Growth Factor Measurement and Histological Analysis in Platelet Rich Fibrin: A Pilot Study

Objective

The aim of this study was to compare growth factor amount contained in platelet rich fibrin (PRF) and compare with that in platelet rich plasma (PRP), and in whole blood. And also to investigate distribution of growth factors and cellular components in PRF.

Materials and Methods

PRF and PRP were obtained from the same sample of peripheral blood. Extraction of proteins were done with lysis buffer, accompanied by freeze and thaw procedures. Concentration of two representative growth factors in platelets: platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β), were measured with enzyme-linked immunosorbent assay (ELISA). PRF was cut into three parts: (top, middle and bottom), and growth factor concentration was measured respectively. Paraffin embedded section of PRF was observed with Giemsa stain. Immuno-histochemical analysis with anti-PDGF and anti-TGF-β antibodies was also conducted.

Results

The growth factor levels in PRF was higher than in peripheral blood and comparable to those in PRP. Growth factor levels in bottom part of PRF was much higher than in top and middle part. Microscopically, platelets and mono-nucleated cells were concentrated just above the yellow–red interface. Poly-nucleated cells were concentrated below the interface.

Conclusion

The growth factors were surely concentrated in PRF. This result can support basis of good clinical outcomes. For effective application of PRF, the knowledge that growth factors and cells are not equally distributed in PRF should be utilized.     

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) Analysis for Changes in the Gut Microbiota Profiles of Indomethacin- and Rebamipide-Treated Mice

Background/Aims: We investigated the effects of indomethacin and rebamipide on the gut microbiota profiles using terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and Methods: Female C57BL/6J mice were given indomethacin (10 mg/kg, s.c.) once a day and 2.5 mg rebamipide orally 3 times a day. After 7 days, they were sacrificed, and luminal contents were obtained from the ileum and cecum. The gut microbiota communities were analyzed by T-RFLP analysis with BslI digestion. Results: T-RFLP analyses showed that rebamipide and indomethacin had no significant effects on the gut microbiota profiles in the ileum and cecum. In contrast, the combination of rebamipide + indomethacin induced a significant change in the gut microbiota. The changes in the microbiota composition induced by the combination of rebamipide + indomethacin were characterized by the increase in the orders Bifidobacteriales and Lactobacillales, the genera Bacteroides and Prevotella and the family Clostridiaceae. The diversity of the gut microbiota community generated by the combination of rebamipide + indomethacin was significantly higher than those induced by either rebamipide or indomethacin alone. Conclusion: The combination of rebamipide + indomethacin induces remarkable changes in the gut microbiota composition and diversity. The clinical activity of rebamipide on nonsteroidal anti-inflammatory drug-induced intestinal injury may be exerted through a modulation of the gut microbiota.     

Wnt2 accelerates cardiac myocyte differentiation from ES-cell derived mesodermal cells via non-canonical pathway

The efficient induction of cardiomyocyte differentiation from embryonic stem (ES) cells is crucial for cardiac regenerative medicine. Although Wnts play important roles in cardiac development, complex questions remain as to when, how and what types of Wnts are involved in cardiogenesis. We found that Wnt2 was strongly up-regulated during cardiomyocyte differentiation from ES cells. Therefore, we investigated when and how Wnt2 acts in cardiogenesis during ES cell differentiation. Wnt2 was strongly expressed in the early developing murine heart. We applied this embryonic Wnt2 expression pattern to ES cell differentiation, to elucidate Wnt2 function in cardiomyocyte differentiation. Wnt2 knockdown revealed that intrinsic Wnt2 was essential for efficient cardiomyocyte differentiation from ES cells. Moreover, exogenous Wnt2 increased cardiomyocyte differentiation from ES cells. Interestingly, the effects on cardiogenesis of intrinsic Wnt2 knockdown and exogenous Wnt2 addition were temporally restricted. During cardiomyocyte differentiation from ES cells, Wnt2 didn't activate canonical Wnt pathway but utilizes JNK/AP-1 pathway which is required for cardiomyocyte differentiation from ES cells. Therefore we conclude that Wnt2 plays strong positive stage-specific role in cardiogenesis through non-canonical Wnt pathway in murine ES cells.