| 1. |
- Thorfve, Anna, 1982, et al.
(författare)
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Gene Expression Profiling of Peri-Implant Healing of PLGA-Li+ Implants Suggests an Activated Wnt Signaling Pathway In Vivo
- 2014
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 9:7
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Tidskriftsartikel (refereegranskat)abstract
- Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of beta-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors' knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.
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| 2. |
- Granéli, Cecilia, 1981, et al.
(författare)
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Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.
- 2014
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Ingår i: Stem cell research. - : Elsevier BV. - 1876-7753 .- 1873-5061. ; 12:1, s. 153-165
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Tidskriftsartikel (refereegranskat)abstract
- Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.
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| 3. |
- Nicolaos, Papadimitriou, 1972, et al.
(författare)
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Cell Viability and Chondrogenic Differentiation Capability of Human Mesenchymal Stem Cells After Iron Labeling with Iron Sucrose
- 2014
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Ingår i: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 23:21, s. 2568-2580
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Tidskriftsartikel (refereegranskat)abstract
- For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs) it is important to be able to trace transplanted cells and their distribution in tissues e.g. cartilage over time. The aim with the study was to determine effects on cell viability, traceability and chondrogenic differentiation of hMSCs after iron labelling with iron sucrose. HMSCs were collected (7 donors, 13-57 years), undergoing spinal surgery. Two sub-sets of experiments were performed. 1)Iron labelling of hMSCs: 1 mg/mL Venofer®(iron sucrose) was added(16 hours) to cultures. hMSCs were examined for uptake of iron sucrose(Preussian blue staining) and cell viability(flow cytometry). 2)Iron labelled hMSCs(passage 4)(n=4, pellet-mass), 200 000 cells/tube were cultured(DMEM-HG) with 10 ng/mL TGFβ and compared to controls(from each donor). The pellets were harvested day 7, 14 and 28. Real time-PCR, IHC and histology were used to evaluate SOX9, ACAN, C6S and COL2A1 expression. Results; mean number of cells containing iron deposits was 98.1 % and mean cell viability 92.7 % (no significant difference compared to unlabelled control cells). Pellets containing iron labelled cells expressed COL2A1 on protein level(all time points), in similar levels as controls and glycosaminoglycan accumulation was observed in iron labelled pellets(day 14 or day 28). Results were supported expression of chondrogenic genes, SOX9, ACAN and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer, for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.
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| 4. |
- Skiöldebrand, Eva, et al.
(författare)
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Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
- 2018
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Ingår i: Heliyon. - : Elsevier BV. - 2405-8440. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1 beta and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.
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| 5. |
- Svala, Emilia, et al.
(författare)
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Effects of interleukin-6 and interleukin-1 beta on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes
- 2014
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Ingår i: American Journal of Veterinary Research. - : American Veterinary Medical Association (AVMA). - 0002-9645 .- 1943-5681. ; 75:2, s. 132-140
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Tidskriftsartikel (refereegranskat)abstract
- Objective-To determine the effects of interleukin (IL)-6 and IL-1 beta stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. Sample-Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. Procedures-Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1 beta for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3 beta (GSK-3 beta), and beta-catenin proteins in macroscopically normal cartilage samples and OCFs. Results-Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3 beta and coiled-coil domain containing 88C increased after 1 hour and expression of beta-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. Conclusion and Clinical Relevance-Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator beta-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.
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| 6. |
- Thorfve, Anna, 1982
(författare)
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Bone and Cartilage Regeneration: Wnt Signaling Pathway in Healing
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Wnt signaling pathway plays a central role in bone and cartilage embryonic development, processes that are recapitulated during regeneration. Imbalance in such well conserved and complex system often contributes to numerous diseases, whereas controlled modulation of the Wnt signaling activity is an attractive target e.g. for improved fracture healing therapies. The first aim of the present thesis was to increase the knowledge of the underlying mechanisms that lead to cellular alterations in osteoarthritis (OA), resulting in cartilage degeneration. In particular, we investigated the genome-wide expression profile of Wnt related markers in human OA cartilage and the effect of the pro-inflammatory cytokines IL-1β and IL-6 in the context of Wnt signaling pathway, thereby revealing mechanisms for OA modulation therapies. As a second aim, we studied if a local release of the canonical Wnt activator Li+ from hydroxyapatite (HA) or poly(lactic-co-glycolic acid) (PLGA) modulated the Wnt pathway and subsequently enhanced the bone regeneration around the implants. The results indicated that the Wnt signaling pathways were dysregulated in OA cartilage, with a partly inhibited canonical Wnt signaling and an active non-canonical Wnt cascade. We were able to demonstrate that WNT5A was excessively expressed in degenerative cartilage, and that the pro-inflammatory cytokine IL-6 possessed cartilage protective properties by reducing β-catenin and canonical Wnt signaling. The canonical Wnt pathway was activated by HA but the osteoinductivity of HA itself overridden the Wnt modulating capacity of Li+. Finally, a global gene expression profiling demonstrated that the controlled release of Li+ from PLGA activated the canonical Wnt signaling. In conclusion, the present findings may be used to develop gene targeted OA treatments and serve as a basis for further improvement of Li+ based therapies associated to fracture repair. This thesis sheds further light on the ambiguous influence of Wnt signaling in osteochondral homeostasis and repair mechanisms.
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| 7. |
- Thorfve, Anna, 1982, et al.
(författare)
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Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage
- 2012
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Ingår i: Cartilage. - : SAGE Publications. - 1947-6035 .- 1947-6043. ; 3:1, s. 43-57
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Tidskriftsartikel (refereegranskat)abstract
- Objective: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. Methods: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. Results: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. Conclusions: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage.
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| 8. |
- Thorfve, Anna, 1982, et al.
(författare)
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Hydroxyapatite coating affects the Wnt signaling pathway during peri-implant healing in vivo
- 2014
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Ingår i: Acta Biomaterialia. - : Elsevier BV. - 1742-7061 .- 1878-7568. ; 10:3, s. 1451-1462
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Tidskriftsartikel (refereegranskat)abstract
- Owing to its bio- and osteoconductivity, hydroxyapatite (HA) is a widely used implant material, but its osteogenic properties are only partly evaluated in vitro and in vivo. The present study focused on bone healing adjacent to HA-coated titanium (Ti) implants, with or without incorporated lithium ions (Li+). Special attention was given to the Wnt signaling pathway. The implants were inserted into rat tibia for 7 or 28days and analyzed ex vivo, mainly by histomorphometry and quantitative real-time polymerase chain reaction. HA-coated implants showed, irrespective of Li+ content, bone-implant contact (BIC) and removal torque significantly higher than those of reference Ti. Further, the expressions of OCN, CTSK, COL1A1, LRP5/6 and WISP1 were significantly higher in implant-adherent cells of HA-coated implants, with or without Li+. Significantly higher β-catenin expression and significantly lower COL2A1 expression were observed in peri-implant bone cells from HA with 14ngcm-2 released Li+. Interestingly, Ti implants showed a significantly larger bone area in the threads than HA with 39ngcm-2 released Li+, but had a lower BIC than any HA-coated implant. This study shows that HA, with or without Li+ is a strong activator of the Wnt signaling pathway, and may to some degree explain its high bone induction capacity.
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| 9. |
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| 10. |
- Thorfve, Anna, 1982, et al.
(författare)
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Madindoline A Affects the Osteogenic Potential and the Wnt Signaling
- 2014
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Ingår i: Journal of Bone Marrow Research. - : OMICS Publishing Group. - 2329-8820. ; 2:3
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Tidskriftsartikel (refereegranskat)abstract
- Background: Human mesenchymal stem cells (hMSCs) have the potential to differentiate at least into adipocytes, chondrocytes and osteoblasts. The differentiation capacity can be modulated by drugs, or chemical substances that affect diverse mechanisms essential for e.g. bone formation. The aim of this study was to investigate the osteoinductive capacity of the interleukin-6 (IL-6) inhibitor Madindoline A (MadA) and its relation to the bone-inducing Wnt signaling pathways. Methods: After stimulation with MadA of hMSC from 4 donors (aged 13-33 years) in an in vitro culture, alkaline phosphatase (ALP) activity and extracellular matrix (ECM) mineralization of hMSCs were quantified and calcification visualized by von Kossa staining. The expression of bone- and Wnt related markers was further studied at gene and protein levels. In addition, stimulation with the non-canonical Wnt5a ligand was added as a positive control, and the effect of MadA on IL-6 gene expression and STAT3 phosphorylation was evaluated. Results: Stimulation with MadA induced increased ECM mineralization and upregulated the expression of the bone related genes RUNX2, COL1A1 and Osteocalcin, although large donor-to-donor differences were observed. Further, MadA affected both the canonical and non-canonical Wnt signaling pathways and displayed a superior osteoinducing property compared to Wnt5a in some cases. Conclusion: In summary, all donors displayed higher gene expression of IL-6 and reduced STAT3 phosphorylation after MadA stimulation. The present results provide for the first time indications of an in vitro osteoinduction potential of the IL-6 inhibitor MadA.
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| 11. |
- Thorfve, Anna, 1982, et al.
(författare)
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Three-dimensional analytical techniques for evaluation of osseointegrated titanium implants
- 2015
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Ingår i: Materials Science and Technology. - 0267-0836 .- 1743-2847. ; 31:2, s. 174-179
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Tidskriftsartikel (refereegranskat)abstract
- Osseointegration, the direct bonding of titanium implant materials with bone, is critical for implant success where nanostructured surface features contribute to nano-osseointegration. However, we also know that features and processes on the microscale influence the biocompatibility of implant materials. We highlight the advantages of using mutlilength scale analyses, focusing on three-dimensional techniques, ranging from X-ray microcomputed tomography, to focused ion beam, to high resolution electron tomography to identify markers of osseointegration. A titanium implant with modified biomimetic coating studied in vitro and in vivo at various time points is used to exemplify the complementary information gained from three-dimensional analyses from the micro- to nanoscale.
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