P. M. Bartold

Investigation of multipotent postnatal stem cells from human periodontal ligament

BACKGROUND Periodontal diseases that lead to the destruction of periodontal tissues--including periodontal ligament (PDL), cementum, and bone--are a major cause of tooth loss in adults and are a substantial public-health burden worldwide. PDL is a specialised connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. We investigated the notion that human PDL contains stem cells that could be used to regenerate periodontal tissue. METHODS PDL tissue was obtained from 25 surgically extracted human third molars and used to isolate PDL stem cells (PDLSCs) by single-colony selection and magnetic activated cell sorting. Immunohistochemical staining, RT-PCR, and northern and western blot analyses were used to identify putative stem-cell markers. Human PDLSCs were transplanted into immunocompromised mice (n=12) and rats (n=6) to assess capacity for tissue regeneration and periodontal repair. Findings PDLSCs expressed the mesenchymal stem-cell markers STRO-1 and CD146/MUC18. Under defined culture conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes, and collagen-forming cells. When transplanted into immunocompromised rodents, PDLSCs showed the capacity to generate a cementum/PDL-like structure and contribute to periodontal tissue repair. INTERPRETATION Our findings suggest that PDL contains stem cells that have the potential to generate cementum/PDL-like tissue in vivo. Transplantation of these cells, which can be obtained from an easily accessible tissue resource and expanded ex vivo, might hold promise as a therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.

Periodontal Ligament Stem Cell-Mediated Treatment for Periodontitis in Miniature Swine

Periodontitis is a periodontal tissue infectious disease and the most common cause for tooth loss in adults. It has been linked to many systemic disorders, such as coronary artery disease, stroke, and diabetes. At present, there is no ideal therapeutic approach to cure periodontitis and achieve optimal periodontal tissue regeneration. In this study, we explored the potential of using autologous periodontal ligament stem cells (PDLSCs) to treat periodontal defects in a porcine model of periodontitis. The periodontal lesion was generated in the first molars area of miniature pigs by the surgical removal of bone and subsequent silk ligament suture around the cervical portion of the tooth. Autologous PDLSCs were obtained from extracted teeth of the miniature pigs and then expanded ex vivo to enrich PDLSC numbers. When transplanted into the surgically created periodontal defect areas, PDLSCs were capable of regenerating periodontal tissues, leading to a favorable treatment for periodontitis. This study demonstrates the feasibility of using stem cell‐mediated tissue engineering to treat periodontal diseases.

Periodontitis and Rheumatoid Arthritis: A Review

Periodontitis and rheumatoid arthritis (RA) appear to share many pathologic features. In this review, the common pathologic mechanisms of these two common chronic conditions are explored. Emerging evidence now suggests a strong relationship between the extent and severity of periodontal disease and RA. While this relationship is unlikely to be causal, it is clear that individuals with advanced RA are more likely to experience more significant periodontal problems compared to their non-RA counterparts, and vice versa. A case is made that these two diseases could be very closely related through common underlying dysfunction of fundamental inflammatory mechanisms. The nature of such dysfunction is still unknown. Nonetheless, there is accruing evidence to support the notion that both conditions manifest as a result of an imbalance between proinflammatory and anti-inflammatory cytokines. As a result, new treatment strategies are expected to emerge for both diseases that may target the inhibition of proinflammatory cytokines and destructive proteases. The clinical implications of the current data dictate that patients with RA should be carefully screened for their periodontal status.

Ovine Periodontal Ligament Stem Cells: Isolation, Characterization, and Differentiation Potential

Periodontal disease leads to destruction of the connective tissues responsible for restraining teeth within the jaw. To date, various conventional therapies for periodontal regeneration have shown limited and variable clinical outcomes. Recent studies have suggested that newly identified human periodontal ligament stem cells (PDLSCs) may offer an alternate and more reliable strategy for the treatment of periodontal disease using a cell-based tissue engineering approach. In the present study, we generated enriched preparations of PDLSCs derived from ovine periodontal ligament using immunomagnetic bead selection, based on expression of the mesenchymal stem cell-associated antigen CD106 (vascular cell adhesion molecule 1). These CD106+ ovine PDLSCs demonstrated the capacity to form adherent clonogenic clusters of fibroblast-like cells when plated at low densities in vitro. Ex vivo-expanded ovine PDLSCs exhibited a high proliferation rate in vitro and expressed a phenotype (CD44+, CD166+, CBFA-1+, collagen-I+, bone sialoprotein+) consistent with human-derived PDLSCs. Furthermore, cultured ovine PDLSCs expressed high transcript levels of the ligament/tendon-specific early transcription factor scleraxis. Importantly, ex vivo-expanded ovine PDLSCs demonstrated the capacity to regenerate both cementum-like mineral and periodontal ligament when transplanted into NOD/SCID mice. The results from the present study suggest that ovine PDLSCs may potentially be used as a novel cellular therapy to facilitate successful and more predictable regeneration of periodontal tissue using an ovine preclinical model of periodontal disease as a prelude to human clinical studies.

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds.

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts

BACKGROUND AND OBJECTIVE Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. MATERIAL AND METHODS iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c-MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM-2, TG30 and TRA-1-60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real-time RT-PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. RESULTS Human gingival fibroblast- and periodontal ligament fibroblast-derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell-associated cell-surface antigens, SSEA3, SSEA4, GCTM-2, TG30 (CD9) and Tra-1-60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. CONCLUSION These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.

Expression of peptidylarginine deiminase-2 and -4, citrullinated proteins and anti-citrullinated protein antibodies in human gingiva

BACKGROUND AND OBJECTIVE The presence of citrullinated proteins, and peptidylarginine deiminase types -2 (PAD-2) and -4 (PAD-4) in periodontal tissues, determine the presence of anti-cyclic citrullinated protein antibodies (anti-CCP) in gingival crevicular fluid (GCF) and compare the expression of these proteins between inflamed and non-inflamed sites. MATERIAL AND METHODS Tissue sections were stained using antibodies against citrullinated proteins, PAD-2 and PAD-4. RT-PCR was performed to investigate PAD-2 and PAD-4 mRNA in inflamed and non-inflamed gingival tissues. Anti-CCP antibodies in gingival crevicular fluid were detected by ELISA. RESULTS Citrullinated proteins, PAD-2 and PAD-4 were detected in gingiva. There was a correlation between inflammation and expression of these proteins. mRNAs for PAD-2 and PAD-4 were detected in both inflamed and non-inflamed gingival tissues. Antibodies to CCP were found mostly in the GCF of individuals with periodontitis. CONCLUSION PAD-2 and PAD-4 (protein and mRNA) as well as citrullinated proteins are present in inflamed gingiva, and anti-CCP antibodies can be detected in the GCF of some patients. Tissue expression of citrullinated proteins and PAD increased with the severity of inflammation. The presence of anti-CCP antibodies in GCF was almost exclusive to a subset of patients with periodontitis. Increased expression of these proteins in inflamed gingiva lends support to the notion that periodontal inflammation contributes to the inflammatory burden in a similar way to rheumatoid arthritis.

Stem cells and periodontal regeneration.

Periodontitis is an inflammatory disease which manifests clinically as loss of supporting periodontal tissues including periodontal ligament and alveolar bone. For decades periodontists have sought ways to repair the damage which occurs during periodontitis. This has included the use of a range of surgical procedures, the use of a variety of grafting materials and growth factors, and the use of barrier membranes. To date periodontal regeneration is considered to be biologically possible but clinically unpredictable. Recently, reports have begun to emerge demonstrating that populations of adult stem cells reside in the periodontal ligament of humans and other animals. This opens the way for new cell-based therapies for periodontal regeneration. For this to become a reality a thorough understanding of adult human stem cells is needed. This review provides an overview of adult human stem cells and their potential use in periodontal regeneration.

Generation of functional mesenchymal stem cells from different induced pluripotent stem cell lines.

The therapeutic potential of mesenchymal stem cells (MSC) has highlighted the need for identifying easily accessible and reliable sources of these cells. An alternative source for obtaining large populations of MSC is through the controlled differentiation of induced pluripotent stem cells (iPSC). In the present study, colonies of iPSC were cultured in MSC culture media for 2 weeks. Serial passaging then selected for fast growing MSC-like cells with a typical fibroblastic morphology and the capacity to proliferate on standard culture flasks without feeder cells. MSC-like cells were developed from iPSC lines arising from three different somatic tissues: gingiva, periodontal ligament (PDL), and lung. The iPSC-MSC like cells expressed key MSC-associated markers (CD73, CD90, CD105, CD146, and CD166) and lacked expression of pluripotent markers (TRA160, TRA181, and alkaline phosphatase) and hematopoietic markers (CD14, CD34, and CD45). In vitro iPSC-MSC-like cells displayed the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. In vivo subcutaneous implantation of the iPSC-MSC-like cells into NOD/SCID mice demonstrated that only the PDL-derived iPSC-MSC-like cells exhibited the capacity to form mature mineralized structures which were histologically similar to mature bone. These findings demonstrate that controlled induction of iPSC into fibroblastic-like cells that phenotypically and functionally resemble adult MSC is an attractive approach to obtain a readily available source of progenitor cells for orthopedic and dental-related tissue-engineering applications. However, a detailed characterization of the iPSC-MSC-like cells will be important, as MSC-like cells derived from different iPSC lines exhibit variability in their differentiation capacity.

Multiphasic Scaffolds for Periodontal Tissue Engineering

For a successful clinical outcome, periodontal regeneration requires the coordinated response of multiple soft and hard tissues (periodontal ligament, gingiva, cementum, and bone) during the wound-healing process. Tissue-engineered constructs for regeneration of the periodontium must be of a complex 3-dimensional shape and adequate size and demonstrate biomechanical stability over time. A critical requirement is the ability to promote the formation of functional periodontal attachment between regenerated alveolar bone, and newly formed cementum on the root surface. This review outlines the current advances in multiphasic scaffold fabrication and how these scaffolds can be combined with cell- and growth factor–based approaches to form tissue-engineered constructs capable of recapitulating the complex temporal and spatial wound-healing events that will lead to predictable periodontal regeneration. This can be achieved through a variety of approaches, with promising strategies characterized by the use of scaffolds that can deliver and stabilize cells capable of cementogenesis onto the root surface, provide biomechanical cues that encourage perpendicular alignment of periodontal fibers to the root surface, and provide osteogenic cues and appropriate space to facilitate bone regeneration. Progress on the development of multiphasic constructs for periodontal tissue engineering is in the early stages of development, and these constructs need to be tested in large animal models and, ultimately, human clinical trials.