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The osteocyte is the most abundant cell type of bone. There are approximately 10 times as many osteocytes as osteoblasts in adult human bone, and the number of osteoclasts is only a fraction of the number of osteoblasts. Our current knowledge of the role of osteocytes in bone metabolism is far behind our insight into the properties and functions of the osteoblasts and osteoclasts. However, the striking structural design of bone predicts an important role for osteocytes in determining bone structure. Over the past several years, the role of osteocytes as the professional mechanosensory cells of bone, and the lacunocanalicular porosity as the structure that mediates mechanosensing have become clear. Strain-derived flow of interstitial fluid through this porosity seems to mechanically activate the osteocytes, as well as ensure transport of cell signaling molecules, nutrients, and waste products. This concept explains local bone gain and loss--as well as remodeling in response to fatigue damage--as processes supervised by mechanosensitive osteocytes. Alignment during remodeling seems to occur as a result of the osteocyte's sensing different canalicular flow patterns around the cutting cone and reversal zone during loading, therefore determining the bone's structure.

Osteocytes are the most abundant cells in bone; however, relatively little is known about their properties and functions. The development of monoclonal antibody MAb OB7.3 directed against chicken osteocytes enabled us to purify osteocytes from enzymatically isolated bone cells. Cultures of purified osteocytes were used to gain better insight into the role of osteocytes in bone metabolism. Until now, the antigen of MAb OB7.3 has not been elucidated. In this study, we examined the antigen to which this osteocyte-specific antibody is directed. Immunoprecipitation and purification of the protein, followed by amino acid sequence analysis of two isolated peptides, revealed that the antigen has high homology to human and murine PHEX/Phex protein sequences (PHosphate-regulating gene with homology to Endopeptidases on the X chromosome). The OB7.3 antigen was therefore identified as chicken Phex protein. In addition, using suppression subtractive hybridization, we obtained a complementary DNA (cDNA) sequence of 502 base pairs (bp) with high homology to the human and murine PHEX/Phex genes. This method was applied to identify genes, which are differentially expressed in osteocytes compared with osteoblasts. The results also suggest that Phex is expressed at higher levels in chicken osteocytes compared with osteoblasts. Reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analyses supported these findings. The function of Phex is not completely understood. However, it is known that the gene is preferentially expressed in bone and that mutations in PHEX/Phex lead to X-linked hypophosphatemia and bone mineralization abnormalities. Our findings suggest that osteocytes play an important role in the Phex-regulated phosphate handling in the kidney and in bone.

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts., (Copyright 2000 Academic Press.)

To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2 (PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz), and PGE2 release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2 activity, arachidonic acid production, and ultimately PGE2 release.

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C2GM was shown to be strongly dependent on osteoblast density. In cocultures of C2GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 x 10(4) cells/cm2), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 x 10(4) cells/cm2). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear.

Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally differentiated osteoclasts. Osteoclast formation was analyzed in long-term cocultures of osteoblasts and nonadherent, osteoclast-depleted bone marrow cells. Osteoclast activity was studied in short-term (48 h) cultures of bone marrow cell populations enriched for osteoclasts, on dentine slices. SCF strongly enhanced osteoclast differentiation. The IL-6-related chicken myelomonocytic growth factor (cMGF) had a similar effect, and the effects of SCF and cMGF were additive. SCF, but not cMGF, also stimulated the bone-resorbing activity of existing osteoclasts. As osteoblasts have been found to regulate osteoclast activity and formation, chicken osteoblasts were tested for their ability to express and secrete SCF. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that osteoblasts express SCF mRNA and that parathyroid hormone increases expression levels about fourfold. SCF did not accumulate in the culture medium, but remained cell (osteoblasts) surface associated.

Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading.

It has been postulated that the transduction of mechanical stress signals to bone cells occurs via loading-dependent flow of interstitial fluid through the lacuno-canalicular network of bone. We have shown earlier that chicken osteocytes release enhanced amounts of prostaglandin E2 after 1 h treatment with pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz). Here we study the acute response to PFF on three cell populations derived from fetal chick calvariae, namely periosteal fibroblasts (PF), an osteoblast and osteocyte containing population (OBmix), and osteocytes (OCY), and the involvement of the actin-cytoskeleton in this process. All three cell populations rapidly (OCY: within 5 min, OBmix, PF: within 10 min) increased their release of prostaglandins E2 and I2 in response to PFF, but the response by OCY was 2-4 times higher than that by OBmix or PF. Disruption of the actin-cytoskeleton by cytochalasin B completely abolished the response. We conclude that osteocytes are more sensitive to fluid shear stress than immature bone cells, and that the actin-cytoskeleton is involved in the response to fluid flow.

Different functions have been proposed for osteocytes over time, but it is now generally accepted that their most important task lies in the sensing of strain caused by mechanical loading on bone. The fact that mechanical strain can be sensed as deformation of the extracellular matrix or as fluid shear stress along the cell, in the space between cell membrane and extracellular matrix, requires that osteocytes have close (specialized) contact with the bone matrix. We studied to which extracellular matrix proteins isolated chicken osteocytes adhere and whether this adhesion is mediated by specific cell adhesion receptors called integrins. The adhesive properties of the osteocytes were compared with that of osteoblasts. Osteocytes (and osteoblasts) adhere to the same substrates (i.e., collagen types I and II, collagen fibers, osteopontin, osteonectin, fibronectin, fibrinogen, thrombospondin, and laminin). Cell spreading varied between substrates, from all cells rounded on thrombospondin to all cells fully spread out on osteopontin, osteonectin, vitronectin, fibronectin, fibrinogen, and laminin. The percentage of osteocytes adhered was equivalent to that of osteoblasts adhered on all substrates except osteopontin and vitronectin, where osteocytes adhered less. The adhesion of osteocytes and osteoblasts to osteopontin, osteonectin, vitronectin, and fibrinogen was strongly inhibited, and to fibronectin and laminin moderately, by an RGD peptide. No RGD inhibition was found on collagen. An antibody against chicken integrin alpha v beta 3, the monoclonal antibody (MAb) 23C6, did not interfere with the adhesion of osteocytes and osteoblasts to matrix proteins, whereas an MAb against chicken integrin subunit beta 1 (CSAT) strongly inhibited adhesion to all substrates. Labeling with osteocyte-specific MAbs (OB7.3, OB37.4, and OB37.11) also did not hinder the adhesion of osteocytes to collagen type I, vitronectin, and osteopontin. Adhesion sites on osteocytes were small compared with the large adhesion plaques of osteoblasts, as demonstrated by interference reflection microscopy and immunocytochemically by staining for vinculin. Osteocyte adhesion is analogous to osteoblast adhesion with regard to the range of extracellular matrix proteins to which they adhere. The adhesion is mediated by the integrin subunit beta 1, but other integrins or nonintegrin adhesion receptors are also involved. Osteocytes make contact with the extracellular matrix via small attachment points which colocalize with vinculin. This connection between the bone matrix and the cytoskeleton may be important for osteocytic sensing of mechanical strain, as it supplies a transduction route of extracellular (mechanical) signals into intracellular messages.

Osteocytes are extremely sensitive to fluid shear stress, a phenomenon that may be related to mechanical adaptation of bone (FASEB J 9:441,1995). Here we examined the effect of pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz, 0.4 Pa/sec) on the release of NO, in relation with upregulation of prostaglandin E2 (PGE2). Chicken calvarial osteocytes, but not periosteal fibroblasts, as well as mouse calvarial cells responded to PFF with a rapid and transient 2 to 3-fold stimulation of NO release. The effect was maximal after 5 min and leveled off thereafter. PFF also stimulated PGE2 release. This effect was significant after 10 min and continued throughout 60 min PFF treatment. Inhibition of NO release by NG-monomethyl-L-arginine prevented the effect of PFF on NO as well as PGE2 release. These results suggest that NO is a mediator of mechanical effects in bone, leading to enhanced PGE2 release. They further strengthen the hypothesis that fluid flow through the osteocyte canalicular network provides the physical stimulus for mechanosensation in bone.

A voltage-dependent, fast, transient inward current was characterized in embryonic chicken osteoclasts using the permeabilized patch configuration of the patch-clamp technique. The current was activated by depolarizations to higher than -28 +/- 4 mV from a holding potential of -80 mV. It peaked within 1-1.5 ms, and inactivated within 3.3-6.9 ms. The 50% inactivation voltage was -59 +/- 6 mV with a steepness factor of 0.11 +/- 0.06. The current disappeared with the removal of extracellular Na+ and was reversibly blocked by tetrodotoxin (K0.5 < 15 nM) but not by verapamil (< or = 100 microM). We conclude that this new current in embryonic chicken osteoclasts is a sodium current known from excitable cells.

Although osteocytes are by far the most abundant cell type of bone, they are least understood in terms of function and regulation. Previous studies have concentrated on their possible role as mobilizers of bone calcium, via the process of osteocytic osteolysis. Currently, however, their possible involvement in mechanical adaptation, the process whereby bone tissue maintains maximal functional strength with minimal bone mass, is discussed. We have recently obtained experimental evidence that osteocytes are the mechanosensory cells of bone, involved in the transduction of mechanical loads into biochemical signals. Our results support the hypothesis that flow of fluid through the lacunar-canalicular system as a result of loading provides the physical signal that activates the cells.

It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Many or perhaps all cell types in the body possess extracellular binding sites for nucleosides and nucleotides, the purinoceptors. The binding sites that favor adenosine triphosphate (ATP) have been classified as P2-purinoceptors. One subclass of the P2-purinoceptors is the P2z-purinoceptor that mediates the permeabilizing effect of ATP4- (the fully ionized form of adenosine triphosphate). In the presence of millimolar concentrations of ATP4-, this receptor, which was found on all cells of hemopoietic origin but not on cells of stromal origin, renders the sensitive cells permeable for molecules up to 1 kD. This property has been used to eliminate cells of hemopoietic origin from mixed populations. Skeletal- and blood-forming tissues have a complex cellular composition of predominantly stromal and hemopoietic cells. The 2 cell types influence each others' formation, differentiation, and activities in a largely unclarified manner. Rigorous separation would help the study of the properties and potencies of the 2 cell types and their mutual interactions. A short treatment of cell populations isolated from bone, bone marrow, or cartilage with 2 mM adenosine triphosphate and 1 mM of cytotoxic, but not permeant, potassium thiocyanate obliterates all cells of hemopoietic origin, including macrophages, osteoclasts, and their progenitors from these populations.

It has been known for more than a century that bone tissue adapts to functional stress by changes in structure and mass. However, the mechanism by which stress is translated into cellular activities of bone formation and resorption is unknown. We studied the response of isolated osteocytes derived from embryonic chicken calvariae to intermittent hydrostatic compression as well as pulsating fluid flow, and compared their response to osteoblasts and periosteal fibroblasts. Osteocytes, but not osteoblasts or periosteal fibroblasts, reacted to 1 h pulsating fluid flow with a sustained release of prostaglandin E2. Intermittent hydrostatic compression stimulated prostaglandin production to a lesser extent: after 6 and 24 h in osteocytes and after 6 h in osteoblasts. These data provide evidence that osteocytes are the most mechanosensitive cells in bone involved in the transduction of mechanical stress into a biological response. The results support the hypothesis that stress on bone causes fluid flow in the lacunar-canalicular system, which stimulates the osteocytes to produce factors that regulate bone metabolism.

Many cell types respond to mechanical membrane perturbation with intracellular Ca2+ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretch-evoked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca2+ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca(2+)-activated K+ (KCa) channel activity, membrane potential changes and local and general intracellular Ca2+ ([Ca2+]i) increases. The model we propose is that during membrane stretch, both SA channels and KCa channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca2+. During membrane stretch, Ca2+ influx through SA channels and hyperpolarization due to KCa channel activity serve as positive feedback, leading ultimately to a Ca2+ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca2+ entry mechanism plays a role in regulation of osteoclast activity.

We succeeded in the isolation of osteocytes from parietal bones of 16-day-old chick embryos. Isolated osteocytes showed a typical stellate morphology. More than 95% of these cells reacted with the osteocyte-specific antibody OB 7.3. In culture osteocytes formed gap junctions with each other, as could be established by ACAS. Sixteen-millimeter time-lapse microcinematography of the cells also demonstrated the formation of intercellular connections and gap junctions, and portrayed the interaction between osteocytes and osteoclasts: osteocytes seemed to inhibit osteoclast activity. This cinematography also showed the ability of osteocytes to proliferate after they had been disconnected from each other. Thereafter these cells redifferentiated into osteoblasts that became embedded in bone matrix produced by themselves. These findings suggest that osteocytes might be involved in bone formation during remodeling.

The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (Isatp) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (Isata). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation.

Investigating the potentiality of cord monocytes to differentiate toward osteoclast-like cells (OCL) in vitro, we previously reported that in the presence of 1,25(OH)2 vitamin D3 (1,25-(OH)2D3), multinucleated-cells generated by cord monocyte cultures though displaying morphological features of OCL failed to resorb devitalized bones. We thus hypothesized that full differentiation of cord monocytes toward bone-resorbing cells may require the presence of factors released from and/or direct interactions with living osteogenic cells. In the present study, we tested these hypotheses using two culture systems supporting the development of bone-resorbing cells in the presence of bone matrix. First, cord mononuclear cells were co-cultured with murine fetal metatarsals depleted of osteoclast progenitor cells (stripped metatarsals) in the presence of 1,25-(OH)2D3. We found that cord mononuclear cells failed to differentiate toward OCL as indicated by the absence of the release of 45Ca previously incorporated in fetal bones and by the absence of formation of TRAP-positive (TRAP[+]) multinucleated cells which have invaded mineralized cartilage during the co-culture period. In the same model, we then investigated the effect of some soluble factors known as stimulators of osteoclast differentiation. Whereas exogenous rhIL6 and rhIL3 were ineffective in this assay, rhM-CSF consistently increased both the number of TRAP(+) multinucleated cells inside the mineralized cartilage and the release of 45Ca into the culture media. The effects of rhM-CSF were time-dependent reaching the maximum after 3 weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Although the osteocyte is the most abundant among the highly differentiated cells of mature bone (osteocytes, lining cells, osteoblasts, and osteoclasts), its properties and functions are the least known and understood. Here we isolated osteocytes from mixed populations of bone cells liberated from fetal chick calvariae by alternate treatments with collagenase and EDTA. The osteocytes were removed from the bone cell populations by binding them via an osteocyte-specific antibody (MAb OB 7.3) to magnetic beads and removing the beads together with the coupled osteocytes from the population using a magnet. Isolated osteocytes were found to be highly differentiated, postmitotic cells that required their typical stellate morphology in culture. Osteocyte populations had alkaline phosphatase (ALP) activity somewhat lower than that of the osteoblast-like cell populations from which they were separated by the immunodissection procedure. On the single-cell level, the ALP activity was highly variable. Parathyroid hormone (PTH) receptors were found to be present on osteocytes as well as on osteoblast-like cells, but not on fibroblast-like cells of the outer periosteum. In response to PTH, osteocytes increased their intracellular levels of cAMP, as did the osteoblast-like cells. Osteocytes appeared to be somewhat more sensitive to PTH than osteoblasts. When seeded onto dentin slices, osteocytes did not corrode the dentin surface to any appraisable degree. We therefore found no evidence to support the notion that osteocytes play a role in the calcium homeostasis through osteocytic osteolysis. Whether osteocytes play an important role in perceiving and transducing hormonal and/or mechanical stimuli remains open for future research.

Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5' triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.

Six murine macrophage precursor cell lines, thought to be arrested around the CFU-GM stage of the myeloid differentiation and shown to be negative for acid phosphatase, F4/80 antigen expression and phagocytosis capacity, were tested for their ability to differentiate into osteoclasts. Their differentiation potential was compared with that of the haemopoietic stem cell line FDCP-mix C2GM. None of the macrophage precursor cell lines could be induced to differentiate into osteoclasts when the cells were cocultured with either periosteum-free metatarsal bones of fetal mice, or monolayers of osteoblast-like cells. In contrast, when the haemopoietic stem cell line FDCP-mix C2GM, murine fetal liver cells or murine spleen cells were used as a source of haemopoietic precursor cells, numerous osteoclasts were formed in both culture systems. During cell culture a small percentage of the macrophage precursor cells attached to the bottom of the culture well. These firmly attached cells acquired acid phosphatase activity, F4/80 antigen expression and phagocytosis capacity. Furthermore, when the cell lines were cultured for 2 or 4 days with 1% DMSO, up to 30% of the precursor cells differentiated into metamyelocytes. These results suggest that the macrophage precursor cell lines are able to acquire macrophage and granulocyte characteristics, but are unable to differentiate into osteoclasts. In contrast, the haemopoietic stem cell line FDCP-mix C2GM is able to differentiate into both macrophages and osteoclasts. We therefore suggest that the osteoclast lineage branches off at an early stage of the myeloid differentiation pathway.

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction-coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body-containing lacunae with each other and with the outside world. During differentiation from osteoblasts to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are 1) osteocytes are actively involved in bone turnover; 2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and 3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations.

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3-4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5-6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1 +/- 4.2% (n = 12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9 +/- 5.7% (n = 17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is described that permits the removal of hematopoietic cells and macrophages from mouse bone marrow cultures. The method is based on the difference in effect of extracellular ATP4- ions (ATP in the absence of divalent, complexing cations) on cells of hematopoietic origin, including macrophages, and of nonhematopoietic origin, such as fibroblastlike stromal cells. In contrast to fibroblastlike cells, hematopoietic cells and macrophages form under the influence of ATP4- lesions in their plasma membranes, which allows the entrance of molecules such as ethidium bromide (EB) and potassium thiocyanate (KSCN), which normally do not easily cross the membrane. The lesions can be rapidly closed by the addition of Mg2+ to the incubation medium, leaving the EB or KSCN trapped in the cell. This method allows the selective introduction of cell-toxic substances such as KSCN into hematopoietic cells and macrophages. By using this method, fibroblastlike stromal cells can be isolated from mouse bone marrow cultures.

The present study evaluates the effect of parathyroid hormone (PTH) on intracellular calcium. Intracellular calcium ion concentrations ([Ca2+]i) in fetal rat osteoblasts in primary culture (ROB) and in UMR106-01 osteogenic sarcoma cells were monitored as changes in the ratio (R) of Fura-2 fluorescence intensities in single cells as well as populations of cells. In both single ROB and UMR106-01 cells, addition of 10(-7) M rat PTH1-34 and 3 NIH units/ml human thrombin resulted in heterogeneous responses in R values and therefore [Ca2+]i. PTH-induced calcium responsiveness of ROB was dependent on culture conditions, such that response frequencies were positively correlated with the percentage of fetal calf serum in the culture medium. PTH responsive ROB and UMR106-01 cells had significantly higher resting [Ca2+]i than unresponsive cells. PTH- or thrombin-mediated calcium signalling appeared not to be correlated to alkaline phosphatase activity in single ROB. Low percentages of cells responded to PTH in comparison to thrombin suggesting that an increase in [Ca2+]i is not a common PTH signalling pathway in osteoblasts in primary culture. Our data suggest that activation of this signalling pathway by PTH is culture condition dependent, possibly via a cell-cycle related increase in sensitivity of the pathway.

Calcium-activated potassium channels were found in embryonic chick osteoclasts using the patch-clamp technique. The activity of the channel was increased by both membrane depolarisation and an increase in intracellular Ca2+ concentration in the range 10(-5) to 10(-3) M. In the cell-attached-patch configuration the channel was only active at extreme depolarising potentials. Ca2+ addition to the cytoplasm via ionomycin increased channel activity at the resting membrane potential of the osteoclast. The channel had a single-channel conductance of 150 pS in the inside-out patch under symmetrical K+ conditions (150 mM) and was selective for potassium ions. During sustained application of increased [Ca2+] at the cytoplasmic side of inside-out patches, channel activity sometimes decreased again after the initial increases (desensitization). The results established the properties of the single channels underlying an outward rectifying K+ conductance in chick osteoclasts described previously by us.

Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch-activated channels in OCL locomotion.

Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.

An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1-2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone.

Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3)-dependent multipotent hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day-old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation.

Patch-clamp measurements done on excised membrane patches obtained from 1-5 day cultured embryonic chick osteoblasts, osteocytes, and periosteal fibroblasts revealed the existence of a high-conductance anion channel: 371 +/- 63 pS when measured under symmetrical 158 mM Cl- conditions. The channel frequently displayed subconductance levels. The ion selectivity of the channel expressed as the (an)ion to chloride permeability ratio was as follows: Cl- (1.0) greater than methylsulfate- (0.71) greater than gluconate- (0.25) greater than glutamate- (0.17) greater than Na+ = K+ (0.10). In addition, the channel had a significant permeability for inorganic phosphate ions. The channel was found in about 1% of the cell-attached patches, which indicates that the channel is under the control of as yet unknown intracellular factors. Once activated by patch excision, the channel was voltage dependent and active at potentials close to 0 mV. At potentials outside the range of +/- 10 mV channel activity decreased. This process proceeded faster at increasing membrane potentials of either polarity. Returning to potentials close to 0 mV caused reopening of the channels within seconds if the preceding voltage step led to complete closure of the channels. Channel activity did not depend noticeably on intracellular and extracellular CA2+ ions. The channel is not unique to (chick) osteogenic cells but has been demonstrated in excised patches obtained from excitable and other nonexcitable cells. Although its presence in a wide variety of cell types suggests that the channel plays a general role in as yet unknown cell physiologic processes, the channel may also have specific functions in osteogenic cells, for example providing a pathway for phosphate ions during mineralization.

The method of Barka and Anderson was used for the demonstration of tartrate-resistant acid phosphatase (TRAcP) in cultures of bone marrow, spleen, lung, and peritoneal cells of the mouse. The staining was performed either in the usual way by adding both substrate (naphthol-AS-BI-phosphate) and coupler (hexazonium pararosanilin) together (the simultaneous-coupling technique) or by adding first the substrate and then the coupler (the post-coupling technique). We measured TRAcP-activity fluorometrically after extraction of the product naphthol-AS-BI, using the same staining solution as in cytochemical method, but without the coupler. In bone marrow, spleen, lung, and peritoneal cell cultures a biochemically measurable TRAcP-activity was detected. Post-coupling generally gave a higher level of staining and larger numbers of TRAcP-positive cells than simultaneous-coupling. In bone marrow cultures macrophages, identifiable by their ability to phagocytose microspheres, became TRAcP-positive during culture. In lung cell cultures cells capable of phagocytosis of bacteria were shown to be TRAcP-positive. Peritoneal macrophages remained TRAcP-negative in the simultaneous-coupling technique. Using the post-coupling technique a small number stained TRAcP-positive. In spleen cell cultures TRAcP-positive cells containing hemosiderin were visible. In cultures of all four cell types, F4/80 positive cells staining also for TRAcP were present. F4/80 is a well known marker for macrophages, whereas osteoclasts are negative. In conclusion, mouse macrophages originating from various tissues can become TRAcP-positive in vitro. TRAcP activity alone is not a reliable marker for osteoclasts in bone marrow cultures.

Primary cultures of embryonic chick osteoblasts consist of a heterogeneous cell population. Patch clamp measurements were done on 1- to 5-day-old osteoblasts, osteocytes, fibroblastlike cells, and cells that could not be classified on morphologic criteria. The measurements showed the omnipresence of depolarization-activated high-conductance channels in cell-attached patches. The whole-cell experiments showed an outward rectifying conductance activating at positive membrane potentials. Channels underlying the latter conductance were found to be K+ conducting in outside-out membrane patches. The activation potential of the outward rectifying K+ conductance shifted to negative membrane potentials upon increasing the intracellular Ca2+ concentration within the range of 10(-8)-10(-3.2) M. The same happened with the activation potential of the K+ channels found in outside-out patches. Finally, inside-out patch experiments directly demonstrated the dependency of the activation potential of K+ channels on Ca2+ ions. Thus the identity and main characteristics of Ca2(+)-activated K+ channels expressed by the various cell types present in chick osteoblast cultures have now been established. Decreased input resistances were found in cells of cultures more than 2 days old. This is consistent with the establishment of electrical coupling between the cells. Functions in which Ca2(+)-activated K+ channels could play a role are discussed.

Four different cell populations--designated PF, OB, OC, and PC--were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the "transition state" between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria from removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E1 (PGE1) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.

The effects of alkaline phosphatase inhibitors (levamisole, L-bromotetramisole) on the activity of the enzyme and on calcification in vitro were studied, to find out whether there is a relationship between alkaline phosphatase and calcification. Metatarsal bones of 15 1/4-day-old embryonic mice were dissected and cultured for 40 hours in the presence and absence of inhibitor. Levamisole and L-bromotetramisole fully inhibited calcification in vitro when present in concentrations which almost totally inhibited alkaline phosphatase activity, as measured biochemically or histochemically. However, incorporation of 3H-thymidine and 35S-sulphate was also inhibited. Furthermore, D-bromotetramisole, the dextroform of bromotetramisole which has no effect on alkaline phosphatase, inhibited calcification and 3H-thymidine and 35S-sulphate incorporation as well. The results of this study show that these inhibitors cannot be used to study the relationship between alkaline phosphatase and calcification. In addition, they suggest that although alkaline phosphatase may be important for the process of calcification, it is probably not a critical factor.

Osteoclast progenitors are seeded via the blood stream in the mesenchyme surrounding embryonic long bone models long before the appearance of multinucleated osteoclasts. The proliferation and differentiation of these progenitors in embryonic mouse metatarsal bones was studied with acid phosphatase (AcP) histochemistry and 3H-thymidine autoradiography. In vivo, tartrate-resistant, acid phosphatase-positive, mononuclear cells appear in the periosteum (AcPP-P cells) at the age of 17 days (after conception). On day 18, AcP-positive, multinucleated osteoclasts invade the bone rudiment and start resorbing the calcified cartilage matrix, resulting in the formation of the marrow cavity. The kinetics of osteoclast formation in vitro was studied in metatarsal bones of embryonic mice of different ages cultured in the continuous presence of 3H-thymidine. In young bones (15 days), mainly proliferating, 3H-thymidine-incorporating progenitors gave rise to AcPP-P cell and osteoclast formation. In older bones (16 and 17 days) osteoclasts were progressively more derived from postmitotic, unlabeled precursors. Irradiation of the metatarsal bones with a radiation dose of 5.0 Gy prior to culture resulted in a selective elimination of the proliferating progenitors, whereas the contribution of postmitotic precursors in AcPP-P cell and osteoclast formation remained unchanged. The results demonstrate that in the periosteum of embryonic metatarsal bones a shift occurs from a population composed of proliferating osteoclast progenitors (15 days) to a population composed of postmitotic precursors (17 days) before multinucleated osteoclasts are formed (18 days). Obviously, postmitotic AcP-negative precursors, already present in 16-day-old bones, differentiate into precursors characterized by tartrate-resistant AcP activity, the preosteoclasts (17 days), which in their turn fuse into osteoclasts.

The development of a monoclonal antibody, OB 7.3, directed against a cell surface antigenic site on osteocytes is described. Osteoblast-like cells were enzymatically isolated from calvaria of chicken embryos after removal of the periostea. The cells were cultured for 6 days, harvested and used to immunize mice. One of the monoclonal antibodies obtained, OB 7.3, reacted specifically with the cell surface of osteocytes. In frozen sections of bone only osteocytes were stained, all other cells present, including mature osteoblasts, were negative. Liver, kidney, spleen, intestine, bloodvessel and skin were also completely negative. Using the monoclonal OB 7.3, positive cells could be demonstrated in sparse osteoblast-like cell cultures. The OB 7.3 positive cells had a stellate morphology and were therefore identified as osteocytes. They behaved in culture as osteocytes in bone tissue in that they formed a network of cell processes connecting osteocytes with each other or with other neighbouring cells. Monoclonal OB 7.3 offers the possibility of isolating osteocytes thereby providing the means for a detailed study of their biochemical properties.

Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.

The fully differentiated osteoblast may be easily recognized in bone tissue. Its cuboidal shape, its position directly opposed to the bone surface and its capacity to produce calcified bone matrix are characteristic. Three other differentiation stages are also reasonably well defined--the preosteoblast, the osteocyte and the lining cell. These differentiation stages are preceded by an unknown number of precursor, progenitor and stem cell stages. Little is known about the regulation of the transitions between the various osteogenic phenotypes and their reversibility or irreversibility. One of the reasons for this is the lack of adequate tools with which to recognize the various differentiation stages. We have developed a number of monoclonal antibodies (in bone) specifically directed against osteocytes, osteoblasts and as yet unidentified cells in the periosteum. The anti-osteocyte monoclonals were used to recognize osteocytes in bone cell cultures and we obtained purified osteocyte populations for metabolic studies. Osteocytes were shown to have binding sites for parathyroid hormone. The antibodies directed against osteoblasts showed that at present our culture conditions are inadequate to allow osteoblast differentiation in vitro.

The effects of ionizing irradiation on the differentiation and activity of the osteoclast were investigated. Embryonic mouse metatarsal bones of different ages (14, 15, 16, 17 days) in which no osteoclasts had as yet been formed were irradiated with various x-ray doses and cultured until a marrow cavity became visible in the nonirradiated paired control bones. Bone growth and calcification were followed microscopically during culture. Irradiation caused a dose-dependent stunting of the longitudinal growth. Calcification was inhibited by high radiation doses (10 to 20 Gray (Gy), whereas a dose of 2.5 Gy stimulated the process in the early stages of long bone development. Histologic examination revealed complete inhibition of osteoclast formation in the 14- and 15-day-old bones after irradiation with 2.5 Gy or more. The number of osteoclasts in cultured older bones (16 days) was significantly reduced by irradiation, but osteoclast formation could not be completely prevented even by high dosages. Irradiation of explanted bone rudiments which were in a stage 1 day prior to the appearance of osteoclasts in vivo (17 days) did not significantly influence the formation of osteoclasts. Autoradiographic experiments using young bones showed that differentiation of osteoclast precursors into multinucleated osteoclasts is preceded by one or more divisions of the precursors in the periosteum. Furthermore, it was established from continuous 3H-thymidine-labeling experiments that in older bones (16 days) a part of the osteoclast nuclei originated from postmitotic osteoclast precursors. Irradiation mainly inhibited the appearance of labeled osteoclast nuclei in these bones. The results indicate that the osteoclast precursor, already present in the periosteum at an early stage of embryonic development, first proliferates and then differentiates into a mononuclear postmitotic preosteoclast. The proliferation is probably highly radiosensitive. Subsequently, the preosteoclasts fuse into multinucleated osteoclasts and invade the calcified hypertrophic cartilage zone. The resorbing activity of the osteoclast is less radiosensitive but can be inhibited by 5.0 Gy or more, as was established by morphometric and biochemical methods.

An in vitro co-culture system was applied to study the direct and indirect effects of irradiation on osteoclast formation. Osteoclast precursor-free fetal mouse metatarsal bones were employed as osteoclast-forming inductor and periostea dissected from fetal calvaria as source of proliferating progenitor cells. Direct radiation effects on the formation of osteoclasts were assessed in co-cultures of irradiated periostea and non-irradiated bone rudiments. The results showed that the (blood-borne) periosteal progenitors were rather radiosensitive. A radiation 'survival' curve of osteoclast formation in relation to various doses could be constructed yielding a mean lethal dose (Do value) of 0.94 +/- 0.02 Gy and an extrapolation number of 1.67 +/- 0.01. Irradiation of the fetal long bones by low doses, effective for direct elimination of osteoclast progenitor cells, did not indirectly affect osteoclast development from the non-irradiated periosteal progenitor population. However, at relatively high radiation levels, though not lethal for the long bone rudiments, a significant inhibition of osteoclast formation became evident. The results indicate that radiation primarily affects osteoclast formation via a direct action on radiosensitive, proliferating progenitor cells. Injury to long bone models by relatively high radiation doses may also lead to severe disturbance of osteoclast formation kinetics.

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.