Expression of Evc2 in craniofacial tissues and craniofacial bone defects in Evc2 knockout mouse

ObjectiveOur objectives were to determine the expression of EVC2 in craniofacial tissues and investigate the effect of Evc2 deficiency on craniofacial bones using Evc2 knockout (KO) mouse model.DesignEvc2 KO mice were generated by introducing a premature stop codon followed by the Internal Ribosomal Entry Site fused to β-galactosidase (LacZ). Samples from wild-type (WT), heterozygous (Het) and homozygous Evc2 KO mice were prepared. LacZ staining and immunohistochemistry (IHC) with anti-β-galactosidase, anti-EVC2 and anti-SOX9 antibodies were performed. The craniofacial bones were stained with alcian blue and alizarin red.ResultsThe LacZ activity in KO was mainly observed in the anterior parts of viscerocranium. The Evc2-expressing cells were identified in many cartilageous regions by IHC with anti-β-galactosidase antibody in KO and Het embryos. The endogenous EVC2 protein was observed in these areas in WT embryos. Double labeling with anti-SOX9 antibody showed that these cells were mainly chondrocytes. At adult stages, the expression of EVC2 was found in chondrocytes of nasal bones and spheno-occipital synchondrosis, and osteocytes and endothelial-like cells of the premaxilla and mandible. The skeletal double staining demonstrated that craniofacial bones, where the expression of EVC2 was observed, in KO had the morphological defects as compared to WT.ConclusionTo our knowledge, our study was the first to identify the types of Evc2-expressing cells in craniofacial tissues. Consistent with the expression pattern, abnormal craniofacial bone morphology was found in the Evc2 KO mice, suggesting that EVC2 may be important during craniofacial growth and development.     

Tissue inhibitor of metalloproteinase-2 inhibits ameloblastoma growth in a new mouse xenograft disease model

J Oral Pathol Med (2010) 39 : 94–102
Background:  Ameloblastomas are odontogenic neoplasms characterized by local invasiveness. This study was conducted to develop a new animal model of ameloblastoma and to address the role of tissue inhibitor of metalloproteinase-2 (TIMP-2) and matrix metalloproteinase-2 (MMP-2) in the growth and invasiveness of ameloblastomas.
Method:  Donated fresh human ameloblastoma tissue was finely minced, screened, and subcutaneously implanted in three locations on each of 10 BALB/c-nu/nu nude mice. Newly established tumors on each mouse were injected with: (i) transfection reagent; (ii) liposome and transfection reagent; or (iii) liposome, transfection reagent, and the expression plasmid pcDNA3.1(+)/green fluorescent protein (GFP)-TIMP-2. Tumors were monitored for 5 weeks and excised for histopathology, RNA, and protein analyses.
Results:  The ameloblastoma xenografts were established with high frequency and contained a variety of typical features, validating this new model system. Xenografts injected with the TIMP-2 expression plasmid showed reduced growth, increased TIMP-2 mRNA and protein, and decreased MMP-2 protein compared with the control groups.
Conclusions:  We successfully established a new experimental model of ameloblastoma consisting of subcutaneous human xenografts in nude mice. In addition, we demonstrated the successful introduction of the TIMP-2 gene in tumor xenograft cells in vivo , resulting in xenograft growth inhibition. This growth inhibition may have resulted from TIMP-2 overexpression specifically inhibiting MMP-2 protein expression and activity.     

肿瘤相关巨噬细胞和MMP-2在成釉细胞瘤中的表达及意义

目的：探讨肿瘤相关巨噬细胞（ TAMs）和基质金属蛋白酶?2（ MMP?2）在人成釉细胞瘤（ ABs）中的表达及意义。方法：采用免疫组化通用二步法,分别检测43例ABs和10例正常口腔黏膜中CD68标记的巨噬细胞数目及MMP?2的表达情况。结果：TAMs和MMP?2在ABs中的表达均高于正常黏膜,差异有统计学意义（ P＜0．01）。在ABs中,TAMs主要表达于肿瘤间质,MMP?2主要表达于肿瘤实质,MMP?2表达强度随着TAMs计数增加而增加,二者呈正相关关系（ r＝0．331,P＜0．05）。结论：ABs中TAMs和MMP?2的共同表达可能在其发生发展中发挥重要作用。     

Salivary matrix metalloproteinase (MMP-8) levels and gelatinase (MMP-9) activities in patients with type 2 diabetes mellitus

We studied the salivary levels and activities of the matrix metalloproteinases (MMP) -8 and -9 in 45 type 2 diabetic patients and 77 control subjects. The patients' mean glycosylated haemoglobin (HbA1c) was 8.7%, indicating an unsatisfactory metabolic control of the disease. The MMP levels were further related to the clinical and microbiological periodontal findings as well as to salivary flow rate and other factors. The salivary flow rate, albumin and amylase concentrations were similar in type 2 diabetic patients to those in the control group. The mean gingival and periodontal pocket indexes were higher in the diabetes group. The number of potential periodontopathogenic bacteria was lower, however, in the diabetic than in the control group. Zymography and immunoblotting revealed that the major MMPs in the type 2 diabetic patients' saliva were MMP-8 and MMP-9. Salivary MMP levels and activities in type 2 diabetic patients were in general similar to those in the control group. However, the correlation coefficients using multiple regression analysis revealed that gingival bleeding, pocket depths and HbA1c were associated with increased MMP-8 levels which, in turn, were negatively predicted by elevated plasma lipid peroxide levels in the diabetic group. Our data on salivary MMP-8 and -9 do not support the concept of generalized neutrophil dysfunction in unbalanced diabetes. Moreover, plasma lipid peroxidation levels reflecting the increased oxidative burden, which is generated mainly by triggered neutrophils, do not indicate neutrophil dysfunction due to diabetes, but may rather be related to the increased tissue damage in an uncontrolled disease. However. advanced periodontitis in type 2 diabetes seems to be related to elevated salivary MMP-8 levels which might be useful in monitoring periodontal disease in diabetes.     

Runt相关基因2/核心结合因子a1在小鼠牙周组织发育中的免疫组化定位研究

目的探讨Runt相关基因2/核心结合因子a1(Runx2/Cbfa1)在小鼠牙周组织发育过程中的时空表达及意义。方法建立BALB/c小鼠牙周发育动物模型,采用免疫组化方法检测Runx2/Cbfa1在小鼠出生后各期牙周组织发育中的表达及特点。结果Runx2/Cbfa1在牙齿发育过程中的表达具有时空特异性,在牙根开始发育之前,仅在牙槽骨及成骨细胞中表达;当牙根开始发育即从第11天以后各期,在根部牙周膜细胞、成牙骨质细胞及成骨细胞中均呈阳性表达,但牙槽骨呈阴性表达。结论Runx2/Cbfa1在成牙骨质细胞及成骨细胞的分化及牙植骨的形成具有重要作用,可能在牙周组织的发育中发挥作用。     

DCN与BGN在发育期小鼠牙髓牙本质复合体形成中的作用研究

目的:研究富含亮氨酸的蛋白多糖类物质:核心蛋白聚糖(DCN)及双糖蛋白聚糖(BGN)在小鼠牙髓牙本质复合体不同发育时期的分布特点,探讨其在牙髓牙本质复合体形成过程中的作用.方法:取出生后第5 d、10 d、15 d、25 d的BALB/℃小鼠36只,引颈处死,解剖分离含下颌第一磨牙区的下颌骨,新鲜配制4%多聚甲醛固定24 h,甲酸-甲酸钠复合脱钙液脱钙1～3周,石蜡包埋,近远中向5μm连续切片.免疫组化PV两步法,观察各发育时期第一磨牙牙本质、成牙本质细胞及牙髓细胞中DCN、BGN的组织学分布特点.结果:牙本质形成初期,BGN在前期牙本质与成牙本质细胞强阳性表达.随后,前期牙本质与成牙本质细胞染色开始减弱,而牙髓细胞阳性表达呈逐渐增强趋势.第5 d,DCN在前期牙本质中强阳性表达,而在牙本质、成牙本质细胞中一直为阴性;第13 d,牙髓细胞开始呈弱阳性表达,随着牙齿发育而逐渐增强.在观察期间,两者在前期牙本质中持续阳性表达,并且随着牙本质的发育成熟,其染色逐渐减低.结论:DCN与BGN在牙体组织和牙发育过程中特定阶段聚集在特定的部位,二者可能在细胞与细胞、细胞与基质相互作用中发挥特异作用并参与牙本质的矿化过程.     

Analysis of the MMP-9 (C-1562 T) and TIMP-2 (G-418C) gene promoter polymorphisms in patients with chronic periodontitis

BACKGROUND: Matrix metalloproteinases (MMP)-9 is an important member of the matrix metalloproteinase family. A functional polymorphism has been described in the promoter region of the human MMP-9 gene. A C-to-T base exchange at -1562 creates two different alleles, and the C/T and T/T genotypes promote high activity of the MMP-9 gene promoter, increasing the risk for inflammatory diseases. The metalloproteinase-2 tissue inhibitor (TIMP-2) regulates the activity of MMPs in the extracellular matrix, and a polymorphism at the -418 position of the TIMP-2 gene promoter has been found in a Sp-1 binding site. In this study we have investigated the association between the above-mentioned polymorphisms and chronic periodontitis severity. METHODS: Genomic DNA from oral mucosa of 100 subjects was amplified by polymerase chain reaction and analysed by restriction endonuclease digestion. The significance of the differences in observed frequencies of polymorphisms in moderate and severe disease and healthy groups was assessed by chi(2) test (p<0.05). RESULTS: No association was observed between the polymorphism in the promoter region of MMP-9 (p=0.6693) and chronic periodontitis. The analysis of TIMP-2 showed that the G/G genotype was found at a frequency of 99%. CONCLUSION: The results show that the polymorphism in the promoter region of MMP-9 gene is not associated with chronic periodontitis. The high frequency of GG genotype in the TIMP-2 gene promoter in the population studied did not allow any conclusion regarding its effect on chronic periodontitis.     

Healing of extraction sockets in collagenase-2 (matrix metalloproteinase-8)-deficient mice

Matrix metalloproteinase-8 (MMP-8) participates in skin wound healing and inflammation. We hypothesized that MMP-8 plays a role in wound healing after tooth extraction and in periapical inflammation. Bone formation, collagen metabolism, and inflammation in tooth extraction socket and in periapical lesions were analyzed in wild-type mice and in MMP-8-deficient (MMP-8^−/−) mice. New trabecular bone area in the extraction sockets and in periapical lesions were similar in both groups. In extraction sockets significantly more type III procollagen was synthesized, and the neutrophil and MMP-9 levels were lower in MMP-8^−/− mice. The amount of Fas ligand, identified as a substrate for MMP-8, was lower in alveolar mucosa but higher in alveolar bone of MMP-8^−/− mice. These results indicate that MMP-8 can modulate inflammation and collagen metabolism of alveolar bone and mucosa.     

A pharmacokinetic study of a topical anesthetic (EMLA®) in mouse soft tissue laceration

The use of topical anesthesia instead of injection of local anesthetics for managing soft tissue lacerations in the emergency situations may be a relief for both patients and surgeons. Topical anesthesia in the form of a cream eutectic mixture of local anesthetics (EMLA^®) containing 2.5% lidocaine and 2.5% prilocaine has been reported as an efficient anesthetic on skin before venipuncture anesthesia and as an alternative to injection anesthesia in some minor surgery situations. The aim of this study was to compare the pharmacokinetics of EMLA^® when applied in a laceration with topical skin application in the mouse. A total of 120 Albino Laboratory‐bred strain mouse (BALB‐c) male mice were divided into three groups with regard to application mode of EMLA^®. Group A: with laceration, 48 mice; Group B: on intact shaved skin, 48 mice; Group C: control group (24 mice) with same procedures but without application of EMLA^®. Blood levels were collected at 0, 10, 20, 30, 45, 60, 75, and 90 min post‐EMLA^® application. Plasma sample analysis was carried out by employing liquid chromatography coupled with tandem mass spectrometric (LC‐MS/MS) method, and the pharmacokinetic analysis of the mouse plasma samples was estimated by standard non‐compartmental methods. The pharmacokinetic parameters of lidocaine and prilocaine were significantly altered following EMLA^® application to lacerated mouse skin in contrast to intact skin. The absorption of lidocaine and prilocaine was rapid following application of EMLA^® to lacerated and intact mouse skin. Maximum drug plasma concentration (C_max) and area under the drug plasma concentration–time curve (AUC) values of lidocaine were significantly increased by 448.6% and 161.5%, respectively, following application of EMLA to lacerated mouse skin in comparison with intact mouse skin. Similarly, prilocaine's C_max and AUC values were also increased by 384% and 265.7%, respectively, following EMLA application to lacerated mouse skin, in contrast to intact skin. Further pharmacokinetic studies on different carriers of lidocaine/prilocaine are warranted before any firm conclusions for the clinic can be drawn.     

Expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue

Previous studies have demonstrated that (at least) matrix metalloproteinase (MMP)-2, -8, -9, -14 and -20 are expressed by human odontoblasts. Here, we analysed the expression of 19 MMPs and their specific tissue inhibitors (TIMP)-1, -2 and -3) -1, -2 and -3 in mature human odontoblasts and pulp tissue. Since MMP-20 is almost exclusively expressed by the dentin-pulp complex cells, we further analysed the effect of transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMPs)-2 on its expression. Matrix metalloproteinase-9 served as a positive control for growth factor responsiveness. It was found that MMP-1, -2, -9, -10, -11, -13, -14, -15, -16, -17, -19, -20 and -23, in addition to TIMP-1, -2 and -3 were expressed by both odontoblasts and pulp tissue. Neither MMP-3 nor MMP-12 were expressed in odontoblasts or pulp tissue, and MMP-7, -8, -24 and -25 were expressed only in the odontoblasts; MMP-2, -10, -11, -14 and -20 were expressed more abundantly by odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Transforming growth factor-beta1 (1 ng ml(-1)) and BMP-2 (100 ng ml(-1)) did not markedly affect MMP-20 mRNA expression. In contrast, TGF-beta1 alone and with BMP-2 significantly upregulated MMP-9 mRNA by 2.4-fold and by 2.6-fold, respectively, in odontoblasts, while in pulp tissue no effects could be detected. The wide-scale expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling by differentially regulating individual MMPs.     

Tissue-specific expression of Fgfr2b and Fgfr2c isoforms, Fgf10 and Fgf9 in the developing chick mandible

Experimental evidence has demonstrated the importance of FGF signalling in morphogenesis of the mandibular processes. FGFs transmit their signals through four tyrosine kinase transmembrane receptors (FGFRs). Alternative splicing in FGFRs including FGFR2 generates different isoforms that exhibit different ligand-specificities, exclusive tissue distributions and specific biological functions. Despite extensive information regarding the isoform-specific patterns of expression Fgfr2c and Fgfr2b during morphogenesis of many organs, a comparative analysis of these specific isoforms in the chick mandible has not been reported. To better understand the function of FGFR2 in mandibular morphogenesis, we have analysed the expression Fgfr2b, Fgfr2c and their putative ligands Fgf10 and Fgf9, in the developing chick mandibular processes by in situ hybridisation and RT-PCR. Our observations show that Fgfr2b was primarily expressed in the mandibular epithelium while Fgfr2c was expressed in the mandibular mesenchyme including Meckel's cartilage. Fgf9 and Fgf10 were expressed in a variety of craniofacial regions including the mandibular epithelium and mesenchyme respectively. The temporal and spatial distributions of Fgfr2b, Fgfr2c, Fgf10 and Fgf9 in the developing mandible reported in this study make them attractive candidates for involvement in epithelial-mesenchymal signalling interactions that are known to be necessary for proper mandibular outgrowth and morphogenesis.     

口腔鳞癌中VEGF和MMP-2的表达及其相关性

目的：探讨MMP-2、VEGF在口腔鳞状细胞癌（OSCC）中的表达、两者相关性及其作用。方法：采用免疫组化法检测MMP-2和VEGF在86例OSCC和20例正常非肿瘤组织中的表达情况及两者的相关性。结果：MMP-2和VEGF在OSCC中的阳性表达率分别为85．9％和88．4％，均高于正常组织的10．0％，15．0％；差异均有显著性差异如〈0．01）：MMP-2和VEGF在OSCC中的表达呈正相关（CAMMA值0．476，p=0．007）。结论：MMP-2和VEGF在OSCC中呈过表达，二者之间的相互作用与OSCC增殖、侵袭和转移密切相关。     

糖尿病大鼠牙齿移动后牙周组织基质金属蛋白酶-2表达的变化

目的探讨糖尿病大鼠正畸牙齿移动过程中基质金属蛋白酶- 2（MMP- 2）的分布和表达变化，研究糖尿病对牙周组织胶原代谢的影响。方法选用80只雄性SD大鼠, 牵引左侧上颌第一磨牙近中移动。实验组以链脲佐菌素腹腔注射制备1型糖尿病模型，3周后开始实验，分别于加力后0、3、7、14、21 d处死大鼠，应用两步免疫组化法检测大鼠牙周组织MMP- 2的表达。结果MMP- 2免疫组化结果显示牙齿移动后，牙周膜两侧MMP- 2表达增加，破骨细胞、骨细胞、成纤维细胞和部分成骨细胞MMP- 2染色阳性。图像分析结果显示实验组变化较对照组小。平均光密度表现动态变化，7 d最低，而后缓慢升高。加力后积分光密度逐渐升高，7 d达到顶峰，而后缓慢下降，21 d时仍维持在较高水平。结论未加力时，糖尿病大鼠颌骨胶原代谢增强。在正畸牙齿移动过程中，糖尿病大鼠骨质反应能力降低，胶原代谢较弱，牙齿移动时MMP- 2呈规律性变化，与牙齿正畸骨改建关系密切，在牙齿移动中起着重要的作用。     

小鼠牙胚发育帽状期Msx2、BMP—4基因的表达和意义

目的：探讨Msx2、BMP-4基因在小鼠牙胚发育帽状期的表达及其与牙胚形态发育的关系。方法：利用原位杂交的方法观察小鼠牙胚帽状期Msx2、BMP-4基因的表达方式;利用原位末端标记法（TUNEL法）研究牙胚细胞凋亡情况。结果：Msx2、BMP-4基因在牙胚的发育吸 表达。帽状期牙胚细胞的凋亡主要集中于釉结处。结论：Msx2、BMP-4参与了牙胚的发育。     

成骨特异转录因子 Runx2 在去势大鼠正畸牙移动牙周组织中的表达

目的：研究成骨特异转录因子Runx2在去势大鼠正畸牙移动过程中牙周组织内的表达规律。方法：选用3月龄雌性未孕SD大鼠50只,随机分为去势组（OVX）与假手术组（Sham）各25只。全麻下行大鼠去势术或假手术,术后饲养3个月。构建大鼠上颌第一磨牙正畸牙移动模型,并分别于牙移动0d、1d、3d、7d与15d处死去势组大鼠和假手术组大鼠各5只,取上颌第一磨牙周围牙周组织,行H-E染色和免疫组织化学染色后观察正畸牙移动牙周组织的改建。结果：去势大鼠与假手术大鼠正畸牙移动过程中牙周组织的改建基本相似,即张力区以成骨为主,压力区以破骨为主。去势组大鼠牙移动15d张力区成骨细胞及压力区破骨细胞均显著多于假手术组大鼠（尸〈0．05）。大鼠正畸牙移动过程中牙周组织Runx2表达先升后降。牙移动1d、3d、7d张力区与压力区Runx2表达均显著高于0d（P〈0．05）,15d与0d则无显著差异。去势组大鼠牙移动牙周组织Runx2表达变化与假手术组大鼠基本一致,但是去势组1d压力区和7d张力区Runx2表达均显著高于假手术组大鼠（P〈0．05）。结论：去势大鼠正畸牙移动过程中的牙周组织改建符合正畸牙移动的基本规律,但其牙周组织的改建更为活跃。去势大鼠正畸牙移动牙周组织内Runx2的表达增强,这可能与活跃的牙周组织改建相关,其机制需进一步研究。     

Genetic variations in the human gelatinase A (matrix metalloproteinase-2) promoter are not associated with susceptibility to, and severity of, chronic periodontitis

BACKGROUND: Gelatinase A (matrix metalloproteinase-2 [MMP-2]) has been shown to play an important role in the pathogenesis of several disorders, including periodontal diseases. In this study, we test the hypothesis that variations in this gene influence the development and severity of chronic periodontitis. METHODS: Four promoter polymorphisms (-1575G/A, -1306C/T, -790T/G, and -735C/T) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 149 patients with mild to severe chronic periodontitis and 127 age-matched controls in the Czech population. RESULTS: No significant differences in distribution of the -1575G/A, -1306C/T, and -735C/T variants between periodontitis and control groups were detected in our study. However, a trend to decreased frequency of the -790 GG homozygotes was observed in patients with chronic periodontitis compared to healthy controls (P = 0.036, P (corr) >0.05). Haplotype analysis of four single nucleotide polymorphisms (SNP) in the MMP-2 gene showed no significant association of any haplotype with chronic periodontitis. CONCLUSION: Our findings suggest that polymorphisms in the MMP-2 gene promoter do not contribute significantly to the interindividual periodontitis susceptibility and/or severity in European Caucasians, and they are not regulatory variants in this disease.