Mineralised Tissue Research Group, Tissue Engineering and Regenerative Dentistry, School of Dentistry Cardiff University, Cardiff, UK. sloanaj@cardiff.ac.uk

INTRODUCTION: It is now accepted that progenitor/stem cells reside within the post-natal dental pulp. Studies have identified several niches of multipotent mesenchymal progenitor cells, known as dental pulp stem cells, which have a high proliferative potential for self-renewal. These progenitor stem cells are now recognized as being vital to the dentine regeneration process following injury. Understanding the nature of these progenitor/stem cell populations in the pulp is important in determining their potentialities and development of isolation or recruitment strategies for use in regeneration and tissue engineering. Characterization of these cells, and determination of their potentialities in terms of specificity of regenerative response, may help direct new clinical treatment modalities. Such novel treatments may involve controlled direct recruitment of the cells in situ and possible seeding of stem cells at sites of injury for regeneration or use of the stem cells with appropriate scaffolds for tissue engineering solutions. Such approaches may provide an innovative and novel biologically based new generation of clinical materials and/or treatments for dental disease. AIM: This study aimed to review the body of knowledge relating to stem cells and to consider the possibility of these cell populations, and related technology, in future clinical applications.

Tissue Engineering and Reparative Dentistry, School of Dentistry, Cardiff University, Cardiff, UK. waddingtonrj@cardiff.ac.uk

The present study compared the cellular characteristics of progenitor stem cell populations present in adult dental pulp, isolated by different methods utilizing 2 different features of stem cell biology. One population expressing high levels of beta1 integrin was isolated by preferential selection of adherent cells to fibronectin over 20 min. In an alternative approach, cells expressing the embryonic neural crest cell marker, low-affinity nerve growth factor receptor (LANGFR), were selected by magnetic-activated cell sorting. For each method, clonal cell lines were established and expanded in culture. One clone derived via the respective methods was examined for embryonic/progenitor cell markers by immunocytochemistry and RT-PCR. Both clonal populations demonstrated the expression of stro-1 and stained positive for vimentin, demonstrating mesenchymal lineage. Of note, cells selected for LANGFR cells demonstrated the additional expression of CD105 and Notch 2. For both clonal populations, expanded cultures demonstrated the ability to differentiate into osteoblasts, adipocytes and chondrocytes. These results would suggest the potential isolation of 2 progenitor cell populations exhibiting different cellular characteristics in terms of their embryonic nature. The potential for both cell populations to derive from a common origin is discussed. Copyright 2008 S. Karger AG, Basel.

Institute of Human Genetics, University of Regensburg, Regensburg, Germany. christian.morsczeck@klinik.uni-regensburg.de

Complex human tissues harbour stem cells and/or precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as periodontal ligament (PDL), dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. The dental stem cell biology might provide meaningful insights into the development of dental tissues and cellular differentiation processes. Dental stem cells could also be feasible tools for dental tissue engineering. Constructing complex structures like a periodontium, which provides the functional connection between a tooth or an implant and the surrounding jaw, could effectively improve modern dentistry. Dental precursor cells are attractive for novel approaches to treat diseases like periodontitis, dental caries or to improve dental pulp healing and the regeneration of craniofacial bone and teeth. These cells are easily accessible and, in contrast to bone-marrow-derived mesenchymal stem cells, are more closely related to dental tissues. This review gives a short overview of stem cells of dental origin.

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, TESLab, Secondo Ateneo di Napoli, Napoli, Italy.

Dental pulp stem cells (DPSCs) can be found within the "cell rich zone" of dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, two groups have studied these cells extensively, albeit with different results. One group claims that these cells produce a "dentin-like tissue", whereas the other research group has demonstrated that these cells are capable of producing bone, both in vitro and in vivo. In addition, it has been reported that these cells can be easily cryopreserved and stored for long periods of time and still retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells: several scaffolds have been used to promote 3-D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. These results enforce the notion that DPSCs can be used successfully for tissue engineering. J. Exp. Zool. (Mol. Dev. Evol.) 310B, 2008. (c) 2008 Wiley-Liss, Inc.

Tissue Engineering and Reparative Dentistry, School of Dentistry, Cardiff University, Cardiff, UK. waddingtonrj@cardiff.ac.uk

The present study compared the cellular characteristics of progenitor stem cell populations present in adult dental pulp, isolated by different methods utilizing 2 different features of stem cell biology. One population expressing high levels of beta1 integrin was isolated by preferential selection of adherent cells to fibronectin over 20 min. In an alternative approach, cells expressing the embryonic neural crest cell marker, low-affinity nerve growth factor receptor (LANGFR), were selected by magnetic-activated cell sorting. For each method, clonal cell lines were established and expanded in culture. One clone derived via the respective methods was examined for embryonic/progenitor cell markers by immunocytochemistry and RT-PCR. Both clonal populations demonstrated the expression of stro-1 and stained positive for vimentin, demonstrating mesenchymal lineage. Of note, cells selected for LANGFR cells demonstrated the additional expression of CD105 and Notch 2. For both clonal populations, expanded cultures demonstrated the ability to differentiate into osteoblasts, adipocytes and chondrocytes. These results would suggest the potential isolation of 2 progenitor cell populations exhibiting different cellular characteristics in terms of their embryonic nature. The potential for both cell populations to derive from a common origin is discussed. Copyright 2008 S. Karger AG, Basel.

Grace Dental Clinic, Kaohsiung City, Taiwan

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune-suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells, which express Nanog, Rex-1, Oct-4, and various cell surface antigens, and have multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre-existing Nestin(+) neural progenitor cells (NPCs) and beta-tubulin-III(+) mature neurons to the site of the graft. Additionally, many cells born during the first 7 days after implantation proliferated, forming NPCs and neurons, and, to a lesser extent, underwent astrogliosis, forming astrocytes and microglia, by 30 days after implantation. Although the DPSC graft itself was short term, it had long-term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of ciliary neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor for up to 30 days after implantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the central nervous system. DPSCs would be a preferable cell source for therapy due to the possibility of a "personalized" stem cell, avoiding the problems associated with host immune rejection. Disclosure of potential conflicts of interest is found at the end of this article.

Departamento de Estomatologia, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Brazil. gustavocoura@hotmail.com

BACKGROUND AND OBJECTIVE: The periodontal ligament is a specialized connective tissue, derived from dental follicle and originated from neural crest cells. Recently it has been suggested, based on animal models, that periodontal ligament could be a niche for neural crest stem cells. However, there is still little knowledge on this subject. The identification of neural crest adult stem cells has received much attention based on its potential in tissue regeneration. The objective of the present work was to verify the human periodontal ligament as a niche for neural crest stem cells. MATERIAL AND METHODS: Cells from human periodontal ligament were isolated from 10 teeth of seven individuals (periodontal ligament pool group) and also from four teeth of one individual (periodontal ligament single group), after enzymatic digestion. The cells were cultured in specific inductive medium. Analyses of protein and gene expression were performed through immunocytochemistry and reverse transcription-polymerase chain reaction techniques, respectively. RESULTS: Mesodermal phenotypes (adipogeneic, osteogenic and myofibroblastic) were identified after culture in inductive medium. Immunocytochemistry analyses showed the presence of the nestin marker of neural stem cells and also markers of undifferentiated neural crest cells (HNK1, p75). When cultured in inductive medium that allowed neural differentiation, the cells showed markers for beta-tubulin III, neurofilament M, peripherin, microtubule-associated protein 2 and protein zero. The results were similar between the two study groups (the periodontal ligament pool group and the periodontal ligament single group). CONCLUSION: This research provides evidence that human periodontal ligament, in addition to its mesodermal derivatives, produces neural crest-like cells. Such features suggest a recapitulation of their embryonic state. The human periodontal ligament revealed itself as a viable alternative source for possible primitive precursors to be used in stem-cell therapies.

Department of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522, Japan.

Center of Advanced European Studies and Research, Bonn, Germany. degistirici@caesar.de

The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans.

Department of Stomatology, Chinese PLA General Hospital and Postgraduate Military Medical School, Beijing 100853, China; Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.

Two crucial growth factors, FGF2 and TGFbeta(1), were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta(1) and FGF2+TGFbeta(1). The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta(1) or FGF2+TGFbeta(1) can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta(1) on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta(1) and FGF2+TGFbeta(1) mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta(1).

Department of Oral and Maxillofacial Science and.

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs.

Department of Pediatric Dentistry, Peking University School & Hospital of Stomatology, Beijing 100081, China.

OBJECTIVE: To isolate and culture the pulp cells from human young permanent teeth (pDPC), and to observe their biological characteristics and the expression of some specific markers, and to induce these pulp cells to differentiate into osteoblast, adipocyte, neuron and chondrocyte lineages. METHODS: Pulp cells were isolated and cultured from orthodontic extracted premolars of children. The attached cells after at least 3 passages were used for the following experiments: 1. Morphology and ultrastructure analysis; 2. Cell cycle and phenotype were analyzed by flowcytometry; 3. Growth curve were recorded; 4. pDPC were induced to differentiate into osteoblast, adipocyte, neuron in vitro, and were identified by histochemical methods and RT-PCR. RESULTS: 1. Attached pDPCs were fibroblast-like cells, which were distinguished from BMSC. 2. The cell organs in dDPCs were well developed. 3. pDPCs were highly positive for CD90, CD44, CD147, which are mesenchymal stem-cell markers, but were negative for other markers including CD34, CD38, CD45, HLA-DR. 4. pDPCs showed high growth rate. 5. pDPCs could be induced to differentiate into osteoblast, adipocyte, and neuron lineages, but not chondrocyte lineages. CONCLUSION: pDPCs were characterized by their ability to proliferate with high growth rate in vitro. The expression of some BMSC markers in these cells were observed. They showed the potential to differentiate into multiple mesenchymal lineages such as osteoblast, adipocyte, neuron lineages under specific conditions in vitro.

Departamento di Scienze Odontostomatologiche, Universitá La Sapienze, Romae, Italy.

OBJECTIVE: Bone tissue engineering is a promising approach for bone reconstruction in oral-maxillofacial surgery. This study investigates the suitability of oral skeletal tissues as convenient and accessible sources of osteogenic progenitors as an alternative to the iliac crest bone marrow. STUDY DESIGN: Samples of maxilla tuberosity (MT) and maxillary and mandibular periosteum (MP) were obtained during routine oral surgery, and donor site morbidity was assessed using a "split-mouth" approach. Cells isolated from MT (bone marrow stromal cells; MT-BMSCs) and from MP (periosteal cells; M-PCs), were analyzed for clonogenicity, phenotype, expression of osteogenic markers, and ability to form bone in vivo. RESULTS: Both MT-BMSCs and M-PCs included clonogenic cells, showed comparable phenotypic profiles, and expressed early osteogenic markers. Most importantly, both cell populations formed bone upon ectopic in vivo transplantation. CONCLUSION: MT-BMSCs and M-PCs behaved as osteoprogenitor cells in vitro and in vivo. MT and MP may be considered as suitable sources of cells for bone tissue engineering in humans.

General BioTechnology, LLC, Indianapolis, Indiana.

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here, we investigated the collection, processing, and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly, all expressed appropriate cell surface markers and underwent osteogenic, adipogenic, and chondrogenic differentiation in appropriate differentiation medium, thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction, and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further, the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC.

Inserm, JPA Center, Inserm U837, Lille, cedex, France; gaelique.stevens@wanadoo.fr.

Adult tissues contain highly proliferative, clonogenic cells that meet criteria of multipotent stem cells and are potential sources for autologous reparative and reconstructive medicine. We demonstrated that human dental pulp contains self renewing human dental pulp stem cells (hDPSCs) capable of differentiating into mesenchymal-derived odontoblasts, osteoblasts, adipocytes, chondrocytes and striated muscle, and interestingly, also into non-mesenchymal melanocytes. Furthermore, we showed that hDPSC cultures include cells with the label-retaining and sphere-forming abilities, traits attributed to multipotent stem cells, and provide evidence that these might be multipotent neural crest stem cells.

Laboratory of Molecular Signaling, Department of Biologic and Materials Sciences, School of Dentistry, The University of Michigan, Ann Arbor, MI 48109, USA.

Dental pulp stem cells (DPSCs) are a unique precursor population isolated from postnatal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through beta-catenin. In this study, the regulation of odontoblast-like differentiation of DPSCs by canonical Wnt signaling was examined. DPSCs were stably transduced with canonical Wnt-1 or the active form of beta-catenin, with retrovirus-mediated infection. Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin, and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules in DPSCs. Moreover, over-expression of beta-catenin was also sufficient to suppress the differentiation and mineralization of DPSCs. In conclusion, our results suggest that canonical Wnt signaling negatively regulates the odontoblast-like differentiation of DPSCs.

Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

To isolate high-quality human postnatal stem cells from accessible resources is an important goal for stem-cell research. In this study we found that exfoliated human deciduous tooth contains multipotent stem cells [stem cells from human exfoliated deciduous teeth (SHED)]. SHED were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. After in vivo transplantation, SHED were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Here we show that a naturally exfoliated human organ contains a population of stem cells that are completely different from previously identified stem cells. SHED are not only derived from a very accessible tissue resource but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem-cell therapies including autologous stem-cell transplantation and tissue engineering.

Abstract:
Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation.

To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45-. These cells were capable of differentiation of osteoblasts in vitro.

By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements.

Laboratório de Genética, Instituto Butantan, São Paulo, Brasil. ikerkis@butantan.gov.br

We report the isolation of a population of immature dental pulp stem cells (IDPSC), which express embryonic stem cell markers Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as several other mesenchymal stem cell markers during at least 25 passages while maintaining the normal karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was maintained in subclones obtained from these cells. Moreover, in vitrothese cells can be induced to undergo uniform differentiation into smooth and skeletal muscles, neurons, cartilage, and bone under chemically defined culture conditions. After in vivo transplantation of these cells into immunocompromised mice, they showed dense engraftment in various tissues. The relative ease of recovery and the expression profiles of various markers justify further exploration of IDPSC for clinical therapy. Copyright 2007 S. Karger AG, Basel.

Suchánek J, Soukup T, Ivancaková R, Karbanová J, Hubková V, Pytlík R, Kucerová L.

Charles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Department of Dentistry, Czech Republic. suchanekj@lfhk.cuni.cz  Acta Medica (Hradec Kralove). 2007;50(3):195-201.

Radboud University Nijmegen Medical Centre, Periodontology & Biomaterials, Nijmegen, The Netherlands.

The current study aimed to prove that human dental pulp stem cells (hDPSCs) isolated from the pulp of third molars can show multilineage differentiation after cryopreservation. First, hDPSC were isolated via enzymatic procedures, and frozen in liquid nitrogen until use. After defrosting, cells were analyzed for proliferative potential and the expression of the stem cell marker STRO-1. Subsequently, cells were cultured in neurogenic, osteogenic/odontogenic, adipogenic, myogenic, and chondrogenic inductive media, and analyzed on basis of morphology, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR) for specific marker genes. All data were replicated, and the results of the primary cells were compared to similar tests with an additional primary dental pulp stem cell strain, obtained from the National Institutes of Health (NIH). Results showed that our cell population could be maintained for at least 25 passages. The existence of stem/ progenitor cells in both cell strains was proven by the STRO-1 staining. Under the influence of the 5 different media, both cell strains were capable to advance into all 5 differentiation pathways. Still differences between both strains were found. In general, our primary culture performed better in myogenic differentiation, while the externally obtained cells were superior in the odontogenic/osteogenic and chondrogenic differentiation pathways. In conclusion, the pulp tissue of the third molar may serve as a suitable source of multipotent stem cells for future tissue engineering strategies and cell-based therapies, even after cryopreservation.

Dipartimento di Medicina Sperimentale, Sezione Istologia ed Embriologia, Secondo Ateneo di Napoli, Napoli, Italy.

Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) was selected, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and then in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue, which is markedly positive for several bone antibodies. This tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone-containing osteocytes. INTRODUCTION: Recently it has been reported that human dental pulp stem cells (DPSCs) are detectable, in humans, only up to the age of 30 years and that they are able to produce in vitro only sporadic calcified nodules and to form, after transplantation in vivo, a mineralized tissue. MATERIALS AND METHODS: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. Light microscope, histochemistry, immunofluorescence, and RT-PCR analyses were performed to study both stem and differentiating cells. RESULTS AND CONCLUSIONS: A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) has been selected by FACSorting, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue. This new-formed tissue is markedly positive for several antibodies for bone, including osteonectin, bone sialoprotein, osteocalcin, fibronectin, collagen III, and bone alkaline phosphatase (BALP). Cells producing LAB can be stored at -80 degrees C for a long period of time and are an extraordinary source of osteoblasts and mineralized fibrous bone tissue. In this study, we also showed that, in aged humans, stem cells can be detected from their pulps. The produced LAB is a fibrous bone tissue resembling the human bone during mineralization, with an external layer formed by osteoblasts markedly positive for osteocalcin. This newly formed tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone containing osteocytes.

Transplantation. 2005 Sep 27; 80(6):836-42.

Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, Becchetti E, Marchionni C, Alviano F, Fossati V, Staffolani N, Franchina M, Grossi A, Bagnara GP.

Department of Histology, Embryology, and Applied Biology, University of Bologna, Bologna, Italy.

Department of Periodontology and Biomaterials, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. J Tissue Eng Regen Med. 2008 Mar 14

Journal of Cellular Physiology. 2006 Aug; 208(2):319-25.

Papaccio G, Graziano A, d'Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G.

Dipartimento di Discipline Odontostomatologiche, Ortodontiche e Chirurgiche, Università Secondo Ateneo di Napoli, Napoli, Italy.

Stromal stem cells from human dental pulp (SBP-DPSCs) were used to study osteogenic differentiation in vitro and in vivo. We previously reported that SBP-DPSCs are multipotent stem cells able to differentiate into osteoblasts, which synthesize three-dimensional woven bone tissue chips in vitro. In this study, we followed the temporal expression pattern of specific markers in SBP-DPSCs and found that, when differentiating into osteoblasts, they express, besides osteocalcin, also flk-1 (VEGF-R2). In addition, 30% of them expressed specific antigens for endothelial cells, including CD54, von-Willebrand (domain 1 and 2), CD31 (PECAM-1) and angiotensin-converting enzyme. Interestingly, we found endotheliocytes forming vessel walls, observing that stem cells synergically differentiate into osteoblasts and endotheliocytes, and that flk-1 exerts a pivotal role in coupling osteoblast and endotheliocyte differentiation. When either SBP-DPSCs or bone chips obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone; in fact, a large number of HLA-1+ vessels were observed either within the bone or surrounding it in a periosteal layer. This study provides direct evidence to suggest that osteogenesis and angiogenesis mediated by human SBP-DPSCs may be regulated by distinct mechanisms, leading to the organization of adult bone tissue after stem cell transplantation.

Centre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy.

Stem cells derived from human dental pulp are able to differentiate into osteoblasts and are a potential source of autologous bone. The aim of this study was to compare genes differentially expressed in osteoblastoids from human dental pulp (OHDP) to osteosarcoma cells (OCs). Human dental pulp was extracted and immersed in a digestive solution. Cells were cultured and selected using c-kit, CD34, CD45 and STRO-1 antibodies. In parallel, two OCs (i.e., SAOS2 and TE85) were cultured. RNA was extracted from different populations of cells and cDNA was used for the hybridisation of human 19.2K DNA microarrays. We identified several differences in gene expression between OHDP and OCs. Some down-regulated OHDP genes, such as RUNX1, MAP4K4 and PRDM2, are involved in bone development, cell motility and transcript regulation. Gene expression in OHDP is significantly different from that in OCs, suggesting differences in cell function and activity between these cells.

Grace Dental Clinic, Kaohsiung, Taiwan.

Background: Dental pulp stem cells (DPSCs) were primarily derived from the pulp tissues of primary incisors and permanent third molar teeth, whereas no report to our knowledge has yet been documented on deriving DPSCs from the other tooth types. The aim of this study is to present a novel approach of harvesting stem cells from a supernumerary tooth (a mesiodens). Materials and methods: The pulp tissues from a mesiodens of a 20-year-old healthy male patient and the left lower deciduous canine of a healthy 10-year-old boy (the positive control) were extracted and cultured for DPSCs, which were examined with stem cells (Oct-4, Nanog and Rex-1) and differentiation (Osteonectin and Nestin) markers. Furthermore, DPSCs were directionally differentiated to osteogenic and adipogenic cell lineages. Results: Dental pulp stem cells derived from the mesiodens were capable of differentiating into adipogenic and osteogenic lineages. The mesioden's DPSCs also expressed stem cell and differentiation markers, which suggested their stem cell origin and differentiation capability. All the aforementioned results for the mesiodens were consistent with those of the DPSCs derived from the positive control. Conclusion: We have demonstrated the feasibility of deriving DPSCs from a usually discarded tissue such as a supernumerary tooth.

Dipartimento di Medicina Sperimentale, Sezione Istologia ed Embriologia, TESLab, Secondo Ateneo di Napoli, Napoli, Italy.

OBJECTIVES: The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step. MATERIALS AND METHODS: Dental germ pulp, collected from 25 healthy subjects aged 13-20 years, were subjected to magnetic-activated cell sorting to select a CD34(+) stem cell population capable of differentiating into pre-osteoblasts. These cells were allowed to adhere to an absorbable polylactic-coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34(+) cell-colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously. RESULTS: After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone-specific proteins were detected by immunofluorescence, within the nodules, and X-ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo-angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA-1(+) and, thus, clearly human in origin. CONCLUSIONS: This study indicates that CD34(+) cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Oral and Maxillofacial Department, Qilu Hospital of Shangdong University, Jinan 250012, Shandong province, China. lshabccba@sdu.edu.cn

PURPOSE: This study was aimed to investigate the localization of dental pulp stem cells (DPSCs) by comparing the characteristics of cultured dental pulp cells in coronal and root pulp. METHODS: Human dental coronal and root pulp cells were cultured in tissue-explant method, the cell culture successfulness, attachment efficiency, cell viality, morphology, proliferation pattern, and the mineralization ability were observed, the localization of DPSCs was investigated in the functional respect of DPSCs. RESULTS: The human dental root pulp cells have more culture successfulness,more attachment efficiency,more cell viality, more primary characteristics, and stronger induced mineralization ability than that of coronal pulp cells. Root and coronal pulp cells showed same proliferation patterns. CONCLUSIONS: DPSCs may exist in both dental root and coronal pulp, and the density of DPSCs in the root pulp may be higher than the coronal pulp.

Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles, California, USA.

Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and growth factor receptor gene profiles. Double-staining experiments showed that STRO-1 coexpressed with dentinogenic markers such as bone sialophosphoprotein, osteocalcin, and growth factors FGFR1 and TGFbetaRI in cultured SCAP. Additionally, SCAP express a wide variety of neurogenic markers such as nestin and neurofilament M upon stimulation with a neurogenic medium. We conclude that SCAP are similar to DPSCs but a distinct source of potent dental stem/progenitor cells. Their implications in root development and apexogenesis are discussed.

Mund Kiefer Gesichtschir. 2007 Sep 6

Morsczeck C, Reichert TE, Völlner F, Gerlach T, Driemel O.

Institut für Humangenetik, Universität Regensburg, Franz-Josef-Strauß-Allee Regensburg, Germany

International Journal Immunopathology & Pharmacology. 2006 Jul-Sep; 19(3):451-60.

Trubiani O, Orsini G, Caputi S, Piatelli A.

Department of Stomatology and Oral Science, Ce.SI. Foundation G. d'Annunzio, Chieti, Italy.