Electrospun Cellulose Acetate Phthalate polymers potential as cell scaffolds

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Introduction

Electrospinning is an electro-physical technique commonly used for the fabrication of nanofibers from homogenous polymer solutions. The nanofibers produced can be used in tissue engineering and regenerative therapy as artificial scaffold that mimic the natural extracellular matrix (ECM). Over the years, several biocompatible scaffolds have been developed from biomaterials for tissue engineering. However, they have several limitations due to the difficulty of 3D in vitro models accurately representing the complexity of cell physiology in vivo. The biomaterials used are selected based on the requirements of the cells involved in regeneration. Polymers that support and enhance the growth of chondrogenic cells are HA and PHBV [Ito Y. et al. 2005], polyurethane [Grad S. et al. 2003], PGA and PLGA [J Z et al. 2007], PCL [Yoshimoto H et al. 2003], PLCL [Y J et al. 2008], silk fibroin [Jin H-J et al. 2004; Kim K-H et al. 2005], PLLA and PLLA/collagen [Ngima M et al. 2009], and HA/CS [Zhang Y. et al. 2008]. They have already been proven as promising biocompatible biomaterials but have had limited success in clinical trials in repairing damaged tissue.

Till date there is continuous research for developing biocompatible polymer scaffolds for the bone/cartilage tissue engineering that can overcome the limitations of the previously studied scaffolds. The numerous features of cellulosic polymers like biocompatibility, biodegradability, and mechanical support to the cells etc. are potential factors enhancing its use as tissue engineering scaffolds. Keeping this in mind, the possibility of using one of the most interesting cellulose derivatives (Cellulose Acetate Phthalate) as a biomaterial in tissue engineering has been considered in this study. CAP is gaining research interest with high value for its ability to exhibit antimicrobial and antiviral properties [Olaru N. et al. 2012]. Detailed studies on the properties of CAP have already been done in 1998 regarding the molecular mass distribution, SEC-MALLS, thermal degradation and glass transition temperature [Roxin P et al. 1998]. Previously, CAP has been used as a pharmaceutical excipient, for drug delivery, and also to prevent infection of human immunodeficiency virus (HIV-1), herpes viruses (HSV I and II), and non-viral sexually transmitted disease pathogens [Kabanov A et al. 2003; Stone A et al. 2002; Gyotoku et al. 1999; Neurath A et al. 2001; Duncan R et al.2003; Neurath A et al. 2003; Neurath A et al. 2002; Manson K et al. 200; Neurath A et al. 2002]. This study attempts to use CAP as a biomaterial for tissue engineering.

CAP is a material known for its characteristic pH dependent solubility (pH>6.0) and is used as an enteric coating material [Malm CJ et al. 1951; Balamuralidhara V et al. 2011]. This property of CAP is beneficial in the case of pharmaceuticals where the pH dependent dissolution helps in the targeted delivery of the drug. Recent studies by Olaru have been performed on the production of micro/nanofibres via electrospinning from several solvent systems such as; 2-methoxyethanol, acetone−water mixture, and 2-methoxyethanol/acetone/water mixture [Olaru N et al. 2010]. The recent study by Huang et al. shows prevention of HIV transmission using CAP nanofibers electrospun from an acetone/dimethylformamide solvent mixture [Huang C et al. 2012].

However, in this study, CAP has been used as a polymer for electrospinning nanofibres for use as a 3D scaffold. Here the instability of the material in aqueous solutions with a pH>6 has been overcome by cross-linking. In the present study the novel solvents mixture such as, acetone/ethanol, dimethylformamide/tetrahyrofuran/acetone, tetrahyrofuran/acetone, THF/ethanol, and chloroform/methanol has been developed for fabrication of nanofibers. In this paper, the first time cross-linking of CAP nanofibers by EDC and EDC/NHS has been reported leading to resistance to pH dependent solubility. Furthermore, this is the first, preliminary study to show the biocompatibility of cross-linked CAP scaffolds for chondrocytes growth which need further, more detailed study for maximizing its use in bone/cartilage tissue engineering.

Materials and methods

2.1 Materials

Cellulose acetate phthalate extrapure (Mw = 2534.12, catalogue no - 034896), EDC (Mw = 191.70, catalogue no - 054886), NHS (Mw =115.09, catalogue no - 084718), tetrahydrofuran (THF, Mw = 72.11g/mol, catalogue no - 2027245) were purchased from SRL, India. Acetone (Mw = 58.08g/mol, catalogue no - SK0SF01088), chloroform (Mw = 119.38g/mol, catalogue no - ID0IF60198), dimethylformamide (Mw = 73.10g/mol, catalogue no - AL9A590603), formaldehyde (Mw = 30.03g/mol, catalogue no - AD2AF62125) were procured from Merck. Carbinol (chemical name - methanol, Mw = 32.04g/mol, catalogue no - 029192) and ethanol (Mw = 46.07, Catalogue no - XK 13-011-00009) was obtained from CDH and CSS respectively. Phosphate-buffered saline (PBS, pH 7.4) was prepared in 18 MΩ Milli-Q ultrapure water (UPW) in the laboratory (Department of Human Genetics, SRU, Chennai, India). Dulbecco’s Modified Eagle’s Medium was purchased from HIMEDIA (Chennai, India). Fetal Bovine Serum (FBS) and Antibiotic/Antimycotic (A/A) solution (10,000 U/mL penicillin, 10mg/mL streptomycin and 25 ug/mL amphotericin B) were obtained from HIMEDIA. All the other chemicals were of reagent grade and used as received, without further purification.

2.2 Preparation of CAP solution

CAP solution was prepared from CAP powder using different combination of solvents such as acetone, ethanol chloroform, methanol, tetrahyrofuran and dimethylformamide etc. The polymer-solvent mixture composition was mixed homogenously using a vortex. The concentration of CAP tested was 7, 10, and 15 wt%.

2.3 Electrospining

The schematic diagram of electrospining hardware setup in shown in Figure 1. The typical electrospining setup was assembled in-house and consists of a peristaltic pump (Ravel Hiteks Pvt. Ltd., Perungudi,Chennai), DC high voltage power pack(Meco), sealed amplifier, needle, metal collector and silicon tube. The CAP solution was prepared in a 50ml centrifuge tube kept on a stand. One end of the silicon tube was passed through a hole in the lid of the centrifuge tube and immersed into the CAP solution. The other end was connected to the needle (21G) with the help of a connector. The needle was clamped horizontally in the metal stand. The power supply was connected to the sealed amplifier from which the positive electrode was linked to the needle while the negative electrode was connected to the metal collector wrapped in aluminium foil. The applied voltage causes the pendant polymer drop to turn into a cone, jet and finally disperse into fibers that were collected on the aluminium foil. Various parameters of electrospining such as voltage, distance between needle and collector and flow rate etc. were optimized. The voltage was tuned at the range of 10-15kV, distance between needle and collector at range of 12 -18 cm and flow rate of 0.04 -0.10 rpm.

2.4 Crosslinking of CAP fiber mats

The CAP fiber mats were subsequently crosslinked by immersion in 90% ethanol containing the cross-linking agents (50mM EDC and 50/20mM EDC/NHS). The ratio of EDC/NHS was 2.5:1. Both variations in the cross-linking reaction were allowed to proceed at 4oC for 2 and 24 hours. The resulting samples were washed in 0.1M NaH2PO4 (pH 9.1) for an hour and then rinsed in 1X PBS. The cross-linked fiber mats that peeled out from the aluminium foil were further washed in 1X PBS, twice for an hour each. The crosslinked fibers were sterilized in ethanol (70, 50, and 25% for 30 minutes each) and washed with 1X PBS overnight. The sterilized cross-linked fibers were stored in 1X PBS with antibiotics at 4oC until cell seeding.

2.5 Fourier-Transform Infrared spectrophotometric analysis of CAP

FTIR was performed for the non-crosslinked and crosslinked scaffold and the non-crosslinked scaffold dissolved in PBS (fibers were allowed to dissolve and then dried into a powder at 45-50oC) by making a pellet with 100mg of potassium bromide (KBr).The mixture was then compressed into a pellet by applying a force of 10.0 tons for 2 minutes in a pelletizer (KBr press model M-15). The spectra were recorded from 4000cm-1 to 5000cm-1 using the FTIR spectrometer with a DTGS (Deuterated Triglycine Sulphate) detector. Horizon MB software was employed to analyse the spectra and all measurements were made using 32 scans and 16cm-1 resolution. The FTIR analysis was performed in IGCAR, Department of Atomic Energy, Kalpakkam, India.

2.6 Characterization of CAP fibers

Scanning electron microscopy was used for the analysis of fiber morphology, fiber diameter, diameter distribution and fiber alignment & entanglement. The SEM sample holder with small pieces of CAP fiber were sputter-coated with gold and then examined with SEM (TS 5130 VEGA, TESCAN) at an accelerating voltage of 10kV to take the photographs. SEM analysis was performed at the Department of Nano Science and Technology, Alagappa College, Anna University, Chennai, India.

2.7 Biological assays

To analyse the biocompatibility, cell adhesion and cell proliferation of CAP scaffolds, L6 myoblast cell lines and primary chick embryo chondrocytes were seeded onto the scaffolds. For cell proliferation study, MTT assay was performed at three time points (2, 4, and 7 days) using L6 myoblast cell lines and scaffolds were further observed by H & E Staining and SEM.

Cell viability and proliferation: The MTT assay was done for measurement of cell viability and proliferation. Crosslinked scaffolds of an appropriate size were coated with 2% gelatin in 12 well plates for 24 hours at room temperature. 20μl of concentrated L6 Myoblast cells in complete DMEM were seeded on the scaffolds. It was then incubated at 37oC for an hour. After the incubation, 250 μl of complete DMEM was pipetted into the well and incubated at 37oC for 24 hours. The next day, 1 ml of complete medium was added to the wells and incubated at 37oC, 5% CO2. The MTT assay was performed at three time points (2, 4, and 6 days). 120 μl of MTT solution (5mg/ml; SRL, India) was added per well and incubated at 37oC, 5% CO2 for 4 hours. The scaffolds with attached cells were transferred into microfuge tubes with 600 μl DMSO. The pale yellow MTT solution is reduced by viable cells on the scaffolds to formazan crystals which are then dissolved by DMSO to give a dark purple color. The intensity of this color was measured by spectrometer at 595nm.

Results

CAP solution with electrospinability

Homogenous solutions of CAP polymer with (electrospinability) viscosity was obtained with different solvent combinations such as, acetone/ethanol (9:1), DMF/THF/acetone (3:3:4), THF/acetone (1:1), THF/ethanol (1:1) and chloroform/methanol (1:1) at room temperature. The suitable polymer concentration for all solvent combinations was found to be 15%. The ratio of solvents is shown in table.

Table Different solvent combination for polymer solution

S.N

Solvent

combination

Ratio

Polymer Concentration (%)

Average Time (minutes)

Viscosity

Remarks

1.

Chloroform/Methanol

1:1

15

10-12

Viscous

Clear solution

2.

THF/Ethanol

1:1

15

10-12

Moderate

Clear solution

3.

Acetone/Ethanol

9:1

15

6-7

Moderate

Clear solution

4.

THF/Acetone

1:1

15

4-5

Moderate

Clear solution

5.

DMF/THF/Acetone

3:3:4

15

4-5

Viscous

Clear solution

*DMF – Dimethylformamide, THF – Tetrahyrofuran, Moderate (+), Viscous (++)

Fabrication of CAP nanofibers using electrospining

Review of literature has revealed only a few reports regarding nanofibre production from CAP with no evidence of its use as a biocompatible scaffold. The few published reports have shown the fabrication of nanofibres using the same solvent combinations for different applications such drug delivery for HIV treatment and….. In the present study, we have formulated different solvent combination which have not been reported so far and have been shown in table. The nanofibres were (fig.) fabricated with the aim of using it as a 3D in vitro model for cell culture. Several mixtures of solvents at different ratios were made to make (spinnibility) homogenous solution of proper viscosity. The various parameters of electrospining such as voltage, flow rate, and distance between collector and needle tip were optimized to obtain nanofibres as shown in table

Table optimized electrospining parameters for fabrication of CAP fibers with different solvent mixture

Collector-needle tip distance [cm]

Height of needle from ground [cm]

Voltage applied

[kV]

Flow rate

[rpm]

Collection time

[min]

Nature of fibers

Chloroform/Methanol [1:1]

15.5

15.5

12

14

15

8

10

10

10

17.5

0.04

0.04

0.04

0.07

0.10

30

30

45

30

60

Beaded fibers

Beaded fibers

Fine fibers, few beads

Broken fibers

Fine fibers

Tetrahydrofuran/Ethanol [1:1]

8.5

14

14

15

10

10

9.5

0.04

0.07

0.08

45

90

30

Breaks in fibers

Ribbon-like fibers

Broken fibers with ribbon defects

Tetrahydrofuran/Acetone [1:1]

13

13

15

9.5

10

0.03

0.07

90

90

Fine fibers

Fine fibers

Dimethylformamide/Tetrahydrofuran/Acetone [3:3:4]

13.5

14

15

15

12

12

14-18

0.03

0.07

0.05

120

120

Breaks in fibers

Beaded fibers

Fine fibers

Acetone/Ethanol [9:1]

13

10

15

6

15-16

0.09

0.07

30

90

Fibers with breaks and

bead defects

Fine fibers

Cross-linking of CAP fibers

There has been no evidence reported for the crosslinking of CAP fibers. However, in this study the various cross-linking agents have been tested for the stabilization of CAP (pH> 6.0). The different chemical (glyoxal, citric acid, maleic acid, formaldehyde, and gluteraldehyde) and physical (heat, UV, and Gamma radiation) cross-linking agents did not show any significant crosslinking effect on the fibers (data not shown) apart from EDC and EDC/NHS. The chemical crosslinking agents (EDC and EDC/NHS) were tested and were shown to cause the crosslinking of fiber mats resulting in resistance to aqueous solutions (pH>6.0).

Effects of EDC and EDC/NHS on nanofibers

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alone, and in combination with N-hydroxysuccinimide (NHS) were employed for cross-linking CAP nanofibres to enhance the resistance to hydrolysis in pH above 6.0. The hydrolysis test (table) clearly demonstrated that EDC and EDC/NHS cross-links CAP nanofibres efficiently at a normal pH of 7.4. The exposure time was 4 hours and 24 hours for both EDC and EDC/NHS. At 4 hours cross-linking exposure the nanofibres were stable with normal morphological appearance under the microscope. However, after a period of 20 days, the beginning of transparency in nanofibres was observed macroscopically in both cases of cross-linking (table). During the exposure time of 24 hours cross-linked fibres, the fibres were stable for more than 3 months and are expected to remain stable for a much longer period. It exhibited normal morphological appearance on microscopy and retained a normal pattern of original thickness macroscopically (mentioned in table) The optimal crosslinking exposure time was found to be 24 hours as the crosslinked fibres exhibited stability for longer time periods in environments with pH>6.0.

Table Comparasion between EDC and EDC/NHS cross-linking of two different solvent combinations for 15% CAP

Basic test

Exposure

time

Solvent ratio and polymer concentration (15% CAP)

Acetone /Ethanol (9:1)

DMF/THF/Acetone (3:3:4)

EDC (50mM)

EDC/NHS

(50mM/20mM)

(2.5:1)

EDC

(50mM)

EDC/NHS

(50mM/20mM)

(2.5:1)

Hydrolysis test in alkaline pH (7.4)

4 hours

Resistance to degradation for >20 days, after onward started to become thin transparent

Resistance to degradation for >20 days, after onward started to become thin transparent

2 4 hours

Resistance to degradation for >3 months, normal pattern and apperance

Resistance to degradation for >3 months, normal pattern and apperance

Microscopy of both 4 and 24 hours exposure time

Normal morphological appearance

Normal morphological appearance

SEM analysis of 24 hours exposure time

Grainy appearance and increase in thickness of fibers

Non-uniform with proper bonding between fibers

Smooth, uniform without grains.

Grainy appearance of smooth fibers.

FTIR interpretation

FTIR is commonly used for the analysis of the functional group present in any compounds. The use of FTIR spectroscopy in this study is to know the effect of PBS on the functional group (ester) of CAP. The analysis reveals the hydrolysis of ester bonds of CAP when it reacts with PBS. This hydrolysis behavior is interpreted based on the peak produced during spectroscopy in which peak of bonds matches with the incoming IR radiation frequency and displayed as a specific peak spectrum for each bond type.

Table IR results of CAP

Peaks

Due to functional groups

Indicated in structure

1731.62 cm-1

C=O stretching of ester groups ( acetate and phthalate groups)

1

1129.6 cm-1,

1249.80 cm-1,

1377.84 cm-1

C-O stretching of ester groups ( acetate and phthalate groups) and ether groups

2

1071.67 cm-1

C-O stretching of cyclic ether (cellulose ring)

3

689.18 cm-1

=C-H stretching of benzene ring of phthalate

4

Table FTIR of CAP nanofibres dissolved in PBS

Peaks

Due to functional groups

1637.77 cm-1

C=O bond stretching of acid group

Broad band 3200- 3600 cm-1

O-H bond stretching of acid group

Characterization of CAP fiber morphology

Fiber morphology, fiber diameter, fiber diameter distribution was elucidated by SEM analysis. The characteristics of crosslinked and non-crosslinked fibers is brief out,

Characteristics of non-crosslinked fibers

Table SEM characterization of non-crosslinked CAP fibers

Solvent combination

Nature of fibers

Average diameter

Standard deviation

Standard error of mean

Chloroform/Methanol

Rough, uneven textured fibers

1.147µ

0.5540

0.1752

THF/Ethanol

Few ribbon and break defects

2.272 µ

0.8367

0.3416

THF/Acetone

Satisfactory fibers

2.310 µ

0.6878

0.2600

DMF/THF/Acetone

Smooth, fine fibers

0.475 µ

0.2745

0.0761

Acetone/Ethanol

Homogenous, smooth, fine fibers

0.545 µ

0.1763

0.0531

Characteristics of cross-linked nanofibres

The characterization of non-crosslinked fiber reveals the appropriate and satisfactory fiber at nanometer range only with two solvents combination out of five such as acetone/ethanol and DMF/THF/ acetone.

Table SEM based characterization of EDC and EDC/NHS cross-linked CAP nanofibres

Solvent combination

Crosslinking agent used

Nature of fibers

Average diameter

Standard deviation

Standard error of mean

Acetone/Ethanol

EDC

Swollen fibers with grainy deposition

1.67µ

0.3439

0.1300

EDC/NHS

Smooth fibers with swelling

0.733 µ

0.1722

0.0609

DMF/THF/Acetone

EDC

Slightly swollen fibers with grainy deposition

0.426 µ

0.1619

0.0488

EDC/NHS

Smooth fibers with slight swelling

0.568 µ

0.1753

0.0716

Table Difference in average fiber diameter post cross-linking

Solvent combination

Average diameter before cross-linking

Change in average diameter after cross-linking

EDC

EDC/NHS

Acetone/Ethanol

0.545 µ

1.67 µ

0.733 µ

DMF/THF/Acetone

0.475 µ

0.426 µ

0.568 µ

Cell viability and proliferation

The proliferation of L6 myoblasts cells cultured in CAP scaffolds after 2, 4, and 6 days were analysed by [3-(4, 5-dimethylthiazol-2-yl)-1, 5-diphenyltetrazolium bromide] (MTT, SRL, India) assay and show in fig. The viability and proliferation of cells on scaffolds were compared with control samples.

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