Vibrational spetroscopy of amphiphilic biomaterials.

The technique of ATR-FTIR has been used to study the rates of diffusion of water and two different protein solutions into hydrogels, in the form of thin films, synthesised "in-situ" onto the surface of an ATR crystal. The work involved making copolymers and crosslinked gels of glycerol monomethacrylate (GMMA), lauryl methacrylate (LMA) and ethylene glycol dimethacrylate (EGDMA).Bands characteristic of the individual monomers could not be identified once a stock solution was made up. The rates of reaction were determined from the integrated area of the broad band at 815 cm[-1], which was designated as a monomer band. This band decreased in intensity as the polymerisation progressed. Similarly, a band at 750 cm[-1], designated as a polymer band, made an appearance and increased in intensity throughout the course of the polymerisation. Attempts were made to fit the reaction kinetics to first and second order reactions, using the linear part of the plot of the integrated area of the 815 cm[-1] band. However no particular trend could be found with increase in amounts of crosslinker for each formulation. The reaction possibly followed complex kinetics and this simplistic approach was not really applicable.Diffusion of deionised water into the synthesised gels was studied for three differentformulations. It was found that the diffusion coefficient was of the order of 10[-5] cm[2] s[-1] for the diffusion of water into the gels and decreased with increasing crosslinker concentration, except in the case of 100% GMMA with 0% crosslinker. The dissolution of the GMMA segments in the GMMA-EGDMA copolymer, was further confirmed by using ESEM. The gels with all other formulations were found to be morphologically featureless when looked at in the ESEM chamber. Four water bands were fitted to the &ugr;(OH) stretching region of each spectrum during diffusion and diffusion coefficients of each types of water were calculated..Diffusion of a 30% saline solution of bovine serum albumin was studied for the synthesised gels. It was found in each case, that only the water from the saline solution permeated through the polymer, leaving the protein in the form of a deposit on the top of the gel. The molecular weight of the protein (65kDa) was responsible for the latter not diffusing through the gel. The pores on the gel surface could not swell to an extent large enough to allow the passage of this bulky protein. Hence the gel acted as a selectively permeable membrane allowing passage of molecules based on their molecular weights. The order of magnitude of the diffusion coefficient for the saline water was the same as that of pure water. Diffusion of a solution of lysosyme containing 1% by volume of lysosyme, 49% by volume of water and 50% by volume of glycerol was characterised by a typical two step diffusion process. The first step was rather rapid and could possibly be due to water uptake from the solution. The second step showed evidence of glycerol permeating through the gel. It was impossible to distinguish the protein from the water and glycerol due to its concentration. Diffusion experiments were also carried out with a 10% solution of lysosyme in D[2]O. It was found that a single stage diffusion profile resulted in this case for the D2O . The protein definitely permeated into the gel in this case and it was possible to distinguish the amide I band. All the D[2]O first permeated into the gel and increased the pore size by swelling of the gel. The protein was then seen to permeate into the polymer

Epithelialization of hydrogels achieved by amine functionalization and co-culture with stromal cells

The aim of this study was to develop a hydrogel which would be suitable for corneal cell re-epithelialization when used as a corneal implant. To achieve this, a series of hydrogels were functionalized with primary amines by post-polymerization reactions between amine compounds and glycidyl ether groups attached to the hydrogels. We report a strong correlation between the structure of the amine and the viability of stromal cells and epithelial cells cultured on these hydrogels. Subsequent co-culture of epithelial and stromal cells on the amine modified hydrogels allowed successful expansion of epithelial cells on surfaces functionalized with alkyl α–ω diamines with carbon chain lengths of between 3 and 6. Analysis of variance showed that corneal epithelial cells had a strong preference for surfaces functionalized by the reaction of excess 1,3 diaminopropane with units of glycidyl methacrylate compared to the reaction products of other amines (ammonia; 1,2-diaminoethane; 1,4-diaminobutane or 1,6-diaminohexane). We suggest this approach of amine functionalization combined with stromal/epithelial co-culture offers a promising new approach to achieving a secure corneal epithelium. Keywords: Epithelial cell

Vibrational Spectroscopy of Amphiphilic Biomaterials

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