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  • 1
    In: Cells, MDPI AG, Vol. 12, No. 7 ( 2023-03-27), p. 1026-
    Abstract: The present study aimed to establish novel canine osteosarcoma cell lines (COS3600, COS3600B, COS4074) and characterize the recently described COS4288 cells. The established D-17 cell line served as a reference. Analyzed cell lines differed notably in their biological characteristics. Calculated doubling times were between 22 h for COS3600B and 426 h for COS4074 cells. COS3600B and COS4288 cells produced visible colonies after anchorage-independent growth in soft agar. COS4288 cells were identified as cells with the highest migratory capacity. All cells displayed the ability to invade through an artificial basement membrane matrix. Immunohistochemical analyses revealed the mesenchymal origin of all COS cell lines as well as positive staining for the osteosarcoma-relevant proteins alkaline phosphatase and karyopherin α2. Expression of p53 was confirmed in all tested cell lines. Gene expression analyses of selected genes linked to cellular immune checkpoints (CD270, CD274, CD276), kinase activity (MET, ERBB2), and metastatic potential (MMP-2, MMP-9) as well as selected long non-coding RNA (MALAT1) and microRNAs (miR-9, miR-34a, miR-93) are provided. All tested cell lines were able to grow as multicellular spheroids. In all spheroids except COS4288, calcium deposition was detected by von Kossa staining. We believe that these new cell lines serve as useful biological models for future studies.
    Type of Medium: Online Resource
    ISSN: 2073-4409
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2661518-6
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  • 2
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 22, No. 23 ( 2021-11-30), p. 12969-
    Abstract: Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 3
    In: Veterinary Ophthalmology, Wiley, Vol. 24, No. 4 ( 2021-07), p. 400-407
    Abstract: Transplantation of minor salivary glands (MSGs) to the conjunctiva is a treatment option for patients suffering from dry eye disease. As there is not enough information about labial and buccal MSGs in dogs, the aim of this study was to provide evidence of the presence of these glands and to investigate their spatial arrangement and excretory ducts. Methods The oral mucosa of the lower lip of 4 dogs and the whole lower jaw of 1 dog were used for histological and microCT analysis. Presence, number, volumes and the tissue depth of MSGs were assessed. Results Histological analysis showed that compact tubulo‐acinar glands were located in the submucosal connective tissue. MicroCT images revealed that 9 to 21 MSGs were arranged in a single row at the level of the dental alveolae. The volume of the MSGs increased from rostral to caudal and the total volume of glandular tissue per animal ranged from 35.01 mm 3 to 549.43 mm 3 . The mean tissue depth of MSGs ranged from 0.57 mm to 1.37 mm (upper surface of glands) and between 1.43 mm and 3.09 mm (lower surface of the glands). Excretory ducts left the dorsal part of the glands and ran in dorso‐rostral direction. Conclusions The location, number and volume of the labial and buccal MSGs in the dog could be detected and described using microCT scans and histology. The present results can provide valuable information for future transplantation of labial MSGs as therapeutic measure against keratoconjunctivitis sicca.
    Type of Medium: Online Resource
    ISSN: 1463-5216 , 1463-5224
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2021
    detail.hit.zdb_id: 2011043-1
    SSG: 22
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  • 4
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 22, No. 11 ( 2021-05-25), p. 5619-
    Abstract: Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2019364-6
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  • 5
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  • 6
    In: The Journal of Gene Medicine, Wiley, Vol. 18, No. 8 ( 2016-08), p. 154-164
    Abstract: Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease‐modifying therapies of osteoarthritis, cell‐based treatments offer a promising new approach. Owing to their paracrine, differentiation and self‐renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regulated by natural disease‐dependent occuring substances, is of high interest. Methods Bone marrow‐isolated equine MSCs were stably transduced with an HIV‐1 based lentiviral vector expressing the luciferase gene under control of an inducible nuclear factor κB (NFκB)‐responsive promoter. Marker gene expression was analysed by determining luciferase activity in transduced cells stimulated with different concentrations of interleukin (IL)‐1β or tumour necrosis factor (TNF)α. Results A dose‐dependent increase in luciferase expression was observed in transduced MSCs upon cytokine stimulation. The induction effect was more potent in cells treated with TNFα compared to those treated with IL‐1β. Maximum transgene expression was obtained after 48 h of stimulation and the same time was necessary to return to baseline luciferase expression levels after withdrawal of the stimulus. Repeated cycles of induction allowed on–off modulation of transgene expression without becoming refractory to induction. The NFκB‐responsive promoter retained its inducibility also in chondrogenically differentiated MSC/Luc cells. Conclusions The results of the present study demonstrate that on demand transgene expression from the NFκB‐responsive promoter using naturally occurring inflammatory cytokines can be induced in undifferentiated and chondrogenically differentiated equine MSCs. Copyright © 2016 John Wiley & Sons, Ltd.
    Type of Medium: Online Resource
    ISSN: 1099-498X , 1521-2254
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2016
    detail.hit.zdb_id: 2002203-7
    SSG: 12
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  • 7
    In: The Journal of Gene Medicine, Wiley, Vol. 20, No. 5 ( 2018-05)
    Abstract: A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease‐induced substances is highly desirable. Methods Bone marrow‐derived equine MSCs were transduced with a lentiviral vector expressing interleukin‐1 receptor antagonist (IL‐1Ra) gene under the control of an inducible nuclear factor‐kappa B‐responsive promoter and IL‐1Ra production upon pro‐inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)‐1β] was analysed. To assess the biological activity of the IL‐1Ra protein that was produced and the therapeutic effect of IL‐1Ra‐expressing MSCs (MSC/IL‐1Ra), cytokine‐based two‐ and three‐dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real‐time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin‐1β, interleukin‐6, interleukin‐8, matrix metalloproteinase‐1 and matrix metalloproteinase‐13. Results A dose‐dependent increase in IL‐1Ra expression was found in MSC/IL‐1Ra cells upon TNFα administration, whereas stimulation using IL‐1β did not lead to IL‐1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL‐1Ra protein present in the conditioned medium from MSC/IL‐1Ra cells blocks OA onset in cytokine‐treated equine chondrocytes and co‐cultivation of MSC/IL‐1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes. Conclusions Thus, pro‐inflammatory cytokine induced IL‐1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.
    Type of Medium: Online Resource
    ISSN: 1099-498X , 1521-2254
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2018
    detail.hit.zdb_id: 2002203-7
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  • 8
    In: Microscopy Research and Technique, Wiley, Vol. 75, No. 4 ( 2012-04), p. 408-415
    Type of Medium: Online Resource
    ISSN: 1059-910X
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2012
    detail.hit.zdb_id: 1474912-9
    SSG: 11
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  • 9
    Online Resource
    Online Resource
    Wiley ; 2019
    In:  Veterinary Ophthalmology Vol. 22, No. 6 ( 2019-11), p. 778-790
    In: Veterinary Ophthalmology, Wiley, Vol. 22, No. 6 ( 2019-11), p. 778-790
    Abstract: The morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation‐, differentiation‐, stem cell‐associated markers and cell junction proteins were assessed. Methods Corneal samples from 12 horses (six foals and six adult horses) were analyzed after H & E staining and immunohistochemistry using the following antibodies: E‐cadherin, β‐catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium. Results The height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea‐specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E‐cadherin, β‐catenin, and Cx43 revealed a similar apico‐lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface. Conclusions This study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.
    Type of Medium: Online Resource
    ISSN: 1463-5216 , 1463-5224
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2011043-1
    SSG: 22
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  • 10
    Online Resource
    Online Resource
    Wiley ; 2020
    In:  Journal of Anatomy Vol. 236, No. 1 ( 2020-01), p. 165-170
    In: Journal of Anatomy, Wiley, Vol. 236, No. 1 ( 2020-01), p. 165-170
    Abstract: Cells use different cell adhesion and communication structures to promote tissue development, maintenance of tissue integrity as well as repair and regenerative processes. Another recently discovered way of information exchange is long‐distance thin cellular processes called nanotubes (NTs), mainly studied in vitro . Information on the existence and relevance of NTs in vivo is sparse. Building on two references which hint at the potential existence of longitudinally directed cell processes resembling NTs, we investigated tendons from young (3 weeks) and adult (9 weeks, 4 and 8 months) Fisher rats. Whole mounts of rat tail tendon fascicles (RTTfs) and sections of Achilles, flexor, extensor and patellar tendons were stained with Deep Red plasma membrane and DAPI nuclear stain and immunolabelled with Connexin43 (Cx43). In addition, 3‐D reconstruction of serial semithin sections and TEM was used to verify the presence of NTs. We were able to demonstrate NTs as straight thin longitudinal processes (Ø 100–500 nm) reaching up to several 100 μm in length, mainly originating from lateral sheet‐like cell processes or cell bodies in all tendon types investigated. NTs were observed to distend between tenocyte rows at the same level but also connect cells of different rows, thus leading to a complex 3‐D cellular scaffold. Shorter NTs connected lateral cell sheets of tenocytes in the same row, omitting one or two cells. In addition, we detected links or potential branching of NTs. Cx43 immunostaining for the detection of gap junctions revealed Cx43‐positive foci at the end‐to‐end contacts of tenocyte cell bodies as well as along their contacting sheet‐like processes. Only rarely, we found clear Cx43 signals at their potential contact points between NTs and tendon cells as well as along the course of NTs, and most NTs appeared completely devoid of Cx43 signals. Therefore, we conclude that NTs in tendons could have a twofold function: long‐distance communication as well as stabilization of a mechanically challenged tissue. From in vitro studies it is known that NTs allow intercellular transmission of various cell components, offering potential protective effects for the respective tissue. Further studies on functional properties of NTs in tendons under changing mechanical loading regimens are required in the future. The fact that NTs are present in tendons may necessitate the reconsideration of our traditional understanding of cell‐to‐cell communication.
    Type of Medium: Online Resource
    ISSN: 0021-8782 , 1469-7580
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 1474856-3
    SSG: 12
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