PUBLISHED RESEARCH 2007

Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2% of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflick's limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60-90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflick's limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

In this study we investigated the in vitro behaviour, morphostructure and extracellular matrix synthesis of human dental follicular stem cells (hDFSCs) isolated from human dental bud, which resulted to be positive for mesenchymal markers (CD29, CD90, CD146 and CD166) by FACS analysis. Cells were analysed by light and electronic microscopy to evaluate their biological response either at week 1, that is before differentiation, or at weeks 3-6, when they had been cultured in osteogenic medium onto a highly porous natural scaffold material (Bio-Oss). Microscopy analysis of primary culture cells showed they had a mesenchymal stem cell-like morphostructure, spindle shaped, similar to the culture of mesenchymal stem cells derived from adult bone marrow. Also, after osteogenic differentiation, these analyses indicate typical osteoblast morphostructure and reveale a tri-dimensional organization of the cells and deposition of extracellular matrix (ECM) in close contact with biomaterial. This approach would allow to personalize the scaffold for bone tissue engineering in order to accelerate the process of osteogenesis.

OBJECTIVE: To investigate the heterotopic odontogenesis ability of Delta1 gene transfected human dental pulp stem cell (DPSC) and nano-hydroxyapatite/collagen (nHAC) composite scaffold.
METHODS: The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant,and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation, the composites were taken out and tested with the histological and the immunohistological methods.
RESULTS: Green fluorescence was observed in the cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue, and only a little dentin-like structure was found in 2 of the 8 control transplants.
CONCLUSION: DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentin-pulp composite engineering.

Recent studies have shown that the pulp of human teeth contains a population of cells with stem cell properties and it has been suggested that these cells originate from pericytes. Molecules of the Notch signaling pathway regulate stem cell fate specification, while Rgs5 represents an excellent marker for pericytes. Pathological conditions such as dental trauma and carious lesion stimulate pulp stem cells to elaborate reparative dentin. Previous studies have shown that genes involved in the Notch pathway are activated in response to pulp injury in rodent and humans. To demonstrate the importance of pericytes as a source of stem cells during dental repair, we have studied Rgs5 and Notch3 mRNA expression by in situ hybridization in developing, adult intact and injured rodent teeth. Furthermore, we have examined the distribution of Notch3 protein in carious and injured human teeth using immunohistochemistry. Overlapping expression patterns of Rgs5 and Notch3 were observed during rodent tooth development as well as immediately after injury. Both genes were expressed in vascular structures during development and in perivascular and single capillary cells of injured teeth. However, the expression patterns of Rgs5 and Notch3 were different during tooth repair, with relatively extensive Rgs5 expression along the pericyte-vascular smooth muscle cell axis in central pulp arterioles. These results show co-expression of Rgs5 and Notch3 in pericytes of developing and injured teeth and furthermore indicate the importance of vascular-derived stem cells during pulp healing.

Stem cells are undifferentiated cells that have the capacity to self-renew. They have been discovered in many adult tissues, including teeth. Dental Pulp Mesenchymal Stem Cells (DP-MSCs) are involved in dental repair by activation of growth factors, released after caries and have the ability to regenerate a dentin-pulp-like complex. The molecular/cellular research gives the possibility to grow new tissues and biological structures for clinical applications, providing cells for therapies including cell transplantation and tissue engineering. In this study DP-MSCs were derived from dental pulp of 10 donors. To evaluate material toxicity, after in vitro isolation, the cells were seeded on mineral trioxide aggregate (MTA). Initial light microscopy investigation of cells revealed no signs of cell death due to toxicity or infection, on the contrary the scaffolds supplied an excellent support for cell structures, the cells proliferated and adhered to substrate. Similar observation was seen in scanning electron microscopy, in particular the cells had proliferated and spread, covering a considerable part of the surface of the biomaterials investigated, with an elaborate form of attachment, in fact, the cells formed a continuous layer on the upper surface of the MTA. In conclusion, the aim of this study is to demonstrate that DP-MSCs combined with MTA could be a potential source for regenerative medicine, encouraging further study to evaluate the new dentin formation.

This review article arranges the current results of stem cell biology for their use in dentistry. There are different types of stem cells, which are applicable for dental treatments. The use of embryonic stem cells, whose possibilities for breeding an artificial tooth were hardly evaluated, is however ethically precarious. On the other side the ethically harmless adult stem cells, which were isolated for example from bone marrow, were little examined for their capability of differentiation into dental tissues. Therefore their forthcoming use in dentistry is rather improbable. However, dental ectomesenchymal stem cells are more promising for dentistry in future. For example dental pulp stem cells (DPSCs) are capable to differentiate into dentin under in vitro conditions. Moreover it is possible to use periodontal ligament (PDL) stem cells and dental follicle precursors for periodontal tissue differentiations in vitro. Recently new populations of stem cells were isolated from the dental pulp and the PDL. These cells distinguish from the initially isolated DPSCs and PDL stem cells in growth and cell differentiation. Therefore stem cell markers are very important for the characterization of dental stem cells. A significant marker for dental stem cells is STRO-1, which is also a marker for bone marrow derived mesenchymal stem cells. Nonetheless dental stem cells are CD45 negative and they express rarely hematopoietic stem cell markers. These research results plead for the participation of dental stem cells in dental practice in future.

Any clinician dreams to obtain the regeneration of the destroyed organ for his patient. In the human being, the regeneration of complex structures is not possible, except the liver and the bone marrow, which can be regenerated because of the presence of adult stem cells in these tissues. The stem cells have two principal properties: they ensure their self-renewal and they have the ability to differentiate into several cellular types. Using specific markers allowing the identification of the stem cells in bone marrow, stem cells were observed in dental pulp tissues. Although the origin, the identification, and the localization of these stem cells of dental pulp remain under consideration, the optimism in research on stem cells permits to believe that the knowledge on dental stem cells will lead to their use in therapeutics.

Experiments with animal models have shown that the tooth crown structure can be regenerated using tissue engineering techniques that combine tooth bud cells and biodegradable materials, or by using embryonic tissue and adult stem cells. Moreover, tooth roots and periodontal tissues have been reconstructed by grafting dental stem cells, which leads to the recovery of tooth function, suggesting that tooth regeneration will become possible in humans in the near future. The present article reviews current research on tooth regeneration, discusses a model of tooth replacement that could be used clinically, and proposes a new tooth regeneration approach that overcomes the difficulties associated with the tooth replacement model. Tooth regeneration is an important stepping stone in the establishment of engineered organ transplantation, which is one of the ultimate goals of regenerative therapies.

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.

It was reported that postnatal stem cells are present in adult tissues such as bone marrow, liver, muscle, dental pulp, and periodontal ligament. We isolated postnatal stem cells from human dental tissues such as dental pulp (DPSC), periodontal ligament (PDLSC), periapical follicle (PAFSC), and the surrounding mandibular bone marrow (MBMSC) to ascertain their properties. Immunocytochemistry proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. These cells also expressed the mesenchymal stem cell (MSC) markers CD29 and CD44. The isolated cells showed self-renewal capabilities and colony-forming efficiency. Almost all of the dental stem cells showed optimal growth when they were cultured in alpha modification of Eagle's medium (alpha-MEM) supplemented with 10% fetal calf serum (FCS) and 100 microM ascorbic acid. Only the PAFSC showed increased proliferation in 20% FCS and 50 microM ascorbic acid. All of the dental stem cells were capable of differentiating into adipocytes and mineral nodule forming cells. MBMSC, in particular, showed much better mineralization compared to the others. These results indicate that MSCs exist in various tissues of the teeth and can differentiate into osteoblasts, adipocytes, and other kinds of cells with varying efficiency.

Millions of teeth are saved each year by root canal therapy. Although current treatment modalities offer high levels of success for many conditions, an ideal form of therapy might consist of regenerative approaches in which diseased or necrotic pulp tissues are removed and replaced with healthy pulp tissue to revitalize teeth. Researchers are working toward this objective. Regenerative endodontics is the creation and delivery of tissues to replace diseased, missing, and traumatized pulp. This review provides an overview of regenerative endodontics and its goals, and describes possible techniques that will allow regenerative endodontics to become a reality. These potential approaches include root-canal revascularization, postnatal (adult) stem cell therapy, pulp implant, scaffold implant, three-dimensional cell printing, injectable scaffolds, and gene therapy. These regenerative endodontic techniques will possibly involve some combination of disinfection or debridement of infected root canal systems with apical enlargement to permit revascularization and use of adult stem cells, scaffolds, and growth factors. Although the challenges of introducing endodontic tissue engineering therapies are substantial, the potential benefits to patients and the profession are equally ground breaking. Patient demand is staggering both in scope and cost, because tissue engineering therapy offers the possibility of restoring natural function instead of surgical placement of an artificial prosthesis. By providing an overview of the methodological issues required to develop potential regenerative endodontic therapies, we hope to present a call for action to develop these therapies for clinical use.

In spite of the vast knowledge of tooth development and of the various kinds of specialized bone/tooth-associated cells, the characteristics and properties of their precursor cell populations present in the postnatal organism are little known, as is their possible therapeutic use. Taken together dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation. Regenerative medicine is based on stem cells, signals and scaffolds. Transplantation of those cells, which can be obtained from an easily accessible tissue resource and expanded in vitro, holds promise as a therapeutic approach for reconstruction of tissues and bone in vivo.

The dentine-pulp complex displays exquisite regenerative potential in response to injury. The postnatal dental pulp contains a variety of potential progenitor/stem cells, which may participate in dental regeneration. A population of multipotent mesenchymal progenitor cells known as dental pulp stem cells with high proliferative potential for self-renewal has been described and may be important to the regenerative capacity of the tissue. The nature of the progenitor/stem cell populations in the pulp is of importance in understanding their potentialities and development of isolation or recruitment strategies, and allowing exploitation of their use in regeneration and tissue engineering. Various strategies will be required to ensure not only effective isolation of these cells, but also controlled signalling of their differentiation and regulation of secretory behaviour. Characterization of these cells and determination of their potentialities in terms of specificity of regenerative response will form the foundation for development of new clinical treatment modalities, whether involving directed recruitment of the cells and 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 will provide an innovative and novel biologically based new generation of clinical treatments for dental disease.

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.