Location of putative stem cells in human periodontal ligament

BACKGROUND AND OBJECTIVE: The origin of cells in the mature periodontium, and the location of their progenitors, are still unknown. It is also unknown whether inflammation influences the number and distribution of these cells within the periodontium. Molecules such as STRO-1, CD146 and CD44 have been identified on a variety of mesenchymal stem cells. The aim of this study was to identify and localize putative stem cells in diseased and healthy human periodontal ligament using cell-surface markers for mesenchymal stem cells. MATERIAL AND METHODS: Healthy and periodontitis-affected teeth were collected, fixed in 10% neutral-buffered formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry. Antibodies against STRO-1, CD146 and CD44 were used to identify putative stem cells in the periodontal ligament. RESULTS: Putative stem cells were identified in both healthy and diseased periodontal ligament. They were mainly located in the paravascular region and small clusters of cells were also found in the extravascular region. Wider distributions of these cells were detected in sections of diseased ligament. CONCLUSION: Within the periodontal ligament of both healthy and diseased teeth, cells have been identified consistent with their identification as putative stem cells. The presence of an inflammatory reaction associated with periodontitis may enhance the number of these cells.     

Identification of multipotent stem cells from adult dog periodontal ligament

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease.     

High numbers of T cells in gingiva from patients with human immunodeficiency virus (HIV) infection

A quantitative, immunohistologic evaluation of CD3⁺, CD4⁺and CD8⁺ cells was carried out on gingival biopsies from 25 HIV-infected persons with gingivitis or periodontitis and 13 HIV-seronegative persons with periodontitis. CD3⁺ T cells were found in all biopsies. CD8⁺ cells were significantly more numerous and the CD4⁺/CD8⁺ ratio was significantly decreased in the gingival connective tissue of the HIV⁺ patients (P < 0.05). The number of CD4⁺ lymphocytes subjacent to the pocket epithelium was moderately lower in the HIVH patients as compared to the HIV⁺ patients (P < 0.05). HIV⁺ patients with a history of necrotizing periodontal disease had fewer CD4⁺ cells subjacent to the oral gingival epithelium than patients without such disease (P < 0.05). The general HIV-related changes in T lymphocyte numbers were therefore reflected in inflamed gingival tissues. HIV⁺ patients had, however, significantly higher CD4⁺/CD8⁺ ratios in gingiva than in peripheral blood (P < 0.05), indicating that CD4⁺ T cells are actively recruited to gingiva, even in cases of extreme CD4⁺ T lymphocytopenia.     

The changes in the T-lymphocyte subsets in a population of Turkish children with puberty gingivitis

Objective.   The aim of the study was to investigate the number of CD4 and CD8 T lymphocytes, analyse subjects with gingivitis and those without, and determine the role of T lymphocytes in the pathobiology of puberty gingivitis.
Material and methods.   Fifty individuals with and without puberty gingivitis were recruited for this study. The CD4⁺ and CD8⁺ T-lymphocyte counts were determined using flow cytometry on the biopsy samples, and the CD4⁺/CD8⁺ ratio was calculated. At the same time, periodontal index scores were recorded to assess the periodontal status. Acquired data were analysed statistically using a paired t -test to compare laboratory values obtained before and after the treatment in individuals with puberty gingivitis and disease-free individuals. In addition, Pearson's correlation analysis was performed to investigate the relation between laboratory values and clinical measurements.
Results.   The CD4⁺/CD8 ratio in gingival tissues obtained from test group was significantly higher ( P <  0.05) than that found in the gingival tissue obtained from control group. We found that the CD4⁺ and CD8⁺ lymphocyte counts continued to increase significantly ( P <  0.001) and the CD4⁺/CD8⁺ ratio continued to drop significantly ( P <  0.05) after treatment in test group.
Conclusions.   T lymphocytes could play a significant role in the pathobiology of puberty gingivitis     

Immunocompetent cells in rat periodontal ligament and their recruitment incident to experimental orthodontic tooth movement

The aims of this study were to evaluate the number and distribution of immunocompetent cells in normal rat periodontal ligament (PDL) and to quantify their recruitment incident to experimental tooth movement. 27 young animals had the 1st right maxillary molar moved mesially by an orthodontic appliance for 1, 3, 7 and 14 days, respectively. 4 animals served as untreated controls. An immunohistochemical procedure was carried out on alternate serial cryostat sections, and monoclonal antibodies against CD1 II (macrophages, dendritic cells), CD43⁺ (lymphocytes, polymorphs), CD4 (helper T-lymphocytes), and class II MHC molecules were used. Mean counts of the immunolabeled cells in the control group showed the highest number of GDI lb⁺ and class II molecule expressing cells, while CD4⁺ and CD43⁺ cells were scarcely found. Significant increase in the number of CD1 lb⁺, CD43⁺ cells and class II molecules was found in the PDL of the experimentally moved 1st molars compared with the contralateral side and the control group, while CD4⁺ cells showed no significant increase. CD11b⁺ and cells expressing class II molecules were found around hyalinized tissue, between dentin and cellular cementum and close to Malassez'epithelial cells. In conclusion, normal rat PDL has high number of macrophage and dendritic-like cells, but few lymphocytes and granulocytes. Furthermore, experimental tooth movement leads to significant recruitment of cells belonging to the mononuclear phagocytic system, but has no significant effect on the number of lymphocytes and granulocytes in the rat PDL.     

人乳牙牙周膜干细胞的生物学特性研究

目的:体外分离纯化人乳牙牙周膜组织来源的干细胞,研究其生物学特性、表面标记物及多向分化能力。方法:用酶消化法和有限稀释法对正常人乳牙牙周膜组织进行原代培养,并通过免疫细胞化学方法和流式细胞仪对其来源进行鉴定,进而从增殖能力、克隆形成能力和多向分化能力等方面对乳牙牙周膜干细胞的生物学特性进行研究。结果:分离培养获得的人乳牙牙周膜干细胞在形态上与恒牙牙周膜干细胞相似,均为长梭形成纤维细胞样;免疫细胞化学和流式细胞仪分子表型鉴定显示乳牙牙周膜干细胞阳性表达间充质来源的表面标志STRO-1,CD146,CD29和CD90,造血系来源的标志物CD34为阴性;细胞周期,MTT和克隆形成率的检测结果显示,人乳牙牙周膜干细胞的增殖能力强于恒牙牙周膜干细胞;人乳牙牙周膜干细胞在体外诱导条件下可以向骨,脂肪细胞方向分化;同时RT-PCR检测发现,矿化诱导后细胞成骨相关基因(ALP,OCN,Col I)的表达上调,成脂诱导后细胞成脂相关基因(PPAR-γ,C/EBPα)的表达上调。结论:人乳牙牙周膜中确实存在间充质来源的干细胞,并且具有较强的增殖能力和多向分化能力,为牙周组织工程种子细胞提供了新的可能来源。     

Human cytomegalovirus in peripheral giant cell granuloma

Background:  The peripheral giant cell granuloma is a relatively common non-neoplastic inflammatory lesion of gingiva, but the etiopathogeny remains unknown. This study aimed to evaluate the importance of human cytomegalovirus and Epstein–Barr virus in a peripheral giant cell granuloma of a 47-year-old female.
Methods:  The lesion was studied clinically, histopathologically, immunologically and virologically using established procedures.
Results:  The gingival growth was located at the mesial surface of the maxillary left canine having a vital pulp. The mass was 12 × 21 mm in size and exhibited a smooth surface with no evidence of fluctuation on palpation. An excisional biopsy revealed giant cells in a fibrohistiocytic stroma with areas of haemorrhage. Serum protein levels and lymphocyte subsets were within normal limits, except CD3⁺ and CD4⁺ cells were below normal ranges. Polymorphonuclear leukocytes expressed p150,95 (CD11c/CD18) and CXCR-2 receptors within normal ranges, but the CXCR1 receptor showed decreased density, and CD15 were below normal range. A virological sample of the tooth surface adjacent to the gingival swelling yielded 7.6 × 10³ copy-counts of cytomegalovirus and 4.3 × 10³ copy-counts of Epstein–Barr virus.
Conclusions:  The clinical and histological findings were consistent with the diagnosis of peripheral giant cell granuloma. Cytomegalovirus has the potential to induce multinucleated giant cells, and the possibility that the virus contribute to the development of peripheral giant cell granuloma merits further study.     

Stem cell properties of human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. MATERIAL AND METHODS: PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. RESULTS: Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. CONCLUSION: The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.     

Serum sCD14, polymorphism of CD14−260 and periodontal infection

Background and Aims:  CD14 is a co-receptor involved in the recognition of Gram-negative and positive bacteria. Infections are known to influence serum sCD14 levels, and CD14 gene promoter polymorphism (CD14 C−260T) has been reported to be associated with many infectious diseases. Our aim was to investigate whether serum sCD14 concentration is associated with periodontal infection and the CD14⁻²⁶⁰ genotype.
Subjects and Methods:  The periodontal status of 56 subjects with chronic periodontitis and 28 controls was clinically examined. Serum sCD14 concentration was analyzed using ELISA and CD14⁻²⁶⁰ genotype using polymerase chain reaction (PCR).
Results:  The mean concentration of sCD14 in serum was significantly higher in subjects with periodontitis than in control subjects (4.9  μ g ml⁻¹ vs 3.8  μ g ml⁻¹, P  < 0.001). Serum sCD14 concentration associated significantly with the extent of advanced periodontal disease. In a regression analysis including both subject groups, the CD14⁻²⁶⁰ genotype was a significant determinant for serum sCD14 concentration. After stratification by periodontal health status (periodontitis vs controls), the influence of the CD14⁻²⁶⁰ genotype on serum sCD14 concentration was seen only in the control group.
Conclusions:  Periodontal infection is associated with the serum concentration of sCD14. Moderate to severe periodontal infection overshadows the influence of the genotype on serum sCD14 concentration.     

大鼠正畸牙移动中牙周组织内STRO-1动态变化的研究

目的:通过观察大鼠正畸牙移动中牙周组织内STRO-1的动态变化,研究大鼠牙周膜干细胞(periodontal ligament stem cell,PDLSC)在正畸牙周组织改建中的作用及表达变化的规律.方法:选用幼年SD大鼠,建立正畸牙移动的模型,分别在加力后1、3、5、7、10、14 d处死动物,制备标本.应用EnVision系统两步法和图像分析进行半定量分析.结果:正常大鼠牙周组织中STRO-1表达较低.STRO-1在加力组牙周膜中的表达与对照组比较,差异有显著性,在正畸牙移动过程中STRO-1阳性显色细胞明显增多.结论:STRO-1的表达强度与牙周改建的活跃程度相关,揭示了在正畸力作用下牙周组织改建的可能机制和牙周膜干细胞的作用.     

Identification of marker genes distinguishing human periodontal ligament cells from human mesenchymal stem cells and human gingival fibroblasts

BACKGROUND AND OBJECTIVE: Molecular gene markers, which can distinguish human bone marrow mesenchymal stem cells from human fibroblasts, have recently been reported. Messenger RNA levels of tissue factor pathway inhibitor-2, major histocompatibility complex-DR-alpha, major histocompatibility complex-DR-beta, and neuroserpin are higher in human bone marrow mesenchymal stem cells than in human fibroblasts. However, human bone marrow mesenchymal stem cells express less apolipoprotein D mRNA than human fibroblasts. Periodontal ligament cells are a heterogeneous cell population including fibroblasts, mesenchymal stem cells, and progenitor cells of osteoblasts or cementoblasts. The use of molecular markers that distinguish human bone marrow mesenchymal stem cells from human fibroblasts may provide insight into the characteristics of human periodontal ligament cells. In this study, we compared the molecular markers of human periodontal ligament cells with those of human bone marrow mesenchymal stem cells and human gingival fibroblasts. MATERIAL AND METHODS: The mRNA expression of the molecular gene markers was analyzed using real-time polymerase chain reaction. Statistical differences were determined with the two-sided Mann-Whitney U-test. RESULTS: Messenger RNA levels of major histocompatibility complex-DR-alpha and major histocompatibility complex-DR-beta were lower and higher, respectively, in human periodontal ligament cells than in human bone marrow mesenchymal stem cells or human gingival fibroblasts. Human periodontal ligament cells showed the lowest apolipoprotein D mRNA levels among the three types of cells. CONCLUSION: Human periodontal ligament cells may be distinguished from human bone marrow mesenchymal stem cells and human gingival fibroblasts by the genes for apolipoprotein D, major histocompatibility complex-DR-alpha, and major histocompatibility complex-DR-beta.     

Inflammation, heat shock proteins and periodontal pathogens in atherosclerosis: an immunohistologic study

Background:  Inflammation is a significant component of atherosclerosis lesions. Bacteria, including periodontopathogens, have been demonstrated in atherosclerotic plaques and cross-reactivity of the immune response to bacterial GroEL with human heat shock protein 60 has been suggested as a link between infections and atherosclerosis.
Methods:  In this study, the nature of the inflammatory infiltrate and the presence of human heat shock protein 60 and GroEL were examined in 31 carotid endarterectomy specimens. Additionally, monoclonal antibodies were used to detect the presence of six bacteria, including those implicated in periodontal disease.
Results:  The inflammatory cell infiltrate of the lesions was dominated by CD14⁺ macrophages and CD4⁺ T cells. Most cells of the infiltrate as well as the endothelium were HLA-DR⁺, indicating activation; however, there was an absence of CD25 expression, demonstrating that the activated T cells were not proliferating. Few CD1a⁺ and CD83⁺ cells were noted. Human heat shock protein 60 expression was evident on endothelial cells and cells with the appearance of smooth muscle cells and lymphocytes. GroEL and bacteria were detected within intimal cells. Chlamydia pneumoniae, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Prevotella intermedia , and Actinobacillus actinomycetemcomitans were found in 21%, 52%, 34%, 34%, 41%, and 17% of arteries, respectively.
Conclusion:  These results give evidence for a specific immune response associated with atherosclerosis. Whether bacteria initiate the observed inflammation in atherosclerotic lesions is not clear; however, the present study shows that maintenance of inflammation may be enhanced by the presence of periodontopathic bacteria.     

Increased number of CD25+ FoxP3+ regulatory T cells in oral squamous cell carcinomas detected by chromogenic immunohistochemical double staining

Background:  The role of tumor-infiltrating regulatory T cells (Treg) compromising antitumor effects of immune cells in oral squamous cell carcinoma (OSCC) is largely unknown.
Purpose:  The presence of CD25⁺ FoxP3⁺ Treg as well as of CD3⁺ FoxP3⁺ and of CD8⁺ FoxP3⁺ tumor-infiltrating lymphocytes (TIL) was verified in OSCC and compared with non-cancerous lymphoepithelial tissue.
Method:  Three double stainings (CD3/FoxP3, CD8/FoxP3 and CD25/FoxP3) were performed on tissue sections of 15 OSCC and compared with 15 human tonsils.
Results:  OSCC biopsy samples provide evidence for a strong infiltration of TIL, in particular, naturally occurring CD25⁺ FoxP3⁺ Treg. Whereas a comparison of OSCC and control tissue did not show significant changes in the number of CD3⁺ FoxP3⁺ TIL and of CD8⁺ FoxP3⁺ TIL, a significantly higher frequency of CD25⁺ FoxP3⁺ TIL (Treg) could be observed in OSCC ( P  < 0.001, two-sided t -test). Given the small number of specimens, a significant correlation with tumor stage could not be verified.
Conclusion:  Chromogenic double staining of CD4/FoxP3 is a promising tool for the detection of Treg in paraffin-embedded tissue of OSCC.     

牙周膜干细胞的表型分析

目的:分析人牙周膜干细胞(PDLSCs)的表型特点,为分离、鉴定和应用PDLSCs提供有效途径.方法:采用免疫磁珠法分离PDLSCs,制备细胞爬片.分别采用免疫荧光、免疫细胞化学染色法检测PDLSCs表面蛋白STRO-1、CD44、Vimentin、CK、COL-Ⅰ、BSP的表达.收集PDLSCs,采用RT-PCR法检...     

Recovery of stem cells from cryopreserved periodontal ligament

Human post-natal stem cells possess a great potential to be utilized in stem-cell-mediated clinical therapies and tissue engineering. It is not known whether cryopreserved human tissues contain functional post-natal stem cells. In this study, we utilized human periodontal ligament to test the hypothesis that cryopreserved human periodontal ligament contains retrievable post-natal stem cells. These cryopreserved periodontal ligament stem cells maintained normal periodontal ligament stem cell characteristics, including expression of the mesenchymal stem cell surface molecule STRO-1, single-colony-strain generation, multipotential differentiation, cementum/periodontal-ligament-like tissue regeneration, and a normal diploid karyotype. Collectively, this study provides valuable evidence demonstrating a practical approach to the preservation of solid-frozen human tissues for subsequent post-natal stem cell isolation and tissue regeneration. The present study demonstrates that human post-natal stem cells can be recovered from cryopreserved human periodontal ligament, thereby providing a practical clinical approach for the utilization of frozen tissues for stem cell isolation.     

Periodontitis lesions are the main source of salivary cytomegalovirus

Background:  Herpesviruses play causal or cooperative roles in childhood infections, tumorigenesis, ulcerogenesis, and periodontitis. Saliva is a common vehicle of herpesvirus horizontal transmission, but the source of salivary herpesviruses remains obscure. To evaluate the significance of periodontal disease in shedding of oral herpesviruses, this study determined the genome-copy counts of human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV) in whole saliva of subjects with periodontitis, gingivitis, or no natural teeth.
Methods:  Whole saliva was collected from 14 periodontitis patients, 15 gingivitis patients and 13 complete denture wearers. The study subjects were systemically healthy and had not received periodontal treatment in the past 3 months. Real-time TaqMan polymerase chain reaction was used to determine the salivary load of HCMV and EBV.
Results:  Salivary HCMV was detected in seven (50%) periodontitis patients, but not in any gingivitis or edentulous subjects ( P  < 0.001). Salivary EBV was detected in 11 (79%) periodontitis patients, in five (33%) gingivitis patients, and in seven (54%) edentulous subjects ( P  = 0.076). Salivary samples showed copy counts of HCMV in the range of 3.3 × 10³–4.2 × 10⁴/ml and of EBV in the range of 3.6 × 10²–1.6 × 10⁹/ml.
Conclusions:  HCMV and EBV are commonly present in the saliva of periodontitis patients. Periodontitis lesions of systemically healthy subjects seem to constitute the main origin of salivary HCMV, but do not comprise the sole source of salivary EBV.     

Autoradiographic study of 3H-proline incorporation by rat periodontal ligament, gingival connective tissue and dental pulp

The rates of ³H-proline incorporation by the rat periodontal ligament, the gingival connective tissue and the dental pulp were studied by autoradiography. The rate of ³H-proline incorporation by the periodontal ligament was 2.8 times higher than by the gingival connective tissue and 5 times higher than by the dental pulp. These differences were significant (p > 0.01). The pattern of ³H-proline incorporation by the periodontal ligament was significantly different (p > 0.01) from that by the gingival connective tissue and the dental pulp. The latter two tissues exhibited a similar pattern of ³H-proline incorporation. The ratio of the rates of ³H-proline incorporation by the three tissues did not correlate with the ratio of the cellular densities in the same three tissues.     

The effect of platelet-rich plasma on osteoblast and periodontal ligament cell migration, proliferation and differentiation

Background and Objective:  Platelet-rich plasma is used to deliver growth factors, in a safe and convenient manner, for enhancing bone and periodontal regeneration. However, conflicting reports regarding its effectiveness suggest that further study of the relevant cellular mechanisms is required. The aim of this study was to investigate the in vitro effect of platelet-rich plasma on osteoblasts and periodontal ligament cell function.
Material and Methods:  Various concentrations of platelet-rich plasma (100, 50, 20 and 10%) and platelet-poor plasma, obtained from human donors, were applied to primary cultures of human osteoblasts and periodontal ligament cells. [³H]-Thymidine incorporation, crystal violet staining and MTT assays were utilized to assess DNA synthesis and proliferation. Migration was determined by assessing the cell response to a concentration gradient, while differentiation was assessed using Alazarin Red staining.
Results:  Platelet-rich plasma and platelet-poor plasma had stimulatory effects on the migration of both human osteoblasts and periodontal ligament cells. At 24 h, DNA synthesis was suppressed by the application of the various concentrations of platelet-rich plasma, but over a 5-d period, a beneficial effect on proliferation was observed, especially in response to 50% platelet-rich plasma. Platelet-poor plasma resulted in the greatest enhancement of cellular proliferation for both cell types. At a concentration of 50%, platelet-rich plasma and platelet-poor plasma facilitated differentiation of both cell types.
Conclusion:  Platelet-rich plasma can exert a positive effect on osteoblast and periodontal ligament cell function, but this effect is concentration specific with maximal concentrations not necessarily resulting in optimal outcomes. Platelet-poor plasma also appears to have the ability to promote wound healing-associated cell function.     

人牙周韧带细胞和牙髓细胞表型特征的比较

目的研究人牙周韧带细胞（PDLCs）和牙髓细胞（DPCs）表型特征以及体外传代对其表型特征的影响,为选择适宜的表面标记分选生物性状同源性的细胞亚群提供依据。方法采用酶消化法体外培养PDLCs和DPCs,免疫组化检测和流式细胞仪分析细胞表面标记的表达。结果PDLCs和DPCs的STRO-1和CD146免疫组化染色阳性。流式细胞仪分析显示：获得的第1代PDLCs和DPCs表达间充质干细胞表面标记STRO-1、CD146、CD29、CD44和CD106,基本不表达CD34。PDLCs的STRO-1、CD29和CD44阳性表达百分比与DPCs间的差异无统计学意义（P>0.05）,PDLCs的CD106阳性表达百分比高于DPCs,CD146阳性表达百分比低于DPCs,且差异有统计学意义（P<0.001）。PDLCs和DPCs表达STRO-1和CD146的阳性表达百分比随传代而逐渐减低。结论PDLCs表面抗原表达与DPCs相似,而且其中成体干细胞的成分随传代逐渐减少。