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Dry polycaprolactone diol (Mr 2000) and trans-cyclohexanechloroydrinisobutyl-POSS (Hybrid Plastics Inc) were placed in a 250ml reaction flask equipped with mechanical stirrer and gas inlet. The mixture was heated to 135Â°C to dissolve the POSS cage into the polyol and then cooled to 60Â°C. Flake Methylene-Diphenylisocyanate (MDI) was added to the polyol blend and then reacted, under nitrogen, at 70Â°C - 80Â°C for 90 minutes to form a pre-polymer. Subsequently dry dimethylacetamide (DMAC) was added slowly to form a solution which was then cooled to 40Â°C. A mixture of Ethylenediamine and Diethylamine in dry DMAC was added drop wise to allow chain extension of the pre-polymer. 1-butanol in DMAC was then added to the polymer solution to form an 18% POSS-PCL solution. All chemicals and reagents were purchased from Sigma-Aldrich Ltd., Gillingham, UK.
Synthesis of POSS-PCL Coagulated Sheet
The intention was to develop a POSS-PCL scaffold with a range of different porosities. Sodium Bicarbonate (NaHCO3) to make the scaffold porous was used as it is commercially available in three different sizes, 40, 65 and 105 microns (Âµm) (Brunner Mond Ltd., Cheshire, UK.) which should correspond with an increasing porosity of the scaffold. 50% of each size NaHCO3, 48% POSS-PCL and 2% of the surfactant Polyoxyethylene Sorbitan Monolaurate, which was used as a stabiliser, were put into a conditioning mixer (ARE-250; Thinky, Tokyo, Japan) for 2 minutes at 2000 RPM and then poured over a stainless steel plate. Based on the literature, it was decided that 800 Âµm was an appropriate thickness for this scaffold to be (Insert References) To obtain this, the appropriate mass (grams, g) of the polymer-salt mixture required was calculated using the formula:
Where m is the required mass of the polymer-salt mixture, d is the density of the polymer-salt mixture and v is the calculated volume required based on the area of the plate and the thickness. To further ensure the thickness, the sides of the stainless steel plate were measured at 800 Âµm using a digital electronic outside micrometer (UKAS Calibration, Corby, U.K) and a mandrel was used to spread the polymer the plate. The polymer was formed using the extrusion-coagulation process where an exchange between solvent and non-solvent occurs resulting in pore formation. The plates containing casting solution (polymer and solvent) is immersed in to a nonsolvent coagulation bath. In this situation, NaHCO3 was used to increase porosity of scaffolds which also diffused out into the nonsolvent, deionized water. The sheets of polymer were kept in in de-ionised water for 48 hours with a regular change of water at 4 hourly intervals. (Young and Chen, 1995) The complete removal of NaHCO3 was confirmed through inductively coupled plasma optical emission spectrometry (Warwick Analytical Services, UK) The samples were then studied for their physical and chemical properties.
POSS-PCU with Silver Nanoparticles
Synthesis of POSS PCU polymer
The method of POSS-PCU synthesis is identical to the method for POSS-PCL, except that Polycarbonate polyol (Mr 2000)(Bayer Material Science GmbH) is added instead of the polycaprolactone diol (Mr 2000)
Synthesis of Silver Nanoparticles
150mg of hydrophilic fumed silica (AEROSIL Â® No(grade?), Evonik Degussa GmbH, Germany) was added to 64mg of Silver Nitrate (AgNO3) (from where, UK). To this 7.5g of Dimethylformamide (DMF) (from where, UK) was added and the solution was put into a sonicating bath (Telsonic AG, Switzerland) for 5 minutes. This was added to 30g of POSS-PCU polymer to form a ??0.75%?? concentration and mixed using a hand held homogeniser (Ultra-Turrax T25 from IKA labortechnik, Staufen, Germany). This is left for 24hours for nanoparticles to form??? Additional DMAC diluted the final concentration of POSS-PCU to 15%, which was an ideal thickness for pouring.
58% POSS-PCU with silver nanoparticles, 40% 40Âµm NaHCO3 and 2% Polyoxyethylene Sorbitan Monolaurate were mixed together as above and the appropriate mass (g) required for a thickness of 100 Âµm was calculated. 40% 40Âµm NaHCO3 was used because this layer is being used as a temporary covering and need not be as porous as the POSS-PCL layer. It is one of the reasons that this layer was fabricated using the solvent casting/particulate leaching method. After the polymer-salt mix is put into the mould, the solvent is removed by evaporation in an oven at 60Â° C for 24 hours. (Liao et al. 2002).Removal of the solvent by this method changes the final structure and appearance of the scaffold as the DMAC does not leave pores on its removal as it does in the coagulation method. Pores are only created when the casted sheet is immersed into deionised water removing the sodium bicarbonate. This structure seemed more appropriate for a temporary epidermal layer of skin. we want it porous but not too porous.
A sample of ag pcl was coagulated to superficially compare the difference - see pictures. Less easy to handle with the thickness being only 50um and
Integra Bilayered Dermal Regeneration Template (Integra Life Sciences, New Jersey, USA) which is the current commercial leader was obtained to perform the same tests and to compare results.
Mechanical tests were performed in uniaxial tension on a modified Instron 4442?? tester (Instron Inc., Massachusetts, USA) unit at room temperature, where symmetrical deformation was carried out. Samples of the polymer were cut into a rectangular shape, with a 40-mm-long working part which were loaded at a constant tension rate of 50 mm/min. Thickness of samples was measured using a digital electronic outside micrometer (UKAS Calibration, Corby, U.K) at three places and averaged. Stress-strain relationships were obtained and graphs were plotted. Stress was calculated by dividing the force generated during stretching by the initial cross-sectional area, and strain was calculated as the ratio of the change in length in reference to the original sample length. Eight tests were performed per sample.
Scanning Electron Microscopy (SEM)
The samples were attached to aluminium stubs with double sided adhesive tape and then coated with gold using an SC500 (EMScope) sputter coater before being examined and photographed using a Philips 501 scanning electron microscope at 15KV. SEM was also carried out after 14 days of in vitro cell culture.
Porosity and Permeability
We used scanning electron microscopy as our main tool to aid characterisation of the porous scaffolds, particularly to look into the pore morphology like pore size, pore-pore interconnectivity, and pore shape. To measure porosity in percentage, we used the formulae given below, where density of the polymer was first calculated and then of the scaffold
4.6.1 Porosity of Scaffolds
The porosity of the scaffolds was calculated by using simple formulae:
Where P is the porosity of the scaffold sample, d is the density of the scaffolds, and dp is the density of the nonporous polymer. Density of the scaffold d was calculated from:
Where, m is the mass and v is the volume of the scaffold. The volume, v was calculated using thickness and diameter of the scaffolds obtained when casted over circular steel plates.
4.6.2 Scanning Electron Microscopy (SEM)
SEM is a useful tool to examine pore-morphology in terms of pore size, pore shape and structure, and pore-interconnectivity. Scaffold samples are sputter coated with gold prior to analysis. SEM provides a high resolution, highly detailed views of a surface of scaffold, however it is limited to the 2D measurements. Moreover, although the pore-interconnectivity can be seen in the 2D views, its quantification is not easy to perform unless the number and size of pores inside the pores, is measured (Murphy, Dennis, Kileny, & Mooney 2002). Pore interconnectivity has been better quantified using micro-Computed Tomography (micro CT) (Moore et al. 2004).
4.6.5. Fourier transform infrared spectroscopy (FTIR)
Infrared spectra of scaffolds were measured on a Perkin-Elmer 1750 FTIR spectrometer equipped with a triglycine sulphate detector. Spectral data were acquired from a 10 Âµl volume gas tight CaF2 cell (path length 6 Âµm). A sample shuttle was employed to permit the sample to be signal-averaged with the background. For each sample, 200 scans were signal averaged at a resolution of 4 cm-1.
Captive bubble technique
4.8 Cell Work
Source of ADMSC's
4.8.1 Scaffold preparation for cell seeding
Scaffold sheets of were cast by solvent casting and salt leaching as described earlier, and thoroughly washed in distilled water. Eight circular discs of 15mm diameter from each sample were then cut using a metal die. Discs were then autoclaved to sterilise them.
4.8.2 Cell Seeding of scaffold samples
Discs of scaffold samples prepared as above were placed in a 24 well plate (BD Falcon, Oxford, U.K.) and seeded with rat's small intestine epithelial cells (IEC 6 cell line, ECACC, Salisbury, U.K.) in 1 ml cell culture medium (DMEM supplemented with 0.1 IU/ml Insulin and 5% Foetal Bovine Serum plus penicillin at 100U/ml and streptomycin at 10Î¼g/ml (all Invitrogen, Paisley, U.K.). Tissue culture plastic wells with no scaffold in were seeded with an identical amount of cells as a positive control. Wells with scaffold but no cells were employed as a negative control. Cells were allowed to attach for 24 hours after which the medium containing unattached cells was removed for lactate dehydrogenase (LDH) analysis to assess initial cell damage. The seeded scaffold discs were then transferred to a fresh 24 well plate (to avoid the possibility of measuring cells seeded onto the initial well bottom during the seeding process) and cell metabolism assessed using an Alamar blueTM assay. Cell metabolism was then further measured at days 3, 6, 10, 14 and 21 post initial seeding. Samples of seeded scaffolds were also taken for analysis by scanning electron microscopy at Day 7 and Day 21 as above.
4.8.3 Optimising seeding density for IEC-6 cells
Rat's intestinal epithelial cells (IEC-6) were counted and diluted to obtain 2, 1, 0.5, 0.125, 0.0625, 0.03125 x 105 cells per ml. A six well plate was seeded for each concentration above where five wells were seeded and one left as blank. They were seeded overnight. The next day (Day 1), medium was removed, washed with PBS and 1 ml 10% Alamar Blue (AB) was added. AB was left for four hours and then removed. Duplicate samples of 100Âµl were read on fluorescent plate reader. AB assay was repeated on Day 4, Day 7, and Day 14.
4.8.8 Assessment of Initial Cell Damage by LDH analysis
LDH was measured using a CytoTox 96Â® Non-Radioactive Cytotoxicity assay kit (Promega, Southampton, U.K.). LDH is a stable cytosolic enzyme released upon cell lysis into the cell culture medium. The amount of LDH released is measured using a 30-minute coupled enzymatic assay based on the conversion of a tetrazolium salt INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium
chloride) into a red formazin product, with the amount of colour formed being proportional to the number of lysed cells. 50Âµl cell culture medium from each sample was transferred to a 96 well plate (Helena Biosciences, Sunderland, U.K). 50Âµl Substrate Mix (1 vial substrate plus 12mls assay buffer) was added to each well and the plate covered in foil to prevent light access. Samples were then incubated at room temperature for 30 minutes after which the reaction was stopped by the addition of 50Âµl stop solution (1M acetic acid). Absorbance was then read at 450nm using a Multiscan MS UV visible spectrophotometer (Labsystems, Ashford, U.K.).
4.8.9 Assessment of Cell Growth and Metabolism by Alamar blueTM assay
Alamar blueTM (Serotec, Kidlington, U.K.) is a commercially available assay which aims to measure quantitatively cell proliferation, cytotoxicity and viability. This is achieved by incorporating resazurin and resarfurin as colorimetric oxidation reduction indicators. These indicators respond to chemical reduction resulting from cell metabolism by changing colour. This colour change may be measured by monitoring fluorescence (excitation at 530nm, emission at 620nm). The advantages of this assay are that it is soluble in media, stable in solution, minimally toxic to cells and produces changes that are easily monitored.
Alamar blue was added to cell culture medium at a concentration of 10% (v/v). At each time point polymer samples were washed with 1 ml PBS and transferred to a fresh 24 well plate to prevent the possibility of measuring cells growing on the bottom of the plate. 0.5 ml of the Alamar blue/cell culture medium mixture was then added to each sample and the positive control wells. 0.5 ml of the mixture was placed into each of the negative control samples. After 4 hours a 100Î¼l sample of the mixture was removed and the absorbance at fluorescence (excitation at 530nm, emission at 620nm) measured in a 96-well plate (Helena Biosciences, Sunderland, U.K.) using a Fluoroscan Ascent FL spectrophotometer (Thermo Labsystems, Ashford, U.K).
4.8.10 Statistical analysis
Statistical analysis of the results was performed using GraphPad Prism version 5 software. The groups were analysed for statistical significance by one- or twoway
ANOVA tests. P<0.05 was considered statistically significant.
Cells attachment to the polymer were spectrally imaged using Nuance Camera System (CRi, Woburn, MA)
Liao, C.J., Chen, C.F., Chen, J.H., Chiang, S.F., Lin, Y.J., & Chang, K.Y. 2002. Fabrication of porous biodegradable polymer scaffolds using a solvent merging/particulate leaching method. J.Biomed.Mater.Res., 59, (4) 676-681 available from: PM:11774329
Young T-H, Chen L-W. 1995. Pore formation mechanism of membranes from phase inversion process. Desalination 103:233-247.