Abstract-A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In the present study, an efficient method for generating bone tissue from cultured human dental pulp-and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, -glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human (rh) BMP2 to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility.
A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 -6) were used as substrates, PaChiB degraded (GlcNAc)4 -6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 -4) fell in the following order: pNp-(GlcNAc)3≫ pNp-(GlcNAc)2 pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%-185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.
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