The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture

Saminathan A, Vinoth KJ, Wescott DC, Pinkerton MN, Milne TJ, Cao T, Meikle MC. The effect of cyclic mechanical strain on the expression of adhesion‐related genes by periodontal ligament cells in two‐dimensional culture. J Periodont Res 2012; 47: 212–221. © 2011 John Wiley & Sons A/S Background and Objective: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion‐related genes by human periodontal ligament cells. Material and Methods: Cultured periodontal ligament cells were subjected to a cyclic in‐plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6–24 h in a Flexercell FX‐4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase‐3 and ‐7 activity; and (iii) the expression of 84 genes encoding adhesion‐related molecules using real‐time RT‐PCR microarrays. Results: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase‐3 and ‐7 activity at 6 and 12 h. Seventy‐three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α‐chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76‐fold induction of SPP1 at 12 h to a 2.49‐fold downregulation of COL11A1 at 24 h. Conclusion: The study has identified several mechanoresponsive adhesion‐related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis ‘executioner’ caspases.     

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

Wada N, Wang B, Lin N‐H, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblasts. J Periodont Res 2011; 46: 438–447. © 2011 John Wiley & Sons A/S Background and Objective: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. Material and Methods: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c‐MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM‐2, TG30 and TRA‐1‐60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real‐time RT‐PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. Results: Human gingival fibroblast‐ and periodontal ligament fibroblast‐derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell‐associated cell‐surface antigens, SSEA3, SSEA4, GCTM‐2, TG30 (CD9) and Tra‐1‐60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. Conclusion: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.     

Heme oxygenase‐1 protects human periodontal ligament cells against substance P‐induced RANKL expression

Lee H‐J, Jeong G‐S, Pi S‐H, Lee S‐I, Bae W‐J, Kim S‐J, Lee S‐K, Kim E‐C. Heme oxygenase‐1 protects human periodontal ligament cells against substance P‐induced RANKL expression. J Periodont Res 2010; 45: 367–374. © 2010 John Wiley & Sons A/S Background and Objective: Although substance P (SP) stimulates bone resorption activity and this is reported to be correlated with the degree of periodontal inflammation, it is unclear how human periodontal ligament cells regulate neuropeptide‐induced osteoclastogenesis or the possible involvement of heme oxygenase‐1 (HO‐1) might be. This study examines how SP affects osteoprotegerin (OPG) and RANKL expression via HO‐1. Material and Methods: Using immortalized human periodontal ligament cells, the effects of SP on the expression of HO‐1, RANKL and OPG mRNA and proteins were determined by RT‐PCR and western blotting, respectively. Various concentrations of SP (10^?7, 10^?8, 10^?9 and 10^?10 m ) were added to the medium, and the cells were treated for 0, 0.25, 0.5, 1, 2 and 3 d. Results: Substance P upregulated RANKL and HO‐1 and downregulated OPG mRNA and protein expression in periodontal ligament cells, in a concentration‐ and time‐dependent manner. A HO‐1 inducer inhibited both the upregulation of RANKL expression and downregulation of OPG expression by SP in periodontal ligament cells. By contrast, treatment with a HO‐1 inhibitor or HO‐1 small interferring RNA (siRNA) enhanced SP‐stimulated RANKL expression. Inhibitors of ERK and p38 MAP kinases, phosphoinositide 3‐kinase and nuclear factor‐κB blocked the effects of SP on RANKL expression in periodontal ligament cells. Conclusion: These results suggest that SP stimulates osteoclastic differentiation by increasing the expression of RANKL vs. OPG via the HO‐1 pathway in periodontal ligament cells. The HO‐1 pathway may be an effective therapeutic target for inhibiting chronic periodontitis involving alveolar bone resorption.     

Lactone form 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) stimulate the osteoblastic differentiation of mouse periodontal ligament cells via the ERK pathway

Kim IS, Jeong BC, Kim OS, Kim YJ, Lee SE, Lee KN, Koh JT, Chung HJ. Lactone form 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase inhibitors (statins) stimulate the osteoblastic differentiation of mouse periodontal ligament cells via the ERK pathway. J Periodont Res 2011; 46: 204–213. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies reported that the lactone forms of 3‐hydroxy‐ 3‐methylglutaryl‐coenzyme A reductase inhibitors, which are also known as statins, have a bone stimulatory effect. However, there are few reports on the effect of statins on periodontal ligament cells. This study examined the statin‐induced osteoblastic differentiation of mouse periodontal ligament cells as well as its mechanism. Material and Methods: Mouse periodontal ligament cells were cultured with lovastatin or simvastatin, and their viability was measured. The levels of alkaline phosphatase (ALP), osteocalcin, bone sialoprotein and bone morphogenetic protein‐2 mRNA expression were evaluated by RT‐PCR. The osteoblastic differentiation was characterized by the ALP activity and Alizarin Red‐S staining for calcium deposition. The activity of the osteocalcin gene (OG2) and synthetic osteoblast‐specific elements (6×OSE) promoter with statins was also measured using a luciferase assay. For the signal mechanism of statins, the ERK1/2 MAPK activity was determined by western blot analysis. Results: A statin treatment at concentrations < 1 μm did not affect the cell viability. Lovastatin or simvastatin at 0.1 μm increased the levels of ALP, osteocalcin, bone sialoprotein and bone morphogenetic protein‐2 mRNA in mouse periodontal ligament cells. In addition, the ALP activity, mineralized nodule formation and OG2 and OSE promoter activity were higher in the lovastatin‐ or simvastatin‐treated cells than the control cells. Western blot analysis confirmed that the statins stimulated the phosphorylation of ERK1/2. Conclusion: Lovastatin and simvastatin may stimulate the osteoblastic differentiation of periodontal ligament cells via the ERK1/2 pathway. This suggests that the statins may be useful for regenerating periodontal hard tissue.     

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor–Associated Genes in PDLSCs

Background: This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor–associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs’ identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor–associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. Results: ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin‐2, bone morphogenetic protein‐10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain‐derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor–associated genes screened, 35 showed high cycle threshold values (≥35). Conclusions: ASA modulates the expression of growth factor–associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell‐surface components through the CD14/Toll‐like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin‐8 (IL‐8) responses of the cells to Salmonella lipopolysaccharide, a water‐soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll‐like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR‐related molecules, i.e. TLR2, TLR4, MD‐2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL‐8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL‐8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2‐ and TLR4‐independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti‐CD14 MAb.     

External apical root resorption concurrent with orthodontic forces: the genetic influence

Root resorption is a pathological process of multifactorial origin related to the permanent loss of dental root structure in response to a mechanical, inflammatory, autoimmune or infectious stimulus. External apical root resorption (EARR) is a frequent clinical complication secondary to orthodontic tooth movement; apart from variables related to treatment, environmental factors and/or interindividual genetic variations can confer susceptibility or resistance to its occurrence. In this context, genetic predisposition has been described as an etiological factor, together with mechanical factors derived from orthodontic treatment. In recent years, international research groups have determined the degree of influence of some genetic biomarkers in defining increased/reduced susceptibility to postorthodontic EARR. The influences of the IL1 gene cluster (IL1B, IL1A, IL1RN, IL6), P2RX7, CASP1, OPG (TNFRSF11B), RANK (TNFRSF11A), Osteopontin (OPN), TNFα, the vitamin D receptor (TaqI), TNSALP and IRAK1 have been analyzed. The objective of the present review study was to compile and analyze the latest information about the genetic background predisposing to EARR during orthodontic treatment. Genetics-based studies along with other basic science research in the field might help to clarify the exact nature of EARR, the influence of genetic inheritance and possibly lead to the prevention or even eradication of this phenomenon during orthodontic treatment.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.     

Correction of hypophosphatasia-associated mineralization deficiencies in vitro by phosphate/pyrophosphate modulation in periodontal ligament cells

Background: Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non‐specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP_i) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P_i) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP‐diagnosed patients, by correcting the P_i/PP_i ratio and modulating expression of genes involved with P_i/PP_i metabolism. Methods: Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP‐associated PDL/tooth root deficiencies. Constitutive expression of PP_i‐associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P_i was provided to correct the P_i/PP_i ratio. Results: PDL tissues obtained from healthy individuals featured higher basal expression of key PP_i regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP‐PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P_i. Dysregulated expression of PP_i regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P_i, although other matrix markers evaluated in our study remained downregulated. Conclusion: These findings underscore the importance of controlling the P_i/PP_i ratio toward development of a functional periodontal apparatus and support P_i/PP_i imbalance as the etiology of HPP‐associated cementum defects.     

Spheroid formation and stemness preservation of human periodontal ligament cells on chitosan films

Objective

The therapeutic potential of periodontal ligament cells for periodontal regeneration gradually decreases when cultured as a monolayer in vitro. Three‐dimensional cell culture models provide an alternative to traditional monolayer cell culture. This study aimed to comparatively evaluate the influence of spheroid culture on periodontal ligament cells.

Materials and Methods

Chitosan films were used to culture three‐dimensional periodontal ligament cell spheroids. The proliferation, self‐renewal, and osteogenic capacity of periodontal ligament cells derived from spheroids were evaluated and compared with cells cultured on a monolayer.

Results

Viable spheroids of periodontal ligament cells were formed on chitosan films. Compared to monolayer cell culture, periodontal ligament cells exhibited decreased proliferation upon spheroid formation. In contrast, their expression of genes related to self‐renewal was significantly higher comparison with cells cultured in a monolayer. Moreover, the formation of periodontal ligament cell spheroids increased their colony‐forming unit ability and osteogenic differentiation capacity.

Conclusion

The results demonstrate the successful use of chitosan films for the culture of periodontal ligament cell spheroids. Compared to cells cultured in monolayer, periodontal ligament cells in spheroids did not proliferate, but exhibited higher self‐renewal gene expression, colony‐forming unit and osteogenic capacity.