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Tissue engineering is a steadily growing field of research due to its wide-ranging applicability in the field of regenerative medicine. Application-dependent mechanical properties of a scaffold material as well as its biocompatibility and tailored functionality represent particular challenges. Here the properties of fibrin-based hydrogels reinforced by functional cytocompatible poly(N-vinylcaprolactam)-based (PVCL) microgels are studied and evaluated. The employment of temperature-responsive microgels decorated by epoxy groups for covalent binding to the fibrin is studied as a function of cross-linking degree within the microgels, microgel concentration, as well as temperature. Rheology reveals a strong correlation between the mechanical properties of the reinforced fibrin-based hydrogels and the microgel rigidity and concentration. The incorporated microgels serve as cross-links, which enable temperature-responsive behavior of the hydrogels, and slow down the hydrogel degradation. Microgels can be additionally used as carriers for active drugs, as demonstrated for dexamethasone. The microgels' temperature-responsiveness allows for triggered release of payload, which is monitored using a bioassay. The cytocompatibility of the microgel-reinforced fibrin-based hydrogels is demonstrated by LIVE/DEAD staining experiments using human mesenchymal stem cells. The microgel-reinforced hydrogels are a promising material for tissue engineering, owing to their superior mechanical performance and stability, possibility of drug release, and retained biocompatibility.
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Stimuli-responsive microgels with ionizable functional groups offer versatile applications, e.g., by the uptake of oppositely charged metal ions or guest molecules such as drugs, dyes, or proteins. Furthermore, the incorporation of carboxylic groups enhances mucoadhesive properties, crucial for various drug delivery applications. In this work, we successfully synthesized poly{N-vinylcaprolactam-2,2'-[(5-acrylamido-1-carboxypentyl)azanediyl]diacetic acid} [p(VCL/NTAaa)] microgels containing varying amounts of nitrilotriacetic acid (NTA) using precipitation polymerization. We performed fundamental characterization by infrared (IR) spectroscopy and dynamic and electrophoretic light scattering. Despite their potential multiresponsiveness, prior studies on NTA-functionalized microgels lack in-depth analysis of their stimuli-responsive behavior. This work addresses this gap by assessing the microgel responsiveness to temperature, ionic strength, and pH. Morphological investigations were performed via NMR relaxometry, nanoscale imaging (AFM and SEM), and reaction calorimetry. Finally, we explored the potential application of the microgels by conducting cytocompatibility experiments and demonstrating the immobilization of the model protein cytochrome c in the microgels.
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Microgéis , Microgéis/química , Ácido Nitrilotriacético , Sistemas de Liberação de Medicamentos , Temperatura , CalorimetriaRESUMO
Periodontal defects' localization affects wound healing and bone remodeling, with faster healing in the upper jaw compared to the lower jaw. While differences in blood supply, innervation, and odontogenesis contribute, cell-intrinsic variances may exist. Few studies explored cell signaling in periodontal ligament stem cells (PDLSC), overlooking mandible-maxilla disparitiesUsing kinomics technology, we investigated molecular variances in PDLSC. Characterization involved stem cell surface markers, proliferation, and differentiation capacities. Kinase activity was analyzed via multiplex kinase profiling, mapping differential activity in known gene regulatory networks. Upstream kinase analysis identified stronger EphA receptor expression in the mandible, potentially inhibiting osteogenic differentiation. The PI3K-Akt pathway showed higher activity in lower-jaw PDLSC. PDLSC from the upper jaw exhibit superior proliferation and differentiation capabilities. Differential activation of gene regulatory pathways in upper vs. lower-jaw PDLSC suggests implications for regenerative therapies.
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Osteogênese , Ligamento Periodontal , Osteogênese/genética , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco/metabolismo , Diferenciação Celular/fisiologia , Mandíbula , Células Cultivadas , Proliferação de CélulasRESUMO
Hydrogels as scaffolds in tissue engineering have gained increasing attention in recent years. Natural hydrogels, e.g., collagen or fibrin, are limited by their weak mechanical properties and fast degradation, whereas synthetic hydrogels face issues with biocompatibility and biodegradation. Therefore, combining natural and synthetic polymers to design hydrogels with tunable mechanical stability and cell affinity for biomedical applications is of interest. By using fibrin with its excellent cell compatibility and dextran with controllable mechanical properties, a novel bio-based hydrogel can be formed. Here, we synthesized fibrin and dextran-methacrylate (MA)-based hydrogels with tailorable mechanical properties, controllable degradation, variable pore sizes, and ability to support cell proliferation. The hydrogels are formed through in situ gelation of fibrinogen and dextran-MA with thrombin and dithiothreitol. Swelling and nuclear magnetic resonance diffusometry measurements showed that the water uptake and mesh sizes of fabricated hydrogels decrease with increasing dextran-MA concentrations. Cell viability tests confirm that these hydrogels exhibit no cytotoxic effect.
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Fibrina , Hidrogéis , Hidrogéis/farmacologia , Dextranos , Porosidade , Engenharia Tecidual , Alicerces TeciduaisRESUMO
BACKGROUND: A rigorous search for alternatives to autogenous bone grafts to avoid invasiveness at the donor site in the treatment of maxillomandibular bone defects. Researchers have used alloplastic, allogeneic, and xenogeneic bone graft substitutes in clinical studies with varying degrees of success, although their in vitro effects on stem cells remain unclear. Dental pulp stem cells (DPSCs) can potentially enhance the bone regeneration of bone graft substitutes. The present in vitro study investigates the osteogenic capability of DPSCs on alloplastic (biphasic calcium phosphate [BCP]), allogeneic (freeze-dried bone allografts [FDBAs]), and xenogeneic (deproteinized bovine bone mineral [DBBM]) bone grafts. METHODS: Human DPSCs were seeded on 0.5 mg/ml, 1 mg/ml, and 2 mg/ml of BCP, FDBA, and DBBM to evaluate the optimal cell growth and cytotoxicity. Scaffolds and cell morphologies were analyzed by scanning electron microscopy (SEM). Calcein AM and cytoskeleton staining were performed to determine cell attachment and proliferation. Alkaline phosphatase (ALP) and osteogenesis-related genes expressions was used to investigate initial osteogenic differentiation. RESULTS: Cytotoxicity assays showed that most viable DPSCs were present at a scaffold concentration of 0.5 mg/ml. The DPSCs on the DBBM scaffold demonstrated a significantly higher proliferation rate of 214.25 ± 16.17 (p < 0.001) cells, enhancing ALP activity level and upregulating of osteogenesis-related genes compared with other two scaffolds. CONCLUSION: DBBP scaffold led to extremely high cell viability, but also promoted proliferation, attachment, and enhanced the osteogenic differentiation capacity of DPSCs, which hold great potential for bone regeneration treatment; however, further studies are necessary.
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Substitutos Ósseos , Transplante de Células-Tronco Hematopoéticas , Humanos , Animais , Bovinos , Osteogênese , Polpa Dentária , Regeneração ÓsseaRESUMO
Mechanical compression simulating orthodontic tooth movement in in vitro models induces pro-inflammatory cytokine expression in periodontal ligament (PDL) cells. Our previous work shows that TLR4 is involved in this process. Here, primary PDL cells are isolated and characterized to better understand the cell signaling downstream of key molecules involved in the process of sterile inflammation via TLR4. The TLR4 monoclonal blocking antibody significantly reverses the upregulation of phospho-AKT, caused by compressive force, to levels comparable to controls by inhibition of TLR4. Phospho-ERK and phospho-p38 are also modulated in the short term via TLR4. Additionally, moderate compressive forces of 2 g/cm2, a gold standard for static compressive mechanical stimulation, are not able to induce translocation of Nf-kB and phospho-ERK into the nucleus. Accordingly, we demonstrated for the first time that TLR4 is also one of the triggers for signal transduction under compressive force. The TLR4, one of the pattern recognition receptors, is involved through its specific molecular structures on damaged cells during mechanical stress. Our findings provide the basis for further research on TLR4 in the modulation of sterile inflammation during orthodontic therapy and periodontal remodeling.
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Ligamento Periodontal , Receptor 4 Toll-Like , Técnicas de Movimentação Dentária , Células Cultivadas , Humanos , Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligamento Periodontal/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Mecânico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.
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Fosfatos de Cálcio/química , NF-kappa B/genética , Fosfatidiletanolaminas/química , Regiões Promotoras Genéticas , Estrôncio/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Regeneração Óssea , Substitutos Ósseos , Osso e Ossos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Alicerces Teciduais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mechanical stimulation affects growth and differentiation of stem cells. This may be used to guide lineage-specific cell fate decisions and therefore opens fascinating opportunities for stem cell biology and regenerative medicine. Several studies demonstrated functional and molecular effects of mechanical stimulation but on first sight these results often appear to be inconsistent. Comparison of such studies is hampered by a multitude of relevant parameters that act in concert. There are notorious differences between species, cell types, and culture conditions. Furthermore, the utilized culture substrates have complex features, such as surface chemistry, elasticity, and topography. Cell culture substrates can vary from simple, flat materials to complex 3D scaffolds. Last but not least, mechanical forces can be applied with different frequency, amplitude, and strength. It is therefore a prerequisite to take all these parameters into consideration when ascribing their specific functional relevance-and to only modulate one parameter at the time if the relevance of this parameter is addressed. Such research questions can only be investigated by interdisciplinary cooperation. In this review, we focus particularly on mesenchymal stem cells and pluripotent stem cells to discuss relevant parameters that contribute to the kaleidoscope of mechanical stimulation of stem cells.
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Elasticidade , Células-Tronco Mesenquimais/citologia , Resistência ao Cisalhamento , Diferenciação Celular/efeitos dos fármacos , Dimetilpolisiloxanos/farmacologia , Humanos , Hidrogéis/química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Plásticos/farmacologiaRESUMO
A new route for the synthesis of functional aqueous nanogels decorated with a controlled amount of surface-drafted isoeugenol molecules has been developed. Obtained nanogels exhibit two key functions: a)â antibacterial activity against different oral pathogens and b)â cell-adhesive and -growth-promoting properties. Functional nanogels can be potentially used as building blocks in the design of bioactive coatings on various implants preventing infections and accelerating tissue regeneration.
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Antibacterianos/química , Antifúngicos/química , Materiais Revestidos Biocompatíveis/química , Eugenol/análogos & derivados , Géis/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Infecções Bacterianas/prevenção & controle , Candida albicans/efeitos dos fármacos , Candidíase/prevenção & controle , Materiais Revestidos Biocompatíveis/farmacologia , Eugenol/química , Eugenol/farmacologia , Géis/farmacologia , Humanos , Boca/microbiologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologiaRESUMO
BACKGROUND: Teeth and supporting oral tissues are attractive and accessible sources of stem cells. Periodontal ligament stem cells (PDLSC) are readily isolated from extracted third molars, and exhibit the ability to self-renew and differentiate into multiple mesodermal cell fates. Clinical experience suggests that the exact location of periodontal defects affects the oral bone remodeling and wound healing. Compared to the mandible, the maxilla heals quicker and more efficiently. Angiogenesis is key in tissue regeneration including dental tissues, yet few studies focus on the angiogenic potential of PDLSC, none of which considered the differences between upper and lower jaw PDLSC (u-PDLSC and l-PDLSC, respectively). METHODS: Here we studied the angiogenic potential of u-PDLSC and l-PDLSC and compared the results to well-established mesenchymal stem cells (MSC). Cells were characterized in terms of surface markers, proliferation, and vascular endothelial growth factor (VEGF) secretion, and angiogenic assays were performed. Newly formed capillaries were stained with CD31, and their expression of platelet endothelial cell adhesion molecule (PECAM-1), angiopoietin 2 (ANGPT2), and vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2) were measured. RESULTS: Periodontal stem cells from the upper jaw showed a higher proliferation capacity, secreted more VEGF, and formed capillary networks faster and denser than l-PDLSC. Gene expression of angiogenesis-related genes was significantly higher in u-PDLSC than in l-PDLSC or MSC, given that culture conditions were suitable. CONCLUSION: The oral cavity is a valuable source of stem cells, particularly PDLSC, which are promising for oral tissue engineering due to their robust growth, lifelong accessibility, low immunogenicity, and strong differentiation potential. Notably, u-PDLSC exhibit higher VEGF secretion and accelerate capillary formation compared to l-PDLSC or MSC. This study suggests a potential molecular mechanism in capillary formation, emphasizing the significance of precise location isolation of PDLSC.
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Neovascularização Fisiológica , Ligamento Periodontal , Fator A de Crescimento do Endotélio Vascular , Humanos , Projetos Piloto , Ligamento Periodontal/citologia , Ligamento Periodontal/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/análise , Células-Tronco Mesenquimais , Maxila , Mandíbula , Proliferação de Células , Células-Tronco/fisiologia , Masculino , Diferenciação Celular , Adulto , Feminino , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Células Cultivadas , Adulto JovemRESUMO
A novel scaffold design has been created to enhance tissue engineering and regenerative medicine by optimizing the controlled, prolonged release of Hepatocyte Growth Factor (HGF), a powerful chemoattractant for endogenous mesenchymal stem cells. We present a new stacked scaffold that is made up of three different fibrin gel layers, each of which has HGF integrated into the matrix. The design attempts to preserve HGF's regenerative properties for long periods of time, which is necessary for complex tissue regeneration. These multi-layered fibrin gels have been mechanically evaluated using rheometry, and their degradation behavior has been studied using D-Dimer ELISA. Understanding the kinetics of HGF release from this novel scaffold configuration is essential for understanding HGF's long-term sustained bioactivity. A range of cell-based tests were carried out to verify the functionality of HGF following extended incorporation. These tests included 2-photon microscopy using phalloidin staining to examine cellular morphology, SEM analysis for scaffold-cell interactions, and scratch and scatter assays to assess migration and motility. The analyses show that the novel stacking scaffold promotes vital cellular processes for tissue regeneration in addition to supporting HGF's bioactivity. This scaffold design was developed for in situ tissue engineering. Using the body as a bioreactor, the scaffold should recruit mesenchymal stem cells from their niche, thus combining the regenerative abilities of HGF and MSCs to promote tissue remodeling and wound repair.
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AIMS: To investigate the effect of tumor necrosis factor (TNF) on the growth of human periodontal ligament (PDL) cells, their osteogenic differentiation and modulation of their matrix secretion in vitro. METHODS: The influence of 10â¯ng/ml TNF on proliferation and metabolic activity of PDL cells was analyzed by cell counting (DAPI [4',6-diamidino-2-phenylindole] staining) and the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In addition, cells were cultured under control conditions and osteogenic conditions (media containing 10â¯mM ß-glycerophosphate). Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALP), collagen type I alpha 1 chain (COL1A1), osteoprotegerin (OPG), and osteopontin (OPN) was performed after 7 and 14 days of cultivation. Calcium deposits were stained with alizarin red. RESULTS: Our studies showed that 10â¯ng/ml TNF did not affect the survival and metabolic activity of PDL cells. Quantitative expression analysis revealed that long-term cultures with TNF impaired osteogenic cell fate at early and late developmental stages. Furthermore, TNF significantly reduced matrix secretion in PDL cells. CONCLUSION: The present data confirm TNF as a regulatory factor of proinflammatory remodeling that influences the differentiation behavior but not the metabolism and cell proliferation of the periodontium. Therefore, TNF represents an interesting target for the regulation of orthodontic remodeling processes in the periodontium.
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This study focuses on enhancing controllable fibrin-based hydrogels for tissue engineering, addressing existing weaknesses. By integrating a novel copolymer, we improved the foundation for cell-based angiogenesis with adaptable structural features. Tissue engineering often faces challenges like waste disposal and nutrient supply beyond the 200 µm diffusion limit. Angiogenesis breaks through this limitation, allowing the construction of larger constructs. Our innovative scaffold combination significantly boosts angiogenesis, resulting in longer branches and more capillary network junctions. The copolymer attached to fibrin fibers enables precise adjustment of hydrogel mechanical dynamic properties for specific applications. Our material proves effective for angiogenesis, even under suppression factors like suramin. In our study, we prepared fibrin-based hydrogels with and without the copolymer PVP12400-co-GMA10mol%. Using a co-culture system of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), we analyzed angiogenetic behavior on and within the modified hydrogels. Capillary-like structures were reproducibly formed on different surfaces, demonstrating the general feasibility of three-dimensional endothelial cell networks in fibrin-based hydrogels. This highlights the biomaterial's suitability for in vitro pre-vascularization of biohybrid implants.
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Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple lineages and can be harvested relatively easily from adults, making them a promising cell source for regenerative therapies. While it is well-known how to consistently differentiate MSCs into adipose, chondrogenic, and osteogenic lineages by treatment with biochemical factors, the number of studies exploring how to achieve this with mechanical signals is limited. A relatively unexplored area is the effect of cyclic forces on the MSC differentiation. Recently, our group developed a thermoresponsive N-ethyl acrylamide/N-isopropylacrylamide (NIPAM/NEAM) hydrogel supplemented with gold nanorods that are able to convert near-infrared light into heat. Using light pulses allows for local hydrogel collapse and swelling with physiologically relevant force and frequency. In this study, MSCs are cultured on this hydrogel system with a patterned surface and exposed to intermittent or continuous actuation of the hydrogel for 3 days to study the effect of actuation on MSC differentiation. First, cells are harvested from the bone marrow of three donors and tested for their MSC phenotype, meeting the following criteria: the harvested cells are adherent and demonstrate a fibroblast-like bipolar morphology. They lack the expression of CD34 and CD45 but do express CD73, CD90, and CD105. Additionally, their differentiation potential into adipogenic, chondrogenic, and osteogenic lineages is validated by the addition of standardized differentiation media. Next, MSCs are exposed to intermittent or continuous actuation, which leads to a significantly enhanced cell spreading compared to nonactuated cells. Moreover, actuation results in nuclear translocation of Runt-related transcription factor 2 and the Yes-associated protein. Together, these results indicate that cyclic mechanical stimulation on a soft, ridged substrate modulates the MSC fate commitment in the direction of osteogenesis.
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Células-Tronco Mesenquimais , Osteogênese , Adulto , Humanos , Osteogênese/fisiologia , Hidrogéis/farmacologia , Hidrogéis/metabolismo , Células Cultivadas , Diferenciação Celular/fisiologiaRESUMO
At the time when pathogens are developing robust resistance to antibiotics, the demand for implant surfaces with microbe-killing capabilities has significantly risen. To achieve this goal, profound understanding of the underlying mechanisms is crucial. Our study demonstrates that graphene oxide (GO) nano films deposited on stainless steel (SS316L) exhibit superior antibacterial features. The physicochemical properties of GO itself play a pivotal role in influencing biological events and their diversity may account for the contradictory results reported elsewhere. However, essential properties of GO coatings, such as oxygen content and the resulting electrical conductivity, have been overlooked so far. We hypothesize that the surface potential and electrical resistance of the oxygen content in the GO-nano films may induce bacteria-killing events on conductive metallic substrates. In our study, the GO applied contains 52 wt% of oxygen, and thus exhibits insulating properties. When deposited as a nano film on an electrically conducting steel substrate, GO flakes generate a Schottky barrier at the interface. This barrier, consequently, impedes the transfer of electrons to the underlying conductive substrate. As a result, this creates reactive oxygen species (ROS), leading to bacterial death. We confirmed the presence of GO coatings and their hydrolytic stability by using X-ray photoelectron spectroscopy (XPS), µRaman spectroscopy, scanning electron microscopy (SEM), and Kelvin probe force microscopy (KPFM) measurements. The biological evaluation was performed on the MG63 osteoblast-like cell line and two selected bacteria species: S. aureus and P. aeruginosa, demonstrating both the cytocompatibility and antibacterial behavior of GO-coated SS316L substrates. We propose a two-step bactericidal mechanism: electron transfer from the bacteria membrane to the substrate, followed by ROS generation. This mechanism finds support in changes observed in contact angle, surface potential, and work function, identified as decisive factors. By addressing overlooked factors and effectively bridging the gap between understanding and practicality, we present a transformative approach for implant surfaces, combating microbial resistance, and offering new application possibilitie.
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Antibacterianos , Grafite , Staphylococcus aureus , Espécies Reativas de Oxigênio/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Metais/farmacologia , Oxigênio/farmacologiaRESUMO
For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type.
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Diferenciação Celular , Epitélio/crescimento & desenvolvimento , Morfogênese/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Pele/crescimento & desenvolvimento , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Pele/citologia , Engenharia TecidualRESUMO
BACKGROUND: Clinical experience indicates that wounds in alveolar bone and periodontal tissue heal faster and more efficiently in the maxilla compared with the mandible. Since stem cells are known to have a decisive influence on wound healing and tissue regeneration, the aim of this study was to determine whether differences in proliferation and differentiation of periodontal ligament stem cells (PDLSC) from upper (u-PDLSC) and lower jaw (l-PDLSC) contribute to the enhanced wound healing in the maxilla. METHODS: u-PDLSC and l-PDLSC from the same donor were harvested from the periodontal ligament of extracted human maxillary and mandibular third molars. Cell differentiation potential was assessed by analyzing stem cell markers, proliferation rate, and multilineage differentiation among each other and bone marrow-derived mesenchymal stem cells (MSC). Successful differentiation of PDLSC and MSC toward osteoblasts, adipocytes, and chondrocytes was analyzed via reverse transcriptase-quantitative polymerase chain reaction and histochemical staining (Alizarin Red, Oil Red O, Toluidine Blue). RESULTS: u-PDLSC and l-PDLSC expressed the MSC-markers CD73+ , CD90+ , and CD105+ and lacked expression of CD34- and CD45- . Proliferation was significantly higher in u-PDLSC than in l-PDLSC, regardless of the culture conditions. Osteogenic (ALP, RunX2, and osteocalcin) and chondrogenic (SOX9 and ACAN) related gene expression as well as staining intensities were significantly higher in u-PDLSC than in l-PDLSC. No difference in adipogenic differentiation was observed. CONCLUSION: u-PDLSC showed a significantly higher proliferative and differentiation potential than l-PDLSC, offering a possible cell-based explanation for the differences in periodontal wound healing efficacy between maxilla and mandible.
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Maxila , Ligamento Periodontal , Humanos , Células-Tronco/metabolismo , Diferenciação Celular , Osteogênese , Dente Molar , Células Cultivadas , Proliferação de CélulasRESUMO
Hemocompatibility is the most significant criterion for blood-contacting materials in successful in vivo applications. Prior to the clinical tests, in vitro analyses must be performed on the biomaterial surfaces in accordance with the ISO 10993-4 standards. Designing a bio-functional material requires engineering the surface structure and chemistry, which significantly influence the blood cell activity according to earlier studies. In this study, we elucidate the role of surface terminations and polymorphs of SiC single crystals in the initial stage of the contact coagulation. We present a detailed analysis of phase, roughness, surface potential, wettability, consequently, reveal their effect on cytotoxicity and hemocompatibility by employing live/dead stainings, live cell imaging, ELISA and Micro BCA protein assay. Our results showed that the surface potential and the wettability strongly depend on the crystallographic polymorph as well as the surface termination. We show, for the first time, the key role of SiC surface termination on platelet activation. This dependency is in good agreement with the results of our in vitro analysis and points out the prominence of cellular anisotropy. We anticipate that our experimental findings bridge the surface properties to the cellular activities, and therefore, pave the way for tailoring advanced hemocompatible surfaces.
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Coagulação Sanguínea , Ativação Plaquetária , Materiais Biocompatíveis/química , Molhabilidade , Propriedades de Superfície , Teste de Materiais , Adesividade PlaquetáriaRESUMO
One of the major components in cementum extracellular matrix is bone sialoprotein (BSP). BSP knockout (Ibsp) mice were reported to have a nonfunctional hypo-mineralized cementum, as well as detachment and disorganization of the periodontal ligament tissue. However, studies investigating the influence of Ibsp in cementoblasts are missing yet. This study investigates the influences of Bsp in three cementoblasts cell lines (OCCM.30-WT,IbspΔNterm, and IbspKAE). The mRNA expression of cementoblast and osteoclast markers (Col1a1, Alpl, Ocn, Runx2, Ctsk, Rankl and Opg) and the cell morphology were compared. Additionally, a functional monocyte adhesion assay was performed. To understand the influence of external stimuli, the effect of Ibsp was investigated under static compressive force, mimicking the compression side of orthodontic tooth movement. Cementoblasts with genotype IbspΔNterm and IbspKAE showed slight differences in cell morphology compared to OCCM.30-WT, as well as different gene expression. Under compressive force, the Ibsp cell lines presented expression pattern markers similar to the OCCM.30-WT cell line. However, Cathepsin K was strongly upregulated in IbspΔNterm cementoblasts under compressive force. This study provides insight into the role of BSP in cementoblasts and explores the influence of BSP on periodontal ligament tissues. BSP markers in cementoblasts seem to be involved in the regulation of cementum organization as an important factor for a functional periodontium. In summary, our findings provide a basis for investigations regarding molecular biology interactions of BSP in cementoblasts, and a supporting input for understanding the periodontal and cellular cementum remodeling.