BIOCHEMICAL MEDICINE l&283-290 (1977)

Local Low-Collagen Content May Allow Herniation of Intervertebral Disc: Biochemical Studies

NELLY BLUMENKRANTZ, JESPER SYLVEST, AND GUSTAV ASBOE-HANSEN

Department of Dermatology (with Connective Tissue Research Laboratories). University of Copenhagen, Rigshospital. Copenhagen, Denmark

Received February IO. 1977

The human intervertebral disc consisting of the central nucleus pul- posus (NP) and the peripheral annulus fibrosus (AF) has been studied in detail as to the content of acid mucopolysaccharides (AMP) and collagen (l-6). We studied the topographic differences in the collagen content in the two different structures of the nonprolapsed human intervertebral disc by determining the two amino acids characteristic of collagen, i.e., hy- droxyproline (Hyp) and hydroxylysine (Hyl).

The present studies were undertaken with the aim of detecting a possi- ble systematic heterogeneity in the distribution of collagen, which might be responsible for differences in the tensile strength of the intervertebral disc (7). Low values of collagen in a certain part of the AF might explain the occurrence of disc herniation in that area (8-10).

MATERIAL AND METHODS

Preparation of Samples

The corpses of three young men, aged 29 (subject 1), 20 (subject 2) and 30 (subject 3) were brought to the University of Copenhagen’s Depart- ment of Forensic Medicine after death from cerebral contusion. At au- topsy, the spines were dissected free. No evidence of trauma or patholog- ical processes appeared at the examination, and the history of the men did not reveal any metabolic or spinal disorders. The spinal segments includ- ing the third lumbar disc in subject I and the first and second lumbar discs in subject 2 were removed with a bone saw. The discs were carefully dissected free from the cartilage end-plates. The specimens were fixed onto a cork brick, and punch necropsies were taken in the vertical plane from different locations all over the discs. From both persons, samples of other connective tissues were collected. In subject 3, the sampling of the disc specimens was made in situ. A 3-mm punch biopsy drill was intro-

283

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284 BLUMENKRANTZ. SYLVEST, AND ASBOE-HANSEN

duced into the right anterior part of the chosen disc. It was passed through the horizontal midplane at an angle of 45’ in relation to the sagittal plane. and appeared at the postero-lateral corner in the intervertebral for-amen. Only a gentle pressure was needed for this procedure to be carried out. The borderline between the annulus and nucleus appeared extremely well defined because of the swelling and glossy appearance of the NP contrast- ing with the dull and lamellar appearance of the AF. Under a dissection microscope, the specimen was carefully divided between the NP and the AF. Thereafter, the annular and the nuclear parts were subdivided into two to five specimens each. This way, five lumbar discs from subject 3, i.e., L,, L,, L,, L,, and L, were examined. A total of I28 samples of AF and NP of all three subjects were analyzed for Hyp and Hyl.

Biochemical Analysis

Tissues were defatted with acetone (three changes per day for three days), acetone-ether (I: 1, three changes, 1 day), and ether (one change). The ether was evaporated, and the samples were placed in a stainless- steel vacuum disiccator (Nikortanks, Catalog No. 800) to constant weight. The dry weight of the samples was determined by a Mettler ultramicro balance. The tissues were submitted to hydrolysis in 6 N HCI at 110°C for I6 hr. After hydrolysis, two aliquots of the hydrolyzed sample were separated, and each one was evaporated. Then the samples were dis- solved in phosphate-citrate buffer, pH 6.0 and 7.0, for determination of Hyp (I 1) or Hyl (12) by our assays as adapted to the Technicon au- toanalyzer.

Calculations

The results were expressed as micrograms of Hyp or Hyl per 10 mg of dried defatted tissue. The moles of Hyp and Hyl per dry weight were also calculated, and with these data their molar ratio was obtained (13).

The Kruskal-Wallis and the Mann-Whitney tests were used to evaluate the statistical significance of the variation of Hyp and Hyl. The t test with paired samples was used to determine if the difference in the content of Hyp and Hyl between the first and second sample from the external postero-lateral surface of AF was significant. The same compari- son was made between the first and second sample from the external surface of the anterior AF. A similar test was performed to evaluate the significance of the difference between the Hyp and Hyl content in the inner anterior and the inner posterior AF.

RESULTS

Skin. Ligament, Aorta, Cartilage, Bone, and Synovial Membrane

The contents of Hyp and Hyl are presented in Table I.

TABL

E I

COLL

AGEN

IN

HU

MAN

TI

SSUE

S (A

UTO

PSIE

S)

Mat

eria

l

Skin

D

orsa

l as

pect

of

finge

r D

orsa

l as

pect

of

finge

r Ax

illa

Liga

men

t lo

ngitu

dina

le

ant.

Aorta

th

orac

ica

Car

tilage

En

d-pl

ate

Ear

Nas

al

sept

um

Knee

jo

int

Bone

Ve

rtebr

al

com

pact

a C

lavi

cula

Th

eta

cran

ii Fe

mur

Sy

novi

al

mem

bran

e

HYP

HY~

HYP

/HY~

(&

IO

mg

DDTn

) @

g/IO

m

g DD

T)

(mol

ar

ratio

)

663

52.2

16

.32

641

40.9

20

.37

1003

49

.7

25.5

3 88

6 72

.2

15.7

2 36

6 28

.5

16.4

1

782

141.

7 7.

01

484

84.9

7.

23

498

85.0

7.

45

818

136.

4 7.

70

384

22.3

22

.54

369

22.7

20

.14

328

19.2

22

.72

417

28.9

18

.71

940

103.

6 II.

58

Subj

ect

I 2 I I I 1 2 2 2 I 2 2 2 2

a D

DT,

dr

ied,

de

fatte

d tis

sue.

286 BLUMENKKANTZ. SYLVEST. AND ASBOE-HANSlN

Intervertebral Dis(,

Subject 1. The Hyp and Hyl content expressed as micrograms per 10 mg of dried defatted tissue was higher in AF than in NP (not shown).

Subject 2. In L, and LS, a higher content of Hyp and Hyl was observed in AF than in NP. In the AF, the Hyp and Hyl contents decreased from the periphery toward the NP (Table 2). The analytical results of double samples of punch necropsies from L, showed no statistically significant difference.

Subject 3. L,, L,, L,, L,. and L5 showed the same differences in the content of Hyp and Hyl as mentioned above, i.e., the values were de- creasing from outer to inner AF and from inner AF to NP (Figs. 1 and 2). A consistently lower content of Hyp was found at the inner posterior than in the inner anterior AF in all five discs studied (Figs. I and 2).

Using the t test of paired samples, the differences in the content of Hyp between the first and second samples of AF from the external postero- lateral surface were statistically significant (P < 0.01). The same is valid for the correspondent samples of the antero-lateral surface.

When using the same test, values of Hyl in samples of similar locations were not significantly different (P > 0.05).

TABLE 2

Hydroxyproline content” (PgilO mg DDT”) of L, (subject 2) Outer AF Inner AF NP

786 445 371 819 444 301 834 541 200 823 550 249 830 521 217 858 478 270

233 825 2 21 446 t 41 263 i: 54

Hydroxylysine content” (&IO mg DDT) of L, (subject 2) Outer AF Inner AF NP

140 64 43 I IO 54 40 II5 60 25 139 72 42 I I5 52 32

52 31 32

124 % I3 59 + 7 35 5 6

n Differences between means of columns I and 2. and between 2 and 3 are statistically significant (p < 0.01). Calculations were made by the Mann-Whitney test.

b DDT. dried defatted tissue.

LOCAL LOW-COLLAGEN CONTENT 287

FIG. I. Hydroxyproline content in necropsies of different locations of annulus fibrosus and nucleus pulposus from L,. L,. L,. L,. and L5 (subject 3).

The differences in the content of Hyp between the inner postero-lateral and the inner antero-lateral AF observed in the five intervertebral discs examined were statistically significant (P < 0.05). There were no signifi- cant differences in the content of Hyl (P > 0.05).

The difference in Hyp to Hyl molar ratio between AF and NP was statistically significant (P < 0.01) (Fig. 3).

Efects of the Sampling Techniques

The horizontal sectioning followed by individual vertical punching of AF or NP gave the variation of the parameters studied in outer to inner areas or in concentric areas of the disc (Subjects I and 2).

The horizontal punching followed by vertical sectioning of needle nec- ropsies allowed studies of the variation of the parameters between neighboring locations in succession from the outer anterior to the outer posterior AF through the NP (Subject 3).

DISCUSSION

The lower content of Hyp found in the inner postero-lateral AF indi- cates a lower collagen content. It is clinically well known that the men-

288 NLUMENKKANT%. SYLVEST. AND ASBOE-HANShN

FIG. 2. Hydroxylysine content in necropsies of different locations of annulus fibrosus

and nucleus pulposus from L,. L,. L,. L,. and L, (subject 3).

tioned location is susceptible to a disc herniation (7-9). The low collagen hydroxyproline at that particular location may be one of the factors in- volved in the mechanism of this condition.

A nonparallel variation of the two amino acids has been found by analysis of other tissues under certain pathologic and experimental condi- tions (I 5- 17). The decreased Hyp content from the outer to the inner AF with relative constancy of Hyl, is an expression of a nonsimultaneous variability of the two amino acids in this area of the disc.

Adams and Muir (18), studying the Hyp content of AF of the lumbar area of a 44-year-old subject, found a higher collagen content in the outer than in the inner area of each disc, and an increasing content from the upper to the lower discs. Our findings of topographic differences within one disc are in accordance with those of these authors, but we did not find differences between different discs. The mentioned difference in results may have originated either in the age differences between the three subjects analyzed by us (third decade of life) and the subject reported by Adams and Muir (fifth decade of life) or, more probably. in the different sampling technique.

Dickson et af. (5) studied the percentage collagen in dry NP and AF in relation to age. For their 3- to I9-year-old group and the 28- to 50-year-old

LOCAL LOW-COLLAGEN CONTENT 289

FIG. 3. Molar ratio Hyp to Hyl at different locations of annulus fibrosus and nucleus pulposus from L,, L,, La, L4, and L, (subject 3).

group, these authors found Hyp values similar to the average values of our Hyp results. However, the mentioned authors used total AF or NP in their studies. The similar content of Hyp and Hyl found in NP and in cartilage is in agreement with the fact that a similar type of collagen is present in the two tissues (19).

SUMMARY

Topographic differences in the content of collagen in 3-mm punch necropsies of nucleus pulposus and annulus fibrosus were studied. A lower collagen content was found at the postero-lateral part of the annulus fibrosus, the weak point where a disc herniation often occurs. The amino acids hydroxyproline and hydroxylysine were used as parameters. Sig- nificantly higher contents of hydroxyproline were found in the outer than in the inner part of the annulus fibrosus. Nucleus pulposus had a lower collagen content than the annulus fibrosus. The content of hydroxyproline and hydroxylysine in nucleus pulposus was very close to that of ear and nasal septum cartilage. Other tissues of two of the subjects were also studied.

290 BLUMENKRANTZ. SYLVEST. AND ASBUE-HANSEN

ACKNOWLEDGMENT

This work was supported by the Danish Rheumatism Association. The able technical assistance of Mr. Ole Christiansen is acknowledged.

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