The effect of calcium phosphate composite scaffolds on the osteogenic differentiation of rabbit dental pulp stem cells.

The objective of this study is to compare the effects of the two calcium phosphate composite scaffolds on the attachment, proliferation, and osteogenic differentiation of rabbit dental pulp stem cells (DPSCs). One nano-hydroxyapatite/collagen/poly (l-lactide) (nHAC/PLA), imitating the composition and the micro-structure characteristics of the natural bone, was made by Beijing Allgens Medical Science & Technology Co., Ltd. (China). The other beta-tricalcium phosphate (ß-TCP), being fully interoperability globular pore structure, was provided by Shanghai Bio-lu Biomaterials Co, Ltd. (China). We compared the absorption water rate and the protein adsorption rate of two scaffolds and the characterization of DPSCs cultured on the culture plate and both scaffolds under osteogenic differentiation media (ODM) treatment. The constructs were then implanted subcutaneously into the back of severely combined immunodeficient (SCID) mice for 8 and 12 weeks to compare their bone formation capacity. The results showed that the ODM-treated DPSCs expressed osteocalcin (OCN), bone sialoprotein (BSP), type I collagen (COLI) and osteopontin (OPN) by immunofluorescence staining. Positive alkaline phosphatase (ALP) staining, calcium deposition and calcium nodules were also observed on the ODM-treated DPSCs. The absorption water rate and protein adsorption rate of nHAC/PLA was significantly higher than ß-TCP. The initial attachment of DPSCs seeded onto nHAC/PLA was significantly higher than that onto ß-TCP; and the proliferation rate of the cells was also significantly higher than that of ß-TCP on 1, 3, and 7 days of cell culture. The ALP activity, calcium/phosphorus content and mineral formation of DPSCs + ß-TCP were significantly higher than DPSCs + nHAC/LA. When implanted into the back of SCID mice, nHAC/PLA alone had no new bone formation, newly formed mature bone and osteoid were only observed in ß-TCP alone, DPSCs + nHAC/PLA and DPSCs + ß-TCP, and this three groups displayed increased bone formation over the 12-week period. The percentage of total bone formation area had no difference between DPSCs + ß-TCP and DPSCs + nHAC/PLA at each time point, but the percentage of mature bone formation area of DPSCs + ß-TCP was significantly higher than that of DPSCs + nHAC/PLA. Our results demonstrated that the DPSCs on nHAC/PLA had a better proliferation, and that the DPSCs on ß-TCP had a more mineralization in vitro, much more newly formed mature bones in vivo were presented in DPSCs + ß-TCP group. These findings have provided a further knowledge that scaffold architecture has different influence on the attachment, proliferation and differentiation of cells. This study may provide insight into the clinical periodontal bone tissue repair with DPSCs + ß-TCP construct.

Relationship of advanced oxidative protein products in human saliva and plasma: age- and gender-related changes and stability during storage.

The blood levels of advanced oxidative protein products (AOPP) elevate in aging and age-related diseases. However, saliva AOPP in healthy humans have been unexplored. Thus, we investigated 143 Chinese healthy adults to assay age- and gender-related changes in saliva and plasma AOPP levels and the stability of saliva AOPP stored both at - 20°C and - 80°C. We found the mean AOPP levels in saliva and plasma of 119 subjects were 7.51 ± 3.20 and 28.31 ± 5.53 µmol/L (µM). An age-dependent increase was observed in both saliva and plasma AOPP levels. This increase was particularly significant in the elderly subjects compared with that in the young and middle-aged ones. A significant positive correlation among age, saliva and plasma AOPP levels was observed. No gender-dependent difference was observed in either saliva or plasma AOPP levels during the aging process. Furthermore, AOPP levels in the 24 saliva samples showed no significant change at every successive determination during 4 weeks at - 80°C, whereas those levels significantly increased after 7 days of storage at - 20°C. These results indicate the feasibility to screen aging biochemical indicators using saliva AOPP as an alternative to blood AOPP. Saliva AOPP samples are suitable to be stored at - 80°C.

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.

Experimental study on co-culture of salivary adenoid cystic carcinoma cells and ganglia / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct the co-culture models of salivarya denoid cystic carcinoma (SACC) cells and dorsal root ganglia (DRG) of chickens and investigate the promotive effects of SACC on neural tissue.</p><p><b>METHODS</b>Glass-base culture dish was adopted to construct co-culture model of SACC-83 cells and DRG. SACC-83 cells were seeded in the medium pore with DRG around them. Outgrowth of neuronal processes was observed. Then DRG was cultured in the conditioned medium of SACC-83, with the groups of conditioned medium of MC3T3-E1 and HGF, the group of cell lysis buffer, the groups of serum-free medium and serum-plus medium as the controls. Outgrowth of neuronal processes was also recorded and compared with control groups.</p><p><b>RESULTS</b>In the co-culture model of tumor and neuronal tissue, SACC-83 cells produced a suitable microenvironment in which neuronal processes remarkably grow. Neuronal processes of most DRG displayed growth tendency toward SACC. The group of conditioned medium from SACC-83 manifested obvious promotive effects on DRG.</p><p><b>CONCLUSIONS</b>Co-culture model of tumor and neuronal tissue was successfully constructed, with which the promotive effects of tumor on outgrowth of neuronal processes could be observed. So hypothesized that SACC could secrete some neurotrophic factors to guide peripheral nerves gemmating and to trigger the cascade of the neural invasion in succession.</p>

Soft and hard tissue changes in Class II division 1 patients treated with Tip-Edge plus appliance / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the soft and hard tissue changes in Class II division 1 patients treated with Tip-Edge plus technique.</p><p><b>METHODS</b>Sixteen Class II division 1 patients (7 boys and 9 girls) with mandibular retrusion in permanent dentition were selected and treated with Tip-Edge plus appliance. Lateral cephalometric films were analyzed before and after treatment. The effects were evaluated with Holdaway soft tissues analysis and routine cephalometric analysis methods. The arithmetic mean and standard deviation were calculated for each variable. Paired t-test was performed.</p><p><b>RESULTS</b>The average treatment time was 16 months. Normal overjet and overbite were established with retroclination of upper incisors and proclination of lower incisors. U1-NA(°) and U1-NA (mm) decreaed by (15.40 ± 5.31)° and (4.16 ± 1.82) mm (P < 0.01). NLA showed an average increase of (-16.60 ± 5.29)° (P < 0.01). Remarkable soft tissue change was noted after the treatment.</p><p><b>CONCLUSIONS</b>The profile in Class II division 1 patients could be quickly and efficiently improved after treatment with Tip-Edge plus technique.</p>

Effect of ultrasound on osteoprotegerin and receptor activator nuclear factor kappaB ligand expression during root resorption in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of pulsed ultrasound on the expressions of osteoprotegerin (OPG) and receptor activator nuclear factor kappaB ligand (RANKL) during root resorption in a mouse model of orthodontic tooth movement.</p><p><b>METHODS</b>Thirty-two male Wistar rats (6-8 weeks old) were randomly assigned into 4 equal groups, including the blank control group, two ultrasound exposure groups with daily local LIPUS stimulation (100 and 150 MW/cm(2)) for 10 days during mechanical loading, and the control group with mechanical loading but not LIPUS exposure. Nickel-titanium closed-coil springs were used to generate 100 g mesial force for 10 days to move the maxillary right first molars. The expression of OPG and RANKL proteins at the compression sites was detected by immunohistochemistry.</p><p><b>RESULTS</b>Ultrasound stimulation significantly up-regulated the expression of OPG and down-regulated RANKL expression (P<0.05). The expressions of OPG and RANKL showed significant differences between the two ultrasound exposure groups (P<0.05).</p><p><b>CONCLUSION</b>Ultrasound stimulation might be useful to protect against root resorption and accelerate its repair by regulating the expressions of OPG and RANKL.</p>

Experiment on inducing human periodontal ligament stem cells into adipose cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the capability of human periodontal ligament stem cells (PDLSCs) differentiating into adipose cells in vitro and to determine their changes in cell morphology, structure and function during differentiation.</p><p><b>METHODS</b>PDLSCs isolated by magnetic-activated cell selection were treated continuously with adipogenic medium for 21 d. Then the cell morphology, ultrastructure, adipose specific markers of low density lipoprotein (LPL) and peroxisome proliferator activated receptor-gamma (PPAR-gamma) were analyzed by inverted contrast microscope, trans mission electron microscope (TEM), flow cytometry, immunofluorescence, RT-PCR and Western blot, respectively. These adipose-like cells were also identified by oil red O staining to determine the formation of lipid droplet, and the non-induced cells were used as control.</p><p><b>RESULTS</b>After continuous induction, the treated cells differentiated into adipose-like cells with round shape, and large amount of lipid drop in cytoplasm. 96.54% of the PDLSCs were found to differentiate into adipose cells as showed by flow cytometry, the specific markers of LPL mRNA and PPAR-gamma mRNA, and oil red O staining, respectively. Further, PPAR-gamma protein was detected in the induced cells in a time-dependent manner.</p><p><b>CONCLUSION</b>Human PDLSCs have the potential of differentiating into adipose cells under appropriate condition, and the differentiated cells exhibited characteristics of adipose cells both from cell morphology and from their functions.</p>

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.

Integrity of multilayer of human periodontal ligament fibroblasts and human gingival fibroblasts grown on filter membrane of Transwell / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the integrity of multilayer of human periodontal ligament fibroblasts (HPDLF) and human gingival fibroblasts (HGF) on filter membrane of Transwell and to provide basis for the drug transcellular transport by the HPDLF and HGF in the hypothesis of delivering medicine to the periodontium and whole body through the root canal.</p><p><b>METHODS</b>HPDLF and HGF derived from the primary culture were seeded on polycarbonate filter membrane of transwell respectively. After 1, 2, 3 and 4 weeks of culture, transepithelial electrical resistance (TEER) was detected and the growth of HPDLF and HGF observed by light microscope. After 2 weeks of culture, section of filter membrane where HPDLF and HGF lived was observed with light microscope and transmission electron microscope (TEM), and the permeability of the drug transport cell models was measured with fluorescein sodium.</p><p><b>RESULTS</b>HPDLF and HGF converged 1 week after inoculation, and the cells connected each other tightly and completely 2 weeks later. Observation of section of filter membrane by light microscope and TEM revealed a stratified cell growth of HPDLF and HGF 2 weeks after inoculation, and TEER of HPDLF and HGF were (56.14 +/- 7.43) and (57.34 +/- 7.62) ohm.cm(-2) respectively. The values of TEER remained the same level until 4 weeks later. Two weeks after inoculation, the paracellular transport of fluorescein sodium was less than 1% after the cell models were incubated for 30 min.</p><p><b>CONCLUSIONS</b>Stratified cell layers of HPDLF and HGF grown on filter membrane of Transwell are analogous to periodontal membrane and gingiva 2 weeks after inoculation, the test results of permeability and TEER were consistent with the demands of development of cell models. HPDLF and HGF grown on filter membrane of Transwell could be used to study drug transcellular transport by HPDLF and HGF in vitro.</p>

Changes of the ultrastructures of temporomandibular joint after removal of emotional stress factors in rats / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To examine the changes of the ultrastructures of temporomandibular joint after removal of the emotional stress factors in rats.</p><p><b>METHODS</b>Thirty-two 12-week-old male Wistar rats were divided into two groups randomly, experimental group and control group. Each group was divided into two subgroups according to execution time, 9-week subgroup and 12-week subgroup with eight rats in each subgroup. Chronic unpredictable stress animal model were firstly established in experimental group in the first 6 weeks, then all the stimulation factors removed and breed normally. After 9 weeks, rats in 9-week subgroup were killed. After 12 weeks, rats in 12-week subgroup were killed. All condyles and articular discs were dissected and observed by scanning electron microscope.</p><p><b>RESULTS</b>There was some recovery in condyles and articular discs in experimental group under scanning electron microscope. The gelatum on the surface of condyles increased, collagen fibrils became regular and deep layer collagen fibrils less exposed. There were no such obvious changes on the surface of condyles and articular discs in control group.</p><p><b>CONCLUSIONS</b>The ultrastructure injures of temporomandibular joint in rats induced by emotional stress could be reversed if the stress factors were removed.</p>

In vitro analysis of intake of minocycline by mature rat mandibular osteoblasts / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the intake of minocycline and its amount in mature rat mandibular osteoblasts (MRMOB) in vitro, and to identify the feasibility of intracellular anti-bacterial activity of minocycline.</p><p><b>METHODS</b>Four groups of MRMOB were incubated in 100 mg/L minocycline for 15, 30, 45 and 60 minutes respectively, and the accumulation of minocycline within MRMOB was measured using a fluorescence spectrophotometer.</p><p><b>RESULTS</b>The intracellular accumulation amount of minocycline in the four groups of MRMOB was (17.29 +/- 1.49), (16.87 +/- 1.57), (16.96 +/- 1.67) and (17.94 +/- 1.63) mg/g respectively after osteoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained minocycline for 15, 30, 45 and 60 min. There was no significant difference in amount of minocycline among the four groups of MRMOB.</p><p><b>CONCLUSIONS</b>The mature rat mandibular osteoblasts can ingest minocycline, and the accumulation amount of minocycline in MRMOB is irrelevant with the exposure time of MRMOB to minocycline.</p>

Biological transport of minocycline by human periodontal ligament fibroblasts / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate biological transport of minocycline by human periodontal ligament fibroblasts (HPDLF). To verify the hypothesis of delivering medicine to periodontium and the whole body through the root canal.</p><p><b>METHODS</b>HPDLF and MC3T3-E1 cells were incubated in minocycline solutions. The intracellular antibiotics contents were measured by high performance liquid chromatography (HPLC) and the cell total protein was measured by Bradford protein assay.</p><p><b>RESULTS</b>HPLC was an accurate, sensitive method for measurement of the intracellular minocycline. The incubation time and cell property had significant effect on the intracellular minocycline contents (P< 0.01). The intracellular contents increased with extracellular concentration.</p><p><b>CONCLUSION</b>Minocycline can be transported by HPDLF. The transport is concentration-dependent, time-dependent and cell specific.</p>

Biological transport of tetracycline hydrochloride by human periodontal ligament fibroblasts / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate biological transport of tetracycline hydrochloride by human periodontal ligament fibroblasts (HPDLF) for verifying the hypothesis of delivering medicine to the periodontium and whole body through the root canal.</p><p><b>METHODS</b>HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by high performance liquid chromatography (HPLC) and the cell total protein was measured by bradford protein assay.</p><p><b>RESULTS</b>The intracellular contents increased with incubation time. The extracellular medicine concentration had effect on the intracellular contents.</p><p><b>CONCLUSIONS</b>Tetracycline hydrochloride can be transported into HPDLF with incubation and this transport is time-dependent and concentration-dependent.</p>

Preliminary studies on histological changes after repairing the facial nerve defect with acellular facial nerve / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the morphological changes after chemically extracted acellular nerve allografts transplant.</p><p><b>METHODS</b>Seventy-two rabbits were divided into four groups. Acellular allografts of facial nerve were used in experimental group, and facial nerve autografts, acellular peroneal nerve allografts and peroneal nerve autografts respectively used in three control groups. The morphological changes after transplant were evaluated by modified trichrome staining, immunohistological staining and transmission electron microscope.</p><p><b>RESULTS</b>The two facial nerve grafts showed numerous regenerated nerve fibers, vessels and as well as a spindle schwann cells arranged longitudinally. No significant difference was observed in the fiber number and myelin thickness between the two groups,while the two peroneal nerve groups showed poor regeneration 6 months after operation.</p><p><b>CONCLUSIONS</b>The facial nerve allografts showed more neurite regeneration six months after transplant, and the regenerated axons passed through the distal stoma and there were well revascularized and proliferated schwann cells in the grafts.</p>