Effects of ultrasound on the proliferation and differentiation of cementoblast lineage cells

BACKGROUND: The purpose of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on the proliferation and differentiation of cementoblast lineage cells. METHODS: An immortalized human periodontal ligament cell line (HPL) showing immature cementoblastic differentiation was used. Cultured HPL cells were subjected to LIPUS exposure (frequency = 1 MHz; pulsed 1:4; intensity = 30 mW/cm(2)) or sham exposure for 15 minutes per day. Expression levels of alkaline phosphatase (ALP), type I collagen (Col-I), runt-related gene 2 (Runx2), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OPN) mRNA were analyzed with real-time polymerase chain reaction analysis. Furthermore, ALP activity, collagen synthesis, and protein level of Runx2 were examined after 6 days of LIPUS exposure. RESULTS: mRNA and protein levels of ALP, Col-I, and Runx2 were significantly increased by LIPUS exposure compared to controls, whereas BSP, OCN, and OPN mRNA expression could not be detected in HPL cells, irrespective of LIPUS exposure. CONCLUSION: LIPUS enhanced ALP activity, collagen synthesis, and Runx2 expression of HPL cells, which provides important insight into the promotion of early cementoblastic differentiation of immature cementoblasts.     

Isolation of murine cementoblasts: unique cells or uniquely-positioned osteoblasts?

While cementoblasts express a number of mineral-related proteins, including bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), these proteins do not appear to be expressed by cells of the intermediate dental follicle/periodontal ligament (PDL). This information was utilized in an experimental strategy to isolate presumptive cementoblasts from the root surface of day 24 murine mandibular first molars. Using microscopic dissection techniques, molars were carefully extracted from their alveolar crypts and subjected to trypsin-collagenase digestion to remove adherent cells. Primary cultures were established and assayed for expression of proteins known to be expressed by cementoblasts at this timepoint in vivo (i.e. BSP, OPN, OC) and also an odontoblast-specific protein (i.e. DSP) to rule out contamination by pulpal cells. A subgroup of cells were found to express Type I collagen (89% of cells), BSP (46%), OPN (23%) and OC (30%); DSP was not detected within these cultures. We propose that cells within this heterogeneous population, which express this profile of osteogenic proteins, represent cementoblasts. The availability of a cementoblast cell line will make possible rigorous and controlled in vitro analysis of these cells and allow for determination of the unique characteristics of these cells not shared with other cells, particularly osteoblasts.     

Bisphosphonate modulates cementoblast behavior in vitro

BACKGROUND: Cementum formation is deemed to be instrumental for the successful regeneration of periodontal tissues, and thus events and modifiers of cementum formation and mineralization need to be determined. This study aimed to determine whether the bisphosphonate 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) altered the behavior of immortalized cementoblasts (osteocalcin-cementoblasts [OCCM]). METHODS: OCCM from transgenic mice were exposed to HEBP at concentrations ranging from 0.01 to 10.0 microM. The assays performed included the count of cell number for proliferation, Northern blot analysis for gene expression (up to 10 days for core binding factor alpha-1 [Cbfa1], bone sialoprotein [BSP], osteocalcin [OCN], and osteopontin [OPN], markers for cementoblast/osteoblast maturation/mineralization), von Kossa stain and alizarin red S stain for mineralization, and enzyme assay (p-nitrophenol phosphate cleavage) for alkaline phosphatase (ALP) activity. RESULTS: Mineral nodule formation was inhibited at the higher doses of HEBP (1.0 and 10.0 microM) only. At early stages (1, 3, and 6 days), gene expression assays revealed only subtle changes in treated cells versus untreated cells, but by day 10, groups treated with lower doses (0.01 and 0.1 microM) were markedly different at the gene expression level. OCN was significantly downregulated (70%) at the lowest dose, with less pronounced effects at higher doses. In concurrence, the master switch gene for osteoblasts, Cbfa1, was also downregulated at the lower doses. Inversely, OPN mRNA was enhanced at the lower doses. ALP activity was not altered by HEBP. CONCLUSION: Bisphosphonate alters cementoblast function in vitro through the regulation of gene expression and mineral formation.     

Cementoblast delivery for periodontal tissue engineering

BACKGROUND: Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model. METHODS: The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens. RESULTS: Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line. CONCLUSIONS: This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogen-     

Amelogenin: a potential regulator of cementum-associated genes

BACKGROUND: Studies suggest that enamel matrix proteins induce differentiation and mineralization of a variety of mesenchymal cells, including odontoblasts, osteoblasts, and cementoblasts. It has been postulated that this activity could be due to amelogenin-like proteins, known to be present in some mixtures of enamel matrix derivatives. Amelogenins have been reported to induce expression of a mineralized tissue-specific marker, bone sialoprotein (BSP), indicating that epithelial products can regulate the activity of mesenchyme-derived cells. METHODS: To explore the molecular mechanisms involved in BSP regulation, a clonal population of immortalized murine cementoblasts (OCCM-30) was exposed to full-length murine amelogenin protein (rp(H)M180), 0.1 microg/ml to 10.0 microg/ml, for 8 days in vitro. To further investigate the potential epithelial-mesenchymal interaction, an amelogenin knockout mouse model was used to examine expression of BSP and other markers, including Type I collagen, in tissue samples. RESULTS: The lowest dose of amelogenin slightly enhanced BSP expression, whereas at the highest dose, a dramatic decrease (three-fold) in BSP expression was observed. Parallel experiments showed a corresponding decrease in mineral nodule formation in vitro for cells treated with the higher dose of rp(H)M180. In situ hybridization and immunohistochemical analysis of sections from amelogenin null mice revealed a dramatic reduction in expression of BSP mRNA and protein in cementoblasts and surrounding osteoblasts in comparison to age-matched controls. In contrast, the expression of Type I collagen was not significantly different from controls. CONCLUSION: These data suggest that amelogenin may be a critical signaling molecule required for appropriate development of the periodontium.     

人脐带间充质干细胞诱导为牙周韧带样细胞的实验研究

目的 建立人牙周韧带细胞(human periodontal ligament cell,hPDLC)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cell,hUCMSC)体外非接触式共培养模型,研究hUCMSC定向分化为hPDLC的可能性,探索新的可用于牙周组织工程的种子细胞.方法 利用跨室培养装置(Transwell)培养板建屯hPDLC与hUCMSC体外非接触式共培养模型,免疫组织化学方法 检测其骨桥蛋白(osteopontin,OPN)、骨钙素(ostocalcin,OCN)及骨涎蛋白(bone sialopmtein,BSP)的表达情况,并采用蛋白质印迹法从蛋白水平定量分析诱导后hUCMSC在分子水平的改变.结果 hUCMSC在非接触式共培养体系中可以被hPDLC诱导为多角形或梭形,蛋白质印迹法检测结果 显示,共培养3、7、14及21 d后的hUCMSC在蛋白水平OCN和OPN表达上调[OCN共培养前(0.88±0.21),共培养21 d(1.42±0.17);OPN共培养前(0.93±0.13),共培养21 d(1.43±0.22)];BSP表达逐渐下调[共培养前(1.60±0.09),培养21 d(0.75±0.20)],与共培养前相比差异均有统计学意义(P＜0.05).结论 hUCMSC在一定条件下可向hPDLC定向分化,并有望成为牙周组织工程的种子细胞.     

Emdogain promotes osteoblast proliferation and differentiation and stimulates osteoprotegerin expression

PURPOSE: The purpose of this study was to investigate the effects of EMD on the growth and differentiation of osteoblastic cells (MC3T3-E1) and on the expression of osteoprotegerin (OPG), a key cytokine that inhibits osteoclastogenesis and osteoclast function. STUDY DESIGN: MC3T3-E1 cells were treated with 100 microg/mL EMD in serum-free medium for 1, 2, 3, 5, and 7 days, or in 2% fetal bovine serum (FBS) for 3 weeks. Cells incubated without EMD served as negative control. At the end of each incubation period, cell numbers were counted and total cellular mRNA was extracted. Northern blot analysis and RT-PCR were performed to determine the mRNA levels of core binding factor alpha (Cbfa1), collagen alpha1 (I), bone sialoprotein (BSP), osteocalcin (OC), insulin-like growth factor I (IGF-I), and OPG. Alkaline phosphatase (ALP) activity was also determined and compared between treatment and control groups. RESULTS: A marked increase in cell numbers was observed in EMD-treated groups from day 2 to day 7 (P < .01). mRNA expression of collagen alpha1 (I), BSP, OC, OPG, and IGF-I were up-regulated in cells treated with EMD. ALP activity was significantly increased by EMD treatment after 3-week culture under differentiating conditions (P < .05). The expression of Cbfa1 was not affected by EMD treatment from day 1 to day 5; the levels were elevated after culturing for 3 weeks in EMD-treated cells. CONCLUSIONS: EMD promotes both proliferation and differentiation of MC3T3-E1 cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.     

小鼠牙囊细胞的体外培养及表型特征

目的　建立小鼠牙囊细胞(DFC)的体外分离培养方法,研究牙囊细胞的表型特征。方法　用1%的胰蛋白酶消化分离9 d龄Balb/c小鼠的下颌第一磨牙牙胚的牙囊组织进行体外培养,倒置显微镜下观察细胞的形态,扫描电镜观察细胞的表面形貌,SP免疫组化法检测细胞碱性磷酸酶(ALP)、骨涎蛋白(BSP)、骨桥蛋白(OPN)的表达。结果　分离培养的细胞有3种类型:立方形或多角形;长梭形;非常细长的细胞形状。扫描电镜下细胞具有多形性,有大量的线状胞浆突和微绒毛;根据细胞表面有无纤维细丝覆盖,可将细胞分为有纤维细丝、无纤维细丝两个亚群。免疫组化染色显示部分牙囊细胞的ALP、BSP、OPN的表达阳性。结论　牙囊细胞具有多种细胞的表型特征, 有分化为成牙骨质细胞、牙周膜细胞和成骨细胞的潜能。