Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Background: Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. Methods: PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte‐like functions of PDLSCs were examined in co‐culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Results: Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146‐ and NG2‐positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary‐like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary‐like structure. Conclusions: PDLSCs possess pericyte‐like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell–based periodontal therapy.     

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.     

Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC’s from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults’ extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).MethodsThe PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.ResultsThe cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.ConclusionsThe periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC’s derived from bone-marrow.     

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.     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

Doxycycline Counteracts Bone Morphogenic Protein 2–Induced Osteogenic Mediators

Background: Microbial colonization during wound healing may exaggerate the inflammatory response and could adversely affect the outcome of periodontal regeneration. Bone morphogenic proteins (BMPs) directly augment bone regeneration. Interestingly, inhibitors of tissue collagenases, such as sub‐antimicrobial–dose doxycycline, also indirectly promote hard‐tissue regeneration. In this study, it is hypothesized that BMP2‐mediated bone regeneration would be positively affected by simultaneous treatment of sub‐antimicrobial–dose doxycycline. Methods: Human periodontal ligament (PDL) cells were stimulated with: 1) 10 ng/mL BMP2; 2) 1 μg/mL doxycycline; or 3) a combination of the two. The expressions of alkaline phosphatase, osteocalcin, osteonectin, and osteopontin were analyzed along with in vitro mineralized nodule formation and calcium accumulation. Results: BMP2 was a potent inducer of osteocalcin/osteopontin (statistically significant at P <0.01) and osteonectin in PDL cells relative to stimulation with doxycycline. However, doxycycline relative to BMP2 (statistically significant at P <0.001) upregulated the expression of alkaline phosphatase and in vitro mineralized nodule formation. Contrary to expected results, combined BMP2 and doxycycline induced a statistically significant (P <0.001) downregulation of alkaline phosphatase, osteocalcin, osteonectin/osteopontin, and in vitro mineralized nodule formation compared to stimulation with either BMP2 or doxycycline alone. Conclusions: Combined treatment of BMP2 and doxycycline in PDL cells counteracts the osteogenic mediators. Molecular interaction of growth factors should be explored before using a combination of these biologic molecules. It is important and clinically relevant to determine whether tetracycline and its other derivatives also counteract BMP functions. Animal models should be used to confirm these in vitro results.     

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.     

Cytokine expression profile in the bone‐anchored hearing system: 12‐week results from a prospective randomized,controlled study

Objective

To study the effect of implanting the percutaneous bone‐anchored hearing system (BAHS) itself and inflammation of the peri‐abutment skin warrant clarification. In this study, we aimed to acquire further insight into the immune responses related to BAHS surgery and peri‐implant skin inflammation.

Materials and Methods

During surgery and 12 weeks post‐implantation, skin biopsies were obtained. If applicable, additional biopsies were taken during cases of inflammation. The mRNA expression of IL‐1β, IL‐6, IL‐8, TNFα, IL‐17, IL‐10, TGF‐ß, MIP‐1α, MMP‐9, TIMP‐1, COL1α1, VEGF‐A, FGF‐2 TLR‐2, and TLR‐4 was quantified using qRT‐PCR.

Results

Thirty‐five patients agreed to the surgery and 12‐week biopsy. Twenty‐two patients had mRNA of sufficient quality for analysis. Ten were fitted with a BAHS using the minimally invasive Ponto surgery technique. Twelve were fitted with a BAHS using the linear incision technique with soft‐tissue preservation. Five biopsies were obtained during episodes of inflammation. The post‐implantation mRNA expression of IL‐1β (P = .002), IL‐8 (P = .003), MMP9 (P = .005), TIMP‐1 (P = .002), and COL1α1 (P < .001) was significantly up‐regulated. IL‐6 (P = .009) and FGF‐2 (P = .004) mRNA expression was significantly down‐regulated after implantation. Within patients, no difference between post‐implantation mRNA expression (at 12 weeks) and when inflammation was observed. Between patients, the expression of IL‐1β (P = .015) and IL‐17 (P = .02) was higher during cases of inflammation compared with patients who had no inflammation at 12‐week follow‐up.

Conclusions

As part of a randomized, prospective, clinical trial, the present study reports the molecular profile of selected cytokines in the soft tissue around BAHS. Within the limit of this study, the results showed that 12 weeks after BAHS implantation the gene expression of some inflammatory cytokines (IL‐8 and IL‐1β) is still relatively high compared with the baseline, steady‐state, expression. The up‐regulation of anabolic (COL1α1) and tissue‐remodeling (MMP‐9 and TIMP1) genes indicates an ongoing remodeling process after 12 weeks of implantation. The results suggest that IL‐1β, IL‐17, and TNF‐α may be interesting markers associated with inflammation.     

Cells isolated from inflamed periapical tissue express mesenchymal stem cell markers and are highly osteogenic

Introduction

We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.

Methods

Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.

Results

Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.

Conclusions

Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.     

Gene profile in periodontal ligament cells and clones with enamel matrix proteins derivative

AIM: Evaluate enamel matrix proteins derivative effect on gene expression profiles in cultured human periodontal ligament cell population and its clones. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were explanted. Cell cloning was performed and clones classified into fibroblastic (FB) and mineralized tissue forming (MTF) according to their capacity to express alkaline phosphatase and form mineralized tissue. All cell cultures were grown for 7 days, with and without enamel proteins added to the medium. Following RNA extraction, expression profiling was performed by hybridization with a DNA micro-array. Selected genes differed from the control at a significant level smaller than p<0.01. RESULTS: Enamel proteins induced major qualitative changes in mRNA expression in all PDL cell populations, differently affecting the entire PDL cell population and its clones. In the entire PDL cell population, enamel proteins significantly enhanced PDL cell function, with a general effect on enhanced cell functional metabolism. CONCLUSIONS: Enamel proteins enhanced gene expression responsible for protein and mineralized tissue synthesis in the entire PDL population. In the MTF clones, nucleic acid metabolism, protein metabolism and signal transduction related genes were up-regulated, while in the FB clones, up-regulated genes were related to cell adhesion, nucleic acid metabolism and signal transduction.     

矿化胶原膜浸提液对MG-63人骨肉瘤细胞分化相关基因表达的影响

目的 研究新型矿化胶原膜对MG-63人骨肉瘤细胞(简称：MG-63细胞)成骨分化相关基因表达的影响。方法 将 MG-63细胞与新型矿化胶原膜浸提液(实验组)共培养,以市售的胶原膜(Bio-gide)浸提液作为同类产品对照组,以不加材料的细胞培养液为空白对照组,采用荧光实时定量 PCR法检测碱性磷酸酶(ALP)、I型胶原(COL I)、骨保护素(OPG)和骨钙素(OC)mRNA表达水平;采用SPSS 17.0 软件对数据进行统计学分析。结果 在ALP与OPG mRNA相对表达量检测中,3组成骨细胞的表达量差异无统计学意义(P>0.05);在COL I与 OC基因相对表达量检测中,14天时Bio-gide组和矿化胶原膜组表达均明显上调,与空白对照组相比,差异均具有统计学意义(P<0.05),而Bio-gide组和矿化胶原膜组相比,差异无统计学意义(P>0.05)。结论 矿化胶原膜和Bio-gide膜的浸提液均可上调MG-63细胞COL I和OC的基因表达,在一定程度上促进了成骨细胞的分化。