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PubMed2001

[Effect of sensitized lymphocytes on rabbit calvarial osteoblasts].

OBJECTIVE: To study the effects of sensitized T lymphocytes on osteoblastic proliferation and differentiation. METHODS: Sensitized lymphocytes were obtained from a rabbit sensitized by chromium, and lymphocyte cultured mediums (LCM) were collected. Osteoblasts were obtained from new-born rabbit calvaria. The osteoblasts were identified with alkaline phosphatase staining and mineralization in vitro. By osteoblast cultured in vitro, osteoblastic proliferation, and production of alkaline phosphatase and osteocalcin were evaluated in three conditions: without LCM, with LCM not stimulated by Cr(6+) and with LCM stimulated by Cr(6+). RESULTS: The cells from rabbit calvaria were positive to ALP staining and could form calcium node in vitro. The sensitized LCM could inhibit proliferation of osteoblasts, increase alkaline phosphatase production and decrease osteocalcin production. These results all had statistically significant difference by ANOVA. CONCLUSION: Sensitized T lymphocytes could decrease the number of mature osteoblasts and impede the formation of mature mineralized bone.

Alkaline Phosphatase↗

PubMed2002

Differential expression of Notch genes in human osteoblastic cells.

Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types. In the present study we investigated the expression of four Notch genes in primary human osteoblasts and in human osteoblastic osteosarcoma cell line SaOS-2 by RT-PCR. We found a strong constitutive expression of Notch-1 and a weak constitutive expression of Notch-2 in both cell types. After stimulation with Dexamethasone or Vitamin D(3), two factors known to induce differentiation in osteogenic cells, both Notch receptors were downregulated, however, with a different time course. Notch-1 and Notch-2 showed a transient induction after 2 days and a decrease after 7 days in osteoblasts and after 28 days in SaOS-2 cells. Notch-4 expression could only be detected after stimulation with Dexamethasone and Vitamin D(3). However, in osteoblasts a transient induction after 2 days could be detected in osteoblasts, whereas Notch-4 expression increased after 14 and 28 days in SaOS-2 cells. In contrast, Notch-3 was not expressed in human osteoblasts and SaOS-2 osteosarcoma cells. These data show, that Notch genes are expressed in human osteoblastic cells and that the expression is differentially regulated upon stimulation with osteogenic factors.

Cell Differentiation↗

PubMed2002

[Dexamethasone inhibits osteoblastic differentiation by down-regulation of LIM mineralization protein 1].

To investigate the mechanisms of the inhibition of osteoblastic differentiation by dexamethasone (DEX), the effects of different doses of DEX on the activity of alkaline phosphatase (ALP), the synthesis of osteocalcin (OC) and the expression of collagen type I were observed in the cultured rat osteoblasts. The LIM mineralization protein-1 (LMP-1) mRNA, a positive regulator of osteoblasts, was semi-quantified by RT-PCR. The results showed that a lower dose (10(-9) mol/L) of DEX could enhance the activity of ALP, the synthesis of OC and the expression of collagen type I. However, a higher dose (10(-7) mol/L) of DEX inhibited them and down-regulated the expression of LMP-1 mRNA in osteoblasts. It is suggested that DEX stimulates osteoblast differentiation at lower dose, while at higher dose it inhibits osteoblast differentiation. The inhibitory action of DEX on osteoblast differentiation might be mediated by the down-regulation of LMP-1 mRNA.

Animals↗

PubMed2002

Apoptosis of rat osteoblasts in process of calcification in vitro.

AIM: To establish a cell model of osteoblasts retaining their differentiated phenotype in culture and observe the apoptosis of osteoblasts in the process of calcification using a novel acetylcholinesterase (AChE) staining method. METHODS: Osteoblasts were isolated enzymatically from skull of newborn SD rats; alkaline phosphatase (AKP) activity was determined by reformed cobalt method and azo dye method; mineral deposition was assessed with Von Kossa staining and Fluo-3 staining; a novel AChE staining method was used to assay cellular apoptosis based on the higher expression of AChE in apoptotic cells. RESULTS: During the 44 d of cells cultured, primary rat skull-derived osteoblasts progressively developed into a bone-like tissue of multi-layered nodules of cells with mineralized extra-cellular matrix and the apoptotic cells increased while the matrix calcificated. CONCLUSION: The phenotype of developmental sequence of rat skull-derived osteoblasts can reflect the maturation of osteoblasts in vitro. It is a convenient model for the research of osteoblasts biology.

Alkaline Phosphatase↗

PubMed2003

Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro.

OBJECTIVE: To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro. METHODS: Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37 degrees C in a relative humidity of 95% and 5% CO2. Then, the cells were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10(-8) mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro. RESULTS: In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survival, stable biochemical indices and excellent purification. Under the condition of dexamethasone 10(-8) mol/L and osteoblasts 10,000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis. CONCLUSIONS: Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.

Adult↗

PubMed2003

The expression of insulin-like growth factor-I mRNA and polypeptide in rat osteoblasts with exposure to parathyroid hormone.

OBJECTIVE: To investigate the insulin-like growth factor-I (IGF-I) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF-I in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH). METHODS: The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2) Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours. RESULTS: The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF-I mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF-I concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF-I antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm. CONCLUSIONS: PTH rapidly and constantly stimulates the IGF-I gene transcription of osteoblast. There was an obvious discrepancy between the IGF-I mRNA content within the osteoblast and the free IGF-I level around the osteoblast in either mode of PTH action. The IGF-I might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts.

Animals↗

PubMed2004

Expression of albumin in bone tissues and osteoblastic cells: involvement of hormonal regulation.

The expression of albumin in bone-related cells was investigated. Bone marrow cells and femoral-diaphyseal and -metaphyseal tissues were obtained from normal rat femur. Western blot analysis for protein in the lysate of bone marrow cells showed expression of albumin. The production of albumin was found in the bone marrow cells and femoral-diaphyseal and -metaphyseal tissues obtained from rat femur. When the femoral-diaphyseal and -metaphyseal tissues were cultured for 48 h in a serum-free medium in vitro, albumin was greatly released into culture medium from the bone tissues. The expression of albumin in osteoblastic MC3T3-E1 cells was investigated by Western blot analysis. Osteoblastic cells were cultured for 48 h in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (human PTH; 10(-7) M), 17beta-estradiol (10(-9) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or zinc sulfate (10(-4) M). The production of albumin in osteoblastic cells was significantly enhanced in the presence of PTH, IGF-I, 17beta-estradiol or zinc sulfate. These factors stimulated significantly the release of albumin from osteoblastic cells into culture medium. The effect of IGF-I (10(-8) M) in increasing the production of albumin in osteoblastic cells was completely prevented in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcriptional activity. This study demonstrates that albumin is expressed in bone marrow cells and osteoblastic cells, and that the production of albumin in osteoblastic cells is enhanced by various bone anabolic factors in vitro.

3T3 Cells↗

PubMed2004

[Effects of kidney-tonifying Chinese herbal drugs on human osteoblast Ca2+ intake and mineralization in vitro].

OBJECTIVE: To study the effects of kidney-tonifying Chinese herbal drugs on Ca2+ intake and mineralization of human osteoblasts in vitro. METHODS: Human osteoblasts were isolated from the iliac trabecular bone followed by purification and culture at 37 degrees Celsius with 5% CO2. The cells were identified by cell morphology, calcium nodule formation and alkaline phosphatase (ALP) activity assay. The third passage of the cultured osteoblasts were treated with 10% scrum from rat fed with the decoction of the kidney-tonifying Chinese herbal drugs of different concentrations for 30 min, 3 d and 28 d, respectively. The cells treated with 10% rat serum without the drugs served as the control. Flow cytometry was used to observe the changes in cell proliferation and intracellular Ca2+ concentration, and von Kossa staining employed for quantification of the mineral nodules. RESULTS: The osteoblasts obtained were positive for ALP staining and could form calcium nodules in vitro. Flow cytometry showed that the drugs at different concentrations all increased Ca2+ influx, as compared with the control cells. The drugs also increased the relative proliferation index of the osteoblasts, and high concentration of the drugs resulted in greater number of the mineral nodules in the osteoblasts (P<0.05). CONCLUSION: The kidney-tonifying Chinese herbal drugs may increase Ca2+ influx and stimulate proliferation and differentiation of adult osteoblasts in vitro.

Alkaline Phosphatase↗

PubMed2005

[The changes of cytoskeleton F-actin in rat bone marrow mesenchymal stem cells and calvarial osteoblasts under mechanical strain].

OBJECTIVE: To explore the response of rat bone marrow mesenchymal stem cells (MSCs and calvarial osteoblasts to mechanical strain and the consequent changes of cytoskeleton F-actin. METHODS: Bone marrow MSCs and calvarial osteoblasts were isolated from SD rats and cultured in vitro. Mechanical stretch was performed on passage 3 cells at 2 000 microepsilon for 0, 2, 6 and 12 hours using four-point bending system. The response of cells and the distribution of F-actin were observed using fluorescent staining under laser scanning confocal microscope and the morphological parameters were quantified using image analysis software Laserpix. RESULTS: Under mechanical stretch, the fluorescent staining decreased obviously at both MSCs and osteoblasts, and F-actin filaments were rearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleus became unclear and apoptotic changes were observed at some of both cells. Cellular size decreased more significantly in MSCs than in osteoblasts. Quantity analysis showed that total area of cells, total fluorescent density and green fluorescent density (F-actin) were all significantly decreased in MSCs (P < 0.05 or P < 0.01), and total fluorescent density, green fluorescent density and red fluorescent density (nuclei) did also in osteoblasts (P < 0.05 or P < 0.01). CONCLUSION: Mechanical stretch caused extensive response on both MSCs and osteoblasts which led to the rearrangement of F-actin filament and apoptosis in some of these cells. MSCs were more sensitive to mechanical strain than osteoblasts.

Actin Cytoskeleton↗

PubMed2005

[Effect of raloxifene and estradiol on the biological function and osteoprotegerin expression of osteoblasts in vitro].

OBJECTIVE: To study the effect of raloxifene and estradiol on the biological function and osteoprotegerin expression of osteoblasts in vitro. METHODS: Different doses of raloxifene and estradiol were added into the medium of the second generation osteoblasts from the shull of newborn SD rats. The proliferation, differentiation, mineralization and osteoprotegerin expression of the osteoblasts in different groups were observed. RESULTS: Raloxifene had no significant effects on stimulating the proliferation, the expression of osteoprotegerin and the formation of mineral nodes of the cultured osteoblasts (the trial vs the control, P>0.05). However, raloxifene can significantly improve the alkaline phosphatase activity of the cultured osteoblasts (the trial vs the control, P<0.05). Estradiol significantly increased the proliferation of osteoblasts, and differentiation, mineralization, expression of osteoprotegerin (P<0.01 vs the control). CONCLUSION: The effects of raloxifene and estradiol on the cultured osteoblasts are different, which implied their different mechanisms in inducing osteoporosis.

Alkaline Phosphatase↗

PubMed2005

[The study of interaction between the osteoblast and bio-derived material by detecting the gene expression].

OBJECTIVE: To study the gene expressions of human osteoblasts during the construction of tissue engineered bone with the bio-derived material. METHODS: The fetal osteoblasts were used to construct tissue engineered bone with the bio-derived material and then were cultured 2, 4, 6, 8 and 10 days in vitro. Real-time PCR analysis indicated that Cbfa 1, Osterix, Collagen type I, osteocalcin (OC) and Integrin alpha5 and beta1 were present in osteoblasts with bio-derived materials. Results The change of Cbfa1 was consistent with the change of Osterix. On 2nd day and 8th day, the expression of Osterix in experimental group was higher than that in control group, P<0.05. Collagen type I's change was consistent with change of OC expression, and its expression was higher in experimental group than that in control group on 2nd, 4th, 6th and 8th day. The Integrin expression was high all along. Conclusion The important genes can be expressed normally by integrating osteoblasts with bio-derived scaffolds. As skeleton tissue engineering scaffold, the bio-derived bone is conducive to keep the osteoblast's phenotype and differentiation with osteo-conductive ability. The osteoblast can enter proliferation stage favorably and the scaffold materials exert no effects on it. Bio-derived bone can also supply more space for cells to proliferate. The bio-derived materials promote osteoblasts adhesion.

Adult↗

PubMed2006

[Effects of osteoblasts and inductor on proliferation and osteodifferentiation of marrow stromal stem cells].

OBJECTIVE: To find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissue engineering. METHODS: There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone, 10 mmol/L beta-glycero-phosphate, 50 microg/ml retinoic acid) on growth of MSCs isolated from the rat femur and the tibia. MSCs were cultured in the DMEM medium (the control group) and in the osteoinductive culture medium (the inductor group); furthermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group). The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured. The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymerase chain reaction (RT-PCR). RESULTS: There were more cells in the osteoblast group than in the control group (31.73 +/- 3.31 x 10(4) VS. 24.33 +/- 3.04 x 10(4), P < 0. 05), but there were fewer cells in the inductor group (16.23 +/- 2.44 x 10(4), P < 0.05). There was no significant difference in the cell number between the combined treatment group (21.54 +/- 2.29 x 10(4)) and the control group (P > 0.05). The ALP activity was higher in the combined treatment group (2.01 +/- 0.56 U) than in the control group (1.27 +/- 0.43 U), in the inductor group (1.27 +/- 0.43 U), and in the osteoblast group (0.77 +/- 0.19 U). The osteocalcin mRNA was expressed in the three treatment groups but was not expressed in the control group. The significantly higher level of the osteocalcin mRNA was expressed in the inductor group (0.783 +/- 0.094) and in the combined treatment group (0.814 +/- 0.071) than in the osteoblast group (0.302 +/- 0.026) (P < 0.05). CONCLUSION: The combined use of the osteoblast and the inductor can induce marrow stromal cells. Their combined use does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering.

Animals↗

PubMedDate not supplied

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Analysis of Variance↗

PubMed1991

Gene expression during skeletal development in three osteopetrotic rat mutations. Evidence for osteoblast abnormalities.

Osteopetrosis is a group of metabolic bone diseases characterized by reductions in osteoclast development and/or function. These aspects of osteoclast biology are known to be influenced by osteoblasts and their products. To ascertain whether osteoblast dysfunction contributes to aberrations in the structural and functional properties of osteoclasts in osteopetrosis, we systematically examined gene expression as reflected by mRNA levels for a series of cell growth- and tissue-related genes associated with the osteoblast phenotype during skeletal development in normal and mutant rats of three different osteopetrotic stocks. We show that the methods used permit the reproducible isolation of undegraded total cellular RNA from bone and that mRNA levels can be reliably quantitated in these preparations. Each osteopetrotic mutation exhibits a distinct aberrant pattern of osteoblast gene expression that may be correlated with and explain some abnormalities in extracellular matrix composition, mineralization, osteoclast development, and effects of elevated serum levels of 1 alpha,25-dihydroxyvitamin D3, depending upon the mutation. Normal rats show minor variations in gene expression that reflect the genetic background (stock). This, the first comprehensive molecular analysis of osteoblast gene expression in osteopetrosis, suggests that some osteopetroses, particularly in the toothless rat, are associated with and potentially related to mechanisms associated with aberrations in osteoblast function. More generally, the present studies demonstrate alterations in gene expression as reflected by mRNA levels that are associated with functional properties of the osteoblast, particularly those contributing to the recruitment and/or differentiation of osteoclasts, thereby influencing skeletal modeling.

Animals↗

PubMed1989

[Comparative test of the soluble toxicity of hydroxyapatite ceramics using human and animal osteoblasts].

The soluble toxicity of 5 different hydroxylapatite granulates was tested in osteoblast cell cultures. We established a human osteoblast-like cell culture and a culture of rat (Lewis) osteoblast-like cells. The granulates were also tested in cultures of human gingival fibroblasts as a conventional system. It is demonstrated that the osteoblast-like cell cultures are of higher sensitivity than fibroblast cultures which do not show any reaction to the extracts of hydroxylapatite granulates. Only 2 hydroxylapatite granulates show some toxic. The human osteoblast-like cells reacted slightly more sensitive to toxic substances of the materials tested than rat osteoblast-like cells. It is demonstrated that the osteoblast-like cell cultures are a highly sensitive test system for materials with low toxicity.

Animals↗

PubMedDate not supplied

Three-dimensional type I collagen gel system containing MG-63 osteoblasts-like cells as a model for studying local bone reaction caused by metastatic cancer cells.

We inoculated the KLE human endometrial cancer, MCF-7 and ZR-75 human breast cancer, and PC-3 human prostate cancer cells into three-dimensional type I collagen gel system that contained uniformy dispersed MG-63 osteoblast-like cells. Then, we analyzed the morphological evidence of osteoblasts reaction, local invasion around the inoculated cancer cells and expression of the cathepsin D and urokinase-type plasminogen activator (uPA) around the sites of inoculation using immunocytochemistry. The prostate cancer cells produced morphological evidence of blastic reaction presented as an increased number of MG-63 osteoblasts and increase density of type I collagen around the sites of inoculation with PC-3 cells. The inoculated MCF-7 and ZR-75 cells decreased the density of type I collagen and number of osteoblasts and invaded the collagen gel around the sites of inoculation. The KLE endometrial cancer cells and cell-free media produced no reaction at the inoculation sites suggestive of cancer cell-specific interactions with osteoblasts in this system. The expression of uPA was remarkably higher at the inoculation sites of PC-3 cells as compared with those of the other cancer cells. Cathepsin D expression was higher at the sites of inoculation with KLE, MCF-7 and PC-3 cancer cells. MG-63 osteoblasts contained relatively low expression of uPA and cathepsin D. We conclude that this collagen gel system is a useful model for studying the morphological evidence of local invasion and osteoblasts reaction produced in response to local growth of metastatic cancer cell in vitro.

Bone Neoplasms↗

PubMedDate not supplied

Growth factors mediate glucocorticoid receptor function and dexamethasone-induced regression of osteoblastic lesions in hormone refractory prostate cancer.

We investigated the ability of important regulators of osteoblast function, such as insulin-like growth factor I (IGF-I), transforming growth factor beta 1 (TGF beta 1), and urokinase-type plasminogen activator (uPA) to act as mediators in cell-cell interactions between osteoblast-like cells and metastatic prostate cancer cells, in vitro. In addition, we assessed whether these growth substances can (a) mediate glucocorticoid receptor (GR) function and (b) be implicated in dexamethasone-induced regression of osteoblastic tumors. Exogenous IGF-I, rat/human uPA, and PA-III (rat)/PC-3 (human) prostate cancer cells conditioned media (CM) stimulated the proliferation of rat (UMR 106 cells) and human (MG-63 cells) osteosarcoma cells. This mitogenic activity was completely neutralized by anti-IGF-I specific antibody. In addition, dexamethasone decreased cell growth, up regulated TGF beta 1 mRNA, and down regulated uPA mRNA expression in prostate cancer cells. Furthermore, it inhibited cell growth by activating latent-TGF beta 1 in osteoblast-like cells. In addition, dexamethasone down regulated the expression of IGF-I mRNA in osteoblast-like cells. Therefore, it is conceivable that uPA, TGF beta 1 and IGF-I mediate at least in part cell-cell interactions and GR function in osteoblastic metastases. Conceivably, regression of the osteoblastic tumors produced by high-dose dexamethasone treatment in hormone-refractory prostate cancer patients is been mediated by differential regulation of growth factors, locally.

Animals↗

PubMed1997

Human prostatic tumor cells in culture produce growth and differentiation factors active on osteoblasts: a new biological and clinical parameter for prostatic carcinoma.

Prostate cancer (PRCA) cells metastasize to bone with high frequency, inducing typical osteosclerotic lesions. To establish if local stimuli on the bone tissue may derive from metastatic colonies of prostatic origin, we evaluated the biologic activities secreted by human prostatic epithelium and effective on osteoblast-like cells in vitro. Supernatant from short-term tissue cultures of human prostatic tissue samples obtained from PRCA (35 cases) and benign prostatic hyperplasia (BPH, 12 cases) patients were applied to three models of cells with osteoblastic phenotype: two normal [rabbit osteoblasts (OB) and rat periosteal cells (PO)] and one transformed (human osteosarcoma cell line, MG63). Proliferative activity was monitored through enzymatic reduction of tetrazolium salts and expressed as relative mitogenic activities (RMA). Analysis of proliferation and alkaline phosphatase (ALP) activity, a marker of osteoblast function, demonstrates that conditioned media (CM) from PRCA cultures stimulate both growth and activity of osteoblast-like cells to a greater extent compared to CM from BPH. Furthermore, cell growth and activity of osteoblast-like cells are progressively increased by CM derived from patients with stage B (tumor confined within the prostate capsule), stage C (locally invasive tumor), and stage D (invasive tumor with distant metastasis) disease. One of the mechanisms potentially underlying the CM-stimulated effects on bone cells is associated with the urokinase (uPA) enzyme route, whose release progressively increases with the stage of disease. However, antibodies against uPA and p-aminobenzamidine (a low molecular weight urokinase inhibitor) treatment, which both inhibit the proliferative and differentiative effects induced by exogenous urokinase, partially slow down the effects of CM from PRCA tissue cultures, suggesting that additional factors are secreted by prostatic tumor cells in vitro. In conclusion, we show that the mitogenic and differentiative activities for osteoblasts produced by prostatic tumor cells in short-term tissue cultures are related to PRCA stage and may predict the behavior of skeletal metastases in single cases of tumor. In addition, the culture methods used may represent a valid model to study prostatic and bone cellular interactions, which may indicate new therapeutic approaches in metastatic prostate tumors.

Alkaline Phosphatase↗