Emdogain stimulates matrix degradation by osteoblasts

Emdogain has been used clinically for periodontal regeneration, although the underlying molecular mechanisms are not clear at present. In this study, we hypothesized that Emdogain stimulated degradation of type I collagen via osteoblasts. We showed that Emdogain enhanced cell-mediated degradation of type I collagen in an MMP-dependent manner. Although MG-63 cells spontaneously produced a zymogen form of MMP-1, treatment with Emdogain significantly induced the generation of the active form of this enzyme. We demonstrated that MMP-3 was produced from MG63 cells in response to Emdogain in a MEK1/2-dependent manner. Concomitantly, blocking of MEK1/2 activation by U0126 significantly inhibited the generation of the active form of MMP-1 without affecting the total production of this collagenase. These results suggest that Emdogain facilitates tissue regeneration through the activation of the collagenase, MMP-1, that degrades matrix proteins in bone tissue microenvironments.     

中药桂皮醛对成骨细胞增殖及成骨功能的影响

牙周炎和种植体周围炎是牙菌斑引起的发生在牙周支持组织上的慢性感染性炎症,通常可导致牙周组织结构破坏,引起牙和种植体松动脱落。课题组前期研究发现中药桂皮醛能杀灭牙周可疑致病菌并具有良好的抗炎作用。目的：研究桂皮醛对成骨细胞增殖及成骨功能的影响。方法：以小鼠成骨细胞MG63细胞株为研究对象,通过MTT法检测128,64,32,16,8ugml-1五种浓度的桂皮醛对成骨细胞增殖的影响;通过碱性磷酸酶活性、钙含量的测定及矿化结节的观察等检测五种浓度的桂皮醛对成骨细胞成骨功能的影响。利用SPSS13.0软件对数据进行方差分析和t检验。结果：与空白对照组相比,16,8ugml-1组成骨细胞的增殖无明显抑制,碱性磷酸酶活性、钙含量及矿化结节数有升高（P〈0.05）;128,64,32ugml-1组成骨细胞增殖及成骨功能均有一定抑制,且抑制作用随着浓度升高而加强。结论：桂皮醛对成骨细胞增殖及成骨功能的影响与浓度有关,体现出效应剂量依赖性。较低浓度的桂皮醛（16,8ugml-1）对成骨细胞的成骨功能有促进作用。     

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells

BACKGROUND: Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. METHODS: This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). RESULTS: EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. CONCLUSIONS: EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.     

促矿化液对大鼠成骨细胞增殖分化的影响

目的比较大鼠成骨细胞在矿化条件与非矿化条件下增殖和分化的过程,评价促矿化液对其生理功能的影响,明确促矿化液加入细胞培养环境的适宜时间。方法取SD大鼠头盖骨做成骨细胞原代培养,将增殖稳定后的第4代细胞分别在矿化条件和非矿化条件下培养,检测其形态、碱性磷酸酶活性、细胞周期等。结果大鼠成骨细胞增殖基本稳定后促矿化液组与无促矿化液组细胞分裂增殖指数相似,但前者碱性磷酸酶活性明显较高且持久。结论成骨细胞增殖基本稳定后加入促矿化液对细胞增殖无影响,但可明显促进细胞矿化功能,是加入促矿化液诱导细胞矿化功能的较好时机。     

The W9 peptide directly stimulates osteoblast differentiation via RANKL signaling

Objective

A RANKL-binding peptide, WP9QY (W9), is known to inhibit mouse osteoclastogenesis by stimulating the production of autocrine factors such as bone morphogenetic proteins (BMPs) to induce osteoblast differentiation. In the present study, we investigated whether osteoblastic differentiation is mediated by RANKL signaling.

Enhanced proliferation, attachment and osteopontin expression by porcine periodontal cells exposed to Emdogain

Emdogain^® (EMD) is an enamel matrix derivative extracted from developing porcine teeth with demonstrated periodontal regenerative potential. EMD has been shown to influence a number of properties of periodontal ligament cells including proliferation, cell attachment and matrix synthesis. To date, the effect of EMD on the epithelial cell rests of Malassez (ERM) is unknown.

In this study, periodontal ligament fibroblasts, ERM, alveolar bone cells and gingival fibroblasts were obtained from porcine periodontal ligament, alveolar bone and gingiva. This study investigated, in vitro, the effect of EMD at three concentrations on proliferation, cell attachment and expression of mRNA for two mineralised tissue-related proteins (osteopontin and bone sialoprotein).

As for other periodontal cells, the ERM proliferative response was enhanced by EMD. Attachment assays revealed a highly significant increase for ERM and gingival fibroblasts after EMD treatment at all concentrations. This study has also shown that EMD stimulated expression of osteopontin mRNA by ERM and alveolar bone cells.

The results from this study provide evidence that EMD enhanced cellular events related with proliferation, attachment and osteopontin mRNA expression by porcine periodontal cells, in a manner consistent with its role in periodontal regenerative therapy.     

Sodium butyrate stimulates mineralized nodule formation and osteoprotegerin expression by human osteoblasts.

OBJECTIVE: Butyric acid (sodium butyrate; BA) is a major metabolic by-product of main periodontopathic bacteria present in subgingival plaque. In the present study, we examined the effects of BA on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, extracellular matrix protein expression, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) in normal human osteoblasts. METHODS: The cells were cultured with 0, 10(-8), 10(-6) or 10(-4)M BA for up to 12 days. Mineralized nodule formation was detected by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium assay kit. The gene and protein expression levels for type I collagen, bone sialoprotein (BSP), osteopontin (OPN), M-CSF, and OPG were examined using real-time PCR and ELISA, respectively. RESULTS: Mineralized nodule formation and the calcium content of mineralized nodules were increased by BA in a dose-dependent manner. Cell proliferation and ALPase activity were not affected by the addition of BA. Following the addition of 10(-4)M BA, the expression levels of BSP, OPN, and OPG increased, whereas the expression levels of type I collagen and M-CSF were not markedly affected. CONCLUSION: These results suggest that BA stimulates bone formation by increasing the production of BSP and OPN, whereas it suppresses osteoclast differentiation by increasing the production of OPG by human osteoblasts.     

Effect of cerium chloride application on fibroblast and osteoblast proliferation and differentiation

ObjectiveThis study investigated effects of cerium-chloride on fibroblast and osteoblast differentiation and proliferation.MethodsMC3T3-E1 cells were plated for an alkaline phosphatase (ALP) activity test. On day 3, CeCl₃-solutions (1, 5 or 10, w/v%) were added. After 10 s, the solutions were aspirated and washed to remove residual CeCl₃. On day 6 ALP activity was determined. Cell activity and proliferation was assessed by thiazolyl blue tetrazolium dye reduction assay (MTT-test) also 3 days after exposure to the CeCl₃-solutions. Calcium deposition by preosteoblastic cells was determined 4 weeks after the exposure of the cells by alizarin red staining. Furthermore, in all experiments the influence of adding rhBMP-2 was tested. Statistical analysis was performed by repeated-measures ANOVA using the post hoc Fisher least significant difference (LSD) test. Statistical significance was set at p < 0.05.ResultsExposure to a Ce-solution of 1% or higher reduced ALP activity significantly. The addition of rhBMP-2 was able to elevate ALP activity above control level. MTT-test showed a significant decrease in cellular activity by 5% Ce or higher. The addition of rhBMP-2 had no positive effect. For human foreskin fibroblasts, exposure to even 10% Ce yielded a significant increase in cellular activity. Ce reduced calcium deposition to a level of below 50% of the control. The addition of rhBMP-2 restored mineral deposition to control levels for all Ce concentrations.ConclusionCeCl₃ had a stimulating effect on fibroblasts but a depressing influence on osteoblasts. However, adding rhBMP-2 could compensate the latter influence.     

Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.     

半导体激光(GaAlAs)对大鼠成骨细胞增殖和分化的影响

目的检测半导体激光(GaAlAs)照射对大鼠成骨细胞增殖和分化的影响,探讨半导体激光促进种植体骨结合及治疗种植体周围炎的作用.方法体外培养SD大鼠成骨细胞,分为7组,A组,连续激光200mW,照射lmin;B组,连续激光20mW,照射2min;C组,脉冲激光200mW,照射lmin;D组,脉冲激光200mW,照射2min;E组,连续激光300mW,照射lmin;F组,连续激光300mW,照射2min;G组,不予激光照射作为对照.用四唑盐(MTT)法检测细胞增殖情况,用碱性磷酸酶(ALP)试剂盒检测ALP活性,用细胞免疫实验检测细胞外基质中羟脯氨酸浓度,观察细胞胶原蛋白的合成分泌.结果激光照射组与对照组比较,成骨细胞的增殖能力无显著性变化;羟脯氨酸浓度显著升高,除F组外,照射时间2min组(B、D)均较相应能量的lmin组(A、C)升高更为明显(P＜0.05);除F组外,实验组细胞ALP活性均显著增加(P＜0.05),各实验组之间无明显差异(P＞0.05).结论适当能量和时间条件下,半导体激光照射可以促进大鼠成骨细胞的分化成熟,对细胞增殖功能无明显影响.     

Effects of fluoride-modified titanium surfaces on osteoblast proliferation and gene expression

PURPOSE: The objective of this study was to test the hypothesis that fluoride-modified titanium surfaces would enhance osteoblast differentiation. Osteoblast growth on a moderately rough etched fluoride-modified titanium surface (alteration in cellular differentiation) was compared to osteoblast growth on the same surface grit-blasted with titanium dioxide. The potential role of nanometer-level alterations on cell shape and subsequent differentiation was then compared. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM) cultures were incubated on the respective surfaces for 1, 3, and 7 days, followed by analysis for cell proliferation, alkaline phosphatase (ALP) -specific activity, and mRNA steady-state expression for bone-related genes (ALP, type I collagen, osteocalcin, bone sialoprotein [BSP] II, Cbfa1, and osterix) by real-time polymerase chain reaction (PCR). RESULTS: The different surfaces did not alter the mRNA expression for ALP, type I collagen, osterix, osteocalcin, or BSP II. However, Cbfa1 expression on the fluoride-modified titanium surface was significantly higher (P < .001) at 1 week. The number of cells on this surface was 20% lower than the number of cells on the surface TiO2-blasted with 25-microm particles but not significantly different from the number of cells on the surface TiO2-blasted with 125-microm particles. Cells grown on all the titanium surfaces expressed similar levels of ALP activity. CONCLUSIONS: The results indicated that a fluoride-modified surface topography, in synergy with surface roughness, may have a greater influence on the level of expression of Cbfa1 (a key regulator for osteogenesis) than the unmodified titanium surfaces studied.     

牙髓卟啉单胞菌脂多糖对小鼠成骨细胞增殖和分化的抑制作用

目的 观察牙髓卟啉单胞菌(Porphyromonas endodontalis,Pe)脂多糖(lipopolysaccharides,LPS)对成骨细胞MC3T3-E1增殖、碱性磷酸酶(alkaline phosphatase,ALP)活性和白细胞介素(IL)-6分泌情况的影响,探讨Pe-LPS在成骨细胞增殖与分化过程...     

Emdogain regulation of cellular differentiation in wounded rat periodontium

Emdogain is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for alpha-smooth muscle actin, a myofibroblastic marker. Morphometry and 3H-proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain. Staining for alpha-smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain. In conclusion, Emdogain does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.     

The effect of platelet-rich plasma on osteoblast and periodontal ligament cell migration, proliferation and differentiation

Background and Objective:  Platelet-rich plasma is used to deliver growth factors, in a safe and convenient manner, for enhancing bone and periodontal regeneration. However, conflicting reports regarding its effectiveness suggest that further study of the relevant cellular mechanisms is required. The aim of this study was to investigate the in vitro effect of platelet-rich plasma on osteoblasts and periodontal ligament cell function.
Material and Methods:  Various concentrations of platelet-rich plasma (100, 50, 20 and 10%) and platelet-poor plasma, obtained from human donors, were applied to primary cultures of human osteoblasts and periodontal ligament cells. [³H]-Thymidine incorporation, crystal violet staining and MTT assays were utilized to assess DNA synthesis and proliferation. Migration was determined by assessing the cell response to a concentration gradient, while differentiation was assessed using Alazarin Red staining.
Results:  Platelet-rich plasma and platelet-poor plasma had stimulatory effects on the migration of both human osteoblasts and periodontal ligament cells. At 24 h, DNA synthesis was suppressed by the application of the various concentrations of platelet-rich plasma, but over a 5-d period, a beneficial effect on proliferation was observed, especially in response to 50% platelet-rich plasma. Platelet-poor plasma resulted in the greatest enhancement of cellular proliferation for both cell types. At a concentration of 50%, platelet-rich plasma and platelet-poor plasma facilitated differentiation of both cell types.
Conclusion:  Platelet-rich plasma can exert a positive effect on osteoblast and periodontal ligament cell function, but this effect is concentration specific with maximal concentrations not necessarily resulting in optimal outcomes. Platelet-poor plasma also appears to have the ability to promote wound healing-associated cell function.