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Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.
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Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Diferenciação Celular/fisiologia , Humanos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Células-Tronco , Fluxo de TrabalhoRESUMO
The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.
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Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Sarcômeros/metabolismo , Actinina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Aprendizado de Máquina , Camundongos , Microscopia de Fluorescência/métodos , Músculo Esquelético/citologia , Miocárdio/citologia , Fenótipo , RNA/genética , RNA/isolamento & purificaçãoRESUMO
PURPOSE: Zirconium dioxide ceramic has been successfully introduced as a framework material for fixed dental prostheses. To reduce manufacturing constraints, joining of subcomponents could be a promising approach to increase the mechanical performance of long-span fixed dental prostheses. In this experimental study, the biomechanical behavior of monolithic and soldered framework specimens for fixed dental prostheses made of Y-TZP was investigated. MATERIALS AND METHODS: Framework specimens (n = 80) of 5-unit fixed dental prostheses made of Y-TZP were prepared and divided into 10 equal groups. The specimens were monolithic or composed of subcomponents, which were joined using a silicate-based glass solder. Thereby, three joint geometries (diagonal, vertical with an occlusal cap, and dental attachment-based) were investigated. Moreover, the groups differed based on the mechanical test (static vs. dynamic) and further processing (veneered vs. unveneered). The framework specimens were cemented on alumina-based jaw models, where the canine and second molar were acting as abutments before a point-load was applied. In addition, µCT scans and microscopic fractography were used to evaluate the quality of soldered joints and to determine the causes of fracture. RESULTS: The determined fracture loads of the different unveneered framework specimens in static testing did not vary significantly (p = 1). Adding a veneering layer significantly increased the mechanical strength for monolithic framework specimens from 1196.29 ± 203.79 N to 1606.85 ± 128.49 N (p = 0.008). In case of soldered specimens with a dental attachment-based geometry, the mechanical strength increased from 1159.42 ± 85.65 N to 1249.53 ± 191.55 N (p = 1). Within the dynamic testing, no differences were observed between monolithic and soldered framework specimens. µCT scans and fractography proved that the dental attachment-based joining geometry offers the highest quality. CONCLUSION: Using glass soldering technology, subcomponents of 5-unit framework specimens made of Y-TZP could be joined with mechanical properties comparable to those of monolithic frameworks.
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Materiais Dentários , Porcelana Dentária , Resistência à Flexão , Falha de Restauração Dentária , Facetas Dentárias , Teste de Materiais , Análise do Estresse Dentário , Cerâmica , ZircônioRESUMO
BACKGROUND: The treatment of acute pain is part of everyday dental practice. Often, these symptoms result from years of patients' inadequate or missing dental routines and lead to a reduction in the quality of life or health of the patients and to high costs for the health care system. Despite the enormous advantages of modern dentistry, many patients avoid going to the dentist. Therefore, the study aimed to determine the reasons and behaviours that cause patients to avoid visits to the dentist. METHODS: We conducted semi-structured interviews with patients who had an above-average DMFT index and had been going to the dentist only irregularly for years. The sample participants were recruited from the northern German region of Mecklenburg-Western Pomerania. 20 individual interviews were recorded, transcribed verbatim and coded. We used a qualitative framework approach to code the transcripts in order to establish a consensus among the researchers. Ultimately, through discussions and reviews of the attributes and meaning of the topics, a typology could be established. RESULTS: A typology of patients who avoid the dentist was developed. Four independent characteristic patterns of dentist avoidance could be developed: avoiding the dentist due to "distance" (type A; includes subtype A1 "avoiding the dentist due to negligence" and subtype A2 "dental avoidance due to neutralization"), "disappointment" (type B), "shame" (type C), and "fear" (type D). Using the typology as a generalised tool to determine the minimum and maximum contrasts, it was possible to capture the diversity and multidimensionality of the reasons and behaviours for avoidance. All patients had negative dental experiences, which had led to different avoidance patterns and strategies. CONCLUSIONS: The identified avoidance characteristics represent a spectrum of patients from Northern Germany who avoid going to the dentist. This is the first comprehensive study in Germany representing avoidance behaviour of dentist patients in the form of a typology. The results suggest that dentistry also needs qualitative research to better understand patient characteristics and provide direct access to patients who avoid regular dental visits. Thus, the results make a potentially fundamental contribution to the improvement of dental care and enrich its understanding.
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Assistência Odontológica , Qualidade de Vida , Atenção à Saúde , Alemanha , Humanos , Pesquisa QualitativaRESUMO
Mesenchymal stem cells (MSCs) are widely recognized as critical players in tissue regeneration. New insights into stem cell biology provide evidence that MSCs may also contribute to host defence and inflammation. In case of tissue injury or inflammatory diseases, e.g. periodontitis, stem cells are mobilized towards the site of damage, thus coming in close proximity to bacteria and bacterial components. Specifically, in the oral cavity, complex ecosystems of commensal bacteria live in a mutually beneficial state with the host. However, the formation of polymicrobial biofilm communities with pathogenic properties may trigger an inadequate host inflammatory-immune response, leading to the disruption of tissue homoeostasis and development of disease. Because of their unique characteristics, MSCs are suggested as crucial regulators of tissue regeneration even under such harsh environmental conditions. The heterogeneous effects of bacteria on MSCs across studies imply the complexity underlying the interactions between stem cells and bacteria. Hence, a better understanding of stem cell behaviour at sites of inflammation appears to be a key strategy in developing new approaches for in situ tissue regeneration. Here, we review the literature on the effects of oral bacteria on cell proliferation, differentiation capacity and immunomodulation of dental-derived MSCs.
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Bactérias/metabolismo , Boca/microbiologia , Regeneração , Células-Tronco/citologia , Diferenciação Celular , Humanos , ImunomodulaçãoRESUMO
Probiotic microorganisms are used in a variety of food supplements and medical formulations to promote human health. In periodontal therapy, probiotics are mainly used in the form of gels, tablets or rinses that often tend to leak from the periodontal pocket, resulting in a strongly reduced therapeutic effect. In this pilot in vitro study, we present biodegradable alginate-based particles as an alternative, highly efficient system for a periodontal delivery of probiotic bacteria to the inflammation site. For this purpose, Lactococcus (L.) lactis was encapsulated using a standardized pump-controlled extrusion-dripping method. Time-dependent bacterial release in artificial saliva was investigated over 9 days. The effect of freeze drying was explored to ensure long-term storage of L. lactis-loaded particles. Additionally, the particles were bound to dentin surface using approved bioadhesives and subjected to shear stress in a hydrodynamic flow chamber that mimics the oral cavity in vitro. Thus, round particles within the range of 0.80-1.75 mm in radius could be produced, whereby the diameter of the dripping tip had the most significant impact on the size. Although both small and large particles demonstrated a similar release trend of L. lactis, the release rate was significantly higher in the former. Following lyophilization, particles could restore their original shape within 4 h in artificial saliva; thereby, the bacterial viability was not affected. The attachment strength to dentin intensified by an adhesive could resist forces between 10 and 25 N/m2. Full degradation of the particles was observed after 20 days in artificial saliva. Therefore, alginate particles display a valuable probiotic carrier for periodontal applications that have several crucial advantages over existing preparations: a highly stable form, prolonged continuous release of therapeutic bacteria, precise manufacturing according to required dimensions at the application site, strong attachment to the tooth with low risk of dislocation, high biocompatibility and biodegradability.
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Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL-6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well-studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll-like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down-regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up-regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS-treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin-6 (IL-6), a potent stimulator of cell migration. Interestingly, the levels of IL-6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental-derived progenitor cells in sites of periodontal infection.
Assuntos
Saco Dentário/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Porphyromonas gingivalis/química , Adolescente , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Movimento Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Saco Dentário/citologia , Saco Dentário/metabolismo , Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Adulto JovemRESUMO
BACKGROUND: Efforts to improve the performance of rotary NiTi instruments by enhancing the properties of NiTi alloy, or their manufacturing processes rather than changes in instrument geometries have been reported. The aim of this study was to compare in-vitro the shaping ability of three different rotary nickel-titanium instruments produced by different manufacturing methods. METHODS: Thirty simulated root canals with a curvature of 35Ë in resin blocks were prepared with three different rotary NiTi systems: AK- AlphaKite (Gebr. Brasseler, Germany), GTX- GT® Series X (Dentsply, Germany) and TF- Twisted Files (SybronEndo, USA).The canals were prepared according to the manufacturers' instructions. Pre- and post-instrumentation images were recorded and assessment of canal curvature modifications was carried out with an image analysis program (GSA, Germany).The preparation time and incidence of procedural errors were recorded. Instruments were evaluated under a microscope with 15 × magnifications (Carl Zeiss OPMI Pro Ergo, Germany) for signs of deformation. The Data were statistically analyzed using SPSS (Wilcoxon and Mann-Whitney U-tests, at a confidence interval of 95%). RESULTS: Less canal transportation was produced by TF apically, although the difference among the groups was not statistically significant. GTX removed the greatest amount of resin from the middle and coronal parts of the canal and the difference among the groups was statistically significant (p < 0.05). The shortest preparation time was registered with TF (444 s) and the longest with GTX (714 s), the difference among the groups was statistically significant (p < 0.05). During the preparation of the canals no instrument fractured. Eleven instruments of TF and one of AK were deformed. CONCLUSION: Under the conditions of this study, all rotary NiTi instruments maintained the working length and prepared a well-shaped root canal. The least canal transportation was produced by AK. GTX displayed the greatest cutting efficiency. TF prepared the canals faster than the other two systems.
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Preparo de Canal Radicular/instrumentação , Instrumentos Odontológicos , Humanos , Níquel , Avaliação de Resultados em Cuidados de Saúde , Preparo de Canal Radicular/métodos , Estatísticas não Paramétricas , TitânioRESUMO
Therapy for large-scale bone defects remains a major challenge in regenerative medicine. In this context, biodegradable electrospun nonwovens are a promising material to be applied as a temporary implantable scaffold as their fibre diameters are in the micro- and nanometre range and possess a high surface-to-volume ratio paired with high porosity. In this work, in vitro assessment of biodegradable PLLA-co-PEG nonwovens with fetuin A covalently anchored to the surface has been performed in terms of biomineralisation and the influence on MG-63 osteoblast cell metabolic activity, biosynthesis of type I collagen propeptide and inflammatory potential. Our finding was that covalent fetuin A funtionalisation of the nonwoven material leads to a distinct increase in calcium affinity, thus enhancing biomineralisation while maintaining the distinct fibre morphology of the nonwoven. The cell seeding experiments showed that the fetuin A functionalised and subsequently in vitro biomineralised PLLA-co-PEG nonwovens did not show negative effects on MG-63 growth. Fetuin A funtionalisation and enhanced biomineralisation supported cell attachment, leading to improved cell morphology, spreading and infiltration into the material. Furthermore, no signs of increase in the inflammatory potential of the material have been detected by flow cytometry experiments. Overall, this study provides a contribution towards the development of artificial scaffolds for guided bone regeneration with the potential to enhance osteoinduction and osteogenesis.
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Engenharia Tecidual , alfa-2-Glicoproteína-HS , Poliésteres , Osteogênese , Ácido Láctico , Alicerces TeciduaisRESUMO
Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of [Formula: see text]-actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.
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Apêndice Atrial , Sarcômeros , Humanos , Sarcômeros/metabolismo , Microscopia , Miocárdio , Miócitos Cardíacos/metabolismoRESUMO
BACKGROUND: Inflammation triggered by bacterial biofilms in the surrounding tissue is a major etiological factor for peri-implantitis and subsequent implant failure. However, little is known about the direct effects of bacterial corrosion and recolonization on implant failure PURPOSE: To investigate the influence of oral commensals on bacterial corrosion and recolonization of titanium surfaces. MATERIALS AND METHODS: Streptococcus sanguinis (S. sanguinis) and Porphyromonas gingivalis (P. gingivalis), which are key bacteria in oral biofilm formation, were cultured on commercially pure titanium and titanium-aluminum-vanadium (Ti6Al4V) plates in artificial saliva/brain heart infusion medium under aerobic or anaerobic conditions. Biofilm formation was examined after 7 and 21 days by crystal violet and live/dead staining. Titanium ions released into culture supernatants were analyzed over a period of 21 days by atomic absorption spectrometry. Visual changes in surface morphology were investigated using scanning electron microscopy. Biofilm formation on sterilized, biocorroded, and recolonized implant surfaces was determined by crystal violet staining. RESULTS: S. sanguinis and P. gingivalis formed stable biofilms on the titanium samples. Bacterial corrosion led to a significant increase in titanium ion release from these titanium plates (p < 0.01), which was significantly higher under aerobic conditions on pure titanium (p ≤ 0.001). No obvious morphological surface changes, such as pitting and discoloration, were detected in the titanium samples. During early biofilm formation, the addition of titanium ions significantly decreased the number of live cells. In contrast, a significant effect on biofilm mass was only detected with P. gingivalis. Bacterial corrosion had no influence on bacterial recolonization following sterilization of titanium and Ti6Al4V surfaces. CONCLUSION: Bacterial corrosion differs between oral commensal bacteria and leads to increased titanium ion release from titanium plates. The titanium ion release did not influence biofilm formation or bacterial recolonization under in vitro conditions.
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Implantes Dentários , Titânio , Ligas , Alumínio , Biofilmes , Corrosão , Implantes Dentários/microbiologia , Violeta Genciana , Porphyromonas gingivalis , Saliva Artificial , Propriedades de Superfície , Titânio/química , VanádioRESUMO
Dentists account for up to 10% of all prescribed antibiotics in primary care, with up to 80% being inappropriate. Targeted approaches to change prescription behavior are scarce. This study aimed at identifying specific barriers and facilitators for prudent antibiotic use in German dentistry by using qualitative methods. Nine in-depth interviews and two focus group discussions with another nine dentists were conducted and analyzed thematically. Dentists described being conflicted by the discordance of available treatment time and the necessity of thorough therapy. Lacking the opportunity of follow-up led to uncertainty. Dentists felt a lack of medical competency concerning prophylaxis for infectious endocarditis. A lack of empowerment to make therapeutic decisions interfered with guideline-conformity. The communication with fellow physicians is conflictual and improvement was wished for. In consequence, dentists felt pressure by potential medico-legal liability. Patients demanding quick and easy pain relief put extra strain on the interviewed dentists. Our hypotheses concord with preliminary data, mainly from the UK, but highlighted specifically medico-legal concerns and interprofessional communication as even greater barriers as described before. Tailored interventional concepts based on our findings may have the potential to lower antibiotic prescriptions in German primary dental care.
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The purpose of the present investigation was to identify the most frequent therapies and, in particular, the prescription patterns for occlusal splints for the management of craniomandibular disorders (CMDs) used by German general dentists and specialists. Additionally, the knowledge and opinion of the practising dentists were examined. All active members of the statutory dental insurance providers of the German North Rhine (n = 5,500) and the Westphalia-Lippe area (n = 4,984) were surveyed with a questionnaire by mail. Results indicated that occlusal splints were the first-choice therapy followed by physiotherapy and occlusal equilibration. In the preceding year, both general dentists and specialists made 30 occlusal splints on average. With regard to high-quality evidence-based recommendations, some statistically significant discrepancies between general dentists and specialists were detected. On the basis of the present data, it seems useful to consider intensifying the topic of CMDs and orofacial pain in future undergraduate dental curricula and in postgraduate training.
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Atitude do Pessoal de Saúde , Transtornos Craniomandibulares/terapia , Odontólogos , Planejamento de Prótese Dentária/estatística & dados numéricos , Educação em Odontologia , Odontologia Baseada em Evidências , Feminino , Odontologia Geral/estatística & dados numéricos , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Ajuste Oclusal/estatística & dados numéricos , Placas Oclusais/estatística & dados numéricos , Modalidades de Fisioterapia/estatística & dados numéricos , Prescrições/estatística & dados numéricos , Encaminhamento e Consulta/estatística & dados numéricos , Terapia de Relaxamento/estatística & dados numéricos , Especialidades Odontológicas/estatística & dados numéricos , Inquéritos e QuestionáriosRESUMO
We investigated the influence of syngeneic cardiomyocyte transplantation after myocardial infarction (MI) on the immune response and cardiac function. Methods and Results: We show for the first time that the immune response is altered as a result of syngeneic neonatal cardiomyocyte transplantation after MI leading to improved cardiac pump function as observed by magnetic resonance imaging in C57BL/6J mice. Interestingly, there was no improvement in the capillary density as well as infarct area as observed by CD31 and Sirius Red staining, respectively. Flow cytometric analysis revealed a significantly different response of monocyte-derived macrophages and regulatory T cells after cell transplantation. Interestingly, the inhibition of monocyte infiltration accompanied by cardiomyocyte transplantation diminished the positive effect of cell transplantation alone. The number of CD68+ macrophages in the remote area of the heart observed after four weeks was also different between the groups. Transcriptome analysis showed several changes in the gene expression involving circadian regulation, mitochondrial metabolism and immune responses after cardiomyocyte transplantation. Conclusion: Our work shows that cardiomyocyte transplantation alters the immune response after myocardial infarction with the recruited monocytes playing a role in the beneficial effect of cell transplantation. It also paves the way for further optimization of the efficacy of cardiomyocyte transplantation and their successful translation in the clinic.
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Infarto do Miocárdio/terapia , Miocárdio/imunologia , Miócitos Cardíacos/transplante , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Coração/fisiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/imunologia , Receptores CCR2/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Multipotent adult mesenchymal stromal cells (MSCs) could represent an elegant source for the generation of patient-specific cardiomyocytes needed for regenerative medicine, cardiovascular research, and pharmacological studies. However, the differentiation of adult MSC into a cardiac lineage is challenging compared to embryonic stem cells or induced pluripotent stem cells. Here we used non-integrative methods, including microRNA and mRNA, for cardiac reprogramming of adult MSC derived from bone marrow, dental follicle, and adipose tissue. We found that MSC derived from adipose tissue can partly be reprogrammed into the cardiac lineage by transient overexpression of GATA4, TBX5, MEF2C, and MESP1, while cells isolated from bone marrow, and dental follicle exhibit only weak reprogramming efficiency. qRT-PCR and transcriptomic analysis revealed activation of a cardiac-specific gene program and up-regulation of genes known to promote cardiac development. Although we did not observe the formation of fully mature cardiomyocytes, our data suggests that adult MSC have the capability to acquire a cardiac-like phenotype when treated with mRNA coding for transcription factors that regulate heart development. Yet, further optimization of the reprogramming process is mandatory to increase the reprogramming efficiency.
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Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Miócitos Cardíacos/citologia , RNA Mensageiro/genética , Tecido Adiposo/citologia , Adulto , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Saco Dentário/citologia , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , TranscriptomaRESUMO
Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.
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Perda do Osso Alveolar/prevenção & controle , Artrite Reumatoide/microbiologia , Artrite Reumatoide/prevenção & controle , Metotrexato/farmacologia , Periodontite/terapia , Animais , Artrite Experimental/microbiologia , Artrite Experimental/prevenção & controle , Autoanticorpos/química , Clorexidina/farmacologia , Colágeno/química , Progressão da Doença , Injeções Intraperitoneais , Masculino , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos DBA , Porphyromonas gingivalis , Microtomografia por Raio-XRESUMO
To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies. The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.
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Separação Celular/métodos , Saco Dentário/citologia , Saco Dentário/fisiologia , Engenharia Genética/métodos , MicroRNAs/fisiologia , Células-Tronco Multipotentes/fisiologia , Adulto , Edição de Genes/métodos , Técnicas de Transferência de Genes , Humanos , Transplante de Células-Tronco/métodosRESUMO
Periodontitis (PD) is a widespread chronic inflammatory disease in the human population. Porphyromonas gingivalis is associated with PD and can citrullinate host proteins via P. gingivalis peptidyl arginine deiminase (PPAD). Here, we hypothesized that infection of human dental follicle stem cells (hDFSCs) with P. gingivalis and subsequent interaction with neutrophils will alter the neutrophil phenotype. To test this hypothesis, we established and analyzed a triple-culture system of neutrophils and hDFSCs primed with P. gingivalis. Mitogen-activated pathway blocking reagents were applied to gain insight into stem cell signaling after infection. Naïve hDFSCs do not influence the neutrophil phenotype. However, infection of hDFSCs with P. gingivalis prolongs the survival of neutrophils and increases their migration. These phenotypic changes depend on direct cellular contacts and PPAD expression by P. gingivalis. Active JNK and ERK pathways in primed hDFSCs are essential for the phenotypic changes in neutrophils. Collectively, our results confirm that P. gingivalis modifies hDFSCs, thereby causing an immune imbalance.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Bacteroidaceae/imunologia , Saco Dentário/patologia , Neutrófilos/fisiologia , Periodontite/imunologia , Porphyromonas gingivalis/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Células-Tronco/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Imunomodulação , MAP Quinase Quinase 4/metabolismo , Ativação de Neutrófilo , Transdução de Sinais , Células-Tronco/microbiologiaRESUMO
Increasing evidence supports the association of periodontitis with rheumatoid arthritis. Even though a prominent role has been postulated for Porphyromonas gingivalis, many bacterial species contribute to the pathogenesis of periodontal disease. We therefore investigated the impact of Porphyromonas gingivalis as well as other major pathobionts on the development of both, periodontitis and arthritis in the mouse. Pathobionts used - either alone or in combination - were Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomintans. Periodontitis was induced via oral gavage in SKG, DBA/1 and F1 (DBA/1 × B10.Q) mice and collagen-induced arthritis was provoked via immunization and boost with bovine collagen type II. Alveolar bone loss was quantified via micro computed tomography, arthritis was evaluated macroscopically and histologically and serum antibodies were assessed. Among the strains tested, only F1 mice were susceptible to P. gingivalis induced periodontitis and showed significant alveolar bone loss. Bone loss was paralleled by antibody titers against P. gingivalis. Of note, mice inoculated with the mix of all three pathobionts showed less alveolar bone loss than mice inoculated with P. gingivalis alone. However, oral inoculation with either F. nucleatum or A. actinomycetemcomintans alone accelerated subsequent arthritis onset and progression. This is the first report of a triple oral inoculation of pathobionts combined with collagen-induced arthritis in the mouse. In this interplay and this particular genetic setting, F. nucleatum and A. actinomycetemcomitans exerted a protective impact on P. gingivalis induced alveolar bone loss. By themselves they did not induce periodontitis yet accelerated arthritis onset and progression.
Assuntos
Actinobacteria , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Artrite/etiologia , Artrite/patologia , Fusobacterium nucleatum , Porphyromonas gingivalis , Actinobacteria/fisiologia , Perda do Osso Alveolar/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Artrite/metabolismo , Artrite Experimental , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Fusobacterium nucleatum/fisiologia , Camundongos , Periodontite/etiologia , Periodontite/patologia , Porphyromonas gingivalis/fisiologiaRESUMO
Although prion protein's (PrP) involvement in transmission of degenerative neurological diseases has been subjected to considerable scrutiny, its physiological role is still obscure. The distribution of PrP in dental tissues was investigated using three different methods: immunohistochemistry, cell culture, and scanning electron microscopy. PrP knockout mice were found to have marked anomalies in dentin structure. In human teeth, cementoblasts and odontoblasts showed prominent staining for PrP at levels comparable to those of nerve fibers. Epithelial rests of Malassez, which are remnants of a cell type formerly forming enamel, were also positive. Thus, all PrP-positive cells in human dentition are in some way involved in calcified tissue formation. This suggests a previously undetected function of prion protein in healthy vertebrates as evidenced by an obvious phenotype in PrP knockout mice. Periodontal and pulpal tissue exposed by disease or trauma might represent a clinically relevant entry point for prions incorporated orally and thus a possible mode of infection.