Detection of, and anti-collagen antibody produced by, CD5-positive B cells in inflamed gingival tissues

This study was performed to investigate the frequency and distribution of CD5-positive (CD5+) B cells in inflamed gingival tissues using flow cytometric and immunohistochemical analyses. The ability of CD5+ B cells to produce anti-type I collagen antibody was also examined. CD5+ B cells expressed "low" fluorescence intensity in the peripheral blood of both healthy subjects and patients with adult periodontitis. However, in inflamed gingival tissues the intensity of this surface marker was high. The percentage of B cells bearing CD5 surface marker was statistically higher in gingiva than in peripheral blood obtained from both the patients and healthy subjects. These CD5+ B cells were observed in gingival subepithelial connective tissues from the bottom to the middle of the periodontal pocket. This area showed destruction of collagen fibers and dense cell infiltrations. Anti-collagen IgG antibody level in patients' gingival crevicular fluids (GCF) was higher than that in sera from healthy subjects, and slightly higher than in autologous sera. IgM anti-collagen antibody in GCF was lower than in autologous sera and in sera from healthy subjects. EBV-transformed CD5+ B cells produced considerably more IgM and IgG antibody to collagen than CD5- B cells. Therefore CD5+ B cells may contribute to the pathogenesis of inflamed gingival tissues.     

血小板源性生长因子对人牙周韧带细胞在壳聚糖-磷酸三钙支架材料上附着和增殖的影响

目的:研究血小板源性生长因子(platelet-derived growth factor BB,PDGF-BB)对人牙周韧带细胞(human periodontal ligament cells,hPDLC)在壳聚糖-磷酸三钙(chitosan/tricalcium phosphate,CTCP)支架材料上附着和增殖的影响。方法:将一定体积的CTCP支架材料置96孔培养板内,取第5代hPDLC接种在材料表面。实验组加入含PDGF-BB的DMEM培养液,使得PDGF-BB终浓度分别为1ng/mL、10ng/mL和100ng/mL;对照组仅加入DMEM培养液。孵育24h及72h后分别对材料上的细胞计数;72h后对标本行扫描电镜观察。结果:与对照组相比,各浓度PDGF-BB组培养24h后均可促进hPDLC在CTCP支架材料上的附着,呈浓度依赖关系,差异具有统计学意义(P<0.05),其中100ng/mL为最有效浓度;同时培养72h后各浓度PDGF-BB组均能促进hPDLC在CTCP支架材料上的增殖,差异具有统计学意义(P<0.05);扫描电镜观察发现PDGF-BB组较对照组hPDLC在材料上附着数量增加,伸展更充分。结论:hPDLC在CTCP支架材料上的附着和增殖可被PDGF-BB所增强。     

骨组织工程基因治疗在颅颌面重建中的应用

骨组织工程中直接添加生长因子的方法存在着诸多不足,基因治疗和组织工程技术的结合,为克服这一难题提供了新的方向。本文回顾骨组织工程基因治疗的原理和特点,介绍了脂肪细胞作为骨组织工程靶细胞的研究现状,以及一种大动物模型报告基因的应用;并就颅颌面骨组织工程基因治疗中两种新的组织特异性成骨基因作一综述。     

Hyper-reactive mononuclear cells and neutrophils in chronic periodontitis

OBJECTIVES: Stimulated mono- and polymorphonuclear cells from patients with periodontitis have shown increased release of interleukin-1beta (IL-1beta) and oxygen radicals, respectively. The aim was to study whether this hyper-reactivity could be found both in mono- and polymorphonuclear cells from the same patient, and whether there was a relation to the gene coding for IL-1beta (IL-1beta(+3953)). MATERIAL AND METHODS: Peripheral mononuclear cells from 14 non-smoking and well-treated patients and pair-matched controls were incubated with opsonized Staphylococcus aureus and lipopolysaccharide (LPS). Released IL-1beta and tumour necrosis factor (TNF)-alpha were determined with ELISA. Generation of oxygen radicals from the Fcgamma-receptor-stimulated neutrophils was measured with chemiluminescence and the polymorphism at IL-1beta(+3953) was measured with polymerase chainreaction. RESULTS: The mononuclear cells from the patients released more IL-1beta after incubation with LPS (p<0.001) and with bacteria (p<0.05). The release of TNF-alpha tended to be higher in the patient group. The peripheral neutrophils from the patients generated more oxygen radicals (p<0.06). We found no differences between the study groups regarding the IL-1beta(+3953) polymorphism. CONCLUSION: The similarity in systemic inflammation between patients and controls suggests that the increased release/generation of IL-1beta and oxygen radicals from peripheral leukocytes in periodontitis patients is of a constitutional nature and of pathogenic relevance.     

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells

AIM: To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. METHODOLOGY: The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. RESULTS: The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). CONCLUSIONS: Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.     

Dlx1基因在大鼠外胚间充质细胞中表达的研究

目的 探讨Dlxl基因在牙胚发育过程中的调控作用。方法 采用外胚间充质细胞组织块培养法；地高辛标记cDNA原位杂交检测方法和斑点杂交方法，观察Dlxl基因在大鼠外胚间充质细胞，人牙乳头细胞，人牙髓细胞中的表达情况。结果 Dlxl基因在外胚间充质细胞和人牙乳头细胞中mRNA呈强阳性表达，而在牙髓细胞中弱表达。结论 Dlxl基因是牙胚发育过程中重要的调节因子，这种调控作用具有一定的时间性与空间性，它可能主要参与调控早期未分化细胞，而对终末分化细胞作用不显著。     

Effect of adoptive transfer of antigen-specific B cells on periodontal bone resorption

BACKGROUND AND OBJECTIVES: Host immune responses to periodontal pathogens have been considered to contribute to the alveolar bone destruction in periodontitis. However, the role of B lymphocytes in the pathogenesis of periodontal bone loss is not clear. METHODS: We examined the effect of adoptive transfer of antigen-specific B cells from rat spleens on experimental periodontal bone resorption. Donor rats were immunized intraperitoneally (i.p.) with formalin-killed Actinobacillus actinomycetemcomitans. Antigen-specific B cells were prepared from splenocytes by first binding CD43(+) cells to Petri dishes coated with anti-CD43 antibody to remove T cells, and non-binding cells were passed through a nylon wool column to deplete accessory cells. The retained cells were then collected and bound to A. actinomycetemcomitans-coated Petri dishes for enrichment of A. actinomycetemcomitans-binding B cells (AAB). A. actinomycetemcomitans non-binding B cells (ANB) and B cells from non-immunized donor rats (NIB) were also collected from these procedures. Each type of B cell was injected into a group of recipient rats that were then orally infected with live A. actinomycetemcomitans. RESULTS: At termination, the antibody levels to A. actinomycetemcomitans in serum and gingival wash fluids were significantly higher in the recipients transferred with AAB when compared to the recipients transferred with ANB or NIB. A markedly elevated number of antibody-forming cells were observed in the spleens of the recipients transferred with AAB, and these recipient rats also exhibited significantly increased bone resorption when compared to the other groups. CONCLUSIONS: It is suggested that B cells can contribute to periodontal bone resorption and that antigen-triggering of B cells is required for the bone resorption.     

SHED repair critical-size calvarial defects in mice

Objective:  Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.
Materials and methods:  We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.
Results:  SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor β receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.
Conclusions:  Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.     

The efficacy of mesenchymal stem cells to regenerate and repair dental structures

OBJECTIVES: Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. METHODS: Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. RESULTS: MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. CONCLUSION: Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.