Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

OBJECTIVES: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. RESULTS: EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.     

In vitro studies on periodontal ligament cells and enamel matrix derivative

Abstract The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EOF, fibronectin, bFGF, γ-interferon. IL-1β, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGFβ. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.     

细胞外基质磷酸化糖蛋白mRNA在人牙周韧带细胞成骨分化过程中的表达

目的 检测人牙周韧带细胞(periodontal ligament stem cell,PDLC)诱导矿化过程中碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)和细胞外基质磷酸化糖蛋白(matrix extracellular phosphoglycoprotein,MEPE)mRNA的表达,探讨MEPE能否作为人牙周韧带细胞的分化标记及其功能.方法 酶消化法培养PDLC并鉴定其来源,取未诱导及矿化诱导7、14和21 d的PDLC,茜素红染色检测矿化结节的形成;免疫组织化学染色检测OCN的表达;实时荧光定量反转录聚合酶链反应(RT-PCR)检测ALP、OCN和MEPE mRNA的表达变化,并对结果进行方差分析.结果 PDLC矿化诱导后茜素红染色显示钙化结节形成,免疫染色OCN表达增强;PDLC成骨诱导培养前ALP、OCN和MEPE mRNA表达系数分别为72、1.1和534.随着诱导时间延长,ALP、OCN和MEPE mRNA表达上调,诱导7 d组的表达系数分别为78、9.56和629.6,诱导14 d组的表达系数分别为290、133和638.3,诱导21 d组的表达系数分别为1108、925和2261.1.与对照组相比,各诱导组OCNmRNA的表达、诱导14 d和21 d组ALP mRNA的表达,以及诱导21 d组MEPE mRNA的表达,差异均有统计学意义(P<0.05).结论 在PDLC向成牙骨质或成骨样细胞分化过程中,MEPE mRNA与ALP和OCN呈现相似的表达变化趋势,提示MEPE与PDLC成骨分化有关,有可能作为PDLC向成牙骨质或成骨样细胞分化的标志.     

釉基质衍生物对人牙周膜干细胞成骨分化的影响

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。     

Simvastatin promotes cell metabolism, proliferation, and osteoblastic differentiation in human periodontal ligament cells

BACKGROUND: Simvastatin is one of the cholesterol lowering drugs. Recent studies demonstrated that it has a bone stimulatory effect. Periodontal ligament (PDL) cells are believed to play an important role in periodontal regeneration; that is, they may differentiate into specific cells which make cementum, bone, and attachment apparatus. It would be of interest whether simvastatin has a positive effect on PDL cells. Therefore, effects of simvastatin on cell proliferation and osteoblastic differentiation in PDL cells were analyzed. METHODS: Human PDL cells were cultured in monolayer with simvastatin for 24 and 72 hours and cell metabolism and proliferation were determined. To analyze osteoblastic differentiation, human PDL cells were cultured in organoid culture for 7, 14, and 21 days and alkaline phosphatase (ALP) activity, osteopontin (OPN), bone morphogenetic protein (BMP) -2, osteocalcin (OCN), and calcium contents were measured. They were co-treated by simvastatin and mevalonate. RESULTS: Simvastatin enhanced cell proliferation and metabolism dose-dependently after 24 hours. Simvastatin also stimulated ALP activity of human PDL cells dose-dependently, and maximum effect was obtained at the concentration of 10(8) M. In time dependent analysis, 10(8) M simvastatin stimulated ALP activity and osteopontin content after 7 days and calcium contents after 21 days. BMP-2 and OCN contents were not detected. Moreover this statin-enhanced ALP activity was abolished by mevalonate. CONCLUSION: These results suggest that at low concentration, simvastatin exhibits positive effect on proliferation and osteoblastic differentiation of human PDL cells, and these effects may be caused by the inhibition of the mevalonate pathway.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells

ObjectivePorphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) induces pro-inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration and are expected to have future applications in cellular therapies for periodontitis. However, no studies have examined the effects of P. gingivalis LPS on PDLSCs. The aim of this study was to investigate how P. gingivalis LPS affects the osteoblastic differentiation and pro-inflammatory cytokine production of PDLSCs.DesignPDLSCs were obtained from healthy adult human mandibular third molars. The identification of PDLSCs was confirmed by immunohistochemical evaluations of the mesenchymal stem cell markers STRO-1 and SSEA-4. Cell proliferation and osteoblastic differentiation were investigated by culturing the PDLSCs in a normal or osteogenic medium with P. gingivalis LPS (0, 1, or 10 μg/mL) and then measuring the alkaline phosphatase (ALP) activity and the production of collagen type 1 Alpha 1 (COL1A1), osteocalcin production, and mineralisation. Additionally, we examined the production of IL-1β, IL-6, and IL-8 in the PDLSCs.ResultsP. gingivalis LPS inhibited the ALP activity, COL1A1 and osteocalcin production, and mineralisation in the PDLSCs, which are positive for STRO-1 and SSEA-4. P. gingivalis LPS also promoted cell proliferation and produced IL-1β, IL-6, and IL-8.ConclusionsThis study provides the first findings that P. gingivalis LPS inhibits osteoblastic differentiation and induces pro-inflammatory cytokines in PDLSCs. These findings will help clarify the relationship between periodontitis and periodontal tissue regeneration.     

N-cadherin expression during periodontal ligament cell differentiation in vitro.

BACKGROUND: When confluent periodontal ligament (PDL) cells were cultured in the presence of dexamethasone (Dex), ascorbic acid (AA), and beta-glycerophosphate (GP), they underwent sequential differentiation, demonstrating distinct morphological characteristics. At 1 week, localized cell proliferation led to the formation of multilayers of cells. As cell differentiation progressed, they formed nodules by deposition of matrix in the clusters of cells at 2 weeks, and mineralized the nodules at 3 weeks. These changes implicate extensive cell-to-cell interactions. Cadherins are known to play an important role in establishing cell contacts during tissue formation. METHODS: To determine whether cadherins are involved in PDL cell differentiation, and the formation and mineralization of nodules by the cells in vitro, we investigated the expression of N-cadherin using immunofluorescence labeling and Northern blot analysis. RESULTS: Immunolabeling showed that N-cadherin was expressed in PDL cells in the stages of nodule formation and mineralization. Northern blot analysis demonstrated a 3-fold increase in the expression of N-cadherin mRNA in the stages. However, neither E-cadherin nor P-cadherin was expressed. CONCLUSIONS: Our data suggest that N-cadherin may play an important role in PDL cell differentiation and the formation of mineralized nodules by PDL cells.     

Enamel matrix derivative promotes superoxide production and chemotaxis but reduces matrix metalloproteinase-8 expression by polymorphonuclear leukocytes

Background: Polymorphonuclear leukocyte (PMN) is the predominant innate immune cell type activated in acute inflammation. The aim of this study is to determine the impact of enamel matrix derivative (EMD) on superoxide (O₂^?) generation, chemotaxis, and matrix metalloproteinase‐8 (MMP‐8) secretion by PMN in vitro to better understand the role of EMD in surgical wound healing. Methods: PMNs were isolated from healthy volunteers (n = 14). O₂^? generation was measured using a cytochrome c reduction assay. Chemotaxis was measured in a modified Boyden chamber. MMP‐8 secretion was analyzed by Western blotting. A relative density method was used to determine the percentage of MMP‐8 released from the PMNs in relation to the total cellular MMP‐8 content. Results: O₂^? generation was significantly elevated when PMNs were stimulated with EMD (200 μg/mL) (P <0.01). Secondary stimulation of PMNs with 1 μM N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) triggered earlier and more sustained O₂^? generation with EMD. EMD significantly increased PMN chemotactic activity (P <0.05). Combined stimulation with EMD plus fMLP resulted in significantly higher chemotaxis compared to fMLP alone (P <0.05). Conversely, EMD did not induce MMP‐8 secretion from PMNs. MMP‐8 secretion by PMNs in response to fMLP or serum‐opsonized zymosan stimulation was significantly inhibited by EMD (P <0.05). Conclusions: EMD has specific, differential actions on PMNs that suggest potential for enhancement of wound healing, bacterial and tissue debris clearance (O₂^? generation and chemotaxis), and suppression of tissue damage and degradation (MMP‐8). Together, the data suggest that EMD enhances wound healing and reduces inflammation.     

高浓度唑来膦酸对人牙周膜干细胞凋亡及成骨分化影响的实验研究

目的：研究高浓度唑来膦酸对体外分离的健康人牙周膜干细胞增殖、凋亡和成骨分化的影响。方法：体外分离培养健康人牙周膜干细胞,用不同浓度的唑来膦酸（5μmol/L、10 μmol/L）处理,不加药组为空白对照。 以 CCK8 检测细胞增殖,流式细胞技术检测细胞凋亡,ALP、茜素红染色及半定量分析检测细胞体外成骨分化,荧光定量PCR检测成?标志物I型胶原(COL1A1)和骨钙素（OCN）基因的表达,免疫荧光检测 OCN和 COL1A1 蛋白的表达。结果： 唑来膦酸可呈计量依赖性抑制人牙周膜干细胞增殖;唑来膦酸用药组细胞的凋亡率显著?于对照组,且随着药物浓度的增加,凋亡百分比逐渐上升;ALP和茜素红染?结果表示唑来膦酸用药组细胞的体外成骨分化能力较对照组显著下降;荧光定量PCR和免疫荧光结果提示,药物处理后OCN和 COL1A1的基因表达和蛋白表达均降低。结论：高浓度唑来膦酸显著抑制人牙周膜干细胞的增殖及体内外成骨分化,并诱导其凋亡。     

淫羊藿苷对人牙周膜细胞增殖和骨保护素mRNA表达的影响

目的：研究淫羊藿苷对人牙周膜细胞（periodontal ligament cells，PDLCs）增殖和骨保护素（osteoprotegerin，OPG）mRNA表达的影响。方法：体外培养人PDLCs，用MTT法检测不同浓度的淫羊藿苷（0．001、0．01、0、1μg／mL）、不同时间（24、48、72、96h）作用下人PDLCs的增殖水平；RT-PCR检测OPG mRNA的表达。结果：淫羊藿苷（0、001-0．1μg／mL）对人PDLCs增殖和OPG mRNA表达具有促进作用（P〈0．01），0．01μg／mL浓度作用最明显。结论：淫羊藿苷能够促进人PDLCs增殖和OPG mRNA表达。     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

诱导成体人牙周韧带干细胞向软骨细胞分化的实验研究

目的从成体人牙周组织中分离培养牙周韧带干细胞，研究其分化为软骨细胞的可行性。方法选取10～15岁的年轻患者因正畸拔除的健康牙齿，刮取根中1／3的牙周膜，采用组织块培养法得到牙周韧带细胞，待细胞达一定量后用有限元稀释法进行单细胞克隆，筛选牙周韧带干细胞（PDLSC），体外采用离心管聚集体诱导法进行软骨化诱导培养，光镜下观察诱导细胞形态学的改变，甲苯胺蓝染色、免疫组化、RT-PCR等方法检测胶原和糖蛋白的体外表达情况。结果体外离心管聚集体诱导培养3周后，实验组呈白色半透明状，甲苯胺蓝染色显示在外周区域有大量软骨基质成分沉积，组织学显示有较为明显的软骨陷窝。免疫组化及RT-PCR检测到Ⅱ型胶原表达，Ⅱ型胶原染色呈强阳性。对照组细胞团块逐渐收缩、崩解，组织学检测无软骨陷窝结构，Ⅱ型胶原免疫组化结果阴性。结论从成体人牙周组织中可分离培养出干细胞，在体外能有效增殖且保持低分化状态，具有作为软骨组织工程种子细胞来源的可能，也为牙周组织工程的应用奠定了实验基础。     

人牙周膜细胞群多向分化潜能的实验研究

目的研究体外培养的人牙周膜细胞群（hPDLP）向成骨和成脂方向分化的潜能,为牙周组织工程提供可靠的种子细胞来源。方法组织块法分离培养人牙周膜细胞群,流式细胞术检测间充质干细胞标记CD146和STRO-1的表达;利用茜素红、油红O染色、免疫组化以及反转录聚合酶链反应（RT-PCR）等检测hPDLP的多向分化标志。结果第1代hPDLP的CD146和STRO-1阳性率分别是27.20%±3.98%和4.23%±4.08%;经矿化诱导可以形成矿化结节,有钙盐沉积;经成脂诱导可见特异性脂滴形成,特异性转录因子过氧化物酶体激活物增生受体2（PPARγ2）和脂蛋白脂酶（LPL）表达上调。第8代hPDLP相对第1代的矿化能力没有差异,但成脂方向分化潜能减弱。结论体外培养的hPDLP具有向成骨和成脂样细胞分化潜能,第1~3代细胞群明显具有牙周膜干细胞的多向分化潜能优势。