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P J Nijweide

Publications and source records attributed to P J Nijweide.

At least 19 recordsLinked to original sources
PubMed1992

Identification of osteopontin in isolated rabbit osteoclasts.

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.

Amino Acid Sequence↗
PubMed1992

Isolation and purification of osteocytes.

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.

Animals↗
PubMed1992

Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

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.

Animals↗
PubMed1991

High-conductance anion channels in embryonic chick osteogenic cells.

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.

Animals↗
PubMed1991

Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro.

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.

Acid Phosphatase↗
PubMed1991

Tartrate-resistant acid phosphatase is not an exclusive marker for mouse osteoclasts in cell culture.

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.

Acid Phosphatase↗
PubMed1990

Identification of Ca(2+)-activated K+ channels in cells of embryonic chick osteoblast 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.

Animals↗
PubMed1989

Osteoclast formation from cloned pluripotent hemopoietic stem cells.

In the present report osteoclast formation from cloned pluripotent hemopoietic stem cells (PHSC) is described. Populations enriched in hemopoietic stem cells were cloned (1 cell/well) and cultured in the presence of different colony-stimulating factors, or combinations of these growth factors. In cultures containing interleukin-3 (Il-3) or pregnant mouse uterus extract (PMUE) alone, cloning efficiency was low. Cultures containing Il-3 and Il-1 or Il-3 and PMUE showed a somewhat higher cloning efficiency, whereas cultures containing Il-3, Il-1 and PMUE had the highest cloning efficiency. All colonies of cloned PHSC, tested for their osteoclast formation capability in cocultures with periosteum-free metatarsal bones of fetal mice, gave rise to osteoclast formation. Other hemopoietic cells could also be demonstrated. In control cultures in which the bones were kept without stem cells, no osteoclast formation was observed. In conclusion, we have demonstrated that the osteoclast is derived from the pluripotent hemopoietic stem cell. A combination of various growth factors is important for stem cell proliferation in vitro.

Animals↗
PubMed1989

Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts.

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.

4-Aminopyridine↗
PubMed1988

Osteoblastic differentiation.

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.

Animals↗
PubMed1988

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures.

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.

Animals↗
PubMed1988

Cell-cell interactions in the osteogenic compartment of bone.

The interactions between two different cell populations within the osteogenic compartment have been examined. The proliferation of periosteal fibroblasts (PF) in the presence or absence of osteoblast-like cells (OB), whose proliferative capacity was inhibited by irradiation, was measured. OB stimulated [3H]thymidine incorporation in PF and parathyroid hormone (PTH) enhanced the stimulation. In the reverse situation, however, PF inhibited OB. Irradiated OB also stimulated 3H-thymidine incorporation in OB, and irradiated PF in PF, but both to a lesser extent. Co-culture experiments showed that direct cell-cell contact was a prerequisite for stimulation of PF. Medium mediated contact between physically separated OB and PF did not stimulate, but rather inhibited PF proliferation. These results demonstrate that OB regulate the proliferation of cells in the PF population and can transmit the proliferation stimulating message of PTH to PF. This implies that in vivo the mature osteoblast may play a pivotal role in the (hormonal) regulation of osteoprogenitor cell proliferation and therefore bone formation.

Animals↗
PubMed1987

Direct and indirect radiation effects on osteoclast formation in vitro.

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.

Acid Phosphatase↗
PubMed1987

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources.

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.

Animals↗
PubMed1986

Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells.

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.

Animals↗
PubMed1986

Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro.

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.

Animals↗
PubMed1986

Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes.

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.

Animals↗