Proteome response of dental pulp cells to exogenous FGF8.

FGF8 specifies early tooth development by directing the migration of the early tooth founder cells to the site of tooth emergence. To date the effect of the FGF8 in adult dental pulp has not been studied. We have assessed the regenerative potential of FGF8 by evaluating changes in the proteome landscape of dental pulp following short- and long-term exposure to recombinant FGF8 protein. In addition, we carried out qRT PCR analysis to determine extracellular/adhesion gene marker expression and assessed cell proliferation and mineralization in response to FGF8 treatment. 2D and mass spectrometry data showed differential expression of proteins implicated in cytoskeleton/ECM remodeling and migration, cell proliferation and odontogenic differentiation as evidenced by the upregulation of gelsolin, moesin, LMNA, WDR1, PLOD2, COPS5 and downregulation of P4HB. qRT PCR showed downregulation of proteins involved in cell-matrix adhesion such as ADAMTS8, LAMB3 and ANOS1 and increased expression of the angiogenesis marker PECAM1. We have observed that, FGF8 treatment was able to boost dental pulp cell proliferation and to enhance dental pulp mineralization. Collectively, our data suggest that, FGF8 treatment could promote endogenous healing of the dental pulp via recruitment of dental pulp progenitors as well as by promoting their angiogenic and odontogenic differentiation. SIGNIFICANCE: Dental pulp cells (DP) have been studied extensively for the purposes of mineralized tissue repair, particularly for the reconstruction of hard and soft tissue maxillofacial defects. Canonical FGF signaling has been implicated throughout multiple stages of tooth development by regulating cell proliferation, differentiation, survival as well as cellular migration. FGF8 expression is indispensible for normal tooth development and particularly for the migration of early tooth progenitors to the sites of tooth emergence. The present study provides proteome and qRT PCR data with regard to the future application and biological relevance of FGF8 in dental regenerative medicine. AUTHORS WITH ORCID: Rozaliya Tsikandelova - 0000-0003-0178-3767 Zornitsa Mihaylova - 0000-0003-1748-4489 Sébastien Planchon - 0000-0002-0455-0574 Nikolay Ishkitiev - 0000-0002-4351-5579.

Role of PDGF-BB in proliferation, differentiation and maintaining stem cell properties of PDL cells in vitro.

OBJECTIVE: Platelet-derived growth factor-BB (PDGF-BB) is one of the most abundant growth factors in platelet derived products and has been shown to stimulate regeneration after tissue injury. There is a population of mesenchymal stem cells (MSC) in human periodontal ligament (PDL) which can contribute to tissue regeneration under appropriate conditions. DESIGN: PDL cells were isolated and characterized using stem cell and differentiation markers via immunofluorescence and flow cytometry and then cultured in vitro and treated with different concentrations of PDGF-BB. The effect of PDGF-BB on cell proliferation, stem cell and differentiation markers expression, soluble collagen production, lysyl oxidase (LOX) activity, alkaline phosphatase (ALP) activity and calcium nodules formation was assessed. RESULTS: PDGF-BB stimulated the proliferation of cells with the maximum effect at 50ng/mL. The growth factor increased the expression of stem cell markers and SPARC; Col1a2 expression was decreased, whereas the expression of Col3a1 remain unchanged. Soluble collagen production, ALP activity and calcium nodules formation were also significantly decreased by PDGF-BB; LOX activity was significantly increased. CONCLUSIONS: PDGF-BB is a powerful promoter of cell proliferation and increases the expression of stem cell markers; inhibites collagen production and mineraliration but accelerates the maturation of collagen chains through increased LOX activity and SPARC expression.

Use of platelet concentrates in oral and maxillofacial surgery: an overview.

OBJECTIVE: To describe and provide a comprehensive overview on the development, use and efficacy of autologous platelet concentrates in different in vitro and in vivo studies focusing on oral and maxillofacial pathologies. MATERIALS AND METHODS: Present work employs an extensive critical overview of the literature on the development and application of platelet concentrates. RESULTS: Platelet concentrates are innovative endogenous therapeutic agents which gained a lot of interest in different medical and dental disciplines due to their potential ability to stimulate and increase regeneration of soft and hard tissues. The effect of platelet-derived products is considered to be a result of the high number of platelets which contain a wide range of growth factors. They are not just therapeutic products but autologous blood concentrates containing active molecules. The quality of platelet concentrates may vary according to the individual physical state of donors making it difficult to to compare the outcomes of their application. Although, there are many studies analyzing the properties of these biomaterials both in vivo and in vitro, a consensus regarding their efficacy still has to be reached. CONCLUSION: Evidences described in the literature on the efficacy of platelet concentrates in procedures in oral and maxillofacial region are controversial and limited. In order to clarify the real advantages and priorities for the patients, when the blood-derived products are applied, further in vitro and in vivo research about the activity of PRP and PRF on the dental cells biology should be conducted.

From Normal Skin to Squamous Cell Carcinoma: A Quest for Novel Biomarkers.

Squamous cells carcinoma (SCC) is the second most frequent of the keratinocyte-derived malignancies after basal cell carcinoma and is associated with a significant psychosocial and economic burden for both the patient himself and society. Reported risk factors for the malignant transformation of keratinocytes and development of SCC include ultraviolet light exposure, followed by chronic scarring and inflammation, exposure to chemical compounds (arsenic, insecticides, and pesticides), and immune-suppression. Despite various available treatment methods and recent advances in noninvasive or minimal invasive diagnostic techniques, the risk recurrence and metastasis are far from being negligible, even in patients with negative histological margins and lymph nodes. Analyzing normal, dysplastic, and malignant keratinocyte proteome holds special promise for novel biomarker discovery in SCC that could be used in the future for early detection, risk assessment, tumor monitoring, and development of targeted therapeutic strategies.

Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.

Regeneration of insulin-producing pancreatic cells using a volatile bioactive compound and human teeth.

Transplantation of insulin (INS)-secreting cells differentiated in vitro from stem cells promises a safer and easier treatment of severe diabetes mellitus. A volatile bioactive compound, hydrogen sulfide (H2S), promotes cell differentiation; human tooth-pulp stem cells undergo hepatic differentiation. The aim of this study is to develop a novel protocol using H2S to enhance pancreatic differentiation from the CD117(+) cell fraction of human tooth pulp. During the differentiation, the cells were exposed to 0.1 ng ml(-1) H2S. Immunocytochemistry, RT-PCR, determination of INS c-peptide content and flow cytometry of pancreatically related markers were carried out. Expression of WNT and the PI3K/AKT signaling pathway were also determined by PCR array. After differentiation, INS, glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PPY) were positive when examined by immunofluorescence. INS and GCG were also determined flow-cytometrically. Only the cells expressing INS increased after H2S exposure. The number of cells expressing GCG was significantly decreased. Genes involved in canonical WNT and the WNT/calcium pathways were highly expressed after H2S exposure. H2S accelerated INS synthesis and secretion by regenerated INS-producing cells from human teeth. All signaling pathway functions of the PI3K-AKT pathway were extremely activated by H2S exposure. The matured INS-producing cells originating in human teeth were increased by H2S in order to control blood-glucose level.

Pancreatic differentiation of human dental pulp CD117⁺ stem cells.

AIM: Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. MATERIALS & METHODS: CD117⁺ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. RESULTS: 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. CONCLUSION: Our results show that CD117⁺ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.

High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium.

INTRODUCTION: We have previously differentiated hepatocyte like cells from deciduous tooth pulp stem and extracted third molar pulp stem cells with a protocol that used fetal bovine serum, but it showed high contaminations of nondifferentiated cells. Both the lower purity of hepatically differentiated cells and usage of serum are obstacles for application of cell therapy or regenerative medicine. Objective of this study was to investigate the capacity for and purity of hepatocyte-like differentiation of CD117-positive dental pulp stem cells without serum. METHODS: Mesenchymal cells from human deciduous and extracted third molar pulp were isolated and expanded in vitro. We separated CD117-positive cells by using a magnetic-activated cell sorter. The cells were characterized immunofluorescently by using known stem cell markers. For hepatic differentiation, the media were supplemented with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. Expression of hepatic markers alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase was examined immunofluorescently after differentiation. The amount of differentiated cells was assessed by using flow cytometry. Glycogen storage and urea concentration in the medium were defined. RESULTS: Both cell cultures demonstrated a number of cells positive for all tested hepatic markers after differentiation, ie, albumin-positive cells were almost 90% of differentiated deciduous pulp cells. The concentration of urea in the media increased significantly after differentiation. Significant amount of cytoplasmic glycogen storage was found in the cells. CONCLUSIONS: Without serum both cell types differentiated into high-purity hepatocyte-like cells. These cells offer a source for hepatocyte lineage differentiation for transplantation in the future.

The role of p53 in an apoptotic process caused by an oral malodorous compound in periodontal tissues: a review.

Oral malodor is caused by volatile sulfur compounds (VSCs) composed mainly of hydrogen sulfide (H(2)S) and methyl mercaptan. In particular, H(2)S is an important compound, since it is a major component of physiologic halitosis. The toxicity of VSCs is similar to that of hydrogen cyanide, and is well investigated. The role of VSCs in reducing collagen in human gingival fibroblasts is one of the main sources of their toxicity to human oral tissues. It has been reported recently that H(2)S may cause apoptosis in several periodontal tissues. In human gingival fibroblasts, H(2)S inhibits not only cytochrome c oxidase activity but also superoxide dismutase activity. The levels of reactive oxygen species are markedly increased, which causes the release of cytochrome c into the cytoplasm, resulting in caspase-9 activation; finally, the executor caspase, caspase-3, is activated. This pathway is commonly observed in cells from all periodontal tissues. Moreover, p53, an apoptotic factor, and phosphorlylated p53, which is the activated form, are increased by H(2)S in keratinocyte stem cells and osteoblasts. H(2)S also increases the expression of Bax, a primary response gene playing an important role in p53-mediated apoptosis, but maintains a lower expression of Bcl-2, an anti-apoptotic factor, in osteoblasts. It is concluded that the Bax apoptotic pathway and the mitochondrial pathway are activated by H(2)S.

Expression of multiple stem cell markers in dental pulp cells cultured in serum-free media.

INTRODUCTION: Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS: Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS: Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS: SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.

Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro.

INTRODUCTION: Mesenchymal stem cells display extensive proliferative capacity of multilineage differentiation. The stromal compartment of mesenchymal tissues is considered to harbor stem cells. We assessed the endodermal differentiation of mesenchymal cells from deciduous and wisdom tooth pulp. METHODS: Dental mesenchymal cells were isolated and expanded in vitro. After cell cultures had been established, cells were characterized using known stem cell markers. For hepatic differentiation the media was supplemented with hepatic growth factor, dexamethasone, Insulin-Transferrin-Selenium-X, and oncostatin. RESULTS: Both cultures showed a number of cells positive for specific hepatic markers including alpha-fetoprotein, albumin, and hepatic nuclear factor 4alpha after differentiation. Also, small clusters of cells positive for insulin-like growth factor 1 were found. The concentration of urea increased significantly in the media. Moreover, a significant amount of glycogen was found in the cells. CONCLUSION: Because the cells proved to produce specific hepatic proteins and to start functions specific for hepatocytes, such as storing glycogen and urea production, we may state that the mesenchymal cell cultures from wisdom and deciduous tooth pulp acquired morphologic and functional characteristics of hepatocytes.