Kanyaluk Kaewprasit1, Amornrat Promboon2, Siriporn Damrongsakkul1*

1 Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, 10330 Thailand

2 Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, 10900 Thailand

Characteristics and cell responses on silk fibroin film prepared from three

the silkworms, Bombyx Mori

Introduction

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Attractive properties in biomedical applications:

Good mechanical properties

Biocompatibility and biodegradability

Silk is consisted of two main proteins:

Silk fibroin (75-80 wt% of raw silk fiber)

Silk sericin (20-25 wt% of raw silk fiber)

Silk Silk fibroin

• An insoluble fibrous protein

• Secondary structure is betasheet

• G - X sequence (G = Glycine , X = Alanine ,Serine, Tyrosine, Valine)

Beta sheet structure of silk fibroin

Silk & Silk fibroin

3Ref: Vepari. C., Kaplan. D.L. Silk as a biomaterial, Prog. Ploym. Sci. 32(2007): 991 - 1007.

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Silk fibroin races derived from“the silkworms, Bombyx mori”

Several Bombyx mori silkworm races:

- Thai race

- Japanese race

- Chinese race

- Indian race

etc.

The applications in tissue engineering field:

- Bone & cartilage tissue

- Blood vessel

- Wound dressing

- Drug delivery

etc.

Ref: Vepari. C., Kaplan. D.L. Silk as a biomaterial, Prog. Ploym. Sci. 32(2007): 991 - 1007.

• To characterize silk fibroin from three Bombyx mori silkworm races including:

•Thai race (Nangnoi-Srisaket 1; NN) •Japanese race (K1) •Chinese race (K8)

• To compare cell responses on all silk fibroin films.

Objectives

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Experiments

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Thai race (Nangnoi-Srisaket 1; NN)

Chinese race (K8)Japanese race (K1)

The Bombyx mori cocoons were kindly given by Queen Sirikit Sericulture Center, Nakornratchasima province, Thailand.

Silk cocoons

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Silk cocoons Silk fiber degummed in Na2CO3

Wash DI water

Dialysis against DI water for 3 days

Fibroin dissolution in LiBr

Air dry silk fibroin

Ref : Kim, U.J. Park, J., Kim, H.J., and Kaplan, D.L. Effects of biodegradation of gelatin/Thai silk fibroin scaffolds. Biomaterials 26, (2005):2775-2785.

Preparation of silk fibroin solution

Silk fibroin solution

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Preparation of silk fibroin films

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Air dried overnight

Silk fibroin solution (6.5-7.0 wt%)

Diluted silk fibroin solution and casted films

Air dried overnight

Immersed in 70 vol% methanol for 30 min

Physicochemical characterizations

Degradation temperature (Td) Thermogravimetric analyzer (TGA)- Temperature range 30 – 700 ºC- Heating rate = 10 °C/min

Conformations of silk fibroin ATR-FTIR spectra - Wave number range 2500 – 650 cm-1

Amino acid compositions High performance liquid chromatography (HPLC)

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In vitro attachment and proliferation test

Isolation of rat-bone marrow derived

mesenchymal stem cells (MSCs)

Cultured in TCP containing α-MEM at 37 ºC in a 5% CO2

Cultured for 6, 24, 72, 120 h

Seed cells onto the silk fibroin films at 2×104 cells/film

Evaluate the number of MSCs attached and proliferated on films by MTT assay

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Results and discussions

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Silkworm races Td (ºC)

Thai race (Nangnoi-Srisaket 1; NN) 264.8

Japanese race (K1) 264.2

Chinese race (K8) 265.0

Table 1: The degradation temperature (Td) of Bombyx mori silk fibroin films from three

silkworm races.

Degradation temperature

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Ab

sorb

ance

(a.

u.)

Wavenumber (cm-1)

FTIR spectra of silk fibroin film

Amide I(1610-1630 cm-1)

Amide II(1510-1520 cm-1)

Amide III(1230-1270 cm-1)

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Am

ide

I

Am

ide

II

Am

ide

III

Amino acid group NN K1 K8

Acidic 2.98 3.60 3.69

Basic 1.33 1.38 1.76

Polar 14.22 17.95 17.38

Total (% by mole) 18.33 22.93 22.83

Hydrophilic groups

Hydrophobic groups

Amino acid compositions

Amino acid group NN K1 K8

Non- polar 74.73 67.39 68.17

Aromatic 6.94 9.68 9.00

Total (% by mole) 81.67 77.07 77.17

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In vitro attachment and proliferation tests

16Remark : Tissue culture plate (TCP) as a control a - g represented significant difference among samples at p < 0.05.

Substrate Cell attachment (%)

Tissue culture plate (TCP) 95.6±5.1

Thai race (NN) 68.9±1.9

Japanese race (K1) 81.1±5.1

Chinese race (K8) 78.9±6.9

Table 2: The percentage of cell attachment on Bombyx mori silk fibroin films prepared from three silkworm races.

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All silkworm races had the similar physicochemical properties :• Degradation temperature• Molecular conformation

Conclusions

Japanese silk fibroin film (K1) promoted cell attached and proliferated slightly better than the others posssibly due to the most hydrophilic amino acid groups.

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Acknowledgements

• Financial supports from The National Research University Project of Commission on Higher Education (CHE) and the Ratchadaphiseksomphot Endowment Fund (AS615A-55).

• We thank Tanom Bunaprasert, M.D. for the use of cell culture facilities at i-Tissue Laboratory, Faculty of Medicine, Chulalongkorn University.

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Thank you for your attention

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Amorphous domains • are the linkers between each crystalline domains• has almost identical 25 amino acid residue (non-repetitive

sequence), which is composed of charged amino acids not found in the crystalline regions

• GAGAGAGAGAGTGSSGFGPYVANGGYSGYEYAWSSESDFGTGS

Ref: Zhou et. al., Proteins 2001;44(2):119–22

GAGAGY - 14%

GAGYGA - 4%

GAGAGA - 3%

GAGAGS - 49%

Crystalline domains are in β-sheet forms

• The crystalline domains consist of Gly-X repeats, with X being Ala, Ser, Thr &Val

• Each domain consists of sub-domain hexapeptides including: GAGAGS, GAGAGY, GAGAGA or GAGYGA

Ref: Vepari & Kaplan, Prog. Polym. Sci. 32 (2007) 991–1007

• Silk has 12 crystalline domains which are linked by 11 amorphous chains

Heavy chain structure in fibroin proteins

Amino acid group NN K1 K8

Acidic       Aspartic acid 1.83 2.08 2.18 Glutamic acid 1.15 1.52 1.51Basic Arginine 0.30 0.31 0.44 Lysine 0.20 0.26 0.30 Histidine 0.83 0.81 1.02Polar Serine 13.42 16.87 16.30 Threonine 0.80 1.08 1.08 Cysteine - - -

Total 18.33 22.93 22.83

Hydrophilic groups Hydrophobic groups

Amino acid group NN K1 K8

Non- polar      

Glycine 38.32 33.00 35.76

Alanine 34.29 31.26 29.39

Proline 0.42 0.64 0.64

Valine 1.15 1.67 1.53

Leucine 0.27 0.38 0.43

Isoleucine 0.20 0.31 0.32

Methionine 0.08 0.13 0.10

Aromatic Tyrosine 5.75 7.66 7.16

Phenylalanine 0.98 1.69 1.39 Trytophan 0.21 0.33 0.45

Total 81.67 77.07 77.17

Amino acid compositions

25 REF : http://www.learners.in.th/blogs/posts/264172

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MTT Assay

MTT or 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Objective

To evaluate the number of cell viability.

Principle

Enzymes (e.g. mitochondrial reductase) of metabolic cells reduce MTT to a purple formazan crystals. Absorbance indicates the enzyme activity, representing cell activity and viability.

Tetrazolium (yellow colour) Formazan (purple colour)

Ref: T. Mosmann, J. Immunol. Methods.65 (1983) 55-63.

1. Prepare 0.5 mg/ml of MTT in DMEM without phenol red or PBS. PROTECT FROM LIGHT!!

2. Remove old medium, wash with PBS, add 350 µl/well of MTT solution and incubate at 37 ºC, 5% CO2 for 30 min (1-4 h for hydrophobic scaffolds or scaffolds with mass transfer limit)

3. Remove MTT solution, add 1 ml/scaffold of DMSO to elute the purple ice crystals of MTT and homogenize the purple solution

4. Measure the absorbance of the solution at 570 nm using a microplate reader

Method of MTT assay

HPLC conditions

High Performance Liquid Chromatography (HPLC)

Column : Shim-pack ISC-07/S1504Na

Mobile phase:

1.) 0.2N sodium citrate pH 3.2 (containing 7% Ethanol)2.) 0.6N sodium citrate + 0.2M boric acid pH 10.03.) 0.2M sodium hydroxide