Lingling E

Biocompatibility and Osteogenic Capacity of Periodontal Ligament Stem Cells on nHAC/PLA and HA/TCP Scaffolds

This study investigated the effects of a newly-developed scaffold, nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA), on the attachment, proliferation and osteogenic capability of dog periodontal ligament stem cells (PDLSCs) in vitro and in vivo. Hydroxyapatite/tricalcium phosphate (HA/TCP), a commonly used bone substitute, was used as a positive control. PDLSCs isolated from dog molar were incubated in an osteogenic medium to evaluate their osteogenic differentiation in vitro, and then seeded onto nHAC/PLA and HA/TCP scaffolds. In vitro cell attachment, proliferation and differentiation were assessed by scanning electron microscopy (SEM), cell counting, 3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium and alkaline phosphate activity, and reverse transcription-polymerase chain reaction, respectively. Finally, the constructs were implanted subcutaneously into dogs to investigate their osteogenic capacity. After osteogenic induction for 21 days, PDLSCs differentiated into osteogenic lineage, as indicated by the expressions of osteoblastic differentiation genes CoL-I, OCN and OPN mRNA, and the formation of mineral deposits. When seeded onto scaffolds, the cells attached and spread well, and retained their osteogenic phenotypes on both scaffolds. Comparatively, cell number and proliferative viability on nHAC/PLA constructs were greater than those on HA/TCP constructs (P < 0.05). Histological results showed that new bone and osteoid was formed in both groups, and histomorphometric analysis demonstrated that the amount of newly formed bone in the nHAC/PLA group was higher than that in the HA/TCP group (P < 0.05). This study suggests that nHAC/PLA can be used as a potent scaffold for alveolar bone regeneration.

Glimepiride induces proliferation and differentiation of rat osteoblasts via the PI3-kinase/Akt pathway

Glimepiride is a third-generation sulfonylurea agent and is widely used in the treatment of type 2 diabetes mellitus. In addition to the stimulatory effects on pancreatic insulin secretion, glimepiride has also been reported to have extrapancreatic functions including activation of PI3 kinase (PI3K) and Akt in rat adipocytes and skeletal muscle. PI3-kinase and Akt are important signaling molecules in the regulation of proliferation and differentiation in various cells. This study investigated the actions of glimepiride in rat osteoblasts and the role of PI3K/Akt pathway. Cell proliferation was determined by measuring absorbance at 550 nm. Supernatant assay was used for measuring alkaline phosphatase activity. Western blot analysis was used for determining collagen I, insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase expression. We found that glimepiride significantly enhanced proliferation and differentiation of osteoblasts and led to activation of several key signaling molecules including insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase. Furthermore, a specific inhibitor of PI3K abolished the stimulatory effects of glimepiride on proliferation and differentiation. Taken together, these observations provide concrete evidence that glimepiride activates the PI3K/Akt pathway; and this activation is likely required for glimepiride to stimulate proliferation and differentiation of rat osteoblasts.

Effects of Vascular Endothelial Growth Factor 165 on Bone Tissue Engineering

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.

Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional β-TCP

The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10(-8) M) or/and BMP-2 (100 ng ml(-1)) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

High glucose inhibits osteogenic differentiation through the BMP signaling pathway in bone mesenchymal stem cells in mice

Patients with diabetes tend to have an increased risk of osteoporosis that may be related to hyperglycemia. In vitro evidence has shown that high glucose can affect the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). Tissue regeneration depends mainly on MSCs. However, the exact mechanisms involved in high glucose-induced bone loss remain unknown. In this study, we investigated the effects of high glucose on the proliferation and osteogenic differentiation of mice bone MSCs (BMSCs) and determined the specific mechanism of bone morphogenetic protein 2 (BMP-2) in the osteogenic differentiation of mice BMSCs in a high-glucose microenvironment. High glucose (< 25 mM) promoted cell growth but suppressed mineralization. The intracellular BMP-2 level in BMSCs cultured in a high-glucose microenvironment was significantly decreased and suppressed activation of the BMP signaling pathway. Consequently, expression of the osteogenic markers Runx2, alkaline phosphatase, and osteocalcin were decreased. Meanwhile, supplementation with ectogenic BMP-2 reversed the cell osteogenic differentiation and osteogenic marker down-regulation under high glucose. Our data indicate that BMP-2 plays an important role in regulating the osteogenic differentiation of BMSCs in a high-glucose microenvironment. Thus, it is possible that agents modifying this pathway could be used by BMSCs to promote bone regeneration in high-glucose microenvironments.

The transmembrane transport of metformin by osteoblasts from rat mandible

Previous studies have demonstrated that metformin, one of systemic antihyperglycemic drugs, can slow bone loss caused by diabetes mellitus and has an osteogenic action on osteoblasts in vitro. It is tempting to speculate that metformin would be transported into bone tissues around dental implant by topical administration to improve the bone-implant contact in diabetic patients. In this study, the osteoblasts from rat mandible were cultured with 5.5 mM (control) or 16.5 mM d-glucose, then the uptake of metformin by osteoblasts was detected with high performance liquid chromatography (HPLC). Rat organic cation transporter (rOct) expression was characterized by immunocytochemistry, RT-PCR and Western blotting. It was found that, the uptake of metformin was saturable, Na(+)-dependent, affected by extracellular pH and inhibited by both phenformin and cimetidine (an inhibitor of Octs). rOct1 but no rOct2 was expressed extensively in osteoblasts and the protein level of rOct1 could be up-regulated by metformin. The uptake of metformin and phosphorylated-rOct1 at hyperglycaemic cell culture (16.5 mM d-glucose) significantly increased versus 5.5 mM control (p < 0.05). In conclusion, rat osteoblasts have the ability to transport the metformin intra-cellularly, the uptake of metformin by osteoblasts is a secondary active transportation mediated by rOct1 and high-glucose can improve the uptake of metformin by osteoblasts through phosphorylation of rOct1. The current results suggest that metformin could be used for dental implant topically in type 2 diabetic patients to increase the bone formation, therefore, to enhance the success rate of dental implants clinically.

Porous Nanohydroxyapatite/Collagen Scaffolds Loading Insulin PLGA Particles for Restoration of Critical Size Bone Defect

Insulin is considered to be a classical central regulator of energy homeostasis. Recently, the effect of insulin on bone has gained a lot of attention, but little attention has been paid to the application in bone tissue engineering. In this study, porous nanohydroxyapatite/collagen (nHAC) scaffolds incorporating poly lactic-co-glycolic acid (PLGA) particles were successfully developed as an insulin delivery platform for bone regeneration. Bioactive insulin was successfully released from the PLGA particles within the scaffold, and the size of the particles as well as the release kinetics of the insulin could be efficiently controlled through Shirasu porous glass premix membrane emulsification technology. It was indicated that the nHAC/PLGA composite scaffolds possessed favorable mechanical and structural properties for cell adhesion and proliferation, as well as the differentiation into osteoblasts. It was also demonstrated that the nHAC/PLGA scaffolds implanted into a rabbit critical-size mandible defect possessed tissue compatibility and higher bone restoration capacity compared with the defects that were filled with or without nHAC scaffolds. Furthermore, the in vivo results showed that the nHAC/PLGA scaffolds which incorporated insulin-loaded microspheres with a size of 1.61 μm significantly accelerated bone healing compared with two other composite scaffolds. Our study indicated that the local insulin released at the optimal time could substantially and reproducibly improve bone repair.

Sustained local delivery of insulin for potential improvement of peri-implant bone formation in diabetes

Dental implantation is an effective standard treatment modality to restore missing teeth and maxillofacial defects. However, in diabetics there is an increased risk for implant failure due to impaired peri-implant osseous healing. Early topical insulin treatment was recently shown to normalize diabetic bone healing by rectifying impairments in osteoblastic activities. In this study, insulin/poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared by a double-emulsion solvent evaporation method. Microspheres were then incorporated in fibrin gel to develop a local drug delivery system for diabetic patients requiring implant treatment. In vitro release of insulin from fibrin gel loaded with these microspheres was assessed, and sustained prolonged insulin release over 21 days ascertained. To assess the bioactivity of released insulin and determine whether slow release might improve impaired diabetic bone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, mineralized nodule formation, and ELISA (enzyme-linked immunosorbent assay) assays were performed. The insulin released from the drug delivery system stimulated cell growth in previously inhibited cells, and ameliorated the impaired bone-forming ability of human MG-63 cells under high glucose conditions. Fibrin gel loaded with insulin/PLGA microspheres shows potential for improving peri-implant bone formation in diabetic patients.

Uptake of Metronidazole by Human Gingival Fibroblasts

BACKGROUND Metronidazole is an important antimicrobial agent for the therapeutic management of periodontal diseases and dentoalveolar infections. As in other tissues, the metronidazole concentration in gingival crevicular fluid is about equal to the plasma level. Thus, we hypothesized that metronidazole is not actively transported into human gingival fibroblasts. METHODS Using high performance liquid chromatography, the influences of extracellular metronidazole concentrations, temperature, pH, and inhibitors of transporters on the uptake of metronidazole by cultured human gingival fibroblasts were tested. RESULTS Metronidazole was taken up rapidly by fibroblasts; the intracellular metronidazole concentration reached the extracellular level in 3 minutes at 37 degrees C and in 2 minutes at 4 degrees C. The uptake of metronidazole by human gingival fibroblasts was not saturable, and the intracellular metronidazole concentrations increased linearly with the extracellular level. Temperature and pH had no significant influence on the uptake of metronidazole by fibroblasts. Probenecid and adenine had no influence on the uptake of metronidazole by fibroblasts. These findings indicate that metronidazole uptake does not involve a transporter. Metronidazole bound rapidly to human gingival fibroblasts, but the cell-associated drug declined progressively until it reached a stable plateau in 15 minutes. CONCLUSIONS Metronidazole rapidly entered human gingival fibroblasts via simple diffusion. Metronidazole easily reached the minimal inhibitory concentration in fibroblasts and gingiva. Given the fact that intracellular concentrations of metronidazole in other tissues and cells are also close to the plasma level, we speculate that metronidazole enters other tissues and cells via simple diffusion.

Proliferation and differentiation of osteoblasts from the mandible of osteoporotic rats.

The objective of this study was to identify the differences between osteoblasts derived from normal adult rat mandibles and osteoporotic adult rats. An osteoporotic animal model was established by performing a bilateral ovariectomy (ovx group). The proliferation and differentiation abilities of osteoblasts were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide), alkaline phosphatase (ALP) and osteocalcin release (OC) assays. Transmission electron microscopy (TEM) was performed to assess differences in the ultrastructure. Proliferating cell nuclear antigen (PCNA) and uncoupling protein 2 (UCP2) protein concentrations were analyzed by Western blot. In addition, UCP2 protein in osteoblasts was assessed by immunohistochemistry staining. ATP and reactive oxygen species (ROS) concentrations were analyzed separately with ATP and ROS quantification kits. At four and 12 weeks after the operation, osteoblasts of the ovx group showed earlier attachment, fewer dead cells and faster growth compared with cells in the sham group. TEM showed that osteoblasts of the ovx group had fewer folds, lysosomes, peroxisomes and less rough endoplasmic reticulum. The results of the MTT, ALP activity and OC assays were all higher in osteoblasts from the ovx group at four or 12 weeks postsurgery than osteoblasts from the sham group. PCNA protein concentrations in the ovx group increased significantly compared with those of the sham group at four or 12 weeks after the operation, but UCP2 concentrations decreased over the same time period. UCP2 immunohistochemical staining of osteoblasts showed that the protein was concentrated in the cytoplasm and that the osteoblasts from the sham group had higher expression than those from the ovx group. The ATP and ROS concentrations of the ovx groups were significantly higher than the sham groups at four or 12 weeks postsurgery. Therefore, we concluded that there are differences in cell ultrastructure, proliferation, differentiation, ATP and ROS concentrations, and PCNA and UCP2 protein expression levels in osteoblasts from the mandibles of rats of the ovx group compared with those from the sham group.