Guided tissue regeneration may modulate gene expression in periodontal intrabony defects: a human study

Background and Objective: Guided tissue regeneration has been shown to lead to periodontal regeneration; however, the mechanisms involved remain to be clarified. The present study was carried out to assess the expression of genes involved in the healing process of periodontal tissues in membrane‐protected vs. nonprotected intrabony defects in humans. Material and Methods: Thirty patients with deep intrabony defects (≥ 5 mm, two or three walls) around teeth that were scheduled for extraction were selected and randomly assigned to receive one of the following treatments: flap surgery alone (control group) or flap surgery plus guided tissue regeneration (expanded polytetrafluorethylene (e‐PTFE) membrane) (test group). Twenty‐one days later, the newly formed tissue was harvested and quantitatively assessed using the polymerase chain reaction assay for the expression of the following genes: alkaline phosphatase, receptor activator of nuclear factor‐κB ligand, osteoprotegerin, osteopontin, osteocalcin, bone sialoprotein, basic fibroblast growth factor, interleukin‐1, interleukin4, interleukin‐6, matrix metalloproteinase2 and matrix metalloproteinase9. Results: Data analysis demonstrated that mRNA levels for alkaline phosphatase, receptor activator of nuclear factor‐κB ligand, osteoprotegerin, osteopontin, bone sialoprotein, basic fibroblast growth factor, interleukin‐1, interleukin‐6, matrix metalloproteinase‐2 and matrix metalloproteinase ‐9 were higher in the sites where guided tissue regeneration was applied compared with the control sites (p < 0.05), whereas osteocalcin mRNA levels were lower (p < 0.05). No difference was observed in interleukin‐4 mRNA levels between control and test groups. Conclusion: Within the limits of this study, it can be concluded that genes are differentially expressed in membrane barrier‐led periodontal healing when compared with flap surgery alone, and this may account for the clinical outcome achieved by guided tissue regeneration.     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway

1 Background

Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside‐induced osteogenic differentiation of hPDL cells.

2 Methods

hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real‐time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β‐catenin was analyzed by both immunofluorescence and western blot.

3 Results

The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real‐time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose‐dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β‐catenin as evaluated by immunofluorescence staining and western blot analysis. Pre‐treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside‐induced β‐catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside‐induced DMP1 protein expression.

4 Conclusion

The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β‐catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.     

The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model

Kim Y‐T, Park J‐C, Choi S‐H, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. J Periodont Res 2012; 47: 514–524. © 2012 John Wiley & Sons A/S Background and Objective: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well‐standardized assessment tool that excludes the innate healing factors from the animals. Material and Methods: Human periodontal ligament stem cells that were isolated and characterized from teeth (n = 12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n = 20). Animals were killed after several different healing periods: 3 d (n = 4), 1 (n = 4), 2 (n = 4), 4 (n = 4) and 8 wk (n = 4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson’s trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue‐specific markers associated with periodontal regeneration. Results: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid‐like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey’s fibers, was associated with active mineralization of cementum‐like tissues, and in the fourth phase, the maturation of cementum‐like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well‐orchestrated time order. Interestingly, well‐organized cementum‐like and periodontal ligament fiber‐like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell‐rich zone. Conclusion: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell‐based treatment techniques.     

Doxycycline Counteracts Bone Morphogenic Protein 2–Induced Osteogenic Mediators

Background: Microbial colonization during wound healing may exaggerate the inflammatory response and could adversely affect the outcome of periodontal regeneration. Bone morphogenic proteins (BMPs) directly augment bone regeneration. Interestingly, inhibitors of tissue collagenases, such as sub‐antimicrobial–dose doxycycline, also indirectly promote hard‐tissue regeneration. In this study, it is hypothesized that BMP2‐mediated bone regeneration would be positively affected by simultaneous treatment of sub‐antimicrobial–dose doxycycline. Methods: Human periodontal ligament (PDL) cells were stimulated with: 1) 10 ng/mL BMP2; 2) 1 μg/mL doxycycline; or 3) a combination of the two. The expressions of alkaline phosphatase, osteocalcin, osteonectin, and osteopontin were analyzed along with in vitro mineralized nodule formation and calcium accumulation. Results: BMP2 was a potent inducer of osteocalcin/osteopontin (statistically significant at P <0.01) and osteonectin in PDL cells relative to stimulation with doxycycline. However, doxycycline relative to BMP2 (statistically significant at P <0.001) upregulated the expression of alkaline phosphatase and in vitro mineralized nodule formation. Contrary to expected results, combined BMP2 and doxycycline induced a statistically significant (P <0.001) downregulation of alkaline phosphatase, osteocalcin, osteonectin/osteopontin, and in vitro mineralized nodule formation compared to stimulation with either BMP2 or doxycycline alone. Conclusions: Combined treatment of BMP2 and doxycycline in PDL cells counteracts the osteogenic mediators. Molecular interaction of growth factors should be explored before using a combination of these biologic molecules. It is important and clinically relevant to determine whether tetracycline and its other derivatives also counteract BMP functions. Animal models should be used to confirm these in vitro results.     

Fluid Shear Stress Stimulates Osteogenic Differentiation of Human Periodontal Ligament Cells via the Extracellular Signal‐Regulated Kinase 1/2 and p38 Mitogen‐Activated Protein Kinase Signaling Pathways

Background: Fluid shear stress (FSS) is a major type of mechanical stress that is loaded on human periodontal ligament cells (hPDLCs) during mastication and orthodontic tooth movement. This study aims to clarify the effect of FSS on the osteogenic differentiation of hPDLCs and to further verify the involvement of mitogen‐activated protein kinase (MAPK) signaling in this process. Methods: After isolation and characterization, hPDLCs were subjected to 2‐hour FSS at 12 dynes/cm², and cell viability, osteogenic gene mRNA expression, alkaline phosphatase (ALP) activity, secretion of Type I collagen (COL‐I), and calcium deposition were assayed. The levels of phosphorylated p38 and phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) in response to FSS were detected by Western blot, and the involvement of ERK1/2 and p38 MAPK signaling pathways in hPDLC osteogenesis under FSS was investigated using the specific MAPK inhibitors U0126 (2Z,3Z)‐2,3‐bis[amino(2‐aminophenylthio)methylene]succinonitrile,ethanol) and SB203580 (4‐[4‐(4‐fluorophenyl)‐2‐(4‐[methylsulfinyl]phenyl)‐1H‐imidazol‐5‐yl]pyridine). Results: The application of FSS on hPDLCs induced an early morphologic change and rearrangement of filamentous actin. ALP activity, messenger RNA (mRNA) levels of osteogenic genes, COL‐I, and osteoid nodules were significantly increased by FSS. Moreover, ERK1/2 and p38 were activated in different ways after FSS exposure. U0126 and SB203580 completely blocked the FSS‐induced increases in ALP activity and osteogenic gene mRNA expression and osteoid nodules formation. Conclusions: FSS is an effective approach for stimulating osteogenic differentiation of hPDLCs. The ERK1/2 and p38 MAPK signaling pathways are involved in this cellular process.     

High‐power,red‐light‐emitting diode irradiation enhances proliferation,osteogenic differentiation,and mineralization of human periodontal ligament stem cells via ERK signaling pathway

1 Background

Light‐emitting diode (LED) is attracting attention as a new light source for phototherapy. However, its effects on periodontal tissue regeneration remain unknown. The aim of this study was to examine the effects of high‐power, red LED irradiation on human periodontal ligament stem cells (PDLSCs), which play an important role in periodontal tissue regeneration.

2 Methods

PDLSCs were derived from adult human third molars. The light source was red LED (peak wavelength: 650 nm). Energy densities ranging from 0 to 10 J/cm² were tested to determine the optimal dose. PDLSC proliferation was measured using two parameters: live cell protease and ATP levels. After the cells were induced to differentiate, the effect of LED irradiation on osteogenic differentiation and mineralization was examined, with particular focus on the extracellular signal‐regulated kinase (ERK)1/2 signaling pathway using an ERK inhibitor (PD98059).

3 Results

LED irradiation at 8 J/cm² led to a significant increase in PDLSC proliferation and enhanced Runx2 and Osterix mRNA expression, Alkaline phosphatase activity, procollagen type I C‐peptide and osteocalcin production, calcium deposition, and alizarin red S staining. In addition, LED induced the activation of ERK1/2, and the effects of LED on PDLSC proliferation, differentiation, and mineralization could be suppressed by treatment with PD98059.

4 Conclusions

The results of this study show that 650‐nm high‐power, red, LED irradiation increases PDLSCs proliferation, and osteogenic differentiation and mineralization, mediated by ERK1/2 activation. These findings suggest that LED may be a useful tool for periodontal tissue regeneration.     

糖基化终末产物对人牙周膜干细胞骨向分化过程中的Wnt经典信号通路研究

目的 探讨Wnt经典通路相关因子Dickkopf蛋白1（DKK-1）、β-链蛋白（β-catenin）在糖基化终末产物（AGEs）介导的人牙周膜干细胞（hPDLSCs）骨分化过程中的变化。方法 通过体外组织块法和有限稀释法克隆化培养获取hPDLSCs,实验分两组：正常hPDLSCs组（N-hPDLSCs组）和AGEs刺激的正常hPDLSCs组（A-hPDLSCs组）。对2组细胞成骨矿化诱导后行碱性磷酸酶（ALP）和茜素红染色;实时聚合酶链反应（real time PCR）检测成骨基因,以及Wnt经典通路中相关因子DKK-1、β-catenin;Western blot检测相关成骨蛋白、细胞核蛋白β-catenin。结果 成骨诱导后,A-hPDLSCs组ALP染色明显浅于N-hPDLSCs组;茜素红染色A-hPDLSCs组钙化结节少于N-hPDLSCs组;real time PCR与Western blot的结果显示A-hPDLSCs组BSP、ALP、Runx-2的表达都较低,差异有统计学意义（P<0.05）。Wnt经典信号通路中相关因子：A-hPDLSCs组β-catenin表达高于N-hPDLSCs组,A-hPDLSCs组DKK-1表达明显低于N-hPDLSCs组,并且Weston blot显示A-hPDLSCs组核蛋白β-catenin的表达高于N-hPDLSCs组。结论AGEs可以使hPDLSCs骨向分化过程中Wnt经典通路的抑制因子DKK-1下调,细胞核中的β-catenin表达升高,激活了Wnt经典通路,并且抑制了骨分化。     

Effects of platelet-rich fibrin on human periodontal ligament fibroblasts and application for periodontal infrabony defects

Background: Platelet‐rich fibrin (PRF) by Choukroun’s technique is derived from an autogenous preparation of concentrated platelets. Little is known about the effects of PRF on periodontal ligament fibroblasts (PDLFs) and the application of PRF for periodontal regeneration. Methods: PDLFs were derived from healthy individuals undergoing extraction for orthodontic reasons. Blood collection was carried out from healthy volunteers. PRF was obtained from a table centrifuge centrifuged at 3000 rpm for 12 minutes. The effects of PRF on PDLFs were determined by measuring the expression of phosphorylated extracellular signal‐regulated protein kinase (p‐ERK), osteoprotegerin (OPG) and alkaline phosphatase (ALP) activity. Moreover, we retrospectively examined the feasibility and safety of reconstructing the periodontal infrabony defects with PRF in six patients. Results: PRF was found to increase ERK phosphorylation and OPG in PDLFs in a time‐dependent manner (p < 0.05). ALP activity was also significantly upregulated by PRF (p < 0.05). Application of PRF in infrabony defects exhibited pocket reduction and clinical attachment gain after six months. Periapical radiography revealed radiographic defect filled in grafted teeth. Conclusions: The enhancement of p‐ERK, OPG and ALP expression by PRF may provide benefits for periodontal regeneration. Clinical and radiologic analysis showed that the use of PRF is an effective modality for periodontal infrabony defects.     

煅烧牙粉影响人牙周膜干细胞体外矿化的自噬机制研究

目的 探讨自噬在煅烧牙粉调节牙周膜干细胞体外矿化中的作用,为牙周膜干细胞的定向诱导分化和牙周病的治疗提供参考。方法 选用成人完整的牙齿在300 ℃的条件下煅烧后,研磨成粉,与α-MEM混合制备成20 μg/mL牙粉条件培养基,与人牙周膜干细胞共培养。采用流式细胞技术检测其对牙周膜干细胞凋亡的影响,通过Western blot检测、免疫荧光染色、茜素红染色和ALP染色观察检测其对牙周膜干细胞自噬活性及体外矿化的影响。结果 流式细胞技术结果显示牙粉对牙周膜干细胞的凋亡无明显影响（P＞0.05）;Western blot结果显示煅烧牙粉显著上调人牙周膜干细胞的自噬相关蛋白Beclin1,ATG5的表达和LC3Ⅱ/LC3Ⅰ的比值,以及明显下调了P62的蛋白水平（P<0.05）;免疫荧光染色显示牙粉促进人牙周膜干细胞中LC3在胞质内点状聚集;茜素红染色显示牙粉促进牙周膜干细胞的体外矿化;ALP染色显示自噬活性抑制剂氯喹降低牙粉对牙周膜干细胞体外矿化的促进作用。结论 煅烧牙粉通过激活自噬调节牙周膜干细胞的体外矿化作用。     

基质细胞衍生因子-1对炎症和正常来源的 人牙周膜干细胞成骨分化能力的影响

目的 通过体外培养炎症来源的人牙周膜干细胞（iPDLSCs）和正常来源的人牙周膜干细胞（hPDLSCs）,比较基质细胞衍生因子-1（SDF-1）对于两种来源细胞的成骨分化作用。方法 采用组织块酶消化法原代培养iPDLSCs 和hPDLSCs,经有限稀释法纯化,通过流式细胞仪对干细胞表面标记物检测鉴定后,对其进行成骨诱导;MTT法检测并比较SDF-1对两种来源的细胞增殖能力的影响;茜素红染色检测SDF-1作用于两种来源的细胞后钙化骨量的表达;碱性磷酸酶法比较SDF-1作用于两者的成骨分化能力;逆转录聚合酶链反应（RT-PCR）法检测SDF-1作用于两种牙周膜干细胞前后成骨相关基因表达水平的变化。结果 两种来源的牙周膜细胞经纯化后均阳性表达干细胞标记物。hPDLSCs较iPDLSCs增殖能力高;两种细胞经SDF-1成骨诱导培养后,成骨相关基因的表达水平均较诱导前明显上调（P<0.05）,SDF-1在50、200 ng·mL ^-1时分别对iPDLSCs和hPDLSCs细胞成骨分化作用最明显（P<0.05）。结论 正常来源和炎症来源的人牙周膜干细胞均具有成骨分化能力,SDF-1可增强两种来源的牙周膜干细胞的成骨分化能力。     

基质细胞衍生因子-1对炎症和正常来源的人牙周膜干细胞成骨分化能力的影响

目的 通过体外培养炎症来源的人牙周膜干细胞（iPDLSCs）和正常来源的人牙周膜干细胞（hPDLSCs）,比较基质细胞衍生因子-1（SDF-1）对于两种来源细胞的成骨分化作用。方法 采用组织块酶消化法原代培养iPDLSCs 和hPDLSCs,经有限稀释法纯化,通过流式细胞仪对干细胞表面标记物检测鉴定后,对其进行成骨诱导;MTT法检测并比较SDF-1对两种来源的细胞增殖能力的影响;茜素红染色检测SDF-1作用于两种来源的细胞后钙化骨量的表达;碱性磷酸酶法比较SDF-1作用于两者的成骨分化能力;逆转录聚合酶链反应（RT-PCR）法检测SDF-1作用于两种牙周膜干细胞前后成骨相关基因表达水平的变化。结果 两种来源的牙周膜细胞经纯化后均阳性表达干细胞标记物。hPDLSCs较iPDLSCs增殖能力高;两种细胞经SDF-1成骨诱导培养后,成骨相关基因的表达水平均较诱导前明显上调（P<0.05）,SDF-1在50、200 ng·mL ^-1时分别对iPDLSCs和hPDLSCs细胞成骨分化作用最明显（P<0.05）。结论 正常来源和炎症来源的人牙周膜干细胞均具有成骨分化能力,SDF-1可增强两种来源的牙周膜干细胞的成骨分化能力。     

Effects of C‐Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells

Background: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. Methods: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full‐length amelogenin (rh174); 2) amelogenin cleavage product lacking the C‐terminal (rh163); 3) amelogenin cleavage product lacking the N‐terminal (rh128); and 4) the C‐terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast‐like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐ tetrazolium assay and cell proliferation enzyme‐linked immunosorbent assay. Mitogen‐activated protein kinase (MAPK)–extracellular signal‐regulated kinase (ERK) (MAPK–ERK) pathway was examined by Western blot analysis. Results: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). Conclusion: C‐terminal amelogenin cleavage product increased proliferation of HCEM through MAPK–ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.