Influence of cryopreservation on human periodontal ligament cells in vitro

Cryopreservation of teeth before autotransplantation may create new possibilities in dentistry. The purpose of this study was to examine the effect of a standardised cryopreservation procedure on human periodontal ligament (PDL) cell cultures. Human PDL fibroblasts obtained from immature third molars of 11 patients were cultured and divided into two groups. The experimental group was cryopreserved and cultured after thawing. The control group was cultured without cryopreservation. A comparison was made between cryopreserved and control cells. To evaluate possible differences in the characteristics of the fibroblasts, the cells in both groups were tested for viability (membrane integrity), growth capacity and alkaline phosphatase (ALP) expression. The Wilcoxon test for paired comparison between cryopreserved and non-cryopreserved cells was performed for each characteristic. The results showed that membrane integrity of cells was not influenced by cryopreservation. There was no statistically significant difference in growth capacity between cryopreserved and control cells. Non-cryopreserved cells were slightly stronger positive for ALP, but the difference was not statistically significant. From these experiments it can be concluded that the observed parameters are not influenced by cryopreservation.     

Cell death and the regulation of populations of cells in the periodontal ligament

Summary The contribution of cell death in regulating cellular populations of periodontal ligament was studied in young adult rats. Mandibular first molar periodontium was prepared for light-microscopic radioautography after a pulse of ³H-thymidine in 6 rats and for electron microscopy in 4 rats. The labeling index for ³H-thymidine and the density of fibroblast-like cells were computed from radioautographs. The percentages of dying or dead cells and macrophages were computed from electron micrographs. The labeling index of cells within 20 m of bone and cementum was significantly lower (p<0.01) than the labeling index within the body of the periodontal ligament. The patterns of cellular density and indices of death were the inverse of the labeling indices. Macrophages were plentiful (% macrophages = 3.68%±0.30) and were clustered around blood vessels (mean distance from blood vessel=2.3 m). However, only 10% of dying or dead cells were within 10 m of blood vessels. These data show that death of cells in the periodontal ligament may, in part, balance production of cells by mitosis. The relationships between labeling index, index of death, and cellular density suggest that cells born in the middle of the periodontal ligament may migrate to regions of high cellular density near bone and cementum, and that they may die there. Macrophages do not appear to be associated with dying cells of the periodontal ligament.     

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.     

Extracellular matrix is required for muscle differentiation in primary cell cultures of larval Mytilus trossulus (Mollusca: Bivalvia)

Components of the extracellular matrix may modulate the growth factor effects that play important roles in the proliferation and differentiation of precursor cells. We developed an in vitro cultivation protocol for cells of the larval marine bivalve Mytilus trossulus to study the role that extracellular matrix components may play in myodifferentiation and replication-mediated DNA synthesis using immunofluorescence and confocal laser scanning microscopy. Here, we demonstrate that the extracellular matrix regulates the expression of muscle proteins, leading to their assembly and the terminal muscle differentiation of larval cells during cultivation. We further show that the myogenesis process progresses in cells cultivated on fibronectin, carbon or poly-l-lysine but is inhibited in cells grown on a collagen carpet. Consistent with a decrease in muscle protein expression in cells cultivated on collagen, we demonstrate an increase in the number of BrdU-positive cells in comparison with cells cultured on other substrates during the entire cultivation period. Moreover, we demonstrate that the matrix-dependent myogenic differentiation of larval mussel cells is reversible. Round-shaped cells cultivated on collagen were able to differentiate into muscle cells after reseeding on fibronectin, carbon or poly-l-lysine. In addition, cells cultured on collagen and then transplanted to fibronectin exhibited distinct cross-striation and contractile activity. Taken together, our data suggest that the extracellular matrix participates in the regulation of the proliferation and myodifferentiation of mussel trochophore progenitor cells and validate novel approaches for successfully culturing cells from bivalves over extended periods.     

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.     

Reduction of alkaline phosphatase activity in aged human osteogenic periodontal ligament fibroblasts exhibiting short telomeres

The osteogenic cell type of human periodontal ligament fibroblasts (PDLF) undergoes senescence at finite population doubling numbers unrelated to donor ages. This study investigated telomere lengths of osteogenic PDLF from differently aged donors and alterations of the osteoblast-like properties in the aged PDLF with short telomeres. Telomere lengths of osteogenic PDLF were biased towards long or short among all 15- to 51-year-old individuals, and did not show a normal distribution by Pearsons test or a correlation to donor age by simple regression analysis. In osteogenic PDLF, senescence-associated -galactosidase was expressed in 78.5% of cells in the clones with short telomeres (mean 3.02 kbp), and in 9.4% of cells in the clones with long telomeres (mean 13.06 kbp). These results suggest that human periodontium comprises aged osteogenic PDLF without correlation to age. Osteogenic PDLF with long telomeres strongly expressed alkaline phosphatase (ALPase) activity whereas cells with short telomeres expressed ALPase activity to a weaker extent. Total activity of ALPase in the clones of osteogenic PDLF with long telomeres was significantly higher than that in the clones with short telomeres. The produced amounts of both osteopontin and osteocalcin in the clones of osteogenic PDLF with long telomeres were slightly but statistically significantly smaller than those in the clones with short telomeres. These findings suggest that aged osteogenic PDLF reduce the expression of ALPase activity but that there is not a critical alteration in bone-associated protein production. Aged osteogenic PDLF may impair the ability to induce ALPase-dependent calcification.This work was supported by a grant-in-aid for Scientific Research (B) (2) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12470379).     

Isolation,culture, and verification of human sweat gland epithelial cells

Human sweat gland epithelial cells (SGECs) have been isolated and grown in vitro, However, slow proliferation makes the culture of these cells extremely difficult. The present study was carried out to explore the modified culture medium for SGECs in vitro. Full-thickness skin samples were minced (1 mm³) and digested overnight with type II collagenase. The gland coils were removed under an inverted phase-contrast microscope. An adherent culture method was used to isolate and culture SGECs. Staining with hematoxylin and eosin was performed, followed by observation of the morphologic features of these cells. Immunofluorescence staining with antibodies to cytokeratins CK7, CK18, and CK19 and carcinoembryonic antigen (CEA) was performed to verify the presence of SGECs. Growth curves by MTT were created for cells grown in serum-free keratinocyte medium and in modified keratinocyte medium containing 2.5% fetal bovine serum (FBS). One week after culturing, the cells grew well and were polygonal or irregular in shape by inverted phase contrast microscopy. Cell fusion, with a characteristic paving-stone arrangement, reached 100% after approximately 3 weeks in culture. Immunofluorescence staining indicated expression of CK7, CK18, CK19, and CEA. Compared with SGECs grown in serum-free keratinocyte medium, the proliferation of SGECs grown in modified culture medium with low concentration of FBS at days 6, 9, and 12 was significantly accelerated (p < 0.05). This study suggests that keratinocyte medium supplemented with 2.5% FBS is effective and suitable for the culture of SGECs.     

Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation. Received: 12 May 1995 / Accepted: 13 October 1995     

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.     

牙龈卟啉单胞菌感染通过激活NLRP3小体诱导牙周膜细胞炎症反应及凋亡效应

目的 观察牙龈卟啉单胞菌感染通过激活含NLR家族PYRIN域蛋白3(NLRP3)小体诱导人牙周膜细胞(hPDLCs)炎症反应及凋亡的效应。 方法 取健康前磨牙样本并分离培养hPDLCs,分为牙龈卟啉单胞菌感染的感染组和常规处理的对照组,检测细胞中NLRP3小体[NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-1]、凋亡基因[自杀相关因子(Fas)、Fas配体(FasL)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Caspase-3]的表达量及培养基中炎症细胞因子[白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]的含量。 结果 感染组hPDLCs中NLRP3、ASC、Caspase-1、Fas、FasL、Bax、Caspase-3的表达量及培养基中IL-1β、IL-18、TNF-α的含量明显高于对照组,细胞中Bcl-2的表达量明显低于对照组。 结论 牙龈卟啉单胞菌感染能够诱导hPDLCs的炎症反应及凋亡且该作用与NLRP3小体的激活有关。     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by ³H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of α-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for α-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis. Received: 2 March 1998 / Accepted: 16 June 1998