scholarly journals Pancreaticline Cell Differentiation of Bone Marrow Mesenchymal Stromal Cells in Acellular Pancreatic Scaffolds

2020 ◽  
Author(s):  
Zhao Li ◽  
Yue Du ◽  
Xin Wang
Keyword(s):  
Cell Differentiation ◽  
Treatment Group ◽  
Stromal Cells ◽  
Culture System ◽  
Collagen Type ◽  
Collagen Type I ◽  
In Vivo Study ◽  
Type I ◽  
Pancreatic Cell

Abstract Background: To evaluate the potential differentiation ability of bone mesenchymal stromal cells(BMSCs) to pancreatic line Cells on rat acellular pancreatic bioscaffold(APB) and the effect of differentiated BMSCs for chronic pancreatitis(CP) in vivo. Methods: After BMSCs were isolated and identified, they were dynamic cultured on the APB and static cultured in tissue culture flask(TCF),with or without the growth factors (GF) in both the culture system. The cytological behavior such as the proliferation and differentiation of BMSCs in all the above kinds of culture system were assessed by morphological observation, flow cytometry, ELASA analysis, qRT-PCR assay and western blot analysis. For the in vivo study, the pancreatic fibrosis and pathological score were evaluated. And also the expression of α-SMA, collagen type I and III, IL-10 in pancreas tissue were detected by ELASA. Results: 4ml/min was the most appropriate flow rate for the dynamic culture of BMSCs. The proliferation rate of BMSCs in the APB groups were significantly increased compared to TCF system. During the pancreatic line cell differentiation process, APB could induce BMSCs express markers such as PDX-1 and PTF-1 at higher mRNA levels. In contrast, the marker Oct 4 was expressed at a lower level in APB group. For the pancreatic functional cytoketatins including α-Amy, CK7, Flk-1, and C-peptide, they were all expressed at higher level in APB group. And metabolic enzymes secretion such as amylase and insulin were promoted significantly in APB system. By scanning electron microscope(SEM) and transmission electron microscopy(TEM), the ultrastructure of BMSCs in the APB group could further demonstrated the morphological characteristics of pancreatic-like cells. In vivo study,the expression of α-SMA, collagen type I and III in tissues were less in differentiated BMSCs treatment group, while the level of IL-10 in pancreatic tissue were higher in differentiated BMSCs treatment group with significant difference (P<0.05). In addition, in both in vitro and in vivo study, GF could significantly facilitate the function of proliferation, differentiation and pancreatic cell therapy. Conclusion: Together our data show the capacity of APB , 3D pancreatic biomatrix, promoting BMSCs differentiate toward pancreatic line phenotypes, and the considerable potential of using these cells for pancreatic cell therapies and tissue engineering.

scholarly journals Pancreatic Lineage Cell Differentiation of Bone Marrow Mesenchymal Stromal Cells on Acellular Pancreatic Bioscaffold

2020 ◽  
Author(s):  
Zhao Li ◽  
Yue Du ◽  
Xin Wang
Keyword(s):  
Electron Microscopy ◽  
Cell Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Pancreatic Tissue ◽  
In Vivo Study ◽  
Morphological Observation ◽  
Collagen Types ◽  
Pancreatic Cell

Abstract Background:We evaluated(1) the potential differentiation ability of bone mesenchymal stromal cells(BMSCs) into pancreatic lineage cells on rat acellular pancreatic bioscaffold (APB) and (2) the effect of differentiated BMSCs on chronic pancreatitis in vivo.Methods:After BMSCs were isolated and characterized, they were dynamically cultured on APB and statically cultured in tissue culture flasks (TCFs), with or without growth factor(GF) in both culture systems. We assessed cytological behavior, such as the proliferation and differentiation of BMSCs, by morphological observation, flow cytometry, enzyme-linked immunosorbent assay (ELISA), quantitative real-time/reverse transcriptase polymerase chain reaction, and Western blot analysis. For the in vivo study, we evaluated the pancreatic fibrosis and pathological scores.We detected the expression of α-SMA,collagen types I and III,and IL-10 in pancreatic tissue by ELISA.Results:The most appropriate flow rate for the dynamic culture of BMSCs was 4mL/min. The proliferation rates of BMSCs in the APB groups were significantly higher than in the TCF groups.During the pancreatic lineage cell differentiation process, APB induced BMSCs to express mRNA markers such as PDX-1 and PTF-1 at higher levels. In contrast, the marker Oct4 was expressed at a lower level in the APB group. All tested pancreatic cytokeratins, including α-Amy, CK7, Flk-1, and C-peptide, were expressed at higher levels in the APB group. The secretion of metabolic enzymes, such as Amy and insulin, was higher in the APB system. By scanning electron microscopy and transmission electron microscopy, the ultrastructure of BMSCs in the APB group further revealed the morphological characteristics of pancreatic-like cells. In the in vivo study, the expression of α-SMA and collagen types I and III in tissues was significantly lower in differentiated BMSCs group, whereas the levels of IL-10 in pancreatic tissue were higher in differentiated BMSCs with significant difference. In addition, in both the in vitro and the in vivo study, GF significantly improved proliferation, differentiation, and pancreatic cell therapy.Conclusion: Our data showed (1) the capacity of APB, a three-dimensional pancreatic biomatrix, to promote BMSC differentiation toward pancreatic lineage and pancreatic-like phenotypes, and (2) the considerable potential of using these cells for pancreatic cell therapies and tissue engineering.

scholarly journals Properties of a bovine collagen type I membrane for guided bone regeneration applications

e-Polymers ◽  
2021 ◽  
Vol 21 (1) ◽  
pp. 210-221
Author(s):  
Igor S. Brum ◽  
Carlos N. Elias ◽  
Jorge J. de Carvalho ◽  
Jorge L. S. Pires ◽  
Mario J. S. Pereira ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Collagen Type ◽  
Mechanical Stability ◽  
Collagen Type I ◽  
Guided Bone Regeneration ◽  
Bone Volume ◽  
Male Rats ◽  
Toxicity Tests ◽  
Type I

Abstract Dental implant treatment requires an available bone volume in the implantation site to ensure the implant’s mechanical stability. When the bone volume is insufficient, one must resort to surgical means such as guided bone regeneration (GBR). In GBR surgery, bone grafts and membranes are used. The objective of this work is to manufacture and characterize the in vitro and in vivo properties of resorbable collagen type I membranes (Green Membrane®) for GBR. Membrane surface morphology was characterized by SEM and roughness was measured using an interferometric noncontact 3D system. In vivo skin sensitization and toxicity tests have been performed on Wistar rats. Bone defects were prepared in 24 adult male rats, filled with biomaterials (Blue Bone® and Bio Oss®) and covered with collagen membranes to maintain the mechanical stability of the site for bone regeneration. The incisions were closed with simple stitches; and 60 days after the surgery, the animals were euthanized. Results showed that the analyzed membrane was homogeneous, with collagen fiber webs and open pores. It had no sign of cytotoxicity and the cells at the insertion site showed no bone morphological changes. There was no tissue reaction and no statistical difference between Blue Bone® and Bio Oss® groups. The proposed membrane has no cytotoxicity and displays a biocompatibility profile that makes it suitable for GBR.

scholarly journals Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

2021 ◽  
Author(s):  
Michel Haagdorens ◽  
Elle Edin ◽  
Per Fagerholm ◽  
Marc Groleau ◽  
Zvi Shtein ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Fibrils ◽  
Type I ◽  
Safe Alternative ◽  
Human Donor ◽  
Human Collagen ◽  
Donor Corneas

Abstract Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

scholarly journals Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0254557
Author(s):  
Christian Freise ◽  
Hyunho Lee ◽  
Christopher Chronowski ◽  
Doug Chan ◽  
Jessica Cziomer ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Collagen Type ◽  
Stellate Cell ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Collagen Type I ◽  
Serum Starvation ◽  
Type I ◽  
Linear Peptide

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-β, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)—derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

scholarly journals Dopamine D1 receptor stimulates cathepsin K-dependent degradation and resorption of collagen I in lung fibroblasts

2020 ◽  
Vol 133 (23) ◽  
pp. jcs248278 ◽  
Author(s):  
Ana M. Diaz-Espinosa ◽  
Patrick A. Link ◽  
Delphine Sicard ◽  
Ignasi Jorba ◽  
Daniel J. Tschumperlin ◽  
...  
Keyword(s):  
Collagen Type ◽  
Cathepsin K ◽  
Collagen Type I ◽  
D1 Receptor ◽  
Collagen I ◽  
Lung Fibroblasts ◽  
Type I ◽  
Cultured Fibroblasts ◽  
Normal Wound Healing

ABSTRACTMatrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution.This article has an associated First Person interview with the first author of the paper.

Rapid and Large-Scale Formation of Chondrocyte Aggregates by Rotational Culture

2003 ◽  
Vol 12 (5) ◽  
pp. 475-479 ◽  
Author(s):  
Katsuko S. Furukawa ◽  
Hideyuki Suenaga ◽  
Kenshi Toita ◽  
Akiko Numata ◽  
Junzo Tanaka ◽  
...  
Keyword(s):  
Culture System ◽  
Large Scale ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Cell Interaction ◽  
Collagen Type I ◽  
Cell Contact ◽  
Type I ◽  
Pellet Culture ◽  
Cell Cell

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

scholarly journals Proteoglycan-4 Regulates Fibroblast to Myofibroblast Transition and Expression of Fibrotic Genes in the Synovium

2020 ◽  
Author(s):  
Marwa Qadri ◽  
Gregory D. Jay ◽  
Ling X. Zhang ◽  
Holly Richendrfer ◽  
Tannin A. Schmidt ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Stress Fiber ◽  
Type I ◽  
Β Cells ◽  
Smad Pathway ◽  
Antifibrotic Effect ◽  
Fibroblast Migration ◽  
Pathway Activation

Abstract Background: Synovial tissue fibrosis is common in advanced OA with features including presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. An antifibrotic effect in OA synoviocytes was associated with PRG4 secretion and native synovial PRG4 reduced collagen content in OA synoviocytes. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo . Methods: OA synoviocytes were treated with TGF-β ± PRG4 for 24 hours and α-SMA content was determined using immunofluorescence. Rhodamine labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-hour incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I and PLOD2 in Prg4 gene trap ( Prg4 GT ) and recombined Prg4 GTR animals was studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-months old Prg4 GT/GT & Cd44 -/- and Prg4 GTR/GTR & Cd44 -/- animals . Results: PRG4 reduced α-SMA content in OA synoviocytes ( p<0.001 ). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4’s antifibrotic effect ( p<0.05 ). rhPRG4 reduced pSmad3 signal in HEK-TGF-β cells ( p<0.001 ) and TGF-β/Smad pathway activation ( p<0.001 ). rhPRG4 reduced the number of stress fiber-positive myofibroblasts, FAs mean size, and cell migration in TGF-β treated NIH3T3 fibroblasts ( p<0.05 ). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-months old Prg4 GT/GT animals had higher α-SMA, collagen type-I and PLOD2 ( p<0.001 ) content and Prg4 re-expression reduced these markers ( p<0.01 ). Prg4 re-expression also reduced α-SMA and PLOD2 in CD44-deficient mice. Conclusion: PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo .

Electrospun poly(vinyl) alcohol/collagen nanofibrous scaffold hybridized by graphene oxide for accelerated wound healing

2018 ◽  
Vol 41 (8) ◽  
pp. 467-473 ◽  
Author(s):  
Rethinam Senthil ◽  
Robert Berly ◽  
Thimmiah Bhargavi Ram ◽  
Nallathambi Gobi
Keyword(s):  
Wound Healing ◽  
Graphene Oxide ◽  
Vinyl Alcohol ◽  
Collagen Type ◽  
Collagen Type I ◽  
Fish Bone ◽  
Poly Vinyl Alcohol ◽  
Type I

Purpose: In this study, a blend of synthetic polymer (poly(vinyl) alcohol), natural polymer (collagen type I from fish bone), and graphene oxide nanoparticles is used to fabricate a composite nanofibrous scaffold, by electrospinning, for their potential application in accelerated wound healing. Methods: The scaffold was characterized for its physicochemical and mechanical properties. In vitro studies were carried out using human keratinocyte cell line (HaCaT) which proved the biocompatibility of the scaffold. In vivo study using mice model was carried out and the healing pattern was evaluated using histopathological studies. Results: Scaffold prepared from poly(vinyl) alcohol, collagen type I from fish bone, and graphene oxide possessed better physicochemical and mechanical properties. In addition, in vivo and in vitro studies showed its accelerated wound healing properties. Conclusion: The scaffold with required strength and biocompatibility may be tried as a wound dressing material in large animals after getting necessary approval.

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