Investigation of dental pulp stem cells isolated from discarded human teeth extracted due to aggressive periodontitis.

Recently, human dental pulp stem cells (DPSCs) isolated from inflamed dental pulp tissue have been demonstrated to retain some of their pluripotency and regenerative potential. However, the effects of periodontal inflammation due to periodontitis and its progression on the properties of DPSCs within periodontally compromised teeth remain unknown. In this study, DPSCs were isolated from discarded human teeth that were extracted due to aggressive periodontitis (AgP) and divided into three experimental groups (Groups A, B and C) based on the degree of inflammation-induced bone resorption approaching the apex of the tooth root before tooth extraction. DPSCs derived from impacted or non-functional third molars of matched patients were used as a control. Mesenchymal stem cell (MSC)-like characteristics, including colony-forming ability, proliferation, cell cycle, cell surface antigens, multi-lineage differentiation capability and in vivo tissue regeneration potential, were all evaluated in a patient-matched comparison. It was found that STRO-1- and CD146-positive DPSCs can be isolated from human teeth, even in very severe cases of AgP. Periodontal inflammation and its progression had an obvious impact on the characteristics of DPSCs isolated from periodontally affected teeth. Although all the isolated DPSCs in Groups A, B and C showed decreased colony-forming ability and proliferation rate (P < 0.05), the decreases were not consistent with the degree of periodontitis. Furthermore, the cells did not necessarily show significantly diminished in vitro multi-differentiation potential. Only DPSCs from Group A and the Control group formed dentin-like matrix in vivo when cell-seeded biomaterials were transplanted directly into an ectopic transplantation model. However, when cell-seeded scaffolds were placed in the root fragments of human teeth, all the cells formed significant dentin- and pulp-like tissues. The ability of DPSCs to generate dental tissues decreased when the cells were isolated from periodontally compromised teeth (P < 0.05). Again, increased periodontal destruction was not necessarily followed by a decrease in the amount of dentin- and pulp-like tissue formed. These findings provide preliminary evidence that periodontally compromised teeth might contain putative stem cells with certain MSC properties, as long as the vitality of the pulp has not been totally damaged. Whether these cells can serve as a source of autologous multipotent MSCs for clinical regenerative therapies warrants further investigation with larger sample sizes and various types of periodontitis.

[Cloning and transcriptional regulation analysis of amelogenin promoter in different cells].

PURPOSE: To clone different promoter region of amelogenin gene and analyze their transcriptional activity. METHODS: The upstream regulation sequences of amelogenin gene were retrieved and analyzed. Amplified by PCR from the genomic DNA of mouse C57BL/6J and digested with restriction endonucleases enzyme, different transcriptional regulation sequences of 5'flanking of amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3-Basic vector. These report vectors were transiently transfected into CHO, Hela and UMR-106 cells and luciferase assay was performed to analyse the transcription activation of these promoters. RESULTS: 6 promoters different in length were cloned. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Luciferase activity fluctuated with the different promoter lengths in Hela cell as well as in CHO and UMR-106 cells. 975 bp and 532 bp of amelogenin 5'flanking DNA had a strong transcriptional activation, but 285 bp of amelogenin 5'flanking DNA had a weaker transcriptional activation. CONCLUSION: The sequence of amelogenin promoter can be activated in Hela cell. Hela cell can be used as a good model to study the transcriptional regulation of amelogenin promoter. According to the different activities of different lengths, it is suggested that there were some potential siliencer located between -975 and -532, and some potential activator in the region between -532 and -285.

[Cloning and sequencing of the upstream of mouse dentin sialophosphoprotein promoter].

OBJECTIVE: To clone and sequence the upstream of mouse dentin sialophosphoprotein promoter. METHODS: Genomic DNA was obtained from Balb/c mouse blood. The upstream of mouse dentin sialophosphoprotein promoter segments was obtained by PCR. Then the segments were inserted into T-vector. The plasmids were identified by digestion with the restriction enzyme analysis. The positive clone was sequenced and compared with Genebank. RESULTS: The upstream of mouse dentin sialophosphoprotein promoter was divided into three sequences and three different target segments with 997 bp, 1004 bp and 674 bp in length were obtained. After identified, sequenced and compared with Genebank, the sequences of the segments were consistent with those displayed on Genebank by 99%. CONCLUSION: The clone of the upstream of mouse dentin sialophosphoprotein promoter was successful. This work will help to study the regulation of DSPP expression.

[Cbfa1 induces the expression of the mineral-related proteins in human dental papilla cells].

OBJECTIVE: To explicit whether the expression of the mineral-related proteins is regulated by cbfa1 in human dental papilla cells. METHODS: Human dental papilla cells were cultured in vitro and transfected with pcDNA3-cbfa1 recombinant plasmids. After selected with G418 sulfate, a cell clone named PC-3, which could stably express the cbfa1 mRNA and protein, was proved by PCR and western blot. Then the amount of ALP and OC and the expression of OPN, BSP, ON, DMP1, DSP and DSPP were detected by immunohistochemistry, Western blot and PCR methods. RESULTS: We established the human dental papilla cells model PC-3 which could stably express the cbfa1 mRNA and protein. Compared with normal cells, a lot of mineral-related proteins such as ALP, OC, OPN, BSP, ON, DMP1 were upregulated in PC-3 cells. CONCLUSIONS: In human dental papilla cells, cbfa1 can induce the expression of most mineral-related genes and proteins. It may implicate that cbfa1 must play a key role during tooth development and mineralization.