Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction

Background and Objective: Platelet‐derived growth factor‐BB is a potent mediator of tooth‐supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet‐derived growth factor‐BB application is its short half‐life in vivo. Gene therapy has shown strong promise for the long‐term delivery of platelet‐derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet‐derived growth factor‐B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad‐PDGF‐B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell‐surface proteins associated with cellular signaling. Material and Methods: HGFs from human subjects were treated by adenoviral PDGF‐B, PDGF‐1308 (a dominant negative mutant of PDGF) and recombinant human platelet‐derived growth factor‐BB, and then incubated in serum‐free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF‐B was measured by RT‐PCR and Western blot. Cell proliferation was evaluated by [methyl‐³H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF‐B gene transfer. The expression of α and β PDGFR and Akt were measured by Western blot analysis. Results: Sustained in vitro expression of PDGF‐B in HGFs by Ad‐PDGF‐B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF‐BB and Ad‐PDGF‐1308, Ad‐PDGF‐B maintained cell growth in serum‐free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF‐B also prolonged downregulation of the growth arrest specific gene (gas) PDGFαR. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long‐term cell signaling effect as a result of the over‐expression of Ad‐PDGF‐B, compared with pulse recombinant human platelet‐derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad‐PDGF‐B, for at least up to 96 h. Conclusion: These findings further demonstrate that gene delivery of PDGF‐B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet‐derived growth factor‐BB protein. These data on platelet‐derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.     

Type II collagen and aggrecan mRNA expressions in rabbit condyle following disc displacement

The aim of this investigation was to study the remodelling of cartilage in the mandibular condyle following disc displacement (DD) of the temporomandibular joint (TMJ). Forty adult Japanese white rabbits were used in this study. The right joints of 28 of the 40 rabbits had their discs surgically displaced. Four of the 28 were killed at 4 days or 1, 2, 4, 6, 8 and 12 weeks after surgery. The messenger RNA (mRNA) expression levels of aggrecan and type II collagen in cartilages were measured using in situ hybridization techniques. Results showed that aggrecan mRNA expression reduced in the first week after DD. The expression began to recover after 4 weeks and reached a normal level after 6 weeks. Type II collagen mRNA expression reduced from 4 weeks and the expression recovered after 8 weeks. This suggests that the chondrocyte reacting to the displacement of the TMJ disc, alters its matrix gene expression patterns and it is may be the cause of the shape changes of TMJ after DD.     

A comparative evaluation of different compensating curves in the lingual and labial techniques using 3D FEM.

Because adults dislike the visibility of orthodontic appliances, the use of the lingual orthodontic technique has increased over time. But few studies compare tooth movement of the lingual technique with that of the labial technique. In this study, human mandibular left teeth were aligned, and a 3-dimensional finite element model was made (consisting of 19382 nodes and 12150 elements). To compare the effect of compensating curves on canine retraction between the lingual and the labial orthodontic techniques, the compensating curve was increased on the.016-in stainless steel labial or lingual archwire, and a 150-g force was applied distally on the canine. The relative direction and the amount of tooth displacement of the finite element model were compared on a schematic displacement graph (magnified 10,000 times), and the compressive stress distributed on the root surface was observed. The pattern of tooth movement (with or without a compensating curve) was different between the labial and the lingual techniques. As the amount of compensating curve increased (0, 2, and 4 mm) in the archwire, the rotation and the distal tipping of the canine was reduced. The antitip and antirotation action of compensating curve on the canine retraction was greater in the labial archwire than in the lingual archwire.     

CSF-1对体外培养人牙囊细胞OPG表达的影响

目的研究集落刺激因子(CSF-1)对体外培养的人牙囊细胞骨保护素(OPG)表达的影响。方法原代培养人牙囊细胞,传代至第3代,制备细胞爬片,进行OPG免疫组化染色;3~6代人牙囊细胞长至80%时,用胰酶消化,离心,重悬,制成单细胞悬液,接种到细胞培养皿,细胞长满至80%时,培养基中加入25ng/ml的CSF-1,孵育0.5、1、3、6h,分别收集上清液,ELISA法检测OPG蛋白分泌量的变化,提取总RNA进行RT-PCR,检测OPG mRNA的表达变化。结果正常人牙囊细胞OPG蛋白表达阳性;25ng/ml的CSF-1可降低人牙囊细胞OPG的表达,共同孵育1h,OPG表达降至最低(P<0.05)。结论牙囊细胞表达OPG,同时在牙齿萌出的特定时期,CSF-1通过下调人牙囊细胞OPG,减弱对破骨细胞形成的抑制作用,促进破骨细胞分化成熟,调节牙齿萌出。     

Tooth loss in treated periodontitis patients responsible for their supportive care arrangements

AIM: To identify risk indicators associated with tooth loss and periodontitis in treated patients responsible for arranging supportive periodontal care (SPC). MATERIALS AND METHODS: Ninety-seven Chinese subjects (34-77 years) who showed favourable responses to periodontal therapy provided in a teaching hospital 5-12 years previously were recalled. They were advised to seek regular SPC on discharge. Background information, general health status, smoking, oral hygiene habits, follow-up dental care, tooth loss, and periodontal parameters were investigated. Multiple regression analysis was performed. RESULTS: Two hundred and fifty-six teeth had been lost, 195 because of self-reported periodontal reasons. Up to 26.8% sites were with pockets > or =6 mm. Positive correlations were found between total/periodontal tooth loss and (i) smoking pack-years, (ii) time spent on oral hygiene, (iii) years since therapy's conclusion, (iv) age, and negative correlations with (v) inter-dental brush use, and (vi) education levels. Tooth loss by arch was correlated with wearing of removable partial denture in that arch. Percentage sites with pockets > or =6 mm were significantly negatively correlated with percentage sites without bleeding on probing. CONCLUSIONS: Smokers, more elderly patients, removable partial denture wearers, and patients with lower education levels or not using inter-dental brushes ought to be targeted for clinic-based SPC.     

mcpr1基因的真核表达载体的构建与鉴定

目的 :构建腭裂相关基因mcpr1与pcDNA3 .1/V5 HisB融合的高效真核表达载体 ,为研究mcpr1基因的功能奠定基础。方法 :根据mcpr1基因的核苷酸序列 ,设计并合成引物 ,通过PCR方法 ,从包含有mcpr1基因全长的克隆载体T easy/mcpr1中 ,扩增出该基因外显子片段 ,将扩增产物连接到真核表达载体pcDNA3 .1/V5 HisB中。对该重组体进行PCR和酶切鉴定 ,以及测序验证。结果 :以重组体为模板扩增出40 0bp左右的特异性基因片段 ,与mcpr1基因片段大小一致 ,酶切鉴定也显示有 40 0bp左右的基因片断。测序结果显示与已知基因序列一致。结论 :成功构建mcpr1基因的真核表达载体 ,为下一步研究mcpr1基因的功能奠定了基础。     

下颌骨恶性血管内皮与外皮瘤的临床病理观察(附4例报告)

本文根据作者经治发生于下颌骨的4个恶性血管内皮与外皮瘤病例，结合文献复习，探讨其临床病理特点、诊断治疗与预后等问题。发现该病发生于颌骨者十分罕见，临床易发生误诊，因此，临床医生遇有颌骨肿块生长迅速，疼痛呈持续性且逐渐加重，X线片提示有溶骨性破坏，诊断也应考虑患本病的可能，尽早手术治疗，并于术中行冰冻活检协诊。一般认为本病预后差，但如组织学形态分化较好，治疗及时，切除范围适当，也可获得较佳疗效。     

Cloning of hamster osteopontin and expression distribution in normal tissues and experimentally induced oral squamous-cell carcinoma

Osteopontin (OPN) is a non-collagenous extracellular matrix (ECM) protein expressed and secreted by several human cancers. This study investigated the expression pattern of OPN during development of oral squamous-cell carcinoma by using 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous-cell carcinomas in buccal pouch of syrian golden hamsters. We first identified the hamster OPN cDNA sequence by screening of a hamster calvariae cDNA library with a rat OPN cDNA probe. The resulting 1,449 bp of hamster OPN cDNA led to a deduced protein sequence of 305 amino acids containing several putative binding sites to integrins, CD44 receptors, calcium ions and hydroxyapatite, as well as multiple sites for phosphorylation, glycosylation and sulphation. Hamster OPN cDNA was then used as a probe to analyze the expression of OPN mRNA by Northern blot and in situ hybridization analyses of normal and malignant tissues. OPN mRNA was detected in several non-mineralized tissues as well as in mineralized tissues, but was not present in normal hamster buccal epithelium. DMBA-treated hamster buccal pouches expressed OPN mRNA as early as 4 weeks and displayed the highest level of expression at 15 weeks. The specimens treated with DMBA for 15 weeks exhibited histological features of squamous-cell carcinoma, presented microcrystalline deposits and showed OPN expression associated with malignant epithelium and tumor-associated macrophages. To summarize, our results suggest that buccal-pouch carcinogenesis of Syrian golden hamster may constitute an excellent experimental model to study the mechanisms by which OPN is associated with oral cancer pathogenesis, and to validate OPN-based therapeutic approaches to ameliorate oral cancer progression and metastasis.     

Soluble CD14 levels in gingival crevicular fluid of subjects with untreated adult periodontitis

BACKGROUND: This study determined soluble CD14 (sCD14) levels in gingival crevicular fluid (GCF) and their potential relationship to periodontal conditions in adult periodontitis. METHODS: GCF was collected from 15 patients with untreated adult periodontitis. sCD14 levels were determined by ELISA and presented as total amount (ng/site) and concentration (microg/ml). The periodontal examination consisted of plaque index (PI), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). PD and CAL were measured with an electronic probe. RESULTS: sCD14 was detected in all 15 subjects and was found in 59% (62/105) of the sampled sites. The percentage of sites with sCD14 varied greatly, ranging from 14% to 100%. The mean total amount of sCD14 was 1.71+/-0.40, range 0.03 to 5.41 ng/site; the concentration of sCD14 was 14.04+/-4.15, range 0.16 to 51.74 microg/ml. No significant difference in clinical data was found between the sites with and without detectable levels of sCD14. However, on the basis of the individual profile of sCD14 levels, i.e., those individuals with >50% of the sites containing sCD14 and mean levels of sCD14 >5.0 microg/ml, the 15 subjects were divided into a high sCD14 group (9 subjects) and a low sCD14 group (6 subjects). Compared to the high group, the low group showed greater mean PD and a higher percentage of sites with PD > or = 5.0 mm (P <0.05). Consistent with this, sCD14 concentrations showed a negative correlation with PD (r(s) = -0.636, P = 0.0174). CONCLUSIONS: The present study shows that sCD14 levels in GCF varied greatly among subjects with untreated adult periodontitis. Individuals with higher levels of sCD14 in GCF and more sites containing sCD14 had fewer deep pockets. The negative correlation between GCF sCD14 levels and probing depth implies a crucial role of sCD14 in bacterially induced periodontal destruction. The relationship between GCF sCD14 levels and probing depth warrants further investigations.