OBJECTIVETo evaluate the use of alginate three-dimensional culture system for mandibular condylar chondrocytes culture in vitro from human osteoarthritic temporomandibular joint.

METHODSThe cultured mandibular condylar chondrocytes from the operatively removed cartilage from a patient with osteoarthritic temporomandibular joint were harvested by mechanical dissection and enzyme digestion. Partial chondrocytes were suspended in the aqueous sodium alginate beads with high seeding density and cultured for 4 weeks, while the others were cultured in monolayer culture condition for 1 week. Thereafter, the alginate beads were embedded in paraffin and sectioned, then studied immunologically with type II collagen antibody and aggrecan antibody, same studies were adopted for the monolayer cultures.

RESULTSThe monolayer cultures were confirmed as chondrocytes. The chondrocytes cultured in the alginate medium showed well. These cells exhibited the excellent differentiated phenotype after 4 weeks culture in alginate gel.

CONCLUSIONSThe condylar chondrocytes from human osteoarthritic temporomandibular joint were successfully cultured in vitro. The alginate three-dimensional culture system was successfully adopted for in vitro culture of condylar chondrocytes from human osteoarthritic temporomandibular joint, in which the chondrocytes exhibited the excellent differentiated phenotype.

Cartilage, Articular ; Cell Culture Techniques ; Cells, Cultured ; Chondrocytes ; cytology ; Humans ; Mandibular Condyle ; Temporomandibular Joint

ObjectiveTo establish the quality standard of Liangditang benchmark samples. MethodUltra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF-MS） was used to qualitatively analyze the chemical composition of Liangditang on the basis of molecular and fragment ion peak information with cracking law. The mobile phase was methanol （A）-0.05% phosphate aqueous solution （B） for gradient elution （0-10 min， 5%-23.5%A； 10-20 min， 23.5%A； 20-58 min， 23.5%-63%A； 58-60 min， 63%-90%A）， the flow rate was 0.8 mL·min^-1， and the detection wavelength was 254 nm. Electrospray ionization was employed under positive ion mode， the detection range was m/z 100-1 700. Key quality attributes and sources were determined by comparing with single medicine and reference substances. Through mass transfer analysis of multiple batches from decoction pieces to benchmark samples， high performance liquid chromatography （HPLC） for determining the contents of index components and HPLC detection of characteristic maps were established. Through the determination of 15 batches of benchmark samples， the content range of the index components and the common peaks of the characteristic map were determined. Thin layer chromatography （TLC） was applied to the identification of 5 medicines in the formula. Moisture and dry extract yield of the benchmark samples were determined by drying method. ResultA total of 27 compounds were inferred from the benchmark samples of Liangditang， among which 9 compounds were confirmed by comparison with the control， including catalpol， harpagide， gallic acid， albiflorin， paeoniflorin， verbascoside， angoroside C， cinnamic acid and harpagoside. A method for determining the characteristic maps of the benchmark samples were established and 13 peaks were assigned， and the characteristic peaks were mainly derived from wine-processed products of Rehmanniae Radix， Scrophulariae Radix and wine-processed products of Paeoniae Radix Alba. The similarity between the characteristic map of 15 batches of benchmark samples and the control characteristic map was >0.9. Methods for the determination of paeoniflorin， harpagoside， L-hydroxyproline and glycine were established， and the contents of these four components in 15 batches of benchmark samples were within ±30% of the corresponding mean value， and the transfer rate of decoction pieces to the benchmark samples was stable and controllable. TLC was established to identify 5 prescription drugs （except Ejiao） with two kinds of test solutions， and the results showed that the method had good specificity. The average dry extract yield was 48.06%， and the average moisture was 5.58%， which were within the range of ±10% and ±30% of their mean values， respectively. ConclusionThe quality standard of Liangditang benchmark samples was as follows：the similarity between the benchmark samples and the control characteristic map is >0.9， the contents of paeoniflorin， harpagoside， L-hydroxyproline and glycine are 217-403， 24-46， 634-1 178， 1 253-2 328 mg per dose， the dry extract yield is 43.0%-53.0%， the moisture is 4.0%-7.0%， under the set detection conditions， the benchmark samples have corresponding characteristic spots by comparing with the control herbs of 5 medicines. This quality standard is stable and reliable， which fills the gap in the quality control of Liangditang， and can provide a reference for the establishment of the quality standard of Liangditang granules.