Decorin和Ⅰ型、Ⅲ型胶原纤维在牙周损伤愈合中的免疫化学定位和表达

目的:研究decorin和Ⅰ型、Ⅲ型胶原纤维在牙周组织损伤愈合过程中的免疫化学定位.方法:损伤成年鼠磨牙周围的牙周组织,跟踪28 d,对decorin 和Ⅰ型、Ⅲ型胶原在愈合过程中的免疫组织化学分布进行分析.结果:decorin在结缔组织中的免疫组织化学分布与Ⅰ型胶原相似.Ⅲ型胶原分布较散在.在牙周损伤愈合早期,decorin与损伤区周围的成纤维细胞关系密切,特别是牙龈黏膜的固有层,成纤维细胞对decorin的表达非常强.愈合中期,decorin广泛表达于肉芽组织成纤维细胞.愈合晚期,decorin的免疫阳性表达主要位于损伤区周围的上皮下区域.结论:decorin在牙周组织损伤区中的细胞内特征性表达,预示其与胶原纤维相互作用,在牙周损伤愈合过程中起着不可忽视的作用.     

纯钛表面牙周韧带细胞形成物的组织学和免疫组织化学研究

目的：探讨牙周韧带细胞在商用纯钛表面的增殖与分化。方法：牙周韧带细胞在纯钛表面进行培养，14天后刮取形成物，进行HE染色及Ⅰ型胶原免疫组织化学分析。结果：牙周韧带细胞在纯钛表面生长良好，14天后形成单层，形成大量细胞基质，其成份主要为Ⅰ型胶原。结论：牙周韧带细胞能够在纯钛表面生长、增殖，并形成大量细胞外基质，细胞外基质的主要成份为Ⅰ型胶原。     

两种正畸力作用下大鼠牙周组织中胶原相关蛋白的表达

目的观察不同正畸力作用下大鼠牙移动过程中Ⅰ型胶原和基质金属蛋白酶-1（matrix metalloprotein-ase-1,MMP-1）、金属蛋白酶组织抑制因子-1（tissue inhibitor of metalloproteinase-1,TIMP-1）在上颌第一磨牙牙周组织中的表达变化。方法建立大鼠正畸牙移动模型,分为加力50 g组和加力150 g组,随机选择加力侧,对侧不加力作为对照组,分别于术后3、6、12 h和1、3、7、14 d处死,用免疫组织化学方法观察各组中Ⅰ型胶原和MMP-1、TIMP-1表达的变化。结果Ⅰ型胶原和MMP-1、TIMP-1在加力组的阳性表达明显强于对照组,且表达有时间依赖性。多元回归分析发现MMP-1、MMP-1与TIMP-1比值（MMP-1/TIMP-1）有与Ⅰ型胶原表达量呈负相关的趋势,TIMP-1有与牵张区Ⅰ型胶原表达量呈正相关的趋势。结论 MMP-1和TIMP-1参与了正畸牙周组织改建过程中Ⅰ型胶原代谢的调节,其表达的平衡性对维护牙周组织的生理健康、正常改建起着非常重要的作用。     

Ⅲ型胶原和基质金属蛋白酶-1在大鼠移动牙齿牙周组织中的表达

目的观察正畸移动大鼠牙齿过程中Ⅲ型胶原和基质金属蛋白酶(MMP)-1在上颌第一磨牙牙周组织中的表达和变化。方法建立大鼠磨牙移动模型,分为对照组和术后13、、57、、101、4 d组,用免疫组化方法观察各组中Ⅲ型胶原和MMP-1表达的变化。结果Ⅲ型胶原和MMP-1在移动牙牙周组织中的阳性表达强于对照组,且有时间依赖性。结论MMP-1参与了正畸牙周组织细胞外基质的代谢过程;Ⅲ型胶原在牙周组织改建中有重要作用。     

纤维调节素在人类牙周细胞损伤愈合中的免疫组织化学表达

目的研究纤维调节素在牙周细胞损伤愈合中的表达,探讨纤维调节素在牙周损伤愈合中的作用。方法培养人类牙周细胞（牙龈成纤维细胞、牙周膜成纤维细胞和成骨细胞）;在培养了牙周细胞的载玻片上做损伤细胞的圆形创口,建立牙周细胞损伤模型,跟踪观察7 d;用免疫组织化学染色的方法,观察纤维调节素在牙周细胞损伤愈合过程中的表达。结果牙周细胞损伤后第1天,纤维调节素较强表达于损伤边缘新移行的细胞;随后,纤维调节素对损伤区新近移行的细胞的表达强于创口周围的细胞。结论纤维调节素强烈表达于损伤区新近移行的牙周细胞,可能参与牙周损伤愈合的早期活动。     

PGA无纺网与PDLCs的3D共培养物在裸鼠体内的生长观察

目的：通过研究人牙周韧带细胞(PDLCs)与聚羟基乙酸(PGA)无纺网共培养物在裸鼠体内的生长，探讨PGA无纺网作为支架应用于牙周组织工程的可能性。方法：将共培养7d的人PDLCs与PGA无纺网复合物接种于BALB／C裸小鼠一侧背部皮下，另一侧植入空白PGA为对照。分别于1、2、3、4周取材进行大体及HE和Masson‘s染色及Ⅰ型胶原免疫组织化学观察，并进行新生毛细血管记数。结果：随着。PGA无纺网在体内的逐渐降解，PDLCs同步增殖，分泌胞外基质。体内2周时即可见实验侧植入物血管化良好并有胶原束的形成，Ⅰ型胶原表达阳性。结论：PGA无纺网具有良好的生物相容性和生物安全性，并易于新生血管的形成，作为牙周组织工程的细胞支架有良好的应用前景。     

正畸牙齿移动时牙周膜Ⅰ、Ⅲ型胶原变化的实验研究

目的 :观察大鼠牙齿移动过程中Ⅰ、Ⅲ型胶原在上颌第一磨牙牙周组织中的变化。方法 :建立大鼠磨牙移动模型 ,分为对照组和术后 1、3、5、7、10、14d组 ,用苦味酸天狼猩红染色观察各组中Ⅰ、Ⅲ型胶原的分布和构成 ,以图像分析系统分析Ⅰ、Ⅲ型胶原相对含量的变化 ,并进行统计学分析。结果 :正常牙周组织主要由Ⅰ型胶原构成 ,牙齿受力后 ,张力侧牙周组织中Ⅲ型胶原含量相对增加 ,且有时间依赖性。结论 :牙周组织在正畸力的作用下Ⅰ、Ⅲ型胶原的分布和构成发生了变化 ,Ⅲ型胶原在牙周组织改建中发挥了重要的作用     

体外培养人牙囊细胞Ⅰ型胶原与纤维粘连蛋白的表达

目的：通过体外培养人牙囊细胞，研究Ⅰ型胶原与纤维粘连蛋白在细胞中的表达，探讨其在牙齿萌出过程中转化为牙周膜细胞的机制。方法：取12岁患者因正畸需要拔除埋藏阻生牙齿的牙囊，组织块法进行人牙囊细胞的培养，通过免疫组织化学染色技术观察Ⅰ型胶原及纤维粘连蛋白在牙囊细胞中的表达及定位。结果：培养的人牙囊细胞呈梭形、卵圆形及多角形，形似成纤维细胞。在牙囊细胞中Ⅰ型胶原和纤维粘连蛋白的表达呈阳性，定位于细胞的胞质中，围绕细胞核周围染色较强，细胞核内染色阴性。结论：体外培养人牙囊细胞具有成纤维细胞的特性，具有合成与分泌Ⅰ型胶原与纤维粘连蛋白的能力。     

细胞外基质磷酸糖蛋白在大鼠牙周组织缺损愈合过程中的表达

目的:观察细胞外基质磷酸糖蛋白(matrix extracellular phosphoglycoprotein,MEPE)在大鼠牙周组织缺损愈合过程中的表达,初步探讨MEPE在牙周组织再生中可能发生的作用。方法:24只成年健康雄性Wistar大鼠随机分2组:术后14 d组和术后28 d组各12只,行左侧下颌骨开窗术,制备牙周组织缺损模型,HE染色观察牙周组织缺损愈合情况,免疫组织化学染色法观察MEPE在牙周组织缺损愈合过程中各组织的表达。结果:术后14 d,术区有部分新生牙槽骨形成,部分牙周纤维附着于根面牙本质上;术后28 d,术区完全被新生牙槽骨覆盖,在新生牙槽骨和牙本质之间可见牙周膜和部分牙骨质生成,牙周膜大部分附着于根面上;牙周组织缺损愈合过程中,MEPE在健康对照侧仅呈弱阳性表达,而在缺损区牙槽骨和牙周膜相关细胞中均呈阳性表达。结论:在牙周组织缺损愈合过程中,细胞外基质磷酸糖蛋白参与牙槽骨以及牙周膜的再生。     

富含亮氨酸的间质蛋白多糖及其在牙和牙周组织中作用的研究

富含亮氨酸的间质蛋白多糖是细胞外基质蛋白多糖的一个亚类,主要包括biglycan、decorin、fibromodulin和lumican等,在牙和牙周组织中有特殊的分布。因它们具有粘结胶原纤维和转化生长因子β的特性,从而推测它们可能在牙和牙周组织自身稳定、发育、损伤愈合中起重要作用。本文就这类蛋白多糖在牙和牙周组织中的研究概况作一综述。     

The expression of plasminogen activator system in a rat model of periodontal wound healing

BACKGROUND: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. METHODS: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). RESULTS: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. CONCLUSIONS: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.     

An immunohistochemical study of matrix molecules associated with barrier membrane-mediated periodontal wound healing

Guided tissue regeneration (GTR) is a clinical procedure developed to facilitate periodontal regeneration by using barrier membranes to selectively promote the repopulation of a periodontal defect by periodontal ligament and bone cells at the expense of epithelial and gingival connective tissue cells. The aim of this study was to gain insight into the biological events occurring during membrane mediated periodontal wound healing by examining the immunohistochemical expression of a number of extracellular matrix components in tissues treated via the GTR technique. Experimental periodontal defects were created around the second premolar tooth in 4 dogs and wound closure was achieved by application of expanded polytetrafluoroethylene membranes around each tooth and flap positioning coronal to the cementoenamel junction. The dogs were sacrificed after a 4-wk healing period, block dissections of the part of the mandible containing the experimental tooth were obtained and paraffin sections were prepared. Using standard immunohistochemical techniques, the sections were stained with a monoclonal antibody against bone morphogenetic proteins 2 and 4 (BMP-2 and -4) and polyclonal antibodies against collagen I, collagen II, decorin, biglycan, bone sialoprotein, osteopontin and osteocalcin. Collagen I was predominantly localized within the regenerating bone, whereas collagen III staining was more abundant in the soft connective tissues of the defect. Decorin and biglycan staining was faint within the extracellular matrix of the regenerating defect, although both proteoglycans exhibited intense intracellular localization within some of the cells inhabiting the defect. The staining for BMP-2 and -4 was weak within the bone but strong within the extracellular matrix of the regenerating soft tissue. Osteopontin and bone sialoprotein were strongly localized in the regenerating bone and cementum found within the defect. Osteocalcin staining was present in both the regenerating and mature cementum and associated cementoblasts, and it was relatively weaker in the regenerating bone compared to the mature bone. The observed pattern of immunolocalization of the extracellular matrix macromolecules suggests that the heterogeneous cell population filling the GTR wound had created an environment that was conducive to periodontal regeneration.     

Collagen-binding heat shock protein HSP47 expression during healing of fetal skin wounds

Wounds in fetal animals are known to heal without scar formation, but the mechanism involved remains unclear. Scar tissue is characterized by disorganized collagen bundles. The 47-kDa heat shock protein (HSP47) is a molecular chaperone that specifically targets collagen processing. However, the role of HSP47 in scar formation is poorly understood. We studied the relation of HSP47 expression in skin to scar formation during fetal wound healing. Immunohistochemical analysis demonstrated HSP47-positive cells in the epidermal cell layer of fetal and neonatal rat skin and the absence of such cells in subcutaneous tissue. After induction of a wound on the back of fetal and neonatal rats, the message of collagen type I was increased only in neonatal skin but not in fetal skin. HSP47-positive cells consistently increased for 7 days after wound induction in neonatal rats. In contrast, HSP47-positive cells and HSP47 protein were unchanged in fetal rats. We conclude that the scarless healing of fetal skin wounds is related to lack of change in HSP47 expression. HSP47 may thus be an important determinant of scar formation during wound healing.     

Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model

BACKGROUND: The therapeutic success of periodontal regenerative therapy may be compromised by our limited understanding of the wound healing process. Wound healing requires the coordination of complex cellular and molecular interactions. Recently, using an in vitro wound model, our laboratory has shown that gingival fibroblasts (GF) fill an in vitro wound more rapidly than periodontal ligament cells (PDL). This suggests that there may be differences in the levels of proliferation for these 2 cell types during the wound healing process. Such specific cell type differences may be significant in clinical outcomes of regenerative therapy. Therefore, the aim of this research was to characterize and compare the levels of both proliferation and cellular wound fill between GF and PDL using our in vitro wound model. METHODS: Primary cultures of human PDL and GF cells were established from explanted tissue, and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, or 9 days in media containing either 0.1% or 10% fetal bovine serum (FBS). At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU), fixed, and nuclei stained to measure DNA synthesis (as a measure for proliferation). Cells were counter stained with cytoplasmic stain to measure cell number. Quantitative analysis distant from (area of interest [AOI 1]), next to (AOI 2), and within the wound boundaries (AOI 3 and 4) was accomplished using computer-assisted histomorphometry. RESULTS: The levels of proliferation and cellular fill for each cell type were assessed relative to time and AOI. Overall, the PDL displayed greater (P <0.01) levels of proliferation than the GF. For both cell types, proliferation was found to be significantly (P<0.001) greater at day 2 compared to other time points. PDL displayed greater levels of proliferation than GF in all AOI, with this difference reaching significance (P<0.02) within the cell layer (AOI 1 and 2). When comparing levels of cellular fill in 10% FBS, GF displayed greater wound fill than the PDL. This difference was significant at day 6 (P <0.05) for both the marginal (AOI 3) and central (AOI 4) portions of the wound. CONCLUSIONS: These findings, demonstrating unique differences between PDL and GF with respect to proliferation and wound fill in an in vitro model, suggest that there may be cell-specific differences in cellular activity critical to periodontal wound healing. In addition, the results of this study show that the cellular proliferation response may not accurately reflect the overall wound healing response.     

Development of an in vitro wound healing model for periodontal cells

BACKGROUND: Periodontal wound healing and regeneration are influenced by a multitude of factors. While many in vitro investigations have compared the proliferation of periodontal ligament (PDL) cells and gingival fibroblasts (GF), there are no reports directly comparing the abilities of these 2 cell types to fill a wound site. As such, the goals of this research were: 1) to develop an in vitro model of wound healing which would allow for the investigation of the biologic basis of periodontal wound healing and regeneration and 2) to compare the rates of PDL cells and GF to fill an in vitro wound site. METHODS: Using both human PDL cells and GF confluent cultures, in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer. Wounded cultures were then incubated for time periods up to 12 days in media containing fetal bovine serum (FBS) concentrations (0, 0.1, 1, 5, 10, and 20%) as appropriate for each experiment. Slides were fixed, stained, and cells quantified within the wound boundaries by computer-assisted histomorphometry. The effect of wounding a cell layer was determined by comparing wounded cells as described above with a cell layer margin created without physically disrupting the cell layer. RESULTS: The in vitro model for periodontal wound healing established in this study showed that GF fill in the wound site at a significantly (P <0.0025) faster rate than PDL cells over 12 days of healing. In addition, PDL cells and GF were found to have unique concentration-dependent responses to FBS (P<0.0025). It was also shown that wounding resulted in a significant delay (P <0.01) in the initial healing response of an in vitro wound. CONCLUSION: This in vitro model demonstrated that the characteristics of wound healing are dependent on cell type, disruption (wounding) of the cell layer, and serum concentration. In addition, this model has incorporated both proliferation and migration to provide the first direct evidence demonstrating GF has a significantly greater ability to fill a wound site than PDL cells. This in vitro model may be utilized in future investigations of the biologic basis of periodontal wound healing.     

Differential expression and distribution of syndecan-1 and -2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell-cell and cell-matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cells infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell-cell and cell-matrix interactions, while syndecan-2 showed a predilection to associate with cell-matrix interactions during hard tissue formation.     

TGF-β1 influences early gingival wound healing in rats: an immunohistochemical evaluation of stromal remodelling by extracellular matrix molecules and PCNA

The effect of topically applied transforming growth factor β1 (TGF - β1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-β1 solution was applied to the surgical wound experimental sites. Two μg/μl were applied at the time of the operation, and 1 μg/μl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days post-surgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-β1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-β1 application appears to promote granulation tissue formation in periodontal wound healing.     

Critical features of periodontal flaps with regard to blood clot stability: A review

BackgroundWound healing is a multifactorial procedure involving different cell types and biological mediators. The principles of wound healing are also applicable to periodontal tissues. The formation and stability of blood clots play a vital role in successful healing of wounds in periodontal tissues. The aim of the present review was to highlight the vital factors of periodontal flaps associated with blood clot stability.HighlightThe data on periodontal regeneration and wound healing have evolved greatly in light of several factors, including space for blood clots and blood clot stabilization. In periodontal osseous defects, the stability of blood clots seems critical to wound healing. If mechanical forces can be managed by wound stabilization, the gingival flap-tooth root interface may show connective tissue repair. However, compromised adhesion is susceptible to mechanical forces and can cause wound breakage and epithelialization.ConclusionThe presence of a thick blood clot may hinder the plasmatic circulation between the recipient bed and graft during the initial stage of healing, which is critical in cases of mucogingival surgery. Root conditioning can also determine the healing consequence by enhancing blood clot adhesion.     

IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes

ObjectiveWounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity.DesignThe function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22, collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes.ResultsIL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1, which may contribute to scarless healing.ConclusionOur study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing.