人牙龈成纤维细胞与牙周膜细胞的生物活性

采用组织块法分离培养牙周膜细胞和牙龈成纤维细胞,测定二者的增殖特性和ALP活性,利用免疫组化和FCM方法比较Ⅰ、Ⅲ型胶原、BMP的表达情况,以观察对比两种细胞的生物学特性的异同。找出牙龈成纤维细胞和牙周膜细胞在胶原基质合成方面存在差异,发现Ⅰ、Ⅲ型胶原可作为鉴别两种细胞的标志物,ALP与BMP可作为鉴别两种细胞的标志,牙周膜细胞比牙龈成纤维细胞具有较强的成骨能力。从而为今后改良两种细胞成为牙周组织工程的种子细胞奠定基础。     

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.     

In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulateproliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodentalligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of youngand healthy people. The stem cells were cultured in α-MEM Medium in three groups, one group with basic medium contains (α-MEM and FBS 10 % and 50 mmol of β_ gelisrophosphat and L_ ascorbic acid µg/ml), the second group: basic medium withdexamethasone and the third one: basic medium without any osteogenic stimulant. Mineralization of cellular layer was analyzedwith Alizarin red stain method. Osteogenic analysis was done by Alkaline phosphates and calcium test. These analysis indicatedthat the amount of intra-cellular calcium and alkaline phosphates in the Dexamethasone group was far more than the control andbasic group (P<0.05). The results of Alizarin red stain indicated more mineralization of cultured cells in Dexamethasone group(P<0.05). The study results showed that Dexamethasone has significant osteogenic effect on PDL stem cells and further studies arerecommended to evaluate its effect on treatment of bone disorders.     

The inhibition of periodontal ligament stem cells osteogenic differentiation by IL-17 is mediated via MAPKs

Periodontal disease (PD), a degenerative bacterially induced disease of periodontium, can lead to bone resorption and teeth loss. Development of PD includes a strong inflammatory reaction, which involves multiple immune cells and their secreting factors including interleukin-17 (IL-17), which is not only an important modulator of immune and hematopoietic responses but also affects bone metabolism. In the present study we aimed to determine whether IL-17 affects the regenerative potential of periodontal ligament mesenchymal stem cells (PDLSCs) by investigating its ability to modulate osteogenic differentiation of these cells in vitro along with associated signaling pathways. Our results revealed that IL-17 inhibited both the proliferation and migration of PDLSCs and decreased their osteogenic differentiation by activating ERK1,2 and JNK mitogen-activated protein kinases. Obtained data suggested that IL-17 might contribute to alveolar bone loss in PD.     

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y ₁ receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y ₁ receptor antagonist), MRS 2365 (a specific P2Y ₁ agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y ₁ in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca ²⁺ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.     

Therapeutic potential of periodontal ligament stem cells

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques.     

The effect of different platelet-rich plasma concentrations on proliferation and differentiation of human periodontal ligament cells in vitro

OBJECTIVES: The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in periodontal regeneration by regulating cellular activity. The purpose of this study was to evaluate the biological effects of platelet-rich plasma (PRP) on human periodontal ligament cells (hPDLCs) in vitro. MATERIALS AND METHODS: Primary cultures of hPDLCs were obtained from healthy premolars. PRP was isolated by two-step centrifugation. Two main growth factors present in the thrombin-activated PRP (platelet-derived growth factor [PDGF-AB] and transforming growth factor-beta1 [TGF-beta1]) were evaluated using ELISA assay. Activated PRP or the combination of recombined human TGF-beta1 (rhTGF-beta1) and PDGF-AB (rhPDGF-AB) were added to hPDLCs in different concentrations to assess cell proliferation and osteogenic differentiation. RESULTS: PRP contained high levels of TGF-beta1 and PDGF-AB. Cell attachment, proliferation and ALP activity were enhanced by addition of PRP or rhTGF-beta1 and rhPDGF-AB combination to the cell cultures, while the stimulatory potency of PRP was much greater than the latter. These stimulatory effects presented in a dose-dependant manner, it seemed that PRP with 50~100 ng/ml TGF-beta1 was an ideal concentration. CONCLUSIONS: PRP can enhance hPDLC adhesion, proliferation and induce the differentiation of hPDLC into mineralized tissue formation cell; thereby contribute to the main processes of periodontal tissue regeneration. For economical and biological reasons, PRP has more clinical beneficial than analogous growth factors.     

牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的 观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。 方法 培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A405值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。 结果 与对照组比较,P.gingivalis组Wnt3a、βcatenin、pGSK3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A405值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A405值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。 结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

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.     

Immunomodulatory properties of human periodontal ligament stem cells

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell‐based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non‐cell contact dependent suppression of PBMNC proliferation in co‐cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN‐γ partially suppressed PBMNC proliferation when compared to CMs without IFN‐γ stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF‐β1, hepatocyte growth factor (HGF) and indoleamine 2, 3‐dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN‐γ. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667–676, 2009. © 2009 Wiley‐Liss, Inc.     

Mass acquisition of human periodontal ligament stem cells

The periodontal ligament (PDL) is an essential fibrous tissue for tooth retention in the alveolar bone socket. PDL tissue further functions to cushion occlusal force, maintain alveolar bone height, allow orthodontic tooth movement, and connect tooth roots with bone. Severe periodontitis, deep caries, and trauma cause irreversible damage to this tissue, eventually leading to tooth loss through the destruction of tooth retention. Many patients suffer from these diseases worldwide, and its prevalence increases with age. To address this issue, regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells, scaffolds, and cytokines as well as their combined applications. In particular, PDL stem cells (PDLSCs) have been well studied. In this review, I discuss comprehensive studies on PDLSCs performed in vivo and contemporary reports focusing on the acquisition of large numbers of PDLSCs for therapeutic applications because of the very small number of PDLSCs available in vivo.     

The effect of platelet-rich plasma (PRP) combined with a bone allograft on human periodontal ligament (PDL) cells

The prominent purpose of the study was the evaluation of the in vitro mitogenic effect of three different homologous platelet-rich plasma (PRP) preparations (PRPa, PRPb, PRPc) on three different lines of periodontal ligament (PDL) cells (PDL_1,2,3), cultured alone or in combination with a demineralized freeze-dried allograft (DFBA). PDL cell cultures were derived from the mid root of three maxillary caries-free premolars extracted for orthodontic reasons. Cells were grown and reached confluence. To evaluate the mitogenic effect of all exogenous factors (PRPa, PRPb, PRPc and DFBA) on PDL cells, specific number of cells (10.000/well) was cultured in the presence or absence of the above factors. Each PRP preparation (5% v/v) was added in all cell lines, in the absence or presence of 10 mg/ml of DFBA. The cells were also treated with 25 ng/ml bFGF (positive control). The mitogenic effect was evaluated 24 h after incubation, using the Trypan blue exclusion assay. The results revealed that all PRP preparations act as potent mitogens as they significantly induced cell proliferation on PDL_1,2,3 lines. All PRP preparations when added alone in the PDL cell cultures, exhibited a significant advantage over the positive control (bFGF). The addition of DFBA to PRP did not influence significantly cell proliferation in all cell lines, comparatively to PRP alone, at the time -period studied. The findings of this study demonstrate the beneficial role of PRP alone or combined with the bone graft on periodontal ligament cells in vitro, suggesting that it may be considered as a potential biological approach in periodontal regeneration.     

Expression and effects of epidermal growth factor on human periodontal ligament cells

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.     

Role of ferritin in the cytodifferentiation of periodontal ligament cells

This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.