126. LMP1 overexpression in the rabbit intervertebral disc upregulates proteoglycans and collagen type ii synthesis in vivo

Object The goal in this study was to develop a convenient, less-invasive animal model to monitor progression of intervertebral disc (IVD) degeneration for future testing of new treatments for disc degeneration. Methods Level 5/6 and 7/8 IVDs of rat caudal spine were stabbed laterally with 18- or 21-gauge hypodermic needles to a depth of 5 mm from the subcutaneous surface with the aid of fluoroscopy. In vivo MR imaging studies were performed at 4, 8, and 12 weeks postsurgery to monitor progression of IVD degeneration. Histological analysis including H & E and safranin O staining, and immunohistochemical studies of collagen type II and bone morphogenetic protein receptor type II (BMPRII) were assessed at 12 weeks postsurgery. Results The 18- and 21-gauge needle–stabbed discs illustrated decreases in both the T2 density and MR imaging index starting at 4 weeks, with no evidence of spontaneous recovery by 12 weeks. Histological staining demonstrated a decreased nucleus pulposus (NP) area, and the NP–anulus fibrosus border became unclear during the progression of disc degeneration. Similar patterns of degenerative signs were also shown in both safranin O– and collagen type II–stained sections. The BMPRII immunohistochemical analysis of stabbed discs demonstrated an increase in BMPRII expression in the remaining NP cells and became stronger in anulus fibrosus with the severity of disc degeneration. Conclusions After introducing an 18- or 21-gauge needle into the NP area of discs in the rat tail, the stabbed disc showed signs of degeneration in terms of MR imaging and histological outcome measurements. Changes in BMPRII expression in this animal model provide an insight for the effectiveness of delivering BMPs into the region responsible for chondrogenesis for disc repair. This convenient, less-invasive, reproducible, and cost-effective model may be a useful choice for testing novel treatments for disc degeneration.

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Degenerated annulus fibrosus of the intervertebral disc contains collagen type II.

Annals of the Rheumatic Diseases ◽

10.1136/ard.43.2.258 ◽

1984 ◽

Vol 43 (2) ◽

pp. 258-263 ◽

Cited By ~ 20

Author(s):

M Adam ◽

Z Deyl

Keyword(s):

Intervertebral Disc ◽

Annulus Fibrosus ◽

Collagen Type ◽

Type Ii ◽

Collagen Type Ii

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

Journal of Neurosurgery Spine ◽

10.3171/2014.4.spine13870 ◽

2014 ◽

Vol 21 (2) ◽

pp. 303-309 ◽

Cited By ~ 6

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.

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Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis

Journal of Experimental Medicine ◽

10.1084/jem.20081862 ◽

2009 ◽

Vol 206 (2) ◽

pp. 449-462 ◽

Cited By ~ 155

Author(s):

Hüseyin Uysal ◽

Robert Bockermann ◽

Kutty S. Nandakumar ◽

Bettina Sehnert ◽

Estelle Bajtner ◽

...

Keyword(s):

Collagen Type ◽

Diagnostic Value ◽

Type Ii ◽

Collagen Type Ii ◽

Pathogenic Factor ◽

Fab Fragment ◽

Citrullinated Peptide ◽

Insight Into

Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359–369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising β-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.

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

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02286-w ◽

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.

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A25 COLLAGEN TYPE II DEGRADATION AND FORMATION ASSESSED IN EX VIVO AND IN VIVO IN MODELS OF RA

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(07)60478-6 ◽

2006 ◽

Vol 14 ◽

pp. S29-S30

Author(s):

A.K. Olsen ◽

R.H. Nielsen ◽

B.-C. Sondergaard ◽

P. Qvist ◽

C. Christiansen ◽

...

Keyword(s):

Collagen Type ◽

Ex Vivo ◽

Type Ii ◽

Collagen Type Ii

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Stromal Cell Derived Factor-1-Mediated Migration of Mesenchymal Stem Cells Enhances Collagen Type II Expression in Intervertebral Disc

Tissue Engineering Part A ◽

10.1089/ten.tea.2018.0131 ◽

2018 ◽

Vol 24 (23-24) ◽

pp. 1818-1830 ◽

Cited By ~ 4

Author(s):

Catarina Leite Pereira ◽

Graciosa Quelhas Teixeira ◽

Joana Rita Ferreira ◽

Matteo D'Este ◽

David Eglin ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Intervertebral Disc ◽

Stromal Cell ◽

Collagen Type ◽

Type Ii ◽

Collagen Type Ii ◽

Stromal Cell Derived Factor

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Collagen Type I and Type II Expression Evaluation on Cartilage Defect Regeneration Treated with Dwikora–Ferdiansyah–Lesmono–Purwati (DFLP) Scaffold Supplemented with Adipose–Derived Stem Cells (ASCs) or Secretome: An In-Vivo Study

Qanun Medika - Medical Journal Faculty of Medicine Muhammadiyah Surabaya ◽

10.30651/jqm.v4i2.4377 ◽

2020 ◽

Vol 4 (2) ◽

pp. 225

Author(s):

Adrianto Prasetyo Perbowo ◽

Dwikora Novembri Utomo ◽

Lukas Widhiyanto ◽

Primadenny Ariesa Airlangga ◽

Purwati Purwati

Keyword(s):

Stem Cells ◽

Collagen Type ◽

Collagen Type I ◽

Cartilage Defects ◽

Type I ◽

Adipose Derived Stem Cells ◽

Type Ii ◽

Collagen Type Ii ◽

Significant Difference

Abstract Cell-based therapies such as Scaffold, stem cells, and secretome, are one of the alternatives to enhance the regeneration of hyaline-like cartilage in cases of cartilage defects. This study is an in-vivo experiment using animal models, in which we apply a composite of DFLP (Dwikora-Ferdiansyah-Lesmono-Purwati) Scaffold and Adipose-Derived Stem Cells (ASCs) or Secretome to an injury model on the distal femoral trochlea of New Zealand White Rabbits. The animals were divided into four groups: (1) control (K); (2) Scaffold only (S); (3) Scaffold + ASCs (SA); (4) Scaffold + Secretome (SS). Animals were terminated in the 12th week, and an immunohistochemistry (IHC) evaluation for Collagen type I and II were done. Statistical analysis shows that collagen type I IHC between groups shows no significant difference (p = 0.546). Collagen type II IHC shows significant difference between groups (p = 0,016). The findings in this study showed that Scaffold + ASCs group and Scaffold + Secretome have better collagen type II expression compared to the control group. DFLP Scaffold composite with ASCs or Secretome shows potential for cartilage regeneration therapy by increasing type II collagen expression as in hyaline-like cartilage which may be used for regenerative therapy for cartilage defects. Keywords : DFLP Scaffold; Adipose-Derived Stem Cells (ASCs); Secretome; Collagen Type I; Collagen Type IICorrespondence : [email protected]

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

10.21203/rs.3.rs-987486/v1 ◽

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.

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