OBJECTIVETo investigate the timing and location of the expression of bone sialoprotein (BSP) and osteopontin (OPN) in developing dental tissues of rats.

METHODSEvery three neonatal rats were sacrificed at day 1, weeks 1, 2, 3, 5 and 8. The mandibles were dissected and the first molar and the surrounding tissue were fixed, then demineralized with 15% EDTA. Immunohistochemical technique was used to determine the expression of BSP and OPN in dental tissues and the surrounding bone at different time points.

RESULTSImmunoreactivitis of BSP and OPN were present in matured ameloblasts, dentinoblasts, cementoblasts, osteoblasts, and the matrix. The expression of BSP and OPN in cementum and alveolar bone was stronger than that in enamel and dentine. In cementum and alveolar bone, BSP appeared to be concentrated in unmineralized and mineralized tissues, but OPN was concentrated in the mineralizing frontier and reversal line.

CONCLUSIONSBSP and OPN play an important role in the development and mineralization of rat mineralized tissues. The expression of BSP was different from OPN, indicating their different functions.

Animals ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Osteopontin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; analysis ; Tooth ; chemistry

BACKGROUNDTissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.

METHODSbMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.

RESULTSUnder current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).

CONCLUSIONSThis study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.

Alkaline Phosphatase ; metabolism ; Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; metabolism ; ultrastructure ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Microscopy, Electron, Scanning ; Nerve Tissue Proteins ; metabolism ; Osteocalcin ; genetics ; Osteogenesis ; Osteopontin ; genetics ; Rats ; Rats, Inbred F344 ; Reverse Transcriptase Polymerase Chain Reaction ; Sialoglycoproteins ; genetics ; Stromal Cells ; cytology ; metabolism ; ultrastructure ; Tissue Engineering

OBJECTIVETo investigate the osteoblast-like functional characteristics exhibited by human periodontal ligament cells (hPDLCs) under mechanical force.

METHODSHuman PDLCs cultured in vitro were stretched by mechanical force. Radioimmunoassay (RIA) was used to measure the expression of secreting alkaline phosphotase (ALP) and osteocalcin (OCN). The non-secreting ALP, OCN and osteopontin (OPN) in cells were determined by immunohistochemistry.

RESULTSIt exhibited increasing of ALP secreted into conditional media, and in the 24 hour period there were two peaks which appeared at the 2nd and 4th hour and the 24th hour (P < 0.01). While in the late of the 24 hours, expression of OCN in conditional media increased (P < 0.05).

CONCLUSIONMechanical force induces hPDLCs to differentiate into functional osteoblast-like cells and plays a role in bone remodeling.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Humans ; Osteocalcin ; analysis ; Osteoclasts ; physiology ; Osteopontin ; Periodontal Ligament ; cytology ; Sialoglycoproteins ; analysis ; Stress, Mechanical

OBJECTIVETo investigate the expression of matrix extracellular phosphorylated protein (MEPE) in human dental pulp cells (hDPC) undergoing odontogenic induction and explore the role of MEPE in odontoblast-like differentiation.

METHODShDPC were isolated by enzymatic digestion and preceded to odontogenic induction for 7, 14 and 21 days respectively. hDPC before induction served as controls. The expressions of MEPE, dentin sialophosphoprotein (DSPP)/dentin sialoprotein (DSP), bone sialoprotein (BSP) and collagen type I were determined by quantitative real-time RT-PCR and Western blotting.

RESULTSThe mRNA levels of MEPE, DSPP, BSP and type I collagen were increased in a time-dependent manner as hDPC were induced along odontoblastic lineage. Statistical differences were detected for MEPE and BSP mRNA expressions in induced hDPC compared with control group (P < 0.001). For DSPP and type I collagen, the mRNA levels in the induced groups were (12 943.33 + or - 3805.73) and (250.55 + or - 31.86) respectively, which were significantly higher than those in control group on days 21 (P < 0.05). Western blotting also revealed the increased expressions of MEPE, DSP, BSP and type I collagen in the induced DPC.

CONCLUSIONShDPC showed analogously temporal expressions of MEPE, DSPP/DSP, BSP and collagen type I while differentiating along odontoblast lineage. MEPE may play an important role in the odontogenic differentiation of hDPC and may be a potential marker of odontoblast-like differentiation.

Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Collagen Type I ; metabolism ; Dental Pulp ; cytology ; metabolism ; Extracellular Matrix Proteins ; metabolism ; Glycoproteins ; metabolism ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Odontoblasts ; cytology ; Phosphoproteins ; metabolism ; Sialoglycoproteins ; metabolism

OBJECTIVETo test the bovine cementoblasts (CBs) cementum-forming ability in vivo.

METHODSRoot fragments of newborn bovine freshly extracted mandibular incisor were cultured routinely and 4th-5th passages of CBs were harvested. CBs were then cultured in the medium supplemented with 50 mg/L alpha-ascorbic acid and 10 mmol/l beta-glycerolphosphate to form a thick layer as tissue engineering scaffold for cementum formation. Collagen membrane was used as control scaffold. 2 x 10(6) cells were attached to the CBs-made carrier as well as collagen membrane scaffolds and transplanted subcutaneously into immunodeficient mice. Transplants were harvested at 7th week. Histological sections were stained with HE, alizarin red S and van Kossa methods as well as monoclonal Ab against bovine cementum attachment protein (CAP).

RESULTSCBs-made scaffold supported more cementum-like tissue (CLT) formation than collagen-made scaffold. The CLT formed on CBs scaffold was partly calcified with embedded cells. Uncalcified cementoid-like material could be seen on the surface and was encircled by cubical CB-like cells. The CLT was also positive to CAP and van Kossa staining.

CONCLUSIONSThese results suggest that the bovine CBs can form cementum-like tissue. The cell-made carrier is a better scaffold than collagen membrane.

Alkaline Phosphatase ; analysis ; Animals ; Bone Transplantation ; methods ; Cattle ; Cell Adhesion Molecules ; analysis ; Cells, Cultured ; Dental Cementum ; chemistry ; cytology ; transplantation ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Osteocalcin ; analysis ; Osteonectin ; analysis ; Sialoglycoproteins ; analysis ; Tissue Engineering ; methods ; Transplantation, Heterologous

OBJECTIVETo observe the effect of Astragalus-Angelica Mixture (AAM) on osteopontin (OPN) expression in rats with chronic nephrosclerosis.

METHODSChronic nephrosclerosis model rats induced by repeated intraperitoneal injection of puromycin were randomly divided into the model group, AAM group and Irbesartan (an antagonist of angiotensin) group. The experimental course lasted 12 weeks. Blood and urine samples were examined by biochemical method. Kidney tissue was taken for pathological stain and immunohistochemical method and was applied to examine OPN expression, mononuclear macrophage, laminin in extracellular matrix and decorin expressions.

RESULTSAAM showed the effects of decreasing urinary protein and improving renal function similar to that of Irbesartan. It also could alleviate the pathological damage of kidney tissue, especially in decreasing renal tubular mesenchymal damage index. The accumulation of decorin and laminin in the mesenchymal extracellular matrix significantly decreased. Renal tubular OPN expression and mesenchymal infiltration of mononuclear macrophage decreased significantly and in a positive correlated manner (r = 0.885, P < 0.01).

CONCLUSIONAAM has similar renal protective action to that of Irbesartan, this action may be related to the inhibition of up-regulated OPN expression.

Animals ; Astragalus Plant ; Drug Synergism ; Drugs, Chinese Herbal ; pharmacology ; Male ; Nephrosclerosis ; chemically induced ; metabolism ; Osteopontin ; Phytotherapy ; Puromycin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; biosynthesis

OBJECTIVETo investigate the correlation between expression of the osteopontin (OPN) and invasion and metastases in gastric cancer.

METHODSThe expression of OPN, NF-kappaB p65 and matrix metallo-proteinase 9 (MMP-9) was detected by immunohistochemistry in non-cancer gastric tissue (n = 12 cases) and gastric cancer tissue (n = 72 cases).

RESULTS(1) OPN, NF-kappaB p65 and MMP-9 were not expressed in 12 non-cancer gastric tissue samples(group A). Their expression rates were 43.3%, 40.0% and 46.7% respectively in 30 gastric cancer samples without lymph nodes metastasis (group B), but they increased to 76.9%, 73.1% and 80.8% in 26 gastric cancer samples with lymph nodes metastases (group C), and 87.5%, 81.3% and 93.8% respectively in 16 gastric cancer samples with lymph node and distant metastases (group D). (2) There were statistically significant differences in their expressions between group D and group B (P(a) = 0.004, P(c) = 0.007, P(e) = 0.002), and between group C and group B (P(b) = 0.011, P(d) = 0.013, P(f) = 0.009). (3) Despite some differences in positive expression rates, correlations existed between OPN and NF-kappaB p65, and between NF-kappaB p65 and MMP-9 (P(1) = 0.042, P(2) = 0.013; r(1)= 0.67, r(2)= 0.72).

CONCLUSIONOsteopondin espression is closely related to the invasion and metastases of gastric cancer. It may upregulate the expression of metastasis-related molecule MMP-9 by activating NF-kappaB pathway.

Female ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Metastasis ; Osteopontin ; Sialoglycoproteins ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Transcription Factor RelA ; metabolism

Migration of adventitial fibroblasts (AF) is involved in the neointimal formation which is one of the common pathological processes in several vascular diseases. The observation of whether the migratory response of AF from hypertensive animal is different from that of controls may provide an explanation of vascular remodeling in hypertension. We examined whether there is any difference between the migratory activity of AF derived from spontaneously hypertensive rat (SHR) and that from their normotensive counterpart Wistar-Kyoto rats (WKY). In addition, the role of osteopontin (OPN) in cell migration was also examined. Primary cultures of aortic adventitial fibroblasts were derived from SHR and age-matched WKY. Migration of fibroblasts was determined with the Transwell method. The mRNA expression level of OPN was measured by a real-time quantitative PCR. When compared with WKY-derived cells, migration of adventitial fibroblasts from SHR exhibited an increased response when stimulated by 10% serum (cell number per field 35.20+/-5.26 vs 22.2+/-3.27, p<0.05). Chemotaxis induced by 10 ng/ml bFGF showed a similar difference (cell number per field 30.23+/-4.54 vs 19.20+/-4.47, p<0.05). We also found that SHR-derived fibroblasts expressed a higher level of OPN mRNA than the cells from WKY (1863.23+/-43.91 vs 326.24+/-68.29, p<0.01). To verify if OPN is associated with the enhanced migratory ability in AF from SHR, we designed the antisense oligonucleotide of OPN. The results showed that the antisense OPN oligonucleotide significantly inhibited AF migration (cell number per field 38.60+/-5.98 vs 26.61+/-3.84, p<0.05), while sense and mismatch OPN oligonucleotide had no effect on cell migration. Therefore, the migration of adventitial fibroblasts appeared to be enhanced in cultures derived from SHR. OPN might be involved in the difference observed.
Animals ; Aorta ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; Hypertension ; physiopathology ; Male ; Osteopontin ; Phosphoproteins ; pharmacology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Sialoglycoproteins ; pharmacology

To investigate the pattern of expression of osteopontin (OPN) in tissues of the central nervous system (CNS) responding to peripheral immunological stimulation, the expression of OPN was studied in the spinal cord of rats with experimental autoimmune neuritis (EAN). In this model system, the sciatic nerves and spinal nerve roots are the target organs of EAN and the spinal cord is a remote organ that may be indirectly affected. OPN was constitutively expressed in some astrocytes adjacent to the pia mater and neurons in normal rats. In rats with EAN, OPN was increased in the same cells and in some inflammatory cells, including macrophages in the subarachnoid space. Expression of CD44, a receptor of OPN, was weak in normal spinal cord tissue and increased in the entire spinal cord parenchyma in rats with EAN, as well as in inflammatory cells. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of OPN and CD44 in the spinal cord of rats with EAN. Further study is needed to elucidate the functional role of OPN in the spinal cord affected by EAN.
Animals ; Antigens, CD44/metabolism ; Astrocytes/metabolism ; Ectodysplasins ; Female ; Immunohistochemistry ; Macrophages/metabolism ; Membrane Proteins ; Neuritis, Autoimmune, Experimental/*metabolism ; Neuroglia/metabolism ; Neurons/metabolism ; Osteopontin ; Rats ; Rats, Inbred Lew ; Sciatic Nerve/metabolism ; Sialoglycoproteins/*metabolism ; Spinal Cord/*metabolism ; Spinal Nerve Roots/metabolism

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood except the suppression of osteoblast function. To investigate the effect of simulated weightlessness on mRNA level of extracellular matrix proteins, osteoblasts were rotated in horizontal plane as a model of simulated microgravity. Primordial osteoblasts of rats were grown for 2 d and then rotated for 24, 48 and 72 h, respectively. After isolating total RNA in cells, reverse transcription polymerase chain reaction (PT-PCR) was made to examine the mRNA level of osteopontin (OPN) and osteonectin (ON). Meanwhile, the levels of alkaline phosphatase (ALP) and osteocalcin (BGP) in the cultured medium were measured to evaluate the calcific function of cell. The expression of OPN and ON mRNA fell significantly after rotating for 24, 48 and 72 h, respectively. The contents of BGP descended significantly, meanwhile, the activity of ALP also showed a degressive tendency. Horizontal rotation decreased the expression of ON and OPN as well as diminished the secretion of BGP and ALP, which affected the calcific function of osteoblast. The results obtained suggest that depression of extracellular matrix proteins expression plays a key role in bone loss during weightlessness.
Animals ; Animals, Newborn ; Cells, Cultured ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteonectin ; biosynthesis ; genetics ; Osteopontin ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Rotation ; Sialoglycoproteins ; biosynthesis ; genetics ; Skull ; cytology ; Weightlessness Simulation