The platelet-rich fibrin (PRF) is known as a rich source of autologous cytokines and growth factors and universally used for tissue regeneration in current clinical medicine. However, the microstructure of PRF has not been fully investigated nor have been studied the key molecules that differ PRF from platelet-rich plasma. We fabricated PRF under Choukroun's protocol and produced its extract (PRFe) by freezing at À808C. The conventional histological, immunohistological staining, and scanning electron microscopy images showed the microstructure of PRF, appearing as two zones, the zone of platelets and the zone of fibrin, which resembled a mesh containing blood cells. The PRFe increased proliferation, migration, and promoted differentiation of the human alveolar bone marrow stem cells (hABMSCs) at 0.5% concentration in vitro. From the results of proteome array, matrix metalloproteinase 9 (MMP9) and Serpin E1 were detected especially in PRFe but not in concentrated platelet-rich plasma. Simultaneous elevation of MMP9, CD44, and transforming growth factor b-1 receptor was shown at 0.5% PRFe treatment to the hABMSC in immunoblot. Mineralization assay showed that MMP9 directly regulated mineralization differentiation of hABMSC. Transplantation of the fresh PRF into the mouse calvarias enhanced regeneration of the critical-sized defect. Our results strongly support the new characteristics of PRF as a bioscaffold and reservoir of growth factors for tissue regeneration.
Periodontitis is the most common cause for tooth loss in adults and advanced types affect 10-15% of adults worldwide. The attempts to save tooth and regenerate the periodontal apparatus including cementum, periodontal ligament, and alveolar bone reach to the dental tissue-derived stem cell therapy. Although there have been several periodontitis models suggested, the apical involvement of tooth root is especially challenging to be regenerated and dental stem cell therapy for the state has never been investigated. Three kinds of dental tissue-derived adult stem cells (aDSCs) were obtained from the extracted immature molars of beagle dogs (n = 8), and ex vivo expanded periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and periapical follicular stem cells (PAFSCs) were transplanted into the apical involvement defect. As for the lack of cementum-specific markers, anti-human cementum protein 1 (rhCEMP1) antibody was fabricated and the aDSCs and the regenerated tissues were immunostained with anti-CEMP1 antibody. Autologous PDLSCs showed the best regenerating capacity of periodontal ligament, alveolar bone, and cementum as well as peripheral nerve and blood vessel, which were evaluated by conventional and immune histology, 3D micro-CT, and clinical index. The rhCEMP1 was expressed strongest in PDLSCs and in the regenerated periodontal ligament space. We suggest here the PDLSCs as the most favorable candidate for the clinical application among the three dental stem cells and can be used for treatment of advanced periodontitis where tooth removal was indicated in the clinical cases.
The Wnt family of proteins regulates development and cell growth. We identified Wnt3a-based regulatory mechanisms for cell proliferation in NIH3T3 fibroblast cells. The degree of Wnt3a-induced proliferation was reduced by β-catenin small interfering RNA (siRNA) and extracellular signalregulated kinase (ERK) siRNA, indicating that both the ERK and Wnt/β-catenin pathways are involved in Wnt3a-induced proliferation. Wnt3a immediately and transiently activated the Raf-1-MEK-ERK cascade in a manner distinct from that of the β-catenin increase seen in cells treated with Wnt3a. Wnt3a-induced ERK activation was maintained even though basal ERK activities were reduced by β-catenin siRNA, indicating that Wnt3a may activate the ERK pathway independently of β-catenin. The ERK pathway was however, activated by β-catenin transfection, which was abolished by co-transfection with dominantnegative Tcf-4. Therefore, ERK pathway activation by Wnt signaling could occur at multiple levels, including β-catenin-independent direct signaling resulting from a Wnt3a and β-catenin/Tcf-4-dependent post gene transcriptional event. Wnt3a stimulated the G1 to S phase cell cycle progression. This stimulation was reduced by the ERK pathway inhibitor, indicating that Wnt3a promotes proliferation by stimulating the ERK pathway. Wnt3a therefore stimulates the proliferation of fibroblast cells, at least in part, via activation of the ERK and Wnt/β-catenin pathways.
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