Dental pulp stem cells from traumatically exposed pulps exhibited an enhanced osteogenic potential and weakened odontogenic capacity

Objectives

Traumatic pulp exposure can bring about some permanent damages to tooth tissues including dental pulps. This study was designed to evaluate the effects of traumatic pulp exposure on the osteo/odontogenic capacity of dental pulp stem cells (DPSCs).

Methods

Rat incisors were artificially fractured and dental pulps were exposed to the oral environment for 48 h. Then, multi-colony-derived DPSCs from the injured pulps (iDPSCs) were isolated. Their osteo/odontogenic differentiation and the involvement of NF-κB pathway were subsequently investigated.

Results

iDPSCs presented a lower proliferative capacity than normal DPSCs (nDPSCs), as indicated by MTT and FCM assay. ALP levels in iDPSCs were significantly higher (P < 0.01) than those in nDPSCs. Alizarin red staining revealed that iDPSCs exhibited an increased capacity of calcium deposition. Moreover, iDPSCs expressed stronger osteogenic markers (Runx2/RUNX2 and Ocn/OCN) and less odontogenic gene/protein (Dspp/DSP) than nDPSCs in vitro. In vivo transplantation showed that nDPSCs implants generated the typical dentine-pulp complex while all iDPSCs pellets formed the osteodentin-like tissues which were immunopositive for OCN. Mechanistically, iDPSCs expressed the higher levels of cytoplasmic phosphorylated IκBα/P65 and nuclear P65 than nDPSCs, indicating an active cellular NF-κB pathway in iDPSCs. After the inhibition of NF-κB pathway, the osteogenic potential in iDPSCs was significantly down-regulated while odontogenic differentiation was up-regulated, as indicated by the decreased Alp/Runx2/Ocn and uprised Dspp expression.

Conclusions

Pulp exposure for 48 h decreased the odontogenic capacity and enhanced the osteogenic potential of DPSCs via the NF-κB signalling pathway.     

辛伐他汀对人骨髓基质干细胞成骨分化功能的促进作用

目的观察辛伐他汀对体外培养的人骨髓基质干细胞(humanBoneMarrowStromalcells,hMSCs)成骨分化功能的影响,探讨其刺激成骨的作用机制。方法体外培养来自于股骨颈骨折患者的骨髓基质干细胞,传代后实验组加入1×10-7molL的辛伐他汀,于不同时间点采用Westernblot检测核转录因子1(CoreBindingFactor1,Cbfa1)的表达,碱性磷酸酶(AlkalinePhosphatase,ALP)试剂盒检测ALP的比活性,及放射免疫法检测骨钙素(Osteocalcin,OCN)含量。结果辛伐他汀作用后,实验组与对照组比较,实验组Cbfa1蛋白表达水平增高,ALP比活性增高且骨钙素含量增加。结论1×10-7molL辛伐他汀能够促进人骨髓基质细胞成骨分化,此种促进作用可能与辛伐他汀增强其分化过程中相关蛋白的表达有关。     

缺氧环境对成牙骨质细胞成骨功能调节的研究

目的 探讨缺氧环境对成牙骨质样细胞OCCM-30成骨功能的影响.方法 利用三气培养箱构建细胞缺氧模型,以常氧条件培养为对照组,应用RT-PCR检测缺氧后OCCM-30细胞碱性磷酸酶(ALP)、骨钙素(OCN)和骨涎蛋白(BSP)的mRNA表达变化.结果 缺氧环境对ALP和OCN的mRNA表达调控表现为先促进后抑制,随着缺氧的开始,ALP及OCN的mRNA表达均逐步上调并在12 h达到顶峰,然后再逐步下降;缺氧能显著抑制BSP mRNA表达,随着缺氧的开始,BSP mRNA表达逐步下调.结论 缺氧微环境在前12 h能刺激OCCM-30细胞成骨功能的表达,但随着时间的延长,成骨功能受到抑制.     

机械牵张力对成骨细胞碱性磷酸酶及骨钙蛋白的影响

目的:研究不同大小的机械牵张力对成骨细胞碱性磷酸酶(ALP)活性及骨钙蛋白(OCN)的影响。方法:通过自制的多通道细胞牵张应力加载系统对小鼠成骨样细胞MC3T3-E1同时施加6%、12%和18%的机械牵张力,作用24 h和48 h后,用ALP试剂盒检测细胞受力后ALP的变化、用放射免疫的方法检测OCN表达的变化(P<0.01)。结果:细胞受力后,其ALP活性随牵张力值的增大明显降低(P<0.01);12%牵张力可使OCN的表达明显增加,而6%、18%牵张力组和对照组比较,无显著性差异(P>0.05)。结论:不同大小的机械牵张力可以影响成骨细胞的ALP活性及OCN的表达,与力值大小密切相关。     

强直脊柱炎成纤维细胞分泌骨钙素的实验研究

目的 :了解强直性脊柱炎 (ankylosingspondylitis ,AS)成纤维细胞分泌骨钙素的情况 ,探讨该细胞的成骨特性。方法 :采用AS患者髋关节囊体外培养AS成纤维细胞 ,并设正常对照组 ,通过传代培养 ,取第 3代细胞的第 3、6、9、12d培养上清液 ,用12 5Ⅰ标记放射免疫 (RIA)法测定骨钙素 (osteocalcin ,OCN)含量。结果 :AS成纤维细胞组上清中OCN分泌量明显高于正常成纤维细胞组 (P <0 .0 1)。结论 :AS成纤维细胞能分泌骨钙素 ,在生物学特性上更接近成骨细胞     

补肾活血固齿方对小鼠颅顶前成骨细胞骨钙素合成的影响

目的观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1细胞)骨钙素(OCN)合成的影响。方法用10%补肾活血固齿方含药血清、无药血清、胎牛血清分别培养MC3T3-E1细胞24 h、48 h、72 h,125Ⅰ放射免疫法检测3组细胞上清液中OCN的含量。结果含药血清组24、48、72 h OCN含量与无药血清组、胎牛血清组同期比较差异均有统计学意义(P0.05),含药血清组24、48、72 h OCN含量显著高于无药血清组、胎牛血清组同期。结论补肾活血固齿方可提高MC3T3-E1细胞分泌OCN的水平,促进该细胞分化为成骨细胞,加速骨的形成。     

Dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells

Sophora japonica L. fruit prevents bone loss by inhibiting osteoclast activity. We hypothesized that S japonica L. extracts could promote osteoblast differentiation. To test this hypothesis, we investigated the effect of S japonica L. on osteoblast differentiation and identified the bioactive compound(s) from S japonica L. The mature fruit of S japonica L. was partitioned with ethanol, hexane, dichloromethane (DCM), ethyl acetate, and butanol, and their effects were tested on osteoblast differentiation of C3H10T1/2 cells. DCM fractionated extracts were identified as the most osteogenic fractions. DCM fractionated extracts dose-dependently stimulated alkaline phosphatase activity and matrix mineralization. The DCM fractions also induced expression of osteoblast markers such as alkaline phosphatase, osterix, and osteocalcin in C3H10T1/2 and primary bone marrow cells. Genistein was found abundantly in the DCM fractions. Furthermore, the genistein and DCM fractions similarly modulated the expression of estrogen target genes and were both active in transfection assays that measured estrogen agonistic activity. Finally, pharmacological inhibition by treatment with an estrogen receptor antagonist or specific inhibition of gene expression by small interference RNAs targeted to estrogen receptor-β abolished the effects of the DCM extracts, further supporting the idea that the genistein in the DCM extracts mediated the pro-osteogenic effects. Taken together, we identified genistein as the key phytoestrogen responsible for the effects of S japonica L. on osteoblast differentiation.     

Design of electrospun nanofibrous mats for osteogenic differentiation of mesenchymal stem cells

The clinical translation potential of mesenchymal stem cells (MSCs) in regenerative medicine has been greatly exploited. With the merits of high surface area to volume ratio, facile control of components, well retained topography, and the capacity to mimic the native extracellular matrix (ECM), nanofibers have received a great deal of attention as bone tissue engineering scaffolds. Electrospinning has been considered as an efficient approach for scale-up fabrication of nanofibrous materials. Electrospun nanofibers are capable of stimulating cell–matrix interaction to form a cell niche, directing cellular behavior, and promoting the MSCs adhesion and proliferation. In this review, we give a comprehensive literature survey on the mechanisms of electrospun nanofibers in supporting the MSCs differentiation. Specifically, the influences of biological and physical osteogenic inductive cues on the MSCs osteogenic differentiation are reviewed. Along with the significant advances in the field, current research challenges and future perspectives are also discussed.     

重组人骨形态发生蛋白2及制动因素对兔肌腱端重建的影响*

背景：骨-肌腱结合结构的重建可能类似于软骨内成骨,只有形成坚固的止点,重建的韧带才能够真正发挥其生理功能。重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)可使未分化间质细胞、成纤维细胞、成肌细胞及骨髓的基质细胞逆转分化为骨系细胞。 目的：观察rhBMP-2和制动因素对肌腱止点结构重建的影响。 方法：新西兰白兔建立肌腱止点结构重建模型后,随机分成3组。向rhBMP-2短期制动组和rhBMP-2长期制动组实验兔靠近胫骨隧道入口的屈趾总腱内注入重组人骨形态发生蛋白2溶液20 µL,模型组不予注射。术后rhBMP-2短期制动组术肢石膏固定1周后拆除;rhBMP-2长期制动组和模型组兔术肢全程石膏固定。各组分别于手术后2,4,8周通过免疫组化法和RT-PCR测定隧道入口部肌腱中的Ⅰ,Ⅱ型胶原、骨碱性磷酸酶和骨钙素的表达。 结果与结论：rhBMP-2可使肌腱止点结构重建兔Ⅰ型胶原、骨碱性磷酸酶和骨钙素表达水平随时间的延长而增加,而Ⅱ型胶原在术后增加至4周时表达至峰值,8周表达有所下降。注射rhBMP-2未对腱重建兔Ⅰ型胶原mRNA表达产生明显变化;而注射rhBMP-2可使Ⅱ型胶原、骨碱性磷酸酶和骨钙素mRNA表达显著增加(P < 0.05),减少制动时间也能使Ⅱ型胶原、骨碱性磷酸酶和骨钙素mRNA表达显著增加(P < 0.05)。rhBMP-2能促进Ⅰ,Ⅱ型胶原、骨碱性磷酸酶和骨钙素的表达,并具有诱导肌腱内异位软骨成骨的作用,使肌腱端结构重建成为可能;长时间制动阻碍肌腱附着点的重建。 关键词：肌腱止点;重组人骨形态发生蛋白2;胶原;碱性磷酸酶;骨钙素;肌肉肌腱;组织工程     

Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells

Objectives

Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.

Design

Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 μg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain^® (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.

Results

Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain^®. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.

Conclusions

The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.