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. 2013 Jan 13;222(3):341–348. doi: 10.1111/joa.12022

Factors regulating viable cell density in the intervertebral disc: blood supply in relation to disc height

Olga A Boubriak 1, Natasha Watson 1, Sarit S Sivan 2, Naomi Stubbens 1, Jill P G Urban 1
PMCID: PMC3582253  PMID: 23311982

Abstract

The intervertebral disc is an avascular tissue, maintained by a small population of cells that obtain nutrients mainly by diffusion from capillaries at the disc–vertebral body interface. Loss of this nutrient supply is thought to lead to disc degeneration, but how nutrient supply influences viable cell density is unclear. We investigated two factors that influence nutrient delivery to disc cells and hence cell viability: disc height and blood supply. We used bovine caudal discs as our model as these show a gradation in disc height. We found that although disc height varied twofold from the largest to the smallest disc studied, it had no significant effect on cell density, unlike the situation found in articular cartilage. The density of blood vessels supplying the discs was markedly greater for the largest disc than the smallest disc, as was the density of pores allowing capillary penetration through the bony endplate. Results indicate that changes in blood vessels in the vertebral bodies supplying the disc, as well as changes in endplate architecture appear to influence density of cells in intervertebral discs.

Keywords: capillary density, diffusion, endplate, subchondral porosity

Introduction

The intervertebral disc is avascular. Over most of the disc, a small population of cells, vital for maintaining disc health, obtains nutrients from the capillaries at the disc–vertebral body interface. Nutrients, mainly oxygen and glucose, then diffuse from these capillaries to the disc cells where they are consumed; the metabolites produced, mainly lactic acid, then diffuse from the cells through the matrix to the blood supply (reviewed in Grunhagen et al. 2011). Because nutrients are transported through the disc by diffusion, gradients of nutrient concentration develop whose magnitude is governed by the balance between the rate of transport of nutrients to the cells and the rate of cellular consumption. If cellular consumption rates are high compared with transport rates, nutrient levels in the centre may be depleted to levels unable to sustain viable cells.

Disc height is one of the important parameters regulating nutrient gradients as shown by modelling nutrient diffusion into the disc and hence in regulating the density of viable cells in the disc centre (Shirazi-adl et al. 2010; Galbusera et al. 2011; Jackson et al. 2011). Diffusion models, which predict an inverse relationship between diffusion distance and cell density, are in agreement with findings in articular cartilage, which like disc is avascular. Stockwell (1971) examined articular cartilage from a wide range of different joints and animal species, and found that cell density was inversely related to cartilage thickness, and that the total viable cell number supported per unit area of surface was relatively independent of cartilage thickness. A similar inverse relationship between viable cell density and diffusion distance and total cell number supported per surface area has been seen in avascular constructs of articular chondrocytes and disc cells (Freed et al. 1994; Horner & Urban, 2001). These results all show that cell density is regulated by nutrient supply.

There are relatively few published measurements of cell density in the disc (Maroudas et al. 1975; Holm & Nachemson, 1983; Vernon-Roberts et al. 2008; Liebscher et al. 2011; Rodriguez et al. 2011) and, while an inverse relationship between disc height and cell density has been reported (Holm & Nachemson, 1983; Rodriguez et al. 2011), the correlation in humans was poor, possibly because of confounding effects of age and pathology. Nevertheless, we hypothesised that in the disc, as in other avascular systems and as predicted from mathematical models, cell density should decrease with increasing disc height. In order to test this hypothesis, we investigated the relationship between cell density and disc height in the central region of the disc, the nucleus pulposus. We used bovine tail discs as a model system. The discs of the bovine tail are similar to human discs in cell phenotype and in composition, and are regarded as a good model of human disc (Ohshima et al. 1993; Demers et al. 2004). In the bovine tail disc height varies progressively with disc level, and we could thus examine the relationship between disc height and cell density in healthy young animals of very similar age with >95% cell viability (Grunhagen, #b300). We also investigated blood vessels of the vertebral body adjacent to the disc, the area of contact of marrow spaces of the subchondral plate with the cartilaginous endplate as this area correlates with permeability of the subchondral plate (Laffosse et al. 2010), and the rate of disc cell metabolism; these factors could also influence cell density.

Materials and methods

Materials

Tissue-Tek compound was purchased from Sakura Finetek, Thatcham, UK. Chondroitin sulphate no. C8529, hydroxyl-l-proline no.H6002, deoxyribonucleic acid (DNA) sodium salt from calf thymus, Type I no. D1501 were purchased from Sigma-Aldrich, Poole, Dorset, UK. Papain from papaya latex buffered aqueous suspension, 2 × crystallised, no. P3135–100MG was purchased from Sigma Life Science, Gillingham, Dorset, UK. Bisbenzimide H 33258 (Hoechst 33258) no. B2883 and deoxyribonuclease I from bovine pancrease (DNase) no. D5025 were obtained from Sigma-Aldrich. Formic acid bone decalcifier Immunocal no. 1440 was obtained from DECAL Chemical, NY, USA.

Methods

Dissection of spinal motion unit and disc tissue

Spinal motion units and intervertebral discs were dissected from bovine tails of 18–24-month-old cattle. Tails from five animals were used for the work described here. Discs 2, 5, 8 and 11 counting from the cranial to caudal direction (Fig. 1a) were dissected together with adjacent vertebrae and frozen. Each motion unit was cut sagittally and disc height was measured through the centre using callipers (Fig. 1b). One half of each motion segment was used to analyse disc composition biochemically, and the other half was used for histology of the discs and adjacent vertebrae.

Fig. 1.

Fig. 1

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Schematic showing the methods of tail and disc dissection. (a) Schematic shows the numbers of the discs (D) and vertebral bodies (V). Discs 2, 5, 8 and 11 counting from the cranial–caudal direction were dissected together with adjacent vertebrae. (b) Disc height was measured in the centre of sagittally sectioned motion segments. (c) The nucleus was dissected sagittally into inner (i) and outer (ii) nucleus samples. (d) The nucleus also was dissected transversely in the cranial–caudal direction, and sections were numbered from 1 to 6. The sixth section was discarded.

For biochemical analyses, full-depth disc tissue was dissected from the vertebrae using a scalpel. A section was cut through the disc sagittally and the nucleus, taken as the central region with no evidence of annular rings present, was carefully dissected out. It was divided into a central region and a region adjacent to the annulus to assess whether disc composition in the nucleus was constant (Fig. 1c). The cranial face of the nucleus was mounted on cork using Tissue-Tek. The nucleus was then snap-frozen, and cut using a cryo-microtome (Leica, CM 1900, Wetzlar, Germany) into 60-μm sections to assess disc composition in the cranial–caudal direction; 8–18 sections were pooled depending on disc height, to give six combined sections for measuring changes in cranial–caudal composition. These were numbered 1–6 in the caudal–cranial direction. The sixth portion (the most cranial) was discarded because of contamination by Tissue-Tek (Fig. 1d).

Papain digestion

Samples of nucleus were weighed wet and dry, and digested at 70 °C overnight in 1% (v/v) of papain in sodium-free papain buffer (Urban et al. 1998).

Glycosaminoglycan content

Total glycosaminoglycan content was determined in papain digests using the colorimetric assay described by Farndale et al. (1986). Briefly, aliquots of papain digests were mixed with dimethylmethylene blue reagent, and absorbance at 525 nm was immediately determined on a Perkin-Elmer spectrophotometer (Waltham, USA). Chondroitin sulphate from bovine trachea was used as a standard.

Collagen content

Aliquots of papain digests were hydrolysed in 6 m hydrochloric acid for 18 h at 110 °C and neutralised with 10 m sodium hydroxide. Total hydroxyproline content of the hydrolysed samples was determined using a modification of the Stegemann procedure (Stegemann & Stalder, 1967). The concentration of collagen was calculated from the hydroxyproline content, considering that types I and II collagens contain 13% of hydroxyproline by weight (Maroudas et al. 1985).

Lactate production rate

Slices of nucleus from discs 2 and 11 were weighed and incubated in Dulbecco's modified Eagle's medium (22320-022; Invitrogen, Paisley, UK) for up to 48 h. Lactate concentration of the medium was measured and rate of lactate production determined (Zhou et al. 2008).

Cell density

DNA was measured using Hoechst 33258 allowing for tissue autofluorescence (Urban et al. 1998). Cell density of the disc samples was evaluated from DNA content assuming that one cell contains 7 pg DNA (Kim et al. 1988). As the disc nucleus is about 83% water per tissue weight, the density of the disc nucleus is close to 1.0; hence, results approximate to cell density and are reported as such as it is this value that is generally reported in the literature.

Scanning electron microscopy (SEM)

Soft tissues were digested from between the vertebrae V 1–2 and V 11–12 (Fig. 1a) using 1% (v/v) papain. The bone was then degreased using xylene and air-dried overnight. A square section, 12 × 12 mm was cut from the central area of the bone overlying the nucleus pulposus (Fig. 2a). After washing in 100% ethanol the bones samples were air-dried, mounted on stubs and sputter-coated with gold. The samples were examined in a JEOL JSM-630 SEM (JEOL UK, London, UK). For quantitation, similar regions from each sample were photographed at × 100 magnification; the number and diameter of openings were measured in the centre of the nucleus and at a distance equal to one half of the nucleus radius from the centre at the positions shown (Fig. 2b). SEM images (Fig 2c) were subsequently converted into grey-scale mode (Fig. 2d) and then into binary 2D images (Fig. 2e). The effective area of the openings (seen on binary images in black) was evaluated and openings were counted using the ‘analyse particles’ option of imagej (NIH public domain software; http://rsbweb.nih.gov/ij/). The lowest range of effective diameter of opening for capillary penetration was taken as 10 μm (Clark, 1990). Only unobstructed openings (Fig. 2c, black arrows) were evaluated. Closed and partly-closed openings (white arrows; Fig. 2c), together with the cracks (gradient-filled arrows; Fig. 2c), were deleted manually from binary images and excluded from evaluation.

Fig. 2.

Fig. 2

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Sampling of vertebral bodies for SEM measurement. (a) A photograph of a vertebral bone surface showing the area cut for sampling (square); the area underlying the nucleus pulposus is ringed. (b) The samples chosen for SEM measurements showing the central region and the surrounding regions at a distance from the centre of ½ radius of the nucleus. (c) Only fully opened openings (black arrows) were evaluated. Closed and degenerated openings (white arrows) together with the cracks (gradient fill arrows) were ignored. (d) Binary image with cracks, partially closed openings and degraded ones being removed. (e) Outlined openings taken into account by imagej software.

Histology

The vertebrae adjacent to the discs 2, 5, 8 and 11 (Fig. 1a) were fixed in 10% buffered formalin and then decalcified using the formic acid bone decalcifier Immunocal. The areas overlying the nucleus pulposus were dissected, and immersed in Tissue-Tek compound overnight at 4 °C. Samples then were snap-frozen and sectioned at 60 μm using a cryo-microtome (Leica, CM 1900). The sections were stained for visualisation of the vascular network by detection of activity of endogenous peroxidase in red blood cells (Sherman & Paull, 1985).

Statistics

Results were expressed as a mean ± standard deviation (SD) or mean ± standard error (SE) as indicated in the legends. Differences were evaluated using two-sided unpaired t-tests, and were considered statistically significant when the probability value P was less than 0.05.

Results

Disc height vs. disc level

Figure 3 shows the height of discs (mm) varied with their level in the tail, decreasing significantly in the cranial–caudal direction (P < 0.001). The average height of most cranial discs (disc 2) was 6.87 mm, while that of most caudal tail discs used here (disc 11) was 2.91 mm.

Fig. 3.

Fig. 3

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Disc height in the cranial–caudal direction. Disc height decreased significantly in the cranial–caudal direction. Results are shown as mean ± SD.

Nucleus composition and energy metabolism vs. disc level

Figure 4a shows the variation in collagen content of the nucleus with disc level. The collagen content on a dry weight basis was significantly higher in the most caudal disc (disc 11; 50%) than in the other discs (34% in disc 2, the most cranial disc), with no apparent difference between central and peripheral regions of the nucleus. Figure 4b shows the GAG concentration vs. disc level. The GAG content of the most cranial disc (disc 2: 38% dry weight) was significantly greater than that of the most caudal disc (disc 11: 29% dry weight). For all discs, the GAG content per dry weight in the nucleus centre was significantly greater than that at the periphery of the nucleus. Figure 4c shows the variation in percentage GAG/dry weight across the nucleus in relation to the fractional distance from endplate–endplate. For all disc levels, there was a trend for the GAG concentration to be lowest near the vertebral bodies and higher towards the centre of the nucleus, but differences in composition did not reach signifance.

Fig. 4.

Fig. 4

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Variations of disc composition in relation to disc level: nucleus pulposus collagen and GAGs profiles in the nucleus–annulus direction and in discs of different size. (a) Collagen content was not significantly different in the nucleus–annulus direction, and it was significantly higher in the smallest disc 11 in comparison with larger discs. (b) Nucleus GAG content decreased significantly from the central to peripheral region of the nucleus (paired t-test, P < 0.01, not indicated on the figure). (c) Profile of GAG content in the nucleus in relation to disc level and within the discs in the caudal–cranial direction. GAG content of the discs decreased significantly from disc 2 to disc 5 and from disc 5 to disc 11 (paired t-test, P > 0.01). There was a slight but not significant increase in GAG content in the centre of the nucleus. Data are presented as mean ± SD.

There was no significant difference in rate of lactate production, as a measure of total energy production (Guehring et al. 2009), between discs 2 (0.87 ± 0.17 μmol g−1 tissue−1 h−1) and 11 (0.92 ± 0.07 μmol g−1 tissue−1 h−1).

Cell density of the nucleus vs. disc level and disc height

Cell density, calculated from DNA levels, showed no significant difference between disc levels (Fig. 5). There were large variations in absolute cell density from tail to tail, resulting in large standard errors; however, within individual tails, there was also no consistent relationship between disc level and cell density (not shown). Viability of cells in bovine disc tissue was >95% (not shown).

Fig. 5.

Fig. 5

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Nucleus cell density in relation to disc level in the cranial–caudal direction. There were no apparent trends with disc level. Data are presented as mean ± SE.

Profile of capillary networks in the subchondral plates and vertebral bodies

Figure 6 shows the blood vessels of the vertebrae and subchondral bone adjacent to the disc, visualised by staining the haemoglobin of the red blood cells trapped in the capillaries.

Fig. 6.

Fig. 6

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Diaminobenzidine staining of endogenous peroxidase in red blood cells (brown deposit). Discs 2, 5, 8 and 11 are shown schematically in a, b, c and d, respectively, together with the microscopic images of corresponding subchondral bones of the vertebras (V) adjacent to the discs in correct orientation on either side. The density of capillary network as well as the size of capillaries decreased in subchondral bones as disc size decreased in the caudal–cranial direction.

The figure is organised to show a view of each disc examined (discs 2, 5, 8, 11), with the cranial and caudal vertebrae shown in the correct orientation on either side. Very few blood vessels are evident in contact with disc 11 at either the cranial or caudal disc–bone interface. Blood vessel density increased markedly in the caudal–cranial direction, with the density greatest in the vertebrae adjacent to disc 2. In addition, the diameter of the capillaries appeared greater in the more cranial vertebral bodies. Apart from disc 11 where very few vessels were seen, the density of capillaries appeared greater at the cranial than at the caudal bone–disc interface.

Area of pores > 10 μm penetrating the subchondral plate

Figure 7a and b shows typical SEMs at low power of the faces of the vertebral bodies overlying disc 2 and disc 11, respectively. A white star shows the position of the nucleus centre. The density of open pores appeared noticeably higher in disc 2 (Fig. 7a) than in disc 11 (Fig. 7b). Only the fractional area occupied by the open pores was measured (see Fig. 2c–e). Figure 7c and d shows that the fractional area of open pores is significantly larger in the vertebra V2–1 adjacent to disc 2 (the largest disc examined) than in that of vertebra V11–12 adjacent to disc 11 (the smallest disc examined) in both the central region of the nucleus (Fig. 7c) and in the area surrounding it (Fig. 7d).

Fig. 7.

Fig. 7

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Evaluation of opening fraction area of subchondral bone surface overlying the nucleus. (a, b) Typical SEMs of bone surface of vertebra V1–2 (a) and vertebra V 11–12 (b) adjacent to disc 2 and 11, respectively. The density of open pores appeared noticeably higher in bone surface adjacent to disc 2 (a) than that adjacent to disc 11 (b). A white star indicates the position of the nucleus centre. (c, d) The opening fraction area of bone surface of V1–2 is significantly higher than that of V11–12 in both the central region of the nucleus (c) and in the area surrounding it (d). The effective diameter of opening for capillary penetration was taken as ≥ 10 μm. Data are presented as mean ± SD.

Discussion

Here we aimed to examine the relationship between cell density and disc height in bovine tails as within the tail of each animal there are progressive changes in disc height with disc level (Fig. 3). Also, because these discs are non-degenerate and were from animals of very similar age, confounding effects of age and pathology were avoided.

We measured the changes in disc composition with disc level. We found that the composition of discs down the tail was broadly similar, although collagen content was significantly greater in the nucleus of the smallest, most caudal disc than in discs at other levels (Fig. 4a), while GAG content was significantly higher in the largest, most cranial disc, than in the more caudal discs (Fig. 4b). These differences in composition could relate to differences in customary loading, as there are large differences in the musculature surrounding the most cranial and most caudal disc levels.

We examined disc height in relation to cell density and found a 2.3-fold difference in disc height between smallest and largest discs (Fig. 3). Cell density however, showed no significant dependence either on disc level or disc height (Fig. 5); moreover, rates of energy metabolism were similar between the smallest and largest discs. We then examined other factors that could influence nutrient gradients across the disc. Variations in blood supply to the nucleus of the discs might be one such factor. As visualised qualitatively by staining residual haemoglobin, the density of blood vessels supplying the disc decreased markedly along the tail in the cranial to caudal direction (Fig. 6). We also measured the density of the pores that permit capillary penetration in the bony vertebral endplates adjacent to the most cranial (disc 2) and most caudal discs (disc 11). The number of pores per unit area in the region overlying the nucleus was significantly greater for the largest, most cranial disc (Fig. 7) than for the smallest disc. The density of these pores is directly correlated with the effective permeability of the subchondral plate (Laffosse et al. 2010), and hence is an additional indication that the nutrient supply to the largest disc is significantly greater than the supply to the smallest disc.

Based on the difference in disc heights seen in the bovine tail, diffusion theory predicts that the cell density in disc 11 should be twice as great as that in disc 2. However, we found no increase in cell density with a decrease in disc height (Fig. 5); rather the cell density tended to be somewhat greater in the larger discs, though not at the level of statistical significance. Thus, disc height does not appear to be the main regulator of cell density in bovine tail discs, a situation that contrasts strongly with that observed in articular cartilage and avascular constructs (Stockwell, 1971; Freed et al. 1994; Horner & Urban, 2001), where an inverse relationship exists between number of cells per unit tissue volume and the thickness of the tissue. The explanation may lie in an important difference in the way that these various tissues receive their nutrients. In articular cartilage in situ, the entire articular surface is bathed by synovial fluid that supplies the required nutrients; cell-matrix constructs in vitro would normally be surrounded by a nutrient medium. Both articular cartilage and cell-matrix constructs therefore have a large pool of nutrients – carried in fluid of near-constant composition and in close contact with their surfaces. Thus, in articular cartilage or constructs, the nutrient gradients within the tissue that regulate cell viability are governed not by nutrient availability at their surfaces but mainly by the distances that the nutrients have to move by diffusion when inside the matrix. However, in the disc in vivo, the nutrient pathway to the cells of the nucleus pulposus is much more complex, and diffusion distance through the matrix is only one of the factors involved in delivery of nutrients (reviewed by Grunhagen et al. 2011). The nutrient pathway to the disc nucleus begins in the arteries that supply the vertebral body; these feed the capillaries that penetrate the subchondral plate and terminate in loops at the interface with the cartilaginous endplate (Maroudas et al. 1975; Roberts et al. 1989; Crock et al. 1991; Oki et al. 1994). Nutrients move through the capillary walls and the cartilaginous endplate into the disc matrix by diffusion under gradients caused by cellular metabolism. The capillary loops adjacent to the cartilaginous endplate form the only contact between the disc matrix and the animal's bulk blood system; almost every aspect of supply to the capillaries and the architecture and performance of this system of capillaries could critically influence the nutrition of the disc, particularly of the nucleus. Disc nutrition in vivo is thus undoubtedly more precarious than that of articular cartilage. The disc has no covering layer of fluid to provide a constant and essentially unlimited supply of nutrients in direct contact with its surface. Instead, nutrient supply is limited by blood vessel supply and architecture even before entering the disc matrix itself; moreover, nutrients can encounter appreciable resistance to diffusion as they move through the material of the cartilaginous endplate (Roberts et al. 1996).

In the bovine discs studied here, there were no major differences between the discs themselves apart from size; the matrix was non-degenerate and the rates of nutrient consumption were similar between disc levels. However, the density of blood vessels supplying the larger discs was much greater than that supplying the smaller discs (Fig. 6), as was the density of pores through the subchondral plate (Fig. 7) large enough to permit capillary penetration. Hence, nutrient supply to the nucleus appears to fall as disc height decreases. Because cell densities were similar at all disc levels, it seems that similar minimum levels of nutrients were achieved in all the discs; the potentially beneficial effect of a decrease in disc height on nutrient gradients appears to be nullified by a reduction in the supply of nutrients to the disc.

It has long been suggested that restricted delivery of nutrients to the disc is associated with disc degeneration (Nachemson et al. 1970; Ogata & Whiteside, 1981). Recent post-contrast magnetic resonance imaging studies following diffusion of gadolinium salts into the disc have found that interruption of the nutrient supply occurs mainly at the endplate (Rajasekaran et al. 2007), probably because of deleterious changes to the architecture of that region, such as effective fall in permeability of the subchondral plate found in degenerate discs in some studies (Nachemson et al. 1970; Rutges et al. 2011) though not others (Rodriguez et al. 2012), and calcification of the cartilaginous endplates themselves (Roberts et al. 1989; Grignon et al. 2000; Peng et al. 2001; Benneker et al. 2005; Wang et al. 2012). The results of the present study show that, in addition, changes in blood supply must also be considered as an important factor limiting nutrient supply to the disc and hence the number of viable cells that can survive. This area has been less well studied, although the direct influence of changes in blood supply on nutrient transport has been measured by dosing with vasoactive substances (Holm & Nachemson, 1988; Rajasekaran et al. 2008), and atherosclerosis of the lumbar arteries (Kauppila, 2009; Suri et al. 2012) and a fall in vertebral body blood flow (Liu et al. 2009) are associated with disc degeneration.

Conclusions

The density of viable cells in the disc is governed by nutrient supply. Interruption of this supply at any point, whether the result of a restriction in blood supply to the vertebral bodies or because of structural changes at the endplate, will lead to a loss of viable cells. However, while both vertebral blood flow (Liu et al. 2009) and transport from the blood supply across the endplate to the disc (Rajasekaran et al. 2007) can be determined non-invasively by imaging, there is at present no way of studying the quantitative relationship between alterations in nutrient supply, changes in nutrient gradients and long-term effects on cell viability. That relationship can only be assessed by modelling (Soukane et al. 2007; Shirazi-adl et al. 2010; Galbusera et al. 2011; Jackson et al. 2011), yet is of interest for the light it might throw on the processes underlying disc degeneration, and also for the more distant aim of identifying patients who might benefit from disc cell therapies. Models will only be able to predict how measured decreases in nutrient supply influence disc cells of individual patients when accurate numerical data are available on parameters such as the permeability of endplates to different solutes, the effect of changes in vertebral blood flow on nutrient delivery, and the extent to which these factors govern metabolic rates in disc cells. Some effort should be expended to gather this information.

Acknowledgments

This work has received funding from the European Community's Seventh Frame-work Program (FP7, 2007–2013) under grant agreement no. HEALTH-F2- 2008-201626. We thank Dr R.B. Lee and Prof P. Winlove for helpful advice.

Conflict of interest

None of authors has anything to disclose.

Author contributions

Contribution to concept/design: OAB, JPGU. Acquisition of data: OAB, NW, SSS, NS. Data analysis/interpretation: OAB, NW, SSS, NS, JPGU. Drafting of the manuscript: OAB, NW, SSS, NS, JPGU. Approval of the article: OAB, NW, SSS, NS, JPGU.

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