scholarly journals A decellularized human corneal scaffold for anterior corneal surface reconstruction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan J. Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

scholarly journals A Decellularized Human Corneal Scaffold for Anterior Corneal Surface Reconstruction

2020 ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

Abstract Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

scholarly journals A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

2021 ◽  
Vol 22 (18) ◽  
pp. 10067
Author(s):  
Naresh Polisetti ◽  
Benjamin Roschinski ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Günther Schlunck ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Host Tissue ◽  
Fibrin Gel ◽  
Deoxyribonuclease I ◽  
Limbal Stem Cell

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

ACTIVATED INTESTINAL MUSCLE CELLS PROMOTE PREADIPOCYTE MIGRATION: A NOVEL MECHANISM OF CREEPING FAT FORMATION IN CROHN’S DISEASE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S34-S34
Author(s):  
Ren Mao ◽  
Genevieve Doyon ◽  
Ilyssa Gordon ◽  
Jiannan Li ◽  
Sinan Lin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Muscularis Propria ◽  
Dominant Factor ◽  
Stricture Formation ◽  
Intestinal Tissue ◽  
Mesenteric Fat

Abstract Background and Aims Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn’s disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. Methods Tissues from normal, ulcerative colitis, non-strictured and strictured Crohn’s disease intestinal specimens were obtained. Fresh and decellularized tissue, mesenteric fat explants, primary human adipocytes, pre-adipocytes, muscularis propria cells, and native extracellular matrix were used in multiple ex vivo and in vitro systems involving cell growth, differentiation and migration, proteomics, and integrin expression. Results Crohn’s disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by TGF-b1. The muscle cell-derived matrix triggered migration of pre-adipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the pre-adipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularized intestinal tissue extracellular matrix. Conclusion Crohn’s disease creeping fat appears to result from the migration of pre-adipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

Preliminary assessment of an injectable extracellular matrix from decellularized bovine myocardial tissue

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Hatice Ercan ◽  
Ayşe Eser Elçin ◽  
Yaşar Murat Elçin
Keyword(s):  
Content Analysis ◽  
Complete Removal ◽  
Nuclear Material ◽  
Biological Properties ◽  
Myocardial Tissue ◽  
Rheological Characterization ◽  
Sgag Content ◽  
Tissue Matrix

Abstract The goal of this study was to develop an injectable form of decellularized bovine myocardial tissue matrix which could retain high levels of functional ECM molecules, and could gel at physiological temperature. Dissected ventricular tissue was processed by a detergent-based protocol, lyophilized, enzymatically-digested, and neutralized to form the injectable myocardial matrix (IMM). Histochemical analysis, DNA quantification, and agarose gel electrophoresis demonstrated the efficiency of the applied protocol. Chemical, thermal, morphological, and rheological characterization; protein and sulfated glycosaminoglycan (sGAG) content analysis were performed, in vitro biological properties were evaluated. An in vivo histocompatibility and biodegradability study was performed. Histochemistry revealed complete removal of myocardial cells. DNA content analysis revealed a significant decrease (87%) in the nuclear material, while protein and sGAG contents were highly preserved following decellularization. Soluble IMM was capable of turning into gel form at ∼37 °C, indicating selfassembling property. In vitro findings showed the biomaterial was noncytotoxic, nonhemolytic, and supported the attachment and proliferation of mesenchymal stem cells. In vivo study demonstrated IMM was well-tolerated by rats receiving subcutaneous injection. This work demonstrates that the IMM from decellularized bovine myocardial tissue has the potential for use as a feasible regenerative biomaterial in prospective tissue engineering and regenerative medicine studies.

scholarly journals Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

2021 ◽  
Vol 14 (3) ◽  
pp. dmm048116 ◽  
Author(s):  
Aikta Sharma ◽  
Alice Goring ◽  
Peter B. Johnson ◽  
Roger J. H. Emery ◽  
Eric Hesse ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Second Harmonic ◽  
Collagen Fibre ◽  
Matrix Composition ◽  
Label Free ◽  
Fibre Number ◽  
Backscattered Electron ◽  
Matrix Mineralisation

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

scholarly journals Biomimetic gradient scaffold of collagen–hydroxyapatite for osteochondral regeneration

2020 ◽  
Vol 11 ◽  
pp. 204173141989606 ◽  
Author(s):  
Cristian Parisi ◽  
Luca Salvatore ◽  
Lorenzo Veschini ◽  
Maria Paola Serra ◽  
Carl Hobbs ◽  
...  
Keyword(s):  
Biological Properties ◽  
Biological Evaluation ◽  
Mechanical Tests ◽  
Host Cells ◽  
Smooth Transition ◽  
Treatment Modalities ◽  
Chemical Compositions ◽  
Osteochondral Defects

Osteochondral defects remain a major clinical challenge mainly due to the combined damage to the articular cartilage and the underlying bone, and the interface between the two tissues having very different properties. Current treatment modalities have several limitations and drawbacks, with limited capacity of restoration; however, tissue engineering shows promise in improving the clinical outcomes of osteochondral defects. In this study, a novel gradient scaffold has been fabricated, implementing a gradient structure in the design to mimic the anatomical, biological and physicochemical properties of bone and cartilage as closely as possible. Compared with the commonly studied multi-layer scaffolds, the gradient scaffold has the potential to induce a smooth transition between cartilage and bone and avoid any instability at the interface, mimicking the natural structure of the osteochondral tissue. The scaffold comprises a collagen matrix with a gradient distribution of low-crystalline hydroxyapatite particles. Physicochemical analyses confirmed phase and chemical compositions of the gradient scaffold and the distribution of the mineral phase along the gradient scaffold. Mechanical tests confirmed the gradient of stiffness throughout the scaffold, according to its mineral content. The gradient scaffold exhibited good biological performances both in vitro and in vivo. Biological evaluation of the scaffold, in combination with human bone-marrow–derived mesenchymal stem cells, demonstrated that the gradient of composition and stiffness preferentially increased cell proliferation in different sub-regions of the scaffold, according to their high chondrogenic or osteogenic characteristics. The in vivo biocompatibility of the gradient scaffold was confirmed by its subcutaneous implantation in rats. The gradient scaffold was significantly colonised by host cells and minimal foreign body reaction was observed. The scaffold’s favourable chemical, physical and biological properties demonstrated that it has good potential as an engineered osteochondral analogue for the regeneration of damaged tissue.

scholarly journals Analysis of Plasmodium vivax schizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sasha V. Siegel ◽  
Lia Chappell ◽  
Jessica B. Hostetler ◽  
Chanaki Amaratunga ◽  
Seila Suon ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Ex Vivo ◽  
Culture Method ◽  
Surface Proteins ◽  
Parasite Development ◽  
Field Isolates ◽  
Host Parasite Interactions ◽  
Short Period ◽  
Host Parasite

Abstract Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

scholarly journals Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo

2019 ◽  
Vol 8 (5) ◽  
pp. 24 ◽  
Author(s):  
Mario R Romano ◽  
Mariantonia Ferrara ◽  
Claudio Gatto ◽  
Barbara Ferrari ◽  
Laura Giurgola ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Cytotoxicity Test ◽  
Human Donor

scholarly journals Very late antigen 4-dependent adhesion and costimulation of resting human T cells by the bacterial beta 1 integrin ligand invasin.

1993 ◽  
Vol 177 (1) ◽  
pp. 207-212 ◽  
Author(s):  
E Ennis ◽  
R R Isberg ◽  
Y Shimizu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adhesion Molecules ◽  
Cd4 T Cells ◽  
Yersinia Pseudotuberculosis ◽  
Biological Properties ◽  
Host Cells ◽  
Late Antigen ◽  
Integrin Ligand

Bacteria and viruses often use the normal biological properties of host adhesion molecules to infect relevant host cells. The outer membrane bacterial protein invasin mediates the attachment of Yersinia pseudotuberculosis to human cells. In vitro studies have shown that four members of the very late antigen (VLA) integrin family of adhesion molecules, VLA-3, VLA-4, VLA-5, and VLA-6, can bind to invasin. Since CD4+ T cells express and use these integrins, we have investigated the interaction of CD4+ T cells with purified invasin. Although VLA integrin-mediated adhesion of T cells to other ligands such as fibronectin does not occur at high levels unless the T cells are activated, resting T cells bind strongly to purified invasin. The binding of resting T cells to invasin requires metabolic activity and an intact cytoskeleton. Although CD4+ T cells express VLA-3, VLA-4, VLA-5, and VLA-6, monoclonal antibody (mAb) blocking studies implicate only VLA-4 as a T cell invasin receptor. Like other integrin ligands, invasin can facilitate T cell proliferative responses induced by a CD3-specific mAb. These results suggest that the nature of the integrin ligand is a critical additional factor that regulates T cell integrin activity, and that direct interactions of T cells with bacterial pathogens such as Yersinia may be relevant to host immune responses to bacterial infection.

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