PUBLISHED RESEARCH 2010

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ cells DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formeding calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

The use of allogeneic stem cells strongly extends the range of stem cell applications in dentistry; however, immunological rejection remains a major concern. There is little information about the immunological features of dental-related stem cells in the literature. Therefore, we investigated the immunological characteristics of stem cells from the root apical papilla (SCAP) of swine in vitro by measuring T cell immunomodulation and apoptosis. We found that SCAP expressed a low level of swine leukocyte antigen (SLA) class I molecules and were negative for SLA class II DR molecules. Moreover, SCAP could inhibit autologous T cell proliferation stimulated by phytohemagglutinin (PHA) and a one-way mixed lymphocyte reaction in a dose-dependent manner. In addition, SCAP could suppress proliferation of allogeneic T cells in a dose-dependent manner, with or without mitomycin C pretreatment. Moreover, soluble factor(s) may be involved in the SCAP-mediated immune suppression. After a 5-day coculture of SCAP, allogeneic T cells, and PHA, only 1.22% of T cells were apoptotic. These data indicated that SCAP were weakly immunogenic and suppressed T cell proliferation in vitro through an apoptosis-independent mechanism.

Stem cells from apical papilla (SCAP) are a novel population of multipotent stem cells that, although similar to dental pulp stem cells, are a discrete source of dental stem cells. SCAP have potential roles in root development, apexogenesis, pulp/dentin regeneration, and bioroot engineering. However, procedures to store and preserve SCAP for future clinical applications have not been explored. In this study, we compared human freshly isolated SCAP (fSCAP) with cryopreserved SCAP (cSCAP) in terms of cell viability, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, profiles of mesenchymal stem cell (MSC) markers, karyotype analysis, and immunological assays. cSCAP showed a similar viable cell ratio, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, MSC surface markers, apoptotic rate, and G-banded karyotype when compared to fSCAP. There was no significant difference between fSCAP and cSCAP with regard to immune properties. In addition, cSCAP of miniature pig possessed the similar proliferation rate, differentiation potential, and immunomodulatory function as seen in fSCAP. This study demonstrates that cryopreservation does not affect the biological and immunological properties of SCAP, supporting the feasibility of SCAP cryopreservation in nitrogen. J. Cell. Physiol. (c) 2010 Wiley-Liss, Inc.

Dental stem cells such as dental follicle precursor cells (DFPCs) are capable of neural-like differentiation. However, compared to neuroectodermal progenitor cells such as murine retinal progenitor cells (mRPCs) they show only a limited capacity for glial cell differentiation. In this study we tested the influence of cell signaling on glial differentiation of mDFPCs. These cells were treated with inhibitors and activators of the Sonic hedgehog-, the Wnt/beta-Catenin-, and the TGF-beta-pathway. After incubation only an activation of the TGF-beta-pathway showed a remarkable glial-like cell differentiation. In contrast gene expression of neural cell markers was not regulated. In conclusion, TGF-beta improved glial-like, but not neural-like, differentiation of mDFPCs.

INTRODUCTION: Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineage-specific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. METHODS: We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. RESULTS: The proliferation rate of SCD was higher (cell number, 25 x 10(6) cells/mL; percent colony-forming units [CFUs], 151.67 +/- 10.5; percent cells in S/G2 phase, 12.4 +/- 1.48) than that of DPSCs (cell number, 21 x 10(6) cells/mL; percent CFUs, 133 +/- 17.62; percent cells in S/G2 phase, 10.4 +/- 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (>2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression >100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. CONCLUSIONS: This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.

Periodontal disease, a worldwide prevalent chronic disease in adults, is characterized by the destruction of the periodontal supporting tissue including the cementum, periodontal ligament and alveolar bone. The regeneration of damaged periodontal tissue is the main goal of periodontal treatment. Because conventional periodontal treatments remain insufficient to attain complete and reliable periodontal regeneration, periodontal tissue engineering has emerged as a prospective alternative method for improving the regenerative capacity of periodontal tissue. However, the potential of periodontal regeneration seems to be limited by the understanding of the cellular and molecular events in the formation of periodontal tissue and by the insufficient collaboration of multi-disciplinary research that periodontal tissue engineering involves. In this paper, we first reviewed the recent advancements in stem cells, signaling factors, and scaffolds that relate to periodontal regeneration. Then we speculate that specific genes would improve regenerative capacity of these stem cells, which could differentiate into cementoblasts, osteoblasts and fibroblasts. In addition, the 3D scaffolds that mimic the different structure and physiologic functions of natural fibro-osseous tissue could be fabricated by rapid prototyping (RP) techniques. It was therefore hypothesized that gene-modified stem cells combined with rapid prototyping techniques would be a new strategy to promote more effective and efficient periodontal regeneration.

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.

The hypoxia condition was expected to be suitable for the establishment and maintenance of human dental pulp cells (hDPCs), because they reside in a low-oxygen environment in vivo. Therefore, we presently examined the effects of hypoxia on the proliferation and differentiation of hDPCs in vitro. hDPCs grown under 3% O(2) showed a significantly higher proliferation rate than those under 21% O(2). Then, we prepared hypoxic cultures of hDPCs from older patients' teeth having inflammation and succeeded in recovering and expanding a small number of hDPCs. These cells were confirmed to have capability for osteo/odontogenic differentiation. Hypoxia suppressed the osteo/odontogenic differentiation of hDPCs in vitro and increased the number of cells expressing STRO-1, an early mesenchymal stem cell marker. This simple method will increase the possibility to obtain living hDPCs from damaged and/or aged tissues, from which it is ordinarily difficult to isolate living stem cells with differentiation capability.

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.

Dental pulp stem cells (DPSCs) can be easily isolated and cultured in low-serum containing medium supplemented with growth factors PDGF-BB and EGF while exhibiting multipotency and immature phenotypic characteristics. In the present study, we investigated their potential to differentiate towards osteogenic lineages using various culture conditions in order to optimize their therapeutic use. DPSCs were cultured either as a cell monolayer or as three-dimensional (3D) micro-mass structures. Monolayers preincubated with bFGF and valproic acid for one week prior their differentiation were cultured in serum containing standard osteodifferentiation medium for four weeks, which resulted in multilayered nodule formation. Micro-mass structures were cultured for same period either in serum containing medium or under serum-free conditions supplemented with TGF-beta3 with or without BMP-2. Histochemically, we detected massive collagen I and weak calcium phosphate depositions in multilayered nodules. When culture 3D-aggregates in either standard osteodifferentiation medium or serum-free medium containing TGF-beta3, only small amount of collagen I fibres was observed and almost no deposits of calcium phosphate were detected. In contrast, in presence of both TGF-beta3 and BMP-2 in the serum-free medium a significant amount of collagen I fibers/bundles and calcification were detected, which is in line with osteogenic effect of BMP-2. Thus, our data indicate that certain environmental cues can enhance differentiation process of DPSCs into osteogenic lineage, which suggest their possible utilization in tissue engineering.

Bone Tissue Engineering (BTE) and Dental Implantology (DI) require the integration of implanted structures, with well characterized surfaces, in bone. In this work we have challenged acid-etched titanium (AET) and Laser Sintered Titanium (LST) surfaces with either human osteoblasts or stem cells from human dental pulps (DPSCs), to understand their osteointegration and clinical use capability of derived implants. DPSCs and human osteoblasts were challenged with the two titanium surfaces, either in plane cultures or in a roller apparatus within a culture chamber, for hours up to a month. During the cultures cells on the titanium surfaces were examined for histology, protein secretion and gene expression. Results show that a complete osteointegration using human DPSCs has been obtained: these cells were capable to quickly differentiate into osteoblasts and endotheliocytes and, then, able to produce bone tissue along the implant surfaces. Osteoblast differentiation of DPSCs and bone morphogenetic protein production was obtained in a better and quicker way, when challenging stem cells with the LST surfaces. This successful BTE in a comparatively short time gives interesting data suggesting that LST is a promising alternative for clinical use in DI.

Stem cells are a promising tool for bone tissue regeneration. Dental pulp stem cells (DPSCs) can be easily obtained even in human young adults. In this study we investigated the capability of DPSCs, to express the osteoblastic phenotype when cultured with osteogenic medium. DPSCs isolated from the dental pulp of impacted third molar teeth were cultured with appropriate medium to induce osteoblast differentiation. Using Western-Blot, RT-PCR and microarray analysis, we studied the expression of osteoblastic parameter, and by Von Kossa staining we evaluated the production of mineralized matrix nodules. The results were compared with controls represented by undifferentiated DPSCs. DPSCs, differentiated into osteoblast-like cells, express large amount of alkaline phosphatase (ALP), collagen I (Coll I), osteopontin (OPN) and osteocalcin (OCN), all these parameters characterizing the osteoblastic phenotype. Differentiated DPSCs express Runx2 and JunB, a member of the AP-1 complex; both the transcription factors are associated with osteoblast differentiation and skeletal morphogenesis. Moreover, DPSCs express insulin growth factor-binding protein 5 (IGFBP-5), one of the regulating proteins of IGFs function. Finally, DPSCs can form mineralized matrix nodules that are a feature exclusive to osteoblasts. DPSCs could represent a potential source of osteoblasts to be used for bone regeneration.

Post-natal mesenchymal stem/stromal-like cells (MSCs) including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs) and bone marrow stromal cells (BMSCs) are capable of self-renewal and differentiation into multiple mesenchymal cell lineages. Despite their similar expression of MSC-associated and osteoblastic markers, MSCs retain the capacity to generate structures resembling the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical setting. With this in mind, systematic approaches are required to identify the differential protein expression patterns responsible for lineage commitment and mediating the formation of these complex structures. This is the first study to compare the differential proteomic expression profiles of ex vivo-expanded ovine PDLSCs, DPSCs and BMSCs derived from an individual donor. Two-dimensional electrophoresis was performed and regulated proteins were identified by Liquid chromatography -- Electrospray-Ionisation tandem mass spectrometry (MS & MS/MS), database searching and de novo sequencing. In total, 58 proteins were differentially expressed between at least two MSC populations in both sheep, 12 of which were up-regulated in one MSC population relative to the other two. In addition, the regulation of selected proteins was also conserved between equivalent human MSC populations. We anticipate that differential protein expression profiling will provide a basis for elucidating the protein expression patterns and molecular cues that are crucial in specifying the characteristic growth and developmental capacity of dental and non-dental tissue-derived MSC populations. These expression patterns can serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.

AIMS: Our aims were to isolate stem cells from human exfoliated deciduous teeth (SHED), to cultivate them in vitro and to investigate their basic biological properties, phenotype and to compare our findings with dental pulp stem cells (DPSC) isolated from permanent teeth. METHODS: Dental pulp was gently evacuated from exfoliated teeth. After enzymatic dissociation of dental pulp, SHED were cultivated in modified cultivation media for mesenchymal adult progenitor cells containing 2% FCS and supplemented with growth factors and insulin, transferrin, sodium (ITS) supplement. Cell viability and other biological properties were examined using a Vi-Cell analyzer and a Z2-Counter. DNA analyses and phenotyping were performed with flow cytometry. RESULTS: We were able to cultivate SHED over 45 population doublings. Our results showed that SHED cultivated under same conditions as DPSC had longer average population doubling time (41.3 hrs for SHED vs. 24.5 hrs for DPSC). Phenotypic comparison of cultivated SHED to that of cultivated DPSC showed differential expression CD29, CD44, CD71, CD117, CD 166. During long-term cultivation, SHED did not showed any signs of degeneration or spontaneous differentiation. CONCLUSIONS: We isolated stem cells from exfoliated teeth. In comparison to DPSC, SHED proliferation rate was about 50% slower, and SHED showed slightly different phenotype. These cells may be extremely useful for stem cell tissue banking, further stem cell research and future therapeutic applications.

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

This study investigated the effect on bone regeneration with dental pulp stem cells (DPSCs), deciduous tooth stem cells (DTSCs), or bone marrow-derived mesenchymal stem cells (BMMSCs) for clinical study, in hydroxyapatite (HA)-coated osseointegrated dental implants, using tissue engineering technology. In vitro, human DPSCs and DTSCs expressed STRO-1, CD13, CD29, CD 44, CD73, and osteogenic marker genes such as alkaline phosphatase (ALP), RUNX 2, osteocalcin (OCN). In vivo, prepared bone defect model was implanted by graft materials as follows: PRP, PRP and canine BMMSCs (cBMMSCs), PRP and canine DPSCs (cDPSCs), PRP and puppy DTSCs (pDTSCs), and control (defect only). After 8 weeks, the dental implants were installed, and 16 weeks later the sections were evaluated histologically and histometrically. The cBMMSCs/PRP, cDPSCs/PRP, and pDTSCs/PRP groups had well-formed mature bone and neovascularization. Histometrically, the bone implant contact (BIC) was significant differences between the cBMMSCs/PRP, cDPSCs/PRP, pDTSCs/PRP groups, and the control and PRP groups (p<0.01). These results demonstrated that these stem cells with PRP have the ability to form bone, and this bone formation activity might be useful for osseointegrated HA-coated dental implants with good levels of BIC.

The ultimate goal of tooth regeneration is to replace the lost teeth. Stem cell-based tooth engineering is deemed as a promising approach to the making of a biological tooth (bio-tooth). Dental pulp stem cells (DPSCs) represent a kind of adult cell colony which has the potent capacity of self-renewing and multilineage differentiation. The exact origin of DPSCs has not been fully determined and these stem cells seem to be the source of odontoblasts that contribute to the formation of dentin-pulp complex. Recently, achievements obtained from stem cell biology and tooth regeneration have enabled us to contemplate the potential applications of DPSCs. Some studies have proved that DPSCs are capable of producing dental tissues in vivo including dentin, pulp, and crown-like structures. Whereas other investigations have shown that these stem cells can bring about the formation of bone-like tissues. Theoretically, a bio-tooth made from autogenous DPSCs should be the best choice for clinical tooth reconstruction. This review will focus on the location, origin, and current isolation approaches of these stem cells. Their odontoblastic differentiation and potential utilizations in the reconstruction of dentin-pulp complex and bio-tooth will be extensively discussed.

Background: With today's 21st century technological advancements, it is expected that individuals will either retain their natural teeth or obtain functional tooth replacements throughout their entire life. Modern dental therapies for the replacement of missing teeth largely utilize partial or complete dentures and titanium implants capped with prosthetic crowns. Although these prostheses serve a purpose, they are not equivalent, neither in function nor aesthetics, to natural teeth. Recent progress in dental tissue engineering has lent significant credibility to the concept that biological replacement teeth therapies may soon be available to replace missing teeth. Objective: In this review, we summarize the emerging concepts of whole-tooth replacement strategies, using postnatal dental stem cells (DSCs) and dental tissue engineering approaches. Methods: We provide a thorough and extensive review of the literature. Results: Current approaches to achieve clinically relevant biological replacement tooth therapies rely on the cultivation of DSCs capable of relaying odontogenic induction signals, through dental epithelial-mesenchymal cell interactions. DSC expansion and differentiation can be achieved by programming progenitor stem cells to adopt dental lineages, using instructive, bioengineered scaffold materials. Periodontal ligament regeneration in particular has demonstrated significant progress recently, despite the somewhat unpredictable clinical outcomes, with regard to its capacity to augment conventional metallic dental implants and as an important component for whole-tooth tissue engineering. Following recent advances made in DSC and tissue engineering research, various research groups are in the midst of performing 'proof of principle' experiments for whole-tooth regeneration, with associated functional periodontal tissues. This mini-review focuses on recent and promising developments in the fields of pulp and periodontal tissue DSCs that are of particular relevance for dental tissue and whole-tooth regeneration. Conclusion: Continued advances in the derivation of useable DSC populations and optimally designed scaffold materials unequivocally support the feasibility of dental tissue and whole-tooth tissue engineering.

The development of biological and biomaterial sciences profiled tissue engineering as a new and powerful tool for biological replacement of organs. The combination of stem cells and suitable scaffolds is widely used in experiments today, in order to achieve partial or whole organ regeneration. This review focuses on the use of tissue engineering strategies in tooth regeneration, using stem cells and stem cells/scaffold constructs. Although whole tooth regeneration is still not possible, there are promising results. However, to achieve this goal, it is important to understand and further explore the mechanisms underlying tooth development. Only then will we be able to mimic the natural processes with the use of stem cells and tissue engineering techniques.