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Direction-dependent resistance to flow in the endplate of the intervertebral disc: an ex vivo study
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Journal of Orthopaedic Research Journal of Orthopaedic Research 19 (2001) 1073-1077 www.elsevier.com/locate/ort hres
Direction-dependent 'resistance to flow in the endplate of the intervertebral disc: an ex vivo study D.C. Ayotte
a-b,
K. Ito
S. Tepic
a
Abstract
A comparison of the higher hydrostatic pressure in the nucleus of the healthy intervertebral disc during daily loading with the relatively lower osmotic swelling pressure in the disc during rest suggests the existence of direction-dependent flow resistance such that all of the fluid exuded from the disc during loading is recovered during rest. In this study, this direction-dependent resistance was demonstrated for flows through the cartilage endplates and the underlying marrow contact channels in the bony endplates. for flow through the endplate was 39.0 f 3.8 (mean f S.E.).In Using an ex vivo sheep endplate model, the resistance ratio (ROut/R,") addition, a path of fluid flow through the marrow contact holes was revealed using fluorescent staining. 0 1001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction
The daily load-induced pressure in the healthy intervertebral disc is countered in part by the osmotic swelling pressure of the disc tissue [21]. However, under normal daily loads this hydrostatic pressure, 0.6 to > 1.O MPa, remains approximately three to five times higher than the swelling pressure, 0.15-0.2 MPa, resulting in net fluid flow out of the disc [2,10,11,19,22]. Although the decrease in disc hydration causes an increase in the swelling pressure, equilibrium between swelling and hydrostatic pressure is likely never reached [71]. In the daily cycle the disc can lose up to 20% of its volume [5]. During rest, the osmotic swelling pressure draws fluid back into the disc, and as the disc rehydrates, the swelling pressure decreases. The weaker swelling pressure, combined with a relatively short period of rest (approximately 8 h of sleep vs. 16 h of daily loading), results in a lower driving potential for flow into the disc than for flow out of the disc. Therefore, some directiondependent mechanism must exist to ensure that all the fluid exuded from the disc during loading is recovered during rest. Based on an assumption that the dominant flow path is through the cartilage endplates, we propose that the *Corresponding author. Tel.: +41-81-414-M50; fax: +11-81-4112288. E-muil uddress: keita.ito@!ao-asif.ch (I(.Ito).
direction-dependent mechanism is provided by a combination of the strain dependent behavior of the cartilage endplate [ 151 and the presence of holes, i.e., marrow contact channels, through the underlying bony endplate [4,9,13,20]. This direction-dependent resistance mechanism was demonstrated in a physical model and a corresponding finite element model [3,4] and was also observed in exudation flow from cartilage compressed with a porous platen [6]. The objectives of the current study were to establish, ex vivo, that flow through the endplates of the intervertebral disc experiences direction-dependent resistance and that this flow is through the marrow contact channels.
Methods Sheep endplates were used to determine the resistance ratio for exudation vs. imbibition flow in the disc (R<,,,,/R,,,).Three lumbar spines were harvested from healthy, adult female. Swiss alpine sheep and kept frozen at -23°C wrapped in saline soaked gauze and double sealed in a plastic bag for upto 20 months. Spine segments were cut from the frozen spines using a butcher's band saw (FK22. Bizerba. Zurich, Switzerland) as required. The thickness of the vertebrae on each side of the disc was reduced using a grinding wheel (Struers RotoPol 25, E. Merck, Darmstadt, Germany) such that only the compact bone and a thin layer of trabecular bone of the bony endplate remained. Marrow was gently removed from any exposed trabeculae with irrigation, and the spine segments were then cut transversely through the disc using a scalpel. One 10 mm diameter plug was cut from the center of each endplate using a punch and hammer (Fig. 1). As much nuclear tissue as possible was removed with a scalpel in order to isolate the effects of the cartilage endplate. However, this proved
0736-0266/01/$ - see front matter 0 2001 Orthopaedic Research Society. F'ublished by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 0 2 6 6 ( 0 1 ) 0 0 0 3 8 - 9
D. C. Aj'otte t z t ul. I Jou~nuIof Orthopedic Research 19 1-7001i 1073- 1077
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pressure ratio (Pc,L,l/R,,). Steady-state pressure was assumed as the value at which the pressure converged to within 5?0 of the estimated final value over a period of approximately 15 min. A general linear model of the form: 3;) = / L
'trabecular bone Fig 1 Sheep endplate plug harvesting One I0 0 mm diameter plug cut from the center of each endplate using a punch and hammer.
UAS
ditlicult. and some nuclear tissue was preserved in order to avoid damaging the cartilage endplates. A glass column of a HPLC system was used to administer flows through the endplate plugs ( Superfoniiancea Universal glass-cartridge system, E. Merck) (Fig. 2). The plugs were supported within the column on each side by customized cylindrical stainless steel supports, which in turn were held in place and sealed on each side by variable nylon adapters. The steel supports were designed with circumferential grooves, and the variable adapters were threaded into the column ends such that they could compress the supports against the plug with great force in order to avoid leakage around the outside of the plug. In addition. O-rings were placed between the adapters and aluminum supports (in addition to the existing O-rings in the variable adapters) to completely seal the plug within the column. The entire assembly was housed within the outer jacket and maintained at 37°C with a water bath for the duration of the experiments. A constant flow rate pump (LiChroGraph" HPLC Pump, Model L-6000, E. Merck) was used to puinp Ringer's solution through endplate plugs. Eight samples were tested (four plugs from Sheep A, two from Sheep B, and two from Sheep C). The solution was randomly pumped in alternate directions to simulate flow into the disc (flow in: fluid pumped from the bone side of the plug) and flow out of the disc (flow out: fluid pumped from the tissue side of the disc) (Fig. 2). A flow rate of 1 pl/inin was applied in each direction, and a cut-off pressure during flow out was set at 110 bar to avoid damaging the equipment. The pressure was measured using the existing pressure transducer in the pump and recorded using an .Y Y plotter (SEFRAM, Paris, France). The steady-state pressure was used to calculate the average resistance, R: R = P/Q. (1) where Q [m2/s] was the applied flow rate and P [Pa] was the steadystate pressure. The resistance ratio (ROut/R,") was therefore equal to the
glass tube stainless steel supports
Ringeris
variable adapter
\
cartilage
bone
Fig. 2. Schematic cross-section of the experimental set-up. Endplate plugs were supported and sealed within a glass tube. The resultant pressure was measured for fluid pumped in alternate directions (at a flow rate of 1 pI/min) to simulate flow into the disc (flow Bi) and flow out of the disc (flow out).
+ 6 , . direction, +
C&
. sheep,
+ E. . direction, . sheep, + I:,,
(2)
was initially used for statistical analysis (SPSS AG, Zurich, Switzerland) of the direction-dependence of the flow resistance where 3;) =pressure, p = overall mean, = effect of direction, ( i = I . ? ) , Ji = effect of sheep, (i = I , ? . 3 ) , i= interaction effect of direction, x sheep,, and F , / = error of directioti, and sheep,. If the effect of sheep and sheep * direction interaction was not significant ( P 2 0.05) then the data could be pooled, and the ratio Pc,u,/F',,,of each specimen examined for the effect of direction. Otherwise, if an interaction was found, P,,,l/P,nwas calculated using least-square means. In the case of the former. significance ( P < 0.05) of direction-dependence resistance was tested using a one-sample (-test. For both analyses the required assumptions were validated using the Shapiro-Wilk's test for normality and Levene's test for equality of variance. The path of fluid flow through the endplate plugs during pumping was visualized with a fluorescent dye (Procion Red H-8BN. Imperial Chemical Industries PLC, London, U K ) added to the Ringer's solution. Four additional plugs were prepared and pumped as for the resistance ratio tests. One plug each was stained for a time period of 15, 30, 60 and 90 min. The fluid was pumped at a flow rate of 1 ~ I / m i n o n l y in the flow in direction to capture the dye as it entered the cartilage endplate through the marrow contact channels. Upon completion of staining, each plug was immediately removed from the glass column and plunge-frozen in liquid nitrogen [16]. Freeze-substitution was then performed in acetone for ten days at - S O T , with 3% gluteraldehyde and molecular sieve [IS]. The samples were transferred to -23°C for a further six days and to 4°C for one final day to complete fixation. PMMA embedded undecalcified histology sections, 60-80 pm thick, were cut (1600 Saw-Microtome, Leica, Nussloch, Germany) and ground (Wmtzky Flachlappmachine, Stiheli, Pieterlen, Switzerland) for fluorescence microscopy from which the path of Ruid flow through the plugs could be determined according to the location of fluorescent stain [IJ].
Results
The pressure during flow out in all eight endplate plugs was considerably greater than the pressure during ilow in (Table 1). In two cases the pressure during flow out reached the cut-off of 110 bar. Taking a conservative approach, 110 bar was taken as the pressure for flow out in these cases. The general linear model revealed that there was no statistically significant effect of sheep ( P = 0.36) or interaction of sheep and direction ( P = 0.26). That is, plugs from different sheep did not exhibit different resistances, and they did not exhibit differences in direction-dependence (& >> &). Hence, the sheep effect could be ignored, allowing pooling of the data. The one sample t-test revealed that the resistance to flow out was significantly greater to that of flow in ( P < 0.001) with a resistance ratio (RoUt/Rln) of 39.0 f 3.8 (mean iS.E.). Staining with Procion red during flow in revealed fluid flow through the marrow contact channels in all endplate plugs. When examined under the fluorescence microscope, no differences in the location or intensity of stain were observed between plugs infused for 30, 60, and 90 min. Fluorescent stain was observed in the marrow cavities of the trabecular bone, through the
D.C. Ayotte et crl. I Journol qf’ Orthopedic- Resrtrrch I 9 12001) 1073-1077
1075
Table 1 Pressure drop as the 10.0 mm diameter ovine endplate discs with a How rate of 1 PI/ mint Specimen number
Sheep
Pressure out [bar]
Pressure in [bar]
Rout /Rm
1 2 3 4 5 6 7 8
A A A A
110 110 86 80 97 70 67 80
2.0 2.8 2.8 I .9 1.8 2.3 2.6 2.3
55.0 39.3 30.7 19.I 53.9 30.4 25.8 34.8
B B C C
“Resistance ratio (Rc,u,/R,n) = 39.0 f 3.8 (mean
S.E.).
Fig. 3. Ringer’s lactate solution stained with Procion red was pumped through endplate specimens in the flow in direction (from the bone to the cartilage). In this sample, infused for 15 min, a flow path was demonstrated as the stain flowed through the marrow contact channels (C) in the compact bone (BL through the marrow contact openings at the bone-cartilage interface (BC), and just began to flow into the cartilage endplate. Scale bar units are I pm.
marrow contact channels, and in the cartilage endplate of these plugs. In the plug infused for 15 min., the marrow contact channels were observed to fluoresce brightly, with ‘bursts’ of stain in the cartilage endplates originating from the marrow contact holes (Fig. 3).
Discussion This study demonstrates that fluid flow through the endplates of the intervertebral disc experiences a direction-dependent resistance, where resistance to exudation flow out of the disc is much greater than that to imbibition flow in. Our ex vivo experiments revealed that at a steady-state flow rate of 1 pl/min, the pressure drop across the sheep endplate plugs during flow out was significantly greater than that during flow in ( P < 0.001), and that this direction-dependent resistance ratio (Rout/&) was 39.0 h 3.8 (mean f S.E.). Furthermore, staining of the fluid during flow itz clearly demonstrated a flow path through the marrow contact
channels where initially the stain was observed to burst through the marrow contact channels and thereafter to stain the entire depth of the cartilage endplate. The use of quadraped spine as opposed to human spine is persistently controversial in biomechanical experiments of the spine. However, in our experiments, functional spine units were not loaded, and only the isolated endplates were used. Of most importance, in both sheep and human, a cartilaginous layer has been found to cover marrow contact channels in the osseous endplate [4], as is true of other quadrapeds [12]. Although the endplates may differ in the size and number of marrow contact channels resulting in different resistance ratios, the observed direction-dependent resistance is believed in principle to be similar to that in humans. The use of frozen specimens was supported by the lack of mechanical property changes with freezing [7,17]. Since our experiment depended on preservation of the cartilage macro-structure, use of frozen tissue is unlikely to have caused major artifacts. Moreover, the period of freezing did not appear to affect the results, as no obvious differences were observed for samples frozen for 16 and 20 months vs. those frozen for 1 month. The hydrostatic pressure during loading of the human spine is at least three times greater than the osmotic pressure during rest [2,10,19] and occurs for approximately twice as long. Thus, pressure control would have been better suited for our experiments. However, the pump available to us was flow-rate controlled and had a minimum flow rate of 1 pl/min. Although this flow rate is comparable to that which is likely to occur across the entire human endplate [l], due to the relatively small cross-sectional area of the plugs compared to the total area available for flow in vivo, the resistance ratio measured in this study may be exaggerated. Higher fluid velocity through the plugs may have accentuated consolidation and expansion of the cartilage matrix into and out the channels resulting in greater R,,,, smaller R,,,, and higher R,,,/RIn ratios. Nevertheless, even an order of magnitude reduction from our measured resistance ratios is probably of sufficient direction-dependence to maintain a diurnal fluid volume balance.
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D. C. .$yotte et al. I Journal of Orthojiardic Research 19 (-7001) 1073-1077
Another possible artifact of this experimental method was fluid leakage around the plug, especially during the flow in. An attempt was made to limit this flow path by compressing the outer rim of the specimens with serrated edge stainless steel supports (Fig. 2). Furthermore, examination of the cartilage endplates after irrigation with the stained fluid showed that the outer area of cartilage endplate beneath the supports was not irrigated. Hence, flow around the specimens was highly unlikely. Preparation of the endplate plugs involved the reduction of the vertebral bone to a thin layer and gentle removal of trabecular marrow content. This was necessary to isolate the effects of the cartilage endplate, as well as to remove any marrow that was forced into the marrow contact channels during grinding. This removal of the contents, normally partially obstructing the endplate openings, probably lead to a reduction of the resistance to flow in both directions, but would not have significantly affected the direction-dependent nature of the resistance ratio. On the other side of the specimen plugs, the presence of some softer poroelastic nuclear tissue may have biased our results with further increases in the resistance ratio. However, this is still consistent with our hypothesis of a poroelastic material over a constriction hole mechanism responsible for the direction-dependent resistance and is consistent with the in vivo situation. Staining was only conducted during flow in. The objective of irrigation with a stained fluid was to capture the flow as it passed through the marrow contact channels. Although it might have been possible to observe the flow path through the bone with staining during flow out, since the nuclear tissue and cartilage endplate would have been completely stained, the path through the tissue would not have been clear and was thus not attempted. As was the case after 15 min, bursts of stain in the cartilage were observed originating from the marrow contact with flow in. Further irrigation only resulted in complete staining of all structures in the flow path. Direction-dependent flow resistance in the intervertebral disc to ensure complete fluid recovery during rest has been recognized [19], but this mechanism has not been established. We hypothesize that this mechanism is the result of strain-dependent permeability of the cartilaginous endplate. During loading, solid dilatational stresses compress the cartilage matrix causing a decrease in its strain dependent permeability and increasing flow resistance. Conversely, as the disc swells during rest, the cartilage matrix expands decreasing flow resistance. Mansour and Mow [8] showed that both the strain and pressure gradients influence the non-linear strain-dependent permeability of cartilage. With a 20-fold increase in pressure and 30% more compressive strain, the permeability is approximately 10-fold lower. Although
the validity of these absolute values remains controversial, it exhibits that general compression of the cartilage endplate may be insufficient to produce the magnitude of observed direction-dependent resistance. However, the endplate cartilage is not supported by a perfectly permeable endplate of molecular pore size, but rather an endplate with openings giving rise to increased local pressure gradient and strains. During loading, the drag force of the fluid on the cartilage solid matrix and the solid dilatational stresses compress the endplate cartilage matrix into the channel opening, causing a decrease in its strain dependent permeability and increasing flow resistance. Conversely, as the disc swells during rest, the cartilage matrix is expanded away from the channel opening decreasing flow resistance (Fig. 4). Another source of resistance to fluid flow in the intact disc is the nucleus pulposus. In these experiments the nucleus was removed from the plugs, and hence the absolute resistances measured in these experiments are much different to that in the intact disc. However, this was done to isolate the difference in resistances that allows for faster fluid imbibing compared to exuding. In the intact disc, mean positive and negative pressures within the nucleus during activity and rest cause interstitial fluid flows within the nucleus, toward the endplate, and into the vasculature of the vertebral bodies. Due to the loss of fluid and associated compressive volumetric strain, the Permeability within the nucleus decreases. However, as is commonly observed in confined compression tests of poroelastic soft tissue (e.g., cartilage), compression and recovery speeds are similar. Thus the difference in fluid flow velocities cannot be explained by the strain dependent permeability of the nucleus alone, and it was removed in our tests. In addition, the overall additional equal flow resistance of the
,marrow contact channel
Flow out
Flow in Fig. 4. Direction-dependent resistance in the intervertebral disc may be due to constriction flow in the cartilage overlying the marrow contact channels in the endplate. During flow out the fluid is constricted through the channels, resulting in high local velocity and therefore high drag forces. These drag forces and the solid dilatational stresses compress the cartilage matrix into the channels. The resulting consolidation of the cartilage causes high resistance to flow. Conversely, as fluid flows back into the disc, the cartilage matrix is dragged away from the hole, expanding, and the resistance is relatively lower.
D.C. Ayotte t'i al. I Joournul oj' Orthipaedic Rrseurch 19 [?001) 1073-1077
disc would in fact reduce the resistance ratio of the endplate. But this should be a lesser order effect because the fluid flux area of the nucleus is several orders larger than that of the endplate channels, whereas the permeability of the iiucleus is not several orders less than that of endplate cartilage. In summary, the flow resistance in the intervertebral endplate was demonstrated to be highly direction-dependent and probably sufficiently so to ensure that all of the fluid lost during daily loading is recovered during rest. Furthermore, by staining the path of fluid flow through the endplate plugs, we established that the observed direction-dependent resistance occurred during flow through the marrow contact channels in the endplate. These findings, combined with our experimental and analytical modeling studies [3,4], suggest that direction-dependent resistance in the isolated endplate arises from the flow induced local compression of the poroelastic cartilage of strain dependent permeability at the marrow contact channel openings.
Acknowledgements
This project has been supported in part by a research grant from the A 0 Foundation, Switzerland. The authors would like to thank Iris Keller for her assistance in histological preparation, Dr. M. Duerrenberger for his advice in cryofixation and freeze substitution, and Dr. D. Pfluger for aid in statistical analysis.
References [I] Adams MA, Hutton WC. The effect of posture on the fluid content of lumbar intervertebral discs. Spine 1983;8:665-71. [?I A d a m MA, McNally DS, Dolan P. 'Stress' distributions inside intervertebral discs. The effects of age and degeneration. J Bone Joint Surg Br 1996;78:965-72. [ 3 ] Ayotte D, Ito K, Tepic S, Perren SM. Direction-dependent constriction flow in a poroelastic solid: the intervertebral disc valve. J Biomech Eng 2000;122:587~-93. [4] Ayotte D, Tepic S, Perren SM. Non-linear constriction flow in a poroelastic solid: a model of the intervertebral disc valve. Conf Eur Soc Biomech 1996;10:183. [5] Botsford DJ, Esses S1, Ogilvie-Harris DJ. In vivo diurnal variation in intervertebral disc volume and morphology. Spine 1994:19:93540.
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[6] Buschmann MD, Jurvelin JS, Hunziker EB. Comparison of sinusoidal and stress relaxation measurments of cartilage in confined compression: the biphasic poroelastic model and the role of the porous compressing platen. Trans Orthop Res Soc 1995;20:521. 171 Gleizes V, Viguier E, Feron JM, Canivet S, Lavaste F. Effects of freezing on the biomechanics of the intervertebral disc. Surg Radio1 Anat 1998;20:403-7. [8] Mansour JM, Mow VC. The permeability of articular cartilage under compressive strain and at high pressures. J Bone Joint Surg Am 1976;58:509-16. [9] Maroudas A, Stockwell RA, Nachemson A, Urban J. Factors involved in the nutrition of the human lumbar intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat 1975:llO: 113-30. [lo] McNally DS, Adams MA. Internal intervertebral disc mechanics as revealed by stress profilometry. Spine 1992;17:66-73. [I I] Nachemson A, Elfstrom G. Intravital dynamic pressure measurements in lumbar discs. A study of common movements, maneuvers and exercises. Scand J Rehabil Med Suppl 1970;1:140. [I21 Nachemson A, Lewin T, Maroudas A, Freeman MA. In vitro diffusion of dye through the end-plates and the annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand 1970;41:589407. [13] Ogata K, Whiteside LA. 1980 Volvo award winner in basic science. Nutritional pathways of the intervertebral disc. An experimental study using hydrogen washout technique. Spine 1981;6:211-6. [14] Rahn BA. Intra vitam staining techniques. In: von Recum AF, editor. Handbook of Biomaterials Evaluation. New York: McMillan; 1986. p. 471-87. [I51 Setton LA, Zhu W, Weidenbaum M, Ratcliffe A, Mow VC. Compressive properties of the cartilaginous end-plate of the baboon lumbar spine. J Orthop Res 1993;11:228-39. [16] Sitte H, Edelmann L, Neumann K. Cryofixation without pretreatment at ambient pressure. In: Steinbrecht RA, Mueller M, editors. Cryotechniques in Biological Electron Microscopy. Germany: Springer; 1987. p. 87-109. [I71 Smeathers JE, Joanes DN. Dynamic compressive properties of human lumbar intervertebral joints: a comparison between fresh and thawed specimens. J Biomech 1988;21:425-33. [I81 Steinbrecht RA, Zierhold K. Freeze-substitution and freezedrying. In: Steinbrecht RA, Mueller M, editors. Cryotechniques in Biological Electron Microscopy. Germany: Springer; 1987. [I91 Urban JP. Factors influencing the fluid content of intervertebral discs. Adv Microcirc 1987;13:160--70. [20] Urban JP, Holm S, Maroudas A, Nachemson A. Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop 1977;101-1 4. [21] Urban JP, McMullin JF. Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration. Spine 1988;13:179-87. [22] Wilke HJ, Neef P, Caimi M, Hoogland T, Claes LE. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine 1999;24:755-62.
Journal of Orthopaedic Research Journal of Orthopaedic Research 19 (2001) 1073-1077 www.elsevier.com/locate/ort hres
Direction-dependent 'resistance to flow in the endplate of the intervertebral disc: an ex vivo study D.C. Ayotte
a-b,
K. Ito
S. Tepic
a
Abstract
A comparison of the higher hydrostatic pressure in the nucleus of the healthy intervertebral disc during daily loading with the relatively lower osmotic swelling pressure in the disc during rest suggests the existence of direction-dependent flow resistance such that all of the fluid exuded from the disc during loading is recovered during rest. In this study, this direction-dependent resistance was demonstrated for flows through the cartilage endplates and the underlying marrow contact channels in the bony endplates. for flow through the endplate was 39.0 f 3.8 (mean f S.E.).In Using an ex vivo sheep endplate model, the resistance ratio (ROut/R,") addition, a path of fluid flow through the marrow contact holes was revealed using fluorescent staining. 0 1001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction
The daily load-induced pressure in the healthy intervertebral disc is countered in part by the osmotic swelling pressure of the disc tissue [21]. However, under normal daily loads this hydrostatic pressure, 0.6 to > 1.O MPa, remains approximately three to five times higher than the swelling pressure, 0.15-0.2 MPa, resulting in net fluid flow out of the disc [2,10,11,19,22]. Although the decrease in disc hydration causes an increase in the swelling pressure, equilibrium between swelling and hydrostatic pressure is likely never reached [71]. In the daily cycle the disc can lose up to 20% of its volume [5]. During rest, the osmotic swelling pressure draws fluid back into the disc, and as the disc rehydrates, the swelling pressure decreases. The weaker swelling pressure, combined with a relatively short period of rest (approximately 8 h of sleep vs. 16 h of daily loading), results in a lower driving potential for flow into the disc than for flow out of the disc. Therefore, some directiondependent mechanism must exist to ensure that all the fluid exuded from the disc during loading is recovered during rest. Based on an assumption that the dominant flow path is through the cartilage endplates, we propose that the *Corresponding author. Tel.: +41-81-414-M50; fax: +11-81-4112288. E-muil uddress: keita.ito@!ao-asif.ch (I(.Ito).
direction-dependent mechanism is provided by a combination of the strain dependent behavior of the cartilage endplate [ 151 and the presence of holes, i.e., marrow contact channels, through the underlying bony endplate [4,9,13,20]. This direction-dependent resistance mechanism was demonstrated in a physical model and a corresponding finite element model [3,4] and was also observed in exudation flow from cartilage compressed with a porous platen [6]. The objectives of the current study were to establish, ex vivo, that flow through the endplates of the intervertebral disc experiences direction-dependent resistance and that this flow is through the marrow contact channels.
Methods Sheep endplates were used to determine the resistance ratio for exudation vs. imbibition flow in the disc (R<,,,,/R,,,).Three lumbar spines were harvested from healthy, adult female. Swiss alpine sheep and kept frozen at -23°C wrapped in saline soaked gauze and double sealed in a plastic bag for upto 20 months. Spine segments were cut from the frozen spines using a butcher's band saw (FK22. Bizerba. Zurich, Switzerland) as required. The thickness of the vertebrae on each side of the disc was reduced using a grinding wheel (Struers RotoPol 25, E. Merck, Darmstadt, Germany) such that only the compact bone and a thin layer of trabecular bone of the bony endplate remained. Marrow was gently removed from any exposed trabeculae with irrigation, and the spine segments were then cut transversely through the disc using a scalpel. One 10 mm diameter plug was cut from the center of each endplate using a punch and hammer (Fig. 1). As much nuclear tissue as possible was removed with a scalpel in order to isolate the effects of the cartilage endplate. However, this proved
0736-0266/01/$ - see front matter 0 2001 Orthopaedic Research Society. F'ublished by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 0 2 6 6 ( 0 1 ) 0 0 0 3 8 - 9
D. C. Aj'otte t z t ul. I Jou~nuIof Orthopedic Research 19 1-7001i 1073- 1077
1074
pressure ratio (Pc,L,l/R,,). Steady-state pressure was assumed as the value at which the pressure converged to within 5?0 of the estimated final value over a period of approximately 15 min. A general linear model of the form: 3;) = / L
'trabecular bone Fig 1 Sheep endplate plug harvesting One I0 0 mm diameter plug cut from the center of each endplate using a punch and hammer.
UAS
ditlicult. and some nuclear tissue was preserved in order to avoid damaging the cartilage endplates. A glass column of a HPLC system was used to administer flows through the endplate plugs ( Superfoniiancea Universal glass-cartridge system, E. Merck) (Fig. 2). The plugs were supported within the column on each side by customized cylindrical stainless steel supports, which in turn were held in place and sealed on each side by variable nylon adapters. The steel supports were designed with circumferential grooves, and the variable adapters were threaded into the column ends such that they could compress the supports against the plug with great force in order to avoid leakage around the outside of the plug. In addition. O-rings were placed between the adapters and aluminum supports (in addition to the existing O-rings in the variable adapters) to completely seal the plug within the column. The entire assembly was housed within the outer jacket and maintained at 37°C with a water bath for the duration of the experiments. A constant flow rate pump (LiChroGraph" HPLC Pump, Model L-6000, E. Merck) was used to puinp Ringer's solution through endplate plugs. Eight samples were tested (four plugs from Sheep A, two from Sheep B, and two from Sheep C). The solution was randomly pumped in alternate directions to simulate flow into the disc (flow in: fluid pumped from the bone side of the plug) and flow out of the disc (flow out: fluid pumped from the tissue side of the disc) (Fig. 2). A flow rate of 1 pl/inin was applied in each direction, and a cut-off pressure during flow out was set at 110 bar to avoid damaging the equipment. The pressure was measured using the existing pressure transducer in the pump and recorded using an .Y Y plotter (SEFRAM, Paris, France). The steady-state pressure was used to calculate the average resistance, R: R = P/Q. (1) where Q [m2/s] was the applied flow rate and P [Pa] was the steadystate pressure. The resistance ratio (ROut/R,") was therefore equal to the
glass tube stainless steel supports
Ringeris
variable adapter
\
cartilage
bone
Fig. 2. Schematic cross-section of the experimental set-up. Endplate plugs were supported and sealed within a glass tube. The resultant pressure was measured for fluid pumped in alternate directions (at a flow rate of 1 pI/min) to simulate flow into the disc (flow Bi) and flow out of the disc (flow out).
+ 6 , . direction, +
C&
. sheep,
+ E. . direction, . sheep, + I:,,
(2)
was initially used for statistical analysis (SPSS AG, Zurich, Switzerland) of the direction-dependence of the flow resistance where 3;) =pressure, p = overall mean, = effect of direction, ( i = I . ? ) , Ji = effect of sheep, (i = I , ? . 3 ) , i= interaction effect of direction, x sheep,, and F , / = error of directioti, and sheep,. If the effect of sheep and sheep * direction interaction was not significant ( P 2 0.05) then the data could be pooled, and the ratio Pc,u,/F',,,of each specimen examined for the effect of direction. Otherwise, if an interaction was found, P,,,l/P,nwas calculated using least-square means. In the case of the former. significance ( P < 0.05) of direction-dependence resistance was tested using a one-sample (-test. For both analyses the required assumptions were validated using the Shapiro-Wilk's test for normality and Levene's test for equality of variance. The path of fluid flow through the endplate plugs during pumping was visualized with a fluorescent dye (Procion Red H-8BN. Imperial Chemical Industries PLC, London, U K ) added to the Ringer's solution. Four additional plugs were prepared and pumped as for the resistance ratio tests. One plug each was stained for a time period of 15, 30, 60 and 90 min. The fluid was pumped at a flow rate of 1 ~ I / m i n o n l y in the flow in direction to capture the dye as it entered the cartilage endplate through the marrow contact channels. Upon completion of staining, each plug was immediately removed from the glass column and plunge-frozen in liquid nitrogen [16]. Freeze-substitution was then performed in acetone for ten days at - S O T , with 3% gluteraldehyde and molecular sieve [IS]. The samples were transferred to -23°C for a further six days and to 4°C for one final day to complete fixation. PMMA embedded undecalcified histology sections, 60-80 pm thick, were cut (1600 Saw-Microtome, Leica, Nussloch, Germany) and ground (Wmtzky Flachlappmachine, Stiheli, Pieterlen, Switzerland) for fluorescence microscopy from which the path of Ruid flow through the plugs could be determined according to the location of fluorescent stain [IJ].
Results
The pressure during flow out in all eight endplate plugs was considerably greater than the pressure during ilow in (Table 1). In two cases the pressure during flow out reached the cut-off of 110 bar. Taking a conservative approach, 110 bar was taken as the pressure for flow out in these cases. The general linear model revealed that there was no statistically significant effect of sheep ( P = 0.36) or interaction of sheep and direction ( P = 0.26). That is, plugs from different sheep did not exhibit different resistances, and they did not exhibit differences in direction-dependence (& >> &). Hence, the sheep effect could be ignored, allowing pooling of the data. The one sample t-test revealed that the resistance to flow out was significantly greater to that of flow in ( P < 0.001) with a resistance ratio (RoUt/Rln) of 39.0 f 3.8 (mean iS.E.). Staining with Procion red during flow in revealed fluid flow through the marrow contact channels in all endplate plugs. When examined under the fluorescence microscope, no differences in the location or intensity of stain were observed between plugs infused for 30, 60, and 90 min. Fluorescent stain was observed in the marrow cavities of the trabecular bone, through the
D.C. Ayotte et crl. I Journol qf’ Orthopedic- Resrtrrch I 9 12001) 1073-1077
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Table 1 Pressure drop as the 10.0 mm diameter ovine endplate discs with a How rate of 1 PI/ mint Specimen number
Sheep
Pressure out [bar]
Pressure in [bar]
Rout /Rm
1 2 3 4 5 6 7 8
A A A A
110 110 86 80 97 70 67 80
2.0 2.8 2.8 I .9 1.8 2.3 2.6 2.3
55.0 39.3 30.7 19.I 53.9 30.4 25.8 34.8
B B C C
“Resistance ratio (Rc,u,/R,n) = 39.0 f 3.8 (mean
S.E.).
Fig. 3. Ringer’s lactate solution stained with Procion red was pumped through endplate specimens in the flow in direction (from the bone to the cartilage). In this sample, infused for 15 min, a flow path was demonstrated as the stain flowed through the marrow contact channels (C) in the compact bone (BL through the marrow contact openings at the bone-cartilage interface (BC), and just began to flow into the cartilage endplate. Scale bar units are I pm.
marrow contact channels, and in the cartilage endplate of these plugs. In the plug infused for 15 min., the marrow contact channels were observed to fluoresce brightly, with ‘bursts’ of stain in the cartilage endplates originating from the marrow contact holes (Fig. 3).
Discussion This study demonstrates that fluid flow through the endplates of the intervertebral disc experiences a direction-dependent resistance, where resistance to exudation flow out of the disc is much greater than that to imbibition flow in. Our ex vivo experiments revealed that at a steady-state flow rate of 1 pl/min, the pressure drop across the sheep endplate plugs during flow out was significantly greater than that during flow in ( P < 0.001), and that this direction-dependent resistance ratio (Rout/&) was 39.0 h 3.8 (mean f S.E.). Furthermore, staining of the fluid during flow itz clearly demonstrated a flow path through the marrow contact
channels where initially the stain was observed to burst through the marrow contact channels and thereafter to stain the entire depth of the cartilage endplate. The use of quadraped spine as opposed to human spine is persistently controversial in biomechanical experiments of the spine. However, in our experiments, functional spine units were not loaded, and only the isolated endplates were used. Of most importance, in both sheep and human, a cartilaginous layer has been found to cover marrow contact channels in the osseous endplate [4], as is true of other quadrapeds [12]. Although the endplates may differ in the size and number of marrow contact channels resulting in different resistance ratios, the observed direction-dependent resistance is believed in principle to be similar to that in humans. The use of frozen specimens was supported by the lack of mechanical property changes with freezing [7,17]. Since our experiment depended on preservation of the cartilage macro-structure, use of frozen tissue is unlikely to have caused major artifacts. Moreover, the period of freezing did not appear to affect the results, as no obvious differences were observed for samples frozen for 16 and 20 months vs. those frozen for 1 month. The hydrostatic pressure during loading of the human spine is at least three times greater than the osmotic pressure during rest [2,10,19] and occurs for approximately twice as long. Thus, pressure control would have been better suited for our experiments. However, the pump available to us was flow-rate controlled and had a minimum flow rate of 1 pl/min. Although this flow rate is comparable to that which is likely to occur across the entire human endplate [l], due to the relatively small cross-sectional area of the plugs compared to the total area available for flow in vivo, the resistance ratio measured in this study may be exaggerated. Higher fluid velocity through the plugs may have accentuated consolidation and expansion of the cartilage matrix into and out the channels resulting in greater R,,,, smaller R,,,, and higher R,,,/RIn ratios. Nevertheless, even an order of magnitude reduction from our measured resistance ratios is probably of sufficient direction-dependence to maintain a diurnal fluid volume balance.
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D. C. .$yotte et al. I Journal of Orthojiardic Research 19 (-7001) 1073-1077
Another possible artifact of this experimental method was fluid leakage around the plug, especially during the flow in. An attempt was made to limit this flow path by compressing the outer rim of the specimens with serrated edge stainless steel supports (Fig. 2). Furthermore, examination of the cartilage endplates after irrigation with the stained fluid showed that the outer area of cartilage endplate beneath the supports was not irrigated. Hence, flow around the specimens was highly unlikely. Preparation of the endplate plugs involved the reduction of the vertebral bone to a thin layer and gentle removal of trabecular marrow content. This was necessary to isolate the effects of the cartilage endplate, as well as to remove any marrow that was forced into the marrow contact channels during grinding. This removal of the contents, normally partially obstructing the endplate openings, probably lead to a reduction of the resistance to flow in both directions, but would not have significantly affected the direction-dependent nature of the resistance ratio. On the other side of the specimen plugs, the presence of some softer poroelastic nuclear tissue may have biased our results with further increases in the resistance ratio. However, this is still consistent with our hypothesis of a poroelastic material over a constriction hole mechanism responsible for the direction-dependent resistance and is consistent with the in vivo situation. Staining was only conducted during flow in. The objective of irrigation with a stained fluid was to capture the flow as it passed through the marrow contact channels. Although it might have been possible to observe the flow path through the bone with staining during flow out, since the nuclear tissue and cartilage endplate would have been completely stained, the path through the tissue would not have been clear and was thus not attempted. As was the case after 15 min, bursts of stain in the cartilage were observed originating from the marrow contact with flow in. Further irrigation only resulted in complete staining of all structures in the flow path. Direction-dependent flow resistance in the intervertebral disc to ensure complete fluid recovery during rest has been recognized [19], but this mechanism has not been established. We hypothesize that this mechanism is the result of strain-dependent permeability of the cartilaginous endplate. During loading, solid dilatational stresses compress the cartilage matrix causing a decrease in its strain dependent permeability and increasing flow resistance. Conversely, as the disc swells during rest, the cartilage matrix expands decreasing flow resistance. Mansour and Mow [8] showed that both the strain and pressure gradients influence the non-linear strain-dependent permeability of cartilage. With a 20-fold increase in pressure and 30% more compressive strain, the permeability is approximately 10-fold lower. Although
the validity of these absolute values remains controversial, it exhibits that general compression of the cartilage endplate may be insufficient to produce the magnitude of observed direction-dependent resistance. However, the endplate cartilage is not supported by a perfectly permeable endplate of molecular pore size, but rather an endplate with openings giving rise to increased local pressure gradient and strains. During loading, the drag force of the fluid on the cartilage solid matrix and the solid dilatational stresses compress the endplate cartilage matrix into the channel opening, causing a decrease in its strain dependent permeability and increasing flow resistance. Conversely, as the disc swells during rest, the cartilage matrix is expanded away from the channel opening decreasing flow resistance (Fig. 4). Another source of resistance to fluid flow in the intact disc is the nucleus pulposus. In these experiments the nucleus was removed from the plugs, and hence the absolute resistances measured in these experiments are much different to that in the intact disc. However, this was done to isolate the difference in resistances that allows for faster fluid imbibing compared to exuding. In the intact disc, mean positive and negative pressures within the nucleus during activity and rest cause interstitial fluid flows within the nucleus, toward the endplate, and into the vasculature of the vertebral bodies. Due to the loss of fluid and associated compressive volumetric strain, the Permeability within the nucleus decreases. However, as is commonly observed in confined compression tests of poroelastic soft tissue (e.g., cartilage), compression and recovery speeds are similar. Thus the difference in fluid flow velocities cannot be explained by the strain dependent permeability of the nucleus alone, and it was removed in our tests. In addition, the overall additional equal flow resistance of the
,marrow contact channel
Flow out
Flow in Fig. 4. Direction-dependent resistance in the intervertebral disc may be due to constriction flow in the cartilage overlying the marrow contact channels in the endplate. During flow out the fluid is constricted through the channels, resulting in high local velocity and therefore high drag forces. These drag forces and the solid dilatational stresses compress the cartilage matrix into the channels. The resulting consolidation of the cartilage causes high resistance to flow. Conversely, as fluid flows back into the disc, the cartilage matrix is dragged away from the hole, expanding, and the resistance is relatively lower.
D.C. Ayotte t'i al. I Joournul oj' Orthipaedic Rrseurch 19 [?001) 1073-1077
disc would in fact reduce the resistance ratio of the endplate. But this should be a lesser order effect because the fluid flux area of the nucleus is several orders larger than that of the endplate channels, whereas the permeability of the iiucleus is not several orders less than that of endplate cartilage. In summary, the flow resistance in the intervertebral endplate was demonstrated to be highly direction-dependent and probably sufficiently so to ensure that all of the fluid lost during daily loading is recovered during rest. Furthermore, by staining the path of fluid flow through the endplate plugs, we established that the observed direction-dependent resistance occurred during flow through the marrow contact channels in the endplate. These findings, combined with our experimental and analytical modeling studies [3,4], suggest that direction-dependent resistance in the isolated endplate arises from the flow induced local compression of the poroelastic cartilage of strain dependent permeability at the marrow contact channel openings.
Acknowledgements
This project has been supported in part by a research grant from the A 0 Foundation, Switzerland. The authors would like to thank Iris Keller for her assistance in histological preparation, Dr. M. Duerrenberger for his advice in cryofixation and freeze substitution, and Dr. D. Pfluger for aid in statistical analysis.
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