Early changes in the extracellular matrix of the degenerating intervertebral disc, assessed by Fourier transform infrared imaging.

OBJECTIVE: Mechanical overloading induces a degenerative cell response in the intervertebral disc. However, early changes in the extracellular matrix (ECM) are challenging to assess with conventional techniques. Fourier Transform Infrared (FTIR) imaging allows visualization and quantification of the ECM. We aim to identify markers for disc degeneration and apply these to investigate early degenerative changes due to overloading and katabolic cell activity. DESIGN: Three experiments were conducted; Exp 1.: In vivo, lumbar spines of seven goats were operated: one disc was injected with chondroitinase ABC [cABC (mild degeneration)] and compared to the adjacent disc (control) after 24 weeks. Exp 2a: Ex vivo, caprine discs received physiological loading (n = 10) or overloading (n = 10) in a bioreactor. Exp 2b: Cell activity was diminished prior to testing by freeze-thaw cycles, 18 discs were then tested as in Exp 2a. In all experiments, FTIR images (spectral region: 1000-1300 cm-1) of mid-sagittal slices were analyzed using multivariate curve resolution. RESULTS: In vivo, FTIR was more sensitive than biochemical and histological analysis in identifying reduced proteoglycan content (P = 0.046) and increased collagen content in degenerated discs (P < 0.01). Notably, FTIR analysis additionally showed disorganization of the ECM, indicated by increased collagen entropy (P = 0.011). Ex vivo, the proteoglycan/collagen ratio decreased due to overloading (P = 0.047) and collagen entropy increased (P = 0.047). Cell activity affected collagen content only (P = 0.044). CONCLUSION: FTIR imaging allows a more detailed investigation of early disc degeneration than traditional measures. Changes due to mild overloading could be assessed and quantified. Matrix remodeling is the first detectable step towards intervertebral disc degeneration.

The effects of esterified solvents on the diffusion of a model compound across human skin: an ATR-FTIR spectroscopic study.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used to investigate the effects of three fatty acid esters on skin permeation. Propylene glycol diperlargonate (DPPG), isopropyl myristate (IPM) and isostearyl isostearate (ISIS) were selected as pharmaceutically relevant solvents with a range of lipophilicities and cyanophenol (CNP) was used as a model drug. The resultant data were compared with that obtained when water was used as the solvent. The diffusion of CNP, DPPG and IPM across epidermis was successfully described by a Fickian model. When ISIS was used as a solvent Fickian behaviour was only obtained across isolated stratum corneum suggesting that the hydrophilic layers of the epidermis interfere with the permeation of the hydrophobic ISIS. The diffusion coefficients of CNP across epidermis in the different solvents were not significantly different. Using chemometric data analysis diffusion profiles for the solvents were deconvoluted from that of the skin and modelled. Each of these solvents was found to diffuse at a faster rate across the skin than CNP. DPPG considerably increased the concentration of CNP in the stratum corneum in comparison with the other solvents indicating strong penetration enhancer potential. In contrast IPM produced a similar CNP concentration in the stratum corneum to water with ISIS resulting in a lower CNP concentration suggesting negligible enhancement and penetration retardation effects for these two solvents respectively.

ATR-FTIR spectroscopy and spectroscopic imaging of solvent and permeant diffusion across model membranes.

The uptake and diffusion of solvents across polymer membranes is important in controlled drug delivery, effects on drug uptake into, for example, infusion bags and containers, as well as transport across protective clothing. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has been used to monitor the effects of different solvents on the diffusion of a model compound, 4-cyanophenol (CNP) across silicone membrane and on the equilibrium concentration of CNP obtained in the membrane following diffusion. ATR-FTIR spectroscopic imaging of membrane diffusion was used to gain an understanding of when the boundary conditions applied to Fick's second law, used to model the diffusion of permeants across the silicone membrane do not hold. The imaging experiments indicated that when the solvent was not taken up appreciably into the membrane, the presence of discrete solvent pools between the ATR crystal and the silicone membrane can affect the diffusion profile of the permeant. This effect is more significant if the permeant has a high solubility in the solvent. In contrast, solvents that are taken up into the membrane to a greater extent, or those where the solubility of the permeant in the vehicle is relatively low, were found to show a good fit to the diffusion model. As such these systems allow the ATR-FTIR spectroscopic approach to give mechanistic insight into how the particular solvents enhance permeation. The solubility of CNP in the solvent and the uptake of the solvent into the membrane were found to be important influences on the equilibrium concentration of the permeant obtained in the membrane following diffusion. In general, solvents which were taken up to a significant extent into the membrane and which caused the membrane to swell increased the diffusion coefficient of the permeant in the membrane though other factors such as solvent viscosity may also be important.

Local examination of skin diffusion using FTIR spectroscopic imaging and multivariate target factor analysis.

In the context of trans-dermal drug delivery it is very important to have mechanistic insight into the barrier function of the skin's stratum corneum and the diffusion mechanisms of topically applied drugs. Currently spectroscopic imaging techniques are evolving which enable a spatial examination of various types of samples in a dynamic way. ATR-FTIR imaging opens up the possibility to monitor spatial diffusion profiles across the stratum corneum of a skin sample. Multivariate data analyses methods based on factor analysis are able to provide insight into the large amount of spectroscopically complex and highly overlapping signals generated. Multivariate target factor analysis was used for spectral resolution and local diffusion profiles with time through stratum corneum. A model drug, 4-cyanophenol in polyethylene glycol 600 and water was studied. Results indicate that the average diffusion profiles between spatially different locations show similar profiles despite the heterogeneous nature of the biological sample and the challenging experimental set-up.