| 1. |
- Andersson, Elin, et al.
(författare)
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Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study
- 2023
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Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- ObjectiveTo validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples.DesignSynovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry.ResultsUnsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low.ConclusionsThe HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.
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| 2. |
- Andersson, Elin, et al.
(författare)
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Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study
- 2023
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Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. Design: Synovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. Results: Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. Conclusions: The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.
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| 3. |
- Andersson, Elin, et al.
(författare)
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Quantification of chondroitin sulfates and hyaluronan in synovial fluid using high performance liquid chromatography
- 2022
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 30:Suppl 1, s. 106-106
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Konferensbidrag (refereegranskat)abstract
- Purpose: Extracellular proteins such as aggrecan may be primed with specific glycan-patterns which result in their degradation, and hence may play a role in the pathogenesis of osteoarthritis (OA). To be able to use glycans as molecular biomarkers, the method of analysis of these molecules needs to be validated. The primary aim of this study was to validate quantitative high performance liquid chromatography (HPLC) of chondroitin sulfate (CS) and hyaluronan (HA) in synovial fluid samples. The secondary aim was to examine the glycan-pattern in different subject groups, and the correlation between age and the concentration of specific glycans.Methods: OA (n=25, age=36-86 years, 40% women) and recent knee injury patients (0-5 days from injury; n=13, age=36-64 years, 46% women) were selected from a cross-sectional convenience cohort. Individual synovial fluid samples, a synovial fluid pool (SF-control; n=7) and a CS quality control sample (CS-QC; Sigma #C2905) were digested with chondroitinase ABC overnight. Samples and glycan standards (CS [n=8] and HA [n=1] standards from Iduron) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative HPLC assay. Aggrecan from synovial fluid samples (n=6) was purified using density centrifugation (D1 mini-prep). Sulfated glycosaminoglycans (sGAG) were quantified using Alcian blue precipitation. Since the CS, HA and sGAG data were not normally distributed, non-parametric analyses for group comparisons were done. P-values less than 0.05 were considered statistically significant.Results: Synovial fluid samples were digested with varying concentrations of chondroitinase ABC and analyzed with the HPLC assay; 5 mU chondroitinase ABC per μg sGAG gave the highest CS- and HA-signals and were chosen for the rest of the study (data not shown).The CS profiles in synovial fluid and on aggrecan purified from corresponding synovial fluids were assessed from six knee injury patients. Of the six CS-markers that were detected, uronic acid (UA)-N-acetylgalactosamine (GalNAc) was only present in aggrecan samples, while UA2S-GalNAc and UA2S-GalNAc6S were found only in the synovial fluids. Similar proportions of UA-GalNAc4S and UA-GalNAc6S were found in synovial fluids and aggrecan samples (Figure 1), and these CS-glycans accounted for 95% of all glycans in the SF-control sample (Table 1).The technical performance of CS- and HA-markers using HPLC-assay were evaluated (Table 1). Of the nine markers, five were present in the majority of the synovial fluid samples (N=20-38) and included in the investigation of the technical performance. The mean intra coefficient of variation (CV) for the synovial fluid samples was between 1.2 and 12.9%. For the SF-control sample, the mean intra CV was 3.3-12.1% and the inter CV was 11.0-18.5%. For the CS-QC sample, the mean intra CV was 2.5-9.5% and the mean inter CV was 3.3-31.7%. For the glycan standards, the mean intra CV was 0.2-7.0%. With dilution of the SF-control sample up to 1:10, the dilution recovery rate for the five CS- and HA-markers was mainly between 75 and 125%.The synovial fluid concentration of biomarkers UA-GalNAc6S and UA2s-GalNAc6s and sGAG were approximately 2 to 3 times higher for the recent injury group compared to the age-matched OA group, while the HA levels were 3.7 times lower for the recent injury group (data not shown). No difference in biomarker concentrations were found between the sexes in any of the patient groups (data not shown). For correlation assessments, the two patient groups were merged (total N=38, assessment N = between 20 and 38). Synovial fluid concentrations of HA and UA-GalNAc4S,6S correlated positively with age (rS=0.420 and 0.532, respectively) while UA-GalNAc6S and sGAG correlated negatively with age (rS=-0.333 and -0.528, respectively). HA correlated negatively with UA-GalNAc6S (rS=-0.462) and sGAG (rS=-0.472) and positively with UA-GalNAc4S,6S (rS=0.868).Conclusions: The technical performance of the HPLC-assay indicates that the method is suitable for analyzing CS and HA markers in synovial fluid samples. Our results suggest that: the vast majority of CS in synovial fluid derives from aggrecan, the glycan pattern differs between OA and knee injured subjects and that the concentrations of some of the CS-markers seem to be associated with HA and age.
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| 4. |
- Andersson, Elin, et al.
(författare)
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Quantification Of Glycosaminoglycans In Knee Synovial Fluid From Different Patient Groups And Knee-Healthy Subjects Using High Performance Liquid Chromatography
- 2023
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 31:Suppl 1, s. 109-109
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Konferensbidrag (refereegranskat)abstract
- Purpose: The glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are important for the normal function of articular cartilage, and changes of their sulfation and concentration may play a role in the pathogenesis of osteoarthritis (OA). Therefore, these glycans may be clinically useful as biomarkers in OA management. The purpose of this study was to analyze the CS and HA pattern in knee synovial fluid from different subject groups.Methods: OA patients (n=20, age=34-75 years, 45% women), recently knee-injured patients (0-77 days from injury, n=46, age 15-64 years, 15% women), previously knee-injured patients (88 days-21 years from injury, n=30, age 25-65 years, 17% women) and knee-healthy subjects (n=22, age 17-48 years, 23% women) were selected from a cross-sectional convenience cohort. CS and HA in individual synovial fluid samples, a synovial fluid quality control sample (SF-QC; a pool of synovial fluids) and a CS quality control sample (CS-QC) were digested with chondroitinase ABC and glucose oxidase overnight. Samples and glycan standards (CS [n=6] and HA [n=1] standards) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative high performance liquid chromatography (HPLC) assay. In total, 118 synovial fluid samples were run, whereof 34 in duplicates. SF-QC, CS-QC and standards were run in duplicates.Since the CS and HA data were not normally distributed, non-parametric analyses for group comparisons were done (Student’s T-test for age analysis, Chi-square test for sex analysis and Mann-Whitney U test for CS and HA analysis). The significance level was set at pResults: HPLC assay validation: The intra experiment coefficient of variation (CV) for the synovial fluid samples (n=36; including SF-QC) was 0.03-36.9% (median 5.4%) for CS and 0.4-44.9% (median 7.6%) for HA; intra CV for the CS-QC sample was 0.01-3.7%, and for the standards it was 0.2-6.7% for CS and 1.9-7.1% for HA. The inter experiment CV for SF-QC (n=5 experiments) was 9.8-17.5% for CS and 15.1% for HA; the inter CV for CS-QC (n=4 experiments) was 3.4-6.1%, and for the standards (n=5 experiments) it was 0.01-0.07% for CS and 0.1% for HA. Glucose oxidase was added to remove glucose that otherwise co-elutes with non-sulfated CS; it did not affect the CS and HA standards (data not shown). Group comparisons: Comparisons were made between the knee-healthy control group and the OA, recent injury and previous injury groups respectively, as well as between the recent injury and previous injury groups. There was no difference in sex between either of the groups (p=0.074-0.866). There was no difference in age between the knee-healthy group and the recent injury group (p=0.330), but the knee-healthy group was younger than the previous injury group and the OA group (pConclusions: Our data suggests that the groups with knee pathologies have higher concentrations of some CS variants and HA than the knee-healthy group. We also see a trend of higher levels of CS variants in the recent knee injury group compared to the previous knee injury group. This indicates that there is both an acute and chronic increase in the concentrations of CS variants and HA in synovial fluid following knee injury and/or cartilage damage.
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| 5. |
- Angelini, Federico, et al.
(författare)
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Osteoarthritis endotype discovery via clustering of biochemical marker data
- 2022
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 81:5, s. 666-675
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Tidskriftsartikel (refereegranskat)abstract
- Objectives Osteoarthritis (OA) patient stratification is an important challenge to design tailored treatments and drive drug development. Biochemical markers reflecting joint tissue turnover were measured in the IMI-APPROACH cohort at baseline and analysed using a machine learning approach in order to study OA-dominant phenotypes driven by the endotype-related clusters and discover the driving features and their disease-context meaning. Method Data quality assessment was performed to design appropriate data preprocessing techniques. The k-means clustering algorithm was used to find dominant subgroups of patients based on the biochemical markers data. Classification models were trained to predict cluster membership, and Explainable AI techniques were used to interpret these to reveal the driving factors behind each cluster and identify phenotypes. Statistical analysis was performed to compare differences between clusters with respect to other markers in the IMI-APPROACH cohort and the longitudinal disease progression. Results Three dominant endotypes were found, associated with three phenotypes: C1) low tissue turnover (low repair and articular cartilage/subchondral bone turnover), C2) structural damage (high bone formation/resorption, cartilage degradation) and C3) systemic inflammation (joint tissue degradation, inflammation, cartilage degradation). The method achieved consistent results in the FNIH/OAI cohort. C1 had the highest proportion of non-progressors. C2 was mostly linked to longitudinal structural progression, and C3 was linked to sustained or progressive pain. Conclusions This work supports the existence of differential phenotypes in OA. The biomarker approach could potentially drive stratification for OA clinical trials and contribute to precision medicine strategies for OA progression in the future. Trial registration number NCT03883568.
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| 6. |
- Black, Rebecca Mae, et al.
(författare)
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Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model
- 2020
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Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 2:4
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: In this exploratory study, we used discovery proteomics to follow the release of proteins from bovine knee articular cartilage in response to mechanical injury and cytokine treatment. We also studied the effect of the glucocorticoid Dexamethasone (Dex) on these responses.Design: Bovine cartilage explants were treated with either cytokines alone (10 ng/ml TNFα, 20 ng/ml IL-6, 100 ng/ml sIL-6R), a single compressive mechanical injury, cytokines and injury, or no treatment, and cultured in serum-free DMEM supplemented with 1% ITS for 22 days. All samples were incubated with or without addition of 100 nM Dex. Mass spectrometry and western blot analyses were performed on medium samples for the identification and quantification of released proteins.Results: We identified 500 unique proteins present in all three biological replicates. Many proteins involved in the catabolic response of cartilage degradation had increased release after inflammatory stress. Dex rescued many of these catabolic effects. The release of some proteins involved in anabolic and chondroprotective processes was inconsistent, indicating differential effects on processes that may protect cartilage from injury. Dex restored only a small fraction of these to the control state, while others had their effects exacerbated by Dex exposure.Conclusions: We identified proteins that were released upon cytokine treatment which could be potential biomarkers of the inflammatory contribution to cartilage degradation. We also demonstrated the imperfect rescue of Dex on the effects of cartilage degradation, with many catabolic factors being reduced, while other anabolic or chondroprotective processes were not.
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| 7. |
- Black, Rebecca Mae, et al.
(författare)
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Proteomic clustering reveals the kinetics of disease biomarkers in bovine and human models of post-traumatic osteoarthritis
- 2021
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Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 3:4
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: In this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome. Design: Mass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein. Results: Our proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins. Conclusions: We demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis.
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| 8. |
- Cronström, Anna, et al.
(författare)
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Is good muscle function a protective factor for early signs of knee osteoarthritis after anterior cruciate ligament reconstruction? The SHIELD cohort study protocol
- 2020
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Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier. - 2665-9131. ; 2:4
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Knee injury history and increased joint load, respectively, are major risk factors for the development of knee osteoarthritis (OA). Lower extremity muscle function, such as knee muscle strength, influence joint load and may be important for the onset of knee OA. However, the role of muscle function as a possible modifiable protective mechanism for the development of OA after anterior cruciate ligament reconstruction (ACLR) is not clear.Methods and analysis: In this prospective cohort study, 100 patients (50% women, 18-35 years) with ACLR will be recruited from Skåne University Hospital, Sweden and Oslo University Hospital, Norway. They will be assessed with a comprehensive test battery of muscle function including muscle strength, muscle activation, hop performance, and postural orientation as well as patient-reported outcomes, one year (baseline) and three years (follow-up) after ACLR. Primary predictor will be knee extension strength, primary outcome will be patient-reported knee pain (Knee injury and Osteoarthritis Outcome Score, subscale pain) and secondary outcomes include compositional MRI (T2 mapping) and turnover of cartilage and bone biomarkers. Separate linear regression model will be used to elucidate the influence of each baseline muscle function variable on the outcomes at follow-up, adjusted for baseline values. Twenty non-injured individuals will also be assessed with MRI. This study is approved by The Regional Ethical Review Board in Lund (Sweden) and Oslo (Norway).Discussion: This study may have important clinical implications for using muscle function to screen for risk of early-onset knee OA and for optimizing exercise therapy after knee injury.
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| 9. |
- Dwivedi, Garima, et al.
(författare)
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Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system
- 2022
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Ingår i: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6354 .- 1478-6362. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. Methods: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin’s grade 0–2, 23–83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. Results: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. Conclusion: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.
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| 10. |
- Einarsson, Emma, et al.
(författare)
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The role of cartilage glycosaminoglycan structure in gagCEST
- 2020
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Ingår i: NMR in Biomedicine. - : Wiley. - 0952-3480 .- 1099-1492. ; 33:5
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Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (-A and -C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTRasym ) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTRasym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS-A and CS-C compared with phantoms containing mainly CS-A. The difference in response corresponds well to the difference in CS-A concentration. GAG bound in aggrecan from calf articular cartilage, where CS-A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS-A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS-A while for CS-C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo.
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