Mineralization Effect of Hyaluronan on Dental Pulp Cells via CD44.

INTRODUCTION: CD44 is a cell-surface glycoprotein involved in various cellular functions. Recent studies have suggested that CD44 is involved in early mineralization of odontoblasts. Hyaluronic acid (HA) is the principal ligand for receptor CD44. Whether and how HA regulated the mineralization process of dental pulp cells were investigated. METHODS: The effects of high-molecular-weight HA on differentiation and mineral deposition of dental pulp cells were tested by using alkaline phosphatase (ALP) activity assay and alizarin red S staining. Osteogenesis real-time polymerase chain reaction array, quantitative polymerase chain reaction, and Western blotting were performed to identify downstream molecules involved in the mineralization induction of HA. CD44 was knocked down and examined to confirm whether the mineralization effect of HA was mediated by receptor CD44. Immunohistochemistry was used to understand the localization patterns of CD44 and the identified downstream proteins in vivo. RESULTS: Pulse treatment of HA enhanced ALP activity and mineral deposition in dental pulp cells. Tissue-nonspecific ALP, bone morphogenetic protein 7 (BMP7), and type XV collagen (Col15A1) were upregulated via the HA-CD44 pathway in vitro. Immunohistochemistry of tooth sections showed that the staining pattern of BMP7 was very similar to that of CD44. CONCLUSIONS: Results of this study indicated that high-molecular-weight HA enhanced early mineralization of dental pulp cells mediated via CD44. The process involved important mineralization-associated molecules including tissue-nonspecific ALP, BMP7, and Col15A1. The findings may help develop new strategies in regenerative endodontics.

Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats.

BACKGROUND: Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. METHODS: Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-ß1 (TGF-ß1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. RESULTS: The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-ß1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. CONCLUSION: Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats.

Effect of F68, F127, and P85 pluronic block copolymers on odontogenic differentiation of human tooth germ stem cells.

INTRODUCTION: The major challenge in dental pulp engineering is to make a successful combination of stem cells and biomaterials with the aim of providing the differentiation of stem cells into odontogenic cell types. Among biomaterials, some types of pluronics have been reported to increase bone formation of stem cells. The effect of these pluronics on odontogenic differentiation has not been addressed yet. This study aimed to examine the effect of pluronics F68, F127, and P85 on odontogenic differentiation of stem cells derived from third molar tooth germs of young adults. METHODS: Human tooth germ stem cells (hTGSCs) were induced to differentiate into odontogenic cells in the presence of different concentrations of pluronics. Differentiation efficiency was assessed by quantitative real-time polymerase chain reaction for determining expression messenger RNA levels and by immunocytostaining for determining the protein expression of odontogenic markers (ie, dentin sialoprotein, dentin matrix protein 1, bone morphogenic protein 2, bone morphogenic protein 7) by measuring alkaline phosphatase enzyme activity and lastly by von Kossa staining for determining mineralization. RESULTS: The results revealed for the first time that F68 has a great potential to boost odontogenic differentiation of hTGSCs. P85 was found to reduce cell viability during differentiation. F127 was nontoxic to hTGSCs but did not have any effect on differentiation. CONCLUSIONS: The positive effect of F68 on odontogenic differentiation might enable more efficient pulp regeneration. Yet, the exact mechanism of how F68 alters the differentiation pattern of hTGSCs remains to be investigated in the future studies.

Gene delivery of c-myb increases bone formation surrounding oral implants.

Bone regeneration around titanium (Ti) implants is a relatively slow process. The c-myb transcription factor has been associated with high proliferation and differentiation rates in bone. This study analyzed whether c-myb can enhance new bone surrounding the implant. In vitro overexpressed chitosan-gold nanoparticles conjugated with plasmid DNA/c-myb (Ch-GNPs/c-myb)-coated Ti surfaces were associated with enhanced expression of the osteogenic molecules osteopontin (OPN), runt-related transcription factor 2 (RUNX-2), and bone morphogenetic proteins (BMP2/7) in MC-3T3E1 osteoblast cells. Further, to determine its in vivo effect, we inserted Ch-GNPs/c-myb-coated Ti implants into rat mandibles. One and 4 wks post-implantation, mandibles were examined by microcomputed tomography, immunohistochemistry, and hematoxylin & eosin staining. The microcomputed tomography analysis demonstrated that c-myb overexpression increased the density and volume of newly formed bone surrounding the implants, compared with those in controls (p < .05). Further, c-myb increased the number of cells expressing BMP2/7 and aided in the increase of new bone (p < .05). These results support the view that c-myb overexpression accelerates new bone surrounding implants and can serve as a potent molecule in promoting tissue regeneration around dental implants. The recipient rat used in this system provides an excellent in vivo model for studies of bone regeneration.

Vitamin D reduces the expression of collagen and key profibrotic factors by inducing an antifibrotic phenotype in mesenchymal multipotent cells.

Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5'-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0.1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.

9-Cis-retinoic acid reduces ischemic brain injury in rodents via bone morphogenetic protein.

Retinoic acid (RA), a biologically active derivative of vitamin A, has protective effects against damage caused by H(2)O(2) or oxygen-glucose deprivation in mesangial and PC12 cells. In cultured human osteosarcoma cells, RA enhances the expression of bone morphogenetic protein-7 (BMP7), a trophic factor that reduces ischemia- or neurotoxin-mediated neurodegeneration in vivo. The purpose of this study is to examine whether RA reduces ischemic brain injury through a BMP7 mechanism. We found that intracerebroventricular administration of 9-cis-retinoic acid (9cRA) enhanced BMP7 mRNA expression, detected by RT-PCR, in rat cerebral cortex at 24 hr after injection. Rats were also subjected to transient focal ischemia induced by ligation of the middle cerebral artery (MCA) at 1 day after 9cRA injection. Pretreatment with 9cRA increased locomotor activity and attenuated neurological deficits 2 days after MCA ligation. 9cRA also reduced cerebral infarction and TUNEL labeling. These protective responses were antagonized by the BMP antagonist noggin given 1 day after 9cRA injection. Taken together, our data suggest that 9cRA has protective effects against ischemia-induced injury, and these effects involve BMPs.