomed.Mater. Res. 2001, 54, 139.

[32] D. Mawad, P. J. Martens, R. A. Odell, L. A. Poole-Warren,Biomaterials 2007, 28, 947.

[33] Y. Tamada, M. Sano, K. Niwa, T. Imai, G. Yoshino, J. Biomater.Sci. Polym. Ed. 2004, 15, 971.

[34] P. Martens, K. S. Anseth, Polymer 2000, 41, 7715.[35] P. Martens, J. Blundo, A. Nilasaroya, R. A. Odell, J. Cooper-

White, L. A. Poole-Warren, Chem. Mater. 2007, 19, 2641.[36] Y. Takasu, H. Yamada, K. Tsubouchi, Biosci. Biotechnol.

Biochem. 2002, 66, 2715.[37] P. Vaithanomsat, V. Kitpreechavanich, Sep. Purif. Technol.

2008, 59, 129.[38] M. Takahashi, K. Tsujimoto, Y. Kato, H. Yamada, H. Takagi,

S. Nakamori, Biotechnol. Lett. 2005, 27, 893.[39] T. Furuzuno, K. Ishihara, N. Nakabayashi, Y. Tamada, Bioma-

terials 2000, 21, 327.[40] H. R. Mellor, J. Nolan, L. Pickering, M. R. Wormald, F. M. Platt,

R. A. Dwek, G. W. J. Fleet, T. D. Butters, Biochem. J. 2002, 366,225.

[41] E. Yoshii, J. Biomed. Mater. Res. 1997, 37, 517.[42] P. Aramwit, S. Kanokpanont, W. De-Eknamkul, T. Srichana,

J. Biosci. Bioeng. 2009, 107, 556.[43] S. Terada, T. Nishimura, M. Sasaki, H. Yamada, M. Miki,

Cytotechnology 2002, 40, 3.[44] N. Kato, S. Sato, A. Yamanaka, H. Yamada, N. Fuwa,

M. Nomura, Biosci. Biotechnol. Biochem. 1998, 62, 145.[45] J. B. Fan, L. P. Wu, L. S. Chen, X. Y. Mao, F. Z. Ren, J. Food.

Biochem. 2009, 33, 74.[46] N. Minoura, S. I. Aiba, Y. Gotoh, M. Tsukada, J. Biomed. Mater.

Res. 1995, 29, 1215.[47] H. Teramoto, A. Kakazu, K. Yamauchi, T. Asakura, Macromol-

ecules 2007, 40, 1562.[48] C. Vepari, D. L. Kaplan, Prog. Polym. Sci. 2007, 32, 991.[49] M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur,

J. Telser, Int. J. Biochem. Cell Biol. 2007, 39, 44.[50] G. Capar, S. S. Aygun, M. R. Gecit, J. Membr. Sci. 2009, 342, 179.[51] C. R. Nuttelman, D. J. Mortisen, S. M. Henry, K. S. Anseth,

J. Biomed. Mater. Res. 2001, 57, 217.[52] T. Hayami, K. Matsumura, M. Kusunoki, H. Nishikawa,

S. Hontsu, Mater. Lett. 2007, 61, 2667.[53] T. Koyano, N. Minoura, M. Nagura, K.-I. Kobayashi, J. Biomed.

Mater. Res. 1998, 39, 486.

12, 12, 322–332


Documents

The influence of silkworm species on cellular interactions with novel pva/silk sericin hydrogels