Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment – both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors – epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF- β ₁) and interleukin-1 β (IL-1 β ). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF- β ₁, on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 β significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis. These findings further illustrate the usefulness of fibroblast-populated collagen gels as a model system for studying the modulatory effects of soluble factors and extracellular matrix molecules on fibroblast function in vitro .     

Cytokine-induced endogenous procollagenase stored in the extracellular matrix of soft connective tissue results in a burst of collagen breakdown following its activation

Numerous data strongly suggest the involvement of cytokines and the matrix metalloproteinase collagenase (MMP-1) in the pathogenesis of periodontitis. Recently, we have demonstrated that, upon culturing under the influence of IL- lα+EGF, a large amount of inactive procollagenase (MMP-1) is stored in the extracellular matrix of periosteal tissue. We now show that this endogenous reservoir of proenzyme can be operative after activation with plasmin and is able to induce a rapid and almost complete breakdown of the collagenous extracellular matrix. The level of collagen degradation following activation showed a strong correlation with the amount of proenzyme that was incorporated in the tissue. The highest levei of degradation (70% of the total amount of collagenous proteins) was found with the IL-lα+EGF-treated explants, followed by those treated with IL-1α alone (35%). Explants cultured with EGF or in the absence of cytokines, containing only small amounts of procollagenase, showed little collagen breakdown following plasmin activation (7%). Inhibition of metalloproteinases by EDTA, or blockage of plasmin by PMSF, prevented the degradation in all explants irrespective of the amount of proenzyme present in the tissue. Our findings demonstrate that endogenous proenzyme stored in a native connective tissue matrix can be activated at a later time interval which results in a massive breakdown of the tissue. This study shows a possible pathway of collagenase-induced breakdown without recent de novo synthesis of the enzyme. Such a sequence may be operative in chronic inflammatory diseases, such as periodontitis, where production of procollagenase under the influence of cytokines spans a longer time period, whereas breakdown is often characterized by a cyclic behaviour.     

Effects of transforming growth factor beta 1 on the plasminogen activation system,collagen and integrin synthesis,and proliferation of rabbit mandibular condylar chondrocytes

The objective of this study was to identify the mechanism by which mandibular condyle chondrocytes regulate the extracellular matrix. Primary rabbit condylar chondrocytes were isolated, cultured, and treated with transforming growth factor beta 1 (TGF-β1). Cells were then assayed for the following: urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1), collagen types I and II, β1 integrin expression, and proliferative activity. TGF-β1 induced synthesis of collagen type II, αVβ1 integrin, and PAI-1. TGF-β1 induced the growth of chondrocytes and suppressed the synthesis of uPA. Chondrocyte regulation of the extracellular matrix is mediated by TGF-β1. Synthesis of collagen type II, αVβ1 integrin, and PAI-1 is induced, while uPA is suppressed. Also, TGF-β1 induces cellular growth.     

Alendronate induces postnatal maxillary bone growth by stimulating intramembranous ossification and preventing premature cartilage mineralization in the midpalatal suture of newborn rats

Cleft palate is a common malformation of craniofacial development, and postnatal deficiencies in palate formation may occur. The aim of this study was to determine whether alendronate treatment could induce maxillary mineralization and thus reduce the need for surgical procedures. The effects of alendronate on maxillary bone development, the midpalatal suture, and the levels of transforming growth factor beta-1 (TGF-β1), bone morphogenetic protein 2 (BMP-2), collagen I and II, and V-ATPase were evaluated in newborn rats. Thirty newborn rats were placed in a control group and 30 in a group that received intraperitoneal alendronate (2.5 mg/kg/day). The animals were euthanized on day 7 or 12, and the heads were subjected to histological and immunohistochemical analyses. Specimens from rats that received alendronate presented larger bone matrix deposition in areas of intramembranous ossification of the maxillary bone when compared to controls. Furthermore, higher levels of TGF-β1, BMP-2, and collagen I were observed, whereas osteoclasts showed no V-ATPase. The alendronate group also showed higher levels of TGF-β1 and collagen II in the midpalatal suture, whereas BMP-2 levels were lower than in controls. These results coincided with an expansion of the chondroid. In conclusion, alendronate increased the intramembranous ossification in the maxillary bone in association with increased expression of TGF-β1, BMP-2, and collagen I and decreased V-ATPase. The drug induced an expansion of chondrocytes and a decrease in mineral bone deposition despite the high levels of TGF-β1 in this area. Alendronate may therefore be useful in the treatment of diseases affecting bone growth.     

Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases

Objective: Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. Materials and methods: Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. Results: Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. Conclusions: Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.     

Regulation of matrix metalloproteinase production by cytokines,pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures

Matrix metalloproteinases (MMPs). produced by both infiltrating and resident cells of the periodontium, play a role in physiologic and pathologic events. It is recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. To date, little is known about the regulation of MMP synthesis and secretion in human periodontal ligament fibroblasts (PDLFs). The purpose of this study was to examine the effects of cytokines, pharmacological agents (protein synthesis inhibitor and protein kinase C inhibitors) and predominant periodontal pathogens (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) on MMP production in human PDLFs using gelatin zymography. The gelatin zymograms revealed that the main gelatinase secreted by human PDLFs migrated at 72 kDa and represents MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. We found that A. actinomycetemcomitans, P. gingivalis and IL-1alpha can elevate MMP-2 secretion in human PDLFs. These results indicate that periodontal pathogens and inflammatory cytokines play an important role in tissue destruction and disintegration of extracellular matrix in periodontal diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of periodontitis. In addition, H7, staurosporine, cycloheximide and TGF-beta could suppress MMP-2 production. Agents that target protein synthesis or the protein kinase C pathway in human PDLFs inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of periodontal inflammation. Taken together, these findings suggest a possible new therapeutic approach, involving the use of drugs that modify host-response mechanisms to suppress or inhibit MMP-mediated tissue destruction.     

转化生长因子β3对脂多糖诱导的成骨细胞中IL-6表达的影响

目的:探讨转化生长因子β3 (transforming growth factor β3,TGF-β3)对成骨细胞内炎性细胞因子IL-6表达的影响,及其发挥抗炎作用的机制.方法:20 μg/mL牙髓卟啉单胞菌脂多糖(lipopolysaccharide of Porphyromonas endodontalis,Pe-LPS)刺激人成骨肉瘤细胞系MG63,构建成骨细胞炎症模型.取不同浓度(5～20 ng/mL)的人重组蛋白生长因子TGF-β3和TGF-β1,分别与20 μg/mL P.e-LPS共同作用于MG63细胞24 h后,利用实时荧光定量PCR检测细胞内IL-6 mRNA的表达,ELISA法检测培养液上清中IL-6的表达水平.以10 ng/mL TGF-β3预处理细胞30 ain后,再与20 μg/mL P.e-LPS共同作用20 min,Western印迹法检测细胞内ERK1/2蛋白磷酸化的表达.采用SPSS13.0软件包对数据进行统计学分析.结果:实时荧光定量PCR结果显示,单独20 μg/mL P.e-LPS作用下,MG63细胞内IL-6的表达显著增高(P＜0.01);TGF-β1在低浓度条件下(5～10 ng/mL)对IL-6的表达无显著作用,仅在20 ng/mL时可显著抑制IL-6的表达(P＜0.05).不同浓度的TGF-β3与Re-LPS共同作用均可显著抑制IL-6的表达(P＜0.01).ELISA结果显示,10～20 ng/mL TGF-β3可在蛋白水平上对IL-6有显著抑制作用(P＜0.05).单独P.e-LPS作用时,可使MG63细胞内ERK1/2蛋白的磷酸化水平升高(P＜0.01);而当TGF-β3与P.e-LPS共同作用时,ERK1/2的磷酸化被抑制(P＜0.05).结论:相同浓度条件下,TGF-β3比TGF-β1对成骨细胞炎症的抑制作用更为显著,ERK1/2信号机制参与了TGF-β3的抗炎过程.     

Periodontal ligament and gingival fibroblasts participate in the production of TGF-β, interleukin (IL)-8 and IL-10

The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 μg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 μg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 μg/mL and 10 μg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.     

TGF-β1 is essential for the homeostasis of the dentin-pulp complex

Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-β1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix production. These properties underscore the importance of this molecule in maintaining the homeostasis of the dentin-pulp complex after injury. The purpose of this paper is to describe new findings of our phenotypic analysis of dentition in mice in which the TGF-β1 gene has been disrupted. The major phenotype of TGF-β1 (?/?) offspring is one of diffuse immune system activation with progressive inflammation, wasting and death. Our studies of adult TGF-β1 (?/?) dentition show widespread pulpal and periapical inflammation and necroses. In addition, the coronal surfaces of occluding molars show marked attrition. To determine whether the phenotypic changes in TGF-β1 (?/?) dentition are directly linked to the loss of TGF-β1 rather than the inflammatory process itself, we studied adult dentition in TGF-β1 (?/?) mice backcrossed into immunodeficient backgrounds. Results of our histopathologic and radiographic analyses show that teeth of TGF-β1 (?/?) immunodeficient mice retain vitality in pulpal and periapical regions but show excessive wear of occlusal surfaces.     

转化生长因子-β1和白细胞介素-10单核苷酸多态性与复发性口腔溃疡易感性的研究

目的 研究转化生长因子-β1（TGF-β1）-509T/C位点与白细胞介素-10（IL-10）-1082A/G位点的单核苷酸多态性与复发性口腔溃疡（RAU）易感性的相关性。方法 采用限制性片段长度多态性-聚合酶链式反应（RFLPPCR）RFLPPCR）法和序列特异性引物-聚合酶链式反应（SSP-PCR）法对138例RAU患者和124例健康对照者进行TGF-β1-509位点与IL-10-1082位点的单核苷酸多态性的检测分析,用比值比（OR）和95%可信区间（95%CI）估计相对危险度。结果 TGF-β1-509位点与IL-10-1082位点在基因型频率与等位基因频率的分布上,病例组与正常对照组之间均存在明显差异（P<0.05）。TGF-β1-509位点基因型CT（OR=1.231,95%CI=0.702~2.160）与TT（OR=2.482,95%CI= 1.250~4.927）为高风险基因型,T等位基因为高风险等位基因（OR=1.465,95%CI=1.036~2.074）。IL-10-1082位点基因型AG（OR=1.391,95%CI=0.808~2.396）与GG（OR=4.165,95%CI=1.944~8.924）为高风险基因型,G等位基因为高风险等位基因（OR=2.134,95%CI=1.474~3.089）。结论 TGF-β1-509位点与IL-10-1082位点是RAU患者的易感基因位点。TGF-β1-509位点携带T等位基因患RAU的风险性是携带C等位基因者的1.465倍。IL-10-1082位点携带G等位基因患RAU的风险性是携带A等位基因者的2.134倍。     

The role of matrix metalloproteinases in the oral environment

This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.     

TGF-β在下颌髁突软骨的发育和改建中作用研究进展

［摘要］ 转化生长因子β(transforming growth factor beta,TGF-β)是一种多功能的细胞因子,广泛参与细胞的多种生物学过程,如细胞增殖分化、胞外基质的合成和组织修复等。近年来的研究发现,髁突软骨的发育必须有TGF-β的参与,TGF-β可介导局部的功能刺激作用,参与髁突软骨的适应性改建,与髁突软骨退行性病变相关。本文就TGF-β在髁突软骨发育、改建和退行性关节病中的作用作一综述。     

脂多糖和转化生长因子-β1对牙髓细胞Toll样受体4的表达及信号通路的影响

目的研究牙髓炎症过程中,在促炎因子脂多糖（LPS）和抑炎因子转化生长因子-β1（TGF-β1）同时存在的情况下,牙髓细胞表面Toll样受体4（TLR4）的表达水平及相关信号分子的变化情况。方法LPS、TGF-β1作用于体外培养的牙髓细胞,用流式细胞术检测牙髓细胞表面TLR4的表达;实时荧光定量聚合酶链反应（real-time PCR）、Westernblot方法检测相关信号分子的表达水平,包括进化保守的Toll信号中介分子（ECSIT）和核转录因子-κB（NF-κB）;酶联免疫吸附试验（ELISA）检测前炎症因子白细胞介素-6（IL-6）的分泌水平;然后进一步通过real-time PCR法检测临床炎症牙髓组织中相应指标的变化。结果体外培养的牙髓细胞在LPS、TGF-β1共同作用下,细胞表面TLR4的表达水平没有明显变化,但是IL-6分泌增加,ECSIT表达增加,NF-κB入核增加。临床标本的real-time PCR结果表明：炎症状态下的牙髓组织中TGF-β1 mRNA表达增加,TLR4 mRNA表达没有明显变化,ECSIT及IL-6 mRNA表达增加。结论牙髓炎症发展过程中,虽然牙髓组织中TGF-β1表达增加,抑制细胞表面TLR4的表达,但TLR4的信号通路仍然被活化,主要机制可能是LPS引起信号分子ECSIT的活化,从而抑制TGF-β1信号通路的活化。     

The Effect of Transforming Growth Factor Beta 1 on the Mineralization of Human Cementoblasts

IntroductionTransforming growth factor beta 1 (TGF-β1) plays an important role in bone mineralization and has been reported to promote osteoblast proliferation and differentiation. However, there is no report about the effects of TGF-β1 on human cementoblasts. The purpose of this study was to clarify the effect of TGF-β1 on the proliferation and differentiation of the human cementoblast cell line (HCEM) in vitro.MethodsHCEM cells were stimulated with TGF-β1 at concentrations of 0.05, 0.5, 5, and 10 ng/mL. A proliferation assay was performed from 24–72 hours. The effect of TGF-β1 on mineralization was analyzed by quantifying the area stained with alizarin red on days 7 and 14. Real-time polymerase chain reaction was used to assess the effect of TGF-β1 on the mineralization-related genes alkaline phosphatase, bone sialoprotein, and type I collagen on days 3, 7, and 14.ResultsTGF-β1 did not affect cell proliferation. TGF-β1 together with the mineralization medium (consisting of ascorbic acid, dexamethasone, and β-glycerophosphate) increased the alizarin red–stained area on days 7 and 14. Real-time polymerase chain reaction revealed that alkaline phosphatase messenger RNA expression was increased in TGF-β1–stimulated HCEM cells in mineralization medium on days 3 and 7, whereas bone sialoprotein and type I collagen messenger RNA expression was increased on day 7.ConclusionsAlthough TGF-β1 does not affect cell proliferation, it does promote cell differentiation and mineralization of HCEM cells.     

Evaluation of peri-implant mucosa: Clinical,histopathological and immunological aspects

Objective

The aim was to compare the inflammatory response in peri-implant mucosa between patients with peri-implantitis (PP-group) and patients with healthy peri-implant tissues (HP-group).

Materials and methods

Two fragments of peri-implant mucosa of 18 patients were collected and serial sections were performed for histological and immunohistochemical analysis.

Results

When compared with HP-group, PP-group showed higher immunostained cell density for TGF-β, IL-17 and CD31, beyond greater density of red cells, leukocytes, mast cells chymase (MCC) and mast cell tryptase (MCT). HP-group patients showed higher IL-13 expression and increased amount of collagen fibres when compared with PP-group. In PP-group there was significant positive correlation between MCT density and density of blood vessels immunostained, and between MCC density and density of blood vessels immunostained. There was significant negative correlation between the IL-17 density and collagen percentage.

Conclusions

This study demonstrated that in patients with peri-implantitis there was higher of TGF-β and IL-17, indicating that these cytokines are directly involved in the inflammatory process. Thus, understanding the influence of cytokines in the peri-implantitis installation, new therapies could be developed in order to inhibit the synthesis of IL-17 and induce synthesis of IL-13 in peri-implant tissue, contributing to increase the longevity of the implant.     

TGF-β1-induced connexin43 promotes scar formation via the Erk/MMP-1/collagen III pathway

Wound healing can be divided into different phases, and timely initiation and cessation of these stages is key to successful wound healing; otherwise, scar tissue forms in the wounded area. Connexins (Cxs) were confirmed to influence scar formation, and Cx43, an indispensable member of the Cx family, was shown to be involved in this process. Our study investigated the regulatory role of Cx43 in scar formation and the possible cell signalling pathways. We established oral mucosa and skin wound healing models in C57BL/6J mice. RT-PCR, western blotting, immunohistochemistry and immunofluorescence were used to examine the expression of ECM components and key proteins in cell signalling pathways (TGF-β1, Smad2/3, Cx43, Erk1/2 MMP-1 and collagen III). After injury, buccal mucosa wounds healed with no scar, whereas skin wounds healed with an evident scar. Nevertheless, TGF-β1 expression gradually increased by the 5th day after injury; Cx43 expression showed a similar response, with a progressive increase in the skin and a peak on day 14. In contrast, TGF-β1 and Cx43 expression in the oral mucosa remained low. The high level of TGF-β1 increased p-Smad2/3 levels and then induced Cx43, whereas increased expression of Cx43 antagonised the phosphorylation of Erk1/2, a protein downstream of Cx43, which affected MMP-1 synthesis. MMP-1 deficiency led to collagen III accumulation and facilitated scar formation. We demonstrated that TGF-β1-induced Cx43 promotes scar formation via the Erk/MMP-1/collagen III pathway.