A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels.

Design

hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays.

Results

LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter.

Conclusions

Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. In this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation.     

Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro

Oral Diseases (2011) 17 , 785–793 Objective: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and epiregulin cooperatively participate in the wound‐healing process in the gingival epithelial and fibroblast cells of the oral mucosa. Material and Methods: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real‐time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. Results: The different regulation of expressions of HB‐EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB‐EGF exerted a cell migration‐inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB‐EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. Conclusion: These results indicated that both growth factors might function importantly in the wound‐healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.     

Helicobacter pylori and Campylobacter rectus share a common antigen

AIM: The aim of this study was to investigate the presence of antigens with immunological cross-reactivity in periodontopathogenic bacteria and Helicobacter pylori, the pathogen associated with gastritis and peptic ulcers in human. MATERIALS AND METHODS/RESULTS: Among the putative periodontopathogens tested (Actinobacillus actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola), cross-reactive bands were only detected in C. rectus by SDS-PAGE/Western immunoblotting analysis using a polyclonal antibody directed to H. pylori cells. One of these cross-reactive antigens, a 64-kDa band antigen, also reacted with a monoclonal antibody directed to the human heat shock protein (HSP) 60. The N-terminal amino acid sequence of this C. rectus protein revealed a high degree of homology with corresponding regions of other HSPs belonging to the HSP60 family, indicating that the 64-kDa antigen was a GroEL protein. The nucleotide sequence of the C. rectus GroEL protein coded for a 547 amino acid protein with a predicted size of 57.8 kDa. Comparison of the alignment of the deduced amino acid sequence of the GroEL protein of C. rectus with that of H. pylori showed a high degree of similarity throughout its length (76.8%). GroEL protein from C. rectus possessed the ability to stimulate production of IL-6 by a confluent monolayer of human gingival epithelial cells and was cytotoxic when used at a high concentration. CONCLUSIONS: This study reveals an immunological relationship between H. pylori and C. rectus, and clearly indicates that one of the shared antigens is a GroEL protein possessing a biological activity that might play a role in the initiation and progression of periodontal disease.     

Enamel matrix derivative alone or in combination with a bioactive glass in wide intrabony defects

This controlled clinical study investigated the clinical and radiographic outcome of wide intrabony periodontal defects treated by enamel matrix derivatives alone or in combination with a bioactive glass over a period of 8 months. Twenty-three chronic periodontitis patients, who received initial therapy and had radiographical interproximal defects with an associated probing depth of 6 mm or more and an intrabony component of at least 4 mm, were included. Each of the patients, contributing at least one intrabony defect, was treated with either enamel matrix derivative alone (group 1, n=10) or the combination (group 2, n=13). In both groups, all clinical and radiographical parameters were improved. Groups 1 and 2 presented a mean pocket reduction of 5.03±0.89 and 5.73±0.80 mm, recession of 0.97±0.24 and 0.56±0.18 mm, relative attachment gain of 4.06±1.06 and 5.17±0.85 mm, and radiographic bone gain of 2.15±0.42 and 2.76±0.69 mm, respectively. An intergroup comparison revealed significant differences for all of the parameters, yielding a more favorable outcome towards the combined approach. Within the limits of the study, both treatments resulted in marked clinical and radiographical improvements, but combined treatment seemed to enhance the results in the treatment of wide intrabony defects.     

煅石膏、骨形成蛋白及羟基磷灰石三元复合人工骨修复骨缺损的实验研究

以煅石膏(PLP）作为颗粒型羟基磷灰石(HA)人工骨粘接成形剂和骨形成蛋白(BMP)的载体，制成三元复合人工骨．分别用HA—bBMP、HA—PLP和单纯HA植入狗下颌骨实验性骨缺损中，采用组织学、定量组织学、免疫组织化学、X线摄片和扫描电镜观察的方法评价该复合人工骨的生物学性能。术后1，2，4，8和16周观察发现，HA—bBMP—PLP复合人工骨具有明显的骨诱导活性．PLP可充当BMP缓慢释放系统载体．增强BMP骨诱导活性，和作为颗粒型HA的粘接成形剂．使复合人工骨具有一定的可塑性和成形性，可达到准确的植入，植入后早期可有效防止HA颗粒移动。本研究证实．HA—bBMP—PLP三元复合人工骨不但可限制植入后HA颗粒的早期移动，更重要的是可以迅速增加新骨形成量．从形态和功能上大大提高了复合人工骨修复骨缺损的质量。     

骨形成蛋白家族与牵引成骨术

牵引成骨术(DO)中新骨的形成具有胚胎骨的发生和正常骨折愈合的某些特征,骨形成蛋白家族(BMPs)在其中扮演了重要角色。本文就BMPs与DO的生物学联系、作用调节机制、外源性BMPs的应用等方面作一综述。     

Use of a composite pedicled muscle flap and rhBMP-7 for mandibular reconstruction

The aim of this study was to investigate the feasibility of reconstructing critical size continuity osteoperiosteal defects of the mandible using a composite of recombinant BMP-7 contained in a bovine type-1 collagen carrier wrapped in a pedicled sterno-occipitalis muscle flap. At 3 months following surgery, bridging of the surgical defect was noted in three subjects (60%). Histologically, the induced bone regenerate showed maturation from woven to lamellar bone. Islands of cartilage were distributed throughout the defect. Replacement ossification of the degenerated muscle was a common feature in all specimens. Microradiography showed a gradual increase in the calcification of mineralized tissue from the margin to the centre of the newly generated bone. This research represents a proof of the concept that bone can be satisfactorily formed within a muscular scaffolding at the site of the created defect in a one-stage procedure.     

Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti-Porphyromonas gingivalis heat-shock protein 60 antisera

The present study has been performed to evaluate Porphyromonas gingivalis heat shock protein (HSP) 60 as a candidate vaccine to protect against multiple putative periodontopathic bacteria. Mouse anti-P. gingivalis HSP antisera demonstrated the elevated IgG antibody titers against the multiple bacteria tested and cross-reacted with heat-induced bacterial proteins of the target bacteria. The antisera also demonstrated a significantly higher opsonophagocytosis function against all the target bacteria than the control sera (P<0.01). We concluded that P. gingivalis HSP 60 could potentially be developed as a vaccine against multiple periodontopathic bacteria.     

Comparative immunohistochemical analysis of the peptidergic innervation of human primary and permanent tooth pulp

This immunohistochemical study sought to determine whether there are any differences in the peptidergic innervation of these pulps and whether dental caries is associated with changes in neuropeptide expression. Mandibular first permanent molars and second primary molars (n=120) were obtained from children requiring dental extractions under general anaesthesia. Extracted teeth were split longitudinally, placed in fixative, and categorized as intact, moderately carious or grossly carious. The coronal pulps were removed and 10-microm frozen sections were processed for indirect immunofluorescence. Double labelling employed combinations of the following antisera: (1) protein gene product 9.5, a general neuronal marker; (2) one of the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), enkephalin (ENK) and somatostatin (SOM). Image analysis was then used to determine the percentage area of immunostaining for each label within different anatomical regions of the coronal pulp. Sparse or absent immunoreactivity for GAL, ENK and SOM made analysis impossible. Analysis of CGRP, SP and VIP revealed significant interdentition differences, with their expression being significantly greater in permanent teeth, but this was not the case for NPY, with primary and permanent teeth demonstrating a similar amount of label for this peptide. Both dentitions showed significant increases in CGRP, SP, VIP and NPY expression with caries progression. These findings could have biological and clinical importance in connection with nociception, inflammation and healing.     

Detection of antigenic proteins in Porphyromonas gingivalis using two-dimensional electrophoresis and western blots

Using two-dimensional (2D) electrophoresis and western blot assay, we analyzed antigenic proteins in Porphyromonas gingivalis uniquely recognized by antibodies in sera of periodontitis subjects. Proteins in the total membrane fraction of P. gingivalis 381 were resolved into at least 70 protein spots by 2D electrophoresis. In the gel stained with silver, the substance around 47 kDa protein on the acidic side (at an isoelectric point of about 4.5) was stained as a smear. Antigenic substances were characterized using purified IgGs from sera of 16 adult periodontitis (AP), 19 rapidly progressive periodontitis (RPP) and 14 periodontally healthy volunteers. Western blots demonstrated that 75 kDa protein reacted with IgGs from 75% of AP patients (p>0.001), the antigenic substance around acidic 47 kDa protein reacted with IgGs from 81.3% of AP (p>0.01) and 68.4% of RPP patients (p>0.01) and the acidic 47 kDa protein reacted with 87.5% of AP (p>0.01) and 78.9% of RPP patients (p>0.01). The reaction frequency was significantly different from that of the healthy volunteers. Also 51 kDa and 41 kDa proteins reacted with 47 and 43 of 49 IgG samples, respectively. The substance around acidic 47 kDa protein reacted with mouse antiserum to P. gingivalis-LPS. After treatment with pronase or heat, the antigenic reactions disappeared not only from the proteins, but also from the area around the acidic 47 kDa protein. When the fraction was digested with lipase, the antigenic reaction of the area decreased. It appeared that the acidic 47 kDa protein and the LPS around it formed a complex, and that the LPS contained the lipid portion. These results suggested that the acidic 47 kDa protein can be classified among the lipid A-associated proteins, and that it is the major antigenic protein which is recognized by antibodies in sera of almost periodontal patients in P. gingivalis 381.