Effect of Molecular Weight and External Loading on Particle Diffusion within Intervertebral Disc: An in Vitro Porcine Model
Date Issued
2010
Date
2010
Author(s)
Wang, Ruei-An
Abstract
Abstract
Background:
Intervertebral Disc (IVD) is the biggest avascular cartilage in human body. The degeneration of disc would results in the calcification of endplate and fiborsis of nucleus pulposus; which both affect the molecular transportation within disc. The degradation of solute transportation would induce a vicious circle of disc degeneration. The study of diffusion of nutrition exchange in IVD and convection due to daily activities can be helpful for the understanding of disc degeneration and treatment.
Objective:
Step I: Set up a fluorescent imaging system for the study of molecular transport within disc.
Step II: Find the effect of molecular weight on the diffusion capability within the disc.
Methods:
A cost effective fluorescence macroscopic photographic apparatus to detect the dextran molecule within the disc matrix was developed. This apparatus includes one digital camera (Canon 450D) with removal of IR-Cut filter, and two paired light sources and filters to detect “red” and “green” fluorescent reagent. All these components are fitted into a box of 31.5cm x 26.5cm x 55.5cm. Three fluorescent reagents, Fluorescein sodium salt (FS, 0.4 kDa, F6377, Sigma-Aldrich, Saint Louis, Missouri, USA), Tetramethylrhodamine isothiocyanate–dextran (TRITC-dextran, 4.4 kDa, T1037, Sigma-Aldrich), Fluorescein isothiocyanate–dextran (FITC-dextran, 40 kDa, FD40S, Sigma-Aldrich) were used for three groups of solution. The light source for excitation and filter for detection are fine tuned for the detection of emission of these three fluorescent reagents.
Three groups of solutions were formulated. The solution A includes the FS (0.4 kDa, 100μM) only. The solution B includes both FS (0.4 kDa, 100μM) and TRITC-dextran (4.4 kDa, 100μM), and the solution C includes both TRITC-dextran (4.4 kDa, 100μM) and FITC-dextran (40 kDa, 100μM). A 0.25 ml solute was injected into the center of disc before the loading. Four types of loading, which include no load, 1 hr 420 N creep loading, 0.5 hr, 5 Hz, 190 N to 590 N, and 1 hr, 5 Hz, 190 N to 590 N peak-to-peak fatigue loading were applied for these three groups of solutions.
After the loading, the discs were cryopreserved. All specimens were sagittally cut in half using a diamond blade saw. The fluorescent images of specimens were photographed using the developed fluorescent photographic system. For the solution A, green light and filter is used to detect FS. For the solution B and C, the “green” and “red” lights and filters were used to differentiate the FS from TRITC, and FITC from TRICT. After the subtraction of disc and vertebrae images, the fluorophore was identified by the gray scale above 20. The area covered by the fluorophore was calculated to represent the penetration of solutions.
Results:
Step I. We successfully build a cost effective fluorescent imaging system. The noises due to high temperature are found under long-time exposure when using high ISO mode (ISO-1600). After assembling the cooling system, the working temperature of CMOS decreases from 30 ℃ to 5℃, and the hot noise is reduced (noise ratio: reduced from 5.5% to 0.2%). This system is capable for adjusting band wavelengths and focal length by changing the filters and lens.
Step II. The diffusion area (no load) and convention area (creep and fatigue) of FS were similar. The convection area of FS is not affected by the existence of medium size solute i.e., the TRITC. However, the convection area of TRITC was entangled by the existence of larger solute, i.e., the FITC. The 1 hr creep and 0.5 hr fatigue loading increased the transport of FITC, hence the TRITC as well. The effect of 1 hr creep and 0.5 hr fatigue on the large solute transport was similar. The transport area of solute of 1 hr fatigue loading is smaller than the one of 0.5 hr fatigue loading.
Conclusion:
In this study, we found that the small (0.4 kDa) and medium (4.4 kDa) solute is not affected by the load induced convection. However, the external loading induced convention does affect the transport of large solute (40 kDa). The large molecule induces a steric hindrance within the disc space (including both endplate and anulus fibrosus) hence entangles the medium molecule. However, it is not sure if the large molecule would affect the transport of small one. Few more phenomena should be studied in the near future, for example, the inward flow mechanism of the fluorescent solute and the quantitative effect of longer creep or fatigue loading.
Subjects
fluorescence
diffusion
convection
external loading
IVD metabolism
Type
thesis
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