Mechanisms of Ageing and Development, 4 (1975) 431-448

431

© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

H I S T O C H E M I C A L STUDIES ON T H E D I S T R I B U T I O N OF ACIDIC G L Y C O S A M I N O G L Y C A N S IN H U M A N RIB C A R T I L A G E D U R I N G T H E A G I N G PROCESS

K. SAMES

Department of Anatomy of the University Erlangen-Niirnberg, and Department of Pathology of the Faculty of Clinical Medicine, Mannheim ( F.R.G.) (Received August 25, 1975)

SUMMARY

In human cadavers of different age five zones of basic substances can be distinguished around the cartilage cell by using hyaluronidase digestion and Alcian blue stain combined with various MgC12-concentrations. This treatment produces different reactions in the various zones. The zones are numbered according to their distance from the cell; thus zone 1 (Z1 ---- pericellular zone) is the nearest to the cell. Z 1 and Z 2 (inner territorial zone) contain hyaluronidase-digestible substance but Z 1 stains at higher electrolyte concentrations than does Z 2. The third zone (Z 3 = outer territorial zone) which first appears in childhood always contains hyaluronidase-resistant material and, besides, hyaluronidase-digestible material in adolescence. Since the distance between the cells increases with the proceeding process of aging, Z 4 (periterritorial zone) and Z 5 (interterritorial zone) appear in the cartilage centre. In adolescence only Z 5 can be found; it is slightly hyaluronidase-sensitive and stains even at high MgClzconcentrations. In adult and old cartilage a weakly basophilic zone 4 appears. The comparison of fixed and unfixed tissues renders the distinction between the zones feasible as the hyalurinodase resistance is increased in fixed tissue and, on the other hand, the structures appear less outlined in unfixed tissue. As far as the distribution of acidic glycosaminoglycans (GAG) is concerned the territories and interterritories are not clearly defined units, for part of the zones mentioned above can be arranged circumcellularly or, in addition, interstitially depending on the various cartilage regions and on the different periods of life. We assume that the pericellular as well as the inner and outer territorial zones belong to the cell itself and that their step by step appearance is due to a process of development whereas the periterritorial and interterritorial zones result from cell degeneration caused by aging, and expand, mainly, without cellular control.

432 INTRODUCTION Stockwell and Scott2aa, b and Quintarelli and Dellovo 15 examined the agerelated changes of acidic glycosaminoglycans (GAG) by using Alcian blue stain with differing electrolyte-concentrations according to the "critical electrolyte concentration (CEC) method" (Scott and Dorling 20a) and, as well, after hyaluronidase-digestion. The application of these methods results in clearly isolated keratan sulfate (KS) achieved by blocking of the staining of chondroitin sulfate (CS) or digestion of chondroitin sulfate. The loss of colour which can now be observed in comparison with the control sections provides evidence on the localization of chondroitin sulfate. CS is contained in infant rib cartilage as well as in the territories in the periphery of older cartilages whereas KS is contained in the interterritories from youth on. In the centre of adult and old rib cartilages, on the contrary, the staining typical for KS is found more in the territories than in the interterritories. When dealing with these results we should not forget to discuss the following problems: Firstly there is only a small amount of CS in the old cartilage. Only in a narrow peripheral zone do the territories show a clear loss of colour contrasted with a quite large amount of intensely stained KS-containing structures. Biochemical tests have shown that the ratio of CS- and KS-content in the old cartilage is 1:1, this status being the result of a continual relative increase of KS with growing age (Gower and Pedrini6; Kaplan and Meyeff; Kresse, Stein and Buddeckeg; Mathews and Glagovl4; SameslTa; Shetlar and Masters21; Stidworthy, Masters and Shetlar2~). Secondly, the varying kinds of storage of KS cause confusion. According to the above-mentioned authors KS is stored away from the cell in youth and in the periphery of adult cartilage; in the centre of old cartilages, however, it is stored near the cell. These results imply the question as to whether the cells of the centrally located chondrones do no longer produce CS or that it is stored further away from the cell especially since the basic substances around the territories here stain similarly to CS when the CEC method is used (SamesX7b). The aim of these investigations is to compare fixed and unfixed cartilages. We expect the precipitation of the acidic glycosaminoglycans by the stain itself to contribute to a reduction of losses of these substances in native cartilage. Furthermore we intend to test whether the effectiveness of the enzyme digestion is increased in native tissue. Rib cartilage, which - - by use of the CECmethod - - shows the described, age-related distribution pattern of acidic glycosaminoglycans, is used for the investigations. We want to find out whether this pattern is also different in the immediate surrounding of the cells of adolescent and adult cartilage, and whether the composition of acidic glycosaminoglycans corresponds. MATERIAL AND METHODS The Department of Pathology of the Faculty of Clinical Medicine, Mannhelm, provided human rib cartilages of the second and third rib taken from cadavers of the following age groups: 6 weeks (infancy), 3 years (childhood), 16 years (adolescence), 35 years (adulthood) and 71 years (old age). Two samples were taken from

433 each cadaver. One was frozen at --20 ° C or with CO2-snow to be able to make cryostat sections out of it afterwards. The other one was fixed in 10% formol for longer than 3 days and then embedded in paraffin. Cryostat and paraffin sections 7-10 # m thick were treated according to the method developed by Scott and Dorling 19; they were stained in a solution of 0.05 % A1cian blue 8GX (ICI), in 0.025 M-acetate buffer (pH 5.7) with 0.05-, 0.1-, 0.2-, 0.3-, 0.4-, 0.6-, 0.65-, 0.7-, 0.8-, 0.9-, 1.0, 1.1- and 1.2 M MgClz. Subsequeotly they were washed in aqueous MgC12 solution of the same concentration as used for staining and in two changes of distilled water each for five minutes. Cryostat and paraffin sections of the cartilage were incubated in hyaluronidase (Serva 500 IE/mg from sheep testis) 1 mg/ml in 0.1 M-phosphate buffer (pH 5.5-5.6) at 37°C for six hours. Controls were incubated in the same buffer but without enzyme, and others were stained without preliminary treatment. After the incubated sections had been washed in three fractions of distilled water for five minutes each time all sections were stained in the above-mentioned Alcian blue solution with 0.4 M MgC12 for 24 hours and washed afterwards. The sections were dehydrated in increasingly higher concentrations of alcohol and in xylol, and layed in Eukitt (Kindler, Freiburg). Of the 16 year old cartilage only the paraffin sections were used. 1 digested section as well as the controls were stained with Alcian blue and 0.1-, 0.3-, 0.8- and 1.0 M MgC12 without pretreatment on the one hand and after incubation in hyaluronidase or buffer solution on the other. Purified samples of chondroitin sulfate, heparin and keratan sulfate were put on filter paper (Scott and Dorling 20a) and, like the cartilage sections, stained in Alcian blue MgC12 solution and washed. Finally all were treated with 10% acetic acid to reduce the background stain. In preliminary experiments with heparin the samples were washed in distilled water only and, as well, with and without acetic acid (sources and preparation of the samples as given earlier, SameslTeI). RESULTS

Staining of purified glucosaminoglycans on filter paper Chondroitin sulfate no longer stains with Alcian blue when the MgCI2 concentration is higher than 0.6-0.7, heparin when it is higher than 0.7, keratan sulfate when it is higher than 0.8 M. After washing with water a slightly blue staining of heparin can be observed at an even higher concentration, which completely disappears after washing with a MgClz solution of the same concentration as used for staining. The use of acetic acid reduces the background stain quite considerably.

Staining of cartilage sections 1. General aspects Different regions can be distinguished in the cross sections of rib cartilage. The region immediately adjoining the perichondrium is built up by long, thin cells arranged parallel to the surface and, as usual, designated as the subperichondrial region. In addition we distinguish between a peripheral region including the sub-

434 perichondrial region and a central region. Up to childhood the two separate regions cannot be distinguished. From adolescence onward the central region can be identified by a contrasting display of the territories (Z1 to Z3) from the interterritories (Z4, Z5--see below). Both regions are vaguely outlined against one another by a more or less wide transition one. A concentric arrangement of substances around the cell is called circumcellular, an arrangement in the interspaces interstitial. Usually a solution of Alcian blue and 0.3 to 0.4 M MgC12 provides a complete and intense staining of all cartilage structures in fixed and unfixed tissue of all ages. A concentration lower than 3.0 M results in an unsatisfactory staining of the structures in paraffin sections, especially in the cartilage centre. Cryostat sections of younger cartilages show complete staining even at the lowest concentrations. In older cartilages the staining is incomplete if the concentration is lower than 0.3 M. 2. The cells After hyaluronidase digestion the nuclei are always stainable with Alcian blue. The cytoplasm is only stained where hyaluronidase-resistant material is detectable among the basic substances; the intensity of staining is quite similar to that of the basic substances present. In adult and old cartilage the cells but not the capsules show an evident loss of co]our after byaluronidase digestion, even in the hyaluronidaseresistant territories of the cartilage centre. The capsules are always stained more than the cells. When using Alcian blue staining with high MgC12 concentrations the cell stains according to the stainability of the surrounding basic substance. In old age the intensity of staining of the cells increases compared with that of other structures. In old cartilage, therefore, the intensity appears too strong, relatively speaking. Control sections which were incubated with buffer solution without hyaluronidase revealed no difference in colour compared with untreated sections. 3. Intercellular zones Excepting the cell capsule up to five zones of basic substances can be distinguished around the cartilage cell. The acidic glycosaminoglycans or proteoglycans in these zones react differently to our methods of treatment. The zones are numbered according to their distance from the cell, the nearest one being called Z 1, the farthest one Z 5 (cf. Fig. 5). Particularly in the cartilage centre the number and arrangement of the zones change with growing age. For definition purposes we should like to suggest the following terms for the zones : Z 1 = pericellular zone Z 2 = inner territorial zone Z 3 = outer territorial zone Z 4 = periterritorial zone Z 5 = interterritorial zone. Zone 1 ( Z 1 ) . This zone lies concentrically around the cartilage cell capsules; it cannot be found in the subperichondrial region. If the MgCI~ concentration is higher than 0,8 M the Alcian blue staining of Z1 weakens. Infant cartilage can no longer be

435

Fig. 1. (a) Cartilage taken from three year old child; stained with Alcian blue and 0.4 M MgCI2 after hyaluronidase digestion; cryostat section. (b)As in (a) but after fixing in formol and embedding in paraffin. In unfixed cartilage zone 3 is stained at single points; in fixed cartilage it is stained completely and lies exclusively interstitially. ( × 87) stained with 1.0 M MgClz, or child cartilage with 1.1 M (Fig. 1). Using higher MgCI2 concentrations than 0.8 M Z1 stains more in the cartilage centre than it does in the periphery, but this can only be observed from childhood onwards. The centre of adult and old cartilages can be stained even at the highest MgC12 concentrations whereas the outer part of the periphery remains unstained with 1 M already. The hyaluronidase digestibility corresponds to that of Z 2 (Fig. 2 (a), (b)).

Zone 2 (Z2). In adult cartilages Z2 lies circumcellularly io the subperichondrial region, directly around the cell capsule. Towards the centre it surrounds Z1. In infant cartilage where only Z1 and Z2 are to be found, Z2 lies interstitially and is broadened in the centre. In cryostat sections of infant and child cartilages Z 2 shows an evident loss of colour in the cartilage centre with 0.05 M to 0.4 M MgCI2. With increasing MgC12 concentrations, in particular 0.6 M and higher, the subperichondrial regions of all age groups show a growing loss of colour. In infant cartilage Z2 is colourless with 0.8 M MgCI2. After infancy the staining intensifies from the periphery towards the centre when using increasingly higher salt concentrations. In some cases the circumcellular matrix, in others the interstitial matrix, in a child's cartilage is stained lighter. In this case it is quite difficult to outline Z 2. It remains colourless with 1.0 M MgCI2. In older cartilages the best distinction between Z1 and Z2 is achieved by using 0.6-

436

Fig. 2. (a), (b) Rib cartilage; age: 16 years; incubated in phosphate buffer and stained with Alcian blue and 1.0 M MgCI2; fixed in formol: (a) periphery, (b) cartilage centre. (c) + (d) Same cartilage and same fixation as mentioned above; stained with Alcian blue and 0.3 M MgCI2 after hyaluronidase digestion. (c) periphery, (d) cartilage centre. (e), (f) Same cartilage and same fixation as above; stained with Alcian blue and 1.0 M MgCI2 after hyaluronidase digestion: (e) periphery, (f) cartilage centre. When staining with Alcian blue and 1.0 M MgCI2 zones 1 to 3 can be distinguished in the periphery, zones 1 and 2 in the centre whereas Z3 and Z4 can hardly be distinguished. After digestion (c) and (d), Z1/Z2 and Z3 are clearly visible in the periphery as well as ZI/Z2, Z3 and Z4 in the centre. Zone 2 can be clearly outlined against Z3 when using high salt concentrations; Z4 after hyaluronidase digestion. ( × 87)

437 0.8 M MgC12. When using 1.0 M MgCle Z2 remains colourless in the largest part of the periphery of the cartilage (Fig. 2 (a), (b)). From adulthood onwards this distinction can only be made in the periphery. In early adulthood a diminished staining of the areas of Z1 and Z2 can still be observed in the cartilage centre; a distinction between the two zones, however, is not possible. In the centre of old cartilage the slight staining does not occur any longer. Because of their identical sensitivity to the enzyme Z1 and Z2 are to be considered as a unit after hyaluronidase digestion; the hyaluronidase resistance of this unit increases with age and with the distance from the perichondrium. Sections of infant cartilage remain completely colourless. In clear contrast with staining with high MgCI2 concentrations without digestion both zones are clearly light in the cartilage centre of adults as well, partly even in old cartilage. All these observations are applicable to unfixed cartilages exclusively. In fixed tissue Z1 and Z2 never remain completely colourless. The two zones can, however, only be 'distinguished in infant cartilage because of the slightly higher resistance of Z1, the stainability after hyaluronidase digestion being minimal in both zones. Compared with unfixed tissue the enzyme resistance is increased in all cartilages. From adulthood onwards in the cartilage centre only the cells are hyaluronidase-sensitive whereas the circumcellular matrix remains unaffected. Similar observations can be made when using 0.8 to 1.0 M MgCI2. In this case zone 1 is stained more intensely than after hyaluronidase digestion; zone 2 is considerably deprived of colour (Figs. 3(a) - (d) and 2 (c) - (f)). Zone 3 (Z3). Zone 3 lies closely to zone 2. In the centre of a child's cartilage Z 3 can only just be distinguished from Z 2 by staining. Z 3 does not extend to the perichondrium. In older cartilages Z 3 lies interstitially in the periphery and forms a meshwork which extends to the perichondrium. In the centre of adolescent cartilage this zone lies partly circumcellularly, partly interstitially. In the centre of adult and old cartilage Z 3 is situated circumcellularly in general and only vaguely outlined against ZI and Z2, the three zones thus forming an undivided area around the cell. Z3 is stained completely with up to 0.6 M MgCI2; with 0.8 M the staining gets weaker, and it is completely unstained with 1.2 M in the periphery. In the centre it remains stainable at even the highest MgCI2 concentrations applied from adulthood onwards. In the centre of old cartilage a varying size and stainability of the cells and the areas around the cells can be observed with 0.4 M MgCI2 already. This being the case the whole circumcellular matrix can be unstained; the cell can be isolated and stained weakly. It can also be surrounded by a matrix of varying size and degree of staining. Finally light, cell-free concentrically arranged matrices are to be found. The changes in the cells and matrices represent the well-known phenomenon of "Verd/immern" (Schaffed9). In early adulthood similar observations can be made in the cartilage centre when using high salt concentrations of 1.0 M MgC12 and higher. Under the highest concentrations used, only part of the cells and matrix zones is displayed in the centre of old cartilages. In cryostat sections the staining is more intense at all salt concentrations and the areas around the ceils appear larger (Fig. 4 (b), (d), (f)).

438

Fig. 3. (a), (b) Thirty five year old rib cartilage; stained with Alcian blue and 0.4 M MgCI2 after hyaluronidase digestion; crysotat section: (a) periphery, (b) cartilage centre. (c), (d) Same cartilage as mentioned above (m.a.) but paraffin section: (c) periphery of (d). In fixed cartilage Z1 and Z2 are more resistant to hyaluronidase. In the centre no effect due to digestion can be traced. ( × 87).

439

~e •

60

.

O

O Qq

e

Fig. 4. (a), (b) Rib cartilage; age: 71 years; stained with Alcian blue and 0.4 M MgCI2 after incubation in phosphate buffer; cryostat section: (a) periphery, (b) cartilage centre (section used for comparison with (c) and (d). (c), (d) Cryostat section of the cartilage m.a.; stained in the same way after hyaluronidase digestion: (c) periphery, (d) cartilage centre. (e), (f) Paraffin section of the cartilage m.a. ; stained with Alcian blue in the same way as m.a. after hyaluronidase digestion : (e) periphery, (f) cartilage centre. Hyaluronidase produces a clearly visible effect in cryostat sections which is a bit vaguer in the centre of the cartilage. Z1/Z2, Z3, Z4 and Z5 can be clearly distinguished. In the centre of paraffin sections an effect can only rarely be observed outside the cells. ( × 87)

440 After hyaluronidase digestion Z 3 is stained less in the periphery of the cartilage than it is in the centre. In cryostat sections Z 3 is incompletely exhibited in the subperichondrial region (Figs. 3 (a) and 4 (c)). In contrast with staining at high electrolyte concentrations it can be clearly outlined against Z 4 after digestion. In this case the transition from Z 2 to Z 3 is less clear-cut than that observed with the CECmethod. The differences become evident when comparing the stainings with 0.3and 1.0 M MgCI2 after digestion (Fig. 2 (c) - (f)). In the fixed cartilage of children and adolescents Z 3 is stained completely but a good deal lighter than it is in control sections. In unfixed tissue, however, it cannot be traced completely. In adult and old cartilage Z 3 is hyaluronidase-sensitive in the periphery only. Zone 4 (Z4). This zone lies circumcellularly and peripherally to zone 3. Only in the centre of adult and old cartilage it appears clearly. Z 4 is displayed in a particularly clear way in fixed cartilage, and it is colourless with 0.4 M MgC12 in some parts, completely colourtess with 0.8 M. In the cryostat section, however, it is still stained partly with 1.2 M MgC12. It appears wider in old cartilage and is, after fixation, unstained with 0.65 M MgC12 already, in the cryostat section with 1.2 M. (Figs. 3 (b), (d) and 4 (b), (d), (f)). Z4 is hyaluronidase-resistant. Zone 5 (Z5). Z 5 lies peripherally to Z4 or, if Z4 has not yet appeared (in youth), to Z3 in the cartilage centre. Z5 is the only zone which is always interstitial and can be traced clearly from youth on. It forms a shapeless irregular meshwork. In adolescent cartilage it shows a clear loss of colour beginning at 0.8 M MgC12, in adult and old cartilage it is unstained with 1.1 M in fixed tissue except of some points which contain asbestos fibers. On the contrary coherent staining at even 1.2 M MgClz can be observed in cryostat sections. In adolescence Z5 is clearly affected by hyaluronidase. The effect hyaluronidase produces in this zone is not visible that clearly in older cartilages but it can be concluded from the fact that the other zones are outlined better against one another after digestion (Figs. 2-4, bottom pictures).

Results achieved by other histochemical methods Previous results (Sames 17eI) partly obtained by using other stains but on the same material as dealt with here were used for comparison. Staining patterns similar to those obtained with Alcian blue and 0.4 M MgCI2 have been observed with acridine orange, toluidine blue and haematoxilin-eosin. Staining with Alcian blue 8 GS (Mowry method) for 30 minutes and subsequent periodic acid-Schiff reaction (PAS)-staining result in an incomplete Alcian blue stain. The increase in PASpositive material with growing age, however, becomes obvious particularly in Z4 and Z5. DISCUSSION The results of our experiments prove that the cells of human rib cartilage are, in general, surrounded by a hyaluronidase-sensitive substance and that the cells themselves contain that substance, except for part of the chondrones in the centre of old cartilage. These hyaluronidase-sensitive substances can, however, only be detected in unfixed rib cartilage of adults and old men. As similarly described by

441

Fig. 5. Distribution diagram of zones 1 to 5 taken from the centre o f rib cartilage of different age groups: (a) cartilage aged 6 weeks, (b) cartilage aged 16 years (a section o f Fig. 2(d)), (c) cartilage aged 71 years (a section of Fig. 4(d)). ............,;:~.,,,Z2 , I r o n - - I l l Z3 , .F.~. . . . . . . . . . . . . . .Zl . Z4, Z5,

442 Quintarelli and Dellovo 15 as well as by Dearden, Bonucci and Cuicchio 5 the cartilage centre in this case does not exhibit any hyaluronidase effects after fixation. Stockwell and Scott z~b made similar observations on joint cartilage. On the other hand a loss of colouring can be observed in the cells in the centre of old cartilages when using paraffin sections. In rib cartilage chondroitin sulfate and keratan sulfate are the only substances to be positively identified as acidic glycosaminoglycans. Chondroitin sulfate should not be stained if the method of Scott and Dorling z0a is used with Alcian blue at high electrolyte concentrations as well as after hyaluronidase digestion. We should expect that keratan sulfate containing structures would display the same staining pattern with both methods. In fact, however, it becomes evident that zone 1 which lies closely around the cell as well as zone 2 are hyaluronidase-sensitive but Z1 stains at higher MgCl2 concentrations than does Z2. Similar observations can be made in the centre of adult and old cartilage where zone 1 and zone 2 are mixed. In this case the two zones are hyaluronidase-digestible in cryostat sections but stain at even the highest MgCI2 concentrations. Because of the increased hyaluronidase resistance these differences cannot be observed in fixed tissue. Experiments in which chondroitin sulfate was stained specifically by toluidine blue after blocking of keratan sulfate by cetylpyridinium chloride resulted in the opposite picture: Z2 is stained, Z1 is not (Samesl7eII; Kelly, Bloom and ScottS). The fact that, especiaJly in old age, Z1 and Z2 react deviatingly to staining according to the CEC-method in comparison with hyaluronidase digestion is quite difficult to explain. The CEC-method is, of course, influenced by molecular weight as well as by the density of reactive groups of the molecule (Scott and Dorling 20a,b) but with growing age the molecular weight displays a clear decrease (KrSz and Buddecke 10) whereas the sulfate content does not change (SameslVa). Therefore the CEC-method with Alcian blue and MgCI2 provides misleading results in the rib cartilage, which can be improved under appropriate conditions by hyaluronidase digestion. Comparison of the results of both methods provides at the same time a definition of ZI and Z2. In addition to that Z3 can be clearly outlined after hyaluronidase digestion; thus five zones can be distinguished around the cartilage cell (Table I). After fixation Mason 13 obtained similar results in bronchial cartilage by using hyaluronidase. He contrasted them with the data of rib cartilage available at that time. Mason, however, distinguished between a smaller number of zones. According to the results we have obtained up to now rib cartilage and bronchial cartilage exhibit the same changes as far as aging is concerned. Because of differevt reactions to staining Schaffer~9 distinguished, except of the cell capsule, an inner, strongly basophilic and an outer, weakly basophilic zone around the cell as well as the "interterritorial substance" in the centre of rib cartilage. The inner zone is clearly basophilic and does not stain with acidic stains; the outer one is weakly basophilic and can be stained with acidic stains as well. Recent studies have shown that the latter stains less with Alcian blue and high MgCI~ concentrations (Sames~Vb). The "interterritories" are more clearly basophilic but also stainable with acidic stains. The application of histochemical methods reveals non-collagen proteins with a high tyrosine and tryptophane content in these zones (Beneke, Goubaud and Schmitt~).

Periph. Centr. Periph. Centr. Periph. Centr. + Periph. ( + ) Centr. ( + ) + Periph. + Centr. ( + ) + +

+

+

CS

+ + + + + ÷ + + + + ++++ + + + + +++ (+)+++ (+)++ + + + (7)+

CS'

+ (+) (+)+ + (+)++

KS

Z 2

+ + + + + + + + + + + + ++++ + + + + +++ (+)+++ (+)++ + + + (+)+

CS

CS'

+ + +÷ ++ +++ (+)-~- + (+)+++

KS

Z 3

++ + (+)

CS

+ + + (+)+ ~+ + (+)+ (+)

CS'

++

(+)++

KS

K S CS CS'

+++

+ + ~

++

Z5

Z4

+

CS

+

+

+

CS"

KS: keratan sulfate; hyaluronidase-resistant substance; stainable with high concentrations of MgC12. CS: chondroitin sulfate; hyaluronidase-sensitive substance; not stainable with high concentrations of MgCI2. CS': hyaluronidase-sensitive substance stainable with high concentrations of MgCl2 or hyaluronidase-resistant substance not stainable with high concentrations of MgCI2. Periph. = periphery; Centr. - centre.

Aged

Adult

Adolescent

Child

Region

Infant

KS

Substance

Age

Z 1

Zone

QUALITATIVE DISTRIBUTION OF G L Y C O S A M I N O G L Y C A N S A N D C H A N G E S WITH A G E

TABLE I

4~ 4~ ta~

444 Similar results were obtained with Alcian blue- PAS-staining by Quintarelli and Dellovo 15. With age and with the distance from the periphery the zones of basic substances increase in number. In the periphery and in younger cartilage Z2 and Z3 are situated interstitially, in the centre of older cartilage, however, circumcellularly. Therefore the zones should not be named according to their arrangement but according to their distance from the cell. To mark the arrangement we used the terms "circumcellular" and "interstitial" (Tables II and III). With growing age Z1, Z2 and Z3 reveal a continually increasing hyaluronidaseresistance, particularly in the old cartilage. The resistance increases from the periphery towards the centre. Biochemically this can be explained by the fact that the content of chondroitin sulfate decreases from childhood onwards on one hand and, on the other, that the proteoglycans become more heterogeneous with age, whereas the keratan sulfate content increases (Buddecke, Sames4; KrSz and Buddeckel0). These proteoglycans form hybrid complexes which might, morphologically, be difficult to define. In the electron microscope chondroitin sulfate appears in a socalled "matrix granula" which decreases in number and size with growing age (Thyberg, Lohmander and Friberg24; Thyberg, Nilson and FribergZS). The appearance of keratan sulfate in early childhood and its regular, structurebound arrangement running parallel to an absolute increase in keratan sulfate (Sames 17a) can be thought of as a process of differentiation. Bonucci, Cuicchio and Dearden 2 are of the opinion that the production of keratan sulfate is connected with filament-like structures which partly mask the collagen fibres and are to be traced - by use of an electron microscope - - in the aging rib cartilage of rats where they lie predominantly territorially. At the same time that the meshwork, formed by Z3,

TABLE II AGE-DEPENDENT DISTRIBUTION OF THE ZONES IN THE TERRITORIES AND INTERTERRITORIES. IN ADULTHOOD AND OLD AGE THERE ARE NO DIFFERENCES TO BE OBSERVED. IN THESE AGE GROUPS THE PROCESS OF AGING IS CHARACTERIZED BY AN INCREASE IN THE AMOUNT OF SUBSTANCE IN Z4 AND Z5 Zone

1

Age

Region

Infant

Periph. Centr. Periph. Centr.

Child Adolescent Adult Aged

2

3

Circurnc. Circumc. Circumc. Circumc.

Interst.

Periph. Centr.

Interst. Interst. Interst. Interst./circumc. C i r c u m c . Circumc. C i r c u m c . Circumc.

Periph. Centr.

C i r c u m c . Circumc. C i r c u m c . Circumc.

Interst. Interst./circumc. Interst. Circumc.

Circumc. = circumceUular, Interst. = interstitial.

4

5

Interst. Circumc.

Interst.

445 TABLE llI Zo ne

Terms

Usltal terms

No.

Age-changes appearance o f the zone in the cartilage centre, MPS In/ant

ZI

Z2

Z3

Z4

Z5

Pericellular Zone Inner territorial Zone Outer territorial Zone

Chron one}

]

Adolescent

]

}

"Zellhof"ln the

Adult/aged

(Main compound)

cs l

Territory (chondrone)

CS l

KS

periphery : interterritory

Periterritorial Zone lnterterritorial Zone

ChiM

NeutraIMPs I

"/:iusserer Zellhof"

o.a.

substances

}

Interterritory

"3

L.

J

KS CS

decays and a greater number of chondrones are undergoing "Verd/immerung" in the cartilage centre, the KS content decreases. But between youth and early adulthood there is a time when the absolute content of KS increases whereas the changes in the structure of Z3 and the loss of chondrones in the centre are already to be observed. Nearly up to this time (4th decade) the hybridization of the KS-CS-proteoglycan proceeds (Buddecke, Sames4). An increase in KS can also be observed in other tissues whereas C-6-S decreases. A hybridization of KS and CS in the proteoglycan can also be observed in the artery wall as well (Lindner12e,d). The areas of basic substances which mark the areas organized by the cell and which are, always, built up identically seem to extend from Z1 to Z3. The differentiated cartilage cell is no !onger capable of division (Le Gros Clark 11, BucherZ). Therefore the existing cells age and the decayed cells are not replaced. The density of cells continually decreases with age in all cartilage regions although the periphery remains always richer in cells than the cartilage centre (Rahlf16). In the cartilage of children and in the periphery of older cartilage Z1 to Z3 fill in the space to zone 3 of the neighbouring cell completely. In the centre of old cartilage, on the other hand, wide interstitial and periterritorial zones appear. These zones contain a small amount of hyaluronidase-sensitive material and a lot of hyaluronidase-resistant material which is, partly, short of acidic glycosaminoglycans and exhibits intensified staining with PAS. The origin of this material can hardly be explained. If we assume that the modest density of cells is due to a dying off of cells as is the case in the centre of old cartilage, the acidic glycosaminoglycans of Z5 could derive from decayed chondrones. According to Schaffer 19 the expansion of the interterritories is the result of the absorption of chondrones that have died off.

446 By virtue of the appositional growth the periphery appears younger than the cartilage centre. The aging of the cell can also be traced from the growing tendency to be basophilic. This, perhaps, indicates an increasing content of degradation products (Lindner12b). The fact that it is hardly possible or is even impossible to trace CS in the centre of old cartilage raises the question on the metabolism of CS. Lindner 12a found an increase in 35S-incorporation related to the DNA content, which is a criterion for the number of cells in the rib cartilage of a rat. Using the means of autoradiography he found out that in older rats it takes a longer period of time until the incorporation of 3aS begins in the cells of the cartilage centre than it does in younger ones. In the human rib cartilage a decrease in the specific zsS-activity of CS - - with KS remaining constant - - was made probable even in relation to the DNA content. These investigations do not include a distinction between periphery and centre (Sames17a). On the whole a decrease or even a closing down of the CS-synthesis in the cartilage centre of men is to be assumed. According to Lindner the CS-synthesis decreases in the cartilage and in the artery with growing age, whereas the keratan sulfate synthesis increases in the cartilage. The half life period of the acidic glycosaminoglycans is prolonged and the turnover is diminished correspondingly (Lindner

12~,d).

Thus the consequences of aging and decay of the cells are changes in the extracellular milieu which in turn result in changes of the cells themselves, thus creating a kind of vicious circle. The exact mechanisms of the extracellular changes are, for the most part, unexplained. Therefore the question whether the changes in the basic substance cause the aging of the cells or that the cells age because of endogenous influences cannot yet be met by an answer. The length of the diffusion stretch may play a part in the nutrition of the cartilage tissue. In other tissues, such as heart valves which float freely in the blood stream, similar age-dependent changes of the acidic mucopolysaccharides are to be found although the diffusion stretches through such thin tissue are very short (Sames, Stegmann and RebellS). Similar changes in connective tissues of differently compounded basic substances and sufficient nutrition by diffusion point to a general process of aging in the glycosaminoglycan metabolism in the mesenchymal cell. Considering the relationship between an aging cell and the extracellular transit routes a general aging problem is pointed out by this situation. The prominence and regularity of aging change in the cartilage cells and their surrounding basic substance provide an outstanding model for the studies of this problem. ACKNOWLEDGEMENTS All technical work for the experiments was carried out by Mrs. J. Riemann and technical drawings by Miss W. Schlicher. The author would very much like to thank Prof. J. W. Rohen for his helpful advice with regard to text and nomenclature. This work was supported by a grant from the Stiftung Volkswagenwerk, Hannover, Federal Republic of Germany.

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