The epidemiology of muskuloskeletal disease Molecules, structures, patients and society
Musculoskeletal diseases are a rapidly growing cause of disability, chronic pain, and reduced quality of life in our ageing populations worldwide. Our aims are to gain novel insights into these diseases - their etiology, occurrence, natural history, treatments, prediction, disease monitoring, and disease burden - to allow for better healthcare decision-making and disease prevention.
To accomplish these goals, we use a multidisciplinary approach. For research on osteoarthritis, a chronic degenerative joint disease, we use human tissue biobanking, proteomics, and MR imaging to characterize the molecular and structural aspects of tissue degradation associated with the earliest stages of the disease. We are especially interested in the role of the meniscus in early knee osteoarthritis. Further, in population-based epidemiologic and health economic studies of musculoskeletal disease, we use physician-coded healthcare data from Sweden to understand the impact of these diseases on patients and society. Our previous work has contributed to improved understanding of musculoskeletal disease and impactful changes in their clinical management. To learn more, read about our projects.
Our secondary analysis of an RCT of ACL injuries found MRI appearance of ACL healing after rupture in one in three adults randomised to initial rehabilitation and one in two who did not cross-over to delayed ACLR. The potential for spontaneous healing of the ACL to facilitate better clinical outcomes may be greater than previously considered.
In this longitudinal register based cohort study, we find that incident physician-diagnosed knee and hip OA is associated with increased risk of depression, cardiovascular diseases, back pain, osteoporosis, and diabetes, with the latter association only found for knee OA.
Symptomatic early-stage OA of the knee urgently needs to be identified and defined, as efficient early-stage case diagnosis in primary care would enable health-care providers to proactively and substantially reduce the burden of disease through proper management. We review efforts to define patient populations with symptomatic early-stage knee OA on the basis of validated classification criteria.
In this prospective cohort study of adults in Norway with and without SARS-CoV-2 infection, we found that the acute and sub-acute burden of post-covid complaints on health services is similar for Omicron and Delta. The chronic burden may be lower for Omicron vs Delta when considering musculoskeletal pain, but not when considering other typical post-covid complaints.
Molecular pathogenesis We perform biobanking from orthopaedic surgeries to target better understanding of osteoarthritis aetiology and pathogenesis using proteomics, towards identification of novel biomarkers of the disease.
Structural imaging We use multiple cohort data sets with repeat magnetic resonance imaging and post processing of conventional radiographs to gain new knowledge of early stage osteoarthritis and its prediction.
Burden of disease Using population-based health care registries in Sweden covering in excess of 20 million person-years, we study the epidemiology and burden of musculoskeletal disease, including health economic aspects.
We're recruiting volunteers. In January 2020, we launched an ambitious new project to follow the earliest changes in the knees of patients at risk for OA, compared to healthy volunteers. If you have healthy knees and would like to participate in our study, follow this link to find out more and participate!
Proteomill is launched! We have developed an interactive, easy-to-access proteomic analysis tool, ProteoMill, which contains a complete pipeline from dataset upload, to differential expression, enrichment- and network analysis. Read our article decribing Preoteomill in Bioinformatics or start using it at proteomill.com.
2023-01-30: Congratulations Andrea Dell'Isola, who has been promoted to Docent in Orthopaedics by the Medical Faculty in Lund University!
2022-12-16: Congratulations Karin Magnusson, who has been named Professor at the Norwegian Institute of Public Health!
2022-11-22: Andrea Dell'Isola gave a public lecture about osteoarthritis, symptoms, treatment methods and exercise to a packed audience of more than 300 at the Helsingborg hospital.
Andrea Dell'Isola talks to the public about osteoarthritis in Helsingborg
2022-09-14: We have had an informative and inspiring symposium on osteoarthritis and Alzheimer's disease, organised by the Kock Foundation in Trelleborg, Sweden. Great discussions from bringing together researchers in these two diseases, which share many challenges!
The Kock symposium on osteoarthritis and Alzheimer's disease in Trelleborg, Sweden
2022-09-12: Our group kicked off the new academic year with a hike to Nimis, a towering network of driftwood and planks one can climb through, here on the southern coast of Sweden. We look forward to an exciting new year of research with colleagues, following this adventurous hike!
Through the portal.
A towering network.
Right on the coast.
2022-05-05: We celebrate our wonderful colleague and Collaborator Prof. Tuhina Neogi, who was awarded an honorary doctorate by the Lund University Faculty of Medicine.
From left to right: Martin Englund, vice dean Martin Olsson, and our guest of honor Prof. Tuhina Neogi.
2022-04-10: We've had an interesting and informative OARSI 2022 meeting in Berlin, presenting our work and meeting our collaborators! Great talks by Karin Magnusson, Clara Hellberg and Ali Kiadaliri, as well as posters by Martin Rydén, Andrea Dell'Isola, Aleksandra Turkiewicz, Patrik Önnerfjord and Martin Englund.
Emma presenting her work for the public at CRC, Malmö.
2022-03-01: We welcome our new communicator Ebba Andersson to the Clinical Epidemiology Unit. We look forward to working with her and the rest of the Lund University Osteoarthritis Division (LOAD) to drive the public outreach initiative Arthritis Portal (Artrosportalen)!
Evidence of ACL healing on MRI following ACL rupture treated with rehabilitation alone may be associated with better patient-reported outcomes: a secondary analysis from the KANON trial.
Stephanie Filbay, Frank Roemer, Stefan Lohmander, Aleksandra Turkiewicz, Ewa Roos, Richard Frobell, Martin Englund. British Journal of Sports Medicine, 57(2):91-98, November 2022.
Objectives: Evaluate the natural course of anterior cruciate ligament (ACL) healing on MRI within 5 years of acute ACL rupture and compare 2-year and 5-year outcomes based on healing status and treatment group.
Methods: Secondary analysis of 120 Knee Anterior Cruciate Ligament Nonsurgical vs Surgical Treatment (KANON) trial participants randomised to rehabilitation and optional delayed ACL reconstruction (ACLR) or early ACLR and rehabilitation. ACL continuity on MRI (Anterior Cruciate Ligament OsteoArthritis Score 0-2) was considered evidence of ACL healing. Outcomes included Knee Injury and Osteoarthritis Outcome Score (KOOS), KOOS patient acceptable symptomatic state (PASS) and treatment failure criteria. Linear mixed models were used to estimate adjusted mean differences (95% CIs) in patient-reported sport and recreational function (KOOS-Sport/Rec) and quality of life (KOOS-QOL) at 2 and 5 years, between participants with MRI evidence of ACL healing and those who had (1) no evidence of ACL healing, (2) delayed ACLR or (3) early ACLR.
Results: MRI evidence of ACL healing at 2-year follow-up was observed in 16 of 54 (30%, 95% CI 19 to 43%) participants randomised to optional delayed ACLR. Excluding participants who had delayed ACLR, 16 of 30 (53%, 36-70%) participants managed with rehabilitation-alone displayed MRI evidence of ACL healing. Two-year outcomes were better in the healed ACL group (n=16) compared with the non-healed (n=14) (mean difference (95% CI) KOOS-Sport/Rec: 25.1 (8.6-41.5); KOOS-QOL: 27.5 (13.2-41.8)), delayed ACLR (n=24) (KOOS-Sport/Rec: 24.9 (10.2-39.6); KOOS-QOL: 18.1 (5.4-30.8)) and early ACLR (n=62) (KOOS-Sport/Rec: 17.4 (4.1-30.7); KOOS-QOL: 11.4 (0.0-22.9)) groups. Five-year KOOS-QOL was better in the healed versus non-healed group (25.3 (9.4-41.2)). Of participants with MRI evidence of ACL healing, 63-94% met the PASS criteria for each KOOS subscale, compared with 29-61% in the non-healed or reconstructed groups.
Conclusions: MRI appearance of ACL healing after ACL rupture occurred in one in three adults randomised to initial rehabilitation and one in two who did not cross-over to delayed ACLR and was associated with favourable outcomes. The potential for spontaneous healing of the ACL to facilitate better clinical outcomes may be greater than previously considered.
Phase-contrast enhanced synchrotron micro-tomography of human meniscus tissue. Emma Einarsson, Maria Pierantoni, Vladimir Novak, Jonas Svensson, Hanna Isaksson, Martin Englund. Osteoarthritis and Cartilage, 30(9):1222-1233, September 2022.
Objective: To investigate the feasibility of synchrotron radiation-based phase contrast enhanced micro-computed tomography (SR-PhC-μCT) for imaging of human meniscus. Quantitative parameters related to fiber orientation and crimping were evaluated as potential markers of tissue degeneration.
Design: Human meniscus specimens from 10 deceased donors were prepared using different preparation schemes: fresh frozen and thawed before imaging or fixed and paraffin-embedded. The samples were imaged using SR-PhC-μCT with an isotropic voxel size of 1.625 μm. Image quality was evaluated by visual inspection and spatial resolution. Fiber voxels were defined using a grey level threshold and a structure tensor analysis was applied to estimate collagen fiber orientation. The area at half maximum (FAHM) was calculated from angle histograms to quantify orientation distribution. Crimping period was calculated from the power spectrum of image profiles of crimped fibers. Parameters were compared to degenerative stage as evaluated by Pauli histopathological scoring.
Results: Image quality was similar between frozen and embedded samples and spatial resolutions ranged from 5.1 to 5.8 μm. Fiber structure, including crimping, was clearly visible in the images. Fibers appeared to be less organized closer to the tip of the meniscus. Fiber density might decrease slightly with degeneration. FAHM and crimping period did not show any clear association with histopathological scoring.
Conclusion: SR-PhC-μCT is a feasible technique for high-resolution 3D imaging of fresh frozen meniscus tissue. Further work is needed to establish quantitative parameters that relate to tissue degeneration, but this imaging technique is promising for future studies of meniscus structure and biomechanical response.
Mineral crystal thickness in calcified cartilage and subchondral bone in healthy and osteoarthritic human knees. Mikko Finnilä, Shuvashis Das Gupta, Mikael Turunen, Iida Hellberg, Aleksandra Turkiewicz, Viviane Lutz-Bueno, Elin Jonsson, Mirko Holler, Neserin Ali, Velocity Hughes, Hanna Isaksson, Jon Tjörnstrand, Patrik Önnerfjord, Manuel Guizar-Sicairos, Simo Saarakkala, Martin Englund. Journal of Bone and Mineral Research, 37(9):1700-1710, September 2022.
Osteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state-of-the-art microfocus small-angle X-ray scattering with a 5-μm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage.
Proteomics Profiling of Human Synovial Fluid Suggests Increased Protein Interplay in Early-Osteoarthritis (OA) That Is Lost in Late-Stage OA. Neserin Ali, Aleksandra Turkiewicz, Velocity Hughes, Elin Folkesson, Jon Tjörnstrand, Paul Neuman, Patrik Önnerfjord, Martin Englund. Molecular and Cellular Proteomics, 21(3):100200, March 2022.
The underlying molecular mechanisms in osteoarthritis (OA) development are largely unknown. This study explores the proteome and the pairwise interplay of proteins in synovial fluid from patients with late-stage knee OA (arthroplasty), early knee OA (arthroscopy due to degenerative meniscal tear), and from deceased controls without knee OA. Synovial fluid samples were analyzed using state-of-the-art mass spectrometry with data-independent acquisition. The differential expression of the proteins detected was clustered and evaluated with data mining strategies and a multilevel model. Group-specific slopes of associations were estimated between expressions of each pair of identified proteins to assess the co-expression (i.e., interplay) between the proteins in each group. More proteins were increased in early-OA versus controls than late-stage OA versus controls. For most of these proteins, the fold changes between late-stage OA versus controls and early-stage OA versus controls were remarkably similar suggesting potential involvement in the OA process. Further, for the first time, this study illustrated distinct patterns in protein co-expression suggesting that the interplay between the protein machinery is increased in early-OA and lost in late-stage OA. Further efforts should focus on earlier stages of the disease than previously considered.
Elastic, dynamic viscoelastic and model-derived fibril-reinforced poroelastic mechanical properties of normal and osteoarthritic human femoral condyle cartilage. Mohammadhossein Ebrahimi, Mikko Finnilä, Aleksandra Turkiewicz, Martin Englund, Simo Saarakkala, Rami Korhonen, Petri Tanska. Annals of Biomedical Engineering, 2021.
Osteoarthritis (OA) degrades articular cartilage and weakens its function. Modern fibril-reinforced poroelastic (FRPE) computational models can distinguish the mechanical properties of main cartilage constituents, namely collagen, proteoglycans, and fluid, thus, they can precisely characterize the complex mechanical behavior of the tissue. However, these properties are not known for human femoral condyle cartilage. Therefore, we aimed to characterize them from human subjects undergoing knee replacement and from deceased donors without known OA. Multi-step stress-relaxation measurements coupled with sample-specific finite element analyses were conducted to obtain the FRPE material properties. Samples were graded using OARSI scoring to determine the severity of histopathological cartilage degradation. The results suggest that alterations in the FRPE properties are not evident in the moderate stages of cartilage degradation (OARSI 2-3) as compared with normal tissue (OARSI 0-1). Drastic deterioration of the FRPE properties was observed in severely degraded cartilage (OARSI 4). We also found that the FRPE properties of femoral condyle cartilage related to the collagen network (initial fibril-network modulus) and proteoglycan matrix (non-fibrillar matrix modulus) were greater compared to tibial and patellar cartilage in OA. These findings may inform cartilage tissue-engineering efforts and help to improve the accuracy of cartilage representations in computational knee joint models.
The impact of first and second wave of COVID-19 on knee and hip surgeries in Sweden. Andrea Dell'Isola, Ali Kiadaliri, Aleksandra Turkiewicz, Velocity Hughes, Karin Magnusson, Jos Runhaar, Sita Bierma-Zeinstra, Martin Englund. Journal of Experimental Orthopaedics, 2021.
Purpose: TTo investigate the impact of COVID-19 in Sweden on rates of knee and hip surgeries.
Methods: We used healthcare data for the population of the southernmost region in Sweden (1.4 million inhabitants). We did an interrupted time-series analysis to estimate changes in rates and trends of joint replacements (JR), arthroscopies, and fracture surgeries for knee or hip in April-December 2020 compared to pre-COVID-19 levels adjusting for seasonal variations.
Results: We found a drop of 54% (95% CI 42%; 68%) and 42% (95% CI 32%; 52%), respectively, in the rate of JRs and arthroscopies in April 2020 when compared to the counterfactual scenario. This was followed by an increase that brought the rates of JRs and arthroscopies back to their predicted levels also during the beginning of the second wave (November-December 2020). Acute fracture surgeries were largely unaffected, i.e. did not show any decrease as observed for the other surgeries.
Conclusions: HIn southern Sweden, we observed a marked decrease in elective knee and hip surgeries following the first wave of Covid-19. The rates remained close to normal during the beginning of the second wave suggesting that important elective surgeries for patients with end-stage osteoarthritis can still be offered despite an ongoing pandemic provided adequate routines and hospital resources.
The relationship between MRI features and knee pain over 6 years in knees without radiographic osteoarthritis at baseline. Karin Magnusson, Aleksandra Turkiewicz, Jaanika Kumm, Fan Zhang, Martin Englund. Arthritis Care & Research, 73(11):1659-1666, November 2021.
To explore whether MRI features suggestive of knee OA are associated with presence of knee pain in possible early-stage OA development.
We included 294 participants from the Osteoarthritis Initiative (mean [SD] age 50 (3) years, 50% women), with baseline Kellgren and Lawrence grade=0 in both knees, and who had all obtained knee MRIs from 4 different time points over 6 years (baseline, 24, 48 and 72 months). Using a linear mixed model (knees matched within individuals), we studied whether MRI features: meniscal body extrusion (millimeter), cartilage area loss (score 0 to 39), cartilage full thickness loss (0-16), osteophytes (0-29), meniscal integrity (0-10), bone marrow lesions (BML) including bone marrow cysts (0-20), Hoffa or effusion synovitis (absent/present) and popliteal cysts (absent/present) were associated with knee-specific pain as reported on the Knee injury and Osteoarthritis Outcomes Score (KOOS) questionnaire on 0-100 scale (worst-best).
The difference in KOOS knee pain for a knee with a one unit higher score on MRI feature was: for meniscal extrusion -1.52 (95% CI -2.35,-0.69), cartilage area loss -0.23 (-0.48,0.02), cartilage full thickness loss: -1.04 (-1.58,-0.50), osteophytes -0.32 (-0.61,-0.03), meniscal integrity -0.28 (-0.58,0.02), BMLs including potential cysts -0.19 (-0.55,0.16), synovitis 0.23 (-1.14,1.60) and popliteal cysts 0.86 (-0.56,2.29).
Meniscal extrusion, full thickness cartilage loss and osteophytes are associated with having more knee pain. Although these features may be relevant targets for future trials, the clinical relevance of our findings is unclear because no feature was associated with a clinically important difference in knee pain.
Arthroscopic partial meniscectomy for a degenerative meniscus tear: a 5 year follow-up of the placebo-surgery controlled FIDELITY (Finnish Degenerative Meniscus Lesion Study) trial. Raine Sihvonen, Mika Paavola, Antti Malmivaara, Ari Itälä, Antti Joukainen, Juha Kalske, Heikki Nurmi, Jaanika Kumm, Niko Sillanpää, Tommi Kiekara, Aleksandra Turkiewicz, Pirjo Toivonen, Martin Englund, Simo Taimela, Teppo Järvine, FIDELITY (Finnish Degenerative Meniscus Lesion Study) Investigators. British Journal of Sports Medicine, 54(22):1332-1339, November 2020.
To assess the long-term effects of arthroscopic partial meniscectomy (APM) on the development of radiographic knee osteoarthritis, and on knee symptoms and function, at 5 years follow-up.
Multicentre, randomised, participant- and outcome assessor-blinded, placebo-surgery controlled trial. Setting: Orthopaedic departments in five public hospitals in Finland.
146 adults, mean age 52 years (range 35-65 years), with knee symptoms consistent with degenerative medial meniscus tear verified by MRI scan and arthroscopically, and no clinical signs of knee osteoarthritis were randomised.
APM or placebo surgery (diagnostic knee arthroscopy).
Main outcome measures:
We used two indices of radiographic knee osteoarthritis (increase in Kellgren and Lawrence grade ≥1, and increase in Osteoarthritis Research Society International (OARSI) atlas radiographic joint space narrowing and osteophyte sum score, respectively), and three validated patient-relevant measures of knee symptoms and function (Western Ontario Meniscal Evaluation Tool (WOMET), Lysholm, and knee pain after exercise using a numerical rating scale).
There was a consistent, slightly greater risk for progression of radiographic knee osteoarthritis in the APM group as compared with the placebo surgery group (adjusted absolute risk difference in increase in Kellgren-Lawrence grade ≥1 of 13%, 95% CI -2% to 28%; adjusted absolute mean difference in OARSI sum score 0.7, 95% CI 0.1 to 1.3). There were no relevant between-group differences in the three patient-reported outcomes: adjusted absolute mean differences (APM vs placebo surgery), -1.7 (95% CI -7.7 to 4.3) in WOMET, -2.1 (95% CI -6.8 to 2.6) in Lysholm knee score, and -0.04 (95% CI -0.81 to 0.72) in knee pain after exercise, respectively. The corresponding adjusted absolute risk difference in the presence of mechanical symptoms was 18% (95% CI 5% to 31%); there were more symptoms reported in the APM group. All other secondary outcomes comparisons were similar.
APM was associated with a slightly increased risk of developing radiographic knee osteoarthritis and no concomitant benefit in patient-relevant outcomes, at 5 years after surgery.
Risk of knee osteoarthritis after different types of knee injuries in young adults: a population-based cohort study. Barbara Snoeker, Aleksandra Turkiewicz, Karin Magnusson, Richard Frobell, Dahai Yu, George Peat, Martin Englund. British Journal of Sports Medicine, 54(12):725-730, June 2020.
Objectives: To estimate the risk of clinically diagnosed knee osteoarthritis (OA) after different types of knee injuries in young adults.
Methods: In a longitudinal cohort study based on population-based healthcare data from Skåne, Sweden, we included all persons aged 25-34 years in 1998-2007 (n=149 288) with and without diagnoses of knee injuries according to International Classification of Diseases (ICD)-10. We estimated the HR of future diagnosed knee OA in injured and uninjured persons using Cox regression, adjusted for potential confounders. We also explored the impact of type of injury (contusion, fracture, dislocation, meniscal tear, cartilage tear/other injury, collateral ligament tear, cruciate ligament tear and injury to multiple structures) on diagnosed knee OA risk.
Results: We identified 5247 persons (mean (SD) age 29.4 (2.9) years, 67% men) with a knee injury and 142 825 persons (mean (SD) age 30.2 (3.0) years, 45% men) without. We found an adjusted HR of 5.7 (95% CI 5.0 to 6.6) for diagnosed knee OA in injured compared with uninjured persons during the first 11 years of follow-up and 3.4 (95% CI 2.9 to 4.0) during the following 8 years. The corresponding risk difference (RD) after 19 years of follow-up was 8.1% (95% CI 6.7% to 9.4%). Cruciate ligament injury, meniscal tear and fracture of the tibia plateau/patella were associated with greatest increase in risk (RD of 19.6% (95% CI 13.2% to 25.9%), 10.5% (95% CI 6.4% to 14.7%) and 6.6% (95% CI 1.1% to 12.2%), respectively).
Conclusion: In young adults, knee injury increases the risk of future diagnosed knee OA about sixfold with highest risks found after cruciate ligament injury, meniscal tear and intra-articular fracture.
Does early anterior cruciate ligament reconstruction prevent development of meniscal damage? Results from a secondary analysis of a randomised controlled trial. Barbara A. Snoeker, Frank W. Roemer, Aleksandra Turkiewicz, Stefan Lohmander, Richard B. Frobell, Martin Englund. British Journal of Sports Medicine, 54(10):612-617, May 2020.
Objectives: To determine development of new and worsening meniscal damage over 5 years after acute anterior cruciate ligament (ACL) injury comparing rehabilitation plus early ACL reconstruction ('early-ACLR') versus rehabilitation with optional delayed ACL reconstruction ('optional-delayed-ACLR').
Methods: We used knee MRIs from the only randomised controlled trial in the field including 121 young adults. One musculoskeletal radiologist read baseline and 5-year follow-up images using the Anterior Cruciate Ligament Osteoarthritis Score (ACLOAS). We defined development (ie, new and worsening) of meniscal damage both dichotomously and as a sum score representing severity (based on the reclassified ACLOAS meniscus grades). In the full analysis set, we analysed development of meniscal damage (yes/no) with logistic regression and severity with zero-inflated Poisson regression and adjusted for age, sex and baseline meniscal damage.
Results: Over 5 years, new or worsening meniscal damage developed in 45% of subjects with early-ACLR and in 53% of subjects with optional-delayed-ACLR. The relative risk for development of meniscal damage on knee level was 1.3 (95% CI 0.9 to 1.9) in optional-delayed-ACLR versus early-ACLR. For medial and lateral meniscal damage, respectively, the relative risks were 2.1 (95% CI 1.1 to 3.9) and 1.0 (95% CI 0.6 to 1.5). The mean severity score was 1.5 higher (more severe damage) on knee level in optional-delayed-ACLR versus early-ACLR (95% CI 1.1 to 1.9) among those with meniscal damage at 5 years. For medial and lateral meniscal damage, respectively, the corresponding scores were 1.7 (95% CI 1.2 to 2.5) and 1.1 (95% CI 0.8 to 1.4).
Conclusion: A strategy of early-ACLR may reduce development of medial meniscal damage following acute ACL injury. For the lateral meniscus, ACLR seems neither to be protective nor to increase the risk of damage.