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Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate
Biochi~mic~a
et Biophysica A~ta ELSEVIER
Biochimica et BiophysicaActa 1290(1996) 250-256
Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate Colin Farquharson a,,, Alexander Duncan b Elaine Seawright ~ Colin C. Whitehead Simon P. Robins b
a
" Roslin Institute [Edinburgh/. Roslin, Midlothian EH25 9PS, U K b Biochemical Sciences Dirision, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
Received 22 February 1996:accepted 28 February 1996
Abstract In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxpyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur. Keywords:
Collagen; Pyridinium cross-link; Growth plate: Cartilage: Chondrocyte:Dyschondroplasia:(Tibia): (Chick)
1. Introduction The epiphyseal growth plate of long bones comprises both chondrocytes and matrix components, of which collagen and proteoglycans are the major constituents. Chondrocytes exhibit a series of well defined stages [I] which are distinguished by changes in proliferation rate, morphology and the synthesis of extracellular matrix proteins [2]. As a result, growth plates can be separated into distinct zones containing chondrocytes of different maturational age [31. The matrix provides structural integrity to the growth plate and each zone contains a unique mixture of various collagens at different concentrations. The collagenous matrix together with the cellular components are responsible for the initial steps in endochondral ossification. The control of endochondral ossification is complex and involves a
* Corresponding author. Fax: +44 131 4400434: e-mail: [email protected]. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0304-4165(96)00026- 8
variety of local and systemic factors regulating chondrocyte proliferation and differentiation and ultimately cellular hypertrophy. Concomitant changes also occur within the chondrocyte extracellular matrix and evidence suggests that chondrocytes must provide the correct extracellular network [4,5] and establish cell-matrix interactions [6] to allow progressive differentiation. Disruption of this orderly progression of chondrocyte differentiation is observed in a number of conditions such as rickets [7] and tibial dyschondroplasia (TD) in growing chickens [8] (a condition which resembles osteochondrosis in mammals). It is now generally accepted that TD is a consequence of an inability of the maturing chondrocytes to differentiate into the fully hypertrophic state [8,9]. This results in an accumulation of unmineralized, avascular cartilage extending distally from the proximal tibiotarsal growth plate [10,11]. Collagen type I1 is the principal structural protein and the predominant collagen of the growth plate. It interacts with collagen types IX and XI to form collagen fibrils [12] where 80% of the total collagen is type II and the remain-
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
der comprises equal amounts of IX and XI [12,13]. Type IX collagen may serve as a molecular coupling protein between type II collagen and other matrix proteins [14], whereas type XI collagen regulates collagen type II fibril diameter [15]. Collagen types II, IX and XI are assembled together in heterotypic fibrils and distributed throughout the cartilage matrix. Collagen type X is a non-fibrillar, short-chain collagen that is restricted to the hypertrophic region of the growth plate [16] where it is associated with fibrils containing collagens II, IX and XI [17]. As collagen type X-null mice demonstrated no gross abnormalities in long bone development [18] this collagen may have a structural and supportive role rather than being involved in mineralization. Collagen undergoes a number of post-translational modifications, one of which is the action of lysyl oxidase to initiate formation of the intermolecular cross-links which confer strength and stability to the growth plate cartilage [19]. Pyridinoline is a maturation product of the reducible, lysine-derived intermolecular bonds [20,21] and is the major cross-link of cartilage [21] and, to a lesser extent, of bone [22]. A deoxy-derivative, deoxypyridinoline [23], is present mainly in bone and dentine collagen [22,24]. In addition to the changes in collagen type and amount [16,25] within the different zones of the growth plate there are alterations to the integrity of the collagen network [26]. The exact nature of these structural changes to the collagen network are however unclear. In the present study we investigated the distribution of the pyridinium cross-links and related their location to specific zones of the normal and dyschondroplastic growth plate. We hoped that the changes seen in defective chondrocyte differentiation in TD would provide further insight into the relationship between pyridinium cross-links and cartilage maturation in normal and abnormal growth plates. Such information might also help to explain the etiology of this disorder.
251
core of tissue (omitting the epiphyseal curvature present at its periphery) was obtained from the tibiotarsi which included the articular (hyaline) cartilage, growth plate and metaphysis; the tissue was briefly immersed in 5.0% polyvinyl alcohol (Sigma, Poole, UK) and chilled immediately in n-hexane at - 7 0 ° C [9]. The tissue was mounted on metal chucks with the long axis of the bone oriented 90 ° to the cryostat knife. This enabled serial transverse sections to be cut sequentially through the articular cartilage followed by the growth plate and then in the case of TD chicks, the lesion tissue. Three 10 I~m thick sections were taken from the surface of the articular cartilage and each was picked up on separate microscope slides. This was followed by six 30 izm thick sections which were pooled in a pre-chilled hydrolysis tube. The combined cartilage segment 210 ~m thick resulting from this protocol was designated N! or TD1 when cut from normal or TD tissue, respectively, with the following (210 Ixm) repeats being classified as N2, N3 or TD2, TD3, etc. This procedure of taking sections for microscopical analysis followed by pooled sections for chromatographic analysis was continued throughout the tissue until bone, underlying the growth plate or the lesion, was detected. One 10 I~m thick section from each zone of cartilage was either reacted for alkaline phosphatase (ALP) activity, immunostained for collagen type X or stained by haematoxylin and eosin (H and E). The pooled 30 ~m thick sections were analysed for pyridinium cross-link concentrations.
2.3. Alkaline phosphatase histochemistry Sections were reacted for 2 min at 37°C in the following medium: 2 mM napthyl acid phosphate, magnesium chloride (2 mM) and Fast Blue RR (1 m g / m l ) in 0.1 M barbitone buffer (pH 9.4). Sections were rinsed in 0.1 M acetic acid, washed in distilled water and mounted in aqueous mountant [9].
2. Materials and methods
2.4. Collagen type X immunolocalisation:
2.1. Animals
Sections of cartilage were fixed for 5 min in ice-cold acetone and air dried. After pre-treatment with hyaluronidase a monoclonal antibody to chick collagen type X (MA3: a kind gift from Dr A. Kwan, University of Cardiff, Cardiff, UK) was added to the sections and left at 4°C for 18 h. A peroxidase-labelled goat anti-mouse IgG (Sigma) second antibody was then added followed by incubation in diaminobenzidine/H20 2 for 5 min. Finally, tissues were dehydrated, cleared and mounted in DePeX. [28] Control procedures consisted of the substitution of the primary antibody with an appropriate dilution of normal mouse serum.
Broiler chicks [male: Ross I strain] were fed from hatching on a diet that included 7.5 g / k g calcium, 7.6 g / k g phosphorus and 25 p.g/kg vitamin D3, which is known to result in a high incidence of TD [27]. At three weeks of age the birds were killed by cervical dislocation. Both proximal tibiotarsi were dissected and examined for the presence of the dyschondroplastic lesion by sectioning lengthways through the joint.
2.2. Tissue preparation After performing a series of pilot experiments, the following procedure was developed to provide biochemical information on well-defined morphological zones covering the full thickness of cartilage and growth plate. A central
2.5. Analytical procedures The tissue was hydrolysed in 6 M HC1 at 107°C for 24 h and the concentrations of pyridinoline and deoxypyridi-
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
252
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sequential sections down growth plate Fig. 1. Distribution of pyridinium cross-links of collagen within the (A) normal and (B) dyschondroplastic growth plates. Pyridinoline concentrations are high in the proliferating zone (p) but decrease with increasing chondrocyte differentiation and are low in the hypertrophic zone (h). Deoxypyridinoline is first detected in the prehypertrophic zone (ph). Higher concentrations of pyridinium crosslinks are found with increasing distance through the lesion. The sites of first detection of alkaline phosphatase (ALP) and collagen type X are shown.
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
noline were estimated by reverse-phase high-pressure liquid chromatography [29,30]. The results were expressed as residues per collagen molecule using values for hydroxyproline measured in aliquots of the hydrolysate by HPLC essentially as described by Einarsson [31 ].
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3.1. Microscopical analysis 0
Articular cartilage, characterised by small clusters of chondrocytes within lacunae embedded in a strongly staining extracellular matrix, was present in the uppermost sections of both normal ( N I - N 8 ) and TD (TD1-TDI3). As expected no type X collagen or ALP activity was detected in the articular cartilage. The proliferating zone of growth plate cartilage, characterized by its increased cellularity (flattened, discoid cells) and decreased matrix, was first observed in sections N9 and TD14 but markers of chondrocyte differentiation were not detected. ALP activity was first detected in sections (N11 and TD16) where the matrix staining was increased and the cells were more voluminous and spherical in shape, characteristic of the prehypertrophic phenotype. Neither chondrocyte hypertrophy nor collagen type X staining were observed in these sections. In the hypertrophic zone of normal tissue ( N I 2 N16) the cells were large and occupied a prominent lacunae. There was strong ALP activity and metaphyseai blood vessels penetrated the matrix which stained strongly for collagen type X. Increased chondrocyte hypertrophy in this zone was associated with a decrease in ALP activity although extracellular type X collagen staining remained strong. Strong ALP activity was detected proximal to the TD lesion (characterized by decreased vascularity) but enzyme activity was not strong within the lesion and declined with increasing distance towards the distal region. Collagen type X was present above and at the top of the lesion (TD20-TD28) and was confined to the extracellular matrix. This staining pattern for collagen type X changed in sections further down the lesion where intracellular staining was observed although matrix staining was still evident.
3.2. Pyridinium collagen cross-link analysis The results of the pilot experiments showed that deoxypyridinoline was invariably restricted to the hypertrophic region of the growth plate whereas pyridinoline was present throughout the tissue, but more prominently in sections comprising hyaline cartilage. A more detailed study of normal and TD tissue (Fig. 1A and B) confirmed the different distribution patterns for the two cross-links. In normal tissue the pyridinoline concentration of articular
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Fig. 2. Pyridinoline/deoxypyridinolineratio through the (A) normal and (B) dyschondroplastic growth plates. There is a progressive decrease in the cross-link ratio with distance through the mature zone of the normal growth plate whereas there is great variation in the cross-link ratio through the dyschondroplasticlesion.
cartilage was similar in the uppermost sections (N1-N6) but maximal concentrations were present in the more mature regions (N7 and N8) of the articular cartilage. A similar trend was also noted in the TD tissue. In contrast, deoxypyridinoline was not detected in articular cartilage or the upper part of the normal or TD growth plate. Deoxypyridinoline was first noted in sections N11 and TD20 which had strong ALP activity indicative of the differentiated phenotype. In normal growth cartilage (N9-N16) an inverse relationship between pyridinoline concentration and chondrocyte differentiation was observed. There was approximately a 10-fold greater pyridinoline cross-link concentration in sections that did not contain collagen type X ( N 9 - N I 1 ) than those that did (N14-N16). In contrast, deoxypyridinoline concentration was highest in the most differentiated regions of the growth plate (NI5 and N16). This disparity in the distribution of the growth plate crosslinks was reflected by the pyridinoline/deoxypyridinoline ratio (Fig. 2A).
254
c. F~wquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
In the growth cartilage above the TD lesion the pyridinoline concentration was higher in the proliferating zone ( T D l 4 and T D I 5 ) than the more mature, collagen type X containing, tissue (TD20 and TD21). There was a progressive increase in pyridinium cross-link concentrations through the lesion and also a progressive change in the pyridinoline/deoxypyridinoline ratio. (Fig. 2B). The concentrations of collagen (ixg/mm~), calculated from the known volume of tissue analysed in each section, were shown to vary approx. 5-fold (Fig. 3A and B) with the highest concentrations in both normal and TD samples being about 500 mm from the cartilage surface. The TD
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articular cartilage
lesion shown no significant differences in collagen concentration.
4. Discussion The principal aim of this study was to examine the distribution of pyridinium cross-links of collagen in avian growth plate cartilage. By employing both microscopical and analytical methodologies we have been able to assess accurately whether the cascade of chondrocyte maturational changes that occur in endochondral ossification [I]
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sequential sections down growth plate Fig. 3. Collagen concentrationthrough the (A) normal and (B) dyschondroplasticgrowth plates. Highest concentrationsare in the articular cartilage. There were no significant differences between the dyschondroplasticlesion and normal growth plate.
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
are associated with concomitant changes in the amount and/or distribution of pyridinium cross-links. The results demonstrate that the pyridinoline concentration was lowest in growth plate zones that displayed the mature phenotype, a finding which is in agreement with indirect evidence of Dean and Howell [26]. These authors [26] indicated that the integrity of the collagen network within the hypertrophic zone was less than that in cartilage from the less mature resting/proliferating zone. Contrasting data were, however, obtained using immunocytochemistry which suggested that collagen cross-linking was highest in cartilage from the hypertrophic region of the growth plate [32]. The high amounts of collagenase and insufficient amounts of its inhibitor (TIMP) within the hypertrophic zone [33,34] may account for the increased collagen type II degradation [35] and the low concentrations of pyridinoline noted in this present study. A poorly cross-linked collagenous matrix within the hypertrophic zone cartilage may be a required for the rapid osteoclastic resorption of growth cartilage during endochondral ossification. The high concentration of pyridinoline in the articular cartilage may reflect its low level of cartilage remodelling combined with slow turnover and lack of osteoclastic resorption (secondary ossification centers are not present in 3-week-old chicks). The increased maturational age of the collagen in the deep mature zones of the articular cartilage [36] would explain the high cross-link concentration in this area. Although adult avian articular cartilage is known to contain relatively large amounts of collagen type I, predominantly towards the surface of the cartilage [37], the young age of the birds in the present study suggests that the proportion of this collagen type would be small. Furthermore, no deoxypyridinoline, a characteristic feature of collagen type I in bone, was detected in the surface layers of the articular cartilage. In agreement with other studies, deoxypyridinoline was not detected in articular cartilage [22,24,38] but has been detected in the hypertrophic cartilage of one-day-old chicks [39]. The present results confirm and extend those of Orth et al [39] and show that deoxypyridinoline is present only in the mature regions of the growth plate and, in contrast to pyridinoline, is at its highest concentration within the hypertrophic zone where it is the principal pyridinium cross-link. The divergence in pyridinoline and deoxypyridinoline concentrations with increasing cartilage development in the growth plate is reflected by the progressive decrease in the pyridinoline/deoxypyridinoline ratio. Collagen types II and IX of articular cartilage are covalently cross-linked by pyridinoline residues [40] but the nature of the lysyl oxidase-mediated cross-links that are formed [17,41] between newly formed type X collagen and pre-existing heterotypic (II + IX + XI) collagen fibrils is unknown. Our results indicate that deoxypyridinoline cross-links are formed in the growth plate matrix before collagen type X is detected and therefore do not fully
255
support the suggestion that deoxypyridinoline formation correlates with collagen type X concentration [39]. Direct analysis of the lysine-derived cross-links in porcine collagen type X have shown that neither pyridinoline nor deoxy-pyridinoline are present in significant amounts, despite relatively high concentrations of the bifunctional, borohydride-reducible precursors [42]. The formation of deoxypyridinoline cross-links in the mature growth plate cartilage may be an adaptive alteration which is necessary, or at least provides a permissive environment, for the mineralization process to take place. Other factors must prevail, however, as mineralization is not observed in the dyschondroplastic cartilage [28,32] despite high concentrations of deoxypyridinoline. The hypertrophic chondrocytes are the principal collagenase secreting cells of the growth plate [33,34] and a reduction in collagenase production occurs in TD [43]. As previously suggested [44], this is likely to result in decreased collagen degradation but increased collagen crosslinking of the existing fibrils. Analysis of tissue collagen concentrations in the present study showed no differences to normal growth plate so that the higher cross-link concentration in the dyschondroplastic tissue presumably resuited in a structurally more stable collagen network. A highly cross-linked matrix may be more impervious to vascular penetration and osteoclastic resorption. Such a hypothesis is in accord with the observed pathology in TD and would also explain the linear increase in the pyridinium cross-link concentration through the lesion. This observation is consistent with the results of others [44,45] but contrasts with immunocytochemical studies which showed impaired collagen cross-linking within the dyschondroplastic lesion [32]. The similar amounts of pyridinium cross-links in cartilage above the lesion to that in the normal growth plate suggests that the high concentration noted in the distal lesion may exacerbate the condition but is unlikely to be the primary cause of TD. Similarities have been drawn between TD and osteochondrosis [46] but the higher amounts of pyridinium cross-links and the abnormal collagen type X metabolism [47] and staining [28] noted in dyschondroplastic tissue have not been reported in osteochondrotic growth plate cartilage [48]. The pyridinoline concentration was lower and deoxypyridinoline concentrations unaltered in osteochondrosis whereas the proportion of collagen type X and its distribution were normal [48] These differences may suggest that both disorders have discrete etiologies despite both having focal areas of impaired endochondral ossification and cartilage accumulation. The results of this study indicate that there are specific changes in the integrity of the collagenous network of the growth plate during cartilage development. The significance of alterations in both the distribution and concentration of pyridinium cross-links of collagen is not known but, together with previous findings [35], it is clear that substantial remodelling of the chondrocyte extracellular
256
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
matrix occurs in the events leading up to matrix mineralization and endochondral ossification.
Acknowledgements This study was funded by the Ministry of Agriculture, Fisheries and Food, the CEC Directorate-General for Agriculture and the Scottish Office Agriculture, Environment and Fisheries Department. The authors are grateful for the gift of collagen type X monoclonal antibody from Dr. A. Kwan, University of Cardiff, Cardiff, Wales, UK.
References [1] Brighton, C.T. (1978) Clin. Orthop. Rel. Res. 136. 22-32. [2] Hunziker, E.B., Schenk, R.K. and Cruze-Orive, L.M. (1987) J. Bone Joint Surg. 69A, 162-179. [3] Loveridge, N., Thomson, B.M. and Farquharson C. (1992) in Bone Biology and Skeletal Disorders in Poultry (Whitehead, C.C., ed.). pp. 3-17, Ca_flax, Abingdon. [4] Bruckner, P., Horler, I., Mendler, M., Houze, M., Winterhalter, K.H., Eich-Bender, S.G. and Spycher, M.A. (1989) J. Cell Biol. 109, 2537-2545. [5] Gerstenfeld, L.C. and Landis, W.J. ( 1991 ) J. Cell Biol. 112, 501-513~ [6] Sommarin, Y., Larssen, T. and Heinegard, D. (1989) Exp. Cell Res. 184, 181-192. [7] Appleton, J. (1988) Arch. Oral Biol. 33, 379-381. [8] Hargest, T.E., Leach, R.M. and Gay, C.V. (1985) Am. J. Pathol. 119, 175-190. [9] Farquharson, C., Whitehead, C.C., Rennie, J.S., Thorp, B.H. and Loveridge, N. (1992) Bone 13, 59-67. [10] Leach, R.M. and Nesheim, M.C. (1965) J. Nutr. 86, 236-244. [11] Riddell, C., Howell, J. and Kaye, M.M. (1971) Avian Dis. 15, 557-565. [12] Vaughan, L., Mendler, M., Huber, H., Bruckner, P., Winterhalter, K.H., Irwin, M.I. and Mayne, R. (1988) J. Cell Biol. 106, 991-997. [13] Mendler, M, Eich-Bender, S.G., Vaughan, L., Winterhalter, K.H. and Bruckner, P. (1989) J. Cell Biol. 108, 191-198. [14] Bruckner, P., Vaughan, L. and Winterhalter. K.H. (1985) Proc. Natl. Acad. Sci. USA 82, 2608-2612. [15] Poole, A.R. (1986) Biochem. J. 236, 1-14. [16] Linsenmayer, T.F., Gibney, E. and Schmid, T.M. (1986) Dev. Biol. 113, 467- 473. [17] Chen, Q., Fitch, J.M., Linsenmayer, C. and Linsenmayer, T.F. (1992) Bone Miner. 17, 223-227. [18] Rosati, R., Horan, G.S.B., Pinero, G.J., Garofalo, S., Keene, D.R.. Horton, W.A., Vuorio, E., De Crombrugghe, B. and Behringer, R.R. (1994) Nature Genet. 8, 129-135. [19] Robins, S.P. (1988) Baillieres Clin. Rheumatol. 2. 1-36.
[20] Fujimoto, D., Moriguchi, T., lshida, T. and Hayashi, J. (1978) Biochem. Biophys. Res. Commun. 84, 52-57. [21] Robins, S.P. (1983) Biochem. J. 215, 167-173. [22] Robins, S.P. and Duncan, A. (1987) Biochim. Biophys. Acta 914, 233-239. [23] Ogawa, T., Ono, T., Tsuda, M. and Kawanishi Y. (1982) Biochem. Biophys. Res. Commun. 107. 1252-1257. [24] Eyre, D.R., Koob, T.J. and Van Ness, K.P. (1984) Anal. Biochem. 137, 380- 388. [25] Sandell, L.J., Sugai, J.V. and Trippel, S.B. (1994) J. Orthop. Res. 12, 1-14. [26] Howell, D.S. and Dean, D.D. (1992) in Disorders of Bone and Mineral Metabolism (Coe, F.L. and Favus, M.J., eds.), pp. 313-352, Raven Press, New York. [27] Rennie, J.S., Whitehead, C.C. and Thorp, B.H. (1993) Br, J. Nutr. 69, 809-816. [28] Farquharson, C., Berry, J.L., Mawer, E.B. and Whitehead, C.C. (1995) Bone 17, 279-286. [29] Black, D., Duncan, A. and Robins, S.P. (1988) Anal. Biochem. 169, 197-203. [30] Black, D., Farquharson C. and Robins, S.P. (1989) Calcif. Tissue Int. 44, 343-347. [31] Einarsson, S. (1985)J. Chromatogr. 348, 213-220. [32] Chen, Q., Gibney, E.P., Leach, R.M. and Linsenmayer, T.F. (1993) Dev. Dyn. 196, 54-61. [33] Dean, D.D. Muniz, O.E., Berman, I., Pita, J.C., Carreno, M.R., Woessner, J.F. and Howell, D.S. (1985) J. Clin. Invest. 76, 716-722. [34] Dean, D.D., Muniz, O.E. and Howell, D.S. (1989) Matrix 9, 366375. [35] Alini, M., Matsui, Y., Dodge, G.R. and Poole, A.R. (1992) Calcif. Tissue Int. 50, 327-335. [36] Banks, W.J. (1986) in Applied Veterinary Histology, 2nd Ed. (Stamathis, G., ed.), pp. 259-284, Williams and Wilkins, Baltimore, MD. [37] Eyre, D.R., Brickley-Parsons, D.M. and Glimcber, M.J. (1978) FEBS Lett. 85,259-263. [38] Farquharson, C., Duncan, A. and Robins, S.P. (1989) Proc. Soc. Exp. Biol. Med. 192, 166-171. [39] Orth. M.W., Martinez, D.A., Cook, M.E. and Vailas, A.C. (1993) Biochim. Biophys. Acta 1157, 229-232. [40] Eyre, D.R., Apon, S., Wu, J.-J., Ericsson, L.H. and Walsh, K.A. (1987) FEBS Lett. 2, 337-341. [41] Reginato, A.M. and Jimenez, S.A. (1991) Biochem, J. 273, 333-338. [42] Rucklidge, G.J., Milne, G. and Robins, S.P. (1995) Matrix Biol. (in press), [43] Cook, M.E. and Bai, Y. (1994) Poultry Sci. 73, 889-896. [44] Orth, M.W., Martinez, D.A., Cook, M.E. and Vailas, A.C. (1991) Biochem. Biophys. Res. Commun. 179, 1582-1586. [45] Orth, MW., Leach, R.M., Vailas, A.C. and Cook, M.E. (1994) Avian Dis. 38, 44-49. [46] Rejno, S. and Stromberg, B. (1978) Acta Radiol. Suppl. 358, 153-178. [47] Bashey, R.I., Leach, R.M., Gay, C.V. and Jimenez, S.A. (1989) Lab. Invest. 60, 106-112. [48] Wardale, J. and Duance, V. (1994) J. Cell Sci. 107, 47-59.
et Biophysica A~ta ELSEVIER
Biochimica et BiophysicaActa 1290(1996) 250-256
Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate Colin Farquharson a,,, Alexander Duncan b Elaine Seawright ~ Colin C. Whitehead Simon P. Robins b
a
" Roslin Institute [Edinburgh/. Roslin, Midlothian EH25 9PS, U K b Biochemical Sciences Dirision, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
Received 22 February 1996:accepted 28 February 1996
Abstract In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxpyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur. Keywords:
Collagen; Pyridinium cross-link; Growth plate: Cartilage: Chondrocyte:Dyschondroplasia:(Tibia): (Chick)
1. Introduction The epiphyseal growth plate of long bones comprises both chondrocytes and matrix components, of which collagen and proteoglycans are the major constituents. Chondrocytes exhibit a series of well defined stages [I] which are distinguished by changes in proliferation rate, morphology and the synthesis of extracellular matrix proteins [2]. As a result, growth plates can be separated into distinct zones containing chondrocytes of different maturational age [31. The matrix provides structural integrity to the growth plate and each zone contains a unique mixture of various collagens at different concentrations. The collagenous matrix together with the cellular components are responsible for the initial steps in endochondral ossification. The control of endochondral ossification is complex and involves a
* Corresponding author. Fax: +44 131 4400434: e-mail: [email protected]. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0304-4165(96)00026- 8
variety of local and systemic factors regulating chondrocyte proliferation and differentiation and ultimately cellular hypertrophy. Concomitant changes also occur within the chondrocyte extracellular matrix and evidence suggests that chondrocytes must provide the correct extracellular network [4,5] and establish cell-matrix interactions [6] to allow progressive differentiation. Disruption of this orderly progression of chondrocyte differentiation is observed in a number of conditions such as rickets [7] and tibial dyschondroplasia (TD) in growing chickens [8] (a condition which resembles osteochondrosis in mammals). It is now generally accepted that TD is a consequence of an inability of the maturing chondrocytes to differentiate into the fully hypertrophic state [8,9]. This results in an accumulation of unmineralized, avascular cartilage extending distally from the proximal tibiotarsal growth plate [10,11]. Collagen type I1 is the principal structural protein and the predominant collagen of the growth plate. It interacts with collagen types IX and XI to form collagen fibrils [12] where 80% of the total collagen is type II and the remain-
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
der comprises equal amounts of IX and XI [12,13]. Type IX collagen may serve as a molecular coupling protein between type II collagen and other matrix proteins [14], whereas type XI collagen regulates collagen type II fibril diameter [15]. Collagen types II, IX and XI are assembled together in heterotypic fibrils and distributed throughout the cartilage matrix. Collagen type X is a non-fibrillar, short-chain collagen that is restricted to the hypertrophic region of the growth plate [16] where it is associated with fibrils containing collagens II, IX and XI [17]. As collagen type X-null mice demonstrated no gross abnormalities in long bone development [18] this collagen may have a structural and supportive role rather than being involved in mineralization. Collagen undergoes a number of post-translational modifications, one of which is the action of lysyl oxidase to initiate formation of the intermolecular cross-links which confer strength and stability to the growth plate cartilage [19]. Pyridinoline is a maturation product of the reducible, lysine-derived intermolecular bonds [20,21] and is the major cross-link of cartilage [21] and, to a lesser extent, of bone [22]. A deoxy-derivative, deoxypyridinoline [23], is present mainly in bone and dentine collagen [22,24]. In addition to the changes in collagen type and amount [16,25] within the different zones of the growth plate there are alterations to the integrity of the collagen network [26]. The exact nature of these structural changes to the collagen network are however unclear. In the present study we investigated the distribution of the pyridinium cross-links and related their location to specific zones of the normal and dyschondroplastic growth plate. We hoped that the changes seen in defective chondrocyte differentiation in TD would provide further insight into the relationship between pyridinium cross-links and cartilage maturation in normal and abnormal growth plates. Such information might also help to explain the etiology of this disorder.
251
core of tissue (omitting the epiphyseal curvature present at its periphery) was obtained from the tibiotarsi which included the articular (hyaline) cartilage, growth plate and metaphysis; the tissue was briefly immersed in 5.0% polyvinyl alcohol (Sigma, Poole, UK) and chilled immediately in n-hexane at - 7 0 ° C [9]. The tissue was mounted on metal chucks with the long axis of the bone oriented 90 ° to the cryostat knife. This enabled serial transverse sections to be cut sequentially through the articular cartilage followed by the growth plate and then in the case of TD chicks, the lesion tissue. Three 10 I~m thick sections were taken from the surface of the articular cartilage and each was picked up on separate microscope slides. This was followed by six 30 izm thick sections which were pooled in a pre-chilled hydrolysis tube. The combined cartilage segment 210 ~m thick resulting from this protocol was designated N! or TD1 when cut from normal or TD tissue, respectively, with the following (210 Ixm) repeats being classified as N2, N3 or TD2, TD3, etc. This procedure of taking sections for microscopical analysis followed by pooled sections for chromatographic analysis was continued throughout the tissue until bone, underlying the growth plate or the lesion, was detected. One 10 I~m thick section from each zone of cartilage was either reacted for alkaline phosphatase (ALP) activity, immunostained for collagen type X or stained by haematoxylin and eosin (H and E). The pooled 30 ~m thick sections were analysed for pyridinium cross-link concentrations.
2.3. Alkaline phosphatase histochemistry Sections were reacted for 2 min at 37°C in the following medium: 2 mM napthyl acid phosphate, magnesium chloride (2 mM) and Fast Blue RR (1 m g / m l ) in 0.1 M barbitone buffer (pH 9.4). Sections were rinsed in 0.1 M acetic acid, washed in distilled water and mounted in aqueous mountant [9].
2. Materials and methods
2.4. Collagen type X immunolocalisation:
2.1. Animals
Sections of cartilage were fixed for 5 min in ice-cold acetone and air dried. After pre-treatment with hyaluronidase a monoclonal antibody to chick collagen type X (MA3: a kind gift from Dr A. Kwan, University of Cardiff, Cardiff, UK) was added to the sections and left at 4°C for 18 h. A peroxidase-labelled goat anti-mouse IgG (Sigma) second antibody was then added followed by incubation in diaminobenzidine/H20 2 for 5 min. Finally, tissues were dehydrated, cleared and mounted in DePeX. [28] Control procedures consisted of the substitution of the primary antibody with an appropriate dilution of normal mouse serum.
Broiler chicks [male: Ross I strain] were fed from hatching on a diet that included 7.5 g / k g calcium, 7.6 g / k g phosphorus and 25 p.g/kg vitamin D3, which is known to result in a high incidence of TD [27]. At three weeks of age the birds were killed by cervical dislocation. Both proximal tibiotarsi were dissected and examined for the presence of the dyschondroplastic lesion by sectioning lengthways through the joint.
2.2. Tissue preparation After performing a series of pilot experiments, the following procedure was developed to provide biochemical information on well-defined morphological zones covering the full thickness of cartilage and growth plate. A central
2.5. Analytical procedures The tissue was hydrolysed in 6 M HC1 at 107°C for 24 h and the concentrations of pyridinoline and deoxypyridi-
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
252
a.-----
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~-growth plate-~ 0.6
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sequential sections down growth plate Fig. 1. Distribution of pyridinium cross-links of collagen within the (A) normal and (B) dyschondroplastic growth plates. Pyridinoline concentrations are high in the proliferating zone (p) but decrease with increasing chondrocyte differentiation and are low in the hypertrophic zone (h). Deoxypyridinoline is first detected in the prehypertrophic zone (ph). Higher concentrations of pyridinium crosslinks are found with increasing distance through the lesion. The sites of first detection of alkaline phosphatase (ALP) and collagen type X are shown.
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
noline were estimated by reverse-phase high-pressure liquid chromatography [29,30]. The results were expressed as residues per collagen molecule using values for hydroxyproline measured in aliquots of the hydrolysate by HPLC essentially as described by Einarsson [31 ].
A
253
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3.1. Microscopical analysis 0
Articular cartilage, characterised by small clusters of chondrocytes within lacunae embedded in a strongly staining extracellular matrix, was present in the uppermost sections of both normal ( N I - N 8 ) and TD (TD1-TDI3). As expected no type X collagen or ALP activity was detected in the articular cartilage. The proliferating zone of growth plate cartilage, characterized by its increased cellularity (flattened, discoid cells) and decreased matrix, was first observed in sections N9 and TD14 but markers of chondrocyte differentiation were not detected. ALP activity was first detected in sections (N11 and TD16) where the matrix staining was increased and the cells were more voluminous and spherical in shape, characteristic of the prehypertrophic phenotype. Neither chondrocyte hypertrophy nor collagen type X staining were observed in these sections. In the hypertrophic zone of normal tissue ( N I 2 N16) the cells were large and occupied a prominent lacunae. There was strong ALP activity and metaphyseai blood vessels penetrated the matrix which stained strongly for collagen type X. Increased chondrocyte hypertrophy in this zone was associated with a decrease in ALP activity although extracellular type X collagen staining remained strong. Strong ALP activity was detected proximal to the TD lesion (characterized by decreased vascularity) but enzyme activity was not strong within the lesion and declined with increasing distance towards the distal region. Collagen type X was present above and at the top of the lesion (TD20-TD28) and was confined to the extracellular matrix. This staining pattern for collagen type X changed in sections further down the lesion where intracellular staining was observed although matrix staining was still evident.
3.2. Pyridinium collagen cross-link analysis The results of the pilot experiments showed that deoxypyridinoline was invariably restricted to the hypertrophic region of the growth plate whereas pyridinoline was present throughout the tissue, but more prominently in sections comprising hyaline cartilage. A more detailed study of normal and TD tissue (Fig. 1A and B) confirmed the different distribution patterns for the two cross-links. In normal tissue the pyridinoline concentration of articular
I" 15
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Fig. 2. Pyridinoline/deoxypyridinolineratio through the (A) normal and (B) dyschondroplastic growth plates. There is a progressive decrease in the cross-link ratio with distance through the mature zone of the normal growth plate whereas there is great variation in the cross-link ratio through the dyschondroplasticlesion.
cartilage was similar in the uppermost sections (N1-N6) but maximal concentrations were present in the more mature regions (N7 and N8) of the articular cartilage. A similar trend was also noted in the TD tissue. In contrast, deoxypyridinoline was not detected in articular cartilage or the upper part of the normal or TD growth plate. Deoxypyridinoline was first noted in sections N11 and TD20 which had strong ALP activity indicative of the differentiated phenotype. In normal growth cartilage (N9-N16) an inverse relationship between pyridinoline concentration and chondrocyte differentiation was observed. There was approximately a 10-fold greater pyridinoline cross-link concentration in sections that did not contain collagen type X ( N 9 - N I 1 ) than those that did (N14-N16). In contrast, deoxypyridinoline concentration was highest in the most differentiated regions of the growth plate (NI5 and N16). This disparity in the distribution of the growth plate crosslinks was reflected by the pyridinoline/deoxypyridinoline ratio (Fig. 2A).
254
c. F~wquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
In the growth cartilage above the TD lesion the pyridinoline concentration was higher in the proliferating zone ( T D l 4 and T D I 5 ) than the more mature, collagen type X containing, tissue (TD20 and TD21). There was a progressive increase in pyridinium cross-link concentrations through the lesion and also a progressive change in the pyridinoline/deoxypyridinoline ratio. (Fig. 2B). The concentrations of collagen (ixg/mm~), calculated from the known volume of tissue analysed in each section, were shown to vary approx. 5-fold (Fig. 3A and B) with the highest concentrations in both normal and TD samples being about 500 mm from the cartilage surface. The TD
A }
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lesion shown no significant differences in collagen concentration.
4. Discussion The principal aim of this study was to examine the distribution of pyridinium cross-links of collagen in avian growth plate cartilage. By employing both microscopical and analytical methodologies we have been able to assess accurately whether the cascade of chondrocyte maturational changes that occur in endochondral ossification [I]
~----growthplate-~ ] p'lph~--"h |
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13 I
articular cartilage
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100
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75
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sequential sections down growth plate Fig. 3. Collagen concentrationthrough the (A) normal and (B) dyschondroplasticgrowth plates. Highest concentrationsare in the articular cartilage. There were no significant differences between the dyschondroplasticlesion and normal growth plate.
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
are associated with concomitant changes in the amount and/or distribution of pyridinium cross-links. The results demonstrate that the pyridinoline concentration was lowest in growth plate zones that displayed the mature phenotype, a finding which is in agreement with indirect evidence of Dean and Howell [26]. These authors [26] indicated that the integrity of the collagen network within the hypertrophic zone was less than that in cartilage from the less mature resting/proliferating zone. Contrasting data were, however, obtained using immunocytochemistry which suggested that collagen cross-linking was highest in cartilage from the hypertrophic region of the growth plate [32]. The high amounts of collagenase and insufficient amounts of its inhibitor (TIMP) within the hypertrophic zone [33,34] may account for the increased collagen type II degradation [35] and the low concentrations of pyridinoline noted in this present study. A poorly cross-linked collagenous matrix within the hypertrophic zone cartilage may be a required for the rapid osteoclastic resorption of growth cartilage during endochondral ossification. The high concentration of pyridinoline in the articular cartilage may reflect its low level of cartilage remodelling combined with slow turnover and lack of osteoclastic resorption (secondary ossification centers are not present in 3-week-old chicks). The increased maturational age of the collagen in the deep mature zones of the articular cartilage [36] would explain the high cross-link concentration in this area. Although adult avian articular cartilage is known to contain relatively large amounts of collagen type I, predominantly towards the surface of the cartilage [37], the young age of the birds in the present study suggests that the proportion of this collagen type would be small. Furthermore, no deoxypyridinoline, a characteristic feature of collagen type I in bone, was detected in the surface layers of the articular cartilage. In agreement with other studies, deoxypyridinoline was not detected in articular cartilage [22,24,38] but has been detected in the hypertrophic cartilage of one-day-old chicks [39]. The present results confirm and extend those of Orth et al [39] and show that deoxypyridinoline is present only in the mature regions of the growth plate and, in contrast to pyridinoline, is at its highest concentration within the hypertrophic zone where it is the principal pyridinium cross-link. The divergence in pyridinoline and deoxypyridinoline concentrations with increasing cartilage development in the growth plate is reflected by the progressive decrease in the pyridinoline/deoxypyridinoline ratio. Collagen types II and IX of articular cartilage are covalently cross-linked by pyridinoline residues [40] but the nature of the lysyl oxidase-mediated cross-links that are formed [17,41] between newly formed type X collagen and pre-existing heterotypic (II + IX + XI) collagen fibrils is unknown. Our results indicate that deoxypyridinoline cross-links are formed in the growth plate matrix before collagen type X is detected and therefore do not fully
255
support the suggestion that deoxypyridinoline formation correlates with collagen type X concentration [39]. Direct analysis of the lysine-derived cross-links in porcine collagen type X have shown that neither pyridinoline nor deoxy-pyridinoline are present in significant amounts, despite relatively high concentrations of the bifunctional, borohydride-reducible precursors [42]. The formation of deoxypyridinoline cross-links in the mature growth plate cartilage may be an adaptive alteration which is necessary, or at least provides a permissive environment, for the mineralization process to take place. Other factors must prevail, however, as mineralization is not observed in the dyschondroplastic cartilage [28,32] despite high concentrations of deoxypyridinoline. The hypertrophic chondrocytes are the principal collagenase secreting cells of the growth plate [33,34] and a reduction in collagenase production occurs in TD [43]. As previously suggested [44], this is likely to result in decreased collagen degradation but increased collagen crosslinking of the existing fibrils. Analysis of tissue collagen concentrations in the present study showed no differences to normal growth plate so that the higher cross-link concentration in the dyschondroplastic tissue presumably resuited in a structurally more stable collagen network. A highly cross-linked matrix may be more impervious to vascular penetration and osteoclastic resorption. Such a hypothesis is in accord with the observed pathology in TD and would also explain the linear increase in the pyridinium cross-link concentration through the lesion. This observation is consistent with the results of others [44,45] but contrasts with immunocytochemical studies which showed impaired collagen cross-linking within the dyschondroplastic lesion [32]. The similar amounts of pyridinium cross-links in cartilage above the lesion to that in the normal growth plate suggests that the high concentration noted in the distal lesion may exacerbate the condition but is unlikely to be the primary cause of TD. Similarities have been drawn between TD and osteochondrosis [46] but the higher amounts of pyridinium cross-links and the abnormal collagen type X metabolism [47] and staining [28] noted in dyschondroplastic tissue have not been reported in osteochondrotic growth plate cartilage [48]. The pyridinoline concentration was lower and deoxypyridinoline concentrations unaltered in osteochondrosis whereas the proportion of collagen type X and its distribution were normal [48] These differences may suggest that both disorders have discrete etiologies despite both having focal areas of impaired endochondral ossification and cartilage accumulation. The results of this study indicate that there are specific changes in the integrity of the collagenous network of the growth plate during cartilage development. The significance of alterations in both the distribution and concentration of pyridinium cross-links of collagen is not known but, together with previous findings [35], it is clear that substantial remodelling of the chondrocyte extracellular
256
C. Farquharson et al. / Biochimica et Biophysica Acta 1290 (1996) 250-256
matrix occurs in the events leading up to matrix mineralization and endochondral ossification.
Acknowledgements This study was funded by the Ministry of Agriculture, Fisheries and Food, the CEC Directorate-General for Agriculture and the Scottish Office Agriculture, Environment and Fisheries Department. The authors are grateful for the gift of collagen type X monoclonal antibody from Dr. A. Kwan, University of Cardiff, Cardiff, Wales, UK.
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