Quantitative analysis of types I and II collagens in human intervertebral discs at various ages

Biochimica et Biophysica Aeta, 492 (1977) 29-42

© Elsevier/North-Holland Biomedical Press BBA 37658 Q U A N T I T A T I V E ANALYSIS OF TYPES I A N D II C O L L A G E N S IN H U M A N I N T E R V E R T E B R A L DISCS AT VARIOUS AGES

DAVID R. EYRE* and HELEN MUIR Biochemistry Division, Kennedy Institute of Rheumatology, Bute Gardens, Hammersmith, London (U.K.)

(Received December 21st, 1976)

SUMMARY "Ihe molecular species of collagen in the human intervertebral disc have been identified and measured quantitatively by assay for specific peptides produced by digestion of whole tissue with cyanogen bromide. As previously found in the pig, the annulus fibrosus of lumbar intervertebral discs contained two genetically distinct molecular types of collagen, types I and II, whereas predominantly type II collagen was found in the nucleus pulposus. In each tissue the proportion of hydroxylysine residues in the total collagen that were glycosylated gave support to these findings, even though the contents of hydroxylysine alone provided an unreliable index of molecular type. The annulus fibrosus of human lumbar discs as a whole contained a considerably higher proportion of type II collagen (50-65 ~ of total collagen) than did pig annulus fibrosus (20 ~o of total collagen). No significant variations with age were found in the relative proportions of types I and II collagens in the annuli fibrosi from lumbar discs of individuals aged 5, 16, 59 and 66 years. At all ages more than 8 5 ~ of the collagen in the nucleus pulposus was type II. In comparison, using the same procedure in the semi-lunar meniscus of the knee, which is classified as fibrocartilage, at least 95 ~ of the collagen was type I, whereas more than 95 ~o of that in young or old human articular cartilage was type II. The distributions of each type of collagen were assessed in a radial section of the annulus of T 12/L 1 of a 5 year old spine. Their relative proportions varied inversely and smoothly from being almost all type I collagen at the outer edge of the annulus to only type lI on reaching the nucleus pulposus. This distribution already shown in pig annulus fibrosus appears to be a characteristic structural feature of this tissue.

INTRODUCTION Structural degeneration and mechanical failure of the intervertebral disc is a major clinical problem. As yet, however, extensive research has not been directed at * Present address: Harvard Medical School, Orthopaedic Research Laboratories, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115, U.S.A.

30 the fibrous matrix of the disc. Little is known about the relative contributions of different structural macromolecules to mechanical function, or whether changes in quality or distribution of these molecules accompany ageing and contribute to degeneration. The tissue of intervertebral disc is often described as fibrocartilage. Collagen fibres provide the major tensile element and are arranged in a complex lamellar weave in the outer annulus fibrosus, where collagen accounts for about half the dry weight [1]. Collagen is more sparsely distributed and randomly oriented in the highly hydrated nucleus pulposus (10-20 ~ of the dry weight), which in the young disc is gelatinous and considered to act mechanically as a shock absorber or hydrostatic ball-bearing [1]. 7he proteoglycans are similar to those of hyaline cartilage, but contain more keratan sulphate [2, 3] which accounts for much of the remaining dry weight of annulus and nucleus [1]. After maturity in man the nucleus loses its gelatinous texture and progressively becomes more fibrous with ageing [4]. Although the collagen content has been reported to increase slightly in old age [5] the changing texture of the nucleus may be more closely linked to an accumulation of non-collagenous proteins at the expense of water [6]. 3-he collagen contents of both annulus and nucleus of discs of the lumbar spine of an adult increased progressively down the spine [7], although it is not known whether this is relative to age or purely an anatomical feature. It would be valuable to be able to correlate physical signs of ageing and possible predisposition to prolapse, with alterations in particular molecular constituents of of the disc, such as in the quality of the collagen. The molecular polymorphism of collagen is now well established [8]. In addition to basement membrane collagen (type IV) three genetically distinct molecular types of fibrillar collagen [8] have so far been identified in vertebrate connective tissue*. Type I, with composition [al (I)]2a2, is the most abundant and predominates in tissues containing coarse fibres such as skin, bone and tendon. Type II [el (II)]a, is restricted mainly to hyaline cartilages, while type III, [al (III)]3, is widely distributed but especially abundant in vascular tissue, embryonic skin and synovial membrane [9] and may be the collagen of reticulin which is identified histologically. Until recently little was known of the molecular composition of collagen in the intervertebral disc, and to which, if any, of the above molecular categories it belonged. 3-he extreme insolubility of disc collagen has tended to limit extensive biochemical analysis [10]. A recent study of the collagen of the lumbar intervertebral disc of the pig by analysis of derived CNBr-peptides showed that the annulus fibrosus contained both type I and type II molecular species but only type II was detected in the nucleus pulposus [11]. Preliminary analyses of the human intervertebral disc confirmed this general distribution but indicated a higher proportion of type II collagen in the human compared with pig [12] annulus fibrosus. In the present study human discs of various ages have been analysed to determine quantitatively the distribution in the annulus and nucleus of collagen of types I and II, and whether this distribution changes significantly with ageing. The quality of *The differenttypes of collagen are distinguishedby amino acid composition, CNBr derived peptides and degree of glycosylation.

31 the human disc collagen was also compared with that of a more typical fibrocartilage, the semilunar meniscus of the knee joint, and with collagen in human hyaline cartilage. EXPERIMENTAL

Tissue Lumbar regions of human spines were obtained at autopsy from individuals with no known spinal defects or general defects of connective tissue, and stored at --20 °C until needed. Ligamentous sheath was removed from the surface of the discs which were then dissected from the spine, cutting as closely as possible to the adjacent vertebral bodies but rigorously avoiding bone or cartilaginous end plates. Each disc was divided into annulus fibrosus and nucleus pulposus. Tissue of intermediate morphology, the so-called transition zone, was included with the annulus sample. For the analyses of disc collagen from a 66 year old, the samples of annulus and nucleus were pooled from the five lumbar discs, L1/L2 to L5/SI*. For the other specimens, a 5 year old male, a 16 year old male and a 59 year old female, annulus and nucleus were dissected from the single disc, T12/LI. A semilunar meniscus was obtained at surgical operation to correct traumatic injury of the knee of a 42 year old male. Attached soft tissue was cut away and discarded. Articular cartilage was sampled at autopsy from tibial surfaces of knee joints with no obvious signs of osteoarthrosis from 16 year old and 68 year old females. About two-thirds of the cartilage thickness was taken with a scalpel from glistening surfaces.

Preparation The tissue was cut into small pieces and soluble proteoglycans and glycoproteins were extracted sequentially with aqueous solvents [7]. The residues remaining after extraction of the tissue with 4 M guanidine. HC1 were washed, freeze-dried and digested with CNBr for analysis of collagen. Measurement of hydroxyproline showed' that less than 2 ~o of the collagen in any preparation of annulus or nucleus was solubilised by this procedure. A thin radial slice was taken from the central anterior segment of the 5 year old disc at the mid-point between the vertebrae (see Fig. 1). This slice was cut in series into 7 pieces of about equal size (3-5 mg dry weight), from the outer surface of the annulus through to the nucleus pulposus. Samples 1-6 were from distinct annulus and 7 would be classified as transition zone [13]. The relative proportions of type I and II collagens in these samples were analysed semiquantitatively by electrophoresis of derived CNBr-peptides in sodium dodecyl sulphate-polyacrylamide [13]. Methods Digestion with pepsin. In preliminary experiments samples of fresh nucleus and annulus were digested with pepsin (Sigma, twice crystallised)[14] and extracted directly. Subsequently the tissue was extracted with 1 M NaC1 and the collagen purified by reprecipitation [15]. This material was denatured and chromatographed

* Lumbar discs are numbered downwards, the first being TI2/L1 and the last L5/S1.

32

LEUi PUL OSUS ~

~1 TRANSITIONZONE

~_ ~!'~NNUL/~US

FIBROSUS

Fig. 1. Diagram showing where the seven serial samples were dissected from the anterior annulus fibrosus of the 5 year old T12/LI disc.

on a column (1.5 cm x 10 cm) of CM-cellulose at pH 4.8 and 42 °C [16] to isolate a-chain components. Digestion with CNBr. The collagen in freeze-dried tissue residues was solubilized by chemical cleavage into peptide fragments by digestion with cyanogen bromide (CNBr) in 70 ~o formic acid under nitrogen for 16 h at 25 °C [11, 17]. Older tissues were particularly difficult to digest and hence the longer digestion period was selected. Digests were diluted 15-20 fold with distilled water and freeze-dried. The small serial samples taken from the 5 year old disc were freeze-dried without prior extraction and digested in 1 ml of 70 ~ (v/v) formic acid containing 10 mg of CNBr. CM-cellulose chromatography. A "fingerprint" of the complete range of peptides in the digests was obtained by column chromatography on CM-cellulose (0.9 cm x 6 cm; Whatman, CM52) as previously described [11, 15, 16]. The protein content of the column effluent was measured by the absorbance at 230 nm and the hexose content of each fraction by an automated modification of the anthrone-HzSO4 procedure [18]. Phosphocellulose chromatography. The small homologous CNBr-peptides, al (I)CB2 and al (II)CB6 in complete CNBr digests of tissue were isolated by column chromatography on phosphocellulose (1.5 cm x 10 cm; Whatman, P11). Samples of digests weighing from 50--400 mg were applied to the phosphocellulose column. Usually effluent fractions that included both peptides were pooled (e.g. 120-180 ml of effluent in Fig. 3) and desalted on a column of Bio-gel P-2 (Bio-Rad Laboratories) prior to hydrolysis and quantitation by amino acid analysis [11, 15]. The identity of the peptides was established initially for one preparation of annulus fibrosus by recovering each peptide separately from the chromatogram and determining its amino acid composition. Amino acid analysis. The desalted peptides were hydrolysed in 6 M HCI at 110 °C for 24 h and amino acids were quantitated using a single-column, automated instrument (Locarte, London, U.K.) eluted stepwise with three buffers [15]. The composition of al (I)CB2 and al (II)CB6 analysed together as pooled fractions was interpreted as follows. The molar sum of the two peptides was estimated from analyses of glutamic acid and glycine since each peptide contains four residues of glutamic acid and

33 al(I)CB2 contains twelve and aI(II)CB6 eleven residues of glycine. The molar proportion of a 1(I)CB2 was estimated from leucine analyses since leucine is exclusive to this peptide. (Background contamination of leucine was allowed for by subtracting 1.5 times the amount of iso-leucine recovered). The molar proportion of a l (II)CB6 was estimated from analyses of valine and aspartic acid which are exclusive to this peptide which contains one residue of each. Valine was found to be the more reliable measure because of its lower background contamination. Since each peptide could be measured independently they provided an internal check on proportions estimated by difference. A further check was provided by the analyses of serine since al (I)CB2 contains two residues and al(II)CB6 only one. Thus, the molar ratio of the two peptides was the best fit taking all these factors into account. The relative proportions of the parent types I and II collagen molecules were calculated from this ratio assuming the molecular formulae [al(I)]2a2 and [al(II)]3 respectively. Hydroxylysine glycosides and non-glycosylated hydroxylysine in collagen were measured directly by a modified procedure for the analysis of basic amino acids after hydrolysis of tissue in 2 M NaOH for 24 h at 110 °C [15]. For another sample of the same tissue total hydroxylysine was related to hydroxyproline by amino acid analysis after acid hydrolysis. Disc electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. Essentially the procedure of Furthmayr and Timpl was employed using 7.5 ~o (w/v) individual acrylamide gels [19]. Freeze-dried digests of the small serial samples of annulus fibrosus were redissolved at 2 mg/ml in electrophoresis buffer by heating at 100 °C for 10 min, and 40-60/zg portions were applied to each gel. Protein bands were fixed and stained with Coomassie blue in isopropyl alcohol/acetic acid/water [20]. RESULTS

Solubilisation of disc collagen by pepsin digestion The relative proportions of two types of collagen in the intervertebral disc could not be obtained from analysis of pepsin-solubilized collagen. Little of the total collagen could be solubilised and type I collagen was selectively lost during the purification by salt precipitation and chromatography on CM-cellulose. Type I collagen is more basic than type II collagen and such losses might be expected because the sulphated polysaccharides, also present in the extracts, should bind electrostatically to type I collagen and precipitate it. Such selective binding of cartilage proteoglycans to type I collagen has been reported recently [21]. The collagen that could be purified revealed essentially only al chains when chromatographed on CM-cellulose. Amino acid analysis indicated that the al(II) chain was the main a chain present but, as explained later, although useful in distinguishing type II collagen in other species, the high content of hydroxylysine in al(I) could not reliably distinguish al (II) from al (I) in man.

Digestion with CNBr Aged human annulus and nucleus were less readily digested and solubilized by CNBr compared with young tissues. Nevertheless, after 16 h even the oldest specimen was completely digested. Furthermore on comparing the effects of digesting aged annulus for 4 h and 24 h the same relative amounts of the two peptides al (I)CB2

34 and al(II)CB6, were recovered despite a lower total yield at 4 h. The proportions of these two peptides in a CNBr-digest therefore appeared to give a reliable estimate of the type I/type II ratio.

Fractionation of CNBr-digests on CM-cellulose Fig. 2 shows a typical elution profile on CM-cellulose of the CNBr-digest of collagen from young (16 years) h u m a n annulus fibrosus, which in the case of young tissues proved to be one of the best profiles of CNBr-peptides on CM-cellulose in that discrete peaks were partially resolved. But the profile of absorbance at 230 nm allowed nothing to be concluded about the molecular types of collagen present. With older tissue the peaks were barely discernible and essentially a single broad peak was eluted with considerable trailing. This was true for digests of both annulus fibrosus and nucleus pulposus. The relatively high content of hexose, roughly following the profile of 230 nm absorbance, nevertheless suggested that a considerable proportion of the total collagen in both annulus and nucleus was of genetic type II [11, 16]. However, the presence of type I collagen could not be inferred from these analyses, unlike the results with pig annulus fibrosus [11].

15

E

tll(ll)eB

.9,%~o

0

~,l(ll~ll.e

j

12

E

t, jt

0

/ I

k

~.

o

! I I 0

25

50

75

I(NI

1.~

I)

Effluent Volume (ml)

Fig. 2. Chromatography on CM-cellulose of a CNBr digest of human annulus fibrosus. The column (0.9 cm x 6 cm) was equilibrated at 43 °C with 0.02 M sodium citrate adjusted to pH 3.6 with citric acid. Peptides from about 20 mg of a digest were eluted at a flow rate of 40 ml/h by a linear gradient of 0.02-0.15 M NaCI in a total volume of 150 ml of starting buffer.

Phosphocellulose chromatography and quantitation of al (I) CB2 and ttl (II) CB6 We have found that the most convenient and reproducible method for measuring relative proportions of types I and II collagens in a cartilagenous tissue is to isolate and quantitate the homologous peptides a l (I)CB2 (36 residues) and a 1(II)CB6 (33 residues) from a CNBr-digest of tissue [11, 15]. The human peptides have the same amino acid compositions and probably therefore, sequences as their porcine counterparts [11, 22, 23]. Furthermore, the method appears equally applicable to young or aged connective tissues. Fig. 3 shows the resolution of the two peptides from a digest of 66 year old h u m a n annulus fibrosus by chromatography on phosphocellulose. The identities of the two peptides were confirmed by amino acid analysis.

35

.2 0

c 0

.<

lO0

200

3(IH)

Effluent Volume (ml)

Fig. 3. Chromatography on phosphocellulose of a CNBr digest of human annulus fibrosus. The column (1.5 cm × 10 cm) was equilibrated at 43 °C with 1 mM sodium formate adjusted to pH 3.6 with formic acid. Peptides from about 120 mg of digest were eluted at a flow rate of 70 ml/h by a linear gradient of 0-0.3 M NaC1 in a total volume of 800 ml of starting buffer.

Occasionally the peaks were not so well resolved, probably because peptides containing open chain and lactone forms of homoserine were only partially separated. However, amino acid analysis of the total peptides recovered in pooled effluent fractions embracing the region where al(I)CB2 and al(II)CB6 elute, consistently showed that these two peptides accounted for more than 90 ~ of total amino acids in the effluent. Thus, because each peptide a l (I)CB2 and a 1(II)CB6 is distinguishable by one or more amino acids not present in the other (see methods), the relative proportions of the peptides and hence of their parent molecules, could be assessed. Quantitative amino acid analyses of the peptide fraction that would contain al (I)CB2 plus al (II)CB6 derived from annulus fibrosus, nucleus pulposus and fibrocartilaginous meniscus are given in Table I. The meniscus clearly yielded predominantly al(I)CB2 in contrast to the nucleus pulposus which gave essentially only al (II)CB6. Both peptides were isolated from the annulus fibrosus, and their relative molar proportions and that of their assumed parent molecules, [al(I)]za2 and [al (II)] 3 were calculated as described under methods. That the peptides were derived from types I and II collagen molecules having the above chain compositions was supported by the detection and quantitation of CNBr peptides from the a2 chain. Thus al (I)CB2 and a2CB2 were recovered from pig annulus fibrosus in a molar ratio of 2:1 [11]. The yield of g2CB3,5 was also appropriate for type I molecules of composition [al (I)]~a2. No CNBr peptides of type III collagen were detected. The relative proportions of types I and II collagens in whole annulus fibrosus at various ages are shown in Table II. No significant age-related variation was found although the lower proportion of type II in the 5 year old annulus may be real, but proof would require further analysis of several spines of different ages. From Table II type I collagen accounted at the most for 15 ~ of total collagen in any of the samples of nucleus pulposus. In fact, al (I)CB2 and therefore type I collagen, was not positively identified by any of the amino acid analyses of peptides from nucleus pulposus. The compositions showed predominantly al (II)CB6, but in most analyses could not rule out 10-15 ~ type II.

36 TABLE l A M I N O ACID COMPOSITION OF THE PEPTIDE FRACTION ctl(I)CB2 PLUS ctl(ll)CB6, F R O M H U M A N NUCLEUS PULPOSUS, A N N U L U S FIBROSUS AND S E M I L U N A R MENISCUS Hyp, hydroxyproline; Hse, homoserine. Residues/peptide al(I)CB2 a

al(II)CB6 b

Meniscus c 42 yr old

59 year old disc Annulus c

Nucleus c

4-Hyp Asx Ser Glx Pro Gly Ala Val Leu Phe Arg Hse

5 0 2 4 7 12 2 0 1 1 1 1

4 1 1 4 6 11 2 1 0 1 1 1

4.9 0.28 1.91 4.00 6.8 11.90 2.20 0.11 1.I0 0.92 1.01 1

4.4 0.76 1.28 4.00 6.1 11.20 2.15 0.66 0.28 0.94 1.02 1

4.2 1.02 1.14 4.00 5.8 11.00 2.28 0.97 0.14 0.87 1.00 1

Total

36

33

36

--

33

" See ref. 22. b See ref. 23. Molar ratios are related to 4 residues of glutamic acid. No corrections were made for destruction or incomplete release on hydrolysis.

TABLE II RELATIVE MOLAR PROPORTIONS OF TYPES I A N D II COLLAGENS IN H U M A N A N N U L U S FIBROSUS A N D N U C L E U S PULPOSUS AT VARIOUS AGES The proportions were calculated from molar yields of the peptides aI(I)CB2 and al(I1)CB6, isolated by phosphocellulose chromatography and measured by amino acid analysis as described under methods. Type I/Type II was assumed to be proportional to [mol of ctl(I)CB2 × 3/2]/[mol of al(II)CB6] thus allowing for the molecular compositions, [al(I)] 2a2 and [al(II)]3, of types I and I1 collagens. Age

Per cent of total collagen Type I Type II

Annulus fibrosus 5 Single discs 16 ' 59 TI2/L1 66 Pooled lumbar Nucleus pulposus All Ages (cf. fibrocartilaginous meniscus and articular cartilage)

53 36 37 44

47 64 63 54

< 15 >90

>85 --*

-- t

> 95

• Type II was not positively identified, a maximum of about 1 0 ~ could be present. • Type I was not positively identified, a maximum of about 5 ~ could be present.

37 Semilunar meniscus, a typical fibrocartilage, contained essentially only type I collagen (Table II) but the presence of some type II collagen was not completely ruled out even though it was not positively identified either.

Hydroxylysine and hydroxylysine glycoside content of disc collagens The hydroxylysine and hydroxylysine glycoside contents of the total collagen in various human cartilaginous tissues are given in Table III. The values for annulus fibrosus are intermediate between those of nucleus pulposus, which contains essentially only type II collagen, and those of meniscus with mainly type I collagen. As such they support the relative proportions of the two collagen types in the annulus determined by peptide analysis. TABLE III HYDROXYLYSINE AND HYDROXYLYSINE GLYCOSIDE CONTENTS OF COLLAGENS FROM HUMAN INTERVERTEBRAL DISC, ARTICULAR CARTILAGE AND FIBROCARTILAGINOUS MENISCUS Hyl, hydroxylysine; Glc, glucose; Gal, galactose. Residues/100 hydroxyproline residues"

Total Hylt GlcGalHyl GalHyl GlcGalHyl/GalHyl of Hyl glycosylated

Type I Semilunar Meniscus (42 yr)

Type I & Type II Annulus Fibrosus (66 yr)

Type II Nucleus Pulposus (66 yr)

Articular Cartilage (68 yr)

Articular Cartilage (16 yr)

12 2.4 1.8 1.35 35

15 4.4 3.2 1.37 51

17 6.4 4.7 1.36 66

12 3.1 5.1 0.61 68

16 4.6 5.5 0.84 63

" Analyses were performed on hydrolysates of whole tissue and therefore refer to total tissue collagen. t Determined after acid hydrolysis. It is noteworthy that, unlike the situation in other species, type I collagen of human meniscus and type II collagen of aged human articular cartilage both contain a similar amount of hydroxylysine, i.e. 12 residues per 100 hydroxyproline residues. They are distinguished, however, by their differing degrees of glycosylation of hydroxylysine. The hydroxylysine glycoside content of the total collagen of the meniscus is somewhat higher than previously reported for type I collagen of other tissues and could reflect the presence of some type II collagen in the tissue. The higher content of hydroxylysine of the collagen of the single specimen of 16 year old articular cartilage compared with that of the aged cartilage (Table III) may be an age-related difference, but more specimens would be needed to be analysed to prove this and rule out variation between individuals unrelated to age.

Heterogeneous distribution of collagen types within the annulus The serial samples of annulus from the 5 year old disc dissolved completely

38 on digestion with CNBr. The results of disc electrophoresis in sodium dodecyl sulphate polyacrylamide of the whole CNBr-digests indicated a relatively smooth radial gradation from mainly type I on the outside to mainly type 1I collagen on the inside (Fig. 4). The peptide a l ( I I ) C B I 0 (about 360 residues) proved to be an excellent marker on electrophoresis for type II collagen and similarly the peptide a2CB3,5 for type I [13, 15]. In man a2CB3,5 (about 640 residues) is only a partial cleavage product of the a2 chain of type I collagen [22] whereas in the pig a methionine residue is lacking in this sequence [13, 15]. Sufficient of this peptide resisted cleavage to act as a semi-quantitative marker for type I collagen.

~

a,5

0 C L.. 0

I

I 20

I

I 40

I

I 6O

Distance Migrated (mm)

Fig, 4. Sodium dodccyl sulphate-PAGE electrophoresis of collagen CNBr-peptides from radial

samples of human annulus fibrosus. The samples a to g shown diagramatically in Fig. 1 were digested with CNBr. About 50/~g of each were run in cylindrical (60 mm × 4 mm) polyacrylamide (7.5 w/v) gels for 3 h at 6 mA per gel. After staining with Coomassie Blue R, bands were scanned densitometrically. Clean electrophoretic separations were best derived from tissues of young discs. When tissues from aged discs were examined, the profile of peptide bands was difficult to interpret and included broad ill-resolved bands. Possibly the same age-related changes that had prevented resolution of CNBr peptides on CM-cellulose were responsible. Nevertheless, in both young and aged discs the same general distributions of types I and II collagens were found. Thus, the outer anterior edge o f the annulus

39 fibrosus from the disc, T12/L1, of the 5 year old from which ligamentous tissue had been removed, contained only type I collagen (profile a, Fig. 4). Going inwards more type 11 was progressively detected in a relatively smooth gradient until on reaching the intermediate zone (profile g, Fig. 4) only type 11 collagen was found. The nucleus pulposus gave poor profiles of CNBr-peptides on disc electrophoresis, probably because of the low ratio of collagen to non-collagenous protein. Even so, the results were consistent with there being only type 11 collagen present (not shown). DISCUSSION Firm evidence of the identity and relative proportions of different molecular species of collagen is possible only if the entire collagen of the tissue is examined as CNBr-peptides released on complete digestion of the tissue. In this way the presence of a substantial proportion of type I collagen in the human annulus fibrosus was demonstrated in the present work. In several previous studies, however, the identity of the type of collagen in intervertebral discs has relied solely on the characterisation of the small amount of collagen that is solubilised by protease treatment. Interpretation of results can be misleading however. For example, from the amino acid composition and abundant hexose content of collagen solubilised by trypsin treatment of human [24, 25] and whale [26] annulus and nucleus it appeared that the collagen might be type 11. Analyses of pepsin-solubilised collagen from human intervertebral disc [27], also indicated that the collagen was mainly type II with some evidence for a small proportion of type I collagen in the annulus fibrosus. More recently, however, the small amount of collagen of human discs solubilised by pepsin [28] was characterised as being type 11, and there was no evidence for the presence of type I collagen in either nucleus or annulus. This was because most of the collagen (75 ~o) was not extracted and therefore not analysed. In the present study, preliminary results suggest that the collagen solubilised by protease digestion of annulus fibrosus is mainly type II collagen, particularly when analysed by chain fractionation on CM cellulose after purification by salt precipitation. However, when the entire collagen was examined as CNBr-peptides after complete CNBr digestion of the tissue, almost half the total consisted of type I collagen. Confidence in this method of estimating the proportions of type I and type II collagens is provided by the reproducibility of the procedure and by corroboration from the overall hydroxylysine glycoside contents of the collagens. This is a more reliable guide to the type of collagen in human hyaline and fibrous cartilage, than is hydroxylysine content. In general, type I collagen is glycosylated to a lesser degree (17-30~ of hydroxylysine residues) than is type 11 collagen (60-70 ~o of hydroxylysine residues). Previous observations of variation in the level of glycosylation of collagen at different sites in the human disc [24] may now be explained by the inverse distribution of type I and type 1I collagen across the dics, demonstrated here. The degree of glycosylation of hydroxylysine residues may vary to some extent however, according to the site, age and metabolic activity of the tissue. The resolution and yield from phosphocellulose of the small peptides a l (I)CB2 and al(I1)CB6 was equally good from young and old tissues. Since neither peptide contains lysine or hydroxylysine residues they are not modified by being involved in

40 cross-linking of collagen, and they cannot react with hexoses. In contrast, the larger CNBr-peptides of collagens from old discs were poorly resolved on CM-cellulose, perhaps because they had undergone some modifications. Collagen is turned over very slowly in adult tissues and there is evidence that hexoses bind via their carbonyl groups to the e-amino groups of lysine and hydroxylysine residues of collagen [29,30]. The resulting hexosyl derivatives accumulate appreciably in older [31] tissues especially cartilage [29]. Peptides containing such modified amino acids might behave anomalously on ion-exchange chromatography and SDS-disc electrophoresis to give broadened peaks and bands. Other small molecules may perhaps also become covalently bound to collagen and introduce heterogeneities in the derived CNBr fragments. Tanning of collagen with ageing by such naturally occurring compounds as glyceraldehyde, pyruvaldehyde and acetaldehyde has been postulated [32]. In a long lived protein such as collagen, methionine residues may with time become oxidised to the sulphoxide, with the result that they would no longer be susceptible to attack by CNBr [33] and hence different CNBr cleavage products would be produced. This did not appear however, to have affected the yield of the small peptides al (I)CB2 and al (II)CB6. The inverse distribution across the disc of type I and type II collagens has now been shown in both human and pig [13] annulus fibrosus and is probably a feature of this tissue. The contribution of each type of collagen to the mechanical properties of the annulus is not known. Compared with the pig [11, 12] human annuli fibrosi of lumbar discs contained a higher proportion of type II collagen which may reflect an evolutionary adaptation to an upright posture, where the forces in the spine will differ from those in the spine of quadrupeds. It is noteworthy, that in the kangaroo, which has a posture intermediate between that of a biped and a quadruped, the relative proportions of type I and type II collagens in the lumbar annulus fibrosus was midway between the proportions found in human and pig annulus (Eyre, D. R., Ghosh, P. and Muir, H., unpublished findings). It is probable that each type of collagen is segregated into separate fibrils, although two morphological groups of fibrils were not evident by electron microscopy [34, 35]. Collagen fibres in young nucleus pulposus are thinner than those in the annulus fibrosus [25, 28] but this is not so in adult disc [35]. There appears to be no simple relationship between molecular type of collagen, degree of glycosylation[36] and fibril diameter and although type II collagen fibrils are usually thinner than type I fibrils, broad fibrils are seen in mature articular cartilage. Meyer and co-workers [38] have suggested that dermatan sulphate is generally associated with coarse collagen fibres. This is in keeping with a recent finding that in the meniscus of the knee, which is classed as fibrous cartilage, the glycosaminoglycans are mostly co-polymers of dermatan sulphate and chondroitin sulphate [39] and since the meniscus contains predominantly type I collagen (Table II) it is possible that in the annulus type I collagen may also be associated with dermatan sulphate proteoglycans. Each type of collagen may be associated with a different proteoglycan. Whether different types of collagen are produced by different cells and whether they also produce proteoglycans specific for the type of collagen is not known. The intervertebral disc would appear to be an appropriate tissue in which to examine such questions which might throw light on how the extracellular macromolecules of connective tissue in general are assembled.

41 lmmunofluorescent methods, although not quantitative, are extremely sensitive and could well detect small proportions of one type of collagen, that would not be detected chemically amongst a preponderance of another type. Thus fluoresceinlabelled antibodies specific for type I and type II collagens have demonstrated the presence of both collagens in human and bovine nucleus pulposus as well as in the annulus fibrosus [40, 41]. Other tissues in which both types of collagen have been found are chicken articular cartilage and bovine epiphyseal cartilage [42, 43]. Moreover, when chicken articular cartilage was examined by the same procedures as those described here, a considerable proportion of type I collagen was found (Eyre, D. R. and Glimcher, M. J., unpublished findings). It is notable that both avian and reptile articular cartilage have been classified histologically as fibrous cartilages [44], unlike most mammalian articular cartilages with the exception of the cartilage of the sterncclavicular and tempero-mandibular joints, which might therefore contain some type I collagen. In the mature intervertebral disc, collagen must turn over very slowly and the relative proportions of each type of collagen would not normally be expected to change with age, as the present results show. Nevertheless, under certain circumstances, the relative rates of deposition of type I and type II collagen may change gradually with time if, for instance, the diffusion of nutrients into this avascular tissue became abnormal. It is also likely that the collagen laid down in regions where repair has taken place, may differ from that originally present, and may be associated with different non-fibrillar constituents. Mechanical failure of the disc might result from such gradual but accumulative changes in its fibrillar architecture. ACKNOWLEDGEMENTS We thank Mr. R. Brown for expert technical assistance and Mr. R. J. F. Ewins for the amino acid analyses. The tissue from autopsies was kindly provided by Dr. B. Vernon-Roberts of the London Hospital. We are grateful to the Medical Research Council for the support of D.R.E. and to the Arthritis and Rheumatism Council for general support. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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