RESUMEN
Oral mucosa is used in various surgical fields as a graft for the reconstruction of tissue defects. Tissue engineering of oral mucosa equivalents using autologous cells represents a suitable less burdensome alternative. The survival of the multilayered epithelium is essential for the functionality of the tissues in vivo. To ensure its functionality after transplantation, mucosa equivalents in vitro were subjected to extracorporeal shock wave therapy (ESWT) to determine whether this treatment stimulated the formation and differentiation of the epithelium. Mucosa equivalents treated with ESWT were examined for cellular metabolic activity using AlamarBlueTM assay. The formation of vascular structures, basement membrane, and multilayered epithelium were examined using confocal fluorescence microscopy and immunohistochemistry. The potential ingrowth in vivo was simulated using the chorioallantoic membrane model (CAM assay) in ovo. ESWT on culture day 19 of oral mucosa equivalents resulted in slightly increased cellular metabolic activity. The in vitro development of basement membrane and multilayer epithelium was stimulated by ESWT. Additionally, in the CAM assay, ESWT led to a more pronounced multilayered epithelium. Thus, ESWT stimulated the formation of a more distinct and differentiated multilayered epithelium of oral mucosa equivalents in vitro and might increase the chance of efficient ingrowth, survival, and functionality of tissue equivalents in vivo.
RESUMEN
Fetal calf serum (FCS) is used for in vitro cell culture, as it provides the cells with various growth-promoting compounds. For applications in humans, FCS does not meet the required safety standards and should be replaced by an appropriate substitute. This study analyzed the suitability of using human platelet lysate (hPL) as a substitute for FCS in endothelial cell cultures for in vitro and in vivo tissue engineering applications. The focus was placed on standardized, commercially available hPLs (MultiPL'30, MultiPL'100), which are approved for applications in humans, and compared to laboratory-prepared hPLs (lp-hLP). Human umbilical vein endothelial cells (HUVEC) were cultured with FCS or with different hPLs. Cell morphology, proliferation, viability, apoptosis, and necrosis, as well as the organization of vascular structures, were assessed. No morphological changes were noticed when FCS was replaced by standardized hPLs in concentrations of 1-10%. In contrast, the use of lp-hLPs led to irregular cell shape and increased vacuolization of the cytoplasm. HUVEC proliferation and viability were not compromised by using media supplemented with standardized hPLs or pl-hPLs in concentrations of 1-10%, compared to cells grown in media supplemented with 20% FCS. The apoptosis rate using lp-hPLs was higher compared to the use of standardized hPLs. The necrosis rate tended to be lower when FCS was replaced by hPLs. HUVEC formed more pronounced capillary-like structures when the media were supplemented with hPLs instead of supplementation with FCS. Thus, compared to the use of FCS, the use of hPLs was beneficial for the growth and optimal expression of functional endothelial cell characteristics during in vitro experiments. Commercially available hPLs proved to be particularly suitable, as they led to reproducible results during in vitro experiments, while meeting the safety requirements for in vivo use.
Asunto(s)
Albúmina Sérica Bovina , Ingeniería de Tejidos , Plaquetas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Medios de Cultivo/metabolismo , Células Endoteliales/metabolismo , Humanos , Necrosis/metabolismoRESUMEN
Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.
Asunto(s)
Regeneración Ósea , Sustitutos de Huesos/farmacología , Carbonato de Calcio/farmacología , Hidrogeles/administración & dosificación , Macrófagos/inmunología , Cicatrización de Heridas , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Sustitutos de Huesos/química , Carbonato de Calcio/química , Microanálisis por Sonda Electrónica , Hidrogeles/química , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Polietilenglicoles/química , Espectrometría por Rayos XRESUMEN
In addition to their chemical composition various physical properties of synthetic bone substitute materials have been shown to influence their regenerative potential and to influence the expression of cytokines produced by monocytes, the key cell-type responsible for tissue reaction to biomaterials in vivo. In the present study both the regenerative potential and the inflammatory response to five bone substitute materials all based on ß-tricalcium phosphate (ß-TCP), but which differed in their physical characteristics (i.e., granule size, granule shape and porosity) were analyzed for their effects on monocyte cytokine expression. To determine the effects of the physical characteristics of the different materials, the proliferation of primary human osteoblasts growing on the materials was analyzed. To determine the immunogenic effects of the different materials on human peripheral blood monocytes, cells cultured on the materials were evaluated for the expression of 14 pro- and anti-inflammatory cytokines, i.e., IL-6, IL-10, IL-1ß, VEGF, RANTES, IL-12p40, I-CAM, IL-4, V-CAM, TNF-α, GM-CSF, MIP-1α, Il-8 and MCP-1 using a Bio-Plex® Multiplex System. The granular shape of bone substitutes showed a significant influence on the osteoblast proliferation. Moreover, smaller pore sizes, round granular shape and larger granule size increased the expression of GM-CSF, RANTES, IL-10 and IL-12 by monocytes, while polygonal shape and the larger pore sizes increased the expression of V-CAM. The physical characteristics of a bone biomaterial can influence the proliferation rate of osteoblasts and has an influence on the cytokine gene expression of monocytes in vitro. These results indicate that the physical structure of a biomaterial has a significant effect of how cells interact with the material. Thus, specific characteristics of a material may strongly affect the regenerative potential in vivo.
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Materiales Biocompatibles/farmacología , Sustitutos de Huesos/farmacología , Citocinas/metabolismo , Macrófagos/metabolismo , Osteoblastos/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacosRESUMEN
Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-ß1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel-like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins' aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.
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Fosfatasa Alcalina/metabolismo , Movimiento Celular , Colágeno/metabolismo , Endotelio Vascular/fisiología , Fibroblastos/fisiología , Microvasos/fisiología , Neovascularización Fisiológica , Fosfatasa Alcalina/química , Sitios de Unión , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Endotelio Vascular/citología , Fibroblastos/citología , Humanos , Técnicas In Vitro , Microvasos/citología , Conformación ProteicaRESUMEN
Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.
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Células Epiteliales/citología , Fibroblastos/citología , Membranas Artificiales , Mucosa Bucal/citología , Procedimientos de Cirugía Plástica/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Procedimientos Quirúrgicos Urogenitales/métodos , Implantes Absorbibles , Animales , Materiales Biocompatibles , Células Cultivadas , Técnicas de Cocultivo , Colágeno Tipo IV/biosíntesis , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Queratina-13 , Ensayo de Materiales , Porcinos , TenascinaRESUMEN
Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino-modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine.
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Nanopartículas , Corona de Proteínas , Transporte Biológico , Células Endoteliales , Humanos , NanomedicinaRESUMEN
Vaterite, a metastable modification of calcium carbonate, embedded in a flexible microgel packaging with adjustable mechanical properties, functionality, and biocompatibility, provides a powerful scaffolding for bone tissue regeneration, as it is easily convertible to bone-like hydroxyapatite (HA). In this study, the synthesis and physical analysis of a packaging material to encapsulate vaterite particles and osteoblast cells into monodisperse, sub-millimeter-sized microgels, is described whereby a systematic approach is used to tailor the microgel properties. The size and shape of the microgels is controlled via droplet-based microfluidics. Key requirements for the polymer system, such as absence of cytotoxicity as well as biocompatibility and biodegradability, are accomplished with functionalized poly(ethylene glycol) (PEG), which reacts in a cytocompatible thiol-ene Michael addition. On a mesoscopic level, the microgel stiffness and gelation times are adjusted to obtain high cellular viabilities. The co-encapsulation of living cells provides i) an in vitro platform for the study of cellular metabolic processes which can be applied to bone formation and ii) an in vitro foundation for novel tissue-regenerative therapies. Finally, the degradability of the microgels at physiological conditions caused by hydrolysis-sensitive ester groups in the polymer network is examined.
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Microgeles , Huesos , Carbonato de Calcio , Geles , OsteogénesisRESUMEN
MOTIVATION: Deep learning use for quantitative image analysis is exponentially increasing. However, training accurate, widely deployable deep learning algorithms requires a plethora of annotated (ground truth) data. Image collections must contain not only thousands of images to provide sufficient example objects (i.e. cells), but also contain an adequate degree of image heterogeneity. RESULTS: We present a new dataset, EVICAN-Expert visual cell annotation, comprising partially annotated grayscale images of 30 different cell lines from multiple microscopes, contrast mechanisms and magnifications that is readily usable as training data for computer vision applications. With 4600 images and â¼26 000 segmented cells, our collection offers an unparalleled heterogeneous training dataset for cell biology deep learning application development. AVAILABILITY AND IMPLEMENTATION: The dataset is freely available (https://edmond.mpdl.mpg.de/imeji/collection/l45s16atmi6Aa4sI?q=). Using a Mask R-CNN implementation, we demonstrate automated segmentation of cells and nuclei from brightfield images with a mean average precision of 61.6 % at a Jaccard Index above 0.5.
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Algoritmos , Núcleo Celular , Procesamiento de Imagen Asistido por Computador , MicroscopíaRESUMEN
Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Endorribonucleasas/metabolismo , Células Endoteliales/enzimología , Proteínas de Choque Térmico/metabolismo , Hemangioma/enzimología , Hemangiosarcoma/enzimología , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas/metabolismo , eIF-2 Quinasa/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/genética , Células Endoteliales/patología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/genética , Hemangioma/genética , Hemangioma/patología , Hemangiosarcoma/genética , Hemangiosarcoma/patología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents.
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Comunicación Celular , Células Endoteliales/citología , Fibroblastos/citología , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Humanos , Interleucina-8/metabolismo , Microcirculación , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PEG is the gold standard polymer for pharmaceutical applications, however it lacks degradability. Degradation under physiologically relevant pH as present in endolysosomes, cancerous and inflammatory tissues is crucial for many areas. The authors present anionic ring-opening copolymerization of ethylene oxide with 3,4-epoxy-1-butene (EPB) and subsequent modification to introduce acid-degradable vinyl ether groups as well as methacrylate (MA) units, enabling radical cross-linking. Copolymers with different molar ratios of EPB, molecular weights (Mn ) up to 10 000â g mol-1 and narrow dispersities (D<1.05) were prepared. Both the P(EG-co-isoEPB)MA copolymer and the hydrogels showed pH-dependent, rapid hydrolysis at pHâ 5-6 and long-term storage stability at neutral pH (pHâ 7.4). By designing the degree of polymerization and content of degradable vinyl ether groups, the release time of an entrapped protein OVA-Alexa488 can be tailored from a few hours to several days (hydrolysis half-life time t1/2 at pHâ 5: 13â h to 51â h).
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Materiales Biocompatibles/química , Hidrogeles/química , Concentración de Iones de Hidrógeno , Hidrólisis , Metacrilatos/química , Polietilenglicoles/química , Polimerizacion , Proteínas , Compuestos de ViniloRESUMEN
Prevascularization of tissue constructs before implantation has been developed as a novel and promising concept for successful implantation. Since hypoxia might induce angiogenesis, we have investigated the effects of hypoxic treatment on vascularization by using co-cultures of primary human osteoblasts (POBs) and outgrowth endothelial cells. Our results show that: (a) repeated short-term hypoxia (2% O2 for 8 hr), not long-term hypoxia (2% O2 for 24 hr), over 1 or 2 weeks, significantly enhances microvessel formation in co-cultures; (b) sustained hypoxia, not short-term or long-term hypoxia, causes cytotoxicity in mono- and co-cultures; (c) the expression of some angiogenic and inflammatory factors such as vascular endothelial growth factor, platelet-derived growth factor subunit B, insulin-like growth factor 1, interleukin-8, and early growth response protein 1 increases significantly in hypoxia-treated POB monoculture and co-cultures after single or multiple 8- or 24-hr hypoxic treatments; (d) long-term (24 hr) hypoxic treatment induces more angiogenic inhibitors compared with short-term hypoxic treatment. Our findings suggest that hypoxia-induced vascularization/angiogenesis is regulated by a complex balance of angiogenic/antiangiogenic factors, and that repeated short-term hypoxia, but not repeated long-term hypoxia, promotes the vascularization and tissue regeneration of bone tissue constructs.
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Técnicas de Cocultivo , Células Endoteliales/patología , Neovascularización Fisiológica , Osteoblastos/patología , Muerte Celular , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Humanos , Mediadores de Inflamación/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.
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Exosomas/metabolismo , Inflamación/patología , Nanopartículas/toxicidad , Dióxido de Silicio/toxicidad , Células CACO-2 , Selectina E/metabolismo , Impedancia Eléctrica , Exosomas/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/metabolismoRESUMEN
BACKGROUND: Image segmentation and quantification are essential steps in quantitative cellular analysis. In this work, we present a fast, customizable, and unsupervised cell segmentation method that is based solely on Fiji (is just ImageJ)®, one of the most commonly used open-source software packages for microscopy analysis. In our method, the "leaky" fluorescence from the DNA stain DRAQ5 is used for automated nucleus detection and 2D cell segmentation. RESULTS: Based on an evaluation with HeLa cells compared to human counting, our algorithm reached accuracy levels above 92% and sensitivity levels of 94%. 86% of the evaluated cells were segmented correctly, and the average intersection over union score of detected segmentation frames to manually segmented cells was above 0.83. Using this approach, we quantified changes in the projected cell area, circularity, and aspect ratio of THP-1 cells differentiating from monocytes to macrophages, observing significant cell growth and a transition from circular to elongated form. In a second application, we quantified changes in the projected cell area of CHO cells upon lowering the incubation temperature, a common stimulus to increase protein production in biotechnology applications, and found a stark decrease in cell area. CONCLUSIONS: Our method is straightforward and easily applicable using our staining protocol. We believe this method will help other non-image processing specialists use microscopy for quantitative image analysis.
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Antraquinonas/metabolismo , Separación Celular/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , HumanosRESUMEN
IMPACT STATEMENT: We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevertheless reliable method.
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Separación Celular/métodos , Células Epiteliales/citología , Células Epiteliales/fisiología , Mucosa Bucal/citología , Mucosa Bucal/fisiología , Ingeniería de Tejidos , Células Cultivadas , HumanosRESUMEN
Gelatin-modified poly(ethylene terephthalate) (PET) surfaces have been previously realized via an intermediate dopamine coating procedure that resulted in surfaces with superior haemocompatibility compared to unfunctionalized PET. The present study addresses the biocompatibility assessment of these coated PET surfaces. In this context, the stability of the gelatin coating upon exposure to physiological conditions and its cell-interactive properties were investigated. The proposed gelatin-dopamine-PET surfaces showed an increased protein coating stability up to 24 days and promoted the attachment and spreading of both endothelial cells (ECs) and smooth muscle cells (SMCs). In parallel, physisorbed gelatin coatings exhibited similar cell-interactive properties, albeit temporarily, as the coating delaminated within 1 week after cell seeding. Furthermore, no or only minimal immunogenic or inflammatory responses were observed during in vitro cytotoxicity and endotoxicity assessment for all gelatin-modified PET surfaces evaluated. Overall, the combined enhanced biocompatibility reported herein together with the previously proven haemocompatibility show the potential of the gelatin-dopamine-PET surfaces to function as vascular graft candidates.
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Biomimética/métodos , Gelatina/metabolismo , Tereftalatos Polietilenos/metabolismoRESUMEN
Patients diagnosed with osteosarcoma are currently treated with intravenous injections of anticancer agents after tumor resection. However, due to remaining neoplastic cells at the site of tumor removal, cancer recurrence often occurs. Successful bone regeneration combined with the control of residual cancer cells presents a challenge for tissue engineering. Cyclodextrins loaded with chemotherapeutic drugs reversibly release the drugs over time. Hydroxyapatite bone biomaterials coated with doxorubicin-loaded cyclodextrin should release the drug with time after implantation directly at the original tumor site and may be a way to eliminate residual neoplastic cells. In the present study, we have carried out in vitro studies to evaluate such a drug-delivery system and have shown that doxorubicin released from cyclodextrin-coated hydroxyapatite retained biological activity and exhibited longer and higher cytotoxic effects on both cancer (osteosarcoma cells) and healthy cells (primary osteoblasts and endothelial cells) compared to biomaterials without cyclodextrin loaded with doxorubicin. Furthermore, doxorubicin released from biomaterials with cyclodextrin moderately induced the expression of tumor suppressor protein p53 whereas p21 expression was similar to control cells. In addition, hypoxic conditions, which occur after implantation until blood-flow to the area is regenerated, protected endothelial cells and primary osteoblasts from doxorubicin-induced cytotoxicity. This chemo-protective effect was far less prominent for the osteosarcoma cells. These findings indicate that a hydroxyapatite-cyclodextrin-doxorubicin chemotherapeutic strategy may enhance the drug-targeting effect on tumor cells while protecting the more sensitive healthy cells for a period of time after implantation. A successful integration of such a drug delivery system might allow healthy cells to initially survive during the doxorubicin exposure period, while eliminating residual neoplastic cells.
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Antibióticos Antineoplásicos , Neoplasias Óseas/tratamiento farmacológico , Doxorrubicina , Sistemas de Liberación de Medicamentos/métodos , Osteosarcoma/tratamiento farmacológico , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Ciclodextrinas/química , Ciclodextrinas/farmacocinética , Ciclodextrinas/farmacología , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Durapatita/química , Durapatita/farmacocinética , Durapatita/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteosarcoma/metabolismo , Osteosarcoma/patología , Cuidados Posoperatorios/métodosRESUMEN
In vascular tissue engineering, great attention is paid to the immobilization of biomolecules onto synthetic grafts to increase bio- and hemocompatibility-two critical milestones in the field. The surface modification field of poly(ethylene terephthalate) (PET), a well-known vascular-graft material, is matured and oversaturated. Nevertheless, most developed methods are laborious multistep procedures generally accompanied by coating instability or toxicity issues. Herein, a straightforward surface modification procedure is presented engineered to simultaneously promote surface endothelialization and anticoagulation properties via the covalent immobilization of gelatin through a photoactivated azide derivative. A complete physicochemical characterization and biological study including cytotoxicity and endotoxin testing are performed. In addition, biocompatibility toward small (diameter ≤ 6 mm) and/or large caliber (diameter ≥ 6 mm) vessels is assessed by micro- and macrovascular endothelial cell assays. Superior bio- and hemocompatibility properties are seen for the gelatin-covalently modified PET surfaces compared to the conventional surface-modification procedures based on physisorption.
Asunto(s)
Anticoagulantes/química , Materiales Biocompatibles/química , Gelatina/química , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Tereftalatos Polietilenos/química , Anticoagulantes/farmacología , Azidas/química , Materiales Biocompatibles/farmacología , Biomarcadores/metabolismo , Prótesis Vascular , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Supervivencia Celular/efectos de los fármacos , Selectina E/genética , Selectina E/metabolismo , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipopolisacáridos/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Tereftalatos Polietilenos/farmacología , Propiedades de Superficie , Ingeniería de Tejidos/métodos , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
We have previously described a promising alternative to conventional synthetic bone biomaterials using vaterite, a metastable CaCO3 polymorph that increases the local Ca2+ concentration in vitro and leads to an oversaturation of phosphate, the primary bone mineral. This stimulates a natural bone-like mineralisation in a short period of time. In this study, sterile and endotoxin-free vaterite particles were synthesised in a nearly quantitative yield. The 500-1,000 nm vaterite particles did not exhibit any cytotoxic effects as measured by MTS, lactate dehydrogenase, or crystal violet assays on the human osteoblast cell line (MG-63) exposed to concentrations up to 500 µg/ml vaterite up to 72 hr. MG-63, primary human osteoblasts or human umbilical vein endothelial cells in the presence of vaterite up to 500 µg/ml for 7 days exhibited typical growth patterns. Endothelial cells exhibited a normal induction of E-selectin after exposure to LPS and MG-63 cells in osteogenic differentiation medium showed an increased expression of alkaline phosphatase compared with the respective control cells without vaterite. MG-63 cultured on a vaterite-containing degradable poly(ethylene glycol)-hydrogel exhibited strong adhesion and proliferation, similar to cells on cell culture plates. Cells did not attach to gels without vaterite. Our results demonstrate that vaterite particles are biocompatible, do not influence cell gene expression, and that vaterite in hydrogels may be able to serve for adhesion of osteoblasts and as a mineral substrate for natural bone formation by osteoblasts. These characteristics make vaterite particles a highly favourable compound for use in bone regeneration applications.