Osteocalcin is one of the most abundant noncollagenous proteins found in adult bone. It is a highly conserved gamma-carboxyglutamic acid-containing protein that is believed to be produced exclusively by osteoblasts. In this study, intracellular and extracellular localization of osteocalcin in osteosarcoma was examined with anti-osteocalcin antibody and in situ hybridization using a synthetic oligonucleotide. Immunohistochemically, osteoblastic osteosarcomas were all positive for osteocalcin. The chondroblastic osteosarcomas were positive on the neoplastic chondrocytes. The five fibroblastic osteosarcomas out of seven were positive for osteocalcin immunostaining over the neoplastic spindle cells. Five cases of osteoblastic osteosarcomas out of seven were positive for osteocalcin in situ hybridization. Two cases of chondroblastic osteosarcomas and three cases of fibroblastic osteosarcomas were positive for in situ demonstration of osteocalcin. The malignant tumor giant cells were positive for osteocalcin immunostaining 83%. They were also positive for in situ hybridization. The benign giant cells in five giant cell tumors and five aneurysmal bone cysts were negative for osteocalcin immunostaining. The benign giant cells in three chondroblastoma and three Paget's disease were positive for osteocalcin. In this study, the osteocalcin in situ hybridization and immunostaining has very important meaning for making differential diagnoses of, especially giant cell rich bone forming tumors.
Base Sequence ; Bone Neoplasms/*chemistry ; Human ; Immunohistochemistry ; In Situ Hybridization ; Molecular Sequence Data ; Osteocalcin/*analysis ; Osteosarcoma/*chemistry

OBJECTIVETo investigate the modulation of pilose antler extract (PAE) on rat osteogenic cells UMR-106 in vitro.

METHODComponent P2 of PAE was isolated by Sephacryl S-200HR gel filtration chromatography. The proliferative effects of P2 and other components isolated by Sephacryl S-200HR on UMR-106 cells were investigated by MTT assay.

RESULTThe P2 could significantly increase the proliferation rate of osteogenic cells. When the protein concentration of P2 was between 0.972 mg x L(-1) and 97.2 mg x L(-1), it could inhibit the proliferation of UMR-106 cells. But while the concentration was equal to or greater than 97.2 mg x L(-1), the P2 could increase the proliferation rate of cells, especially 477.92% at 9.72 g x L(-1), which was approximated to 499.62% of PAE. The molecular weight of the P2 was about 59 kDa determined by SDS-PAGE. On the other hand, inhibition was also observed in the sample of the P3, P4 and P5.

CONCLUSIONThose regulative factors in PAE which have different molecular weight can affect the proliferation of UMR-106 cells two-wayly. And this adjustment also relies on the dose of those factors. This finding may help us to understand the possible mechanism of Chinese traditional medicine from animal materials.

Animals ; Antlers ; chemistry ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deer ; Materia Medica ; isolation & purification ; pharmacology ; Osteosarcoma ; pathology ; Rats ; Tissue Extracts ; isolation & purification ; pharmacology

AIMTo study the antitumor peptide components in the stems and leaves of mistletoe (Viscum coloratum (Kom.) Nakai), the primary structure of the novel peptide was elucidated.

METHODSCation exchange, gel filtration and HPLC were employed for isolation and purification. Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry was used to determine the mass. The complete amino acid sequence of the novel peptide was obtained by Edman degradation combined with enzyme digestion. The antitumor activity of the peptide in vitro was studied with MTT method.

RESULTSThe primary stucture of the peptide named as viscotoxin B2 is KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK. The IC50 value of viscotoxin B2 on the Rat Osteoblast-like Sarcoma 17/2.8 cells in vitro is 1.6 mg x L(-1).

CONCLUSIONViscotoxin B2 in V. coloratum, which has high similarity with viscotoxins from V. album, showed antitumor activity.

Amino Acid Sequence ; Animals ; Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Bone Neoplasms ; pathology ; Inhibitory Concentration 50 ; Molecular Sequence Data ; Molecular Weight ; Osteosarcoma ; pathology ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Plant Leaves ; chemistry ; Plant Proteins ; chemistry ; isolation & purification ; pharmacology ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Tumor Cells, Cultured ; drug effects ; Viscum ; chemistry

OBJECTIVETo investigate the effect of minocycline hydrochloride ointment on cell attachment and proliferation on titanium disks.

METHODSCommercially pure (grade 4) machined titanium discs with three different kinds of surfaces (smooth, acid-etched and sandblasted combined with acid-etched) were treated with minocycline ointment for 1 week, and then cleaned in ultrasonic cleanser for 10 minutes. Surface properties were examined by scanning electron microscope (SEM) and roughness tester before and after the treatment. Surface roughness was compared by paired t test. MG-63 (human osteoblast-like osteosarcoma cell) cells were seeded on these three kinds of discs with or without minocycline treatment, and methl thiazolyl tetrazolium (MTT) was performed to investigate the attachment in the 1st day and proliferation in the 4th and 7th day. Data were analyzed by double factor analysis of variance.

RESULTSSurface roughness before and after minocycline application was as follows, Smooth: (0.093 ± 0.025) µm, (0.086 ± 0.026) µm; Acid-etched: (1.100 ± 0.095) µm, (1.009 ± 0.196) µm; Sandblasted combined with acid-etched: (2.837 ± 0.283) µm, (2.968 ± 0.206) µm. No significant changes in roughness were found before and after minocycline application (P values were 0.118, 0.436 and 0.692). SEM examination revealed as similar surface configuration after minocycline application as before, except for some remnant of the minocycline ointment in acid-etched and sandblasted combined acid-etched groups. In MTT test, the growth of MG-63 cells in the 1 st, 4th day and 7th day was not different between groups with and without minocycline application (P values were 0.450, 0.848 and 0.835), and among three groups of different surface (P values were 0.184, 0.579 and 0.331).

CONCLUSIONSMinocycline hydrochloride ointment did not affect the surface configuration, surface roughness or the properties for cell attachment and proliferation of titanium discs.

Acid Etching, Dental ; Bone Neoplasms ; pathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Microscopy, Electron, Scanning ; Minocycline ; administration & dosage ; pharmacology ; Ointments ; Osteoblasts ; pathology ; Osteosarcoma ; pathology ; Surface Properties ; Titanium ; chemistry

OBJECTIVEThe activity of deer serum albumin on proliferation of rat osteogenic-like cells UMR-106 and the IGF-I secretion were investigated in order to elucidate pilose antler's bone-strengthening mechanism.

METHODDeer serum albumin was isolated from freeze-dry pilose antler powder extract. The methods were Sephacryl S-200HR gel filtration, POROS 20QE ion-exchange and TSK G3000SW chromatographies. The effect of deer serum albumin on proliferatio of UMR-106 cells was assaied by MTT, and the secretion of IGF-I of UMR-106 cells was assaied by RIA.

RESULTDeer serum albumin, with the molecular weight of 56.3 kDa, significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF-I secretion. When concentration of deer serum albumin reached 0.149 microg x mL(-1), UMR-106 cell proliferation rate was 241.03% and IGF-I secretion was 66.89 ng x mL(-1).

CONCLUSIONThe concentration of deer serum albumin, from 14.9 ng x mL(-1) to 14.90 microg x mL(-1), significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF- I secretion.

Animals ; Antlers ; chemistry ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deer ; Insulin-Like Growth Factor I ; secretion ; Materia Medica ; isolation & purification ; pharmacology ; Osteoblasts ; metabolism ; pathology ; Osteosarcoma ; pathology ; Rats ; Serum Albumin ; isolation & purification ; pharmacology

OBJECTIVETo study the effects of ultraviolet (UV) light-treatment on the physicochemical properties and bioactivity of micro-arc oxidation (MAO) titanium surfaces in vitro.

METHODSThe pure titanium were prepared using MAO. MAO titanium samples were treated with 15 W bactericidal lamp UVC [λ = (250 ± 20) nm] or 15 W mercury lamp [λ = (360 ± 20) nm] for 24 h under ambient conditions. Three sample groups were prepared: MAO, UVA treated after MAO (MAO + UVA), UVC treated after MAO (MAO + UVC). The surface physicochemical properties were evaluated using scanning electron microscopy (SEM), contact angle measuring device, energy dispersive X-ray spectrometer (EDX), and X-ray diffraction (XRD). Bicinchoninic acid (BCA) based colorimetric detection was used to quantify the percentage of albumin adsorption after 2 h, 6 h, and 24 h incubation on the titanium surfaces. The rates of MG-63 cells attached to each group titanium surfaces were calculated by nucleus immunofluorescence using Hoechst 33342 after 1 h, 2 h, and 4 h incubation. SEM was used to observe cell morphology on titanium surfaces in each group.

RESULTSNo obvious differences in surface topography, TiO(2) crystal and elemental composition were detected on titanium surfaces with or without UV treatment. Statistically significant difference in contact angles among MAO + UVC group (65.34 ± 1.16)°, MAO + UVA group (44.64 ± 1.28)°, and MAO group (3.41 ± 0.48)° were found (P < 0.001). The percentage of albumin adsorption reached the plateau after 2 h incubation on MAO + UVC titanium surfaces (48.16 ± 1.24)%, which was higher than those in MAO [(8.22 ± 2.99)%] and MAO + UVA groups [(5.29 ± 2.27)%, P < 0.001]. The rates of cells attached to the surfaces of MAO + UVC titanium was greater than that on MAO surfaces and MAO + UVA surfaces after 1 h [(40.71 ± 4.08)%], 2 h [(53.72 ± 2.38)%], 4 h [(70.32 ± 2.85)%] incubation (P < 0.05). The MAO + UVC surfaces remarkably enhanced the spread of MG-63 cells, however, there was no significant difference between the group of MAO and MAO + UVA.

CONCLUSIONSPretreatment of micro-arc oxidation titanium with UVC light considerably improved the surface bioactivity to MG-63 cells, which showed an increase in cellular attachment and spread.

Absorption ; Albumins ; metabolism ; Cell Adhesion ; Cell Line, Tumor ; Humans ; Microscopy, Electron, Scanning ; Osteosarcoma ; pathology ; Oxidation-Reduction ; Spectrometry, X-Ray Emission ; Surface Properties ; Titanium ; chemistry ; radiation effects ; Ultraviolet Rays ; X-Ray Diffraction

The cajanine (longistylin A-2-carboxylic acid) is isolated and identified from extracts of Cajanus cajan L. (ECC) , which structure is similar to diethylstilbestrol. The regulation properties of the cajanine and other four extracts of Cajanus cajan L. (32-1, 35-1, 35-2, and 35-3) were tested in human osteoblast-like (HOS) TE85 cells and marrow-derived osteoclast-like cells. By using MTT assay to test the change of cell proliferation, 3H-proline incorporation to investigate the formation of collagen, and by measuring alkaline phosphatase (ALP) activity, bone formation in HOS TE85 cell was evaluated after pretreated for 48 hours. Bone marrow cells were cultured to examine the derivation of osteoclast cells (OLCs), which were stained with tartrate-resistant acid phosphatase (TRAP). The long term effect (pretreated for 18 days) on promoting mineralized bone-like tissue formation was tested by Alizarin red S staining in HOS TE85 cells. After the treatment with cajanine (1 x 10(-8) g x mL(-1)) for 48 hours, cell number increased significantly (57.7%). 3H-Proline incorporation also statistically increased (98.5%) in those cells. Significant change of ALP activity was also found (P < 0.01) in 35-1 and 35-3 treated cells (they were 66.2% and 82.4% in the concentration of 1 x 10(-8) g x mL(-1), respectively). The long term (18 days) effects of 32-1 and 35-3 on promoting mineralized bone-like tissue formation in HOS TE85 cell were obvious. There were much more red blots over the field of vision compared with that of control group. After the treatment of cajanine, derived-osteoclast cells appeared later and much less compared with control. The inhibition of cajanine was 22.8% while it was 37.9% in 32-1 treated cells in the dose of 1 x 10(-7) g x mL(-1). It is obvious that cajanine and ECCs promoted the osteoblast cells proliferation and mineralized bone-like tissue formation in HOS TE85 cells, while inhibited derivation of osteoclast cells. All of these suggested that cajanine has the estrogen-like action on osteoblast and osteoclast, which could be developed as anti-osteoporosis drugs.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Bone Neoplasms ; metabolism ; pathology ; Cajanus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Diethylstilbestrol ; analogs & derivatives ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Osteoblasts ; drug effects ; Osteoclasts ; cytology ; metabolism ; Osteogenesis ; drug effects ; Osteosarcoma ; enzymology ; pathology ; Phytoestrogens ; isolation & purification ; pharmacology ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar

Expressions of p53 protein, a product of the tumor suppressor gene were studied in osteosarcomas relating to various prognostic factors. Thirty-four osteosarcomas were investigated immunohistochemically with a monoclonal antibody clone PAb240, which recognizes a common conformational epitope of mutant p53 proteins and another clone PAb1801, which reacts with both wild- and mutant-type p53 proteins. The results were compared with expressions of proliferating cell nuclear antigen (PCNA) and Ki-67 providing a simple method for the assessment of growth fractions of tumors. PAb240 stained nuclei and cytoplasm of tumor cells in 8 of 34 osteosarcomas (23.5%), whereas PAb1801 reacted in all 34 osteosarcomas (100%). Fifteen tumors (44.1%) showed positivity for PAb1801 in more than half of the tumor cells. Twelve patients were alive and thirteen were dead. Tumors from 9 patients (75%) who survived revealed only focal positive immunoreactions with PAb1801 and tumors from 6 patients (46.1%) who died revealed diffuse reactions. Twelve cases (35.3%) showed a high PCNA index (> 40%) and fibroblastic osteosarcomas revealed the highest PCNA positivity. Twenty-two cases (64.7%) revealed a very low Ki-67 index (less than 10%) and Ki-67 index showed a good correlation with PCNA positivity (r = 0.6247). Expressions of both wild-and mutant-type p53 protein, PCNA, and Ki-67 were not correlated with other clinical or pathological parameters.
Adolescent ; Adult ; Aged ; Antibodies, Monoclonal ; Bone Neoplasms/*chemistry/genetics/pathology ; Cell Cycle/physiology ; Child ; Child, Preschool ; Genes, p53 ; Human ; Immunohistochemistry ; Ki-67 Antigen ; Male ; Middle Age ; Mutation ; Neoplasm Proteins/*analysis ; Nuclear Proteins/*analysis ; Osteosarcoma/*chemistry/genetics/pathology ; Proliferating Cell Nuclear Antigen/*analysis ; Protein p53/*analysis ; Support, Non-U.S. Gov't

OBJECTIVETo investigate the effects of 20% fluor-hydroxyapatite (FHA) on proliferation and osteogenic differentiation of human MG63 osteosarcoma cells.

METHODSFHA was prepared by chemical precipitation method, and its structure and surface features were tested by scanning microscope, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy. MG63 cells were cultured and divided into FHA, hydroxyapatite (HA) and control groups (n = 3). The proliferation of the cells was evaluated using methylthiazol tetrazolium (MTT) assay. ALP activity of the cells was assessed. Osteogenic differentiation was evaluated based on the reverse transcription PCR (RT-PCR) of differentiation-related genes, namely, collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OCN) and core binding factor α1 (Cbfα1). The data were analyzed statistically by one-way analysis of variance using SPSS 13.0 software.

RESULTSXRD test showed that the main crystalline phase of FHA was similar to that of HA. Absorptance value of cells exposed to FHA(1.87±0.06) measured by MTT was higher than that of the control(1.25±0.02) on the third day(P < 0.05), and there was no statistically significant difference between the cells exposed to FHA and HA(1.84±0.03) (P > 0.05). ALP activity of the cells exposed to FHA(4.62±0.09)was higher than that of the control (1.92 ± 0.05) (P < 0.05). RT-PCR tests showed that compared with the control, FHA up-regulated the expression of Col I, ALP and OCN mRNA, down-regulated the expression of Cbfα1 mRNA.

CONCLUSIONSFHA enhances the proliferation and osteogenic differentiation-related gene expression, and has good biocompatibility.

Alkaline Phosphatase ; genetics ; metabolism ; Analysis of Variance ; Biocompatible Materials ; Bone Neoplasms ; metabolism ; pathology ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; physiology ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; Durapatite ; chemistry ; pharmacology ; Humans ; Hydroxyapatites ; Osteocalcin ; genetics ; metabolism ; Osteogenesis ; Osteosarcoma ; metabolism ; pathology ; Spectroscopy, Fourier Transform Infrared ; X-Ray Diffraction