Biomedical subjects

R Zohar

Publications and source records attributed to R Zohar.

16 recordsLinked to original sources

PubMed2006

Osteopontin and mucosal protection.

Protection of mucosal tissues of the oral cavity, intestines, respiratory tract, and urogenital tract from the constant challenge of pathogens is achieved by the combined barrier function of the lining epithelia and specialized immune cells. Recent studies have indicated that osteopontin (OPN) has a pivotal role in the development of immune responses and in the tissue destruction and the subsequent repair processes associated with inflammatory diseases. While expression of OPN is increased in immune cells--including neutrophils, macrophages, T- and B-lymphocytes--and in epithelial, endothelial, and fibroblastic cells of inflamed tissues, deciphering the specific functions of OPN has been difficult. In part, this is due to the broad range of biological activities of OPN that are mediated by multiple receptors which recognize several signaling motifs whose activities are influenced by post-translational modifications and proteolytic processing of OPN. Understanding the role of OPN in mucosal inflammation is further complicated by its contributions to the barrier function of the lining epithelia and the complexity of the specialized mucosal immune system. In an attempt to provide some insights into the involvement of OPN in mucosal diseases, this review summarizes current knowledge of the biological activities of OPN involved in the development of inflammatory responses and in wound healing, and indicates how these activities may affect the protection of mucosal tissues.

Animals↗

PubMed2005

Regulation of BMP-induced ectopic bone formation by Ahsg.

alpha2-HS-glycoprotein (Ahsg), also known as fetuin is a serum and bone resident glycoprotein, which binds to TGF-beta superfamily members including bone morphogenetic proteins (BMP) and inhibits dexamethasone-induced osteogenesis in bone marrow cultures in vitro. Here we demonstrate that Ahsg reduces cytokine binding to its cognate receptor in HOS osteocyte cells and suppresses intracellular signaling, while in vivo, we test the hypothesis that Ahsg-deficient mice are hyper-responsive to BMP-induced osteogenesis. Human native BMP was implanted into the hindquarter muscles of Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) mice and 4 weeks later, ossicle formation was analyzed by radiography, bone density scanning (DEXA) and histomorphometry. Alkaline phosphatase (AP) activity was measured in ossicles as a marker for bone cell differentiation, and was significantly higher in Ahsg(-/-) versus Ahsg(+/-) and/or Ahsg(+/+) mice. Ectopic ossicle size in the Ahsg(+/-) mouse was 4-fold greater than that in the wild type (Ahsg(+/+)), and intermediate to that shown in Ahsg(-/-) mouse. Bone mineral density (BMD) was lower in the Ahsg(-/+) and Ahsg(-/-) mice compared to Ahsg(+/+) littermates. The ratio of cortical to cancellous bone was found to be >2-fold higher in Ahsg(-/-) mouse in comparison to the Ahsg(+/+) mice with no significant change in the Ahsg(-/+) mouse. Finally, a significantly higher incidence of satellite ossification; small islands of immature bone, was shown in Ahsg(-/-) mice as compared to Ahsg(+/+) mice. Although Ahsg binds to TGF-beta/BMP and blocks receptor signalling, it may also sequester cytokines in matrix, thereby acting as a reservoir of osteoinductive activity when released. This may explain the non-linear relationship between ectopic bone formation characteristics and Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) genotypes, although the increase in satellite bone formation might also explain this phenomenon. Our results suggest that Ahsg may be useful for prevention of the heterotopic ossification and the regulation of osteoinductive effects of BMP used with grafts.

Alkaline Phosphatase↗

PubMed2004

Inhibition of osteogenesis in vitro by a cigarette smoke-associated hydrocarbon combined with Porphyromonas gingivalis lipopolysaccharide: reversal by resveratrol.

BACKGROUND: Smoking and infection with Gram-negative bacterial pathogens are risk factors for alveolar bone loss. The aims of this study were: 1) to examine the combined effects of an aryl hydrocarbon, benzo[a]pyrene (BaP), that is concentrated in cigarette smoke, and lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis on osteogenesis in a rat bone marrow cell (RBMC) model of osteogenesis; and 2) to determine whether resveratrol (Res), an aryl hydrocarbon receptor antagonist, could reverse the putative inhibitory effects of BaP + LPS on osteogenesis. METHODS: LPS of P. gingivalis strain 2561 was introduced in various concentrations to the RBMC in 96-well plates and kept in culture for 8 to 12 days. The same protocol was used for studying BaP and LPS + BaP combinations. Following the incubation periods, parameters of osteogenesis were measured, including formation of mineralized bone nodules, alkaline phosphatase activity, and total cell protein. Transcription of the pro-inflammatory cytokine interleukin (IL)-1beta in the cultures was determined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Bone nodule formation generally decreased significantly with increasing LPS concentrations (P<0.05), whereas total cell protein decreased only slightly (P>0.05). BaP in previously high concentrations alone also caused a significant dose-dependent decrease in bone nodule formation (P<0.05) but when half maximal doses were used, significant decreases were most often seen when LPS was added. Hence, in combination, the inhibitory effects of LPS + BaP on osteogenesis were additive, inhibiting bone nodule formation up to 9-fold. Resveratrol partially reversed the inhibitory effects of low concentrations of LPS alone, and completely reversed the inhibition of nodule formation when low concentrations of LPS were combined with BaP. IL-1beta expression generally fluctuated inversely to the inhibitory activity of LPS, LPS + BaP, and LPS + BaP + Res combinations. CONCLUSIONS: Smoke-derived aryl hydrocarbons and bacterial LPS may act additively to inhibit bone formation. The findings may explain, in part, why net periodontal bone loss is greater and bone healing is less successful in smokers than non-smokers with periodontal infections. Reversal of the inhibitory effects in vitro by resveratrol suggests that this phytoalexin should be studied further for its potential therapeutic value, given its aryl hydrocarbon receptor antagonism and apparent anti-inflammatory activity.

Alveolar Bone Loss↗

PubMed2001

Alveolar bone resorption following coronal versus apical approach in a mucoperiosteal flap surgery procedure in the rat mandible.

BACKGROUND: In several publications, we have reported that a distinct resorptive phase of alveolar bone developed during 3 weeks following elevation of a mucoperiosteal flap in rats by a coronal surgical approach. In the present study, we compared the alveolar bone loss after mucoperiosteal flap surgery by a coronal approach (COR) to a surgical approach incising the mucosa close to the tooth apices and separating the mucoperiosteum toward the tooth crown (apical approach, AP). METHODS: Eighteen Wistar rats were divided into 2 experimental groups; each group consisted of 9 rats. In the first group, COR was performed on both buccal and lingual aspects on the right side of the rat mandible, while the left side served as a sham-operated control. In the second experimental group, AP was performed. RESULTS: In the AP group, the outer aspect (buccal or lingual) of the alveolar bone was slightly resorbed usually at the level where the mucosa was incised. In many sections, extensive modeling of new trabecular bone was seen, coronal to the surgical incision. On the contrary, the coronal surgical approach revealed an extensive resorptive phase, mainly of the periodontal aspect of the alveolar bone. CONCLUSIONS: The results of this study show that periodontal surgery by a coronal surgical approach stimulates a burst of remodeling of the alveolar bone in a very specific pattern.

Alveolar Bone Loss↗

PubMed2001

Tetracycline modulates collagen membrane degradation in vitro.

BACKGROUND: Structural integrity of implanted bioabsorbable barrier membranes should be preserved for a sufficient time to ensure expected results. Collagen membranes are degraded by metalloproteinases (MMP). Their degradation rate can be altered either by enhancing structural integrity or by delaying the degradation process using MMP inhibitors. Tetracyclines (TTC) present inhibitory effects on matrix MMP. Immersing membranes in TTC solution before implantation can delay their degradation. The purpose of the present study was to evaluate the effect of collagen membranes immersed in varying TTC concentration solutions on the rate of their degradation in vitro. METHODS: Collagen bioabsorbable membranes were prepared as 5 mm diameter membrane discs. Membranes were then incubated at 4 degrees C for 24 hours, in either phosphate buffered saline (PBS, Ca2+ and Mg2+ free) or with TTC-HCl dissolved in PBS concentrations of 5 mg/ml, 50 mg/ml or 100 mg/ml. After rinsing, membranes were incubated with either bacterial collagenase or cultures of human bone lineage cells. Membrane degradation was studied on days 2, 4, 7, and 14. Two- and 3-way analysis of variance was used to analyze results. RESULTS: Samples supplemented with bacterial collagenase exhibited a statistically significant interaction between changes of free protein in the medium, antibiotic concentration used for the immersion, presence of collagenase in the medium, and incubation time (P<0.0001). Membranes incubated with bone cells exhibited similar degradation trends. CONCLUSIONS: Collagen membranes immersed in 50 mg/ml TTC solution exhibited the longest degradation time, both in the clostridial collagenase and the human bone cell lineage assays. Immersion in a 50 mg/ml TTC solution before implantation will delay their degradation.

Absorbable Implants↗

PubMed2000

Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration.

Osteopontin (OPN) is a secreted glycoprotein with mineral- and cell-binding properties that can regulate cell activities through integrin receptors. Previously, we identified an intracellular form of osteopontin with a perimembranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88-98, 1997). Since OPN and CD44 expression are increased in migrating cells, we analyzed the relationship of these proteins with immunofluorescence and confocal microscopy. A distinct co-localization of perimembranous OPN and cell-surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. The co-localization of OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN also co-localized with the ezrin/radixin/moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan-coated beads. The subcortical location of OPN in these cells was verified by cell-surface biotinylation experiments in which biotinylated CD44 and non-biotinylated OPN were isolated from complexes formed with hyaluronan-coated beads and identified with immunoblotting. That perimembranous OPN represents secreted protein internalized by endocytosis or phagocytosis appeared to be unlikely since exogenous OPN that was added to cell cultures could not be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or alpha-actin, was indicated by immunoadsorption and immunoblotting of cell proteins in complexes extracted from hyaluronan-coated beads. The functional significance of OPN in this complex was demonstrated using OPN-/- and CD-/- mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan-coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan-CD44-ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells.

Animals↗

PubMed1999

Stromal mesenchymal progenitor cells.

The precursors of bone, cartilage, fat and muscle cells are likely to be derived from more primitive mesenchymal cells which exhibit some of the characteristics of stem cells. Despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well characterized. Here we describe the use of flow cytometry to isolate from fetal rat periosteum a population of small, relatively agranular cells (S cells) that display stem cell characteristics. After plating, S cells demonstrated extensive self-renewal with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase or osteopontin. However following attachment, S cells became immunopositive for collagens I, II, III, osteopontin and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. S-cells showed two discrete populations of surface-stained protein by sulforhodamine, wheat germ agglutinin and Thy-1. In contrast, large (L) cells that did not exhibit stem cell characteristics exhibited low staining levels for Thy-1 and for wheat germ agglutinin. These studies demonstrate that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods.

Animals↗

PubMed1998

Flow cytometric analysis of recombinant human osteogenic protein-1 (BMP-7) responsive subpopulations from fetal rat calvaria based on intracellular osteopontin content.

The bone morphogenetic proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiation of mesenchymal cells in vivo and in vitro. Primary cultures of fetal rat calvarial cells contain a broad spectrum of osteogenic cells at various stages of differentiation, but the responsive subpopulations are incompletely characterized. To identify responsive cells in osteogenic cell differentiation, we have treated fetal rat calvarial cells with recombinant osteogenic protein-1 and used flow cytometric analyses of intracellular osteopontin, and of cartilage and bone nodule formation, to evaluate the effects. When administered as a single dose at confluence, osteogenic protein-1 stimulated bone nodule formation in fetal rat calvarial cultures in dose-dependently way. To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml recombinant osteogenic protein-1 for 12 h before analysis by flow cytometry. Flow cytometry analyses of cell suspensions revealed that osteogenic protein-1 increased the total protein content of cells, and selectively increased the mean expression of osteopontin and the size and granularity of osteopontin expressing cells, particularly at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation. Pulse administration of 100 ng/ml osteogenic protein-1 to sorted, osteopontin-negative subpopulations enriched for stem cells reduced by more than four-fold the number and size of bone nodules while promoting chondrogenesis and adipogenesis. In contrast, a pulse administration of osteogenic protein-1 to more differentiated, large osteopontin-positive cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopontin-positive and small osteopontin-negative cell populations obliterated bone nodule formation and promoted chondrogenesis. We conclude that pulse administration of osteogenic protein-1 promotes osteogenic differentiation of cells committed to the osteogenic lineage, whereas undifferentiated periosteal cells are induced to differentiate along the chondrogenic pathway. In contrast, continuous exposure to osteogenic protein-1 promotes chondrogenesis in populations of committed osteogenic cells and in undifferentiated periosteal cells.

Adipocytes↗

PubMed1998

Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration.

Formation and repair of the hard and soft connective tissues of teeth and their supporting structures require stem cells to divide, differentiate and migrate to generate specific tissues in a defined temporo-spatial sequence. We have used antibodies to osteopontin (OPN) and fluorescence-activated cell sorting (FACS) to determine the relationship between OPN expression and cell differentiation in cultures of fetal rat calvarial cells. At different stages of osteogenic differentiation, OPN was expressed by 60-98% of cells. Populations of small OPN-negative cells with low cellular granularity (S cells) were isolated and shown to be enriched in stem cells, characterised by a lack of differentiation markers, high proliferative potential, capacity for self renewal and multipotentiality. Within 24 h of plating, S cells attached, spread and started expressing OPN, CD44, and collagens types I, II and III. Confocal microscopy of OPN in differentiating cells revealed two distinct phenotypes; a perinuclear distribution, characteristic of secreted OPN, and an intracellular perimembranous distribution co-localising with the CD44 receptor, characteristic of migrating cells in which OPN was increased > 10-fold as measured by immunoblotting. These studies show that OPN is expressed early in mesenchymal cell differentiation and is related to cell migration as well as osteogenesis.

Animals↗

PubMed1997

Characterization of stromal progenitor cells enriched by flow cytometry.

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.

Animals↗

PubMed1997

Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells.

Osteopontin (OPN), a major component of the bone matrix, is expressed at different stages of bone formation. To determine possible relationships between OPN expression and stages of osteogenic cell differentiation, we have performed single cell analyses of intracellular OPN in early (proliferating), subconfluent (differentiating), and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC) using a combination of flow cytometry and confocal microscopy. At each culture stage, a high proportion (60-98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a small proportion of OPN-ve cells which were characterized by their small size, low granularity, high proliferative capacity, and enhanced osteogenic potential. The OPN+ve cells displayed two distinct patterns of intracellular immunostaining: a perinuclear distribution typical of secreted proteins and a perimembrane distribution in which patches of OPN were concentrated at the cell surface. Perimembranous staining predominated in migrant cells, which contained greater than tenfold higher levels of OPN than nonmigrant cells as separated in a Boyden chamber. When cell proliferation was high (day 2), most cells were OPN + ve. At all culture stages the intensity of OPN staining was increased as cells progressed through the cell cycle. As cells differentiated and started to form matrix (days 4 and 6), the mean cell expression of OPN was also increased (fourfold), independent of changes in total cell protein. However, despite the association of OPN with osteogenic cells, we were surprised to find that a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these cell populations was confirmed by RT-PCR, and a strong correlation was observed between the quantitative flow cytometry data and Western blot analysis of cell extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies, therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: small OPN-ve cell populations enriched in osteogenic precursors, differentiating osteogenic cells that synthesize and secrete OPN, and migrating stromal cells characterized by a perimembranous OPN staining pattern.

Animals↗

PubMed1997

Bone regeneration in extraction sites. Part 2: The staged approach.

The use of guided tissue regeneration in conjunction with implants is a routine procedure in oral implant reconstruction. Three patient reports of the staged approach are presented and discussed. Implants were placed in regenerated bone 9 months after augmentation. Barrier membranes, with and without supporting screws, were used in different types of extraction site defects and followed for 2 years postoperatively.

Adult↗

PubMed1997

Tamoxifen attenuates glucocorticoid actions on bone formation in vitro.

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors.

Alkaline Phosphatase↗

PubMed1997

Periodontal and peri-implant bone regeneration: clinical and histologic observations.

The principle of guided tissue regeneration by barrier membranes to restore lost periodontal tissue around natural teeth has also been used around osseointegrated implants in an attempt to restore alveolar ridge defects. While most periodontal procedures in the literature describe root coverage by mucogingival surgery, which achieves healing through soft tissue attachment, regeneration of denuded root surfaces is performed by guided tissue regeneration using expanded polytetrafluoroethylene barrier membranes and demineralized freeze-dried bone allografts as inductive/conductive materials. In this study the technique is applied in two partially exposed cylindrical hydroxyapatite-coated implants in extraction sites in one patient. Surgical reentry in both sites is presented, with histologic examination revealing new bone formation on the exposed root surface and the hydroxyapatite-coated implants.

Alveolar Bone Loss↗

PubMed1988

Pregnancy outcome in heat-exposed hamsters; the involvement of the pineal.

The effect of high ambient temperature (34 degrees C) on the function of the female reproductive system, on embryonic development and on outcome of pregnancy, was investigated in heat-exposed sham-operated (Sh) and pinealectomized (Px) golden hamsters maintained under short photoperiod. Plasma prolactin levels were reduced in both heat-exposed groups (ShH and PxH) but pituitary prolactin was increased in the pinealectomized groups irrespective of ambient temperature (21 or 34 degrees C). Pituitary weights and LH contents were not affected in any test group. Heat exposure brought about a reduction in the number of corpora lutea and of pups born, the latter being more drastically reduced in absence of the pineal; the depressant effect of heat on ovarian weight was evident only in the pinealectomized animals. Progesterone levels were not affected in any test group and pregnancy was not prolonged, thus, it would seem that pregnant hamsters adapt themselves well to heat. Moreover, high ambient temperature promoted a rise in pineal. HIOMT activity and boosted cortisol levels in presence of the pineal gland only, which, together with the above findings, shows that the pineal can provide protection for pregnant hamsters against adverse effects of high ambient temperature.

Animals↗

PubMed1982

Effect of Sodium Chloride on Fruit Ripening of the Nonripening Tomato Mutants nor and rin.

Tomato (Lycopersicon esculentum Mill) plants of the nonripening mutant nor, the ripening-inhibited mutant rin, and the normal cultivar ;Rutgers' were grown in nutrient solution supplemented with 3 grams per liter NaCl from the time of anthesis. In plants treated with NaCl, all the ripening parameters of the fruits of the nor mutant increased, but those of the rin mutant did not. The ripening of the fruits of the NaCl-treated nor plants was characterized by the development of a red color and taste, increased pectolytic activity, and increased evolution of CO(2) and ethylene. These changes do not normally take place in nor under control conditions. The values of these ripening parameters in nor were lower than those of the normal Rutgers fruits. In addition, both in nor and rin and in the normal variety, exposure of the plants to NaCl shortened the developmental period of the fruit, decreased the fruit size, and increased the concentrations of total soluble solids, Na(+), Cl(-), reducing sugars, and titratable acids in the fruit. The role of NaCl in overcoming the inability of nor to ripen is discussed.

Journal Article↗