Cell culture plates, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antibiotic-antimycotic solution (100×), Griess reagent, Sirius Red F3B and potassium bromide (KBr, IR grade) were purchased from Sigma-Aldrich® (Bangalore, India). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and trypsin were obtained from HiMedia® (Mumbai, India). Lithium bromide was purchased from Loba Chemie® (Mumbai, India). All other chemicals were of AR grade and were obtained from Merck® (Mumbai, India).
2.2.2 Processing and degumming of cocoons
The cocoons were collected from the Silk Mark Organization of India (Central Silk Board), Guwahati, India and were processed according to a previous report with slight modifications (Rajkhowa et al., 2000). Briefly, the cocoons were cooked in 0.5 % (w/v) Sodium carbonate solution for 20 min at 95 oC, with material to liquo r ratio (MLR) of 1:20. They were cooked for another 30 min at 45 oC in 0.25 % (w/v) Sodium carbonate solution with MLR of 1:30, and finally degummed at 90 oC for 3 h with MLR of 1:50 in a solution containing 0.2 % (w/v) Sodium carbonate and 0.025 % (w/v) Triton® X-100. In order to remove residual fats, cocoons were treated with a 3:1 Methanol and Diethyl ether solution for 8 h, followed by treatment with Cyclohexane for 24 h and then with Toluene for 8 h. The fibers were thoroughly washed with distilled water, dried under vacuum and stored at room temperature in moisture-free environment until use.
2.2.3 Fabrication of nonwoven scaffold
About 1 g of pure fibroin fibers were suspended in 100 ml of Formic acid (100% GR grade) solution containing Lithium bromide (1 M). The suspension was subjected to
homogenization (T 25 digital ULTRA-TURRAX®, IKA®) at 5000 rpm for 10 min. The homogenous mixture was cast in a 100 mm Petri dish and the solvent was allowed to evaporate at 37 oC for 48 h. The residual formic acid and traces of salts were removed by washing thoroughly with distilled water. It was finally dried overnight under vacuum at 37 oC and stored in a moisture-free pouch until use. The resulting scaffold was used for future characterization studies. For blood compatibility and cyto-compatibility studies the scaffolds were sterilized by direct autoclaving prior to use.
2.2.4 Characterization studies 2.2.4.1 Morphological properties
The samples were mounted on the specimen holder with electro-conductive tape and then sputter coated under vacuum with gold in a coating unit (Sputter Coater, SC7620, Polaron Range). The surface and cross section morphologies of the scaffold were examined under scanning electron microscope (SEM, LEO 1430 VP and JSM 6380 LA, JEOL).
2.2.4.2 Physical properties
Density and porosity: The density and porosity of the scaffold were measured by liquid displacement method (Nazarov et al., 2004). A sample of weight W was immersed in known volume (V1) of hexane in a graduated cylinder for about 5 min. The total volume of hexane and the hexane impregnated scaffold was recorded as V2. The residual volume after removing hexane impregnated scaffold was recorded as V3. The volume difference of V2-V1 was the volume of the scaffold. The volume of hexane within the scaffold (V1- V3
The total volume of the scaffold was V = (V ) was determined as the void volume of the scaffold.
2 - V1) + (V1 - V3) = V2 - V The density of the scaffold was obtained from d = W / (V
3
2 - V3
The porosity percentage was obtained from ε (%) = (V
)
1 - V3) / (V2 - V3) × 100
Swelling behavior: To study the swelling behavior, measured amounts of scaffold were taken (Wd) and were immersed in water for 24 h at 37 oC. The swollen scaffold was
subjected to a quick spin in a micro-centrifuge, in presence of a filter, to remove excess of water from the scaffold and the wet weight (Ws
Degree of swelling (Q) = (W
) of the scaffold was determined. The degree of swelling (Q) and water uptake (%) were calculated as follows:
s - Wd
Water uptake (%) = (W
) / Wd
s - Wd) / Ws) × 100
2.2.4.3 Chemical / structural properties
Fourier Transform Infra-Red (FTIR) Spectroscopy: About 3 mg of the finely powdered samples were pressed into a pellet with 200 mg of KBr and infrared spectra were recorded in a FTIR spectroscope (Spectrum One, Perkin Elmer, MA, USA), from 4000 cm-1 to 450 cm-1 with a resolution of 2 cm-1
2.2.4.4 Thermal properties
and 5 scans per sample.
Thermo Gravimetric Analysis (TGA): TGA was performed with a thermo gravimetric analyzer (TGA/SDTA851e/LF/1100, Mettler Toledo, USA). The cell was swept with nitrogen for analysis and the thermograph of 5 mg scaffold was obtained for a temperature ranging from 37 oC to 550 oC with a heating rate of 10 oC min-1
Differential Scanning Calorimetry (DSC): DSC measurements were performed with a DSC instrument (DSC 7, Perkin Elmer, MA, USA). The thermograph of 5 mg scaffold was obtained for a temperature ranging from 37
.
oC to 550 oC with a heating rate of 10 oC min-1
2.2.4.5 Mechanical properties
. The cell was swept with argon during analysis.
Mechanical properties of the scaffold were measured on strips of 25 × 2 mm by means of a micro tensile tester interfaced with a computer (MICROTEST, DEBEN UK Ltd.).
Samples were mounted horizontally and clamped in a pair of jaws which were supported on stainless steel sliding bearings. A dual threaded lead screw, drives the jaws symmetrically in opposite directions. The measurements were controlled by the software, and were performed under standard conditions.
2.2.5 Blood compatibility studies
The blood compatibility of the scaffold was evaluated in vitro, and the following categories of blood interactions were studied: thrombogenicity and hemolytic potential.
Also, platelet and leukocyte count, protein adsorption and platelet adhesion studies were performed to gain detailed knowledge about blood compatibility of the scaffold.
Blood processing: Venous blood from healthy individuals was collected in heparin coated siliconized vials and the tests were performed within 3 h post collection (Tamada, 2004).
The scaffolds were conditioned in normal saline for 30 min at 37 o 2.2.5.1 Thrombogenecity
C before testing.
The evaluation of thrombus formation was carried out using a gravimetric method (Imai and Nose, 1972). Briefly, 10 ml of heparinized blood was incubated with scaffold at 37 o
2.2.5.2 Hemolytic assay
C. After 1 h of incubation, 0.2 ml of blood sample was taken and placed onto a clean film. Clotting was initiated by adding 0.02 ml of 10 M Calcium chloride solution and mixing it properly by teflon stick. After 15, 30, 45 min and 60 min, clotting was stopped by adding 5 ml distilled water. The clot formed, was fixed in 5 ml formaldehyde solution (36%) for 5 min, washed with distilled water, blotted between tissue paper and weighed.
The heparinized blood alone, incubated without scaffold, was processed in similar fashion and was regarded as a control.
To test the hemolysis (%), the scaffold was placed in a silicon tube containing 10 ml of heparinized blood (1:10 diluted with phosphate buffered saline – PBS, pH 7.4). 1 ml of heparinized blood diluted with 9 ml of PBS was taken as negative control (0 % hemolysis) and 1 ml of heparinized blood diluted with 9 ml of distilled water was taken as positive (100 % hemolysis) control. The contents were incubated at 37 o
Hemolysis % = (OD
C for 1 h.
Samples were gently inverted twice at every 10 min to maintain contact of the blood with the material. After incubation, each fluid was transferred to a suitable tube and centrifuged (3K30, Sigma) at 2000 rpm for 15 min. The hemoglobin released by hemolysis was measured by the optical densities (OD) of the supernatants at 540 nm using a UV–vis spectrophotometer (Cary 100, Varian Inc.). The percentage of hemolysis was calculated as follows (Singh et al., 1990):
sample – OD -ve control) / (OD +ve control – OD -ve control) × 100
2.2.5.3 Platelet and leukocyte (total and differential) count
To evaluate the effect of scaffold on the platelet and leukocyte count, the heparinized blood was taken in Silicone tubes and was put in contact with scaffold for 30 min at 37 o
2.2.5.4 Protein adsorption
C. Samples were gently inverted twice at every 10 min to maintain contact of the blood with the material. The platelet count, total leukocyte count and differential leukocyte count were determined using a hemocytometer. The heparinized blood alone, incubated without scaffold, was processed in similar manner and was regarded as a control.
For protein adsorption studies, scaffolds were incubated in serum solution (55 % in PBS, simulating plasma content in the blood) for different time periods ranging from 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 12 h, 18 h and 24 h at 37 o
2.2.5.5 Platelet adhesion
C. At the end of each incubation period, the scaffolds were gently washed with PBS followed by distilled water to remove loosely adhered proteins. The scaffolds were placed in 1% Sodium dodecyl sulfate (SDS, w/v in PBS) shaken vigorously to dislodge the adsorbed proteins andthe solution was centrifuged to remove particulate or insoluble matter, if any. Finally, the amount of protein in the suspension was assayed by Folin-Lowry method (Lowry et al., 1951). Bovine serum albumin (BSA) prepared in 1% SDS (w/v in PBS) was taken as the standard.
The amount of platelets adhered on the scaffold was determined as per a previous report (Singh et al., 1990). The heparinized blood was centrifuged at 1000 rpm for 10 min to obtain platelet rich plasma (PRP) and at 2800 rpm for 15 min to obtain platelet-poor plasma (PPP). The platelet concentration of PRP was adjusted to 1 × 105 platelets/μl.
About 1 ml of PRP was incubated with the scaffold at 37 o
Platelet adhesion (%) = [(Total platelets – Unattached platelets) / Total platelets] × 100 C. After 1 h of incubation, the scaffold was removed and the number of unattached platelets in the PRP was counted using a hemocytometer. The heparinized blood alone, incubated without scaffold, was processed in similar manner and was regarded as a control. The percentage of platelets adhered were determined as per the following equation:
2.2.6 Cyto-compatibility studies
To test the cyto-compatibility of the scaffold, in the current investigation, we used four established human cell lines viz. A-549 (derived from lung), KB (derived from oral epidermis; HeLa contaminant), HepG2 (derived from liver) and HeLa (derived from cervix) obtained from the National Centre for Cell Science (NCCS, Pune, India) – the national repository for cell lines in India.
Cell culture: As per the NCCS guidelines, the cell lines were maintained in DMEM containing 2 mM L-glutamine and 1.5 g/l Sodium bicarbonate, supplemented with 10%
FBS and 1% antibiotic antimycotic solution (100×, 1000 U/ml Penicillin G, 10 mg/ml Streptomycin sulfate, 5 mg/ml Gentamycin and 25 µg/ml Amphotericin B). Cells were cultured till 70-80% confluence in T-25 culture flasks (CELLBIND, Corning®) at 37 oC in a CO2
2.2.6.1 Culture of cells on scaffold
incubator (New Braunswick) and were trypsinized to get cell suspension. Cell count was done with a hemocytometer before proceeding for further experiments.
Before cell seeding, sterile scaffolds were conditioned in DMEM supplemented with 10%
FBS for 1 h. In independent experiments run simultaneously, the scaffolds were seeded with A-549, KB, HepG2 and HeLa cells. Typically, about 1 cm2 scaffold and 2 ml of medium (containing 1 × 105 cells/ml) were taken in sterile vials. The cells were allowed to attach in static condition at 37 oC in CO2. After 2-4 h of incubation, the cell-scaffold constructs (3D - three dimensional cultures) were transferred to a new 24 well culture plate and returned back to CO2 incubator. The cells were fed with fresh medium at a regular interval of 24 h. After 24 h, 48 h, 72 h, 1 week, 2 weeks, 3 weeks and 4 weeks of incubation, the cell-scaffold constructs were used for future analysis. Cells seeded and grown in 24 well tissue culture plates (CELLBIND, Corning®
2.2.6.2 Evaluation of cell adhesion, spreading, viability and growth on scaffold
), without scaffold, were considered as the control experiments (2D - two dimensional cultures); growth conditions and incubations periods were similar to the test experiments.
To investigate the attachment and spreading of cells on the scaffold, one week old cell- scaffold constructs were fixed in 2.5 % glutaraldehyde solution, dehydrated in ethanol
gradient, sputter coated with gold and finally examined under SEM (1430 VP, LEO and JSM 6380 LA, JEOL). Also, the viability and growth of seeded cells was evaluated by following a colorimetry based MTT assay (Mosmann, 1983). Briefly, cell-scaffold constructs were incubated with 1 ml of MTT solution (0.5 mg/ml in phenol red free and serum free DMEM) for 2-4 h at 37 oC in a CO2
Viability (%) = (OD of test / OD of control) × 100
incubator. The medium was replaced with equa l volume of Dimethyl sulfoxide to dissolve the formazan complex and the optical density (OD) of the soluble fraction was read at 570 nm in a UV-Vis spectrophotometer.
The cell viability was determined using the following equation:
2.2.6.3 Estimation of collagen content in cell-scaffold constructs
After preset periods of incubation, quantification of collagen deposition by growing cells on the scaffold was performed using a colorimetry based Picro-Sirius Red assay (Tullberg-Reinert and Jundt, 1999). Briefly, cell-scaffold constructs were incubated in Sirius Red F3B solution (0.1 % in saturated picric acid) for 1 h. After gentle washing with distilled water, cell-scaffold constructs were incubated in 1 ml of destaining solution (0.2 M NaOH /Methanol, 1:1) for 15 min to extract the stain. The optical density (OD) was read at 490 nm in UV-Vis spectrophotometer.
2.2.7 Assessment of angiogenic properties
2.2.7.1 Chicken embryo chorio allantoic membrane (CAM) assay
A modified CAM assay (Azzarello et al., 2007; Miller et al., 2004; Zwadlo-Klarwasser et al. 2001; Wilting et al., 1991) was followed to assess the angiogenic potential of the scaffold. Briefly, fertilized chicken eggs were initially incubated for 3 days at 37 oC, 65 % relative humidity with gentle rocking and finally for another 4 h without shaking. The eggs were wiped with 70 % ethanol, carefully cracked out into 100 mm culture dish and subsequently incubated in a CO2 incubator for 4 h. The scaffolds were implanted onto the CAM near large vessels and were returned back to CO2 incubator. The presence of new blood vessels through scaffold was examined under a trinocular stereo zoom microscope
(SMZ 800, Nikon) equipped with a fiber optic light source (Fiber illuminator, Nikon) and a digital camera (Cool pix 5400, Nikon).
2.2.7.2 Nitric Oxide (NO) content
The Griess reagent (1:1 ratio of 0.1 % of N-1 naphthylenediamine and 1 % sulphaniliamide in 5% phosphoric acid) was used to assess the nitrite levels in the spent culture medium, which gives an estimate of NO produced by the growing cells on the scaffold (Giustarini et al., 2008). Briefly, cell-scaffold constructs were grown in DMEM without phenol red, after preset periods of incubation (24 h, 36 h, 72 h, 1 week, 2 weeks, 3 weeks and 4 weeks), the cell-scaffold constructs were removed, the Griess reagent was added to spent medium in a 1:1 ratio, incubated at 37 oC for 10 min and subsequently the optical density (OD) was recorded at 550 nm in a spectrophotometer. The amounts of NO produced in samples were calculated from the standard curve prepared by using sodium nitrite.
2.2.8 Biodegradation properties
The scaffolds were immersed in 5 ml of trypsin (0.5 mg/ml in PBS; pure, from bovine pancreas 3x, activity approx. 2500 NFU/mg) and subsequently incubated at 37 o
Weight loss (%) = (Initial weight – Final weight) / Initial weight × 100
C under gentle rocking (60 rpm). The enzyme solution was exchanged daily with a fresh solution.
After 24 h, 36 h, 72 h, 1 week, 2 weeks, 3 weeks and 4 weeks of incubation periods, the samples were taken out, washed gently with distilled water, air dried and finally weighed to determine the extent of degradation in vitro (Vasconcelos et al., 2008). Native fibroin fibers processed under similar conditions was considered as a control. The weight loss (%) was calculated using following equation:
2.2.9 Statistical analysis
Experiments with quantitative data were done in replicates of four independent experiments and the results were expressed as Mean ± Standard Deviation (n = 4).