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Alizarin red staining of mineralized nodule formation in hPDLF cultures. Control (a and A), 2D collagen (b and B), and 3D collagen (c and C). Bar a-c: 200 µm and Bar A-C: 100 µm
Source publication
To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures.
hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viabi...
Context in source publication
Context 1
... alizarin red-stained cultures did not exhibit areas of calcified matrix on all evaluated groups at 10 days (data not shown). However, at 14 days, stained areas and the beginning of mineralized nodule formation was observed by microscopy in all groups, independently of the presence of the particles ( Figure 6). The calcium content measure by extraction of alizarin red S from the mineralized matrix was greater on the 3D culture at 10 and 14 ...
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Citations
... This effect is particularly important for the immune response and for wound healing, since platelets secrete a multitude of factors involved in the activation of these processes, including platelet-derived growth factor, transforming growth factor beta, and vascular endothelial growth factor. [19][20][21] The presence of these growth factors is associated with the promotion of adhesion, dissemination, and migration of gingival and periodontal fibroblasts. This observation suggests the existence of a synergistic mechanism between blood and peripheral fibroblasts, which contributes to promoting faster tissue regeneration in contact with treated Ti surfaces. ...
Background: The integrity of the protective seal provided by the gingiva in direct contact with the implant surface is one of the main factors involved in the prevention of peri-implantitis. Aim: The aim of this study was to assess the viability of periodontal fibroblasts grown in an osteogenic culture medium in contact with titanium surfaces treated either with acid etching alone or with acid etching + anodizing. Materials and Methods: Periodontal fibroblasts grown in an osteogenic culture medium were distributed in a control group, with cells grown in culture bottles, and two experimental groups, with cells grown in contact with titanium disks measuring 6 mm in diameter. The surface of the disks was subjected to acid etching alone (AEG, n = 25) or to acid etching + anodizing (ANG, n = 25), and then evaluated using scanning electron microscopy (SEM). Cell viability was assessed by the [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium] bromide test on days 1, 2, 3, 7, and 14 of the cell culture. The Mann–Whitney test was used for the statistical analysis (P < 0.05). Results: The SEM assessment revealed that the surface of AEG specimens had micrometric characteristics, whereas the surface of ANG specimens had nanometric characteristics. No significant difference was observed among the groups regarding cell viability at any of the evaluation time points. Conclusion: The titanium surface treatments tested did not affect the viability of periodontal fibroblasts in an osteogenic culture medium.
... Alizarin Red staini- hence, identifies the osteoblastic phenotype. In addition, the expression of ALP and OPN genes are used to identify genes associated with this phenotype [22]. ...
Statement of the problem:
Osteoblastic differentiation of periodontal ligament stem cells (PLSCs) is essential for alveolar bone regeneration.
Purpose:
The purpose of this study was to compare the potential of two β-tricalcium phosphate (βTCP) products to induce osteoblastic differentiation of human PLSCs.
Materials and method:
In this in vitro study, human PLSCs were cultured in mediums supplemented with Guidor Easy-Graft [βTCP+polylactide-co-glycolide (PLCG)+n-methyl-2-pyrrolidone (NMP)] [Sunstar Company, Swiss] or Sorbone [βTCP] [Meta Company, South Korea] as two alloplasts experimental groups, mesenchymal cells differentiated into osteoblasts without alloplast as positive control group, and mesenchymal cells without osteoblastic induction as negative control group. Osteoblastic differentiation was evaluated using Alizarine Red staining and spectrophotometry to assay calcium deposits and real-time polymerase chain reaction to examine expression of alkaline phosphatase (ALP) and osteopontin (OPN) antigens on day 21. Data were analyzed by using SPSS 22 software and one-way ANOVA and Bonferoni tests (p< 0.05).
Results:
Spectrophotometry confirmed that calcium deposits were higher in Guidor Easy-Graft group compared to Sorbone group (p< 0.001) and higher in two experimental groups than controls (p< 0.05). According to real-time polymerase chain reaction, level of ALP expression was higher in Sorbone than Guidor and the levels of Guidor, positive control and negative control were equal; OPN levels of the positive control were more than the other groups. OPN levels of Sobone, Guidor and negative control were the same.
Conclusion:
These findings indicated that Guidor Easy-Graft and Sorbone enhanced differentiation of human PLSCs to osteoblasts, and could be employed as appropriate bone-graft materials.
... On the other hand, TNN, OPN, and OCN were also functionally expressed in PDLFs (Alves et al., 2015;Barczyk et al., 2013) These results indicated that OPN might respond to mechanical stress and promote remodeling in PDL. According to the molecular structure, OCN contained Gla motif which was able to bind to hydroxyapatite (Razny et al., 2017). ...
Objectives:
The periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration.
Materials and methods:
We isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second-generation sequencing technique to create profiling. We also determined the protein expression using Western blot.
Results:
Collagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, β1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed.
Conclusions:
Our study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.
... The denser HPdLF grow, the more they show parallel arrangements with intensive cell-cell contacts and they start building calcified nodules 35,36 . For further investigation of growth characteristics of dense HPdLF, we performed a scratch assay on complete monolayers of HPdLF grown for one week in DMEM prior six-day fatty acid cultivation (Fig. 1d,e). ...
... Interestingly, differentiation processes can also have crucial effects on the metabolic activity of cells 37 , which possibly explains the changes in oleate-cultured HPdLF since we found an increase in osteogenic differentiation of HPdLF in response to oleate treatment. It already had been reported that HPdLF show a distinct osteogenic differentiation potential 31,32,56 , which can be stimulated by several external cues 35,[57][58][59] . In addition, fatty acids can affect the differentiation of osteoblasts 60 . ...
https://rdcu.be/b7EiI
Alveolar bone (AB) remodeling is necessary for the adaption to mechanical stimuli occurring during mastication and orthodontic tooth movement (OTM). Thereby, bone degradation and assembly are strongly regulated processes that can be altered in obese patients. Further, increased fatty acids (FA) serum levels affect bone remodeling cells and we, therefore, investigated whether they also influence the function of periodontal ligament fibroblast (PdLF). PdLF are a major cell type regulating the differentiation and function of osteoblasts and osteoclasts localized in the AB. We stimulated human PdLF (HPdLF) in vitro with palmitic (PA) or oleic acid (OA) and analyzed their metabolic activity, growth, survival and expression of osteogenic markers and calcium deposits. Our results emphasize that PA increased cell death of HPdLF, whereas OA induced their osteoblastic differentiation. Moreover, quantitative expression analysis of OPG and RANKL revealed altered levels in mechanically stimulated PA‑treated HPdLF. Furthermore, osteoclasts stimulated with culture medium of mechanical stressed FA‑treated HPdLF revealed significant changes in cell differentiation upon FA‑treatment. For the first time, our results highlight a potential role of specific FA in the function of HPdLF‑modulated AB remodeling and help to elucidate the complex interplay of bone metabolism, mechanical stimulation and obesity‑induced alterations.
... These results are concordant to the SEM images, which show space available for cell proliferation even after 7 days of cell growth in the 3D scaffolds (Fig. 6). These results are in accordance with previous reports that showed better proliferation of cells in 3D scaffolds compared to the 2D surface culture of the same polymer [40]. In addition, cellulose surface modification by gelatin leads to enhanced biocompatibility of the 3DMP rBC/G scaffolds. ...
This study reports the fabrication of porogen-induced, surface-modified, 3-dimensionally microporous regenerated bacterial cellulose (rBC)/gelatin (3DMP rBC/G) scaffolds for skin regeneration applications. Round shaped gelatin microspheres (GMS), fabricated using a water-in-oil emulsion (WOE) method, were utilized as the porogen. The dissolution of GMS from the solution casted BC scaffolds led to surface-modified microporous rBC. The scaffolds were characterized using field emission scanning electron microscopy (FE-SEM) and elemental analysis. FE-SEM analysis confirmed the regular microporosity of the 3DMP rBC/G scaffolds, while elemental analysis confirmed the successful surface modification of cellulose with gelatin. In vitro tests showed good adhesion and proliferation of human keratinocytes (HaCaT) on the 3DMP rBC/G scaffolds during 7 days of incubation. Confocal microscopy showed penetration of HaCaT cells into the scaffolds, up to 300 μm in depth. In vivo wound healing and skin regeneration experiments, in experimental mice, showed complete skin regeneration within 2 weeks. The wound closure efficacy of the 3DMP rBC/G scaffolds was much higher (93%) than that of the control (47%) and pure BC-treated (63%) wounds. These results indicated that our 3DMP rBC/G scaffolds represent future candidate materials for skin regeneration applications.
... [33] Periodontal ligament fibroblasts have been known to express Runx 2 (the master transcription factor for osteoblast differentiation) and bone-related proteins such as osteopontin, osteocalcin, and alkaline phosphatase. [34,35] Several investigators have shown the in vitro ability of PDLFs to form mineralized bone nodules under the influence of ascorbic acid, beta glycerophosphate, and dexamethasone. [36] Further, the periodontal ligament has been shown to be a niche for mesenchymal stem cells (with expression of stem cell markers CD 34, 109, and STRO 1) which have demonstrated the formation of soft and hard tissue in culture. ...
With the advent of esthetic dentistry, implant treatment has gained much importance, and osseointegrated implant requires appropriate positioning three-dimensionally to achieve optimum functional and esthetic results. The various techniques available for implant site development are mostly invasive with variable outcomes. On resorting to alternate modalities, orthodontic extrusion/forced eruption is the primary choice as it is noninvasive and aids in optimal increase in the volume of residual bone and soft tissue. In orthodontic extrusion, the available periodontal ligament acts as a source of cell supply and signals, while the socket itself acts as a naturally occurring scaffold for regeneration. The presence of gingival inflammation or active periodontal disease is always a negative predictor for periodontal regeneration. Hence, periodontal ligament in an uninflamed state is essential before performing orthodontic extrusion. This article aims to provide an insight into the mechanisms by which the periodontal ligament cells act at a molecular level to provide an improved bone and soft tissue levels for optimum implant placement.
... collagen gel presented an increased proliferation rate over time (Alves et al., 2015). However, compared with the monolayer cell culture and the above-mentioned 3D collagen gel culture system, PDL cells exhibited decreased proliferation after spheroid formation, which is in agreement with some earlier studies performed on spheroids of human ASCs (Cheng et al., 2012) and hepatocellular carcinoma cell line (Meli, Jordan, Clark, Linhardt, & Dordick, 2012). ...
Objective:
The therapeutic potential of periodontal ligament cells for periodontal regeneration gradually decreases when cultured as a monolayer in vitro. Three-dimensional cell culture models provide an alternative to traditional monolayer cell culture. This study aimed to comparatively evaluate the influence of spheroid culture on periodontal ligament cells.
Materials and methods:
Chitosan films were used to culture three-dimensional periodontal ligament cell spheroids. The proliferation, self-renewal, and osteogenic capacity of periodontal ligament cells derived from spheroids were evaluated and compared to cells cultured on a monolayer.
Results:
Viable spheroids of periodontal ligament cells were formed on chitosan films. Compared to monolayer cell culture, periodontal ligament cells exhibited decreased proliferation upon spheroid formation. In contrast, their expression of genes related to self-renewal was significantly higher comparison to cells cultured in a monolayer. Moreover, the formation of periodontal ligament cell spheroids increased their colony forming unit ability and osteogenic differentiation capacity.
Conclusion:
The results demonstrate the successful use of chitosan films for the culture of periodontal ligament cell spheroids. Compared to cells cultured in monolayer, periodontal ligament cells in spheroids did not proliferate, but exhibited higher self-renewal gene expression, colony forming unit and osteogenic capacity. This article is protected by copyright. All rights reserved.
... PDL fibroblasts are different from other fibroblasts because of their function in the formation and maintenance of the PDL in addition to their ability to differentiate to osteoblasts or cementoblasts and thereby contribute to repair, remodeling and regeneration of the adjacent alveolar bone and cementum 30 . They also exhibit characteristics of osteoblasts by showing high basal alkaline phosphatase (ALP) activity 31,32 . Moreover, PDL fibroblasts may participate in the osteoclasts differentiation through their cell-cell interactions with osteoclast progenitor cells 27 . ...
The bone remodeling process in response to orthodontic forces requires the activity of osteoclasts to allow teeth to move in the direction of the force applied. Receptor activator of nuclear factor-κB ligand (RANKL) is essential for this process although its cellular source in response to orthodontic forces has not been determined. Orthodontic tooth movement is considered to be an aseptic inflammatory process that is stimulated by leukocytes including T and B lymphocytes which are presumed to stimulate bone resorption. We determined whether periodontal ligament and bone lining cells were an essential source of RANKL by tamoxifen induced deletion of RANKL in which Cre recombinase was driven by a 3.2 kb reporter element of the Col1α1 gene in experimental mice (Col1α1.CreERTM+.RANKLf/f) and compared results with littermate controls (Col1α1.CreERTM−.RANKLf/f). By examination of Col1α1.CreERTM+.ROSA26 reporter mice we showed tissue specificity of tamoxifen induced Cre recombinase predominantly in the periodontal ligament and bone lining cells. Surprisingly we found that most of the orthodontic tooth movement and formation of osteoclasts was blocked in the experimental mice, which also had a reduced periodontal ligament space. Thus, we demonstrate for the first time that RANKL produced by periodontal ligament and bone lining cells provide the major driving force for tooth movement and osteoclastogenesis in response to orthodontic forces.
... Similar results were obtained in previous studies in three-dimensional scaffolds,[47,48]where ALP activity was higher at 7 days with subsequent reduction due to the beginning of extracellular matrix mineralization. The slight ALP increase at 21 days corroborates with Runx2 expression in the same period, and may be related to the renewal of the cell cycle[49].The expression of Runt-related transcription factor 2 (Runx2) is associated with osteoblastic differentiation, and was decreased at 7 days of cell culture in the scaffold groups, regarding other periods and the control group (p < 0.001). At 14 days there was an increase in scaffold gene expression, but not significant in relation to the control (p > 0.05), although there was an increase in the expression for the PBAT/5%nHAp group in the period from 7 to 14 days, this group showed significantly lower expression of Runx2 compared to the other groups (p < 0.001). ...
... We suggest that the low Runx2 expression observed in the scaffold groups at 7 days may be justified due to the mature phenotype in this period, as confirmed by high cell activity and by elevated ALP expression in the scaffold groups in the same period. Furthermore, the renewal of the cell cycle may explain the increased late expression of Runx2 at 21 days;[49]nHAp-incorporated scaffold groups had higher Runx2 expression due to the influence of nHAp in the modification of the surface wettability and then to promote osteoblastic differentiation.The expression of Collagen type 1 (Col I), an abundant organic component of the extracellular matrix, was similar at 7 and 14 days of cell culture with higher control group expression (p > 0.001). However, at 21 days, the scaffold groups showed a higher Col I expression than the controls (p < 0.001), and, when compared, the PABT group presented similar expression to the PABT/5%nHAp group (p > 0.05), with PABT/3%nHAp showing the highest Col I expression compared to the others (p < 0.001) (Fig. 2B). ...
... The high OPN expression at 14 days, as well as its distribution among the groups, corroborates our findings of mineralized nodules, a period that we evaluated and found in vitro mineralization. Alves et al.[49]reported decreased expression of some genes at 14 days and correlated it with apoptosis during the cell cycle. The constant expression of OC over time is important to support the mineralization process, and similar results were observed[54,55]where OC expression was maintained since the matrix mineralization was initiated with the consequent formation of mineralization nodules. ...
Combining polyester scaffolds with synthetic nanohydroxyapatite (nHAp), which is bioactive and osteoconductive, is a plausible strategy to improve bone regeneration. Here, we propose the combination of PBAT [poly(butylene-adipate-co-terephthalate)] and synthetic nHAp (at 3 and 5 wt%). PBAT is a relatively a new polymer with low crystallinity and attractive biodegradability and mechanical properties for orthopaedic applications, however, with a still underexplored potential for in vivo applications. Then, we performed a careful biological in vitro and in vivo to evaluate the influence of PBAT containning two different nHAp loads. For in vitro assays, osteoblast-like MG63 cells were used and the bioactivity and gene expression related to osteogenesis were evaluated by qRT-PCR. For in vivo experiments, twenty-four male rats were used and a tibial defect model was applied to insert the scaffolds. Micro-computed tomography (Micro-CT) and histological analysis were used to assess the bone neoformation after 6 weeks of implantation. Flexural three point’s test measured the mechanical properties of bone neoformation. All scaffolds showed promising in vitro properties, since they were no cytotoxic against MG-63 cells and promoted highest cell proliferation and nodules matrix mineralization formation. From a mechanistic point-of-view, nHAp loading increased hydrophilicity, which in turn allowed for a better adsorption of proteins and consequent changes in the phenotypic expression of osteoblasts. nHAp induced better cellular response on/in the scaffolds, which was mainly attributed to its osteoconductive and osteoinductive properties. Micro-CT images showed that nHAp at 3% and 5 wt% led to more effective bone formation, presenting the highest bone volume after 6 weeks of implantation. Considereing flexural three points test, 5 wt% of nHAp positively influenced the flexural mode of bone neoformation, but the stiffiness presented similarity between 3% and 5 wt% groups. In summary, this investigation demonstrated great potential for the application of these novel scaffolds towards bone regeneration and, thus, should be further studied.
... Images were quantified using ImageJ. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetra-zolium bromide (MTT) Cell Proliferation Assay Kit [34]. ...
The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2016;00:000-000.
