Micro-Scale Distribution of CA4+ in Ex vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

Contrast-enhanced micro-computed tomography (CE mu CT) with cationic and anionic contrast agents reveals glycosaminoglycan (GAG) content and distribution in articular cartilage (AC). The advantage of using cationic stains (e.g., CA4+) compared to anionic stains (e.g., Hexabrix (R)), is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution mu CT setups. In this study, we use high-resolution mu CT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo mu CT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6 mgI/ml, and 24 mgI/ml) and imaged with high-resolution mu CT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG content detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis in the samples and the relative difference between the interterritorial matrix and segmented chondron area was less than 4%. To conclude, our results suggest that GAG quantification with CE mu CT is not affected by the chondron uptake of CA4+. This further confirms the use of CA4+ for macro-scale assessment of GAG throughout the AC, and highlight the capability of studying chondron properties in 3D at the micro scale.Peer reviewe

Solu- ja kehitysbiologian esseet 2015

Esipuhe Tämä esseekokoelma on prosessi, joka alkoi 2007-vuoden lääketieteen opiskelijoiden tärppilistasta. Saatuani listan käsiini ja havaittuani sekä sen ansiot että puutteet, päätin hyödyntää opportunistisesti hyvää ideaa ja niin tämä kokoelma sai alkunsa. Anatomian opettajat ohjasivat ensimmäiset esseet v 2008 esseet ja näin saatiin koottua ensimmäinen esseekokoelma. Siinä oli jo huomattavasti vähemmän virheitä ja puutteellisia tietoja, kuin alkuperäisessä tärppilistassa. V 2009 versioon opiskelijat pääosin itse tuottivat jo kuvamateriaalin, ja luovuimme kuvavarkauksista, koska kaikenlaiset oikeusprosessit ovat kuitenkin niin aikaa vieviä ja turhanpäiväisiä. Ensimmäisen painetun kokoelman jälkeen esseiden kirjoittaminen on edennyt polveilevasti. Olen aina uusia aiheita kootessani huomioinut kliinikoilta saamani esseevinkit ja näin kokoelma onkin paisunut ja rönsyillyt. Viime vuoden jälkeen on tullut aika tehdä välitilinpäätös ja koota kaikki taas yhdeksi kokonaisuudeksi. Tämä tehdään siksi, että prosessi etenee seuraavaan vaiheeseen, jossa esseekokoelmarungon ympärille aletaan kasata erilaisia oksia. Esseekokoelma on yhdenlainen toteutus yhteisöllisen oppimisen teemasta ja asiantuntijakoulutuksesta, jotka ovat omia pedagogisia johtotähtiäni. Me opettajat olemme osa oppivaa yhteisöä, ehkä hieman kokeneempia, mutta periaatteellisesti saman tiedon valtameren ääressä ammentamassa tieteellisin menetelmin tuotettua uutta tietoa. Esseiden ohjeissa opiskelijoita on perehdytetty tieteellisen tiedon etsimiseen, omaksumiseen ja laadun arviointiin ja omien johtopäätösten tekemiseen. Tämä prosessi on käytännön lääkärille elinehto, joka mahdollistaa maailmanlaajuisesti alan nopean etenemisen potilaiden parhaaksi. Toivon, että tämä kokoelma on inspiroiva johdanto lääketieteelliseen solubiologiaan, joka yhdessä anatomian kanssa on tosiasiallinen ja järkkymätön lääketieteen perusta. Haluan kiittää opiskelijoita ja kaikkia opettajia, erityisesti kollegaani Juha Tuukkasta, joka on kannustanut kehittämään tätä opetusmuotoa edelleen. Lisäksi kiitän erityisesti Piia Rantakokkoa, joka oli version pohjoinen äiti, lujatahtoinen, kärsivällinen ja ymmärtäväinen. Taitosta kiitämme Mari Liimataista. Valtiovallan kallisalaleikkaukset, strategiset ja epäoikeudenmukaiset päältä pois erät yliopiston sisällä ja muut viheliäisyydet ovat harventaneet opetuksen esitaistelijoiden joukkoa laitoksellamme noin kolmasosaan tämän esseekirjoittelun alusta, mutta tämä yksikkö lakkaa taistelemasta viimeisenä. Jos meille ei anneta pateja, heittelemme käpyjä! Ja tämä on oikeastaan ihan hauskaa. Oulussa 1.6.2015 Petri Lehenkar

Predisposing factors for a second fragile hip fracture in a population of 1130 patients with hip fractures, treated at Oulu University Hospital in 2013–2016:a retrospective study

Abstract Objective: The life-time risk of a second fragile hip fracture is 8.4%, but the risk factors that predispose to a second hip fracture remain unresolved. This study aimed to define risk factors that predisposed patients to a second hip fracture. Methods: For this retrospective study, we retrieved clinical data on 1130 patients with fragile hip fractures (67.2% female, mean age: 79.3 years) that underwent surgery at the Oulu University Hospital in 2013–2016. These data included the fracture risk assessment score (measured with the FRAX tool), the bone-mass T-score, laboratory values, ambulatory capacity, and the time of death. Results: In this population, 12.4% of patients sustained a second hip fracture. The predisposing factors for a second hip fracture were: female (p = 0.016), a high FRAX score (p = 0.020), and low physical capacity (p < 0.001). The vitamin D level recommended for treating osteoporosis (i.e., vitamin D > 75 nmol/l) was observed in only 24% of patients, and 42% of patients had ionized calcium levels below the reference range. According to the level of the cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), 37% of patients did not have high bone turnover. We found a positive correlation between age and ICTP (p = 0.001). The risk of death was higher after the second hip fracture (p = 0.005), but we found no difference in age between patients with first and second hip fractures (p = 0.11). Conclusions: After a hip fracture, a second hip fracture is a well-known risk. Nevertheless, we found that only one-third of patients with a second hip fracture had used anti-osteoporosis medication at any time previously. These findings suggested that second hip fractures were most likely to occur in patients with osteopenic T-score values, in women more often than men, and in patients with high FRAX scores and low ambulatory capacity

Volumetric assessment of bone microstructures by a 3D local binary patterns -based method:bone changes with osteoarthritis

Abstract Osteoarthritis (OA) causes progressive degeneration of articular cartilage and pathological changes in subchondral bone, conventionally assessed volumetrically using micro-computed tomography (μCT) imaging in vitro. The local binary patterns (LBP) method has recently been suggested as a new alternative solution to perform analysis of local bone structures from μCT scans. In this study, a novel 3D LBP-based method to provide a new lead in bone microstructural analysis is proposed. In addition to the detailed description of the method, this solution is tested using µCT data of OA human trabecular bone samples, harvested from patients treated with total knee arthroplasty. The method was applied to correlate the distribution of orientations of local patterns with the severity of the disease. The local orientations of the bone fibers changed along the severity of OA, suggesting an adaptation of the bone to the disease. The structural parameters derived from the process were able to provide a new approach for the assessment of the disease, supporting the potential of this volumetric LBP-based method to assess trabecular bone changes

Osteoclastogenesis of human peripheral blood, bone marrow, and cord blood monocytes

Abstract Osteoclasts are multinucleated bone resorbing cells that can be differentiated from human monocytes in vitro. There are few studies comparing osteoclastogenesis of different monocyte sources. We compared monocytes from human bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) and their osteoclastogenic potential by culturing them with RANKL (20 and 80 ng/ml) and M-CSF (10 ng/ml) for 14 days. We also cultured cells without growth factors, as umbilical cord blood monocytes have been reported to be able to fuse spontaneously into osteoclasts. The data was analysed on d4, d8, d11, and d14. After culture with RANKL and M-CSF, all types of cell cultures developed TRACP -positive multinuclear cells that were able to form resorption pits on human bone slices. Only occasional multinuclear cells and small infrequent resorbed areas could be found in PB and CB-derived cultures without growth factors. BM-derived cells formed greater resorption areas than PB- and CB-derived monocytes. The greatest monocyte population in BM samples were intermediate (CD14⁺⁺CD16⁺) and in PB and CB classical monocytes (76.3% and 54.4%, respectively). In conclusion, our data demonstrates that bone resorbing osteoclasts can be differentiated from BM, PB and CB. However, the osteoclast precursor origin can affect the osteoclast properties and function

1,25(OH)₂D₃ and its analogue calcipotriol inhibit the migration of human synovial and mesenchymal stromal cells in a wound healing model:a comparison with glucocorticoids

Abstract Vitamin D analogue calcipotriol is currently used in the local treatment of psoriasis. However, it also has antiproliferative and anti-inflammatory effects in the cells of the joint — suggesting a possible benefit in local treatment of arthritis. In this study, calcipotriol was studied in different in vitro methods to find out its effect on synovial and mesenchymal stromal cells. Primary human cell lines of osteoarthritis or rheumatoid arthritis patients (five mesenchymal stromal cells, MSC, and four synovial stromal cells, SSC) were cultured to study migration and proliferation of the cells in a wound healing model. The media was supplemented with calcipotriol, 1,25(OH)2D3, dexamethasone, betamethasone, methylprednisolone or control solution in 1–100 nM concentrations. To see possible toxic effects of calcipotriol, concentrations up to 10 µM in SSCs and MSCs were studied in apoptosis and necrosis assays in four cell lines. Calcipotriol and 1,25(OH)₂D₃, as well as the three glucocorticoids, reduced the migration of both SSCs and MSCs. In SSCs, the effect of calcipotriol and 1,25(OH)₂D₃ was at least as effective as with glucocorticoids, while with MSCs, the glucocorticoids were stronger inhibitors of migration. The antimigratory of calcipotriol and 1,25(OH)₂D₃ was consistently maintained in 10 µM and 1 µM. Calcipotriol was not toxic to MSCs and SSCs up to concentrations of 10 µM. Calcipotriol, as well as 1,25(OH)₂D₃, exerts antimigratory and antiproliferative effects on human SSCs and MSCs of the joint. These effects are not caused by apoptosis or necrosis. Both calcipotriol and 1,25(OH)₂D₃ have similar effects as glucocorticoids without apparent toxicity, suggesting that calcipotriol might be an eligible candidate to the local treatment of arthritis with a broad therapeutic window

Micro-Scale Distribution of CA4+ in Ex vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

Contrast-enhanced micro-computed tomography (CEμCT) with cationic and anionic contrast agents reveals glycosaminoglycan (GAG) content and distribution in articular cartilage (AC). The advantage of using cationic stains (e.g., CA4+) compared to anionic stains (e.g., Hexabrix®), is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution μCT setups. In this study, we use high-resolution μCT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo μCT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6 mgI/ml, and 24 mgI/ml) and imaged with high-resolution μCT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG content detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis in the samples and the relative difference between the interterritorial matrix and segmented chondron area was less than 4%. To conclude, our results suggest that GAG quantification with CEμCT is not affected by the chondron uptake of CA4+. This further confirms the use of CA4+ for macro-scale assessment of GAG throughout the AC, and highlight the capability of studying chondron properties in 3D at the micro scale

Gravidity, parity and knee breadth at midlife:a population-based cohort study

Abstract Gestation increases the biomechanical loading of lower extremities. Gestational loading may influence anthropometrics of articular surfaces in similar means as bone diaphyseal properties. This study aimed to investigate whether gravidity (i.e. number of pregnancies) and parity (i.e. number of deliveries) is associated with knee breadth among middle-aged women. The study sample comprised 815 women from the Northern Finland Birth Cohort 1966. The median parity count of our sample was 2 and the median gravidity count 3. At the age of 46, questionnaires were used to enquire gravidity and parity, and posteroanterior knee radiographs were used to obtain two knee breadth parameters (tibial plateau mediolateral breadth (TPML) and femoral condylar mediolateral breadth (FCML)) as representatives of articular size. The associations of gravidity and parity with knee breadth were analyzed using general linear models with adjustments for height, weight, leisure-time physical activity, smoking, and education years. Individuals with osteoarthritic changes were excluded from our sample. The mean TPML in our sample was 70.3 mm and the mean FCML 71.6 mm respectively. In the fully adjusted models, gravidity and parity showed positive associations with knee breadth. Each pregnancy was associated with 0.11–0.14% larger knee breath (p < 0.05), and each delivery accounted for an increase of 0.20% in knee breadth (p < 0.01). Between-group comparisons showed that multiparous women had 0.68–1.01% larger knee breath than nulli- and primiparous women (p < 0.05). Pregnancies and deliveries seem to increase the mediolateral breadth of the knee. This increase is potentially associated with increased biomechanical loadings during gestation