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& p.1:Abstract Cultures of isolated osteocytes may offer anappropriate system to study osteocyte function, since iso-lated osteocytes in culture behave very much like osteo-

cytes in vivo. In this paper we studied the capacity of os-teocytes to change their surrounding extracellular matrixby production of matrix proteins. With an immunocyto-chemical method we determined the presence of colla-gen type I, fibronectin, osteocalcin, osteopontin and os-teonectin in cultures of isolated chicken osteocytes, os-teoblasts and periosteal fibroblasts. In osteoblast and pe-riosteal fibroblast cultures, large extracellular networksof collagen type I and fibronectin were formed, but in os-teocyte populations, extracellular threads of collagen orfibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopon-tin in the Golgi apparatus, on the other hand, was highestin the osteocyte population. These results show that os-teocytes have the ability to alter the composition of theirsurrounding extracellular matrix by producing matrixproteins. We suggest this property is of importance forthe regulation of the calcification of the bone matrix im-mediately surrounding the cells. More importantly, as os-teocytes depend for their role as mechanosensor cells ontheir interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their re-sponse to mechanical loading.& bdy:

Introduction

The development of a monoclonal antibody (mAb) di-rected to antigenic sites only present on the cell surfaceof osteocytes (mAb OB7.3; Nijweide and Mulder 1986)has made it possible to identify osteocytes in mixed pop-ulations of cells isolated from chicken bone. The anti-

body has also been used to isolate osteocytes from mixedcalvarial cell populations by applying an immunomag-netic isolation procedure (van der Plas and Nijweide

1992). More recently this method has been used to studysome characteristics and properties of osteocytes in cul-ture (van der Plas et al. 1994): isolated osteocytes whenseeded on a culture substratum formed networks of cellsinterconnected with each other via long, slender cell pro-cesses; osteocytes appeared not to be able to proliferate;alkaline phosphatase activity per cell was variable be-tween the individual cells but their mean activity waslow compared to that of osteoblasts. The conclusionfrom these experiments was that isolated osteocytes be-have in culture, as far as we know at the moment, verymuch like osteocytes in vivo, and that therefore culturesof isolated osteocytes may offer an appropiate system tostudy osteocyte function.

At present little is known about the function of osteo-cytes in bone metabolism (Aarden et al. 1994). A numberof decades ago it was thought that osteocytes were in-volved in blood calcium homeostasis and were capable of local bone resorption, osteocytic osteolysis (Blanger1969). Later, this idea of osteocytic osteolysis was refuted(Parfitt 1977; Boyde 1980; Marotti et al. 1990). Indeed,we observed no sign at all of any resorptive activity whenisolated osteocytes were seeded onto dentine slices (vander Plas et al. 1994). The possibility remains, however,that osteocytes are involved in facilitation of calcium andphosphate exchange between blood and bone, therebycontributing to the fine regulation of blood calcium ho-meostasis (Bonucci 1990; Aarden et al. 1994). A secondpossible function of osteocytes is a role in the maturationof osteoid matrix and regulation of its calcification. Ma-trix mineralisation generally takes place some distancefrom the osteoblastic layer, i.e. in the matrix between re-cently incorporated, osteoid osteocytes. It is feasible thatosteocytes, by secreting (non-collagenous) matrix pro-teins into the matrix around them, prepare or adapt thatmatrix for mineralisation (Mikuni-Takagaki et al. 1995).A third possible role for osteocytes is as mechanosensorycells in the regulation of the process of functional adapta-

E.M. Aarden (u ) A.-M.M. Wassenaar M.J. AlblasP.J. NijweideLaboratory of Cell Biology and Histology,Department of Cell Biology, Faculty of Medicine,Leiden University, PO Box 2156, NL-2301 CD Leiden,The NetherlandsTel. +31 71-5276415; Fax +31 71-5276437& /fn-bloc k:

Histochem Cell Biol (1996) 106:495501 Springer-Verlag 1996

O R I G I N A L PA P E R

& role s:Elisabeth M. Aarden Anne-Marie M. WassenaarMarcel J. Alblas Peter J. Nijweide

Immunocytochemical demonstrationof extracellular matrix proteins in isolated osteocytes

& misc :Accepted: 9 July 1996

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tion to mechanical stress (Lanyon 1993; Aarden et al.1994). The notion that the mechanical function of bonedetermines to a large extent skeletal design and tissue or-ganisation has become widely accepted (Rubin et al.1990). Osteoblasts and osteoclasts are clearly responsiblefor the execution of the necessary adaptation to mechani-cal stress, but the identity of the sensor cell, the cell thattranslates mechanical stimuli into chemical messages

which activate the (re)modelling system (osteoblasts, os-teoclasts), is not known. It is a very attractive idea that os-teocytes play such a role as mechanosensory cells andsupporting evidence for this notion is accumulating (seefor review Aarden et al. 1994). Indeed, we have recentlyacquired experimental evidence that isolated osteocytesare extremely sensitive to mechanical stress (Klein Nu-lend et al. 1995). If osteocytes are the mechanosensorycells of bone, the composition of the matrix around theosteocytes is of extreme importance. Osteocytes have tobe anchored to the matrix in order to perceive strain. Thisanchorage probably takes place via bone extracellularmatrix (ECM) proteins. The nature and the number of an-

choring sites may determine the sensitivity of the cells forstrain and the signal transduction system that translatesstrain into intra- or extracellular signal molecules. Finally,whatever the function of the osteocytes, they have to beactively involved in the adaptation of the matrix directlyaround them for a more general reason. Osteocytes aredependent on the diffusion of oxygen, ions, nutrients,waste products and probably chemical signal moleculesthrough the lacunar and canalicular spaces. They have tobe able to stop the mineralisation front at some distancefrom the cell membrane in order to keep an open space inwhich diffusion is possible.

Involvement of osteocytes in matrix adaptation by pro-

duction of ECM proteins is feasible. From immunohisto-chemical and in situ hybridisation studies on human, ratand porcine bone sections, it is known that ECM proteinsand/or ECM protein mRNA are present in osteocytes, suchas osteocalcin protein (Bronckers et al. 1985; Vermeulen etal. 1989; Boivin et al. 1990) and osteocalcin mRNA (Ikedaet al. 1992), osteopontin protein (Mark et al. 1987; Chen etal. 1991; 1993a) and osteopontin mRNA (Arai et al. 1993;Chen et al. 1993b, 1994), and osteonectin protein (Chen etal. 1991, 1993a) and osteonectin mRNA (Bianco et al.1985; Metsranta et al. 1989). Of special interest is that os-teopontin and bone sialoprotein, besides being concentrat-ed in cement lines and lamina limitans, are also particular-ly present in the perilacunar matrix of some osteocytes (In-gram et al. 1993; McKee et al. 1993).

In the present paper we have studied the production of ECM proteins by isolated chicken osteocytes and com-pared isolated osteocytes with osteoblasts and fibroblastsin this respect. With immunocytochemical methods wehave determined the presence of several proteins in andaround osteocytes: the predominant bone matrix proteincollagen type I (COLL I), proteins that may be involvedin the regulation of mineralisation, such as osteocalcin(OC; Hauschka et al. 1975; Price et al. 1976) and os-teonectin (ON; Termine et al. 1981) and proteins that are

probably involved in cell attachment, such as fibronectin(FN; Hynes and Yamada 1982) and osteopontin (OP;Oldberg et al. 1986). A difference in the matrix aroundosteocytes, when compared to that surrounding osteo-blasts and periosteal fibroblasts, may reflect a differentrelationship and/or adhesion of osteocytes to their sur-rounding matrix and hence be important in the light of their proposed mechanosensory function.

Materials and methods

Isolation of bone cells

Bone cell populations were isolated from 18-day-old foetal chick-en calvariae according to van der Plas and Nijweide (1992). Inshort, calvariae were dissected and the outer fibrous layers werestripped off; from these outer layers periosteal fibroblasts wereisolated with collagenase (245 min; 1 mg/ml crude collagenasetype 1, Sigma, St. Louis, USA) in HEPES buffer (Hefley 1987);the calvariae were submitted to subsequent treatments with 1mg/ml collagenase (10 and 45 min), 4 mM EDTA in phosphate-buffered saline (PBS; 10 min) and collagenase (45 min); the three

last fractions were combined, washed and seeded for culture, thispopulation, a mixture of osteocytes (stellate, mAb OB7.3-positivecells), osteoblasts (roundish or oval, mAb OB7.3-negative cells)and a small number of fibroblasts (elongated, spindle shaped,mAb OB7.3-negative cells) was designated OBmix. From theOBmix population the osteocytes were isolated by an immunomag-netic procedure (van der Plas and Nijweide 1992). Briefly, 1-day-cultured OBmix were harvested with a mixture of 0.05% trypsinand 0.01% EDTA in PBS; the suspension of cells was filteredthrough a 30-m pore nylon filter; after centrifugation the pelletwas resuspended in PBS and subsequently immunomagnetic beads(Dynabeads M-450 coated with sheep anti-mouse IgG1; Dynal, N-0212, Oslo, Norway), coupled to the osteocyte-specific mAbOB7.3 (3 g/ml IgG to 3 mg/ml beads), were added; osteocyteswere allowed to couple to immunolabelled beads for 5 min; osteo-cytes bound to the beads were removed from the cell suspensionwith a magnet, and subsequently detached from the beads with an

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Fig. 1ah The presence of extracellular matrix proteins (ECM)proteins in cultures of osteocytes, osteoblasts and periosteal fibro-blasts.a Extracellular collagen type I network (red ) formed be-tween periosteal fibroblasts after 3 days of culture (magnification365).b Intracellular (orange ) collagen type I in an osteocyte andextracellular (red ) fibres of collagen after 1 day of culture. The os-teocyte is identified with monoclonal antibody (mAb) OB7.3(green ). The Golgi apparatus and the secretory vesicles are stainedorange because of the green fluorescence on top of the red fluores-cence (magnification 1400).c Fibronection network (green ) pro-duced by osteoblasts after 3 days of culturing of mixed cells(OBmix) (magnification 300).d Fibronectin staining ( yellowwhen on thered , mAb OB7.3-positive osteocyte andgreen outsidethe cell profile) on and around an osteocyte and an osteoblast(OBmix after 1 day; magnification 1300).e Identification of os-teopontin ( yellow ) in osteocytes (green ) within the Golgi appara-tus and secretory vesicles (culture of osteocyte population (OCY)for 3 days; magnification 1300).f Extracellularly, osteopontin(red ) shows a collagen-like fibre orientation in OBmix culture (1day; magnification 520).g Osteocalcin ( yellow/red ) in the Golgiapparatus and secretory vesicles of two osteocytes (green ) and oneosteoblast (culture of OCY after 4 days; magnification 570).hIntracellular osteonectin (red ) distribution in the Golgi apparatusand secretory vesicles of OBmix cultured for 3 days (magnification520). Nuclei are stainedblue (4 ,6-diamidino-2-phenylindole) inall cell cultures. ( Asterisk Golgi apparatus,arrow secretory vesi-cle)& /fig.c :

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excess of mAb OB7.3 (250 g/ml) in PBS; this final cell popula-tion consisted of more than 95% osteocytes (van der Plas and Ni- jweide 1992) and was called the osteocyte population (OCY).

Cell culture and immunocytochemistry

The cell populations periosteal fibroblasts, OBmix and OCY werecultured on glass coverslips in MEM (minimal essential medium;Gibco Life Technologies, Gaithersburg, USA) supplemented with2% foetal calf serum (Gibco). After 16 days of culture, the cellswere washed, twice with Hanks Balanced Salt Solution (HBSS)and once with 0.25% bovine serum albumin (BSA) in HBSS(HBSA). For immunocytochemistry with the antibody against OC,1% foetal calf serum in HBSS was used instead of HBSA becauseof the cross-reactivity of the antibody with some batches of BSA.After removal of the culture medium and washing, the cells wereincubated with mAb OB7.3 for 30 min. Again, the cells werewashed with HBSA and HBSS and subsequently fixed in 4%formaldehyde in HBSS for 10 min at 4 C. Washing followed,with PBS for extracellular labelling of proteins or with 0.1% Tri-ton X-100 in PBS (10 min at 0 C) for both extra- and intracellularstaining. For the identification of the mAb OB7.3-labelled osteo-cytes, cell preparations were incubated with biotinylated horse an-ti-mouse antibody (15 g/ml; Vector Laboratories, Burlingame,USA) and subsequently with fluoresceince isothiocyanate (FITC)-

conjugated extravidine (green fluorescence; 20 g/ml, Sigma) orwith Cy3-conjugated donkey anti-mouse antibody (red fluores-cence; 3 g/ml, Jackson Immuno Research Laboratories, WestGrove, USA). The antibodies used for the demonstration of thevarious matrix proteins were: rabbit anti-chicken COLL I (Chem-icon, Temecula, USA; working concentration 1/500); rabbit anti-chicken OC (gift of J. Lian; see Hauschka et al. 1983; concentra-tion 1/200); rabbit anti-chicken OP (gift of Y. Gotoh; see Gersten-feld et al. 1990; Gotoh et al. 1990; concentration 1/300); rabbit an-ti-chicken bone ON (LF8; gift of L. Fisher; see Pacifici et al.1990; concentration 1/300); goat anti-chicken FN (gift of D.R.Garrod; see Roach 1992; concentration 1/1000). Incubation withthese primary antibodies was followed, after thorough washing, byincubation with an appropriate secondary antibody, either donkeyanti-rabbit conjugated to Cy3 (3 g/ml, Jackson) or FITC-conju-gated rabbit anti-goat (5 g/ml, Sigma). Finally, nuclei were

counterstained with 4,6-diamidino-2-phenylindole.2 HCl (DAPI;60 ng/ml; Serva, Heidelberg, Germany). As controls, cell popula-

tions were stained according to the procedures described above,leaving out the primary antibody.

Additionally, the number of cells positive for OP, OC and ONintracellularly was counted. Osteocytes, osteoblasts and periostealfibroblasts were identified as described above in OCY, OBmix andperiosteal fibroblast populations, respectively, and the percentageof positive cells was determined.

Results

Immunocytochemically, the presence of COLL I, FN,OC, OP and ON could be demonstrated in the three cell

types investigated: osteocytes (i.e. mAb OB7.3-positivecells in OBmix and OCY populations), osteoblasts (i.e.roundish or oval mAb OB7.3-negative cells in OBmix)and periosteal fibroblasts. Reactivity was heterogeneous,meaning that not all cells of each type were positive and,when positive, not all to the same extent. COLL I immu-noreactivity was seen as an extracellular network of wo-ven fibres between osteoblasts and periosteal fibroblasts(Fig. 1a). In OCY populations, such a network was onlyvisible in the immediate vicinity of an occasional, con-taminating osteoblast. Around osteocytes, without neigh-bouring non-osteocytes, only a few collagen fibres couldbe observed (Fig. 1b). Intracellularly, collagen produc-tion was demonstrated as an intensive staining of theGolgi apparatus and of secretory vesicles in osteoblastsand periosteal fibroblasts (Fig. 2), and also in osteocytes

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Fig. 2 Simultaneous intra- and extracellular identification of col-lagen type I. After 6 days of culture of OBmix, an extracellular net-work can be stained around osteoblasts together with an intracellu-lar localisation in the Golgi apparatus and secretory vesicles of thecells (magnification 480)& /fig.c :

Table 1 Percentage of osteocytes, osteoblasts and periosteal fi-broblasts intracellularly positive for osteopontin (OP ), osteocalcin(OC ) and osteonectin (ON ). Percentages are expressed asmeansSD. The numbers of cultures are in parentheses. Osteo-cytes were identified as monoclonal antibody (mAb) OB7.3-po-

sitive cells in mixed cell (OBmix) and osteocyte populations, osteo-blasts as roundish or oval mAb OB7.3-negative cells in OBmix, andperiosteal fibroblasts as elongated, spindle shaped, mAb OB7.3-negative cells in periosteal fibroblasts. ( ECM ) Extracellular matrixprotein)& /t bl.c :& t bl.b:

ECM protein identified Osteocytes Osteoblasts Periosteal fibroblasts

OP 65.912.8 (8) 17.86.7 (8) 12.43.9 (4)OC 83.87.4 (5) 24.85.8 (5) 0.81.1 (3)ON 76.55.8 (6) 23.412.7 (6) 33.09.8 (3)

& /t bl.b:

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(Fig. 1b). Osteoblasts (Fig. 1c) and fibroblasts in culturealso formed a web of FN, very similar to the COLL Inetwork. A typical staining pattern could be observed onand around individual osteoblasts (Fig. 1d) and fibro-blasts. On osteocytes, only little dots or very short fibro-nectin fibres could be detected (Fig. 1d). In all popula-tions only occasionally was the Golgi apparatus found tobe positive for FN (not shown).

OP, OC and ON staining showed very similar pat-terns. OP was found in the Golgi apparatus within os-teocytes (Fig. 1e), osteoblasts and periosteal fibro-blasts. Additionally, extracellular threads of anti-OP-positive material were found in OBmix (Fig. 1f) and pe-riosteal fibroblasts, resembling the COLL I and FN net-work orientation. Distinct reactivity for OC was alsofound intracellularly in the Golgi apparatus and vesi-cles of osteocytes, osteoblasts (Fig. 1g) and fibroblasts.ON too could be identified intracellularly in all types of cells, i.e. osteocytes, osteoblasts (Fig. 1h) and perios-teal fibroblasts. The number of osteocytes positive forOP, OC and ON intracellularly was higher than the

number of osteoblasts or periosteal fibroblasts (Table1).

Discussion

The extracellular matrix of bone consists of about 90%COLL I. The collagen fibres form by their many inter-and intramolecular cross-links the basic structural net-work in which mineralisation takes place. Non-collage-nous proteins comprise about 10% of the total bone pro-tein content. A small fraction of these proteins are of ex-tra-osseous origin, but the majority is produced and se-

creted by bone cells. Most of these ECM proteins areable to bind to COLL I, such as FN (Hynes and Yamada1982; Majmudar et al. 1991), OC (Bianco et al. 1985;Vermeulen et al. 1989; Boivin et al. 1990), ON (Termineet al. 1981) and OP (Chen et al. 1992). The precisefunctions of most of the non-collagenous proteins arenot known. The fact that they are deposited into bone atdifferent stages of bone formation may reflect their vari-ous functions. FN is produced during various phases of bone formation (Weiss and Reddi 1981) and clearly hasa function in cell-matrix adhesion (Yamada 1983;Grzesik and Gehron Robey 1994). OP is only incorpo-rated in the bone matrix prior to mineralisation (Mark etal. 1988; McKee et al. 1992; Sodek et al. 1992), and isprobably involved both in cell-matrix adhesion and inthe initiation of mineralisation (Roach 1994). ON isfound in newly laid down osteoid (Bianco et al. 1988)and, together with OC, it probably prevents excessivemineralisation (Roach 1994). OC is not present in oste-oid, only in calcified matrix (Groot et al. 1986). Its syn-thesis has been reported not to take place until aftermineralisation has occurred (McKee et al. 1992; Kasaiet al. 1994).

The production and calcification of bone matrix haslong been considered to be a primary function of the os-

teoblast. However, over the last few years, an increasingnumber of reports have also indicated that other cells inbone, notably osteocytes, may contribute to bone matrixsynthesis (see Introduction). In this paper we studied thepresence of the matrix proteins COLL I, FN, OP, ON andOC in and around isolated and cultured chicken osteo-cytes.

Osteocytes were shown to produce only occasional

COLL I fibres or FN threads, while osteoblasts and peri-osteal fibroblasts strongly secreted COLL I, as is shownby the large networks of collagen fibres that are formedaround these cells during culture. The anti-FN-positivenetworks around the osteoblasts and periosteal fibro-blasts, which are so similar to the COLL I networks,probably represent FN adherence to the collagen fibres.Therefore, the small amount of FN seen around osteo-cytes does not have to signify that osteocytes secrete lit-tle FN, but may be due to the small number of collagenfibres. Intracellularly, FN was less demonstrable in allthree cell types than COLL I, perhaps due to a faster se-cretion of FN compared to COLL I.

Besides COLL I (and to a lesser extent FN) OP, ONand OC could also be immunocytochemically demon-strated in the Golgi apparatus and secretory vesicles of periosteal fibroblasts, osteoblasts and osteocytes in cul-ture. OP, and to a lesser extent ON, were also found ex-tracellularly, in osteoblast and fibroblast, but not in os-teocyte cultures. On the other hand, the percentage of os-teocytes positive for OP, ON and OC intracellularly wasmuch higher than that of osteoblasts or periosteal fibro-blasts. This probably relates to the fact that the OCYpopulation is a homogeneous population of mature, dif-ferentiated cells, while the OBmix and periosteal fibro-blast populations contain a large variety of differention

stages, from actively secreting osteoblasts and fibroblaststo precursor stages that probably do not (yet) producemany matrix proteins.

These results suggest that osteocytes are capable of producing various matrix proteins, although probably toa limited extent as was explicitly shown for insolubleCOLL I. OCY cultures, such as those described here, al-low study of the possible effects of external factors suchas hormones, cytokines and mechanical loading on thematrix production profile of osteocytes in a systematicway. In particular, the study of the effects of mechanicalloading on matrix production by osteocytes will be of ex-treme interest considering their proposed role asmechanosensor cells (see Introduction). The recent pa-pers of Klein Nulend et al. (1995) and Sun et al. (1995)have demonstrated that isolated osteocytes are extremelysensitive to mechanical loading as they respond (in vivo)to mechanical stress with an increased COLL I mRNAcontent. The mechanism proposed here, by which osteo-cytes may regulate their response to mechanical loadingthrough regulation of matrix production and, maybe, theregulation of the attachment to these proteins, is nowopen for investigation.

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