| Part1Pletelet lysate promote the culture of adipose-derived stem cells innon-serum culture mediumObjective To study the changes of platelet lysate as a substitute of fetal bovinserum in culture of ADSC cells. And to provide labtory theories of individual applicationof PL in clinic.Methods Based on two-step centrifugations, PRP was prepared, and PL was preparedby the way of freeze thawing. Then ADSC cells were separated, cultrued and the immunetype markers identified. And the different influence of the ADSC expansion of10%PLgroup,5%PL group and10%FBS were checked.Results PRP was prepared with six fold of pletelet compared to the normal density.The immune type markers were normal. The result of CCK-8test showed that expansionof ADSC with10%PL was better significantly than10%FBS at the day of four.Conclusion PL was safe in the non-serum culture medium when used in the culturefor ADSC. PL would promote the expansion significantly at the concentration of10%.And the immune type markers were stable when PL existed. Part2platelet lysate promote the migration and chondrogenic differentiation ofadipose-derived stem cellsObjective To comfirm the promotion of PL to the migration of ADSC and the valueof PL at the chondrogenic differentiation of ADSC in vitro.Methods Migration test was performed using several kinds of concentration of PL byTranswell,10%FBS as a contrast. Chondrogenic differentiation culture was underwentresectively at three groups (control, TGFB3, PL+TGFB3). Alcine blue was used toevaluate proteoglycan secreted by cells. Immunofluorescence assay for type II collagenwas also used. Message-RNA of type II collagen was evaluated by qPCR at every timepoint. Message-RNA of OCN was evaluated after four weeks.Results More or less ADSC were adhered at the membrane of Transwell after48hours at all PL groups with a best migration at the group of5%. The two groups ofPL+TGFB3and TGFB3were dyeing by alcine blue after chondrogenic differentiationculture. The control group wasn’t response to immunofluorescence assay for type IIcollagen at2w and3w. But the two groups of PL+TGFB3and TGFB3were rich dyeing.Compared with chondrogenic differentiation group, the mRNA value of control group wassignificantly low at2w,3w and4w. The mRNA value of PL+TGFB3was higher thanTGFB3only at every time point. No mRNA value difference of OCN was found in thethree groups.Conclusion PL promote migration of ADSC with a dependent of concentration. Thebest concentration was5%. PL would advance the chondrogenic differentiation in acontroled circumstance without effect of osteogenic differentiation. The effect was betterthan TGFB3only. Part3Pletelet lysate combined with adipose-derived stem cellsimprove rat tendon-bone healingObjective To discuss whether Pletelet lysate combined with adipose-derived stemcells will improve rat tendon-bone healing. To observe fibro cartilage develepment at thetendon bone juncture and evaluate tendon-bone healing from histoloy and biomechanic. Methods The rat model of tendon-bone healing of Achilles tendon was established.Achilles tendon-bone interface after cleanup divided into three groups, sham operationgroup (G1), ADSC group (G2), ADSC+%PL group (G3), respectively, for treatment.Then use the HE staining, Masson staining and Alcian blue staining for histologicalobservation. Tendon bone tissue were extracted when performed qPCR detection of type IIcollagen content at2w,4w,6w. And the determination of biomechanical changes wereperformed at2w,4w,6w.Results Sharpy’s fibers were formed at G1group in HE staining, as the tide markfounded at G2after6w, for G3,4w. The typical structure of four-layer conjunction wasfounded when6w. Masson staining: G1group was loose fibrous tissue disorders, collagenfiber wave-like, messy arrangement. G2group was better aligned fibrous tissue,collagen fibers clear boundary. The G3Group neatly arranged collagen fibers, calcifiedcartilage and fibrocartilage tightly. Alcian blue staining: G1group seen tendon-bonehealing after6w, blue-stained cells around the cartilage, calcified cartilage Department hascalcium deposits, G2group seen more visible blue dye around the cartilage, chondrocytesoval, and G3groups seen around chondrocytes blue dye darker, indicating a richproteoglycan content. Aizen value of each image: G3statistical analysis showssignificantly higher than other groups. QPCR detection in each time period, G1group oftype II collagen mRNA expression were significantly less than the G2and G3, when2w,G2group of type II collagen mRNA expression and G3were similar, but weresignificantly less than the G3group when4w and6w. The maximum tensile load lowestbiomechanical test: G1group, a significant difference compared with the normal tendonbone tissue after2w, while a significant increase in G2and G3than G1. But G2and G3were significantly decreased compared to the maximum tensile load of normal tendon bonetissue. The maximum tensile load in each group increased with time. Maximum tensileload of groups G2and G3is significantly greater than G1after6w. While when6w, G3maximum tensile load was significantly higher than the normal tendon bone tissue.Conclusion Pletelet lysate combined with adipose-derived stem cells will promotemigration and chondrogenic differentiation of ADSC, result in improved rat tendon-bonehealing. |
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