Effect of Particle Size in Diffusion Efficiency within Nucleus Pulposus and Anulus Fibrosus
Date Issued
2011
Date
2011
Author(s)
Liu, Te-Chun
Abstract
Background：Diffusion is one of the nutrient and waste particles transportation mechanisms within intervertebral discs, and thus could be crucial for disc self-recovery from destructive injury. Disc diffusion capacity could be affected by the degeneration change and structural remodeling. However, the underlying mechanism is not well investigated yet. Disc degeneration can be modeled by introducing the biochemical stresses, e.g., trypsin, which may denature the biomolecular compositions within disc. Recently, many reagents have been studied in recovering the disc function. The genipin, a peptide crosslinker, is well proved to restore disc mechanical properties by forming crosslinking between the peptide structures. Determining the diffusion capacity of anulus fibrosus and nucleus fibrosus tissue after denaturation and crosslinking will help to verify the feasibility of peptide crosslinkers for disc regeneration therapy from the aspect of particle transportation function.
Purpose：The purpose of this study is to evaluate the diffusion capacity of anulus fibrosus (AF) and nucleus pulposus (NP) after denaturation and crosslinking with fluorescent image analysis techniques
Material and method：Specimens of AF and NP were harvested from healthy 6-month-old juvenile pigs. The AF was dissected by a home-made scalpel into a cubic shape and NP were store in a inject syringe (1cc.), and 0.09 g of NP was measured and squeezed into the home-made cubic slots. Seventy-two AF along the circumferential direction, Seventy-two AF along the radial direction, and Seventy-two NP (total two hundred and sixteen groups) specimens were prepared. The three types of specimens were further assigned into 3 groups; i.e., intact group, denatured group and crosslinked group. To simulate the denaturation, trypsin (0.25%, 0.5cc.) was injected into the disc and stored at 4℃ for 12 hours and at room temperature for 12 hours, and then the NP was obtained by dissecting the disc. The AF were first dissected and immerged in trypsin solution at 4℃ for 12 hours and at room temperature for 12 hours, then the AF were prepared using the same procedure as intact group. For the crosslinked group, all the specimen were prepared first using the denatured group protocol, and then the specimen were treated with genipin for 24 hours at room-temperature. The Fluorescein Sodium Salt (FS), Tetramethylrhodamine Isothiocyanate-Dextran (TRITC-Dextran) and Fluorescein Isothiocyanate-dextran (FITC-Dextran) were used in this study. Specimens were set in a channel with two sink at both end. The source sink at right hand side was filled with 100μL fluorescence dye at 100 μM intensity and the drain sink at left hand side was filled with same amount of saline. An in-house fluorescent photographic system was used to find the signal changes of both source and drain sink every 5 minutes for 60 minutes. The diffusion coefficient can be calculated from these images using Fick’s first law.
Result：Smaller molecule (FS) always has higher diffusion coefficient than the medium (TRITC) and large (FITC) molecule. The diffusion coefficient of AF along the circumferential direction is higher than the AF along radial direction and NP. The diffusion coefficient of intact AF and NP is higher than the coefficient of denatured and crosslinked one. However, the difference between the denatured and crosslinked disc is not significant.
Conclusion：(1) Increasing the particle size lead to decrease the diffusion coefficient. (2) Higher diffusion coefficient through circumferential specimen in comparison with radial one shows the effect of direction. (3) The diffusion coefficient decreased significantly after denaturation and crosslinking.
Subjects
Diffusion
Fluorescent
Degeneration of Intervertebral Disc
Trypsin
Denature
Genipin
Crosslink
SDGs
Type
thesis
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