A biomechanical study of the human periodontal ligament

The mechanical properties of the normal human periodontal ligament (PDL) were investigated at eight different root levels. One millimetre transverse sections of teeth, PDL and alveolar bone of mandibular premolars were examined in a materials testing machine. During testing bone was supported by metal rings and teeth by metal cylinders of individually adjusted sizes. Having corrected for differences of size and width of the PDL the influence of root level was estimated using a multivariate analysis of variance. The shear strength was almost constant at the upper part of the root, diminishing in apical direction. The shear extensibility and the relative failure energy in shear were higher at the middle of the root, diminishing coronally and apically. Only the elastic stiffness did not vary significantly along the root. These results demonstrate that in order to compare the mechanical properties of PDL care should be taken to compare areas at the same root level.     

Development of a multipotent clonal human periodontal ligament cell line

Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage.     

Experiments to determine the time dependent material properties of the periodontal ligament]

The periodontal ligament is a tissue that attaches the tooth (root) to its alveolar socket, and thus plays an important role in the regulation of tooth movements. Detailed knowledge of the material properties of the periodontal ligament is therefore essential to an understanding of tooth reaction to forces applied during orthodontic treatment. A knowledge of material parameters can also be used in simulations of long-term tooth movements with the aim of improving orthodontic treatment. To this end, this study investigated time-dependent material properties, namely the hysteresis behaviour of the periodontal ligament under constant-velocity loading, the influence of loading velocity on the hysteresis, and its failure under constant loading. Specimens obtained from pigs were used for testing purposes, and the experiments were conducted in a special test setup using a material testing device. The material behaviour of the periodontal ligament was shown to be viscoelastic, and the elastic parameters of material behaviour were also determined. Under constant-velocity loading, material behaviour showed a nonlinear course of the stress-strain curve, also known as hysteresis. When loading was repeated several times, the maximum stress of the hysteresis decreased with each cycle. Determination of the deflection of the specimen at different velocities showed maximum stress to be dependent on the loading rate. The measured stress-strain curves were approximated by bilinear behaviour, permitting the use of finite element calculations. Also investigated was the failure behaviour of the periodontal ligament, which revealed tissue rupture to be inconstant.     

A visco-hyperelastic-damage constitutive model for the analysis of the biomechanical response of the periodontal ligament

The periodontal ligament (PDL), as other soft biological tissues, shows a strongly non-linear and time-dependent mechanical response and can undergo large strains under physiological loads. Therefore, the characterization of the mechanical behavior of soft tissues entails the definition of constitutive models capable of accounting for geometric and material non-linearity. The microstructural arrangement determines specific anisotropic properties. A hyperelastic anisotropic formulation is adopted as the basis for the development of constitutive models for the PDL and properly arranged for investigating the viscous and damage phenomena as well to interpret significant aspects pertaining to ordinary and degenerative conditions. Visco-hyperelastic models are used to analyze the time-dependent mechanical response, while elasto-damage models account for the stiffness and strength decrease that can develop under significant loading or degenerative conditions. Experimental testing points out that damage response is affected by the strain rate associated with loading, showing a decrease in the damage limits as the strain rate increases. These phenomena can be investigated by means of a model capable of accounting for damage phenomena in relation to viscous effects. The visco-hyperelastic-damage model developed is defined on the basis of a Helmholtz free energy function depending on the strain-damage history. In particular, a specific damage criterion is formulated in order to evaluate the influence of the strain rate on damage. The model can be implemented in a general purpose finite element code. The accuracy of the formulation is evaluated by using results of experimental tests performed on animal model, accounting for different strain rates and for strain states capable of inducing damage phenomena. The comparison shows a good agreement between numerical results and experimental data.     

Immunomodulatory properties of human periodontal ligament stem cells

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell‐based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non‐cell contact dependent suppression of PBMNC proliferation in co‐cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN‐γ partially suppressed PBMNC proliferation when compared to CMs without IFN‐γ stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF‐β1, hepatocyte growth factor (HGF) and indoleamine 2, 3‐dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN‐γ. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667–676, 2009. © 2009 Wiley‐Liss, Inc.     

Mass acquisition of human periodontal ligament stem cells

The periodontal ligament (PDL) is an essential fibrous tissue for tooth retention in the alveolar bone socket. PDL tissue further functions to cushion occlusal force, maintain alveolar bone height, allow orthodontic tooth movement, and connect tooth roots with bone. Severe periodontitis, deep caries, and trauma cause irreversible damage to this tissue, eventually leading to tooth loss through the destruction of tooth retention. Many patients suffer from these diseases worldwide, and its prevalence increases with age. To address this issue, regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells, scaffolds, and cytokines as well as their combined applications. In particular, PDL stem cells (PDLSCs) have been well studied. In this review, I discuss comprehensive studies on PDLSCs performed in vivo and contemporary reports focusing on the acquisition of large numbers of PDLSCs for therapeutic applications because of the very small number of PDLSCs available in vivo.     

Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.     

Tensile testing of the mechanical behavior of the human periodontal ligament

Background

The periodontal ligament (PDL) plays a key role in alveolar bone remodeling and resorption during tooth movements. The prediction of tooth mobility under functional dental loads requires a deep understanding of the mechanical behavior of the PDL, which is a critical issue in dental biomechanics. This study was aimed to examine the mechanical behavior of the PDL of the maxillary central and lateral incisors from human. The experimental results can contribute to developing an accurate constitutive model of the human PDL in orthodontics.

Methods

The samples of human incisors were cut into three slices. Uniaxial tensile tests were conducted under different loading rates. The transverse sections (cervical, middle and apex) normal to the longitudinal axis of the root of the tooth were used in the uniaxial tensile tests. Based on a bilinear simplification of the stress–strain relations, the elastic modulus of the PDL was calculated. The values of the elastic modulus in different regions were compared to explore the factors that influence the mechanical behavior of the periodontal ligament.

Results

The obtained stress–strain curves of the human PDL were characterized by a bilinear model with two moduli (E₁ and E₂) for quantifying the elastic behavior of the PDL from the central and lateral incisors. Statistically significant differences of the elastic modulus were observed in the cases of 1, 3, and 5 N loading levels for the different teeth (central and lateral incisors). The results showed that the mechanical property of the human incisors’ PDLs is dependent on the location of PDL (ANOVA, P?=?0.022, P?<?0.05). The elastic moduli at the middle planes were greater than at the cervical and apical planes. However, at the cervical, middle, and apical planes, the elastic moduli of the mesial and distal site were not significantly different (ANOVA, P?=?0.804, P?>?0.05).

Conclusions

The values of elastic modulus were determined in the range between 0.607 and 4.274 MPa under loads ranging from 1 to 5 N. The elastic behavior of the PDL is influenced by the loading rate, tooth type, root level, and individual variation.

     

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial–endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.     

The in vitro life-span of human periodontal ligament fibroblasts

The in vitro life-span of human periodontal ligament fibroblasts (PDLF) was studied on clones from periodontium of teeth extracted due to periodontitis and dental caries (69 clones/192 individuals, aged 20-80 years) and from periodontium of teeth extracted for orthodontic reasons (23 clones/26 individuals, aged 15-19 years). In the primary cultures the ratio of the number of cells expressing senescence-associated beta-galactosidase (SA-beta-Gal) to the total number of cells is significantly larger in PDLF (92 clones; 11.1+/-4.9%) than in human gingival fibroblasts (GF) (10 clones; 0.5+/-0.1 %). The finite population doubling numbers (PD) of PDLF are not age-matched and the mean PD of PDLF (7.1+/-2.9) is significantly smaller than GF (28.5+/-3.2), IMR-90 (human lung fibroblasts, 5 clones; 44.3 +/- 2.2), and human osteoblasts (5 clones; 19.7+/-1.4). Comparing the ratio of the number of SA-beta-Gal positive cells to the total number of cells in primary culture, and the finite PD in PDLF cultures: 1) the ratio of 15-19 years old donor group is significantly smaller than in the other donor groups (20-29, 30-39, 40-49, 50-59 and 60-80 years old), and 2) there were no statistically significant differences among the 20-29, 30-39, 40-49 and 50-59 year old donor groups, and the 30-39, 40-49, 50-59 and 60-80 year old donor groups. These findings suggest that the in vitro life-span of PDLF is shorter than other fibroblasts in the connective tissues and that PDLF may undergo senescence in adult clones without relation to donor's age. There may be more aged fibroblasts in periodontium than in other tissues, such as gingiva and lung.     

Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.     

Influence of cryopreservation on human periodontal ligament cells in vitro

Cryopreservation of teeth before autotransplantation may create new possibilities in dentistry. The purpose of this study was to examine the effect of a standardised cryopreservation procedure on human periodontal ligament (PDL) cell cultures. Human PDL fibroblasts obtained from immature third molars of 11 patients were cultured and divided into two groups. The experimental group was cryopreserved and cultured after thawing. The control group was cultured without cryopreservation. A comparison was made between cryopreserved and control cells. To evaluate possible differences in the characteristics of the fibroblasts, the cells in both groups were tested for viability (membrane integrity), growth capacity and alkaline phosphatase (ALP) expression. The Wilcoxon test for paired comparison between cryopreserved and non-cryopreserved cells was performed for each characteristic. The results showed that membrane integrity of cells was not influenced by cryopreservation. There was no statistically significant difference in growth capacity between cryopreserved and control cells. Non-cryopreserved cells were slightly stronger positive for ALP, but the difference was not statistically significant. From these experiments it can be concluded that the observed parameters are not influenced by cryopreservation.     

Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non‐functional or orthodontic reason and differentiated them towards CSK phenotype using a two‐step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL‐differentiated CSK‐like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14‐day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.     

The response of the human periodontal ligament to torsional loading—I. Experimental methods

A test technique is described in which torsional loads are applied to human maxillary central incisors in vivo. The central axis of the incisor is located by a stereoscopic X-ray method and the tooth loading device is adjusted by means of a setting jig so that torque is applied about this central axis. The maximum torque which can be applied is ± 0·05 Nm and the maximum rotation of the tooth is ± 0·02 radian (± 1·2). A servo-control system allows one to apply any desired torque or deflection history. e.g. a creep or stress relaxation test or a cyclic load of any waveform.

Initial results obtained with this system are presented and show that the torque vs rotation response is initially linear but becomes highly nonlinear at higher torques. Creep tests and cyclic loading tests indicate that the periodontal ligament is viscoelastic in nature. Periods of cyclic loading separated by rest periods of 1–20 min show that the commonly observed decrease in tissue stiffness during the initial few load cycles is not a permanent effect in this in vivo test. Recovery to the initial stiffness takes place rapidly. e.g. 50 per cent recovery in 5 min at zero load.     

Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study

Periodontal-ligament-derived cells (PDL cells) have stem-cell-like properties and, when implanted into periodontal defects in vivo, can induce periodontal regeneration including the formation of new bone, cementum, and periodontal ligament. We have previously demonstrated that PDL cell sheets, harvested from temperature-responsive cell culture dishes, have a great potential for periodontal regeneration. The purpose of this study has been to validate the safety and efficacy of human PDL (hPDL) cell sheets for use in clinical trials. hPDL tissues from three donors were enzymatically digested, and the obtained cells were cultured with media containing autologous serum in a cell-processing center (CPC). The safety and efficacy of hPDL cell sheets were evaluated both in vitro and in vivo. In vitro studies showed that the hPDL cell sheets had high alkaline phosphatase activity and periostin expression (known PDL markers) and no contamination with microorganisms. In vivo studies revealed that hPDL cell sheets, implanted with dentin blocks, induced the formation of cementum and PDL-like tissue in immunodeficient mice. The hPDL cells presented no evidence of malignant transformation. Thus, hPDL cell sheets created in CPCs are safe products and possess the potential to regenerate periodontal tissues.