Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats.

OBJECTIVE: This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. MATERIAL AND METHODS: Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn's post-test. RESULTS: On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. CONCLUSION: The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade's effects.

The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats

Abstract Objective This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. Material and Methods Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn's post-test. Results On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. Conclusion The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade's effects.

Effects of the photobiomodulation using different energy densities on the periodontal tissues under orthodontic force in rats with type 2 diabetes mellitus.

To evaluate the impact of the GaAlAs diode laser with energy densities of 160 J/cm2, 320 J/cm2, and 640 J/cm2 on the periodontal tissues under continuous orthodontic force application and on the rate of orthodontic tooth movement in rats with type-2 diabetes mellitus. The intensity of primary alveolar bone formation was also investigated through the immune-positive osteocytes for OPN antibody. Forty adult male Wistar rats were divided into eight groups of 5 rats: normoglycemic (N), 160 J-laser-normoglycemic (160 J-LN), 320 J-laser-normoglycemic (320 J-LN), 640 J-laser-normoglycemic (640 J-LN), diabetic (D), 160 J-laser-diabetic (160 J-LD), 320 J-laser-diabetic (320 J-LD), and 640 J-laser-diabetic (640 J-LD) rats. Diabetes mellitus was induced by a single intravenous injection of 40 mg/kg monohydrated-alloxan. An orthodontic force magnitude of 20cN was applied. The laser parameters were continuous emission of 780-nm wavelength, output power of 20mW, and fiber probe with a spot size of 0.04 cm in diameter. Radiographic, histomorphological, and immunohistochemical analysis were performed after a period of 21 days. The photobiomodulation using the energy density of 640 J/cm2 strongly stimulated the alveolar bone formation and contributed the reorganization of the soft periodontal tissues, followed by the 320 J/cm2. Extensive alveolar bone loss, intense infiltration of inflammatory cells, and degradation of the PDJ tissue were mainly found in the D and 160 J-LD groups. The rate of orthodontic tooth movement was represented by the interdental distance between the cementoenamel junctions of the right mandibular first and second molars . This distance was larger in the diabetic groups (D: 39.98±1.97, 160 J-LD: 34.84±6.01, 320 J-LD: 29.82±1.73, and 640 J-LD: 35.47±4.56) than in the normoglycemic groups (N: 21.13±1.19; 160 J-LN: 22.69±0.72, 320 J-LN: 22.28±0.78, and 640 J-LN: 24.56±2.11). The number of osteopontin-positive osteocytes was significantly greater in the 640 J-LD (14.72 ± 0.82; p < 0.01) and 640 J-LN (13.62 ± 1.33; p < 0.05) groups than with D (9.82 ± 1.17) and 160 J-LD (9.77 ± 1.10) groups. Therefore, the energy density of 640 J/cm2 provided the best maintenance and integrity of the periodontal tissue microarchitecture under continuous orthodontic force when compared with the other dosages, mainly in the uncontrolled diabetic rats. The interdental distance was greater in the D and 160 J-LD groups due to presence of severe periodontitis caused by diabetes plus the mechanical stress generated by continuous orthodontic forces, implying, thus, an insufficient biostimulatory effect for the dosage of 160 J/cm2.

Effects of the photobiomodulation using different energy densities on the periodontal tissues under orthodontic force in rats with type 2 diabetes mellitus

Abstract To evaluate the impact of the GaAlAs diode laser with energy densities of 160 J/cm2, 320 J/cm2, and 640 J/cm2 on the periodontal tissues under continuous orthodontic force application and on the rate of orthodontic tooth movement in rats with type-2 diabetes mellitus. The intensity of primary alveolar bone formation was also investigated through the immune-positive osteocytes for OPN antibody. Forty adult male Wistar rats were divided into eight groups of 5 rats: normoglycemic (N), 160 J-laser-normoglycemic (160 J-LN), 320 J-laser-normoglycemic (320 J-LN), 640 J-laser-normoglycemic (640 J-LN), diabetic (D), 160 J-laser-diabetic (160 J-LD), 320 J-laser-diabetic (320 J-LD), and 640 J-laser-diabetic (640 J-LD) rats. Diabetes mellitus was induced by a single intravenous injection of 40 mg/kg monohydrated-alloxan. An orthodontic force magnitude of 20cN was applied. The laser parameters were continuous emission of 780-nm wavelength, output power of 20mW, and fiber probe with a spot size of 0.04 cm in diameter. Radiographic, histomorphological, and immunohistochemical analysis were performed after a period of 21 days. The photobiomodulation using the energy density of 640 J/cm2 strongly stimulated the alveolar bone formation and contributed the reorganization of the soft periodontal tissues, followed by the 320 J/cm2. Extensive alveolar bone loss, intense infiltration of inflammatory cells, and degradation of the PDJ tissue were mainly found in the D and 160 J-LD groups. The rate of orthodontic tooth movement was represented by the interdental distance between the cementoenamel junctions of the right mandibular first and second molars . This distance was larger in the diabetic groups (D: 39.98±1.97, 160 J-LD: 34.84±6.01, 320 J-LD: 29.82±1.73, and 640 J-LD: 35.47±4.56) than in the normoglycemic groups (N: 21.13±1.19; 160 J-LN: 22.69±0.72, 320 J-LN: 22.28±0.78, and 640 J-LN: 24.56±2.11). The number of osteopontin-positive osteocytes was significantly greater in the 640 J-LD (14.72 ± 0.82; p < 0.01) and 640 J-LN (13.62 ± 1.33; p < 0.05) groups than with D (9.82 ± 1.17) and 160 J-LD (9.77 ± 1.10) groups. Therefore, the energy density of 640 J/cm2 provided the best maintenance and integrity of the periodontal tissue microarchitecture under continuous orthodontic force when compared with the other dosages, mainly in the uncontrolled diabetic rats. The interdental distance was greater in the D and 160 J-LD groups due to presence of severe periodontitis caused by diabetes plus the mechanical stress generated by continuous orthodontic forces, implying, thus, an insufficient biostimulatory effect for the dosage of 160 J/cm2.

Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo.

Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs) isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine.

Combination of LED light and platelet-derived growth factor to accelerate dentoalveolar osteogenesis.

AIM: This study aimed to evaluate the adjunctive effect of LED light in platelet-derived growth factor (PDGF)-aided dentoalveolar osteogenesis. MATERIAL AND METHODS: Full-thickness osseous wounds were created on rat maxillae and were either unfilled or filled with poly-(D,L-lactide) and poly-(D,L-lactide-co-glycolide) microspheres encapsulating PDGF. Animals received daily 660 ± 25 nm LED light irradiation at 0, 10 (LD), or 20 (HD) J/cm(2) , were killed at days 4-28 (n = 6/group/time) and evaluated by microcomputed tomography (micro-CT), histology, and the expressions of osteopontin and tartrate-resistant acid phosphatase (TRAP). RESULTS: Greater osteogenesis was noted in the PDGF-treated defects at day 14. Under the LED light irradiation, osteogenesis was significantly greater in both LD and HD groups of the non-PDGF-treated defects, but only in the LD group of the PDGF-treated defects. No significant differences in osteogenesis among groups were noted at day 28. Greater bone marrow space was noted in the LED light-irradiated specimens, especially in the PDGF-treated defects at both time points. Osteopontin was significantly promoted in the LD group at both time points, and TRAP was significantly promoted in all LED light-irradiated groups at day 28. CONCLUSION: LED light could an adjunct to promote early PDGF-aided dentoalveolar osteogenesis by facilitating the osteoblast-osteoclast coupling.

Periodontal fibroblasts modulate proliferation and osteogenic differentiation of embryonic stem cells through production of fibroblast growth factors.

BACKGROUND: Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). METHODS: PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. RESULTS: In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. CONCLUSION: PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

Assessment of different decalcifying protocols on Osteopontin and Osteocalcin immunostaining in whole bone specimens of arthritis rat model by confocal immunofluorescence.

Confocal immunofluorescence is a valuable technique for the detection of relevant molecules in the pathogenesis of arthritis in rat models; however, it requires efficient processing of tissues including bone decalcification. The decalcification process must ensure the complete removal of calcium and also a proper preservation of cellular structures and, specially, the antigenicity of the tissue to allow the immunodetection of the molecules of interest. In the present study, we evaluated the effect of four different decalcifying solutions: the Morse´s solution, 10% EDTA (pH 7.4), 7% HCl/2% EDTA and 5% Nitric acid, as well as four different treatments of the tissues (including microwave irradiation) in the processes of decalcification for large pieces of adult rat bones (hind paw, fore paw, knee and column). We assessed the time of decalcification, the easiness of slicing, the morphological preservation and finally, the antigenicity of two different bone proteins (Osteopontin (OPN) and Osteocalcin (OC)) measured by its immunofluorescence intensity under controlled confocal microscopy conditions. Our results showed that the specimen size and the presence of skin are critical factors for the rate of decalcification, and no significant benefit was found if microwave irradiation is applied to the tissue. The comprehensive statistical analysis showed that the optimal solution for the detection of OPN and OC by confocal immunofluorescence is the 5% Nitric Acid, and followed by 10% EDTA (pH 7.4), Ana Morse solution and 7% HCl/2% EDTA.

Catechin prevents the calcium oxalate monohydrate induced renal calcium crystallization in NRK-52E cells and the ethylene glycol induced renal stone formation in rat.

BACKGROUND: Reactive oxygen species play important roles in renal calcium crystallization. In this study, we examined the effects of catechin, which have been shown to have antioxidant properties on the renal calcium crystallization. METHODS: In the vitro experiment, the changes of the mitochondrial membrane potential, expression of superoxide dismutase (SOD), 4-hydroxynonenal (4-HNE), cytochrome c, and cleaved caspase 3 were measured to show the effects of catechin treatment on the NRK-52E cells induced by calcium oxalate monohydrate (COM). In the vivo study, Sprague-Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with catechin (2.5 and 10 mg/kg/day) for 14 days. The urine and serum variables were dected on 7 and 14 days after EG administration. The expression of cytochrome c, cleaved caspase 3, SOD, osteopontin (OPN), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG) in kidney were measured. Furthermore, the mitochondrial microstructure in the kidney was also examined by transmission electron microscopy. RESULTS: Catechin treatment could prevent the changes in mitochondrial membrane potential and expression of SOD, 4-HNE, cytochrome c, and cleaved caspase 3 in NRK-52E cells induced by the COM. For the in vivo experiments, the EG administration induced renal calcium crystallization was also prevented by the catechin. The expression of SOD, OPN, MDA, OPN and 8-OHdG, were increased after EG administration and this increase was diminished by catechin. Moreover, catechin also prevented EG induced mitochondrial collapse in rat. CONCLUSIONS: Catechin has preventive effects on renal calcium crystallization both in vivo and in vitro, and provide a potential therapeutic treatment for this disease.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells.

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

In vitro osteoinduction of human mesenchymal stem cells in biomimetic surface modified titanium alloy implants.

Interaction between cells and implant surface is crucial for clinical success. This interaction and the associated surface treatment are essential for achieving a fast osseointegration process. Several studies of different topographical or chemical surface modifications have been proposed previously in literature. The Biomimetic Advanced Surface (BAS) topography is a combination of a shot blasting and anodizing procedure. Macroroughness, microporosity of titanium oxide and Calcium/Phosphate ion deposition is obtained. Human mesenchymal stem cells (hMCSs) response in vitro to this treatment has been evaluated. The results obtained show an improved adhesion capacity and a higher proliferation rate when hMSCs are cultured on treated surfaces. This biomimetic modification of the titanium surface induces the expression of osteblastic differentiation markers (RUNX2 and Osteopontin) in the absence of any externally provided differentiation factor. As a main conclusion, our biomimetic surface modification could lead to a substantial improvement in osteoinduction in titanium alloy implants.

Corn baked by burning coal triggered overexpression of osteopontin in hepatocytes of rats following fluorosis.

OBJECTIVE: To observe the expression of osteopontin (OPN) in hepatocytes of rats fed with corn baked by burning coal from fluorosis areas and a deficiency of calcium/protein intake following fluorosis. METHODS: A total of 48 Wistar rats as objects were randomly assorted into four groups: dose-free fluorine group, which were mainly fed with fluorine-free corn (56% structurally), dose-free fluorine with biased dietary group, which were fed with lower contents of protein (119.41 g/kg) and calcium (0.68 g/kg), high-dose fluorine group (fluorine contents: 104.2 mg/kg), and high-dose fluorine with biased dietary group. After 180 days of cultivation, the contents of fluorine in the bones of rats were tested for the assessment of construction of fluorosis animal model. And the expression of OPN in hepatocytes of rats in different groups was detected with immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: The present study validated the result that OPN was overexpressed in hepatocytes following fluorosis after oral intake of burning coal-baked corn. OPN was expressed most significantly in high fluorine with biased dietary group, and the high-fluorine group ranked the second most; and dose-free fluorine with biased dietary group ranked the third. The dose-free fluorine group expressed the least OPN. CONCLUSION: Overexpression of OPN in hepatocytes following fluorosis after excess fluorine intake was involved in liver damage process, which was enhanced by deficiency of calcium and protein intake. The results also demonstrated that the development of fluorosis in Guizhou province was correlated with local baking staple corn as a way of excess intake of fluorine and deficiency of calcium/protein intake.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

O objetivo foi avaliar o potencial odontogênico de células indiferenciadas da polpa (OD-21) por meio de indução química in vitro. As células foram divididas em grupos: controle (OD-21), induzido (OD-21 em meio suplementado/OD-21 + OM), e células odontoblastóides (MDPC-23). Após 3, 7, 10 e 14 dias, avaliou-se proliferação e viabilidade celular, proteína total e fosfatase alcalina (ALP), mineralização, imunolocalização da proteína da matriz dentinária 1 (DMP1), ALP e osteopontina (OPN), assim como a expressão dos genes ALP, OSTERIX (Osx), DMP1 e fator de transcrição RUNX2 por PCR em tempo real. Os dados foram avaliados pelo teste de Kruskal-Wallis seguido pelo teste de Mann-Whitney U (p<0.05). Houve diminuição na proliferação celular em OD-21 + OM, com viabilidade celular similar em todos os grupos, exceto aos sete dias. O conteúdo de proteína total foi maior no grupo OD-21 + OM em todos os períodos; o mesmo ocorreu com a atividade de ALP quando comparada com o grupo OD-21, além de apresentar resultados similares ao grupo MDPC-23. A mineralização foi maior em OD-21 + OM quando comparada com o controle negativo. A imunolocalização demonstrou expressão de DMP1 e ALP em MDPC-23 e OD-21 + OM, enquanto que todos os grupos foram positivos para OPN. A expressão gênica de DMP1 e ALP foi maior nas culturas de MDPC-23, enquanto que a de RUNX2 foi menor para estas células e maior no controle negativo. A expressão de OSTERIX foi menor em OD-21 + OM quando comparada aos outros grupos. Sugere-se que as células indiferenciadas da polpa da linhagem OD-21 apresentam potencial odontogênico e poderiam ser usadas para a engenharia tecidual.

In vivo production of mineralised tissue pieces for clinical use: a qualitative pilot study using human dental pulp cell.

Numerous previous studies have investigated the production of mineralised tissues by transplanting human dental pulp cells with calcium based scaffolds. The potential of alternative setups remains largely uninvestigated, therefore in this study, human dental pulp cells were encapsulated into non-calcium based biomaterial - self-assembling peptide nano-fibre hydrogel. The cell-gel constructs were cultured in full medium for 2 weeks. Then they were cultured in full medium supplemented with ß-glycerophosphate, dexamethasone and l-ascorbic acid for 2 more weeks. These cell-gel constructs and plain-gel constructs (with no cells) were transplanted subcutaneously into five nude mice. The gel constructs were retrieved 4 weeks after surgery. The plain-gel constructs were all completely resorbed with no new tissue formation. The cell-gel constructs were transformed into tissue pieces that were mineralised and contained blood capillaries. Immunohistochemistry analysis confirmed the expression of multiple bone markers (osteopontin, osteocalcin, osteonectin and parathyroid hormone receptor) in these tissue pieces. Computerised analysis of the contact radiographs gave the mean radio-opaque area percentage as 78% (N=5, P<0.001 compared with the 0% of the control). The results demonstrate good prospects for using human dental pulp cell plus self-assembling peptide nano-fibre hydrogel to produce mineralised tissue pieces for clinical use.

1,2,3,4,6-Penta-O-galloyl-beta-D-glucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells may be a key event in the pathogenesis of kidney stones associated with marked hyperoxaluria. Previously, we found that 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), isolated from a traditional medicinal herb, reduced CaOx crystal adhesion to renal epithelial cells by acting on the cells as well as on the crystal surface. Here we used the ethylene glycol (EG)-mediated hyperoxaluric rat model and found evidence of oxidant stress as indicated by decreases in the activities of the renal antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, with increased kidney cell apoptosis and serum malondialdehyde levels, all evident by 21 days of EG treatment. These effects of hyperoxaluria were reversed by concurrent PGG treatment along with decreased urinary oxalate levels and CaOx supersaturation. Renal epithelial cell expression of the crystal binding molecule hyaluronan increased diffusely within 7 days of EG initiation, suggesting it is not a result of but precedes crystal deposition. Renal cell osteopontin (OPN) was also upregulated in EG-treated animals, and PGG significantly attenuated overexpression of both OPN and hyaluronan. Thus, our findings demonstrate that PGG reduces renal crystallization and oxidative renal cell injury, and may be a candidate chemopreventive agent for nephrolithiasis.

Responses of periodontal ligament stem cells on various titanium surfaces.

OBJECTIVE: Periodontal ligament has been reported to have adult stem cells (PDLSCs) which are responsible to regenerate the alveolar bone tissue after tooth is removed from its socket. Also PDLSCs may be the stem cells responsible for the osseointegration of titanium implants after installing the implant immediately in the fresh extracted socket. Here we tested cellular responses of PDLSCs on the various titanium surfaces to verify this notion. MATERIALS AND METHODS: Titanium disc were prepared for the different surface textures; smooth machined, blasted with 75 and 125 µm Al(2) O(3) particles, and anodized. PDLSCs were cultured on these titanium discs and tested their proliferation and gene expressions of osteocalcin, osteopontin, type I collagen, and GAPDH. RESULTS: Proliferation of PDLSCs was higher on the rough surface blasted with 75 µm Al(2) O(3) particles. Osteocalcin expression was increased on the Al(2) O(3) particle treated-surface regardless of its particle size. Type I collagen expression was generally decreased with time in 6 days culture. CONCLUSIONS: In this experiment, it was shown that cultured PDLSCs proliferate in higher rate on the rough surface especially at the 75 µm Al(2) O(3) particle treated surface than other surfaces. Also, osteocalcin was highly expressed on the rough surfaces treated with 75 µm and 125 µm Al(2) O(3) particles.

Wnt5a promotes differentiation of human dental papilla cells.

AIM: To investigate the role of Wnt5a in the process of differentiation of human dental papilla cells (HDPCs). METHODOLOGY: Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of Wnt5a on the differentiation of HDPCs. The effect of Wnt5a on HDPCs differentiation was determined by ALP activity assay, ALP staining and mineral induction assay. Mineralization-related gene expressions were assessed by RT-PCR. RESULTS: Immunostaining revealed Wnt5a expression in the odontoblast layer and dental papilla tissue. Over-expression of Wnt5a by transfecting HDPCs with an Wnt5a-carrying construct increased ALPase activity and the formation of mineralized nodules of HDPCs. RT-PCR analysis showed that the expressions of mineralization-related genes, such as bone sialoprotein, collagen type I, osteonectin, osteopontin (OCN), dentine matrix protein-1 were up-regulated by Wnt5a. CONCLUSIONS: Wnt5a promoted differentiation of HDPCs.

Effects of the ErbB1/ErbB2 kinase inhibitor GW2974 on androgen-independent prostate cancer PC-3 cell line growth and NSE, chromogranin A and osteopontin content.

Prostate cancer is one of the most frequently diagnosed cancer in men. Treatment by radical prostatectomy, radiotherapy and anti-androgen drugs is successful in patients with localized cancer. However, prolonged androgen deprivation often leads to hormone refractory condition, associated with disease relapse. ErbB1 and ErbB2 activity has been correlated with androgen-independence. We determined the effects of GW2974, a dual inhibitor of ErbB-1 and ErbB-2 tyrosine kinase activity, on growth, NSE, chromogranin A and osteopontin cytosol content in the androgen-independent prostate cancer cell line PC-3. We found that PC-3 cell growth was inhibited by GW2974, whereas NSE and chromogranin A cell contents were stimulated and osteopontin cytosol level was not affected. The present data may have clinical implications for the treatment of advanced prostate cancer.

Short-term effect of combined therapy with Jinlong Capsule and transcatheter arterial chemoembolization on patients with primary hepatic carcinoma and its influence on serum osteopontin expression.

OBJECTIVE: To observe the clinical combination effect of Jinlong Capsule (JLC) and transcatheter arterial chemoembolization (TACE) on the patients with primary hepatic carcinoma (PHC) and JLC' s influence on serum osteopontin (OPN) expression and elucidate the correlation between the serum OPN level and curative effect of JLC and TACE. METHODS: A total of 98 patients with PHC were observed in a randomized controlled trial (RCT). They were assigned to the Chinese medicine (CM) group (53 patients who were treated with TACE and JLC) and the intervention group (45 patients who were treated with TACE only). The serum OPN levels were measured before and after treatment by quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Forty healthy people were assigned to the control group. The clinical efficacy was observed and Karnofsky score (KPS) was graded. RESULTS: The clinical efficacy of the CM group (60.38%) was better than that of the intervention group (40.00 %), and the KPS (84.35+/-12.19) was higher than the intervention group (69.86+/- 11.58) (P<0.05). The serum OPN levels before and after treatment in the patients with PHC were significantly elevated compared with those in the control group (P<0.01). After treatment, the OPN levels in CM group (117.69 <+/-78.50) were significantly lower compared with those in intervention group (151.09+/-83.90, P<0.05). The OPN levels of responders were remarkably lowered than the non-responders after treatment, and the level of OPN in the CM group was lower than the intervention group (P<0.05). CONCLUSIONS: The short-term clinical efficacy and the quality of life of patients with PHC can be improved by combining JLC with TACE. The serum OPN levels in PHC patients can reflect the curative effect of treatment and the prognosis of the disease.