Development and characterization of a novel meniscal extracellular matrix-derived scaffold

2017 ◽  
Author(s):  
◽  
Farrah Ann Monibi
Keyword(s):  
Extracellular Matrix ◽  
Tissue Repair ◽  
Cellular Proliferation ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Critical Role ◽  
Meniscal Repair ◽  
Biomechanical Properties ◽  
Ex Vivo Model

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Musculoskeletal injuries are a common and significant problem in orthopaedic practice. Despite advances in orthopaedic surgery, effective treatments for injuries to the knee meniscus remain a common and significant clinical challenge. Tissue engineering is a developing field that aims to regenerate injured tissues with a combination of cells, scaffolds, and signals. Many natural and synthetic scaffold materials have been developed and tested for the repair and restoration of a number of musculoskeletal tissues. Among these, biological scaffolds derived from extracellular matrix (ECM) have been developed and tested given the critical role of the ECM for maintaining the biological and biomechanical properties, structure, and function of native tissues. Decellularized scaffolds composed of ECM hold promise for repair and regeneration of the meniscus given the potential for ECM-based biomaterials to aid in cell recruitment, infiltration, and differentiation. The objectives of this research were to decellularize canine menisci in order to fabricate a micronized, ECM-derived scaffold, and to determine the cytocompatibility and repair potential of the scaffold ex vivo by developing an in vitro model for meniscal repair. In the first series of experiments, menisci were decellularized with a combination of physical agitation and chemical treatments. For scaffold fabrication, decellularized menisci were cryoground into a powder and the size and morphology of the ECM particles were evaluated using scanning electron microscopy. Histologic and biochemical analyses of the scaffold confirmed effective decellularization with loss of proteoglycan from the tissue but no significant reduction in collagen content. When washed effectively, the decellularized scaffold was cytocompatible to meniscal fibrochondrocytes, synoviocytes, and whole meniscal tissue based on the resazurin reduction assay, fluorescent live/dead staining, and histologic evaluation. Further, the scaffold supported cellular attachment and proliferation when combined with platelet rich plasma, and promoted an upregulation of genes associated with meniscal ECM synthesis and tissue repair. In an ex vivo model for meniscal repair, radial tears repaired and augmented with the scaffold demonstrated increased cellular proliferation and tissue repair compared to non-augmented repairs. Therefore, a micronized scaffold derived from decellularized meniscus may be a viable biomaterial for promoting avascular meniscal healing. However, further studies are necessary to determine an optimal carrier for delivery of the scaffold, and to examine the potential for the scaffold to induce cellular differentiation and functional meniscal fibrochondrogenesis.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals MT1-MMP–dependent neovessel formation within the confines of the three-dimensional extracellular matrix

2004 ◽  
Vol 167 (4) ◽  
pp. 757-767 ◽  
Author(s):  
Tae-Hwa Chun ◽  
Farideh Sabeh ◽  
Ichiro Ota ◽  
Hedwig Murphy ◽  
Kevin T. McDonagh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Collagenolytic Activity ◽  
Type I ◽  
Ex Vivo Model

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents

1995 ◽  
Vol 73 (02) ◽  
pp. 219-222 ◽  
Author(s):  
Manuel Monreal ◽  
Luis Monreal ◽  
Rafael Ruiz de Gopegui ◽  
Yvonne Espada ◽  
Ana Maria Angles ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Subcutaneous Administration ◽  
In Vitro Study ◽  
Ex Vivo Model ◽  
Suitable Candidate ◽  
Significant Prolongation ◽  
High Doses ◽  
Different Doses

SummaryThe APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

Dipyridamole Inhibits Platelet Aggregation in Whole Blood

1983 ◽  
Vol 50 (04) ◽  
pp. 852-856 ◽  
Author(s):  
P Gresele ◽  
C Zoja ◽  
H Deckmyn ◽  
J Arnout ◽  
J Vermylen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Significant Inhibition ◽  
Oral Drug ◽  
Human Blood Samples ◽  
Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

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.

scholarly journals Interactome Analysis of iPSC Secretome and Its Effect on Macrophages In Vitro

2021 ◽  
Vol 22 (2) ◽  
pp. 958
Author(s):  
Luca Tamò ◽  
Kleanthis Fytianos ◽  
Fabienne Caldana ◽  
Cedric Simillion ◽  
Anis Feki ◽  
...  
Keyword(s):  
Tissue Repair ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Critical Role ◽  
Organ Injury ◽  
Macrophage Phenotype ◽  
Gene Interaction Network ◽  
Tissue Repair And Regeneration ◽  
Secretory Pattern

Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

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 Considerations to Model Heart Disease in Women with Preeclampsia and Cardiovascular Disease

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 899
Author(s):  
Clara Liu Chung Ming ◽  
Kimberly Sesperez ◽  
Eitan Ben-Sefer ◽  
David Arpon ◽  
Kristine McGrath ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Disease ◽  
Ex Vivo ◽  
Myocardial Damage ◽  
Cardiovascular Disorder ◽  
Model Systems ◽  
Ex Vivo Model ◽  
Disease Manifestation

Preeclampsia is a multifactorial cardiovascular disorder diagnosed after 20 weeks of gestation, and is the leading cause of death for both mothers and babies in pregnancy. The pathophysiology remains poorly understood due to the variability and unpredictability of disease manifestation when studied in animal models. After preeclampsia, both mothers and offspring have a higher risk of cardiovascular disease (CVD), including myocardial infarction or heart attack and heart failure (HF). Myocardial infarction is an acute myocardial damage that can be treated through reperfusion; however, this therapeutic approach leads to ischemic/reperfusion injury (IRI), often leading to HF. In this review, we compared the current in vivo, in vitro and ex vivo model systems used to study preeclampsia, IRI and HF. Future studies aiming at evaluating CVD in preeclampsia patients could benefit from novel models that better mimic the complex scenario described in this article.

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