Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds

2021 ◽  
Author(s):  
Camilo Mora-Navarro ◽  
Mario Eduardo Garcia ◽  
Prottasha Sarker ◽  
Emily W Ozpinar ◽  
Jeffrey Enders ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Complex Structure ◽  
Scale Up ◽  
Bioactive Molecules ◽  
Good Representation ◽  
Process Standardization ◽  
Tissue Microenvironment ◽  
Potential Therapeutic Application ◽  
Dna Release

Abstract Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the specific local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g., DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the process standardization, cost-effectiveness, and scale-up. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization system to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allowing for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

scholarly journals Autolysin-Independent DNA Release inStreptococcus pneumoniae in vitroBiofilms

2018 ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Extracellular Matrix ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Lytic Enzymes ◽  
Microbial Biofilms ◽  
Dna Release

ABSTRACTBiofilms are defined as layers of cells of microorganisms adhered to the surface of a substrate and embedded in an extracellular matrix and provide an appropriate environment for increased genetic exchange. Extracellular DNA (eDNA) is an essential component of the extracellular matrix of microbial biofilms, but the pathway(s) responsible for DNA release are largely unknown. Autolysis (either spontaneous or phage-induced) has been proposed the major event leading to the appearance of eDNA. The ‘suicidal tendency’ ofStreptococcus pneumoniaeis well-known, with lysis mainly caused by the triggering of LytA, the major autolytic amidase. However, the LytC lysozyme and CbpD (a possible murein hydrolase) have also been shown involved. The present work examines the relationship between eDNA, autolysins, and the formation and maintenance ofin vitropneumococcal biofilms, via fluorescent labelling combined with confocal laser scanning microscopy, plus genetic transformation experiments. Bacterial DNA release mechanisms other than those entailing lytic enzymes were shown to be involved by demonstrating that horizontal gene transfer in biofilms takes place even in the absence of detectable autolytic activity. It had been previously suggested that the quorum sensing systems ComABCDE and LuxS/AI-2 are involved in the production of eDNA as a response to the accumulation of quorum sensing signals, although our immunofluorescence results do not support this hypothesis. Evidence that the release of DNA is somehow linked to the production of extracellular vesicles byS. pneumoniaeis provided.

scholarly journals Human foreskin-derived dermal stem/progenitor cell-conditioned medium combined with hyaluronic acid promotes extracellular matrix regeneration in diabetic wounds

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu Xin ◽  
Peng Xu ◽  
Xiangsheng Wang ◽  
Yunsheng Chen ◽  
Zheng Zhang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
Matrix Metalloproteinases ◽  
Conditioned Medium ◽  
Progenitor Cell ◽  
Bioactive Molecules ◽  
Published Data ◽  
Collagen Iii ◽  
Diabetic Wounds

Abstract Background Diabetic wounds remain a challenging clinical problem, which requires further treatment development. Published data showed that dermis-derived stem/progenitor cells (DSPCs) display superior wound healing in vitro. The beneficial effects of DSPCs are mediated through paracrine secretion, which can be obtained from conditioned medium (CM). Hyaluronic acid (HA) is especially suitable for skin regeneration and delivering bioactive molecules in CM. This study investigated the effect of human foreskin-derived dermal stem/progenitor cell (hFDSPC)-CM combined with HA on a diabetic mouse model and relevant mechanism in vitro. Methods hFDSPCs and human adipose-derived stem cells (hADSCs) were identified, and the respective CM was prepared. PBS, HA, hFDSPC-CM combined with HA, or hADSC-CM combined with HA was topically applied to mice. HE, CD31, CD68, CD86, and CD206 staining was performed to evaluate gross wound condition, angiogenesis, and inflammation, respectively. Masson and Picrosirius red staining was performed to evaluate collagen deposition and maturation. The effects of hFDSPC-CM and hADSC-CM on human keratinocyte cells (HaCaT) and fibroblasts were evaluated in vitro using CCK-8 and EdU assays to determine cell viability and proliferation, respectively. The scratch assay was performed to evaluate cell migration. Tube formation assay was performed on human umbilical vein endothelial cells (HUVECs) to confirm angiogenesis. Extracellular matrix (ECM) metabolic balance-related genes and proteins, such as collagen I (COL 1), collagen III (COL 3), fibronectin (FN), α-SMA, matrix metalloproteinases 1 (MMP-1), matrix metalloproteinases 3 (MMP-3), and transforming growth factor-beta 1 (TGF-β1), were analysed. Results hFDSPC-CM combined with HA showed superior wound closure rate over hADSC-CM. Histologically, the hFDSPC-CM combined with HA group showed significantly improved re-epithelialisation, angiogenesis, anti-inflammation, collagen regeneration, and maturation compared to hADSC-CM combined with HA group. In vitro assays revealed that hFDSPC-CM displayed significant advantages on cell proliferation, migration, and ECM regeneration through a TGF-β/Smad signalling pathway compared with hADSC-CM. Conclusions hFDSPC-CM combined with HA was superior for treating diabetic wounds. The underlying mechanism may promote proliferation and migration of epidermal cells with fibroblasts, thus leading to ECM deposition and remodelling. Reduced inflammation may be due to the above-mentioned mechanism.

scholarly journals Crosstalk between invadopodia and the extracellular matrix

2020 ◽  
Author(s):  
Shinji Iizuka ◽  
Ronald P. Leon ◽  
Kyle P. Gribbin ◽  
Ying Zhang ◽  
Jose Navarro ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Complex Structure ◽  
Three Dimensional ◽  
3D Culture ◽  
Model Systems ◽  
Type I ◽  
Extracellular Matrix Components

ABSTRACTThe scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native type I collagen, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, collagen I, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

CD44 Signaling Via PI3K/AKT and MAPK/ERK Pathways Protects CLL Cells from Spontaneous and Drug Induced Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 541-541 ◽  
Author(s):  
Yair Herishanu ◽  
Federica Gibellini ◽  
Ndegwa Njuguna ◽  
Keyvan Keyvanfar ◽  
Adrian Wiestner
Keyword(s):  
Extracellular Matrix ◽  
Protein Stabilization ◽  
Lymphocytic Leukemia ◽  
Live Cells ◽  
Drug Induced ◽  
Tissue Microenvironment ◽  
Unstimulated Control ◽  
Induced Apoptosis ◽  
Survival Effect

Abstract Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-cells. CLL cell proliferation and survival depends on host factors that are present in the tissue microenvironment. In–vitro these cells rapidly undergo apoptosis but can be supported by culture on stroma cells. The extracellular matrix is an important component of the microenvironment. Interactions between tumor cells and the extracellular matrix are, in part, mediated by CD44, whose principle ligand is hyaluronic acid. To explore a possible role of this adhesion receptor in CLL, we evaluated the effect of CD44 engagement on the survival of CLL cells and the induction of drug resistance. Dimerization of CD44 activated the PI3K/AKT and MAPK/ERK pathways and resulted in increased levels of MCL-1. There was no increase in MCL mRNA in CD44 stimulated cells, consistent with an effect on protein stabilization. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous apoptosis: CD44 stimulated CLL cells had a 46% increase (range 7% – 181%) in viability over the corresponding unstimulated control cells (n=20, p<0.0001). Furthermore, CD44 activation also protected from fludarabine-induced apoptosis: cell viability for isotype treated cells 76 ±6%, for isotype and fludarabine treated cells 47 ±18% and for CD44 stimulated and fludarabine treated cells 69 ±16% (p=0.005). CLL cells of the IgVH unmutated subtype expressed CD44 more highly than IgVH mutated CLL cells (MFI ratio 224 ±43 to 122 ±44, respectively, p<0.0001) and derived a more pronounced survival advantage from CD44 activation: % live cells (CD44 stimulated - control) 21 ±9% vs. 13 ±6%, respectively (p=0.04), which could contribute to the more progressive nature of this subtype. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44 activation. Furthermore, obatoclax was able to sensitize CLL cells to fludarabine. In conclusion, CD44 engagement protects CLL cells from spontaneous and fludarabine induced apoptosis. This prosurvival effect is mediated through PI3K/Akt and MAPK/ERK pathways and increased MCL-1 levels. Targeting either signaling pathway or MCL-1 with specific inhibitors could overcome the protective effect of the microenvironment in CLL and provides a rationale for combination therapy.

scholarly journals User-defined, temporal presentation of bioactive molecules on hydrogel substrates using supramolecular coiled coil complexes

2021 ◽  
Author(s):  
M. Gregory Grewal ◽  
Vincent P. Gray ◽  
Rachel A. Letteri ◽  
Christopher B. Highley
Keyword(s):  
Extracellular Matrix ◽  
Coiled Coil ◽  
Bioactive Molecules ◽  
Culture Systems ◽  
Significant Attention

The ability to spatiotemporally control the presentation of relevant biomolecules in synthetic culture systems has gained significant attention as researchers strive to recapitulate the endogenous extracellular matrix (ECM) in vitro....

scholarly journals Decellularized Extracellular Matrix as anIn VitroModel to Study the Comprehensive Roles of the ECM in Stem Cell Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Takashi Hoshiba ◽  
Guoping Chen ◽  
Chiho Endo ◽  
Hiroka Maruyama ◽  
Miyuki Wakui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Regenerative Medicine ◽  
Intracellular Signaling ◽  
Complex Structure ◽  
Stem Cell Differentiation

Stem cells are a promising cell source for regenerative medicine. Stem cell differentiation must be regulated for applications in regenerative medicine. Stem cells are surrounded by extracellular matrix (ECM)in vivo. The ECM is composed of many types of proteins and glycosaminoglycans that assemble into a complex structure. The assembly of ECM molecules influences stem cell differentiation through orchestrated intracellular signaling activated by many ECM molecules. Therefore, it is important to understand the comprehensive role of the ECM in stem cell differentiation as well as the functions of the individual ECM molecules. Decellularized ECM is a usefulin vitromodel for studying the comprehensive roles of ECM because it retains a native-like structure and composition. Decellularized ECM can be obtained fromin vivotissue ECM or ECM fabricated by cells culturedin vitro. It is important to select the correct decellularized ECM because each type has different properties. In this review, tissue-derived and cell-derived decellularized ECMs are compared asin vitroECM models to examine the comprehensive roles of the ECM in stem cell differentiation. We also summarize recent studies using decellularized ECM to determine the comprehensive roles of the ECM in stem cell differentiation.

scholarly journals Extracellular Matrix-Derived Hydrogels as Biomaterial for Different Skeletal Muscle Tissue Replacements

Materials ◽  
2020 ◽  
Vol 13 (11) ◽  
pp. 2483 ◽  
Author(s):  
Daniele Boso ◽  
Edoardo Maghin ◽  
Eugenia Carraro ◽  
Mattia Giagante ◽  
Piero Pavan ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Muscle Tissue ◽  
Myogenic Differentiation ◽  
Skeletal Muscle Tissue ◽  
Tissue Microenvironment ◽  
Topographical Cues

Recently, skeletal muscle represents a complex and challenging tissue to be generated in vitro for tissue engineering purposes. Several attempts have been pursued to develop hydrogels with different formulations resembling in vitro the characteristics of skeletal muscle tissue in vivo. This review article describes how different types of cell-laden hydrogels recapitulate the multiple interactions occurring between extracellular matrix (ECM) and muscle cells. A special attention is focused on the biochemical cues that affect myocytes morphology, adhesion, proliferation, and phenotype maintenance, underlining the importance of topographical cues exerted on the hydrogels to guide cellular orientation and facilitate myogenic differentiation and maturation. Moreover, we highlight the crucial role of 3D printing and bioreactors as useful platforms to finely control spatial deposition of cells into ECM based hydrogels and provide the skeletal muscle native-like tissue microenvironment, respectively.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

1997 ◽  
Vol 78 (02) ◽  
pp. 934-938 ◽  
Author(s):  
Hsiun-ing Chen ◽  
Yueh-I Wu ◽  
Yu-Lun Hsieh ◽  
Guey-Yueh Shi ◽  
Meei-Jyh Jiang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Platelet Adhesion ◽  
Treatment Time ◽  
Shape Change ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Apical Surface ◽  
Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Sign in / Sign up

Export Citation Format

Share Document