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1.
Adv Sci (Weinh) ; : e2406608, 2024 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-39324843

RESUMEN

The Food and Drug Administration's recent decision to eliminate mandatory animal testing for drug approval marks a significant shift to alternative methods. Similarly, the European Parliament is advocating for a faster transition, reflecting public preference for animal-free research practices. In vitro tissue models are increasingly recognized as valuable tools for regulatory assessments before clinical trials, in line with the 3R principles (Replace, Reduce, Refine). Despite their potential, barriers such as the need for standardization, availability, and cost hinder their widespread adoption. To address these challenges, the Robotic Enabled Biological Automation (ReBiA) system is developed. This system uses a dual-arm robot capable of standardizing laboratory processes within a closed automated environment, translating manual processes into automated ones. This reduces the need for process-specific developments, making in vitro tissue models more consistent and cost-effective. ReBiA's performance is demonstrated through producing human reconstructed epidermis, human airway epithelial models, and human intestinal organoids. Analyses confirm that these models match the morphology and protein expression of manually prepared and native tissues, with similar cell viability. These successes highlight ReBiA's potential to lower barriers to broader adoption of in vitro tissue models, supporting a shift toward more ethical and advanced research methods.

2.
J Clin Invest ; 134(10)2024 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-38747285

RESUMEN

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.


Asunto(s)
Transdiferenciación Celular , Fibroblastos , Miofibroblastos , Factor de Crecimiento Transformador beta , Proteína Wnt-5a , Fibrosis , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/farmacología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , MAP Quinasa Quinasa 4/metabolismo , Quinasas Asociadas a rho/metabolismo , Ligandos , Piel/metabolismo , Piel/patología , Humanos , Animales , Ratones , Células Cultivadas , Regulación hacia Arriba
3.
Nat Commun ; 14(1): 7660, 2023 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-37996412

RESUMEN

Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly's bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites' development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.


Asunto(s)
Parásitos , Trypanosoma brucei brucei , Trypanosoma , Moscas Tse-Tse , Animales , Humanos , Trypanosoma brucei brucei/genética , Piel/parasitología , Moscas Tse-Tse/parasitología , Mamíferos
4.
Adv Healthc Mater ; 12(30): e2301131, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37660290

RESUMEN

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.


Asunto(s)
Biomimética , Prótesis e Implantes , Humanos , Piel , Titanio , Neovascularización Patológica , Propiedades de Superficie
5.
Elife ; 122023 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-37435805

RESUMEN

Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca2+-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca2+ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca2+ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca2+ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca2+-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg2+-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca2+-myristoyl switch for CHP3. Instead, a Ca2+-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca2+ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.


Asunto(s)
Calcineurina , Proteínas de Unión al Calcio , Humanos , Calcineurina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Conformación Molecular , Protones , Lípidos , Calcio/metabolismo , Unión Proteica , Conformación Proteica
6.
Int J Mol Sci ; 23(24)2022 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-36555855

RESUMEN

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. Unfortunately, rifampicin is sensitive to hydrolysis and oxidation. As a result, no topical drug product for wound-healing applications has been approved. To address this medical need two nanostructured hydrogel formulations of rifampicin were developed. The liposomal vesicles were embedded into hydroxypropyl methylcellulose (HPMC) gel or a combination of hyaluronic acid and marine collagen. To protect rifampicin from degradation in aqueous environments, a freeze-drying method was developed. Before freeze-drying, two well-defined hydrogel preparations were obtained. After freeze-drying, the visual appearance, chemical stability, residual moisture content, and redispersion time of both preparations were within acceptable limits. However, the morphological characterization revealed an increase in the vesicle size for collagen-hyaluronic acid hydrogel. This was confirmed by subsequent release studies. Interactions of marine collagen with phosphatidylcholine were held responsible for this effect. The HPMC hydrogel formulation remained stable over 6 months of storage. Moving forward, this product fulfills all criteria to be evaluated in preclinical and clinical studies.


Asunto(s)
Hidrogeles , Rifampin , Rifampin/farmacología , Hidrogeles/química , Ácido Hialurónico/química , Cicatrización de Heridas , Colágeno/metabolismo , Desarrollo de Medicamentos
7.
Biomedicines ; 10(11)2022 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-36428530

RESUMEN

The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC50 of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.

8.
Sci Rep ; 12(1): 16269, 2022 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-36175453

RESUMEN

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.


Asunto(s)
Melanoma , Neoplasias Cutáneas , Animales , Epidermis , Glucosa , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Ratones , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Microambiente Tumoral , Vemurafenib/farmacología , Melanoma Cutáneo Maligno
9.
Ophthalmologie ; 119(9): 891-901, 2022 Sep.
Artículo en Alemán | MEDLINE | ID: mdl-35925345

RESUMEN

In the case of thermal or caustic burns of the ocular surface, loss of limbal epithelial stem cells leads to compromised self-renewal of the corneal epithelium. This results in permanent loss of vision. In these situations, transplantation of cultured limbal epithelial cells on an amniotic membrane or fibrin gel as substrate (Holoclar®) can help to regenerate the corneal surface. The required cells are obtained from the healthy partner eye, if available. Adult stem cells from other parts of the body potentially serve as alternative cell sources: hair follicles, oral mucosa, mesenchymal stromal cells, or induced pluripotent stem cells (originally, e.g., skin fibroblasts). The reprogramming of such cells can be achieved with the help of transcription factors. In addition, work is being done on biosynthetic or synthetic matrices, which not only serve as substrate material for the transplantation but also support the functional properties of these cells (self-renewal, corneal epithelial-typical phenotype).


Asunto(s)
Quemaduras Químicas , Epitelio Corneal , Quemaduras Químicas/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos , Córnea , Humanos , Medicina Regenerativa
10.
Biomedicines ; 10(6)2022 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-35740308

RESUMEN

A balanced and moist wound environment and surface increases the effect of various growth factors, cytokines, and chemokines, stimulating cell growth and wound healing. Considering this fact, we tested in vitro and in vivo water evaporation rates from the cellulose dressing epicitehydro when combined with different secondary dressings as well as the resulting wound healing efficacy in a porcine donor site model. The aim of this study was to evaluate how the different rates of water evaporation affected wound healing efficacy. To this end, epicitehydro primary dressing, in combination with different secondary dressing materials (cotton gauze, JELONET◊, AQUACEL® Extra ™, and OPSITE◊ Flexifix), was placed on 3 × 3 cm-sized dermatome wounds with a depth of 1.2 mm on the flanks of domestic pigs. The healing process was analyzed histologically and quantified by morphometry. High water evaporation rates by using the correct secondary dressing, such as cotton gauze, favored a better re-epithelialization in comparison with the low water evaporation resulting from an occlusive secondary dressing, which favored the formation of a new and intact dermal tissue that nearly fully replaced all the dermis that was removed during wounding. This newly available evidence may be of great benefit to clinical wound management.

11.
Appl Microbiol Biotechnol ; 106(7): 2541-2555, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35325274

RESUMEN

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.


Asunto(s)
Acuaporinas , Cupriavidus necator , Proteínas de Escherichia coli , Acuaporinas/metabolismo , Dióxido de Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicerol/metabolismo , Glicerol Quinasa/genética , Glicerol Quinasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo
12.
Appl Opt ; 61(32): 9616-9624, 2022 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-36606902

RESUMEN

We present the results of the automated post-processing of Mueller microscopy images of skin tissue models with a new fast version of the algorithm of density-based spatial clustering of applications with noise (FastDBSCAN) and discuss the advantages of its implementation for digital histology of tissue. We demonstrate that using the FastDBSCAN algorithm, one can produce the diagnostic segmentation of high resolution images of tissue by several orders of magnitude faster and with high accuracy (>97%) compared to the original version of the algorithm.


Asunto(s)
Algoritmos , Microscopía , Piel/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos
13.
Adv Mater ; 34(10): e2106780, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-34933407

RESUMEN

The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber-to-fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun-based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen-gel-based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.


Asunto(s)
Ingeniería de Tejidos , Andamios del Tejido , Animales , Tejido Conectivo , Matriz Extracelular/química , Piel , Andamios del Tejido/química
14.
Biomedicines ; 9(9)2021 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-34572338

RESUMEN

Burns affect millions every year and a model to mimic the pathophysiology of such injuries in detail is required to better understand regeneration. The current gold standard for studying burn wounds are animal models, which are under criticism due to ethical considerations and a limited predictiveness. Here, we present a three-dimensional burn model, based on an open-source model, to monitor wound healing on the epidermal level. Skin equivalents were burned, using a preheated metal cylinder. The healing process was monitored regarding histomorphology, metabolic changes, inflammatory response and reepithelialization for 14 days. During this time, the wound size decreased from 25% to 5% of the model area and the inflammatory response (IL-1ß, IL-6 and IL-8) showed a comparable course to wounding and healing in vivo. Additionally, the topical application of 5% dexpanthenol enhanced tissue morphology and the number of proliferative keratinocytes in the newly formed epidermis, but did not influence the overall reepithelialization rate. In summary, the model showed a comparable healing process to in vivo, and thus, offers the opportunity to better understand the physiology of thermal burn wound healing on the keratinocyte level.

15.
Immunity ; 54(4): 702-720.e17, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33789089

RESUMEN

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.


Asunto(s)
Cromatina/inmunología , Linfocitos T Reguladores/inmunología , Cicatrización de Heridas/inmunología , Adulto , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Diferenciación Celular/inmunología , Línea Celular , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/inmunología , Células HaCaT , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores CCR8/inmunología , Células T Auxiliares Foliculares/inmunología
16.
Sci Rep ; 11(1): 7070, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782484

RESUMEN

The replacement of animal models for investigation of inflammation and wound healing has been advancing by means of in vitro skin equivalents with increasing levels of complexity. However, the current in vitro skin models still have a limited pre-clinical relevance due to their lack of immune cells. So far, few steps have been made towards the incorporation of immune cells into in vitro skin and the requirements for immunocompetent co-cultures remain unexplored. To establish suitable conditions for incorporating macrophages into skin models, we evaluated the effects of different media on primary keratinocytes, fibroblasts and macrophages. Skin maturation was affected by culture in macrophage medium, while macrophages showed reduced viability, altered cell morphology and decreased response to pro- and anti-inflammatory stimuli in skin differentiation media, both in 2D and 3D. The results indicate that immunocompetent skin models have specific, complex requirements for supporting an accurate detection of immune responses, which point at the identification of a suitable culture medium as a crucial pre-requisite for the development of physiologically relevant models.


Asunto(s)
Macrófagos/fisiología , Supervivencia Celular , Medios de Cultivo , Técnicas In Vitro , Macrófagos/citología , Macrófagos/inmunología
17.
Foods ; 11(1)2021 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-35010202

RESUMEN

With the rising trend of consumers being offered by start-up companies portable devices and applications for checking quality of purchased products, it appears of paramount importance to assess the reliability of miniaturized sensors embedded in such devices. Here, eight sensors were assessed for food fraud applications in skimmed milk powder. The performance was evaluated with dry- and wet-blended powders mimicking adulterated materials by addition of either ammonium sulfate, semicarbazide, or cornstarch in the range 0.5-10% of profit. The quality of the spectra was assessed for an adequate identification of the outliers prior to a deep assessment of performance for both non-targeted (soft independent modelling of class analogy, SIMCA) and targeted analyses (partial least square regression with orthogonal signal correction, OPLS). Here, we show that the sensors have generally difficulties in detecting adulterants at ca. 5% supplementation, and often fail in achieving adequate specificity and detection capability. This is a concern as they may mislead future users, particularly consumers, if they are intended to be developed for handheld devices available publicly in smartphone-based applications.

18.
PLoS One ; 15(11): e0242615, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33253240

RESUMEN

3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.


Asunto(s)
Ciencias Bioconductuales , Reactores Biológicos , Células Endoteliales , Fibroblastos , Impresión Tridimensional , Ingeniería de Tejidos , Técnicas de Cultivo de Célula , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Masculino
19.
Skin Pharmacol Physiol ; 33(4): 189-197, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32683369

RESUMEN

INTRODUCTION: An accelerated healing of superficial wounds was demonstrated in clinical trials with a topical comfrey preparation (Symphytum × uplandicum Nyman). The effect has previously not been examined in skin models. METHODS: An established in vitro model of epidermal cells with the typical strata was used for the observation of effects of applied substances on skin regeneration. Damage corresponding to a typical abrasion was created on day 1 by punching an opening into the epidermal fine structure down to the stratum basale. Samples were either untreated (controls) or exposed to comfrey cream on days 2, 3, 5, and 6. Tissue samples were taken for light and electron microscopy on days 1, 4, and 7. RESULTS AND CONCLUSIONS: Application of comfrey cream led to a quicker regeneration of skin cells and to an earlier differentiation of the cells towards a normal fine structure with a visible distinction of epidermal strata, keratin, and corneocyte formation within 4-7 days. The study covered the early days of skin regeneration and confirms the benefits observed in published clinical trials and non-interventional studies in patients with abrasions.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Consuelda , Epidermis/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Microscopía Electrónica de Transmisión , Extractos Vegetales/farmacología , Repitelización/efectos de los fármacos , Administración Cutánea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Técnicas de Cocultivo , Consuelda/química , Epidermis/ultraestructura , Humanos , Queratinocitos/ultraestructura , Masculino , Extractos Vegetales/aislamiento & purificación , Crema para la Piel , Factores de Tiempo
20.
ALTEX ; 37(3): 429-440, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32302003

RESUMEN

With cellular products being on the front run there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells finally allowing to seed these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. Afterwards, we were able to induce inflammation-based tissue damage by pre-stimulated mismatched allogeneic lymphocytes and/or inflammatory cytokine containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. The effects could be prevented by the addition of immunosuppressants to the models. Consequently, these models would harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. This would also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells which would otherwise be limited to animal models. Thus, the current test platform developed with the limitation given that no professional APC are in place could highly reduce animal testing for investigation of novel immune therapies.


Asunto(s)
Alternativas a las Pruebas en Animales , Enfermedad Injerto contra Huésped/patología , Inmunosupresores/uso terapéutico , Modelos Biológicos , Piel/patología , Humanos , Linfocitos/fisiología , Andamios del Tejido
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