Báo cáo khoa học: Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line ppt

Eyre1,3 1 Department of Orthopedics and Sports Medicine, University of Washington, Seattle, WA, USA;2Department of Biochemistry, Rush Medical College, Rush University, Chicago, IL, USA;3

Assembly of collagen types II, IX and XI into nascent hetero-fibrils

by a rat chondrocyte cell line

Russell J Fernandes1, Thomas M Schmid2and David R Eyre1,3

1

Department of Orthopedics and Sports Medicine, University of Washington, Seattle, WA, USA;2Department of Biochemistry, Rush Medical College, Rush University, Chicago, IL, USA;3Department of Biochemistry, University of Washington,

Seattle, WA, USA

The cell line, RCS-LTC (derived from the Swarm rat

chondrosarcoma), deposits a copious extracellular matrix in

which the collagen component is primarily a polymer of

partially processed type II N-procollagen molecules

Transmission electron microscopy of the matrix shows no

obvious fibrils, only a mass of thin unbanded filaments We

have used this cell system to show that the type II

N-pro-collagen polymer nevertheless is stabilized by pyridinoline

cross-links at molecular sites (mediated by N- and

C-telo-peptide domains) found in collagen II fibrils processed

nor-mally Retention of the N-propeptide therefore does not

appear to interfere with the interactions needed to form

cross-links and mature them into trivalent pyridinoline

residues In addition, usingantibodies that recognize specific cross-linkingdomains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen

II N-propeptides

Keywords: chondrocyte; type II procollagen; pyridinoline cross-links; collagen fibril; extracellular matrix

The collagen framework of the extracellular matrix of

developinghyaline cartilage is assembled primarily from

three cartilage-specific collagens: type II; type IX; and type

XI [1] These three collagens copolymerize into heterotypic

fibrils and become cross-linked intermolecularly [2,3] The

predominant mature cross-link is the trivalent

hydroxyl-ysyl pyridinoline (HP) residue, which links at two sites

(from N-telopeptide to helix and from C-telopeptide to

helix) between head-to-tail overlappingtype II collagen

molecules packed in fibrils [4] Pyridinolines and divalent

cross-links covalently bond type IX collagen molecules to

N- and C- telopeptides on the surface of type II collagen

fibrils [1,5] Divalent cross-links (keto-amines) also link

type XI collagen molecules to each other and to

C-telopeptides of type II collagen within the

heteropoly-mer [3] All the cross-links are formed by the lysyl

oxidase-catalyzed mechanism This copolymeric fibrillar

network is an essential template for the assembly of the

matrix and normal function of hyaline cartilages

Muta-tions in any one of the genes encoding the three primary

collagen subunits can cause chondrodysplasia syndromes and/or premature osteoarthritis [6–10]

Type II collagen, the major structural protein of cartilage,

is secreted as a procollagen molecule which is processed by removal of its C- and N-propeptides before or duringfibril assembly in the extracellular matrix [11–13] Although propeptide removal is required for the normal growth of fibril diameter [14], fibrillogenesis experiments in vitro, using purified collagens from pig eye vitreous humor, have shown that partially processed N-procollagen can coassemble with fully processed type II collagen into thin fibrils [15] Type IIA N-procollagen, an alternatively spliced product from the type II collagen gene, COL2A1 [16], together with type IIB N-procollagen and fully processed type II collagen molecules, have all been detected in bovine vitreous humor [17] Type IIA N-procollagen has been immunolocalized to the surface of collagen fibrils in vitreous humor [18] To what degree the type II N-procollagen molecules can form fibrils and become cross-linked, however, is unclear

We have pursued this question usinga rat chondrosar-coma cell line, RCS-LTC These cells and the cells from the parental tumor lay down a copious, highly hydrated matrix

of collagen [19], aggrecan [20,21] and noncollagenous proteins, includingcartilage oligomeric matrix protein [22] and matrilin-3 [23] Type II collagen is the major collagen product of the cell line, but a high proportion of collagens

IX and XI are also synthesized and deposited in the extracellular matrix [19] In previous reports we showed that the form of type II collagen in the matrix was the IIB splicingvariant, all molecules of which had retained their N-propeptides [24,25] The RCS-LTC cell line therefore fails

to express an active collagen II N-propeptidase In the

Correspondence to R J Fernandes, Orthopaedic Research

Laborat-ories, Department of Orthopædics and Sports Medicine, Box 356500,

University of Washington, Seattle, WA 98195, USA.

Fax: +1 206 685 4700; Tel.: +1 206 543 4700;

E-mail: rjf@u.washington.edu

Abbreviations: bAPN, beta-aminoproprionitrile; HP, hydroxylysyl

pyridinoline; LP, lysyl pyridinoline; pN a1(II), type II

N-procollagen a-chains.

(Received 11 April 2003, revised 4 June 2003, accepted 9 June 2003)

understand better the temporal sequence and mechanism of

assembly

Materials and methods

Cell culture

The RCS-LTC cell line was maintained in monolayer

culture in high-glucose DMEM (Dulbecco’s modified

Eagle’s medium) (Hyclone) containing 10%

iron-supple-mented bovine calf serum (Hyclone), 10 lgÆmL)1L

-ascor-bate, at 37°C and 5% CO2 for 1–4 weeks [24] Some

cultures were additionally supplemented with

beta-amino-proprionitrile (bAPN) (Sigma) to inhibit lysyl oxidase

Metabolic radiolabeling

After 4 weeks in culture, the medium was replaced with

serum-free DMEM containing25 lCiÆmL)1 [3H]proline,

10 lgÆmL)1ascorbate, 100 lgÆmL)1bAPN, and incubation

was continued for a further 24 h The medium was then

removed and the cell layer extracted with 0.15Mpotassium

phosphate (130 mMK2HPO4and 19 mMKH2PO4, pH 7.6)

containing1 mM phenylmethanesulfonyl fluoride, 1 mM

N-ethylmaleimide, 5 mM EDTA [19,26], for 24 h at 4°C

The medium and the cell layer extract were dialyzed against

0.4M NaCl, 50 mM Tris (pH 7.5), 5 mM EDTA, 2 mM

phenylmethanesulfonyl fluoride, and stored at)20 °C until

analyzed

Collagen extraction and purification

The cell layer was extracted with 0.15Mpotassium

phos-phate containingprotease inhibitors (as described above)

for 20 h at 4°C, to solubilize newly synthesized,

noncross-linked collagen After centrifugation (30 min, 4°C,

30 000 g), the pellet was suspended in buffer comprising

50 mM sodium acetate (pH 6.0), 0.15M NaCl and 2 mM

EDTA, and digested with 0.5 mgÆmL)1porcine testicular

hyaluronidase (Sigma) The cross-linked collagen polymer

in the residue was solubilized by digestion with pepsin

(100 lgÆmL)1in 3% acetic acid) Followingpartial

purifi-cation of type II collagen by 1.2MNaCl precipitation from

3% acetic acid, the a1(II) collagen chains were purified to

homogeneity by C8 reverse-phase HPLC, monitoring

absorbance (at 220 nm) and fluorescence (excitation

297 nm, emission 396 nm) Type II collagen from rat

cartilage was run as a control

Peptide analysis

Purified a1(II) collagen chains from the cell layer and

control tissue were digested with cyanogen bromide in 70%

formic acid The resultingpeptides were fractionated by

molecular sieve HPLC, monitoringabsorbance (at 220 nm)

and fluorescence (excitation 330 nm, emission 396 nm)

[4,8]

quantified by C-18 reverse-phase HPLC (excitation 297 nm, emission 396 nm) [27,28]

Gel electrophoresis and Western blotting Collagen chains and chain fragments were resolved by PAGE [29] and stainingwith Coomassie Blue, or by PAGE and transfer to a poly(vinylidene difluoride) membrane and probingwith monoclonal antibody (mAb) 10F2 (1 : 1000 dilution) The mAb 10F2 is one of several mAbs raised against protease-generated neoepitopes in the collagen a1(II) C-telopeptide It recognizes a cleavage-site (neoepi-tope) in a sequence within the C-telopeptide cross-linking domain of type II collagen [30] This antibody can detect the C-telopeptides of type II collagen (even as short fragments) when cross-linked to collagen triple-helical domains A polyclonal antibody to type IX collagen [31] was used to probe for a3(IX) chains Biotin-labeled goat anti-mouse IgG (Jackson) was used as the secondary antibody and streptavidin-alkaline phosphatase (Sigma) was used for detection

[3H]Proline-labeled proteins were visualized by graphy after gel electrophoresis using Amplify fluoro-graphic reagent (Amersham Pharmacia Biotech) and Biomax MS X-ray film (Kodak)

Electron microscopy The RCS-LTC cell line and chondrocytes from the Swarm rat chondrosarcoma parental tumor, which synthesize type

II collagen [32], were cultured in micromass or in high-density monolayers on Thermonox tissue cover slips (Nalge Laboratories) for 12–14 days The cultures were fixed for

1 h at room temperature in 2% glutaraldehyde, 2% paraformaldehyde in 0.1M sodium cacodylate buffer,

pH 7.4 Post-fixation was carried out using2% osmium tetraoxide in 0.2Mcacodylate buffer, pH 7.4 The cultures were then stained en bloc with 1.25% aqueous uranyl acetate, dehydrated and embedded in plastic Ultrathin sections (60–70 nm) were cut perpendicular to the plane of culture, placed on 300-mesh copper grids and stained with Reynold’s lead citrate (4.4% lead nitrate, 5.9% sodium citrate in distilled water, pH 12.0), and 2.5% uranyl acetate

in 50% ethanol All sections were examined and photo-graphed on a JEOL 100 CX transmission electron micro-scope

Results

Transmission electron microscopy of the cell layer iden-tified cells that retained the organelles and morphology typical of chondrocytes (Fig 1) In Fig 1A, the RCS-LTC cells show oval-shaped mitochondria, a prominent nucleus, abundant rough endoplasmic reticulum and Golgi vacuoles, indicating active synthesis and secretion

of proteins The extracellular matrix, however, featured extensive electron-lucent areas and a distinctive lack of

electron-dense material and collagen fibrils Linear arrays

of extremely thin filaments (<10 nm) were observed in

the extracellular matrix (Fig 1A) This contrasted with

the more abundant network of thin fibrils (17–20 nm),

typical of developingcartilage, surroundingthe

chondro-cytes derived from the cultured parent Swarm rat

chondrosarcoma tumor cells (Fig 1B)

The results of radiolabelingwith [3H]proline showed that the cultured RCS-LTC cells continued actively to synthesize and incorporate type II N-procollagen, and types IX and XI collagens into the matrix, even after 1 month in culture (Fig 2) No fully processed type II collagen chains were detected in the medium (Fig 2, lane 1) or cell/matrix layer (Fig 2, lane 2) Digestion of the cell layer collagen with

A

B

250 nm

250 nm

Fig 1 Transmission electron-microscopy of cultured chondrosarcoma cells and surrounding matrix (A) RCS-LTC chondrocyte Note the presence of thin filamentous fibrils pericellularly (arrowhead) Bar ¼ 1 lm (B) Parent Swarm rat chondrosarcoma chondrocyte Abundant, thicker collagen fibrils (arrowhead) are seen Bar ¼ 1 lm The insets show a higher magnification (digital) of the area enclosed within each box (bar ¼ 250 nm).

pepsin removed the N-propeptides from the a1(II) chains

(Fig 2, lane 3)

No fully processed type II collagen a-chains were detected

at any time in the RCS-LTC cell cultures (Fig 3, lanes 1 and

2) In the presence of bAPN for 1 month, the amount of

type II N-procollagen extractable in 0.15M potassium

phosphate was increased (Fig 3, lane 2) The pepsin extract

of the residual collagen contained less type II collagen

(Fig 3, lane 4) than that of the non-bAPN treated cultures

(Fig 3, lane 3) These results are consistent with the

formation of lysyl oxidase-mediated cross-links in the

polymeric type II N-procollagen

Havingestablished that only unprocessed type II

N-procollagen was deposited in the RCS-LTC cultures, the

cross-linkingproperties of the collagen were analyzed Type

II collagen was extracted with pepsin and component a1(II)

chains were purified by RP-HPLC (Fig 4) A peak of

fluorescence, characteristic of trivalent pyridinolines,

coin-cided with the a1(II) chains from control rat cartilage and

from the RCS-LTC matrix The elution position of the

a-chains was confirmed by SDS/PAGE (Fig 4, inset)

Fractions containingthe a1(II) chains from control tissue

and RCS-LTC cultures were hydrolyzed and analyzed by

C-18 reverse-phase HPLC for HP and LP, the two forms of

pyridinoline

As seen in Fig 5, both HP and LP were present in the

type II collagen deposited in the matrix by 1 week in culture

With increasingtime in culture, the total pyridinoline content increased from 0.06 molÆmole)1 of collagen (1 week) to 0.13 molÆmole)1 of collagen (4 weeks) In comparison, the concentration in rat cartilage type II collagen was 0.24 mol pyridinoline per mol of collagen Figure 6A compares the molecular sieve HPLC pat-terns of cyanogen bromide-derived peptides from type II collagen of the RCS-LTC cell layer (4 weeks in culture) and control rat cartilage monitored for pyridinoline fluorescence Two peaks of fluorescent peptide of similar yield were evident for both They correspond to peptides from the two cross-linkingsites in the type II collagen molecule that have been described previously [4,8] The results indicate that type II N-procollagen molecules are polymerized and cross-linked as in fully processed type II collagen Direct assay for pyridinolines in the hydrolyzed fractions confirmed the presence of HP and LP residues (Fig 6B)

To determine whether types IX and XI collagens, which are also synthesized by these cells [19], can copolymerize with the type II N-procollagen polymer, the collagen network laid down by the cells after 2 weeks

in culture was depolymerized usingpepsin Pepsin cleaves

in the telopeptide domains of type II collagen and in the noncollagenous domains of the minor collagens, type XI and IX, but leaves their triple helical domains intact The short stubs of cleaved telopeptides remain cross-linked to

Fig 2 Type II N-procollagen synthesized by RCS-LTC cells after

1 month in culture SDS/PAGE/fluorography of [ 3 H]proline-labeled

protein No fully processed a1(II) chains were detected in either the

medium (lane 1) or cell layer (lane 2) Lane 3: pepsin treatment of the

cell layer collagen converted the type II N-procollagen molecules to

fully processed a1(II) chains All samples were run under nonreducing

conditions Lanes 1 and 2, 10 nCiÆlane)1; lane 3, 5 nCiÆlane)1.

Fig 3 Effect of beta-aminoproprionitrile (bAPN) on the extractability

of type II collagen from the cell layer No fully processed type II col-lagen molecules are detected with or without bAPN in the RCS-LTC cultures (lanes 1 and 2) The yield of soluble type II N-procollagen was less from cultures in the absence of bAPN (lane 1) Addition of bAPN (lane 2) increased the pool of type II N-procollagen extractable in 0.15 M KH 2 PO 4 , pH 7.6, presumably by inhibitinglysyl oxidase-mediated cross-linking Pepsin-extracted type II collagen from untreated (lane 3) and bAPN-treated (lane 4) cultures Intact disulfide-bonded type IX collagen chains were identified by MS after in-gel trypsin digestion Equal volumes were loaded for each extract and run under nonreducingconditions.

the triple-helical sites to which they were attached in the

matrix, and the mAb 10F2 detects the pepsin-generated

neoepitope in the a1(II) C-telopeptide wherever it is

cross-linked to intact collagen chains or to chain

fragments The various chains and chain fragments of

types II, IX and XI collagen were resolved by SDS/

PAGE (Fig 7A, lanes 4 and 5) Western blot analysis

(Fig 7A, lanes 1 and 2) of the proteins resolved in lanes

4 and 5 usingmAb 10F2 showed a strongreaction with

the a1(II) chain, as expected for the derivative of a

cross-linked type II collagen polymer [C-telopeptide of type II

collagen cross-linked to residue 87 of the a1(II) collagen

chain] The antibody also reacted with the a1(XI) chains,

indicatingthat some of these chains had been

cross-linked to the C-telopeptide of the a1(II) chain and/or the

a3(XI) chain of type XI collagen, as they are the product

of the same g ene, Col2A1 A third immunoreactive band

is evident only after reduction with dithiothreitol (lanes 1

and 2) This band, from its properties on SDS/PAGE (lanes 4 and 5) and reaction with a polyclonal antibody

to type IX collagen [31] (lane 3), is a3(IX), in which the COL2 domain is cross-linked to a cleaved C-telopeptide from type II collagen

Discussion

The rat chondrosarcoma cell line, RCS-LTC, expresses type

II collagen abundantly, but fails to process it beyond the stage of N-procollagen molecules [24] This presents a novel system for studyingwhether chondrocytes can assemble newly synthesized type II N-procollagen molecules into a cross-linked fibrillar network, and whether types IX and XI collagen molecules can be incorporated and cross-linked into the nascent fibril Yang et al [15] have shown, by fibril-formingassays in vitro, that vitreous type II N-procollagen

Fig 4 Purification of pepsin-solubilized type II collagen a-chains from

the cell layer of RCS-LTC cultures Pepsin-solubilized type II collagen

from rat cartilage (upper panel) was resolved by reverse-phase HPLC

as a control for comparison with the RCS-LTC digest (lower panel).

Fractions marked by a bar contained a1(II) chains, as shown by SDS/

PAGE (inset), and were pooled for cross-link analysis.

Fig 5 Detection of pyridinoline cross-links in isolated a1(II) collagen chains Reverse-phase HPLC analysis of the a1(II) chains from 1-week and 1-month RCS-LTC cultures (lower panels) contain both hyd-roxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) cross-links Control type II collagen prepared from rat cartilage contains only HP cross-links (upper panel).

are deposited in an extracellular matrix by the RCS-LTC cells and become cross-linked by the lysyl oxidase mechanism Despite the retained N-propeptides, cross-linking also progressed to the stage of mature pyridin-oline residues by 1 week in culture The best explanation for this is that the procollagen molecules were assembled into microfibrils with the precise molecular stagger and proximities required for complex cross-links to form, even at this early stage of fibril formation (Fig 5) It has been reported that the mature vitreous contains a mix of types IIA and IIB N-procollagens [15,17,18] in its gel-like matrix [18,33]

HP is the predominant cross-link in type II collagen of normal rat cartilage The significant proportion of LP present at the two cross-linkingsites in the RCS-LTC type

II collagen molecule indicates an under-hydroxylation of lysine residues at these two triple-helical cross-linking residues The rapid cell doublingtime of 21 h, and the high rate of synthesis of collagen [19], may be factors contribu-tingto this under-hydroxylation An under-hydroxylation

of cross-links, compared with the equivalent tissue collagen, has been observed for type I collagen synthesized by primary chick osteoblasts in culture [34] In contrast, over-hydroxylation has been observed in type I collagen synthesized by the SAOS-2 cell line in culture, and linked

to an over-expression of lysyl hydroxylase 1 [35] It is unknown whether the presence of LP in place of HP confers any distinctive properties on the collagen fibril Despite the presence of mature pyridinoline cross-links, usually associ-ated with stiff, resilient connective tissues, the matrix was a highly hydrated gel in texture [19], similar in gross appear-ance to vitreous humor of the eye As aggrecan is also deposited in the matrix in large amounts by these cells [20], the fine filamentous collagenous network was presumably distended by the osmotic swellingpressure of entrapped aggrecan

The N-propeptide of type IIB collagen is essentially a short triple-helical domain that folds back onto the N-terminus of the main triple-helix, and so exposes the N-propeptidase cleavage site [36,37] Its presence could sterically interfere with the cross-linkinginteractions of the adjacent N-telopeptide domain The RCS-LTC cell line provides a useful model for studyingwhether this occurs,

as the cells deposit only type II N-procollagen (no fully processed molecules) and the N-propeptide appears to be folded correctly as it is cleaved by conditioned medium from normal chondrocytes [24] and by ADAMTS-2 (the known fibrillar collagen N-propeptidase) and ADAMTS-3 (the putative collagen N-propeptidase of cartilage) [25] The present results indicate that mature trivalent pyridinoline residues are formed in equal amounts at both ends of the molecule, where they link two C-telopeptides to residue 87 and two N-telopeptides to residue 930 Control rat cartilage showed a similar result (Fig 6) This implies that the N-propeptide of type IIB N-procollagen does not sterically interfere with aldehyde formation by lysyl oxidase or the linkage of the N-telopeptide to helical residue 930 and

Fig 6 Analysis of collagen type II cyanogen bromide-derived peptides.

(A) Chromatogram of cyanogen bromide-derived peptides from

digestion of type II collagen purified from rat cartilage (upper panel)

and the RCS-LTC matrix (lower panel), as resolved by molecular sieve

HPLC Pyridinoline fluorescence is detected in peptides that are

derived from the two cross-linkingsites in type II collagen CB12 X

(C-telo) 2 and CB9,7 X (N-telo) 2 (B) Reverse-phase HPLC analysis

confirmingthat hydroxylysyl pyridinoline (HP) and lysyl pyridinoline

(LP) are responsible for the fluorescence in the cross-linked cyanogen

bromide-derived peptides from RCS-LTC type II collagen.

Fig 7 Western blot analysis showing intertype cross-linking between

collagens II, IX and XI (A) Pepsin-solubilized RCS-LTC matrix

col-lagens [in the presence and absence of dithiothreitol (lanes 1 and 2,

respectively)] were probed usingmAb 10F2, which specifically

recog-nizes the C-telopeptide domain of type II collagen Lanes 4 and 5 show

Coomassie Blue-stained samples equivalent to those in lanes 1 and 2.

Lane 3 shows a Western blot of a sample similar to that in lane 1 but

probed with an antibody to type IX collagen (B) Molecular

inter-pretation of the results of Western blot analysis Antibody 10F2

reacted with the a1(II), a1(XI) and a3(IX) chains, showingthat

C-telopeptide domains of type II collagen had become cross-linked to

type II, XI and IX collagen molecules in the matrix These heterotypic

cross-linkingreactions have all been demonstrated in the collag en

heteropolymers that form the matrix of developingcartilage in vivo

[2,3].

subsequent interaction with a second N-telopeptide to form

pyridinoline Similarly, the cross-linkingof C-telopeptides

to helical residue 87 also proceeded to pyridinoline As

electron micrographs of the matrix showed no obvious

collagen fibrils, only fine filaments (Fig 1), we can speculate

that the retained N-propeptides had prevented lateral

growth of the nascent type II N-procollagen assembly

beyond a limitingsize Immunolocalization of

N-propep-tides to thin fibrils of skin type I N-procollagen, but not to

thick fibrils [38], supports this speculation It is probable

that type II N-procollagen forms fine fibrils in developing

cartilage, but this is more obvious with the RCS-LTC cells

because none of the type II N-procollagen is processed

This system demonstrated also the incorporation of

collagens IX and XI into the collagen II heteropolymer that

characterizes developingcartilage [39], as an early, integral

process The results of Western blottingwith antibody 10F2

establish that type II collagen molecules are covalently

linked to both types IX and XI collagens in the forming

matrix (Fig 7) This antibody specifically recognizes a

protease-generated cleavage site in the cleaved C-telopeptide

domain of type II collagen when it is cross-linked to

triple-helical sequences [40] Hence, on electrophoresis of a pepsin

digest of cell layer collagen, the a1(II) chain is heavily

stained, but the a3(IX) COL2 domain and the a1(XI) chain

are also recognized strongly by the antibody These results

are consistent with the known cross-linkingproperties and

chain-specific interactions of the collagen II C-telopeptide

domain with collagens IX and XI in cartilage (Fig 7B) [2,3]

Cross-linkingof the minor collagens, IX and XI, to the type

II N-procollagen polymer at 2 weeks and a nascent stage of

fibril growth, supports the early integration of types IX and

XI collagens during collagen II fibrillogenesis

From the present data we can speculate that the type II

N-procollagen heteropolymer is assembled by RCS-LTC

cells, duringor soon after secretion, in the form of a

microfibril The concept of a microfibril was introduced by

Smith [41] In the Smith microfibril, a unit of five type I

collagen molecules, staggered by 67 nm (234 amino acids),

repeats to form a microfibril of 4-nm diameter [42–46] For

type II collagen it was further speculated that such

microfibrils associate laterally with the minor cartilage

collagen molecules (types IX and XI) [47] On theoretical

grounds it was concluded that type II N-procollagen can

probably form such a microfiber, but that further lateral

growth will be sterically hindered by the N-propeptides [45]

Although microfibrils of 4-nm diameter have not been

visualized convincingly, the concept is consistent with

electron microscopic findings on the RCS-LTC extracellular

matrix, showingthin filaments in an otherwise amorphous

background (Fig 1)

In summary, the findings support the integration and

intermolecular cross-linkingof type II N-procollagen with

types IX and XI collagen molecules at an early stage in the

process of collagen network formation by chondrocytes

Acknowledgements

The authors thank Kae Ellingsen for help in preparing the manuscript

and Tom Eykemans for his expertise with the graphics This work was

supported by grants AR37318, AR36794 and AR39239 from the

National Institutes of Health.

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