scholarly journals In Silico and In Vitro Investigations On Mutated IFNβ-scFv Against Human Collagen Type II

2021 ◽  
Author(s):  
Fatemeh Alimohammadi ◽  
Zohreh Hojati ◽  
Mazdak Ganjalikhani-Hakemi
Keyword(s):  
Fusion Protein ◽  
In Silico ◽  
Collagen Type ◽  
Hek293 Cells ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Human Collagen ◽  
Clinical Experiments

Abstract Current medications for rheumatoid arthritis (RA), a common synovial autoimmune disease, are associated with adverse effects. Interestingly, interferon beta (IFNβ), effective in multiple sclerosis (MS) treatment, also can help decreasing articular destruction in RA. Here, a novel fusion protein was introduced containing human mutated IFNβ (with mutations in 27th and 101th residues; IFNβ27+101) fused to a single chain fragment variable (scFv) antibody against human collagen type II for decreasing IFNβ27+101 off-targets (according to drug targeting benefits) in future in vivo and clinical experiments. After designing, bioinformatic analyses and the recombinant vector transfection into HEK293 cells, the mutated IFNβ-scFv protein confirmation and function were assessed by SDS-PAGE, western blotting, ELISA, and real-time PCR. The fusion protein secondary and tertiary structures had proper folding. Also, the recombinant mRNA secondary structure considered stable. 2.35 fold difference between the test and negative control groups confirmed the scFv attachment to human collagen type II (p= 0.046). MxA 25.68 fold overexpression in peripheral blood mononuclear cells (PBMCs) treated with the recombinant protein compared with the non-treated sample (p= 0.0001), demonstrated IFNβ27+101 bioactivity as the fusion protein. In vitro and in silico studies verified function of mutated IFNβ-scFv, however in vivo studies are proposed for further validation.

Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo

2014 ◽  
Vol 21 (2) ◽  
pp. 303-309 ◽  
Author(s):  
Ganjun Feng ◽  
Li Li ◽  
Ying Hong ◽  
Hao Liu ◽  
Yueming Song ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Collagen Type ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Low Oxygen ◽  
Nucleus Pulposus Cells ◽  
Experimental Conditions ◽  
3D Scaffolds

Object The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo. Methods Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses. Results Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II. Conclusions Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

scholarly journals Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-β3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ilona Uzieliene ◽  
Edvardas Bagdonas ◽  
Kazuto Hoshi ◽  
Tomoaki Sakamoto ◽  
Atsuhiko Hikita ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Collagen Type ◽  
Activin A ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Type Ii ◽  
Collagen Type Ii

Abstract Background Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A. Methods MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. Results MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

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.

Specificity and T cell receptor β chain usage of a human collagen type II-reactive T cell clone derived from a healthy individual

1992 ◽  
Vol 22 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Tan Yan ◽  
Harald Burkhardt ◽  
Thomas Ritter ◽  
Barbara Bröker ◽  
Karl Heinz Mann ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Collagen Type ◽  
Cell Clone ◽  
Cell Receptor ◽  
Type Ii ◽  
Collagen Type Ii ◽  
T Cell Clone ◽  
Human Collagen ◽  
Β Chain

Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

2007 ◽  
Vol 20 (03) ◽  
pp. 185-191 ◽  
Author(s):  
A. O. Oshin ◽  
E. Caporali ◽  
C. R. Byron ◽  
A. A. Stewart ◽  
M. C. Stewart
Keyword(s):  
Articular Cartilage ◽  
Collagen Type ◽  
Articular Chondrocytes ◽  
Soluble Form ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Endogenous Expression ◽  
Chondrocytic Phenotype

SummaryArticular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

scholarly journals Adjuvant properties of a biocompatible thermo-responsive polymer of N -isopropylacrylamide in autoimmunity and arthritis

2011 ◽  
Vol 8 (65) ◽  
pp. 1748-1759 ◽  
Author(s):  
Akhilesh Kumar Shakya ◽  
Ashok Kumar ◽  
Kutty Selva Nandakumar
Keyword(s):  
Antibody Response ◽  
Collagen Type ◽  
Physical Adsorption ◽  
Type Ii ◽  
Specific Igg ◽  
Ifn Γ ◽  
First Time ◽  
Molecular Weight Form

To evaluate the thermo-responsive poly( N -isopropylacrylamide) (PNiPAAm) polymer as an adjuvant, we synthesized PNiPAAm through free radical polymerization and characterized it both in vitro and in vivo . The polymer when mixed with collagen type II (CII) induced antigen-specific autoimmunity and arthritis. Mice immunized with PNiPAAm–CII developed significant levels of CII-specific IgG response comprising major IgG subclasses. Antigen-specific cellular recall response was also enhanced in these mice, while negligible level of IFN-γ was detected in splenocyte cultures, in vitro . PNiPAAm–CII-immunized arthritic mouse paws showed massive infiltration of immune cells and extensive damage to cartilage and bone. As determined by immunostaining, most of the CII protein retained its native configuration after injecting it with PNiPAAm in naive mice. Physical adsorption of CII and the high-molecular-weight form of moderately hydrophobic PNiPAAm induced a significant anti-CII antibody response. Similar to CII, mice immunized with PNiPAAm and ovalbumin (PNiPAAm–Ova) induced significant anti-ovalbumin antibody response. Comparable levels of serum IFN-γ, IL-1β and IL-17 were observed in ovalbumin-immunized mice with complete Freund, incomplete Freund (CFA and IFA) or PNiPAAm adjuvants. However, serum IL-4 levels were significantly higher in PNiPAAm–Ova and CFA–Ova groups compared with the IFA–Ova group. Thus, we show for the first time, biocompatible and biodegradable thermo-responsive PNiPAAm can be used as an adjuvant in several immunological applications as well as in better understanding of the autoimmune responses against self-proteins.

scholarly journals Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro

1999 ◽  
Vol 145 (5) ◽  
pp. 1103-1115 ◽  
Author(s):  
M.M. French ◽  
S.E. Smith ◽  
K. Akanbi ◽  
T. Sanford ◽  
J. Hecht ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Collagen Type ◽  
Heparan Sulfate Proteoglycan ◽  
Surrounding Tissue ◽  
Collagen Type Iv ◽  
Type Ii ◽  
Sulfate Proteoglycan ◽  
Collagen Type Ii ◽  
Murine Embryonic Fibroblast

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

scholarly journals Different Phenotypes and Chondrogenic Responses of Human Menstrual Blood and Bone Marrow Mesenchymal Stem Cells to activin A and TGF-β3

2020 ◽  
Author(s):  
Ilona Uzieliene ◽  
Edvardas Bagdonas ◽  
Kazuto Hoshi ◽  
Tomoaki Sakamoto ◽  
Atsuhiko Hikita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Collagen Type ◽  
Activin A ◽  
Cell Surface Markers ◽  
Type Ii ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis. Menstrual blood-derived mesenchymal stem cells (MenSCs) are much less characterized compared to bone marrow mesenchymal stem cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A, member of the TGF-β superfamily of proteins.Methods: MenSCs (n=6) and BMMSCs (n=5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, Notch1 was analysed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit. Adipogenic differentiation capacity was evaluated according to Oil-Red staining, osteogenic differentiation - Alizarin Red staining. Chondrogenic differentiation (Activin A and TGF-β3 stimulation) was induced in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) and investigated by histologically and by expression of chondrogenic genes (collagen type II, aggrecan). Activin A protein production was evaluated by ELISA.Results: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimisation of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration.Trial registration: Not applicable.

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