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Biomedical subjects

R D Finkelman

Publications and source records attributed to R D Finkelman.

33 records · Page 2Linked to original sources
PubMed1992

Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss.

Previous work showed that production of transforming growth factor beta (TGF-beta) by osteoblast-like rat UMR 106 cells was increased by 17 beta-estradiol at physiological concentrations. To determine whether ovariectomy alters the concentration of TGF-beta in rat long bones, female Sprague-Dawley rats were either sham-operated (n = 19) or ovariectomized (n = 19), pair-fed a semisynthetic diet for 6 weeks, and sacrificed. Tibial and femoral diaphyses were removed and extracted by demineralization. Ovariectomy lowered serum estrogen; did not alter body weight, serum magnesium, or serum 1,25-dihydroxyvitamin D; and produced only modest differences in serum calcium and phosphate concentrations. Hydroxyproline was higher and extractable protein was lower in bones from ovariectomized rats than in bones from sham-operated rats; calcium content did not differ between the two groups of animals. Ovariectomy lowered the concentration of TGF-beta in bone but did not change the concentration of insulin-like growth factors I or II compared with values in bone from control animals. The reduction of bone TGF-beta was evident 6 weeks after surgery but not at 3 weeks. Treatment of ovariectomized rats with estrogen eliminated the TGF-beta deficit. To determine whether 17 beta-estradiol increased TGF-beta production by normal bone cells, mouse osteoblasts were treated for 2 days with 17 beta-estradiol. The production of TGF-beta was increased almost 2-fold by 1 nM 17 beta-estradiol, and short-term treatment stimulated the intracellular accumulation of TGF-beta 1 mRNA. We conclude that ovariectomy reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could play a role in the pathogenesis of bone loss, fractures, and microfractures that occur in estrogen-deficient states. Our results support the possibility that estrogen and bone TGF-beta may be necessary for normal maintenance of the skeleton in female rats.

Animals↗
PubMed1992

Evidence for a lack of functional receptors for nerve growth factor (NGF) in chick bone cells in vitro.

Nerve growth factor (NGF) is essential for the development and differentiation of sympathetic and sensory neurons. Recently, NGF receptors were demonstrated in non-neural cells, and several mesenchymal cell types including lymphocytes and skeletal myotubes were shown to be stimulated to proliferate by NGF. Our purpose was to examine for the presence of functional NGF receptors in osteoblasts. Bone cells from chick calvaria were used as a model; PC-12 cells derived from rat adrenal pheochromocytoma were used as positive controls. NGF was examined for functions in chick bone cells by studying effects on (1) [3H]-thymidine incorporation; (2) alkaline phosphatase (ALP) activity; and (3) protein tyrosine phosphorylation. Effects of NGF on thymidine incorporation and protein tyrosine phosphorylation by PC-12 cells were also measured. A radioreceptor assay was used to test for the presence of receptors. In chick calvarial cells, NGF had no effect on thymidine incorporation, ALP activity or protein tyrosine phosphorylation. Radioreceptor assay with bone cells showed no evidence of NGF receptors. In contrast, in PC-12 cells, NGF (1) decreased thymidine incorporation; (2) increased protein tyrosine phosphorylation; and (3) showed receptor activity by radioreceptor assay. In conclusion, unlike several other mesenchymal cell types, chick bone cells show no evidence of NGF receptors or functional responses to NGF in vitro.

Alkaline Phosphatase↗
PubMed1992

Transforming growth factor-beta 1 mRNA in neonatal ovine molars visualized by in situ hybridization: potential role for the stratum intermedium.

Human dentine contains relatively large amounts of transforming growth factor-beta (TGF-beta), which might originate from odontoblasts. The expression of the TGF-beta 1 message in developing teeth was examined by in situ hybridization. The analysis was made on 5-microns serial sections of mandibular third molars of neonatal sheep cut from tissues that had been fixed in glutaraldehyde and paraffin-embedded. A 35S-labelled cRNA probe, complementary to TGF-beta 1 mRNA, was constructed from human TGF-beta 1 cDNA. Northern analysis of total RNA from sheep placenta and neonatal third molars demonstrated hybridization to a single 2.4 kb TGF-beta 1 transcript from both tissues, indicating cross-reactivity of the human probe in the sheep. In the neonatal molars, in situ hybridization was observed in cells of the inner enamel epithelium, mature ameloblasts and mature odontoblasts, but not within preodontoblasts before dentine matrix formation. TGF-beta 1 mRNA expression was also evident in the cells of the dental papilla but scarcely so in the stellate reticulum. The most striking feature was the appearance of hybridization signal in the cells of the stratum intermedium before hybridization was evident in the inner enamel epithelium. Control sections incubated with RNAase before incubation with probe did not show evidence of hybridization. These findings suggest that TGF-beta 1 may have an important regulatory role in the differentiation of ameloblasts and odontoblasts, perhaps by modulating matrix formation during amelogenesis or odontogenesis. They also suggest a potential novel regulatory role for the cells of the stratum intermedium.

Ameloblasts↗
PubMed1992

PTH stimulates the proliferation of TE-85 human osteosarcoma cells by a mechanism not involving either increased cAMP or increased secretion of IGF-I, IGF-II or TGF beta.

Injections of parathyroid hormone (PTH) result in increased bone formation in several species. Work in our laboratory and others has shown a stimulation of bone cell proliferation and growth factor production by PTH. Our purpose was to study the effects of PTH on a human bone cell line using TE-85 human osteosarcoma cells as a model. After 24 h treatment, PTH caused an increase in cell proliferation as measured by cell counts and [3H]-thymidine incorporation. Proliferation was not inhibited by an anti-transforming growth factor beta (TGF beta) antibody which could abolish stimulation by exogenous TGF beta. PTH did not stimulate cAMP production, alkaline phosphatase activity or production of insulin-like growth factors I or II (IGF-I or IGF-II) in TE-85 cells. Although basal TE-85 proliferation was slowed by incubation with the calcium channel blocking agent verapamil, PTH still caused an increase in growth rate. We conclude that PTH directly stimulates TE-85 proliferation via a mechanism not involving increased adenylate cyclase activity or increased secretion of IGF-I, IGF-II or TGF beta and may stimulate bone formation in vivo by activating some other mitogenic signal to increase bone cell proliferation.

Adenylyl Cyclases↗
PubMed1992

Growth factors in bones and teeth.

Human bone contains an abundance of polypeptide growth factors. These growth factors stimulate the proliferation and activity of bone cells and can stimulate bone formation. Data from this laboratory and others suggest that bone growth factors may act to couple bone formation to resorption to maintain bone mass during remodeling. Research is underway to study these growth factors in bones and teeth, and their possible roles in both the pathogenesis and the treatments of osteoporosis and dental diseases.

Animals↗
PubMed1991

Vitamin D deficiency causes a selective reduction in deposition of transforming growth factor beta in rat bone: possible mechanism for impaired osteoinduction.

We demonstrated previously that implants of bone matrix prepared from vitamin D-deficient (-D) rats were less osteoinductive and contained less extractable mitogenic activity compared with control implants prepared from vitamin D-replete (+D) rats and proposed that bone from -D rats is deficient in one or more specific growth factors. To test this hypothesis, bones from rats that were fed either +D or -D diets and kept in the dark for 8 wk were extracted and assayed for insulin-like growth factors I and II (IGF-I and IGF-II) and transforming growth factor beta (TGF-beta), the three most abundant growth factors in rat bone, and osteocalcin. Serum calcium, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were determined at sacrifice. In -D rats, there were significant reductions in serum calcium, 25-hydroxyvitamin D3, and 1,25(OH)2D3 and skeletal TGF-beta but no differences in extractable skeletal protein, IGF-I, IGF-II, or osteocalcin compared with +D rats. To determine whether 1,25(OH)2D3 increased TGF-beta production by bone cells, we treated mouse calvaria for 6 days and mouse osteoblasts for 2 days with 10 nM 1,25(OH)2D3. Production of TGF-beta was increased almost 100% by 1,25(OH)2D3. We conclude that vitamin D deficiency reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could contribute to the previously observed decrease in osteoinduction in implants from -D rat bone. The findings support the possibility that vitamin D and bone-derived TGF-beta are required for normal repair of the skeleton.

Animals↗
PubMed1990

Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin.

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors. Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulin-like growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-beta) by bioassay. Gu-EDTA extracts stimulated bone cell proliferation. At 10 micrograms/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/micrograms Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-beta, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.

Calcium↗
PubMed1988

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat.

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.

Alkaline Phosphatase↗
PubMed1987

A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone gamma-carboxyglutamic acid (Gla)-containing protein in osteoblasts.

Bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformaldehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.

Animals↗
PubMed1987

Immunocytochemical localization of gamma-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin.

Gla-protein or osteocalcin is one of the most abundant noncollagenous matrix proteins found in bone and dentin. The present study describes, with high resolution, the intracellular and extracellular distribution of Gla-protein in alveolar bone and incisor dentin. Sections of tissues embedded in Lowicryl K4M were incubated with rabbit antibodies to rat dentin Gla-protein. The site of the specific antigen-antibody reaction was revealed by the protein A-gold complex. Labeling was detected over bone and dentin while fewer gold particles were present over prebone and predentin. Gold particles were also seen over the protein synthetic organelles (rough endoplasmic reticulum, Golgi apparatus) of osteoblasts and odontoblasts. These findings confirm, with improved resolution, previous light immunohistochemical studies, and offer the possibility to examine the secretory pathway of the protein.

Alveolar Process↗
PubMed1987

Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement.

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.

Animals↗
PubMed1987

Developmental appearance of Gla proteins (osteocalcin) and alkaline phosphatase in tooth germs and bones of the rat.

The tissue distribution and developmental appearance of alkaline phosphatase and Gla proteins, relative to mineralization were examined. Undecalcified sections of tooth germs, calvaria and alveolar bone of the rat were immunostained for Gla proteins, (indirect immunofluorescence and avidin-biotin-peroxidase-complex) and then stained for alkaline phosphatase activity. In the growth regions of tooth germs, Gla protein was observed in young odontoblasts and in early predentin, at a stage prior to formation of the first mineralized dentin. Similar results were obtained for bone: young osteoblasts as well as osteoid were immunopositive for Gla protein prior to formation of the first mineralized bone. Histochemical staining revealed that differentiating odontoblasts and osteoblasts exhibited alkaline phosphatase activity at stages prior to appearance of Gla proteins and that cells adjacent to odontoblasts and osteoblasts (not directly involved in the formation of predentin/dentin and osteoid/bone) stained for alkaline phosphatase, but not for Gla proteins. We conclude, that in these bone- and dentin-forming cells, alkaline phosphatase activity is expressed before the appearance of Gla proteins, but that both appear before the onset of mineralization. We also conclude that Gla protein is a more specific marker for bone and dentin formation than histochemical alkaline phosphatase activity, since only the cells directly involved in these processes were immunostained for Gla protein.

Alkaline Phosphatase↗
PubMed1985

Vitamin D and skeletal tissues.

It is now accepted that vitamin D is an integral part of a complex endocrine system, one with far-reaching implications in mineral metabolism. Reviews of the sources, functions and metabolism of vitamin D, as currently understood, are presented as a prelude to discussions of the role of vitamin D in calcium and phosphorous homeostatis and possible specific roles for vitamin D in mineralized tissues. Data describing a possible regulatory function for vitamin D in bone and bone protein metabolism are presented. Some of the controversy which presently exists regarding the biochemical mechanism of the action of this vitamin is discussed. Finally, the possible relationship of vitamin D and disorders of skeletal tissues is described.

Animals↗
PubMed1985

Appearance of dentin gamma-carboxyglutamic acid-containing proteins in developing rat molars in vitro.

An in vitro model of mineralization was devised in order to study the developmental appearance of dentin gamma-carboxyglutamic acid-containing proteins (DGPs) in relation to the onset of mineralization. Maxillary third molars from 11-day-old rats were cultured with or without fetal calf serum (FCS) as modified from Navia et al. (1984). Molars were incubated without radiolabel, or with either 45CaCl2 (5 microCi/ml) for 24 hr at various stages of a ten-day culture period or [3H]-leucine (10 microCi/ml) for 24 hr at the eighth day of culture. Molars were lyophilized and extracted with 10% formic acid overnight at 4 degrees C. DGPs in extracts were detected by immunologic and chromatographic techniques; DGPs in molar sections were detected by immunolocalization using indirect immunofluorescence. Molar development was evaluated histologically using the Von Kossa staining technique. Molars cultured with FCS showed histologic evidence for mineralized dentin and enamel and a significant increase in 45Ca uptake after the sixth day in vitro. Eleven-day-old molars in vivo and molars cultured without FCS showed no evidence of the presence of mineralized tissues. [3H]-Leucine-labeled DGPs were isolated and identified by affinity and reversed-phase high-performance liquid chromatography and by gel electrophoresis from both mineralized and unmineralized molars. DGP antigens were localized immunohistochemically using rabbit anti-rat antibodies raised against a highly purified DGP preparation. In the unmineralized molar, antigenicity was seen in odontoblasts but not in predentin matrix, preodontoblasts, or in any other cell type. Antigens in the mineralized molar were localized to odontoblasts and dentin.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗