Suppressing ROS generation by apocynin inhibited cyclic stretch-induced inflammatory reaction in HPDLCs via a caspase-1 dependent pathway.

It has been reported that cyclic stretch could induce inflammatory reaction in human periodontal ligament cells (HPDLCs). Though reactive oxygen species (ROS) has been reported to be involved in pathogen-induced periodontal inflammatory reaction, its role in the force-related periodontal diseases has not been well clarified. This study inspected the role of ROS in the cyclic stretch-induced inflammatory reaction in HPDLCs and studied the inhibitory effect of antioxidant apocynin on this inflammatory reaction. Results confirmed that cyclic stretch induced inflammatory reaction and production of ROS in HPDLCs. This inflammatory reaction was inhibited by apocynin through blocking the production of ROS. The cyclic stretch also induced the expression of caspase-1 and NLRP3 inflammasome, which could also be inhibited by apocynin. Moreover, the cyclic stretch-induced inflammatory reaction was inhibited by caspase-1 inhibitor. Collectively, it is the first time that increased intracellular ROS was proved to play as an intermediate signal in the cyclic stretch-induced inflammatory reaction in HPDLCs, via a caspase-1-dependent pathway. The inhibitory effect of apocynin on the cyclic stretch-induced inflammatory reaction in HPDLCs shows the potential of antioxidants in the treatment of force-related periodontal inflammatory diseases.

ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human periodontal ligament fibroblasts via Rac1/Cdc42.

Rho GTPase-activating protein (Rho-GAP) and Rho GDP dissociation inhibitor (Rho- GDI) are two main negative regulators of Rho GTPase. Our previous work has found that Rho-GDI and Rho GTPase are involved in the response of human periodontal ligament (PDL) cells to mechanical stress. However, whether Rho-GAP also has a role in this process remains unknown. Here, we attempted to find the Rho-GAP gene that may be involved in pathological stretch-induced apoptosis of PDL cells. Human PDL fibroblasts were exposed to 20% cyclic strain for 6 hours or 24 hours, after which the expression levels of ARHGAP10, ARHGAP17, ARHGAP21, ARHGAP24 and ARHGAP28 were determined. Results showed that ARHGAP17 expression decreased the most obviously after treatment of stretch. In addition, ARHGAP17 overexpression abolished 20% cyclic strain-induced apoptosis. Therefore, ARHGAP17 has an important role in pathological stretch-induced apoptosis of human PDL fibroblasts. Moreover, we found that ARHGAP17 overexpression also alleviated cyclic strain-induced activation of Rac1/Cdc42, a major downstream target of ARHGAP17. Furthermore, two Rac1 inhibitors, NSC23766 and EHT 1864, both attenuated ARHGAP17 knockdown-mediated apoptosis in human PDL fibroblasts. Collectively, our data demonstrate that ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human PDL fibroblasts through inactivating Rac1/Cdc42. This study highlights the importance of Rho signalling in the response of human PDL fibroblasts to mechanical stress.

Inhibition of transglutaminase activity in periodontitis rescues periodontal ligament collagen content and architecture.

BACKGROUND AND OBJECTIVE: Periodontal disease (PD) afflicts approximately 50% of the population in the United States and is characterized by chronic inflammation of the periodontium that can lead to loss of the periodontal ligament through collagen degradation, loss of alveolar bone, and to eventual tooth loss. Previous studies have implicated transglutaminase (TG) activity in promoting thin collagen I fiber morphology and decreased mechanical strength in homeostatic PDL. The aim of this study was to determine whether TG activity influenced collagen assembly in PDL in the setting of periodontal disease. MATERIAL AND METHODS: A ligature model was used to induce clinically relevant PD in mice. Mice with ligature were assessed at 5 and 14 days to determine PDL collagen morphology, transglutaminase (TG) activity, and bone loss. The effects of inhibition of TG on PDL were assessed by immunohistochemistry and second-harmonic generation (SHG) to visualize collagen fibers in native tissue. RESULTS: Ligature placement around the 2nd molar resulted in significant bone loss and a decrease in total collagen content after 5 days of ligature placement. A significant increase in thin over thick fibers was also demonstrated in mice with ligature at 5 days associated with apparent increases in immunoreactivity for TG2 and for TG-mediated N-ε-γ-glutamyl cross-links in PDL. Inhibition of TG activity increased total collagen and thick collagen fiber content over vehicle control in mice with ligature for 5 days. SHG of PDL was used to visualize and quantify the effects of TG inhibition on enhanced collagen fiber organization in unfixed control and diseased PDL. CONCLUSION: These studies support a role of TG in regulating collagen fiber assembly and suggest that strategies to inhibit TG activity in disease might contribute to restoration of PDL tissue integrity.

Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells.

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.

Up-regulated ferritin in periodontitis promotes inflammatory cytokine expression in human periodontal ligament cells through transferrin receptor via ERK/P38 MAPK pathways.

OBJECTIVE: Ferritin, an iron-binding protein, is ubiquitous and highly conserved; it plays a crucial role in inflammation, which is the main symptom of periodontitis. Full-length cDNA library analyses have demonstrated abundant expression of ferritin in human periodontal ligament. The aims of the present study were to explore how ferritin is regulated by local inflammation, and to investigate its functions and mechanisms of action in the process of periodontitis. METHODS: Human gingival tissues were collected from periodontitis patients and healthy individuals. Experimental periodontitis was induced by ligature of second molars in mice. The expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH) were assessed by immunohistochemistry. Meanwhile, after stimulating human periodontal ligament cells (HPDLCs) with P. gingivalis-lipopolysaccharide (LPS), interleukin (IL)-6, and tumor necrosis factor-α (TNF-α), the expression of FTH and FTL were measured. Then, IL-6 and IL-8 were measured after incubation with different concentrations of apoferritin (iron-free ferritin) and several intracellular signaling pathway inhibitors, or after knockdown of the transferrin receptor. RESULTS: Both FTH and FTL were substantially higher in inflamed periodontal tissues than in healthy tissues. The location of the elevated expression correlated well with the extent of inflammatory infiltration. Moreover, expression of FTH and FTL were enhanced after stimulation with P. gingivalis-LPS, IL-6, TNF-α. Apoferritin induced the production of IL-6 and IL-8 in a dose-dependent manner partly through binding to the transferrin receptor and activating ERK/P38 signaling pathways in HPDLCs. CONCLUSIONS: Ferritin is up-regulated by inflammation and exhibits cytokine-like activity in HPDLCs inducing a signaling cascade that promotes expression of pro-inflammatory cytokines associated with periodontitis.

Mutual inhibition between HDAC9 and miR-17 regulates osteogenesis of human periodontal ligament stem cells in inflammatory conditions.

Histone deacetylases (HDAC) plays important roles in the post-translational modifications of histone cores as well as non-histone targets. Many of them are involved in key inflammatory processes. Despite their importance, whether and how HDAC9 is regulated under inflammatory conditions remains unclear. The aim of this study was to evaluate the effects of HDAC9 under chronic inflammation condition in human periodontal ligament stromal cell (PDLSCs) and to explore the underlying regulatory mechanism. PDLSCs from healthy or periodontitis human tissue was compared. The therapeutic effects of HDAC inhibitors was determined in PDLSC pellet transplanted nude mice and LPS-induced rat periodontitis. We report that HDAC9 was the most affected HDAC family member under inflammatory conditions in PDLSCs. HDAC9 impaired osteogenic differentiation capacity of PDLSCs under inflammatory conditions. Downregulation of HDAC9 by HDAC inhibitors or si-HDAC9 rescued the osteogenic differentiation capacity of inflammatory PDLSC to a similar level with the healthy PDLSC. In this context, HDAC9 and miR-17 formed an inhibitory loop. The inhibition of miR-17 aggravated loss of calcified nodules in inflamed PDLSCs and interrupted the effect of HDAC inhibitor in rescuing osteogenesis. In vivo experiments using nude mice and LPS-induced periodontitis model confirmed that HDAC inhibitors could improve new bone formation. We conclude that HDAC inhibitors improved osteogenesis of PDLSCs in vitro and periodontitis in vivo.

IL-12-mediated transcriptional regulation of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) remodelling enzymes involved in developmental processes, tissue remodelling and repair, inflammatory and immune diseases and cancer. In a recent issue of Bioscience Reports (vol. 37, issue 6, BSR20170973), Liu and colleagues investigated the expression of MMPs such as MMP-1 (interstitial collagenase), MMP-3 (stromelysin 1) and MMP-13 (collagenase 3) in human periodontal ligament fibroblasts (hPDLFs) regulated by interleukin-12 (IL-12), a cytokine implicated in inflammatory and immune responses. They showed that IL-12 activates canonical nuclear factor-κB (NF-κB) signalling leading to increased expression of MMP-1, MMP-3 and MMP-13, and to a smaller reduction in the expression of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) at both mRNA and protein levels, with corresponding changes in the secreted levels of these ECM-remodelling and immune regulatory metalloproteinases. While canonical NF-κB signalling regulates these MMPs, it also interacts with additional factors to determine whether some of these MMPs are induced or downregulated, in response to IL-12. Here, we comment on the possible mechanisms of IL-12-mediated transcriptional regulation of MMPs.

Mineral trioxide aggregate enhances the osteogenic capacity of periodontal ligament stem cells via NF-κB and MAPK signaling pathways.

Mineral trioxide aggregate (MTA), as a bioactive material, has a widespread application in clinical practice. To date, the effects of MTA on the proliferation and differentiation of human periodontal ligament stem cells (hPDLSCs) remain unclear. hPDLSCs were isolated from human periodontal ligament tissues and cultured with MTA conditioned media. Cell counting kit-8 (CCK-8) assay was performed to assess the proliferation capacity of MTA-treated hPDLSCs. Immunofluorescence assay, alkaline phosphatase (ALP) activity, alizarin red staining, real-time RT-PCR, and western blot analyses were used to investigate the odonto/osteogenic capacity of hPDLSCs as well as the involvement of NF-κB and MAPK pathways. ALP activity assay revealed that 2 mg/ml was the optimal concentration for the induction of hPDLSCs by MTA. The protein expression of DSP, RUNX2, OCN, OSX, OPN, DMP1, ALP, and COL-I in MTA-treated hPDLSCs was significantly higher than those in control group (p < 0.01). When hPDLSCs were treated with the inhibitors of NF-κB and MAPK pathways (U0126, SP600125, SB203580, and BMS345541), the effects of MTA on the differentiation of hPDLSCs were suppressed. Mechanistically, P65 was detected to transfer from cytoplasm to nuclei, as indicated by western blot and immunofluorescence assay. Moreover, MAPK-related proteins and its downstream transcription factors were also upregulated in MTA-treated hPDLSCs. Together, mineral trioxide aggregate can promote the odonto/osteogenic capacity of hPDLSCs via activating the NF-κB and MAPK pathways.

Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell-like cells via Ror2/JNK signaling.

Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-ß, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4+ and the ratio of CD4+ CD25high /CD4+ CD25low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.

Treponema denticola increases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications.

Host-derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP-2 expression and activation in periodontal ligament (PDL) cells, and dentilisin-mediated activation of pro-MMP-2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP-2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP-2 expression, and activation were assessed by immunoblot, zymography, and qRT-PCR, respectively. Chromatin modification enzyme expression in T. denticola-challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola-challenged PDL cells. T. denticola-mediated MMP-2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola-mediated MMP-2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.

Adhesion of human periodontal ligament cells by three-dimensional culture to the sterilized root surface of extracted human teeth.

Residual periodontal ligament (PDL) and cement mass on the roots of extracted teeth are factors that considerably affect tooth transplantation. Therefore, when normal extracted teeth are used for autologous transplantation, it is necessary to regenerate the PDL of the root surface. Here we describe a method to examine human PDL cell adhesion on sterilized root surfaces. Sample teeth were extracted during orthodontic treatment. PDL cells were obtained from healthy periodontal tissue explants from teeth extracted for orthodontic reasons. We developed a method for adhering PDL cells to sterile root surfaces using three-dimensional culture for 3 weeks. We evaluated the adhesion of human PDL cells to the sterilized root surfaces biochemically and histologically. The adherent PDL cells presented new projections on the sterile root surfaces. Therefore, PDL cells can adhere to sterile root surfaces.

[Effects of cell-to-cell communication and histone acetyltransferase on the change of osteogenic differentiation ability among single-cell clones from healthy periodontium with heterogeneity of osteogenic differentiation abilities].

Objective: To investigate the effect of cell-to-cell communication amongst single-cell clones from healthy periodontium with different osteogenic differentiation potentials on change of osteogenic differentiation capabilities and the role histone acetyltransferase partaken in this process. Methods: In order to research the change of osteogenic differentiation ability via cell-to-cell communication, indirect co-culture method was used by placing two single-cell clones with different osteogenesis potentials in each of the 6-well plates. Blank control, weak and strong osteogenic groups were set up, corresponding to Transwell chambers with blank, cells of weak osteogenesis ability and cells of strong osteogenesis ability, respectively. Each group was made in triplicate. After co-culture for four days, Transwell chamber was removed. Quantitative real-time PCR (qPCR) and alizarin red staining were employed to detect the change of osteogenic differentiation ability. The acetylation level of H3 was measured by using Western blotting. Histone acetyltransferases were detected by qPCR. Results: Single-cell clones were ensured from mesenchymal stem cells by flow cytometer, the positive expression of CD29, CD90, CD105, CD146 was (99.80±0.02)%, (99.36±0.18)%, (99.41±0.05)% and (95.10±2.11)%, respectively. And CD31 and CD34 expression were (0.29±0.11)% and (0.22±0.13)%, respectively. Alizarin red and oil red O staining confirmed that single-cell clones had the abilities of adipogenesis and osteogenesis. Alkaline phosphatase (ALP) and alizarin red staining indicated that different single-cell clones were heterogeneity in osteogenesis differentiation. Indirect co-culture indicated that the mRNA expression of osteocalcin (OCN) were 14.24±5.60 and 4.78±2.90, respectively and Runt-related transcription factor 2 (RUNX2) were 2.75±1.44 and 1.61±0.44, respectively, in strong and weak osteogenic groups. They were significantly higher compared to the blank group (the mRNA expression of OCN and RUNX2 were 1.00±0.47 and 1.00±0.39, respectively). The expression of OCN and RUNX2 were also higher in strong osteogenic group than that in weak osteogenic group (P<0.05). The mean gray level of the acetylation of H3 in strong osteogenic group (0.76±0.09) and weak osteogenic group (0.54±0.12) were also higher than that in the blank group (0.30±0.04)(P<0.05). qPCR results showed that KAT6A in strong osteogenic group exhibiting higher expression (P<0.05) compared to weak osteogenic group and the blank group, which were corresponding to the changes of acetylation levels. Conclusions: Single-cell clones from healthy periodontium showed heterogeneity in osteogenic differentiation abilities. Single-cell clones with strong osteogenesis abilities had an advantage over others by promoting others' osteogenesis differentiation and this change mediated by cell-to-cell communication might be caused by modulating KAT6A to affect the acetylation level of histone.

In vitro proliferation of periodontal ligament-like tissue on extracted teeth.

OBJECTIVE: Transplantation of autologous teeth is a routine component of orthodontic treatment. The aim of this study was to develop a method for the regeneration of damaged periodontal ligament (PDL) on extracted teeth using a three-dimensional culture system. DESIGN: We used the maxillary first premolars or third molars extracted from patients for orthodontic treatment. The extracted teeth were stained with toluidine blue to measure the residual PDL area. After confirming damage of the periodontal tissue on the root surface of the extracted teeth, we tried to regenerate the periodontal tissue. Other extracted teeth were inserted into a cell strainer filled with cellulose-based carrier materials to regenerate the periodontal tissue. The strainer was then placed in a 90-mm culture dish filled with culture medium and incubated at 37°C and 5% CO2 for about 1 month. The cultured teeth were observed under a stereomicroscope and examined by scanning electron microscopy (SEM), and were stained to detect alkaline phosphatase (ALP) activity. RESULT: Toluidine blue staining revealed that the residual periodontal membrane covered an average of 50.4% of the root surface area of each tooth. After culturing extracted teeth with our culture system, globular structures were found on the entire tooth root surface by stereomicroscopy, and PDL-like filamentous tissue was also detected by SEM. The entire tooth root surfaces of the cultured teeth were positive for ALP activity. CONCLUSIONS: We have developed a useful culture method to stimulate the proliferation of cells in PDL-like tissue on the roots of extracted teeth.

Doxycycline reduces the expression and activity of matrix metalloproteinase-2 in the periodontal ligament of the rat incisor without altering the eruption process.

BACKGROUND AND OBJECTIVE: Doxycycline is an antibiotic agent that inhibits the activity of matrix metalloproteinases (MMPs) present in the extracellular matrix. In this study, the rat incisor was submitted to a hypofunctional condition, and the effects of doxycycline (80 mg/kg/d) on the expression and activity of MMP-2, as well as on eruption rate, were determined in the odontogenic region and in the periodontal ligament for 14 d. MATERIAL AND METHODS: Rats were distributed into four groups: normofunctional (NF); doxycyline normofunctional (DNF); hypofunctional (HP); and doxycyline hypofunctional (DHP). The left lower incisors of 10 rats were shortened every 2 d, using a high-rotation drill, to produce the HP and DHP groups, after starting doxycycline treatment (80 mg/kg) by gavage. Eruption was measured using a millimeter ocular, from the gingival margin to the top of the tooth in the HP and DHP groups, and also by a mark made in the tooth previously, in the NF and DNF groups. The hemimandibles were removed and the teeth were extracted to collect the periodontal and odontogenic tissues for immunohistochemical analyses and zymography. RESULTS: The eruption rates were higher in the HP and the DHP groups than in the NF and DNF groups, respectively (p < 0.05). In the odontogenic region, neither of the treatments changed the expression and activity of MMP-2. In the HP group, the shortening treatment decreased the expression, but not the activity, of MMP-2, while doxycycline was able to inhibit the increase of expression and activity of MMP-2. CONCLUSION: We conclude that the inhibition of MMP-2 by doxycycline, during incisor shortening, was not enough to alter the eruption rate, which suggests that MMP-2 may have an important role in the turnover of extracellular matrix of the periodontal ligament during the tooth-eruption process.

Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch-Induced Apoptosis in Human Periodontal Ligament Cells.

BACKGROUND: Human periodontal ligament (PDL) cells underwent apoptosis after mechanical stretch loading. However, the exact signalling pathway remains unknown. This study aimed to elucidate how the apoptotic caspases functioned in the cyclic stretch-induced apoptosis in human PDL cells. MATERIALS AND METHODS: In the present study, 20% cyclic stretch was selected to load the cells for 6 or 24 h. The following parameters were analyzed: apoptotic rates, the protein levels of caspase-3, -7, -8 and -9 and the activities of caspase-8 and -9. Subsequently, the influences of caspase-8 and caspase-9 inhibitors on the apoptotic rate and the protein level of the activated caspase-3 were assessed as well. RESULTS: The apoptotic rates increased in response to cyclic stretch, but the cells entered different apoptotic stages after 6 and 24 h stretches. Caspase-3, -7, -8 and -9 were all activated after stretch loading. The stretch-induced apoptosis and the protein level of the activated caspase-3 were inhibited after inhibiting both caspase-8 and caspase-9 in both 6 and 24 h stretched cells and after inhibiting caspase-9 in 24 h stretched cells. CONCLUSION: Caspase-8 and -9 functioned differently at different apoptotic stages in human PDL cells after cyclic stretch.

Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering.

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration-culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.

Activation of canonical Wnt/ß-catenin signaling inhibits H2O2-induced decreases in proliferation and differentiation of human periodontal ligament fibroblasts.

Human periodontal ligament fibroblasts (hPLFs) are exposed to oxidative stress during periodontal inflammation and dental treatments. It is hypothesized that hydrogen peroxide (H2O2)-mediated oxidative stress decreases survival and osteogenic differentiation of hPLFs, whereas these decreases are prevented by activation of the Wnt pathway. However, there has been a lack of reports that define the exact roles of canonical Wnt/ß-catenin signaling in H2O2-exposed hPLFs. Treatment with H2O2 reduced viability and proliferation in hPLFs in a dose- and time-dependent manner and led to mitochondria-mediated apoptosis. Pretreatment with lithium chloride (LiCl) or Wnt1 inhibited the oxidative damage that occurred in H2O2-exposed hPLFs. However, knockout of ß-catenin or treatment with DKK1 facilitated the H2O2-induced decreases in viability, mitochondrial membrane potential, and Bcl-2 induction. Osteoblastic differentiation of hPLFs was also inhibited by combined treatment with 100 µM H2O2, as evidenced by the decreases in alkaline phosphatase (ALP) activity and mineralization. H2O2-mediated inhibition of osteoblast differentiation in hPLFs was significantly attenuated in the presence of 500 ng/ml Wnt1 or 20 mM LiCl. In particular, H2O2 stimulated the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) at protein and mRNA levels in hPLFs, whereas the induction was almost completely suppressed in the presence of Wnt1 or LiCl. Furthermore, siRNA-mediated silencing of Nrf2 blocked H2O2-induced decreases in ALP activity and mineralization of hPLFs with the concomitant restoration of runt-related transcription factor 2 and osteocalcin mRNA expression and ALP activity. Collectively, these results suggest that activation of the Wnt/ß-catenin pathway improves proliferation and mineralization in H2O2-exposed hPLFs by downregulating Nrf2.

Mechanical Loading Stimulates Expression of Collagen Cross-Linking Associated Enzymes in Periodontal Ligament.

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, ß-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.

Focal adhesion kinase activation is required for TNF-α-induced production of matrix metalloproteinase-2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts.

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF-α) and its effects on interleukin (IL)-6 and IL-8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP-2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real-time PCR. Tumor necrosis factor alpha dose-dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF-α-induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL-6, IL-8, and MMP-2 in a dose-dependent manner. Knockdown of FAK significantly suppressed TNF-α-induced expression of IL6 and IL8 mRNA and release of IL-6 and IL-8 protein in HPDLFs. Similarly, MMP-2 down-regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF-α-induced IL-6, IL-8, and MMP-2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.