GAG Coatings

Three methods of synthesising GAG coatings were investigated to examine if the cellular response was affected by the GAG itself or how the GAG was bound to the surface. The three methods were: firstly, BioInteractions Ltd. polymer GAG coatings (2.4.1), secondly, GAGs dissolved in medium then adsorbed onto various substrates (2.4.2) and thirdly, covalently bound GAGs onto amine coated coverslips (2.4.3). Adsorbed GAG coatings and bound GAG coatings only examined hyaluronic acid (HA) and chondroitin sulphate (CS) as the response of heparin (HEP) was well established and no further evaluation was required.

2.4.1

BioInteractions Ltd. Cell Polymer Coatings

BioInteractions Ltd. synthesised HEP, HA and CS, using a proprietary method, directly into 24 well plates, each plate contained all three GAGs (eight wells per coating). An additional uncoated TCPS plate was used as a control.

2.4.1.1 Cell Growth Study

N/N1003A LECs were seeded onto GAG polymer coatings and TCPS at a seeding density of 1x104/cm2. Representative phase contrast micrographs were taken throughout the study starting at day 1 and cells were fed every 2 – 3 days. Cells were fixed at days 1, 4, and 7 with NBF.

2.4.1.2 Cell Staining

GAGs plates were fixed with NBF on days 1, 4, 7 10 and 14. Cells were stained with phalloidin (2.1.15), to examine the cytoskeleton, αSMA (2.1.17) to view myofibroblast cells and give an indication of dedifferentiation and DAPI (2.1.14) to stain the nuclei. Three micrographs per well were taken using an x10 objective. The average cell growth on each coating at each time point was measured by counting cell nuclei using a configured macro to count cells in ImageJ.

2.4.1.3 Surface Analysis

Polystyrene coverslips were coated with HEP, HA and CS for the purpose of surface analysis. The piezo-electric system was used for CA measurements (2.1.18.1), 9 points

per sample were measured, repeated in triplicate. Their topography was investigated using SEM (2.1.18.2) and WLI (2.1.18.3). Five areas per sample were analysed on WLI at x50 objective, repeated in triplicate.

2.4.1.4 Time Lapse Microscopy

GAGs coatings were also examined using the IncuCyte time lapse microscopy (Essen BioScience) for a total of 14 days, micrographs were taken every hour to build up a video of still micrographs. Micrographs were taken in the exact same location each time using a motorised stage and software.

2.4.2

Adsorbed GAG Coatings

Five variations of the adsorbed GAG assay were studied to optimise the conditions (Table 2-4). These included varying the: concentration of HA and CS (0.01mg/ml, 0.1mg/ml, 1mg/ml, 5mg/ml and 10mg/ml), incubation times (3 hours and 24 hours), temperatures (4°C and 37°C) and substrates (TCPS plastic, PEI base polymer and polystyrene coverslips (PS, Goodfellow, UK, roughly cut 10mm x 10mm). In addition HA and CS were added to the cell culture medium.

Table 2-4: To detail the various incubation times and optimisation of the soluble GAG assay.

# Incubation Time Concentrations Substrates

1 3 hour incubation at 37oC – GAGs rinsed off

0mg, 0.01mg, 0.1mg, 1mg, 5mg and 10mg – 500µl p/w

Base polymer, TCPS & PS

2 3 hour incubation at 37oC – GAGs not rinsed off

0mg, 0.01mg and 0.1mg and 1mg – 500µl p/w

Base polymer, TCPS and PS 3 24 hour incubation at

37oC – GAGs not rinsed off

0mg, 0.01mg and 0.1mg and 1mg – 500µl p/w

Base polymer and TCPS

4 24 hour incubation at 4oC – GAGs not rinsed off

0mg, 0.01mg and 0.1mg and 1mg – 500µl p/w

Base polymer and TCPS

5 GAG added in medium 0mg, 0.01mg and 0.1mg and 1mg – added in medium

Base polymer, TCPS and PS

The above table presents all adsorbed GAG assays tested, numbered 1-5. Various incubation times and concentrations were tested to optimise the process of binding the GAGs to the different substrates.

HA and CS were dissolved in medium and mixed well prior to adding either 500µl or 1000µl per/well to the substrates at the above concentrations, temperatures and time periods. HA and CS were removed and wells were either rinsed with fresh medium or not before seeding N/N1003A LECs. To replicate the same environment as the polymer GAG coatings provided by BioInteractions Ltd. HA and CS were adsorbed onto the base polymer, as this was the base material present in the polymer GAG coatings. LECs seeded straight onto the base polymer in the absence of any GAG served as a positive control. To confirm if the HA and CS encouraged cell growth or the base polymer, LECS were seeded onto PS coverslips either in the presence or absence of adsorbed GAG. LECs seeded onto PS in the absence of any GAG served as a negative control as PS does not encourage cell growth. LECs were also seeded onto TCPS in the presence or absence of adsorbed GAG. LECs seeded onto TCPS in the absence of any GAG served as a control.

2.4.2.1 Cell Growth Study

N/N1003A LECs were seeded onto the adsorbed coatings (4 wells per GAG concentration) at a seeding density of 1x104/cm2. Cells were fed every 2 – 3 days and monitored for a total of seven days. Base polymer, PS and TCPS wells with the absence of GAGs served as controls. Representative micrographs were taken on days 1, 4 and 7. Micrographs were taken from the same well in roughly the same area at each time point so cell growth could be monitored throughout the study.

2.4.2.2 Quantifying the Amount of GAG Present

Toluidine blue was used at 0.1% w/w in distilled water to assess whether the GAG was present (2.1.11). Toluidine blue was added to adsorbed GAG coatings for 5 minutes and then removed and rinsed with water. If HA and CS were present the coated wells or coverslips stained blue.

2.4.3

Bound GAG Coatings

HA and CS were immobilised onto modified 13mm glass coverslips (Agar Scientific, UK) coated with amine functionality by silinisation. GAGs were bound onto glass coverslips following the below protocol:

1. 10% 3-aminopropyl-triethoxysilane (APTES, Sigma Aldrich, UK) was made up in isopropanol

2. Glass coverslips were placed inside 24 well plates (4 coverslips per concentration).

3. APTES (10%) solution was mixed well then pipetted onto glass coverslips and left for 10 minutes

4. Coverslips were washed thoroughly with isopropanol 5. APTES coated coverslips were left to air dry overnight

6. 3mg/ml, 1mg/ml and 0.1mg/ml of HA and CS were dissolved in a solution of 0.01M 2-(N-morpholino)ethanesulfonic acid (MES, Sigma Aldrich, UK) buffer 7. GAGs were added to a reaction solution of 0.05M N-hydroxysuccinimide (NHS,

Sigma Aldrich, UK)/0.2M 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma Aldrich, UK) sequentially, made up in MES buffer, to activate the GAGs

8. Activated GAG solutions were stirred for 1 hour at room temperature

9. 1ml of activated GAG solution was added to amine coated coverslips overnight at room temperature on a laboratory rocker

10. The following morning bound GAG coverslips were washed with double distilled ultrapure water (Sigma Aldrich, UK) throughout 24 hours, 2 – 3 changes of water were made

11. 24 hours later the distilled water was removed and bound GAG coverslips were left to air dry for two hours in a class II biological hood

12. Bound GAG coverslips were used in cell culture the same day

13. Prior to seeding cells the coverslips were UV sterilised for 5 minutes at 1500J/cm2

2.4.3.1 Cell Growth Study

N/N1003A LECs were seeded onto the bound GAG coatings (four wells per concentration) at a seeding density of 1x104/cm2 for a total of 7 days. Amine coated coverslips, glass coverslips and untreated TCPS wells served as controls. Representative micrographs were taken on days 1, 4 and 7. Micrographs were taken from the same well in approximately the same area at each time point so cell growth could be monitored throughout the study. On day 7 cells were fixed at methanol for 5 minutes at 4°C and stained with methylene blue (2.1.10) to visualise the LECs attachment and growth.

2.4.3.2 Quantifying the Amount of GAG Present

Additional bound GAG coverslips were synthesised and stained with toluidine blue (2.1.11) to determine the presence of HA and CS.

2.4.3.3 Surface Analysis

Additional bound GAG coverslips were synthesised for the purpose of contact angle analysis. The piezo-electric system was used for CA measurements (2.1.18.1), 9 points per sample were measured, repeated in triplicate.