PREPARATION OF SILK FIBROIN NANOCHITOSAN BLENDS …

www.wjpr.net Vol 7, Issue 8, 2018. 439

Gokila et al. World Journal of Pharmaceutical Research

PREPARATION OF SILK FIBROIN / NANOCHITOSAN BLENDS

TOWARDS HIGH PERFORMANCE TISSUE ENGINEERING

APPLICATIONS

S. Gokila and P. N. Sudha*

Biomaterials Research Lab Department of Chemistry, D.K.M. College for Women, Vellore,

Tamil Nadu, India.

ABSTRACT

Tissue engineering is a powerful tool to treat bone defects caused by

trauma, infection, tumors and other factors. Both Nanochitosan (NCS)

and silk fibroin (SF) are non-toxic and have good biocompatibility, but

are poor biological scaffolds when used alone. In this study, the

structure and related properties of NCS/SF Composites were

examined. The prepared sample was then characterized using advanced

analytical techniques such as FT-IR and XRD. The scaffold material

was most suitable for osteoblast growth were determined, and these

results offer an experimental basis for the future reconstruction of bone

defects. First, via freeze-drying and chemical crosslinking methods,

NCS /SF composites with different component were prepared and their

structure was characterized. Changes in the internal structure of the NCS and SF mixture

were observed, confirming that the mutual modification between the two components was

complete and stable. This favors the early adhesion, growth and proliferation of MC3T3-E1

cells. Some of the assays studied in the cell line- MC3T3-E1 include MTT and Alizarin Red.

In addition to good biocompatibility and satisfactory cell affinity, this material promotes the

secretion of extracellular matrix materials by osteoblasts. Thus, NCS/SF is a good material fo

tissue engineering.

KEYWORDS: Silk fibroin, Nanochitosan, characteristics, cell culture, in vitro studies.

*Corresponding Author

Dr. P. N. Sudha

Biomaterials Research Lab

Department of Chemistry,

D.K.M. College for

Women, Vellore, Tamil

Nadu, India.

World Journal of Pharmaceutical Research SJIF Impact Factor 8.074

Volume 7, Issue 8, 439-450. Conference Article ISSN 2277– 7105

Article Received on

05 March 2018,

Revised on 25 March 2018,

Accepted on 15 April 2018

DOI: 10.20959/wjpr20188-11164



INTRODUCTION

Bone tissue engineering seems to be a promising technology to overcome the limitations of

current bone grafts associated with limited source, immunological rejection and infection.[1]

It

is a technology that combines the principles of medical science and engineering to repair or

recover damaged tissue by the substitution with scaffolds. Scaffold serves as an artificial

extracellular matrix (ECM) which mimics the extracellular environment by providing

appropriate environmental conditions for intercellular contact and signaling. With high

porosity and interconnected network, scaffolds allow cell penetration and adhesion and

support structure.[2]

The use of Chitosan as a potential biomaterial for tissue engineering and regenerative

medicine has been investigated during the past 20 years. Chitosan is produced by

deacetylation of chitin, which is the second most abundant naturally occurring polysaccharide

in nature. Chitosan is a linear polysaccharide copolymer of β –[1-4]

linked D-glucosamine and

N-acetylated-D-glucosamine making up deacetylated and acetylated regions respectively.[3]

The amide groups on the polysaccharide chain of Chitosan can be positively charged and

solubilized when the solution pH is below 6, hence becoming a polycationic polymer.[4]

It has

excellent biocompatibility, antimicrobial and wound healing potential as well as hemostatic

properties and it has found popular use in the management of burns.[5]

By including

nanoscale particles in the polymeric scaffolds, further enhancement in the properties of

biomaterials for bone tissue engineering can be achieved.[6]

The interaction between

nanosized particles and organic polymeric materials may result in improved mechanical and

biological properties of the scaffold[7]

and hence the chitosan can be modified into its

nanoform in this work by ionic cross linking method using sodium tripolyphosphate as a

cross linking agent for various biomedical applications.

Nanosized chitosan possess high performance in tissue engineering due to its large surface-

to-volume ratio, high surface reactivity, quantum size effect, small size and these favorable

properties are being exploited recently in biomedical applications when compared to the

traditional Nanosized chitosan materials.[8]

Importantly, the blended scaffolds based on

chitosan have strong potential to be used in tissue engineering and have been evaluated in

different experimental conditions for bone regeneration.[9]

Many researchers reported that the

blending of chitosan with other polymers has substantially improved the biocompatibility and

other properties (e.g., permeability) critical for a wide-range of biomedical applications.[10]



Silk fibroin, a well-known natural fibre produced by the silkworm Bombyx mori consisting of

fibroin and glue-like sericin acts as a favorable scaffold material for bone tissue engineering.

since it possesses the tunable biodegradation rate, unique excellent mechanical properties and

the ability of supporting the differentiation of mesenchymal stem cells along the osteogenic

lineage.[11]

Silk fibroin has been gradually perceived for its biocompatibility,

biodegradability, optical performance and thus the development and application of silk

fibroin in the field of biomedical materials has gained increasing attention.[12]

The synergistic

combination of various biomaterials with silk fibroin which can be processed into various

scaffold forms leads to the formation of chemically modified composites and this provides an

impressive toolbox which allows silk fibroin scaffolds to be tailored to specific

applications.[13]

In this work it was intended to use the polymeric materials namely nanochitosan(NCS) and

silk fibroin (SF) for preparing the Binary blended scaffolds (NCS/SF) and to utilize the same

for tissue engineering.

2. Materials

Chitosan was purchased from India Sea Foods, Cochin, Kerala. Cocoons of Bombyx mori

were obtained from the sericulture farm in Vaniyambadi, Vellore District and hyaluronic acid

was purchased from Sisco chemicals Pvt, Ltd., Maharastra. MC3T3-E1 cell line was

purchased from National Cell Science Centre Pune. The crosslinking agent sodium

tripolyphosphate and the solvent formic acid and glacial acetic acid were procured from Finar

chemicals, Ahmedabad and Thomas Bakers chemicals Pvt. Ltd., Mumbai. All the chemicals

utilized in this present study were of analytical grade.

2.1. MATERIALS AND METHODS

2.1.1. Preparation of nanochitosan

As per the ionic gelation method reported by Sivakami et al., (2013), the nanochitosan was

synthesized by the interaction of negatively charged sodium tripolyphosphate (TPP) with

positively charged chitosan biopolymer in this work. In order to prepare it, initially a

homogeneous viscous chitosan gel was prepared by completely stirring known amount of

chitosan (1g) dissolved in 200ml of 2% acetic acid for a period of 20 minutes. An ionic

crosslinking agent, sodium tripolyphosphate (0.8 g of sodium tripolyphosphate dissolved in

107 ml of deionized water) was then added drop wise to the above prepared homogeneous

chitosan solution with rapid stirring for over a period of 30 min. A milky emulsion like



appearance of nanochitosan obtained was then allowed to stand overnight to settle as

suspension. The supernantant solution was decanted and finally the thick suspension of

nanochitosan settled at the bottom of the beaker was preserved in the refrigerator for further

use.

2.1.2. Preparation of silk fibroin

Short cutted silk fibers of 3 mm length (0.5g) were dissolved in 100ml of 10% LiCl in formic

acid. This silk fibroin solution was then stirred well under magnetic stirrer for a period of

2hrs. After this process is over, finally the thick emulsion of silk obtained above was

preserved in the refrigerator.

2.1.3. Preparation of nanochitosan / silk fibroin scaffold

The above prepared nanochitosan and silk fibroin solutions were mixed, neutralized and

stirred well for 2 hours to remove the air bubbles completely. This prepared solution mixture

was then freezed to -300C and freeze dried to -80

0C for overnight and the scaffold was

subjected to further studies.

2.2. Physico-chemical characterization.

FT-IR spectrum of the prepared sample was measured in the wavenumber range from 4000-

650 cm-1

using a 100 FT-IR Perkin Elmer spectrophotometer. The powder X-ray

diffractogram (XRD) of binary blended nanochitosan/ silk fibroin scaffold was measured in a

SHIMADZU XRD 6000 (Japan) diffractometer using CuKα radiation (λ = 1.5406Ǻ) with 30

mA, 40 kV and scanning rate of 3°/min.

2.3. Cell culture

Cell types were mainly used to assess the effect of the scaffold composition onto the different

stages of cell differentiation within the osteoblast lineage. MC3T3-E1 cells were cultured in

Iscove’s Medium (Sigma, USA) and MC3T3-E1 cells were added in the culture media to

induce osteoblastic differentiation. The cell-line was then washed, hydrated for 2 h with PBS

prior to cell seeding and thereafter the scaffolds were placed in a 24-well cell culture plate.

After this process, then 2×104 cells/scaffold was seeded in a volume that soaked the scaffold

and were incubated for 3 h. Five hundred µL of culture medium was added into each well.

After 24 h, the scaffolds were changed to new culture wells in order to analyze only the cells

growing into the scaffolds. Empty scaffolds (without cells added) were treated in the same

manner and used as controls, to obtain proliferation and differentiation data.



2.3.1 MTT Assay

Cell adhesion and proliferation rates were estimated by MTT assay. For cell adhesion, the

cells were loaded onto the scaffold and left for 24 h. After 24 hours, the scaffolds were

removed and the attached biomass was measured at 570 nm. The cytotoxicity properties of

the fabricated pure scaffolds were evaluated using a method. Briefly, 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) assay

was used which was prepared in phosphate buffered saline (PBS) at a final concentration of 5

mg/ ml. For cell adhesion, the cells were loaded onto the scaffold and left for 24 h. After 24

hours, the scaffolds were removed and the attached biomass was measured at 570 nm. Then,

the supernatant was withdrawn and centrifuged to prepare the conditioned extracts before the

cytotoxicity test. MC3T3-E1 cells, osteoblast-like cell. Cell viability and proliferation were

measured by MTT assay from which 50% cytotoxic concentration (IC50) was calculated.

(American Type Culture Collection (ATCC, Manassas, VA, USA).

2.3.2. Alizarin Red S Assay (ARS)

By utilizing alizarin red S (ARS) staining of the MC3T3-E1 cells, the calcium deposition can

be determined. Osteoblasts and osteocytes are involved in the formation and mineralization

of bone. Modified (flattened) osteoblasts become the lining cells that form a protective layer

on the bone surface. The mineralised matrix of bone tissue has an organic component of

mainly collagen called ossein and an inorganic component of bone mineral made up of

various salts. This study describes a sensitive method for the recovery and semiquantification

of Alizarin Red S in a stained monolayer by acetic acid extraction and neutralization with

ammonium hydroxide followed by colorimetric detection at 405 nm in a 96-well format.

Cells were cultured in different osteogenic differentiation medium for 7 days, fixed for ARS

staining and quantified for mineral deposit using the kit.

3. RESULTS AND DISCUSSION

3.1. FT-IR spectroscopy

Figure 1: FT-IR spectrum of 3D-porous scaffolds, NCS/SF.

https://en.wikipedia.org/wiki/Osteoblast

https://en.wikipedia.org/wiki/Osteocyte

https://en.wikipedia.org/wiki/Mineralization_(biology)

https://en.wikipedia.org/wiki/Collagen

https://en.wikipedia.org/wiki/Bone_mineral



The FT-IR spectrum of the prepared scaffold (Figure-1) showed the characteristic peaks of

blended moieties. The FT-IR spectrum of NCS/SF binary blended scaffold showing a strong

broad absorption band at around 3305.99 cm-1

is associated with the intra and intermolecular

hydrogen bonded stretching vibrations of OH groups with the stretching vibration of the

hydrogen bond from -NH-group.[13]

Absorption band at 2929.87 cm-1

was assigned to the

asymmetric CH2 stretching vibration attributed to the pyranose ring of Nanochitosan. The

resultant pure SF film showed these Amide I-III bands are conformationally sensitive bands

for polypeptides and proteins.[14]

Their intensity and position of these bands give information

about the molecular conformation of the materials examined in IR spectrum. In amide I and

amide II regions of the IR spectra of the films, instead of a single characteristic peak, bands

were observed. In literature, amide I (-CO- and –CN- stretching) appeared to be in the region

of 1647.21 cm-1, amide II (-NH- bending) in the region of 1544.98 cm-1 and amide III (-CN-

stretching) at 1247.97 cm-1 were attributed to silk I conformation.[15]

The band at 1456.26

cm−1

is correlated with asymmetric vibration of COO−

group while the peak from 1033.85

cm-1

is related with the C-O-C units present in silk-fibroin.[16]

At 1410 cm-1 corresponding to

valency vibration of carboxylate ion and formation of a shoulder at 1095 cm-1 corresponding

to –C=O- stretching for the SF-CH film evidenced the complex formation between the amino

groups of SF and carboxyl groups of CH.

Certain absorption bands observed at 1307.74 cm-1

, 1155.36 cm-1

and 592.15 cm-1

were

indicative of OH in plane bending, P=O stretching and C-O bending respectively.[17]

The

appearance of strong absorption bands at various wave numbers corresponding to P=O

stretching, COO- stretching, amide I, amide II and amide III stretching indicate that all added

polymeric components namely nanochitosan and silk fibroin gets interacted effectively

resulting in binary blend formation.

3.2. X- Ray Diffraction studies

X-ray diffraction technique is mainly used to study the crystallinity and the crystalline

structure of materials. A lower 2θ value indicates larger spacing and in addition broadness of

reflection, high noise and low peak intensities are characteristic of poorly crystalline

material.[18]

The X-ray diffractogram details of the nanochitosan and silk fibroin binary

blended scaffold was represented in Figure-2.



Collected Data-1

0

100

200

300

400

500

20 40 60 80

Theta/2-Theta[deg]

Inte

nsity

[CP

S]

Figure 2: X-ray diffractogram of 3D-porous scaffolds, NCS/SF.

The X-ray diffractogram of nanochitosan and silk fibroin binary blended scaffold showed a

weak and wide pattern around 2θ= 32-40° due to noncrystalline form as the typical

characteristic diffraction pattern of amorphous silk fibroin.[19]

Nanochitosan also showed up a

very broad pattern around 2θ=32° and these findings denoted that the complexation between

NCS and SF occurs through ionic interactions and this phenomenon reduced the crystallinity

which favored the β-sheet conformation for sik fibroin.[20]

In other words we can say that, the

XRD results confirmed co-existence of these polymeric substances in the blend scaffolds.[21]

by appearance of broad peak with low intensity which is also an indication of lowcrystallinity

of the prepared scaffolds.

3.3. Methyl Thiazolyl Tetrazolium (MTT) assay

The response and cytotoxicity of the prepared scaffolds was investigated through the MTT

assay by seeding the known concentration of MC3T3-E1cells onto NCS/SF/HA scaffolds.

The MC3T3-E1 cells were incubated with NCS/SF binary blended scaffold test solutions of

different concentration to determine their effect on cell viability.



1000 ml/µg 100ml/µg 10ml/µg

3 ml/µg 0.3 ml/µg control

Figure-3: Invitro cytotoxicity and cell proliferation of NCS/SF scaffolds by MTT assay

Figure-3(a): Percentage of cell viability of MTT Assay using NCS/SF scaffold.

The obtained results of Figure-3 clearly indicate that the NCS/SF based scaffold are totally

nontoxic to osteoblast-like cells and the scaffolds do not show any adverse effect on the

growth of cells.[22]

The percentage viability of the cells in scaffolds and the control was at the

same level of statistical significance. The results shown in Figure-3(a) also indicate that after

incubation for few minutes, the NCS/SF binary blended scaffold showed cell viability of

80.62% respectively and this result showed that these polymeric excipients are not cytotoxic

and not harmful to the cells.[24]

More adhered MC3T3-E1 cells were observed and it is

beneficial for cell growth and cell–cell communication. In addition, an increase in metabolic

activity with culture period is evident with all the scaffolds with a varied degree of metabolic

activity.

3.4. Alizarin Red S (ARS) assay

Alizarin Red S (ARS), an anthraquinone dye, has been widely used to evaluate calcium

deposits in cell culture. The mineralization is mainly assessed by extraction of calcified



mineral at low pH, neutralization with ammonium hydroxide and colorimetric detection at

405 nm in a 96-well format. The ARS staining is quite versatile because the dye can be

extracted from the stained monolayer of cells and readily assayed. Scien Cell’s ARS Staining

Quantification Assay (ARed-Q) provides a sensitive tool for the recovery and semi-

quantification of ARS in a stained monolayer of cells.

This assay is more sensitive than the cetylpyridinium chloride (CPC) extraction method,

improving the detection of weakly mineralizing monolayers.[25]

The bioactivity of the bone

implants was evaluated by examining the in vitro mineralization of the osteoblasts cultured in

the bone implants. The capacity of minerals deposition is a late stage marker of osteogenic

differentiation that can be used to confirm that MC3T3-E1 cells entered into the

mineralization phase to deposit mineralize ECM.[26]

25 ml/µg 50 ml/µg control

Figure 4: Alizarin Red S (ARS) images of MC3T3-E1 cell cultured on NCS/SF scaffold.

Figure 4(a): Alizarin Red S (ARS) activity of MC3T3-E1 cell culture assessed by 3D

scaffold of NCS/SF.

Figure-4 and Figure-4(a) shows the Alizarin Red S (ARS) images of MC3T3-E1 cell cultured

on NCS/SF scaffold and the ARS activity of the of MC3T3-E1 cell culture assessed by 3D

scaffold of NCS/SF binary blend. The Alizarin Red S staining showed slight reddish dots on



the NCS/SF binary blended scaffold and this indicate that the prepared sample can facilitate

the calcium mineralization of MC3T3-E1 cells.[27]

A recent study reported that the

significantly higher surface of the scaffold could facilitate the binding of cells and calcium[28]

and therefore it was concluded that the prepared binary blended NCS/SF scaffold can

promote the osteogenic differentiation and facilitate the calcium mineralization of MC3T3-E1

cells.

CONCLUSIONS

There has been a remarkable progression during the past two decades in the development of

tissue engineering scaffolds which can be attributed in large measure to novel advanced

materials. In the present study, the NCS/SF scaffold was successfully prepared and

evaluated for its suitability in bone tissue engineering applications. FTIR and XRD results

reveal the presence of specific functional groups in the blend formation and the amorphous

nature of the blended polymer. Cell attachment studies revealed that cells were able to attach

and spread throughout the NCS/SF scaffolds. These results indicated that the binary blended

NCS/SF scaffold support cell adhesion, attachment and proliferation by MTT and Alizarin

Red assays shows the non cytotoxic nature. The present study may further enhance the

understanding of biomineralization through the Alizarin Red and the results showed that the

prepared NCS/SF scaffold is suitable for bone tissue engineering applications. In the near

future, it is most likely that the NCS/SF scaffold based systems would help to reconcile the

clinical and commercial demands in tissue engineering.

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