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1.  Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces 
Stem Cells and Development  2011;20(10):1659-1668.
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.
doi:10.1089/scd.2010.0431
PMCID: PMC3210028  PMID: 21250830
2.  Cementoblast Delivery for Periodontal Tissue Engineering 
Journal of periodontology  2004;75(1):154-161.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
PMCID: PMC2596890  PMID: 15025227
Animal studies; biomimetics; cementoblasts; cementogenesis; dental follicle/anatomy and histology; periodontal regeneration; wound healing
3.  Effects of human relaxin on orthodontic tooth movement and periodontal ligaments in rats 
Introduction
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1016/j.ajodo.2006.06.014
PMCID: PMC2846749  PMID: 17208099
4.  A Three-Dimensional Cell Culture Model to Study the Mechano-Biological Behavior in Periodontal Ligament Regeneration 
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.
doi:10.1089/ten.tec.2011.0367
PMCID: PMC3262976  PMID: 21913838
5.  Heterogeneous Dental Follicle Cells and the Regeneration of Complex Periodontal Tissues 
Tissue Engineering. Part A  2012;18(5-6):459-470.
Dental follicle cells (DFCs) are a heterogeneous population that exhibit a variety of phenotypes. However, it remains unclear whether DFCs can maintain stem cell characteristics, or mediate tissue-regeneration to form single or complex tissues in the periodontium, after long-term culturing. Therefore, DFCs were isolated from human impacted molars (HIM-DFCs), passaged >30 times, and then evaluated for their heterogeneity and multipotential differentiation. Morphology, proliferation, epitope profile, and mineralization characteristics of clones derived from single HIM-DFCs in vitro were also assayed. HIM-DFCs (passage #30) were found to be positive for the heterogeneous markers, Notch-1, stro-1, alkaline phosphomonoesterase (ALP), type I collagen (COL-I), type III collagen (COL-III), and osteocalcine. Moreover, passage #30 of the HDF1, 2, and 3 subclone classes identified in this study were found to express high levels of the mesenchymal stem cells markers, CD146 and Stro1. HDF3 subclones were also associated with the strongest ALP staining detected, and strongly expressed osteoblast and cementoblast markers, including COL-I, COL-III, bone sialoprotein (BSP), and Runx2. In contrast, HDF1 subclone analyzed strongly expressed COL-I and COL-III, yet weakly expressed BSP and Runx2. The HDF2 subclone was associated with the strongest proliferative capacity. To evaluate differentiation characteristics in vivo, these various cell populations were combined with ceramic bovine bone and implanted into subcutaneous pockets of nude mice. The 30th passage of subclone HDF1 and 3 were observed to contribute to fiber collagens and the mineralized matrix present, respectively, whereas HDF2 subclones were found to have a minimal role in these formations. The formation of a cementum-periodontal ligament (PDL) complex was observed 6 weeks after HIM-DFCs (passage #30) were implanted in vivo, thus suggesting that these cells maintain stem cell characteristics. Therefore, subclone HDF1-3 may be related to the differentiation of fibroblasts in the PDL, undifferentiated cells, and osteoblasts and cementoblasts, respectively. Overall, this study is the first to amplify HIM-DFCs and associated subclones with the goal of reconstructing complex or single periodontium. Moreover, our results demonstrate the potential for this treatment approach to address periodontal defects that result from periodontitis, or for the regeneration of teeth.
doi:10.1089/ten.tea.2011.0261
PMCID: PMC3286823  PMID: 21919800
6.  Differentiation and characteristics of undifferentiated mesenchymal stem cells originating from adult premolar periodontal ligaments 
Korean Journal of Orthodontics  2012;42(6):307-317.
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.4041/kjod.2012.42.6.307
PMCID: PMC3542451  PMID: 23323245
Histochemistry; Periodontics; Bone biology
7.  Ameloblastin Regulates Cell Attachment and Proliferation through RhoA and p27 
European Journal of Oral Sciences  2011;119(Suppl 1):280-285.
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.
doi:10.1111/j.1600-0722.2011.00887.x
PMCID: PMC3402544  PMID: 22243257
extracellular matrix signaling; ameloblastin; integrin; RhoA; p27
8.  Reduced functional loads alter the physical characteristics of the bone-PDL-cementum complex 
Journal of periodontal research  2011;46(6):730-741.
Background
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.
Results
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.
Conclusions
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.
doi:10.1111/j.1600-0765.2011.01396.x
PMCID: PMC3200425  PMID: 21848615
functional loads; tissue interfaces; cementum; bone-tooth biomechanics; alveolar bone; periodontal ligament
9.  Periodontal ligament influence on the stress distribution in a removable partial denture supported by implant: a finite element analysis 
Journal of Applied Oral Science  2012;20(3):362-368.
ABSTRACT
Objective
The non-homogenous aspect of periodontal ligament (PDL) has been examined using finite element analysis (FEA) to better simulate PDL behavior. The aim of this study was to assess, by 2-D FEA, the influence of non-homogenous PDL on the stress distribution when the free-end saddle removable partial denture (RPD) is partially supported by an osseointegrated implant.
Material and Methods
Six finite element (FE) models of a partially edentulous mandible were created to represent two types of PDL (non-homogenous and homogenous) and two types of RPD (conventional RPD, supported by tooth and fibromucosa; and modified RPD, supported by tooth and implant [10.00x3.75 mm]). Two additional FE models without RPD were used as control models. The non-homogenous PDL was modeled using beam elements to simulate the crest, horizontal, oblique and apical fibers. The load (50 N) was applied in each cusp simultaneously. Regarding boundary conditions the border of alveolar ridge was fixed along the x axis. The FE software (Ansys 10.0) was used to compute the stress fields, and the von Mises stress criterion (σvM) was applied to analyze the results.
Results
The peak of σvM in non-homogenous PDL was higher than that for the homogenous condition. The benefits of implants were enhanced for the non-homogenous PDL condition, with drastic σvM reduction on the posterior half of the alveolar ridge. The implant did not reduce the stress on the support tooth for both PDL conditions.
Conclusion
The PDL modeled in the non-homogeneous form increased the benefits of the osseointegrated implant in comparison with the homogeneous condition. Using the non-homogenous PDL, the presence of osseointegrated implant did not reduce the stress on the supporting tooth.
doi:10.1590/S1678-77572012000300012
PMCID: PMC3881771  PMID: 22858705
Removable partial denture; Dental implant; Finite element analysis; Periodontal ligament
10.  The biomechanical characteristics of the bone-periodontal ligament-cementum complex 
Biomaterials  2010;31(25):6635-6646.
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.
doi:10.1016/j.biomaterials.2010.05.024
PMCID: PMC2925235  PMID: 20541802
Interfaces; Bone-Tooth Complex; Biomechanics; Fibrous Join Cementum; Alveolar Bone
11.  Strategies for periodontal ligament cell viability: An overview 
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.
doi:10.4103/0972-0707.85789
PMCID: PMC3198546  PMID: 22025820
PDL cell viability; tooth avulsion; storage media
12.  Establishment of Periodontal Ligament Cell Lines from Temperature-Sensitive Simian Virus 40 Large T-antigen Transgenic Rats 
Cytotechnology  2004;44(1-2):55-65.
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.
doi:10.1023/B:CYTO.0000043412.08814.80
PMCID: PMC3449499  PMID: 19003229
Calcification; Cell lines; Fibroblasts; Gene expression; Periodontal ligament; SV40 transgenic rat
13.  Effects of a Low Level Laser on Periodontal Tissue in Hypofunctional Teeth 
PLoS ONE  2014;9(6):e100066.
Malocclusions, such as an open bite and high canines, are often encountered in orthodontic practice. Teeth without occlusal stimuli are known as hypofunctional teeth, and numerous atrophic changes have been reported in the periodontal tissue, including reductions in blood vessels in the periodontal ligament (PDL), heavy root resorption, and reduced bone mineral density (BMD) in the alveolar bone. Low Level Laser (LLL) has been shown to have a positive effect on bone formation and the vasculature. Although the recovery of hypofunctional teeth remains unclear, LLL is expected to have a positive influence on periodontal tissue in occlusal hypofunction. The aim of the present study was to elucidate the relationship between LLL and periodontal tissue in occlusal hypofunction. Twenty-four male rats aged 5 weeks were randomly divided into control and hypofunctional groups. An anterior metal cap and bite plate were attached to the maxillary and mandibular incisors in the hypofunctional group to simulate occlusal hypofunction in the molars. LLL irradiation was applied to the maxillary first molar through the gingival sulcus in half of the rats. Rats were divided into four groups; control, control+LLL, hypofunctional, and hypofunctional+LLL. Exposure to LLL irradiation was performed for 3 minutes every other day for 2 weeks. Animals were examined by Micro-CT at 5 and 7 weeks and were subsequently sacrificed. Heads were resected and examined histologically and immunohistologically. The hypofunctional group had obvious stricture of the PDL. However, no significant differences were observed in the PDL and alveolar bone between the hypofunctional+LLL and the control groups. In addition, the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-positive cells were higher in the hypofunctional + LLL group than in the hypofunctional group. These results indicated that LLL enhanced the production of bFGF and VEGF in the periodontal tissue of hypofunctional teeth.
doi:10.1371/journal.pone.0100066
PMCID: PMC4057341  PMID: 24927024
14.  Bioengineered Dental Tissues Grown in the Rat Jaw 
Journal of dental research  2008;87(8):745-750.
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.
PMCID: PMC3024580  PMID: 18650546
tooth tissue engineering; dental stem cells; mandibular model
15.  Cementum engineering with three-dimensional polymer scaffolds 
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.
doi:10.1002/jbm.a.10058
PMCID: PMC2581746  PMID: 14517861
tissue engineering; cementoblasts; periodontal ligament; dental follicle; PLGA; cell therapy
16.  Extracellular Matrix-Mediated Differentiation of Periodontal Progenitor Cells 
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.
doi:10.1016/j.diff.2009.03.005
PMCID: PMC2744845  PMID: 19433344
growth factors; extracellular matrix; periodontal regeneration; progenitor cells
17.  Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement 
The Angle orthodontist  2012;83(1):48-56.
Objective
To examine changes in microvasculature and the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (VEGFR-2) in rat hypofunctional periodontal ligament (PDL) during experimental tooth movement.
Materials and Methods
Twelve-week-old male Sprague-Dawley rats were divided into normal occlusion and occlusal hypofunction groups. After a 2-week bite-raising period, rat first molar was moved mesially using a 10-gf titanium-nickel alloy closed coil spring in both groups. On days 0, 1, 2, 3, and 7 after tooth movement, histologic changes were examined by micro–computed tomography and immunohistochemistry using CD31, VEGF-A, VEGFR-2, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method.
Results
Hypofunctional molars inclined more than normal molars and did not move notably after day 1 of tooth movement. Blood vessels increased on the tension side of the PDL in normal teeth. Immunoreactivities for VEGF-A and VEGFR-2 in normal teeth were greater than those in hypofunctional teeth during tooth movement. Compressive force rapidly caused apoptosis of the PDL and vascular endothelial cells in hypofunctional teeth, but not in normal teeth.
Conclusions
Occlusal hypofunction induces vascular constriction through a decrease in the expression of VEGF-A and VEGFR-2, and apoptosis of the PDL and vascular cells occurs during tooth movement.
doi:10.2319/011712-45.1
PMCID: PMC4037556  PMID: 22716278
Occlusal hypofunction; Tooth movement; VEGF; VEGFR
18.  Analysis of gene expression during mineralization of cultured human periodontal ligament cells 
Purpose
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.5051/jpis.2011.41.1.30
PMCID: PMC3051055  PMID: 21394295
Microarray analysis; Gene expression profiling; Periodontal ligament; Cell differentiation
19.  LPS Induces Greater Bone and PDL Loss in SPARC-null Mice 
Journal of Dental Research  2011;90(4):477-482.
Individuals with periodontal disease have increased risk of tooth loss, particularly in cases with associated loss of alveolar bone and periodontal ligament (PDL). Current treatments do not predictably regenerate damaged PDL. Collagen I is the primary component of bone and PDL extracellular matrix. SPARC/Osteonectin (SP/ON) is implicated in the regulation of collagen content in healthy PDL. In this study, periodontal disease was induced by injections of lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in wild-type (WT) and SP/ON-null C57/Bl6 mice. A 20-µg quantity of LPS was injected between the first and second molars 3 times a week for 4 weeks, whereas PBS control was injected into the contralateral maxilla. LPS injection resulted in a significant decrease in bone volume fraction in both genotypes; however, significantly greater bone loss was detected in SP/ON-null maxilla. SP/ON-null PDL exhibited more extensive degradation of connective tissue in the gingival tissues. Although total cell numbers in the PDL of SP/ON-null were not different from those in WT, the inflammatory infiltrate was reduced in SP/ON-null PDL. Histology of collagen fibers revealed marked reductions in collagen volume fraction and in thick collagen volume fraction in the PDL of SP/ON-null mice. SP/ON protects collagen content in PDL and in alveolar bone in experimental periodontal disease.
doi:10.1177/0022034510391800
PMCID: PMC3144132  PMID: 21191126
SPARC; periodontal ligament; collagen; BM-40; osteonectin; extracellular matrix; matricellular; periodontal diseases; alveolar bone resorption
20.  Function of Chemokine (CXC Motif) Ligand 12 in Periodontal Ligament Fibroblasts 
PLoS ONE  2014;9(5):e95676.
The periodontal ligament (PDL) is one of the connective tissues located between the tooth and bone. It is characterized by rapid turnover. Periodontal ligament fibroblasts (PDLFs) play major roles in the rapid turnover of the PDL. Microarray analysis of human PDLFs (HPDLFs) and human dermal fibroblasts (HDFs) demonstrated markedly high expression of chemokine (CXC motif) ligand 12 (CXCL12) in the HPDLFs. CXCL12 plays an important role in the migration of mesenchymal stem cells (MSCs). The function of CXCL12 in the periodontal ligament was investigated in HPDLFs. Expression of CXCL12 in HPDLFs and HDFs was examined by RT-PCR, qRT-PCR and ELISA. Chemotactic ability of CXCL12 was evaluated in both PDLFs and HDFs by migration assay of MSCs. CXCL12 was also immunohistochemically examined in the PDL in vivo. Expression of CXCL12 in the HPDLFs was much higher than that in HDFs in vitro. Migration assay demonstrated that the number of migrated MSCs by HPDLFs was significantly higher than that by HDFs. In addition, the migrated MSCs also expressed CXCL12 and several genes that are familiar to fibroblasts. CXCL12 was immunohistochemically localized in the fibroblasts in the PDL of rat molars. The results suggest that PDLFs synthesize and secrete CXCL12 protein and that CXCL12 induces migration of MSCs in the PDL in order to maintain rapid turnover of the PDL.
doi:10.1371/journal.pone.0095676
PMCID: PMC4012992  PMID: 24806431
21.  Effect of Intermittent PTH(1–34) on Human Periodontal Ligament Cells Transplanted into Immunocompromised Mice 
Tissue Engineering. Part A  2012;18(17-18):1849-1856.
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.
doi:10.1089/ten.tea.2011.0626
PMCID: PMC3432899  PMID: 22497226
22.  Utility of PDL progenitors for in vivo tissue regeneration: a report of 3 cases 
Oral diseases  2010;16(1):20-28.
Objective
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.
Results
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.
Conclusions
This study demonstrated clinical and experimental evidences supporting a potential efficacy and safety of utilizing autologous PDL cells in the treatment of human periodontitis.
PMCID: PMC2848819  PMID: 20355278
periodontal ligament progenitors; regeneration; periodontitis
23.  Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse 
Background
The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC.
Tissue samples were obtained from four healthy horses. Primary cell populations were har-vested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical in vitro characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed.
Results
Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages.
Conclusions
Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.
doi:10.1186/1746-6148-7-42
PMCID: PMC3161857  PMID: 21810270
24.  Effect of fibroblast growth factor-2 on periodontal ligament cells derived from human deciduous teeth in vitro 
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.
doi:10.3892/etm_00000052
PMCID: PMC3445930  PMID: 22993547
endothelial cell marker; periodontal ligament; deciduous teeth
25.  Bone Regeneration in Odontostomatology 
Maxillary edentulism, together with periodontal disease, is the condition that most frequently induces disruption of alveolar bone tissue. Indeed, the stimulus of the periodontal ligament is lost and the local bone tissue becomes subject to resorption processes that, in the six months following the loss of the tooth, result in alveolar defects or more extensive maxillary atrophy. In both cases, loss of vestibular cortical bone is followed by reduction in the vertical dimension of the alveolar process, producing effects that upset the morphology of the three-dimensional relations between the dental arches. Maintenance, or restoration, of sufficient bone volume to withstand prosthetic loading and the insertion of an endosseous implant, demands the implementation of operating protocols that bring about bone regeneration in the defect sites. Given the biological principles involved, this requires the implementation of osteogenesis, osteoinduction and osteoconduction protocols.
Osteogenesis is the synthesis of new bone by autologous cells that remain viable, given the capacity of the grafted material to become part of the newly forming bone tissue; osteoinduction is based on the capacity of the grafted material to induce the migration, proliferation and phenotypic conversion, into bone-producing cells, of multipotent undifferentiated cells derived from connective tissue or bone marrow; osteoconduction, meanwhile, provides three-dimensional support and guidance to osteoblast precursors within the defect. The operating procedures implemented take into account the size and morphology of the defect, for the restoration of which guided repair or an out-and-out regenerative protocol may be sufficient. Guided repair exploits the principle of resorption/replacement of the biomaterial with newly-formed bone and consists of restoring the lost bone tissue through the implantation of different, osteointegrative biomaterials. This type of repair requires the application of biocompatible osteoconductors which will gradually be absorbed and replaced by newly formed tissue. Instead, the clinical-surgical basis of bone regeneration is: guided bone regeneration (GBR), the use of growth factors and the application of grafts/osteointegrative materials. GBR, through the use of membranes (resorbable or non-resorbable) allows the filling of a defect, “guiding” the growth only of the osteogenic lines and preventing the invasion of non-osteogenic tissues that compete with the bone. This objective is achieved also thanks to the capacity of the membranes to serve as a filter, thereby strengthening the osteocompetent lines and, at the same time, keeping epithelial cells away. The clinical use of GBR, partly on account of its predictable results, is now very widespread. The growth factors used in bone regeneration are glycoproteins which exert autocrine and paracrine effects on the primordial cells in the site. One of these factors, plasma-rich protein (PRP), is an autologous source of growth factors; obtained by separating and concentrating the platelets in a small volume of plasma, it is immediately utilisable in the surgical site. As regards the osteointegrative materials we can distinguish between autologous, homologous, heterologous, and alloplastic grafts. Of these, autologous bone is the gold standard as it has osteogenic, osteoinductive, and osteoconductive properties and, being fresh, keeps osteoblasts viable. Depending on the size of the defect to be treated, harvesting is from endoral or extraoral sites (calvaria, iliac crest, tibia). The harvested material conserves the embryological characteristics of the site of origin: this principle is reflected in the bone density that develops in the regenerated site. Homologous bone supplied by tissue banks in various formulations is an osteoconductive and partially osteoinductive material that guarantees good mechanical properties even in large defects. Heterologous bone of bovine or equine origin is a carbonate-rich non-stoichiometric apatite. Despite showing low resorption, it does not withstand traction or masticatory loading. Alloplastic materials are osteoconductive materials showing different degrees of resorption; they have biomechanical properties and the speed of their resorption varies, depending on their chemical and stoichiometric formulation. The purpose of bone regeneration thus obtained is to allow the insertion of a titanium implant in the site of the regeneration. This alloplastic implant, whose rough and porous surface allows integration with the bone tissue, will support the prosthesis subsequently applied.
PMCID: PMC3213819

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