Large Molecular-Weight Solute (40 kDa) Entangles the Transport of Medium One (4 kDa) within Intervertebral Disc
1
Wang, R A; 1Hsu, Y C; 2Hsiao, J K;+1Wang, J L
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan; 2Buddhist Tzu Chi General Hospital, Taipei, Taiwan
Senior author: jlwang@ntu.edu.tw
INTRODUCTION:
Solute transport within the intervertebral disc is critical for the disc
functions. Small molecular-weight solutes, such as glucose, lactate and
oxygen, are the components of the nutrient supply and the metabolic
waste removal. Molecular weight of enzymes and chemical drugs for the
disc therapy are usually within the order of thousands of Dalton. Larger
solutes such as cytokines (10-40 kDa), morphogens and protease (30-90
kDa) are critical to regulate the cellular process and disc biological
functions. The solute transport within the disc is affected by the
molecular size, interstitial space of extracellular matrix and the driving
pressure. It is understood that the small solute transport within the disc is
dominated by the diffusion, and the larger solute transport can be helped
by the convection. The disc imbibes and expels the fluid during diurnal
loading. The dynamic external loading, which is practically the origin of
driving pressure of disc fluid, increases fluid flow rate within the disc;
hence increase the solute transport.
Many emerging researches used large molecules (such as morphogens
and proteases) or drug delivery vehicles (such as microsomes or
liposomes) for disc therapy. To warrant the delivery of these particles,
the confounding effects between molecular size, interstitial space and
driving pressure are needed to be cleared. For example, does the larger
molecular-weight solute retard the transport of smaller molecular-weight
solute? Does the dynamic repetitive loading increase the solute transport
than the static creep loading does? The purpose of this study is to answer
these questions by evaluating the solute transport of fluorescent reagent
of small (0.4 kDa), medium (4.4 kDa), and large molecule (40 kDa)
under no load, 1 hr creep and 0.5 hr fatigue loading.
MATERIALS AND 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 400D) with removal of
UR 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 (Table 1).
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. Three types of loading, which include no load, 1 hr
420 N creep loading, and 0.5 hr, 5 Hz, 190 N to 590 N peak-to-peak
fatigue loading were applied for these three groups of solutions (Table 2).
After the loading, the discs were cryopreserved. All specimens
were defrosted for 2 hrs and then 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.
Table 1: Wave length of excitation and emission of fluorescent reagent
Weight
Excitation
Emission
FS
0.4 kDa
490 nm (Blue)
515 nm (Green)
TRITC-Dextran
4.4 kDa
529 nm (Green)
596 nm (Red)
FITC-Dextran
40 kDa
492 nm (Blue)
518 nm (Green)
Table 2: Loading conditions and specimen numbers of three solutions
No load
1 hr creep
0.5 hr fatigue
Solution A (FS only)
V (n=8)
Solution B (FS+TRITC)
V (n=8)
V (n=8)
V (n=8)
Solution C (TRITC+FITC)
V (n=8)
V (n=8)
V (n=8)
RESULTS:
A typical example fluorescent image of Solution B (FS and TRITC) and
Solution C (TRITC+FITC) after 0.5 hr fatigue loading shows the
fluorophore flow out the disc through the anulus fibrosus and endplate
(Figure 1). 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 (Figure 2).
FS
TRITC
Solution B
TRITC
FITC
Solution C
Figure 1: Example images of Solution B (FS and TRITC) and Solution
C (TRITC and FITC) after 0.5 hr fatigue loading.
FS (Sol A)
FS &
TRITC &
TRITC (Sol B)
FITC (Sol C)
*,+: Significant compare to no load,
#: Significant compare to TRITC in Sol B
150
Area (mm2)
1
#
#
*
125
100
+
*
+
#
75
50
25
0
A
B
C
No Load
B
C
Creep 1 h
B
C
Fatigue 0.5 h
Figure 2. Diffusion and convention area of three solutes under no load, 1
hr creep and 0.5 hr fatigue loading.
DISCUSSION:
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.
ACKNOWLEDGEMENT:
National Health Research Institute, Taiwan (NHRI-EX99-9733EI)
National Science Council, Taiwan (NSC 99-2221-E-002-007-MY3)
Poster No. 607 • ORS 2011 Annual Meeting