The regeneration of lost periodontal ligament (PDL) and alveolar bone is the purpose of periodontal tissue engineering. The goal of the present study was to assess the suitability of 3 odontogenic progenitor populations from dental pulp, PDL, and dental follicle for periodontal regeneration when exposed to natural and synthetic apatite surface topographies. We demonstrated that PDL progenitors featured higher levels of periostin and scleraxis expression, increased adipogenic and osteogenic differentiation potential, and pronounced elongated cell shapes on barren root chips when compared with dental pulp and dental follicle cells. When evaluating the effect of surface characteristics on PDL progenitors, natural root surfaces resulted in elongated PDL cell shapes, whereas PDL progenitors on synthetic apatite surfaces were rounded or polygonal. In addition, surface coatings affected PDL progenitor gene expression profiles: collagen I coatings enhanced alkaline phosphatase and osteocalcin expression levels and laminin-1 coatings increased epidermal growth factor (EGF), nestin, cadherin 1, and keratin 8 expression. PDL progenitors seeded on natural tooth root surfaces in organ culture formed new periodontal fibers after 3 weeks of culture. Finally, replantation of PDL progenitor-seeded tooth roots into rat alveolar bone sockets resulted in the complete formation of a new PDL and stable reattachment of teeth over a 6-month period. Together, these findings indicate that periodontal progenitor cell type as well as mineral surface topography and molecular environment play crucial roles in the regeneration of true periodontal anchorage.
Orthodontic tooth movement is controlled by various cell types in the periodontal ligament (PDL). Mechanical stresses, such as orthodontic force, are thought to induce differentiation of the mesenchymal cells in the PDL into osteoblasts and cementoblasts. The details of the process of differentiation, however, are not known, in part because adequate in vitro systems for their study do not yet exist. The purpose of this study was to establish and characterize immortalized PDL cell lines derived from the PDL of transgenic rats harboring the temperature-sensitive simian virus 40 T-antigen gene (TG rats). The PDL was removed from the molar roots of TG rats and incubated in tissue culture. Outgrowth cells from the PDL explant were passaged and cloned, depending on the shape of the colonies formed. The cell lines thus established were analyzed by reverse transcription–polymerase chain reaction for expression of type-I collagen, osteopontin, fibronectin, alkaline phosphatase (bone type), bone sialoprotein, the receptor activator of NF-κ B ligand, and osteoprotegerin. In addition, the capacity for formation of mineralized nodules was assessed by incubating cells in calcification-promoting medium at 37 °C. A total of 15 stable cell lines were successfully established and characterized. These cell lines were classified into six groups based on their pattern of gene expression at 33°C. Moreover, three of these clones were capable of forming calcified nodules. In conclusion, differential gene expression was demonstrated in 15 established PDL cell lines. Some cells had the potential to differentiate into cell types found in mineralized tissues, such as osteoblasts and cementoblasts, as well as cells expressing molecules that regulate osteoclast differentiation.
Calcification; Cell lines; Fibroblasts; Gene expression; Periodontal ligament; SV40 transgenic rat
Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.
tooth tissue engineering; dental stem cells; mandibular model
The purpose of this study was to investigate the isolation and characterization of multipotent human periodontal ligament (PDL) stem cells and to assess their ability to differentiate into bone, cartilage, and adipose tissue.
PDL stem cells were isolated from 7 extracted human premolar teeth. Human PDL cells were expanded in culture, stained using anti-CD29, -CD34, -CD44, and -STRO-1 antibodies, and sorted by fluorescent activated cell sorting (FACS). Gingival fibroblasts (GFs) served as a positive control. PDL stem cells and GFs were cultured using standard conditions conducive for osteogenic, chondrogenic, or adipogenic differentiation.
An average of 152.8 ± 27.6 colony-forming units was present at day 7 in cultures of PDL stem cells. At day 4, PDL stem cells exhibited a significant increase in proliferation (p < 0.05), reaching nearly double the proliferation rate of GFs. About 5.6 ± 4.5% of cells in human PDL tissues were strongly STRO-1-positive. In osteogenic cultures, calcium nodules were observed by day 21 in PDL stem cells, which showed more intense calcium staining than GF cultures. In adipogenic cultures, both cell populations showed positive Oil Red O staining by day 21. Additionally, in chondrogenic cultures, PDL stem cells expressed collagen type II by day 21.
The PDL contains multipotent stem cells that have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. This adult PDL stem cell population can be utilized as potential sources of PDL in tissue engineering applications.
Histochemistry; Periodontics; Bone biology
The long-term success of the reimplanted teeth is related to the maintenance of periodontal ligament (PDL) cell viability. Dental tissues are unique in comparison to most other tissues in the body due to their marked capacity for regeneration. Understanding the circumstances leading to repair and regeneration in oral tissues has been a formidable challenge. Numerous storage media have been introduced by many authors that help to maintain the PDL cell viability. To present an overview of the various available storage media. A literature search for the past 20 years was performed across the Internet database (Pubmed) and relevant citations using the keywords PDL cell viability, tooth avulsion, storage media, and the combination of all to retrieve around (n=225) citations. Articles that included follow-up of intervention for avulsed and re-implanted teeth were considered (n=44) and some literature review from well-known text books were considered. Literature supports that moist storage appears to be a more productive approach to optimize PDL cell survival. However, no medium is ideal and in vivo studies are inadequate.
PDL cell viability; tooth avulsion; storage media
The periodontal ligament (PDL) is a specialized connective tissue that connects the surface of the tooth root with the bony tooth socket. The healthy PDL harbors stem cell niches and extracellular matrix (ECM) microenvironments that facilitate periodontal regeneration. During periodontal disease, the PDL is often compromised or destroyed, reducing the life-span of the tooth. In order to explore new approaches toward the regeneration of diseased periodontal tissues, we have tested the effect of periodontal ECM signals, fibroblast growth factor 2 (FGF2), connective tissue growth factor (CTGF), and the cell adhesion peptide Arg-Gly- Asp (RGD) on the differentiation of two types of periodontal progenitor cells, PDL progenitor cells (PDLPs) and dental follicle progenitor cells (DFCs). Our studies documented that CTGF and FGF2 significantly enhanced the expression of collagens I & III, biglycan and periostin in tissue engineered regenerates after 4 weeks compared to untreated controls. Specifically, CTGF promoted mature PDL-like tissue regeneration as demonstrated by dense periostin localization in collagen fiber bundles. CTGF and FGF2 displayed synergistic effects on collagen III and biglycan gene expression, while effects on mineralization were antagonistic to each other: CTGF promoted while FGF2 inhibited mineralization in PDL cell cultures. Incorporation of RGD peptides in hydrogel matrices significantly enhanced attachment, spreading, survival and mineralization of the encapsulated DFCs, suggesting that RGD additives might promote the use of hydrogels for periodontal mineralized tissue engineering. Together, our studies have documented the effect of three key components of the periodontal ECM on the differentiation of periodontal progenitor populations.
growth factors; extracellular matrix; periodontal regeneration; progenitor cells
Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells.
To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (β-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data.
The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4).
This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.
Microarray analysis; Gene expression profiling; Periodontal ligament; Cell differentiation
Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to examine the effect of mechanical load in vitro. In the current in vitro study, green fluorescent protein labeled periodontal ligament (PDL) cells form rat incisors were embedded in a 3D matrix and exposed to mechanical loading alone, to a chemical stimulus (Emdogain; enamel matrix derivative [EMD]) alone, or a combination of both. Loading consisted of unilateral stretching (8%, 1 Hz) and was applied for 1, 3, or 5 days. Results showed that PDL cells were distributed and randomly oriented within the artificial PDL space in static culture. On mechanical loading, the cells showed higher cell numbers. Moreover, cells realigned perpendicular to the stretching force depending on time and position, with great analogy to natural PDL tissue. EMD application gave a significant effect on growth and upregulated bone sialoprotein (BSP) and collagen type-I (Col-I), whereas Runx-2 was downregulated. This implies that PDL cells under loading might tend to act similar to bone-like cells (BSP and Col-I) but at the same time, react tendon like (Runx-2). The combination of chemical and mechanical stimulation seems possible, but does not show synergistic effects. In this study, a new model was successfully introduced in the field of PDL-related regenerative research. Besides validating the 3D model to mimic an authentic PDL space, it also provided a useful and well-controlled approach to study cell response to mechanical loading and other stimuli.
A blood supply is crucial for tissue healing and regeneration. Periodontal ligament (PDL) tissue is situated between the tooth root and alveolar bone, and cells derived from PDL tissue are reported to have stem cell-like activity. This study aimed to evaluate the potential of PDL cells derived from deciduous teeth to express endothelial cell (EC)-specific markers. Using quantitative PCR, we investigated whether PDL cells derived from human deciduous teeth express mRNA for the EC-specific markers: vascular endothelialcadherin (VE-cadherin), vascular endothelial growth factor receptor 2 (VEGFR2) and CD31 upon treatment with 15 ng/ml heparin or 10 ng/ml fibroblast growth factor (FGF)-2 in vitro. Quantitative PCR showed that PDL cells expressed mRNA for the EC-specific markers, VE-cadherin and VEGFR2, when cultured in the presence of heparin alone or with FGF-2. By contrast, marked CD31 mRNA expression was induced only when PDL cells were cultured with both heparin and FGF-2. Western blot analysis showed that the CD31 protein was induced in PDL cells upon treatment with both heparin and FGF-2 for 3 weeks. PDL cells derived from deciduous teeth inducibly express EC-specific markers and thus have the potential to differentiate into a vascular cell lineage.
endothelial cell marker; periodontal ligament; deciduous teeth
The relative motion between the tooth and alveolar bone is facilitated by the soft-hard tissue interfaces which include periodontal ligament-bone (PDL-bone) and periodontal ligament-cementum (PDL-cementum). The soft-hard tissue interfaces are responsible for attachment and are critical to the overall biomechanical efficiency of the bone-tooth complex. In this study, the PDL-bone and PDL-cementum attachment sites in human molars were investigated to identify the structural orientation and integration of the PDL with bone and cementum. These attachment sites were characterized from a combined materials and mechanics perspective and were related to macro-scale function.
High resolution complimentary imaging techniques including atomic force microscopy, scanning electron microscopy and micro-scale X-ray computed tomography (Micro XCT™) illustrated two distinct orientations of PDL; circumferential-PDL (cir-PDL) and radial-PDL (rad-PDL). Within the PDL-space, the primary orientation of the ligament was radial (rad-PDL) as is well known. Interestingly, circumferential orientation of PDL continuous with rad-PDL was observed adjacent to alveolar bone and cementum. The integration of the cir-PDL was identified by 1 to 2 μm diameter PDL-inserts or Sharpey’s fibers in alveolar bone and cementum. Chemically and biochemically the cir-PDL adjacent to bone and cementum was identified by relatively higher carbon and lower calcium including the localization of small leucine rich proteins responsible for maintaining soft-hard tissue cohesion, stiffness and hygroscopic nature of PDL-bone and PDL-cementum attachment sites. The combined structural and chemical properties provided graded stiffness characteristics of PDL-bone (Er range for PDL: 10 – 50 MPa; bone: 0.2 – 9.6 GPa) and PDL-cementum (Er range for cementum: 1.1 – 8.3 GPa), which was related to the macro-scale function of the bone-tooth complex.
Interfaces; Bone-Tooth Complex; Biomechanics; Fibrous Join Cementum; Alveolar Bone
The matrix adhesion protein ameloblastin (AMBN) is one of the unique components of the mineralizing matrix of bones and teeth. Here we have focused on two cell types with AMBN expression to decipher AMBN function in developing dental, periodontal and bone tissues: mouse dental follicle cells (mDF) and periodontal ligament cells (mPDL). To test AMBN function, cell culture dishes for mDF and mPDL culture were exposed either to full length or C-terminal (AA 137–407) recombinant protein. Alternatively, cells were subjected to transient transfection using an AMBN-shRNA vector. Our cell culture studies documented that full-length AMBN-coated dishes promoted the attachment of mPDL and mDF cells as early as 1 hour post seeding. In order to identify potential intermediaries that might aid the effect of AMBN on adhesion, RhoA expression levels in AMBN coated and uncoated control dishes were assessed. These studies indicated that AMBN induced RhoA expression 4 hours post seeding, especially in mPDL cells. After four hours culture, the cell cycle inhibitor p27 was upregulated as well. In addition, exogenous AMBN and its C-terminal fragment reduced the proliferation of mDF and mPDL. Finally, transient transfection of mDF and mPDL cells with AMBN-shRNA vector resulted in a down-regulation of p27 in mPDL cells. Together, these data indicate that AMBN affects cell adhesion via RhoA and cell cycle progression through p27.
extracellular matrix signaling; ameloblastin; integrin; RhoA; p27
The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.
Ligament; TGF-β; Differentiation; Endothelial Cell; Smooth Muscle Cell
Cloned cementoblasts (OCCMs), periodontal ligament fibroblasts (SV-PDLs), and dental follicle (SV-F) cells obtained from mice were used as a tool to study periodontal tissue engineering. OCCM, SV-PDL, and SV-F cells were seeded onto three-dimensional poly lactic-co-glycolic acid (PLGA) scaffolds and cultured with the use of bioreactors or implanted subcutaneously in severe combined immune deficiency (SCID) mice for up to 6 weeks. We explored the behavior of these cells in porous PLGA sponges by cell growth, expression of mineral-associated genes using reverse transcriptase polymerase chain reaction, and mineralization by histologic analysis in vitro and in vivo. Results indicated that cells attached to PLGA scaffolds under either static or dynamic conditions in vitro. Only OCCM implants, retrieved from both in vitro bioreactors and SCID mice at 3-and 6-weeks post-cell implantation exhibited mineral formation. Types I and XII collagens, osteocalcin, and bone sialoprotein genes were detected in all implants retrieved from SCID mice. These results suggest that delivery of selected cells via PLGA scaffolds may serve as a viable approach for promoting periodontal tissue regeneration.
tissue engineering; cementoblasts; periodontal ligament; dental follicle; PLGA; cell therapy
Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multi-lineage differentiation, and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSC) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multi-lineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1+/CD146+, whereas more than 63% were STRO-1-/CD146-. Colony-forming efficiency of STRO-1+/CD146+ PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly up-regulated under appropriate conditions in PMP cells and MSCs compared to either their non-induced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic or adipogenic conditions in PMP and MSC cultures, respectively, compared to non-induced controls or induced PPDL cells. Thus, STRO-1+/CD146+ PMP cells demonstrate multi-lineage differentiation capacity comparable in magnitude to MSCs, and could potentially be utilized for regeneration of the periodontium and other tissues.
Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1+/CD146+, whereas more than 63% were STRO-1−/CD146−. Colony-forming efficiency of STRO-1+/CD146+ PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1+/CD146+ PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.
Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 μg/kg body weight PTH(1–34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.
The rate-limiting step in orthodontic treatment is often the rapidity with which teeth move. Using biological agents to modify the rate of tooth movement has been shown to be effective in animals. Relaxin is a hormone present in both males and females. Its main action is to increase the turnover of fibrous connective tissues. Thus, relaxin might increase the amount and rate of tooth movement through its effect on the periodontal ligament (PDL). The purpose of this study was to measure the effect of relaxin on orthodontic tooth movement and PDL structures.
Bilateral orthodontic appliances designed to tip maxillary molars mesially with a force of 40 cN were placed in 96 rats. At day 0, the animals were randomized to either relaxin or vehicle treatment. Twelve rats in each group were killed at 2, 4, 7, and 9 days after appliance activation. Cephalograms were taken at appliance placement and when the rats were killed. Tooth movement was measured cephalometrically in relation to palatal implants. Fractal analysis and visual analog scale assessments were used to evaluate the effect of relaxin on PDL fiber organization at the tension sites in histologic sections. The in-vitro testing for PDL mechanical strength and tooth mobility was performed by using tissue from an additional 20 rats that had previously received the same relaxin or vehicle treatments for 1 or 3 days (n = 5).
Both groups had statistically significant tooth movement as functions of time. However, relaxin did not stimulate significantly greater or more rapid tooth movement. Fractal and visual analog scale analyses implied that relaxin reduced PDL fiber organization. In-vitro mechanical testing and tooth mobility assessments indicated that the PDL of the mandibular incisors in the relaxin-treated rats had reduced yield load, strain, and stiffness. Moreover, the range of tooth mobility of the maxillary first molars increased to 130% to 170%, over vehicle-treated rats at day 1.
Human relaxin does not accelerate orthodontic tooth movement in rats; it can reduce the level of PDL organization, reduce PDL mechanical strength, and increase tooth mobility at early time points.
The periodontal ligament (PDL) is a key contributor to the process of regeneration of the periodontium. The heterogeneous nature of the PDL tissue, its development during early adulthood, and the different conditions to which the PDL tissue is exposed to in vivo impart on the PDL unique characteristics that may be of consequence during its cultivation in vitro. Several factors affecting the in vivo setting influence the behavior of PDL fibroblasts in culture. The purpose of this review is to address distinct factors that influence the behavior of PDL fibroblasts in culture — in vivo-in vitro transitions, cell identification/isolation markers, primary PDL cultures and cell lines, tooth-specific factors, and donor-specific factors. Based on the reviewed studies, the authors recommendations include the use of several identification markers to confirm cell identity, use of primary cultures at early passage to maintain unique PDL heterogeneic characteristics, and noting donor conditions such as age, systemic health status, and tooth health status. Continued efforts will expand our understanding of the in vitro and in vivo behavior of cells, with the goal of orchestrating optimal periodontal regeneration. This understanding will lead to improved evidence-based rationales for more individualized and predictable periodontal regenerative therapies.
periodontal; human; fibroblast; cell culture; regeneration
Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model.
The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens.
Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line.
This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogenesis.
Animal studies; biomimetics; cementoblasts; cementogenesis; dental follicle/anatomy and histology; periodontal regeneration; wound healing
Although cells derived from periodontal ligament (PDL) tissue are reported to have stem cell-like activity and are speculated to play a crucial role for tissue healing and regeneration after injury or orthodontic treatment, mechanisms regulating their recruitment and activation remain unknown. Recently, stromal cell-derived factor 1α (SDF-1α) has been reported to be important for stem cell homing and recruitment to injured sites. The aim of this study was to evaluate whether fibroblast growth factor 2 (FGF-2) affects the expression of SDF-1α in PDL cells derived from human permanent teeth in vitro. Using real-time PCR, the expression of SDF-1α mRNA in PDL cells was inhibited by treatment with 10 ng/ml FGF-2. When PDL cells were treated with SU5402 (an inhibitor of FGF receptor 1) in combination with FGF-2, the FGF-2-reduced expression of SDF-1α was inhibited. In the presence of the JNK inhibitor SP600125, SDF-1α mRNA in PDL cells was not suppressed by the FGF-2 treatment. Western blot analysis also showed that SDF-1α production was suppressed by treatment with FGF-2, but it recovered with treatment by FGF-2 + SU5402. These findings suggest that SDF-1α from PDL cells plays an important role in the regeneration and homeostasis of periodontal tissues via the recruitment of stem cells.
stromal cell-derived factor 1α; fibroblast growth factor 2; periodontal ligament cells; permanent tooth
Periodontal disease is an inflammatory disorder with widespread morbidities involving both oral and systemic health. The primary goal of periodontal treatment is the regeneration of the lost or diseased periodontium. In this study, we retrospectively examined feasibility and safety of reconstructing the periodontal intrabony defects with autologous periodontal ligament progenitor (PDLP) implantation in three patients.
Materials and Methods
In this retrospective pilot study, we treated 16 teeth with at least one deep intrabony defect of probing depth (PD) ≥ 6 mm with PDLP transplantation and evaluated clinical outcome measures in terms of probing depth, gingival recession and attachment gain for a duration of 32–72 months. Furthermore, we compare PDLPs with standard PDL stem cells (PDLSCs) and confirmed that PDLPs possessed progenitor characters.
Clinical examination indicated that transplantation of PDLPs may provide therapeutic benefit for the periodontal defects. All treated patients showed no adverse effects during the entire course of follow up. We also found that PDLPs were analogous to PDLSCs in terms of high proliferation, expression of mesenchymal surface molecules, multipotent differentiation, and in vivo tissue regain. However, PDLPs failed to express scleraxis, a marker of tendon, as seen in PDLSCs.
This study demonstrated clinical and experimental evidences supporting a potential efficacy and safety of utilizing autologous PDL cells in the treatment of human periodontitis.
periodontal ligament progenitors; regeneration; periodontitis
To investigate the stem cell-like characteristics of human periodontal ligament (PDL) stromal cells outgrown from orthodontically extracted premolars and to evaluate the potential for myogenic differentiation.
PDL stromal cells were obtained from extracted premolars by using the outgrowth method. Cell morphological features, self-replication capability, and the presence of cell-surface markers, along with osteogenic, adipogenic, and chondrogenic differentiation, were confirmed. In addition, myogenic differentiation was induced by the use of 5-aza-2'-deoxycytidine (5-Aza) for DNA demethylation.
PDL stromal cells showed growth patterns and morphological features similar to those of fibroblasts. In contrast, the proliferation rates of premolar PDL stromal cells were similar to those of bone marrow and adipogenic stem cells. PDL stromal cells expressed surface markers of human mesenchymal stem cells (i.e., CD90 and CD105), but not those of hematopoietic stem cells (i.e., CD31 and CD34). PDL stromal cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages. Myotube structures were induced in PDL stromal cells after 5-Aza pretreatment, but not in the absence of 5-Aza pretreatment.
PDL stromal cells isolated from extracted premolars can potentially be a good source of postnatal stem cells for oromaxillofacial regeneration in bone and muscle.
Human periodontal ligament stem cell; Myogenic differentiation; Cell biology; Molecular biology; Soft tissue
Modifying the balance between resorption and apposition through selectively injuring the cortical plate of the alveolus has been an approach to speed tooth movement and is referred to as periodontally accelerated osteogenic orthodontics. The aim of this study was to investigate the alveolar response to corticotomy as a function of time and proximity to the surgical injury in a rat model.
Maxillary buccal and lingual cortical plates were injured in 36 healthy adult rats adjacent to the upper left first molars. Twenty-four animals were euthanized at 3, 7, or 11 weeks. In one group, the maxillae were removed and stripped of soft tissues, and histomorphometric analysis was performed to study alveolar spongiosa and periodontal ligament (PDL) modeling dynamics. Catabolic activity was analyzed with tartrate-resistant acid phosphatase–positive osteoclasts and preosteoclasts. Anabolic actions were measured using a fluorescent vital bone stain series followed by sacrifice at 30 and 51 days. To further analyze the new bone formation, a separate group of animals were fed with calcein fluorescent stain and processed for non-decalcified fluorescent stain histology.
At 3 weeks, the surgery group had significantly (P <0.05) less calcified spongiosa bone surface, greater periodontal ligament surface, higher osteoclast number, and greater lamina dura apposition width. The catabolic activity (osteoclast count) and anabolic activity (apposition rate) were three-fold greater, calcified spongiosa decreased by two-fold, and PDL surface increased by two-fold. Surgical injury to the alveolus that induced a significant increase in tissue turnover by week 3 dissipated to a steady state by postoperative week 11. The impact of the injury was localized to the area immediately adjacent to the decortication injury.
Selective alveolar decortication induced increased turnover of alveolar spongiosa, and the activity was localized; dramatic escalation of demineralization-remineralization dynamics is the likely biologic mechanism underlying rapid tooth movement following selective alveolar decortication.
Bone formation; osteoclast; tooth movement
Adaptive properties of the bone-PDL-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed PDL-space, changes in mineral level of the cortical bone and alveolar jaw bone, and glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-PDL-cementum complex due to prolonged reduced loads has not been fully explained to date, especially with regards to concurrent adaptations of bone, PDL and cementum. Hence, the temporal effect of reduced functional loads on physical characteristics such as morphology and mechanical properties, and mineral profiles of the bone-periodontal ligament (PDL)-cementum complex using a rat model was investigated.
Materials and Methods
Two groups of six-week-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127–158N/mm for hard pellet or 0.32–0.47N/mm for soft powder forms. Spatio-temporal adaptation of the bone-PDL-cementum complex was identified by mapping changes in: 1) PDL-collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro X-ray computed tomography, 2) mineral profiles of the PDL-cementum and PDL-bone interfaces by X-ray attenuation, and 3) microhardness of bone and cementum by microindentation of specimens at ages six, eight, twelve, and fifteen weeks.
Reduced functional loads over prolonged time resulted in 1) altered PDL orientation and decreased PDL collagen birefringence indicating decreased PDL turnover rate and decreased apical cementum resorption; 2) a gradual increase in X-ray attenuation, owing to mineral differences, at the PDL-bone and PDL-cementum interfaces without significant differences in the gradients for either group; 3) significantly (p<0.05) lower microhardness of alveolar bone (0.93±0.16 GPa) and secondary cementum (0.803±0.13 GPa) compared to the higher load group (1.10±0.17 GPa and 0.940±0.15 GPa respectively) at fifteen weeks indicating a temporal effect of loads on local mineralization of bone and cementum.
Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology and mechanical properties, and mineral variations and of the local load-bearing sites in a bone-PDL-cementum complex. These observed local changes in turn could help explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease, and/or orthodontic and prosthodontic treatments.
functional loads; tissue interfaces; cementum; bone-tooth biomechanics; alveolar bone; periodontal ligament
Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.