George T.-J. Huang

Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources: Their Biology and Role in Regenerative Medicine

To date, 5 different human dental stem/progenitor cells have been isolated and characterized: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAP), and dental follicle progenitor cells (DFPCs). These postnatal populations have mesenchymal-stem-cell-like (MSC) qualities, including the capacity for self-renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dental-tissue-derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells. However, they also have the ability to give rise to other cell lineages similar to, but different in potency from, that of BMMSCs. This article will review the isolation and characterization of the properties of different dental MSC-like populations in comparison with those of other MSCs, such as BMMSCs. Important issues in stem cell biology, such as stem cell niche, homing, and immunoregulation, will also be discussed.

Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study.

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.

The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot Engineering

Some clinical case reports have shown that immature permanent teeth with periradicular periodontitis or abscess can undergo apexogenesis after conservative endodontic treatment. A call for a paradigm shift and new protocol for the clinical management of these cases has been brought to attention. Concomitantly, a new population of mesenchymal stem cells residing in the apical papilla of permanent immature teeth recently has been discovered and was termed stem cells from the apical papilla (SCAP). These stem cells appear to be the source of odontoblasts that are responsible for the formation of root dentin. Conservation of these stem cells when treating immature teeth may allow continuous formation of the root to completion. This article reviews current findings on the isolation and characterization of these stem cells. The potential role of these stem cells in the following respects will be discussed: (1) their contribution in continued root maturation in endodontically treated immature teeth with periradicular periodontitis or abscess and (2) their potential utilization for pulp/dentin regeneration and bioroot engineering.

Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells

ABSTRACT Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, includingBacteroides forsythus, Campylobacter curvus,Eikenella corrodens, Fusobacterium nucleatum,Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatumwere also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.

iPS cells reprogrammed from human mesenchymal-like Stem/Progenitor cells of dental Tissue Origin

Generation of induced pluripotent stem (iPS) cells holds a great promise for regenerative medicine and other aspects of clinical applications. Many types of cells have been successfully reprogrammed into iPS cells in the mouse system; however, reprogramming human cells have been more difficult. To date, human dermal fibroblasts are the most accessible and feasible cell source for iPS generation. Dental tissues derived from ectomesenchyme harbor mesenchymal-like stem/progenitor cells and some of the tissues have been treated as biomedical wastes, for example, exfoliated primary teeth and extracted third molars. We asked whether stem/progenitor cells from discarded dental tissues can be reprogrammed into iPS cells. The 4 factors Lin28/Nanog/Oct4/Sox2 or c-Myc/Klf4/Oct4/Sox2 carried by viral vectors were used to reprogram 3 different dental stem/progenitor cells: stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), and dental pulp stem cells (DPSCs). We showed that all 3 can be reprogrammed into iPS cells and appeared to be at a higher rate than fibroblasts. They exhibited a morphology indistinguishable from human embryonic stem (hES) cells in cultures and expressed hES cell markers SSEA-4, TRA-1-60, TRA-1-80, TRA-2-49, Nanog, Oct4, and Sox2. They formed embryoid bodies in vitro and teratomas in vivo containing tissues of all 3 germ layers. We conclude that cells of ectomesenchymal origin serve as an excellent alternative source for generating iPS cells.

In vitro characterization of human dental pulp cells: various isolation methods and culturing environments

Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens.

Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

BACKGROUND Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). AIMS To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. MATERIALS & METHODS DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. RESULTS The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 + or - 2.9) was lower than that of DPSCs-NPs (58.9 + or - 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. CONCLUSION DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo.

A paradigm shift in endodontic management of immature teeth: Conservation of stem cells for regeneration

OBJECTIVE This article will review the new concept of regenerative endodontics in the management of immature permanent teeth. The potential role of stem cells to regenerate immature permanent teeth after conservative treatment will be discussed. DATA AND SOURCES Two sets of data source are focused in this review: (i) the characterization of various dental stem cells discovered since 2000 and (ii) recent clinical case reports showing that after conservative treatment, severely infected immature teeth with periradicular periodontitis and abscess can undergo healing and apexogenesis or maturogenesis. RESULTS A new protocol of treating endodontically involved immature permanent teeth based on published articles to date is summarized in the review. The key procedures of the protocol are (1) minimal or no instrumentation of the canal while relying on a gentle but thorough irrigation of the canal system, (2) the disinfection is augmented with intra-canal medication of a triple-antibiotic paste between appointments, and (3) the treated tooth is sealed with mineral trioxide aggregate (MTA) and glass ionomer/resin cement at the completion of the treatment. Periodical follow-ups will take place to observe any continued maturation of the root. CONCLUSION While more clinical research is needed, regenerative endodontics promotes a paradigm shift in treating endodontically involved immature permanent teeth from performing apexification procedures to conserving any dental stem cells that might remain in the disinfected viable tissues to allow tissue regeneration and repair to achieve apexogenesis/maturogenesis.

Pulp and dentin tissue engineering and regeneration: current progress.

Dental pulp tissue is vulnerable to infection. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial rubber-like material is employed to treat the infection - commonly known as root-canal therapy. Regeneration of pulp tissue has been difficult as the tissue is encased in dentin without collateral blood supply except from the root apical end. However, with the advent of the concept of modern tissue engineering and the discovery of dental stem cells, regeneration of pulp and dentin has been tested. This article will review the early attempts to regenerate pulp tissue and the current endeavor of pulp and dentin tissue engineering, and regeneration. The prospective outcome of the current advancement in this line of research will be discussed.