Characterization of tissue engineered endothelial cell networks in composite collagen-agarose hydrogels

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either Matrigel™ and rat tail collagen I (Matrigel™/rCOL) or human collagen (hCOL) with SeaPlaque™ agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in Matrigel™/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of Matrigel™/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Decelularizarea de succes a aortei porcine pentru generarea scaffoldului acelular necesar în obținerea grefelor vasculare inginerești

Laboratory of Tissue Engineering and Cell Cultures, Nicolae Anestiadi Department of Surgery no. 1, Nicolae Testemitanu SUMPh, LEBAO, Hannover Medical School, GermanyBackground. Based on limited availability of autologous vascular grafts, the development of tissueengineered blood vessels (TEBVs) has reached significant research interest in the last couples of years. Fabrication of TEBVs from decellulazed scaffolds seems to be an attractive research strategy. Objective of the study. To develop a method for the generation of cell-free vascular scaffolds by combining a detergent-based decellularization (DC) strategy with subsequent nuclease treatment. Material and Methods. Cryopreserved porcine aortae were treated with detergents for 48h under rotation (24h exposure to 0.5% SDS and 0.5% SDC, followed by 24h exposure to 1% TritonX-100) with/without following enzymatic digestion (48h exposure to 300 U/ml DNase I solution). The efficacy of DC was evaluated by 4’,6-Diamidino-2-phenylindole (DAPI) and hematoxylin and eosin (H&E) stainings. Results. H&E staining revealed no persisting cells in all groups, including the samples which were not treated with DNase. The DAPI stain of those specimens, however, uncovered substantial amounts of residual DNA. Additional enzymatic treatment with DNase led to such a reduction of residual DNA that DAPI positive structures could not be detected. Conclusion. A direct stain of DNA, e.g. DAPI, is much more sensitive measure for the presence of cell debris than the H&E stain. In addition, detergent-based decellularization technique per se is not sufficient for complete cell and debris removal and has to be combined with a DNase digestion.Introducere. Din cauza deficiențelor funcționale a grefelor vasculare (GV) existente, dezvoltarea substituenților sanguini, prin tehnicile de inginerie tisulară, a avansat semnificativ în ultimii ani. Producerea GV în baza matrixului acelular pare a fi o strategie atractivă. Scopul studiului: Dezvoltarea unei metode de generare a scaffoldului decelularizat, prin combinarea unei tehnici bazate pe detergenți, suplimentată cu prelucrarea cu nuclează. Material și Metode. Aorta porcină crioprezervată a fost tratată cu detergenți timp de 48h (expunerea la 0.5% SDS și 0.5% SDC pe 24h, urmată de spălarea cu soluție de 1% TX-100) cu/fără următoarea digestie enzimatică (prelucrarea cu soluție 300 U/ml DNază). Eficacitatea decelularizării (DC) a fost evaluată prin colorația 4’,6-Diamidino-2-fenilindol(DAPI) și Hematoxilină-Eozină (H&E). Rezultate. Colorația H&E nu a evidențiat celule persistente în nici un grup, inclusiv în probele tratate fără nuclează. Cu toate acestea, colorația DAPI a acelorași specimene a descoperit cantități substanțiale de ADN rezidual. Prelucrarea suplimentară cu DNază a dus la o reducere semnificativă a ADN-ului subzistent, încât structuri DAPI pozitive nu au fost detectate. Concluzii. Colorațiile cu afinitatea pentru ADN, d.e. DAPI, sunt mai sensibile pentru evaluarea prezenței resturilor celulare, decât H&E. Tehnica DC bazată pe detergenți nu este suficientă pentru îndepărtarea completă a celulelor și a detritului, și trebuie combinată cu prelucrarea enzimatică

Principii de decelularizare a grefelor osoase vascularizate

Department of Orthopedics and Traumatology, Laboratory of Tissue Engineering and Cell Cultures, Nicolae Testemitanu SUMPh, LEBAO, Hannover Medical School, GermanyBackground. By using extracellular matrices (nathural or synthetic), tissue engineering has a final aims to create autologous grafts for the effective use of replacement therapy of the organs or body segments in the absence of an immune response. Objective of the study. Analysis of the decellularization protocol of the composite (vascularized) bone graft in order to obtain a non-immunogenic vascularized extracellular bone matrix. Material and Methods. The effectiveness of the decellularization protocol was checked on 3 vascular grafts of different diameters (large, medium, small) and on bone blocks (cortical and spongy), both of porcine origin. The processed grafts were examined histologically and analyzed for the amount of DNA. The biocompatibility of the grafts was also tested. Results. The decellularization protocol used has been shown to be effective on vascular grafts with different diameters and on cortical and spongy bone blocks. Histological examination showed cell death after graft processing. DNA quantification has shown a decrease in the amount of DNA in bone grafts and the biocompatibility test has demonstrated the biocompatibility of vascular and bone grafts after processing. Conclusion. Even if soft and hard tissues are different histological structures, the decellularization protocol can be adapted for both tissue types in such a way that decellularization of composite grafts can become possible.Introducere. Prin utilizarea matricilor extracelulare (naturale sau sintetice), ingineria țesuturilor are ca scop final crearea de grefe autologe pentru utilizarea eficientă a terapiei de substituție a organelor sau a segmentelor corpului în absența unui răspuns imun. Scopul lucrării. Analiza protocolului de decelularizare a grefei osoase compozite (vascularizate) în vederea obținerii unei matrice osoase extracelulare vascularizate neimunogenă. Material și Metode. Eficacitatea protocolului de decelularizare a fost verificat pe 3 grefe vasculare de diferite diametre (mari, medii, mici) și pe blocuri osoase (cortical și spongios), ambele de origine porcină. Grefele prelucrate au fost examinate histologic și analizate după cantitatea de ADN. Biocompatibilitatea grefelor la fel a fost testată. Rezultate. Protocolul de decelularizare utilizat și-a demonstrat eficacitatea pe grefele vasculare de diferite diametre și pe blocurile osoase corticale și spongioase. Examinarea histologică a demostrat lipa celulară după procesarea grefelor. Coantificarea ADN a demonstrat scăderea cantității de ADN neînsemnată în grefele osoase, iar testul de biocompatibilitate a demontrat biocompatibilitatea grefelor vasculare și osoase. Concluzii. Chiar dacă țesuturile moi și dure reprezintă structuri histologice diferite, protocolul de decelularizare poate fi adaptat pentru ambele tipuri de țesuturi în așa mod încât decelularizarea grefelor compozite să fie posibilă

Experimental study in obtaining of a vascularised composite bone extracellular matrix

Introduction. One of the final products of tissue engineering is the extracellular matrix (ECM), a noncellular component of tissues that can be obtained using different methods of decellularization. Most decellularization protocols are divided into those for soft tissues and those for hard tissues [1-4]. Our study aims to develop and test the universal protocol for decellularization of the composite vascularized bone graft (soft and hard tissue) in order to obtain the vascularized extracellular bone matrix that can be used in reconstruction of the massive bone defects. Material and Methods. The same protocol was used for the decellularization of different diameters vascular grafts, and different structures of bone tissue (soft and hard tissue) porcine origin. Like large diameter vessel, was was taken from the tibial bone. The efficacy of the protocol was demonstrated by histological examinations, DNA quantification and the biocompatibility test. Results. The used protocol has been effective even for small diameter vascular grafts and on cortical and spongy bone blocks. Histological examination (H&E staining) showed cell death after processing. DNA quantification has shown a decrease in the amount of DNA, especially for spongy bone grafts and the biocompatibility test has demonstrated the biocompatibility of vascular and bone grafts after processing. Conclusions: We can obtain an effective decellularization for both soft and hard tissues using the same protocol

Use of sucrose to diminish pore formation in freeze-dried heart valves

© 2018, The Author(s). Freeze-dried storage of decellularized heart valves provides easy storage and transport for clinical use. Freeze-drying without protectants, however, results in a disrupted histoarchitecture after rehydration. In this study, heart valves were incubated in solutions of various sucrose concentrations and subsequently freeze-dried. Porosity of rehydrated valves was determined from histological images. In the absence of sucrose, freeze-dried valves were shown to have pores after rehydration in the cusp, artery and muscle sections. Use of sucrose reduced pore formation in a dose-dependent manner, and pretreatment of the valves in a 40% (w/v) sucrose solution prior to freeze-drying was found to be sufficient to completely diminish pore formation. The presence of pores in freeze-dried valves was found to coincide with altered biomechanical characteristics, whereas biomechanical parameters of valves freeze-dried with enough sucrose were not significantly different from those of valves not exposed to freeze-drying. Multiphoton imaging, Fourier transform infrared spectroscopy, and differential scanning calorimetry studies revealed that matrix proteins (i.e. collagen and elastin) were not affected by freeze-drying

Gain-of-function screen for genes that affect Drosophila muscle pattern formation.

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Identification of New X-Chromosomal Genes Required for Drosophila Oogenesis and Novel Roles for fs(1)Yb, brainiac and dunce

We performed a screen for female sterile mutations on the X chromosome of Drosophila melanogaster and identified new loci required for developmental events in oogenesis as well as new alleles of previously described genes. We present mapping and phenotypic characterization data for many of these genes and discuss their significance in understanding fundamental developmental and cell biological processes. Our screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. We also describe new roles for the genes dunce (dnc), brainiac (brn) and fs(1)Yb, and we identify new alleles of Sex lethal (Sxl), ovarian tumor (otu), sans filles (snf), fs(1)K10, singed (sn), and defective chorion-1 (dec-1)