Objective To establish analytical method for the determination of tramadol and its metabolite O - demethyltramadol in human plasma and urine by RP - HPLC. Methods Separation was carried out ons a zorbax RP - select B column and the mobile phase was a mixture of 0. 05 mol · L~(-1) potassium dihydrogen phosphate ( adjusted to pH 4. 0 with phosphoric acid)-acetonitrile (90: 10). The flow rate was 1 mL · min~(-1). Fluormetric detection was performed at excitation and emission wavelengths of 216 run and 308 nm, respectively. Results In plasma, the calibration curve was linear within 12. 5 - 800. 0 ng · mL~(-1) for tramadol and 5 -320 ng · mL~(-1) for O - demethyltramadol. The absolute recoveries were all higher than 86% , and the relative recoveries were from 93% to 105%. The RSD of the within - day and between - day variations were lower than 9% and 8% , respectively. In urine, the calibration curve was linear within 12. 5 -4 000 ng · mL~(-1) for tramadol and 5 -1 280 ng · mL~(-1) for O -demethyltramadol. The absolute recoveries were all higher than 80% , and the relative recoveries were from 91% to 107%. The RSD of the within-day and between-day variations were lower than 9%. For stability test in plasma and urine, the difference of the results was within 10%. Conclusion This RP - HPLC method is simple, sensitive, accurate and specific.

OBJECTIVETo evaluate the effects of Icariin (ICA) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in vitro and in vivo.

METHODSAn enzymatic digestion block was used in vitro to culture hPDLSCs, which were separated and purified by limited dilution cloning. The hPDLSCs were identified using cell-surface markers and cocultured with 1 x 10(-7) mol.L-1 ICA solution. The proliferation ability of these cells was determined by thiazolyl blue tetrazolium bromide (MTT) assay. After staining with alkaline phosphatase (ALP), osteogenesis was detected by enzyme-linked immunosorbent assay. Osteoblast-related genes were analyzed by reverse transcription-polymerase chain reaction. Alizarin red staining was performed to measure the level of calcium deposition. The hPDLSCs were cocultured with 1 x 10(-7) mol.L-1 ICA and nano-hydroxyapatite scaffolds in vivo before transplantation into subcutaneous tissues of nude mice. Osteogenic abilities were histochemically analyzed after 30 days of induction.

RESULTSThe hPDLSCs were affected by 1 x 10(-7) mol.L-1 ICA, and MTT assay showed that the proliferation of the groups treated with ICA in vitro was better than that of the control groups on the second day. The ALP activity of the treated hPDLSCs was significantly enhanced after cell culture for 3, 5, and 7 days. The gene expression of osteoblastic markers was also significantly enhanced after 7 days. The deposition of mineralization after incubation with 1 x 10(-7) mol.L-1 ICA increased compared with the control after cell culture for 14, 21, and 28 days. Furthermore, the bone expression of the treatment groups in vivo was significantly enhanced compared with that of the control groups.

CONCLUSIONTreatment with 1 x 10(-7) mol.L-1 ICA can significantly promote proliferation and differentiation of hPDLSCs in vitro and in vivo. ICA can effectively function as a bioactive growth factor in periodontal tissue engineering to replace traditional growth factors.

Alkaline Phosphatase ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Coculture Techniques ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; Humans ; Mice ; Mice, Nude ; Osteoblasts ; Osteogenesis ; Periodontal Ligament ; Stem Cells

Objective??To?evaluate?the?effects?of?Icariin?(ICA)?on?the?proliferation?and?osteogenic?differentiation?of?human?periodontal?ligament?stem?cells?(hPDLSCs)?in vitro and?in vivo. Methods??An?enzymatic?digestion?block?was?used?in vitro?to?culture?hPDLSCs,?which?were?separated?and?purified?by?limited?dilution?cloning.?The?hPDLSCs?were?identified?using?cell-surface?markers?and?cocultured?with?1×10?7 mol·L?1 ICA?solution.?The?proliferation?ability?of?these?cells?was?determined?by?thiazolyl?blue?tetrazolium?bromide?(MTT)?assay.?After?staining?with?alkaline?phosphatase?(ALP),?osteogenesis?was?detected?by?enzyme-linked?immunosorbent?assay.?Osteoblast-related?genes?were?analyzed?by?reverse?transcription-polymerase?chain?reaction.?Alizarin?red?staining?was?performed?to?measure?the?level?of?calcium?deposition.?The?hPDLSCs?were?cocultured?with?1×10?7 mol·L?1 ICA?and?nano-hydroxyapatite?scaffolds?in vivo?before?transplantation?into?subcutaneous?tissues?of?nude?mice.?Osteogenic?abilities?were?histochemically?analyzed?after?30?days?of?induction.?Results??The?hPDLSCs?were?affected?by?1×10?7 mol·L?1 ICA,?and?MTT?assay?showed?that?the?proliferation?of?the?groups?treated?with?ICA?in vitro?was?better?than?that?of?the?control?groups?on?the?second?day.?The?ALP?activity?of?the?treated?hPDLSCs?was?significantly?enhanced?after?cell?culture?for?3,?5,?and?7?days.?The?gene?expression?of?osteoblastic?markers?was?also?significantly?enhanced?after?7?days.?The?deposition?of?mineralization?after?incubation?with?1×10?7 mol·L?1?ICA?increased?compared?with?the?control?after?cell?culture?for?14,?21,?and?28?days.?Furthermore,?the?bone?expression?of?the?treatment?groups?in vivo?was?significantly?enhanced?com-pared?with?that?of?the?control?groups.?Conclusion??Treatment?with?1×10?7 mol·L?1 ICA?can?significantly?promote?proliferation?and?differentiation?of?hPDLSCs?in vitro?and?in vivo.?ICA?can?effectively?function?as?a?bioactive?growth?factor?in?periodontal?tissue?engineering?to?replace?traditional?growth?factors.