Histochemical study of the influence of transplanted teeth with periodontal ligament on the binding of peanut agglutinin in rat dorsal skin

Our previous study showed that the expression of carbohydrate residues in junctional epithelium (JE) after resection is closely related to attachment and stratification along the dental root surface. However, the influence of the periodontal ligament on carbohydrate expression has still not been clarified. In this study we examined the relationship between the presence of periodontal ligament and the expression of carbohydrate residues on epithelium regenerating along root surfaces. We transplanted extracted rat molars with or without periodontal ligament tissue, repeatedly frozen and thawed teeth with ligament and demineralized teeth without ligament into the dorsal skin of rats. After 2, 3, 5, 7, 10, 14 or 21 d, dorsal cutaneous tissues containing transplanted teeth were resected, fixed, decalcified and embedded in paraffin. Serial sections were stained histochemically with the HRP-conjugated lectin. peanut agglutinin (PNA) to observe the expression of carbohydrate residues in regenerating epithelium. Histochemical observation revealed that lectin binding reactions were changed from the characteristics of skin to those of JE when the regenerating epithelium was attached and stratified along the tooth with unfrozen or frozen tissue. These results suggested that the structural formation and expression of PNA in regenerating epithelium around the root surface were influenced by not only the tooth but also by the periodontal ligament.     

The potential coronal migration of periodontal ligament tissue following experimental regeneration

The aim of the present study was to determine if the periodontal ligament cells can migrate onto curretted root surfaces following an experimental regeneration procedure. Buccal mucoperiosteal flaps were elevated in all three premolar regions in 5 mongrel dogs. The buccal bone was reduced to approximately 7 mm from its original level on 30 roots. Before the elevated flaps were replaced and sutured, Gore-Tex filters were adjusted to cover exposed root surfaces. No membranes were placed over 10 root surfaces, which served as controls. After 7 weeks of healing, the animals were sacrificed and all the roots were examined for histological evaluation of contralateral experimental and control teeth. With polychrome staining, the new periodontal ligament fibers of the curetted root surfaces were dyed methyl blue and the adjacent gingival collagen fibers were dyed red. On the experimental surface, coronal migration of periodontal ligament cells and connective tissue attachment were identified over long distances of curetted root surface. Periodontal ligament spaces were more prevalent in experimental sites than in control root surfaces. The results of the experiment suggest that the periodontal ligament cells actually migrated onto the curetted root surfaces when a periodontal space was created by physical barriers.     

Altered expression level of calbindin D28k in the periodontal ligament of rat molar in response to changes in occlusal force

The present immunohistochemical study was designed to investigate the alteration in the expression level of calbindin D28k in the periodontal ligament of the rat molar in response to changes in occlusal force to clarify the physiological role(s) of this protein in the ligament. In normal periodontal ligament of the lower first molar, immunoreactivity for calbindin D28k was found in the spindle-shaped cells, presumably fibroblasts, at the alveolar portion of the ligament at the distal side of the mesial root and mesial side of the distal root. Following the overload of occlusal force to the upper first molar by bite-raising, the number and immunoreactivity of the positive cells in the periodontal ligament of the lower first molar increased gradually. A more significant increase was detected at 7 d following the bite-raising compared to the normal animals. When occlusal force was removed by the extraction of the upper first molar, the expression level of calbindin D28k in the periodontal ligament of the lower first molar rapidly decreased, however a subsequent gradual increase was recognized. Statistical analysis of the spatial immunoreactivity of calbindin D28k in the periodontal ligament was performed and showed statistically significant differences. The present results suggest that calbindin D28k may play important roles in the homeostasis and cytoprotection of the periodontal fibroblasts against occlusal force.     

Use of enamel matrix protein derivative before dental reimplantation: a histometric analysis

The use of enamel matrix protein in the treatment of periodontal defects has shown a favorable action on the proliferation of periodontal ligament cells, as well as on collagen formation and mineralization. The goal was to evaluate, histologically and histometrically, periodontal tissue regeneration after dental reimplantation using enamel matrix protein derivative (Emdogain, Biora AB, Malm?, Sweden). Male rats (Albinus, Wistar), weighing between 180 and 200 g, were divided in 3 groups. Animals in group I (control) had the upper right incisors extracted, the root canal was sealed with calcium hydroxide, and teeth were reimplanted in their alveoli. Group II underwent the same procedure, but the remaining periodontal ligament was removed from the root surfaces by root planing before reimplantation. In group III,following removal of the periodontal ligament, Emdogain was applied to the root surfaces. Animals were sacrificed 7, 20, and 60 days after reimplantation, and the alveoli were fixed, processed, and stained with hematoxylin and eosin. Formation of periodontal ligament, resorption areas, and ankylosis were analyzed. The results showed that group I (control) was better than groups II and III, with statistically significant differences on days 7 and 20 after reimplantation for formation of periodontal ligament. It may be concluded that with the methodology used, Emdogain was unable to stimulate tissue repair in reimplanted teeth.     

Differential effects of estrogen on DNA synthesis in human periodontal ligament and breast cancer cells

BACKGROUND: It is important to clarify the biological function of the female sex hormones estrogen and progesterone in periodontal ligament cells, as these hormones may affect periodontal health. We have previously shown that human periodontal ligament cells express estrogen receptor beta (ERbeta) but not ERalpha, whereas human breast cancer cells (MCF7) express both ERalpha and ERbeta. Data on progesterone receptor (PgR) expression in human periodontal ligament cells have not been reported. OBJECTIVES: Determine PgR expression in human periodontal ligament and MCF7 cells and to investigate how estrogen affects DNA and collagen synthesis in these two cell types showing different pattern of expression for ERalpha and beta. METHODS: Periodontal ligament cells were obtained from the periodontal ligament of premolars extracted for orthodontic reasons and MCF7 cells from the American Type Culture Collection (ATCC). PgR expression was determined by immunocytochemistry. DNA and collagen synthesis was determined by [(3)H]thymidine and L-[(3)H]proline incorporation, respectively. RESULTS: PgR immunoreactivity was observed in nuclei of MCF7 but not periodontal ligament cells. Treatment with estrogen (17beta-estradiol, E(2)) at physiological concentrations for 24 h stimulated DNA synthesis by more than two times in MCF7 cells, whereas there was no effect on periodontal ligament cell DNA synthesis. The ER blocker ICI 182780 fully reversed the stimulatory effect of E(2). Not only short-term (24 h) but also long-term (5 days) treatment with E(2) lacked effect on DNA synthesis in periodontal ligament cells. Neither periodontal ligament cell viability nor collagen synthesis was affected by E(2) treatment. Identical results were observed in periodontal ligament cells from male and female subjects. CONCLUSIONS: Human MCF7 but not periodontal ligament cells express PgR, suggesting that progesterone via PgR affects MCF7 but not periodontal ligament cells. Further, estrogen stimulates breast cancer MCF7 cell proliferation, whereas it has no effect on proliferation of periodontal ligament cells, probably reflecting cell type specific ER expression pattern in these two cell types.     

Morphology of Malassez's epithelial rest-like cells in the cementum: transmission electron microscopy, immunohistochemical, and TdT-mediated dUTP-biotin nick end labeling studies

BACKGROUND AND OBJECTIVE: It is known that epithelial islands are embedded in the cementum during tooth root formation, but details of this process remain unknown. The purpose of this study was to investigate the dynamic characteristics of Malassez's epithelial rest cells in the cementum during tooth root formation in pigs in vivo. MATERIAL AND METHODS: The first molars of 6-mo-old pigs were used in this study. Specimens were decalcified before being embedded in paraffin. Paraffin sections were investigated using TdT-mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemical, and ultrastructural techniques. RESULTS: Malassez's epithelial rest cells were located close to the root surface at the apical one-third of the periodontal ligament, and epithelial clusters surrounded by distinct lamina cementia were sometimes observed in the cementum. TUNEL-positive cells were detected only in the cementum. Malassez's epithelial rest cells in the periodontal ligament were completely surrounded by basement membranes, but epithelial clusters in the cementum were only intermittently surrounded by such membranes. Cytokeratin-positive cells in the superstratum of the cementum were directly connected by cementocytes and by desmosome-like structures. However, organelles were scarce in the cytokeratin-positive cells in the substratum of the cementum, and the matrix of the cementum was deposited in the cells. CONCLUSION: These results suggest that the majority of the fragmented Hertwig's root sheath remains in the periodontal ligament and that some cells, which are connected to cementoblasts, are embedded in the cementum and progress to apoptosis.     

Up-regulation of estrogen receptor-beta expression during osteogenic differentiation of human periodontal ligament cells

Background and Objective: Estrogen has been shown to up‐regulate the expression of osteoblastic phenotypes of human periodontal ligament cells via binding to estrogen receptors and may also help periodontal tissue regeneration. However, which subtype of estrogen receptor (α or β) is predominately expressed in human periodontal ligament cells, and how estrogen receptor expression is regulated during the osteogenic differentiation of human periodontal ligament cells, is still unclear. This study aimed to explore the expression and regulation of estrogen receptor subtypes in human periodontal ligament cells and during their osteogenic differentiation. Material and Methods: Human periodontal ligament cells derived from 10 individual age‐matched donors (five male and five female donors) were cultured. Human periodontal ligament cells under osteogenic induction (group M) and the corresponding controls (group C) were harvested on days 7, 14 and 21 for estrogen receptor detection. Results: Both estrogen receptor‐α and estrogen receptor‐β mRNAs were expressed in human periodontal ligament cells from all of the 10 donors. Protein only of estrogen receptor‐β (not of estrogen receptor‐α) was detected and was shown to be located in the nuclei of human periodontal ligament cells. The expression levels of estrogen receptor‐β mRNA and protein from both male and female donors in group M were significantly higher compared with group C during the 21‐d study period. In comparison, the expression level of estrogen receptor‐α mRNA of the donors was not significantly different from that of the controls during osteogenic differentiation and no estrogen receptor‐α protein was detected. Conclusion: The results suggest that estrogen receptor‐β may be the predominant subtype expressed in human periodontal ligament cells and may actively participate in the osteogenic differentiation process of human periodontal ligament cells, both in male and in female subjects.     

Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells' expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.     

T-helper 17 cells mediate the osteo/odontoclastogenesis induced by excessive orthodontic forces

Oral Diseases (2012) 18 , 375–388 Objective: The aim of this study was to investigate how T‐helper 17 cells (Th17 cells), interleukin (IL)‐17, and interleukin‐6 contribute to root resorption during orthodontic tooth movement. Materials and Methods: Fifteen male 6‐week‐old Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. The expression levels of TRAP, IL‐17, the IL‐17 receptor (IL‐17R), and IL‐6 proteins were determined in periodontal ligament (PDL) by immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of IL‐17 on IL‐6 release were investigated using human PDL cells in vitro. The effect of IL‐17 on osteoclastogenesis was evaluated by TRAP staining, actin ring staining, and the pit formation assay. Results: The immunoreactivity for Th17, IL‐17, IL‐17R, and IL‐6 was detected in PDL tissue subjected to the orthodontic force on day 7. IL‐17 increased the release of IL‐6 from human periodontal ligament cells in a time‐dependent manner. Moreover, IL‐17 stimulated osteoclastogenesis from human osteoclast precursor cells, and these effects were partially suppressed by an anti‐IL‐6 antibody. Conclusion: These results suggest that Th17 cells may aggravate the process of orthodontically induced inflammatory root resorption.     

人牙周膜细胞在弹性牵张力作用下核心结合因子mRNA的表达

目的 探讨人牙周膜细胞在应力作用下的成骨特性及核心结合因子(corebinding factor a 1，cbfal)在正畸成骨和正畸牙齿移动中的作用机制。方法 体外原代培养新生SD大鼠颅骨成骨细胞，进行大鼠cbfal cDNA片段的克隆和序列测定，制备大鼠cbfal cDNA探针。人牙周膜细胞在弹性膜上体外培养，采用体外培养细胞加载系统加载弹性牵张力。用原位杂交方法检测cbfal mRNA的表达。结果 正常对照组不加力的人牙周膜细胞未检测到cbfal mRNA阳性表达，实验组加载弹性牵张力的人牙周膜细胞出现cbfal mRNA阳性表达。结论 弹性牵张力可以诱导人牙周膜细胞向成骨样细胞分化；cbfal作为转录因子是正畸成骨的调控途径之一。     

淫羊藿苷对人牙周膜细胞增殖及骨向分化的影响

目的：探讨淫羊藿苷对人牙周膜细胞增殖及骨向分化的影响,为淫羊藿苷在治疗牙周病方面的应用提供一定依据。方法：体外培养人牙周膜细胞,矿化诱导条件下将第3代细胞与10～0.001mg/L,6个浓度的淫羊藿苷作用,通过噻唑蓝（MTT）比色法、ALP活性测定及BSP表达水平,反应其对人牙周膜细胞增殖及分化的影响。结果：作用24h,各药物浓度均能促进人牙周膜细胞增殖（P〈0.05）。作用48h,1～0.001mg/L组可促进人牙周膜细胞增殖（P〈0.05）。作用72h,0.1～0.001mg/L组可促进人牙周膜细胞增殖（P〈0.05）,10mg/L组抑制人牙周膜增殖（P〈0.05）;作用72h,1～0.001mg/L组可促进人牙周膜碱性磷酸酶（ALP）活性（P〈0.05）;作用72h,0.1～0.001mg/L组的BSP表达水平较对照组显著增加（P〈0.05）。结论：淫羊藿苷在一定浓度范围内可促进人牙周膜细胞增殖及骨向分化。     

实验性正畸牙齿移动中RANKL在牙周组织中表达的研究

目的 观察实验性正畸牙齿移动过程中，大鼠牙周组织中转录因子NFKB受体活化剂配体（receptor activator of NF-kB ligand／RANKL）的表达及定位，进一步探讨RANKL的来源、作用机制以及与正畸牙齿移动中骨改建的关系。方法 采用SP免疫组织化学方法检测RANKL在正畸大鼠牙周组织中的表达，并采用计算机图像分析的方法对各组RANKL的表达强度进行半定量分析。结果 实验性正畸牙齿移动过程中：①RANKL主要表达于牙槽骨表达的成骨细胞、破骨细胞和牙周膜成纤维细胞胞浆和胞膜中。②RANKL在牙周组织内的表达和分布在时间上是不均衡的，随时间的增加呈先增高后回降的趋势，峰值在第5天。③RANKL在牙周组织内的表达和分布在部位上是不均衡的，压力侧的表达量高于张力侧，牙槽骨表面的表达量高于近牙骨质侧的牙周膜。结论 RANKL的表达与正畸牙槽骨改建过程中破骨细胞的生命过程密切相关，RANKL通过旁分泌、自分泌机制作用于破骨细胞前体、成熟破骨细胞膜表达的受体RANKL，促进破骨细胞前体的分化、成熟，激活成熟破骨细胞的功能，加速骨改建。     

体外评估不同根管充填材料对牙周膜细胞的毒性

目的通过体外实验评估4种根管充填材料对牙周膜细胞的毒性。方法体外培养牙周膜细胞,取AH Plus、Cortisomol、RoekoSeal和氧化锌丁香油糊剂4种材料的新鲜和陈旧样本,新鲜样本在材料固化后即刻提取浸提液,陈旧样本于37℃恒温水浴箱内放置7d提取浸提液,并分别制成50％稀释液,与人牙周膜细胞接触24h、48h,通过二甲基噻唑二苯基四唑溴盐比色法,比较不同时期、不同浓度、不同材料对牙周膜细胞的毒性。结果新鲜样本：RoekoSeal对牙周膜细胞无毒性,AH Plus有轻度毒性,Cortisomol和氧化锌丁香油糊剂有重度毒性;陈旧样本：RoekoSeal对牙周膜细胞有轻度毒性,AH Plus,Cortisomol有中度毒性,氧化锌丁香油糊剂仍为重度毒性;随着浸提液与细胞作用时间延长,RoekoSeal和AH Plus的毒性增强,统计学上有显著意义;而Cortisomol和氧化锌丁香油糊剂的毒性与细胞作用时间长短无相关性。4种材料浸提液原液和50％稀释液之间无显著差异。结论RoekoSeal不论在何种情况下,是4种材料中对牙周膜细胞影响最小的材料。     

Differential chemotactic effect of cementum attachment protein on periodontal cells

Selective re-population of the root surface by periodontal ligament cells is considered a key factor in Periodontal regeneration. A recently isolated cementum attachment protein (CAP) has been shown to enhance fibroblast attachment. In the present study the potential of CAP to selectively attract periodontal ligament cells (PLC) was studied in vitro in a micro-chemotaxis system. Human periodontal ligament cells and gingival fibroblasts (GF) were compared for their chemotactic response to either cementum attachment protein or to fibronectin. Murine dermal fibroblasts (MDF) served as control, irrelevant to the periodontium. The chemotactic response of PLC to fibronectin at 10^?8 M was of a similar magnitude as that of GF (16 ± 5 and 11 ± 3 cells/field, respectively), but both were significantly lower than the response of MDF (28 ± 3 cells/field). The chemotactic response of periodontal ligament cells to the cementum attachment protein at 10^?7 M was higher (36 ± 5 cells/field) than that of gingival fibroblasts or murine dermal fibroblasts (14 ± 2 and 16 ± 2 cells/field, respectively). These results suggest that cementum attachment protein can influence the selective re-population of root surfaces by periodontal ligament cells.     

A histopathological study of the role of periodontal ligament tissue in root resorption in the rat

Whether periodontal ligament (PDL) tissue is capable of inducing root resorption was examined. The distal root of the rat molar was sectioned at the furcation and the PDL tissue removed from the root (non-PDL group, n=40). The distal root with the PDL intact was also prepared (PDL-intact group, n=40). The roots were transplanted into the dorsal skin of the rat. On the 1st, 3rd, 5th, 7th, 10th, 14th, 21st or 28th day after transplantation, the roots were removed together with surrounding dorsal subcutaneous tissue and were fixed, demineralized and embedded in paraffin. Serial sections from each block were stained with haematoxylin and eosin or by the tartrate-resistant acid phosphatase (TRAP) method to observe root-resorbing cell formation. Cyclo-oxygenase-2 (COX2) was also detected immunohistologically to examine prostaglandin E(2) production. On the 7th day after transplantation, multinucleated root-resorbing cells with TRAP were observed in the PDL-intact group. The number of TRAP-positive cells peaked on the 10th day after transplantation. COX2-positive cells were observed in PDL during the early experimental stages. No root resorption was seen in the non-PDL group. These results suggest that PDL tissue is involved in the formation of root-resorbing cells and root resorption.