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Emerging evidence suggests that fatty acids (FAs) and their lipid mediator derivatives can induce both beneficial and detrimental effects on inflammatory processes and joint degradation in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA). The present study characterized the detailed FA signatures of synovial membranes collected during knee replacement surgery of age- and gender-matched OA and RA patients (n = 8/diagnosis). The FA composition of total lipids was determined by gas chromatography and analyzed with univariate and multivariate methods supplemented with hierarchical clustering (HC), random forest (RF)-based classification of FA signatures, and FA metabolism pathway analysis. RA synovium lipids were characterized by reduced proportions of shorter-chain saturated FAs (SFAs) and elevated percentages of longer-chain SFAs and monounsaturated FAs, alkenyl chains, and C20 n-6 polyunsaturated FAs compared to OA synovium lipids. In HC, FAs and FA-derived variables clustered into distinct groups, which preserved the discriminatory power of the individual variables in predicting the RA and OA inflammatory states. In RF classification, SFAs and 20:3n-6 were among the most important FAs distinguishing RA and OA. Pathway analysis suggested that elongation reactions of particular long-chain FAs would have increased relevance in RA. The present study was able to determine the individual FAs, FA groups, and pathways that distinguished the more inflammatory RA from OA. The findings suggest modifications of FA elongation and metabolism of 20:4n-6, glycerophospholipids, sphingolipids, and plasmalogens in the chronically inflamed RA synovium. These FA alterations could have implications in lipid mediator synthesis and potential as novel diagnostic and therapeutic tools.

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 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). Conclusion 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.

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.

Calcipotriol, a vitamin D analogue, is an antiproliferative and anti-inflammatory drug currently used in psoriasis. Here, our aim was to analyse the safety of calcipotriol for cartilage and bone in alleviated-dose (0.1 mg instead of usual ≥1mg dose) zymosan-induced arthritis in rats. Theoretically, high doses of vitamin D or analogues could have detrimental effects on bone or cartilage. The rats were divided into four groups: vehicle (n = 9), dexamethasone 0.1 mg/kg (n = 9), calcipotriol 0.1 mg/kg (n = 8) and negative control (n = 10) with no injections. Arthritic rats were given phosphate-buffered saline (PBS) injections to left knees as a control. After euthanasia on day 8, all knees were imaged with micro-computed tomography for surface lesions and decalcified for histological analyses. Contrary to our expectations, no significant changes could be observed in the tomography data and histological scores among the three treatment groups or between the vehicle-treated and non-arthritic group. Calcipotriol did not cause adverse effects on cartilage or subchondral bone within a week, suggesting that it could be safely used in local treatment of arthritis. The alleviated model caused synovitis with local and systemic inflammatory response without cartilage erosions, which might be useful in studying self-limiting synovitis where cartilage or bone effects are not of primary interest.

This case-control study aimed to analyze the dynamics of macrophage infiltration in subcutaneous adipose tissue following bariatric surgery or conservative treatment of obesity and to clarify whether these features predict the weight loss outcome after the surgery.Subcutaneous tissue samples taken before and 12 months after laparoscopic Roux-en-Y gastric bypass surgery (n = 39) or conservative (n = 43) treatment for obesity were analyzed. Fat cell size was determined, and with CD68 immunohistochemistry, crown-like structures (CLS) were counted and single macrophages were quantitated.A major decline in CLS density from 4.1 (SD 3.5) to 1.1 (SD 0.8) per 1000 fat cells (p 0.000) was found, regardless of the degree of weight loss after the surgery. Surgery had no effect on the fraction of infiltrating single-cell macrophages in subcutaneous adipose tissue. The abundance of these macrophage populations before the intervention did not predict the degree of postsurgery weight loss or suboptimal response to the surgery.The effect of gastric bypass on adipose tissue inflammatory status associates closely with CLS density even in subjects with suboptimal weight loss. The study suggests that factors related to bypass surgery other than weight loss modify the inflammatory response in adipose tissue.

Background: Periprosthetic bone loss after Total Hip Arthroplasty (THA), detected as an early migration of the prosthesis may predict later loosening of the implant. Objective: We hypothesized that intra-operative bisphosphonate rinsing would reduce bone resorption after THA. It might therefore be possible to achieve better early fixation of the implant. Methods: Nineteen patients suffering from arthrosis were recruited in a prospective, double-blinded, randomized, placebo-controlled clinical pilot trial. Patients were operated with an uncemented Bimetric stem using tantalum markers. The femoral proximal intramedullary canal was rinsed with 1mM clodronate in nine patients and with saline in 10 patients. These patients were followed for two years using radiostereometric analysis (RSA), dual energy x-ray absorptiometry (DXA) and the Harris Hip Score (HHS). Results: We did not found any significant differences between the study groups with regards to the primary output measures (maximum total point motion, MTPM). However, there was evidence that clodronate could affect periprosthetic bone quality; a beneficial effect in BMD in Gruen zone 3 during the two-year follow-up was observed, BMD decreased less in the clodronate group (p = 0.02). The maximal x-translation of the stem at 3-24 months was significantly two-fold, being higher in the placebo group (p = 0.02). The baseline BMD and the maximal total point motion (MTPM) at 3-24 months showed a positive correlation in the clodronate group and a negative correlation in the placebo group. Conclusion: In conclusion, further studies with larger patient groups and longer follow-up periods are needed to estimate the clinical importance of these findings and further to prove if an intraoperative clodronate rinsing prior to application of femoral stem during THA can prevent periprosthetic bone loss. Clinical Trial Registration No.: NCT03803839

Although knee measurements yield high classification rates in metric sex estimation, there is a paucity of studies exploring the knee in artificial intelligence-based sexing. This proof-of-concept study aimed to develop deep learning algorithms for sex estimation from radiographs of reconstructed cadaver knee joints belonging to the Terry Anatomical Collection. A total of 199 knee radiographs were obtained from 100 skeletons (46 male and 54 female cadavers; mean age at death 64.2 years, range 50-102 years) whose tibiofemoral joints were recon-structed in standard anatomical position. The AIDeveloper software was used to train, validate, and test neural network architectures in sex estimation based on image classification. Of the explored algorithms, an MhNet-based model reached the highest overall testing accuracy of 90.3%. The model was able to classify all females (100.0%) and most males (78.6%) correctly. These preliminary findings encourage further research on artificial intelligence-based methods in sex estimation from the knee joint. Combining radiographic data with automated and externally validated algorithms may establish valuable tools to be utilized in forensic anthropology.

Signal amplification by reversible exchange (SABRE) hyperpolarisation is used to enhance the NMR signals of nicotine and acrolein in methanol-d

Objectives Body mass estimation from skeletal dimensions is a useful tool when studying archeological human samples. Bony articular surface dimensions of the lower limb have frequently been utilized to estimate body size. In the present study, we investigated the association between knee breadth and body mass in a Northern European population. Our study aimed to confirm both methodology and results presented in earlier studies. Materials and methods The study sample consists of 1,290 subjects belonging to the Northern Finland Birth Cohort 1966. Three knee breadth dimensions-femoral biepicondylar breadth, mediolateral breadth of femoral condyles, and mediolateral breadth of the tibial plateau-were measured from subjects' knee PA-radiographs. Measurements and their association with body weight at 31 years were utilized for creating body mass estimation equations using linear regression and reduced major axis regression. Correlations between knee measurements and body weight at three different ages (18, 31, and 46) were also analyzed. Results Positive associations were detected between each knee breadth variable and weight in the total sample and both genders separately. Body mass estimation equations were created for the total sample, for males and for females. R values of the models ranged from 0.38 to 0.74. Median absolute percent prediction errors ranged from 6.89 to 9.72%. The highest correlations were obtained between knee breadth and body weight in early adulthood. Discussion Our large sample confirmed that equations derived from knee breadth dimensions are accurate when estimating body mass of modern humans. Knee breadth measurements clearly have a positive association with body weight in early maturity.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are common joint diseases associated with changes in local, as well as systemic bone structure and osteoclast function. We investigated how the different soluble inflammatory stimuli in these diseases can affect osteoclastogenesis and bone resorptionRA serum and synovial fluid increased both osteoclastogenesis and bone resorption. Osteoclastogenesis and activity increased more in the cultures containing OA than RA synovial fluid. The osteoclasts cultured in different culture media exhibited different phenotypes, especially the cells cultured with OA synovial fluid were generally larger and had more nuclei. A general increase in proinflammatory cytokines in RA synovial fluid and serum was found. Surprisingly, OA synovial fluid showed lower levels of osteoclastogenesis inhibiting cytokines, such as IL-4 and IL-10, than RA synovial fluid, which at least partly explains more pronounced osteoclastogenesis. No significant difference was found in RANKL or OPG levels.The proinflammatory stimulus in OA and RA drives the monocyte differentiation towards inflammatory osteoclastogenesis and altered osteoclast phenotype.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600 mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

Sex estimation is an important part of osteological analysis of skeletons and forensic identification process. Traditionally cranial and pelvic traits are utilized for accurate sex estimation. However, post-cranial measurements have also been proven to accurately estimate sex especially from robust bones such as the femur. In this study, we investigated the potential of knee breadth dimensions in sex estimation in a Finnish population. To conduct this study we utilized a study sample (n=1654) belonging to the Northern Finland Birth Cohort 1966. All individuals were 46 years of age at the time of the examination. Three knee breadth dimensions were measured from subjects' knee posteroanterior radiographs: femoral biepicondylar breadth (FBEB), mediolateral breadth of the femoral condyles (FCML), and mediolateral breadth of the tibial plateau (TPML). Sex estimation was performed using logistic regression. The study clearly demonstrated that all three measurements were different between males and females. Sectioning points for individual knee breadth measurements were 82.9 mm for FBEB, 76.6 mm for FCML and 75.4 mm for TPML. The classification rates ranged from 90.9% to 93.6%. The less commonly used measurements of FCML and TPML showed higher accuracy than FBEB in sex estimation. Our study confirmed that knee breadths can be successfully utilized to improve sex estimation in cases where the skeleton is only partially preserved and other major components of sex estimation are absent. We can also provide new standards for sex estimation from the knee joint in a Finnish population. Sex estimation is an important part of osteological analysis of skeletons and forensic identification process. Traditionally cranial and pelvic traits are utilized for accurate sex estimation. However, post-cranial measurements have also been proven to accurately estimate sex especially from robust bones such as the femur. In this study, we investigated the potential of knee breadth dimensions in sex estimation in a Finnish population. To conduct this study we utilized a study sample (n=1654) belonging to the Northern Finland Birth Cohort 1966. All individuals were 46 years of age at the time of the examination. Three knee breadth dimensions were measured from subjects' knee posteroanterior radiographs: femoral biepicondylar breadth (FBEB), mediolateral breadth of the femoral condyles (FCML), and mediolateral breadth of the tibial plateau (TPML). Sex estimation was performed using logistic regression. The study clearly demonstrated that all three measurements were different between males and females. Sectioning points for individual knee breadth measurements were 82.9 mm for FBEB, 76.6 mm for FCML and 75.4 mm for TPML. The classification rates ranged from 90.9 % to 93.6 %. The less commonly used measurements of FCML and TPML showed higher accuracy than FBEB in sex estimation. Our study confirmed that knee breadths can be successfully utilized to improve sex estimation in cases where the skeleton is only partially preserved and other major components of sex estimation are absent. We can also provide new standards for sex estimation from the knee joint in a Finnish population.

Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone. Electronic supplementary material The online version of this article (10.1007/s00418-019-01770-y) contains supplementary material, which is available to authorized users.

Background Bone flap resorption (BFR) is the most prevalent complication resulting in autologous cranioplasty failure, but no consensus on the definition of BFR or between the radiological signs and relevance of BFR has been established. We set out to develop an easy-to-use scoring system intended to standardize the interpretation of radiological BFR findings. Methods All 45 autologous cranioplasty patients operated on at Oulu University Hospital from 2004 to 2014 were identified, and the bone flap status of all the available patients was evaluated using the new scoring system. Derived from previous literature, a three-variable score for the detection of BFR changes is proposed. The variables “Extent” (estimated remaining bone volume), “Severity” (possible perforations and their measured diameter), and “Focus” (the number of BFR foci within the flap) are scored from 0 to 3 individually. Using the sum of these scores, a score of 0–9 is assigned to describe the degree of BFR. Additionally, independent neurosurgeons assessed the presence and relevance of BFR from the same data set. These assessments were compared to the BFR scores in order to find a score limit for relevant BFR. Results BFR was considered relevant by the neurosurgeons in 11 (26.8%) cases. The agreement on the relevance of BFR demonstrated substantial strength (κ 0.64, 95%CI 0.36 to 0.91). The minimum resorption score in cases of relevant BFR was 5. Thus, BFR with a resorption score ≥ 5 was defined relevant (grades II and III). With this definition, grade II or III BFR was found in 15 (36.6%) of our patients. No risk factors were found to predict relevant BFR. Conclusions The score was proven to be easy to use and we recommend that only cases with grades II and III BFR undergo neurosurgical consultation. However, general applicability can only be claimed after validation in independent cohorts.

Supplemental digital content is available in the text., Objectives Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma with increased expression of tenascin C and fibronectin. Their role and tumor-stroma ratio in PDAC are not well known. The aim of this study was to evaluate tenascin C and fibronectin expression and tumor-stroma ratio and their prognostic relevance in PDAC. Methods Ninety-five resected PDACs were immunohistochemically stained for tenascin C and fibronectin, and the expression was separately assessed in tumor bulk and front. Tumor-stroma ratio was determined with sections stained with hematoxylin-eosin. Results Tenascin C and fibronectin were abundantly expressed in the stroma of PDAC, but absent in adjacent normal pancreatic tissue. Fibronectin expression of the bulk was associated with high T class (P = 0.045). In the main analysis, tenascin C and fibronectin expression and tumor-stroma ratio were not associated with patient survival. In a subgroup analysis of early-stage PDAC (T1–T2 tumors), high tenascin C expression in the tumor bulk was associated with poor prognosis (hazard ratio, 8.23; 95% confidence interval, 2.71–24.96). Conclusions Tenascin C and fibronectin are abundantly expressed in PDAC, but they seem to have no major association with patient survival. However, in early-stage PDAC, tenascin C expression of the tumor bulk may have prognostic impact. Tumor-stroma ratio has no prognostic value in PDAC.

Summary Objective To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. Method Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with μCT scanner to determine the subchondral-bone plate thickness over the entire surface. Results Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade ( ρ = 0.86 and ρ = 0.54 respectively), while cartilage thickness slightly increased ( ρ = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. Conclusion The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and μCT assessments.

Objectives We investigated the effects of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), i.e. biologically active vitamin D and calcipotriol, a vitamin D analog, on growth and secretion of inflammatory mediators in synovial stromal cells (SSC) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Methods Synovial stromal cells (SSC) isolated during knee prosthesis surgery from four patients with RA and four with OA were exposed to 1,25(OH)2D3 or calcipotriol with or without stimulation of cells with IL-1β or TNF-α. The proliferation of cells was studied by MTT assay. Levels of cytokines were analyzed by a magnetic bead–based multiplex assay (a panel of 27 important cytokines and IL-6 alone) and RT-PCR was used to validate the concentrations of the key cytokines secreted by SSC. The vitamin D receptor (VDR) was visualized by immunofluorescence in SSC and by immunohistochemistry in the synovial tissues of three RA and three OA patients. Results We detected intense staining for VDR in the synovial lining and vascular endothelium in tissue sections from all our RA and OA patients. Both 1,25(OH)2D3 and calcipotriol inhibited SSC proliferation for a prolonged time (up to 23 days with calcipotriol), but dexamethasone tended to increase SSC proliferation in a 4-day culture. 1,25(OH)2D3, calcipotriol and dexamethasone reduced the secretion of most inflammatory factors. Calcipotriol and dexamethasone additively reduced the secretions of IL-6, IFN-γ, basic FGF and VEGF in TNF-α stimulated SSC. The level of IL-6 was still diminished at 10 days after exposure, emphasizing the long-term impact of calcipotriol on SSC. Conclusions Exposure for 24–48 h to 1,25(OH)2D3 or calcipotriol causes a long-lasting inhibition of cell proliferation and cytokine production in SSC in vitro.

// Joni Leppanen 1 , Sara Bogdanoff 1 , Petri P. Lehenkari 1 , Juha Saarnio 1 , Joonas H. Kauppila 1, 2 , Tuomo J. Karttunen 1 , Heikki Huhta 1, * and Olli Helminen 1, * 1 Cancer and Translational Medicine Research Unit, Medical Research Center Oulu, University of Oulu and Oulu University Hospital, Oulu, Finland 2 Department of Molecular Medicine and Surgery, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden * Equal contribution as senior author Correspondence to: Olli Helminen, email: olli.helminen@oulu.fi Keywords: esophageal adenocarcinoma, Barrett’s esophagus, tenascin-C, fibronectin, extracellular matrix Received: January 06, 2017 Accepted: June 19, 2017 Published: July 12, 2017 ABSTRACT Background: Tenascin-C and fibronectin are adhesive glycoproteins modulating the structure of the extracellular matrix and cellular functions. Their expression and function in esophageal adenocarcinoma is poorly known. The aim of this study was to evaluate the expression of tenascin-C and fibronectin in esophageal adenocarcinoma and its precursor stages. Results: Stromal tenascin-C and fibronectin expression were found in all evaluated lesion types. Expression of both molecules increased from gastric metaplasia towards adenocarcinoma (p

Objectives To investigate whether height at the age of 31 is associated with the incidence of knee and hip osteoarthritis (OA) in the following 15 years. Methods Participants in The Northern Finland Birth Cohort 1966 (NFBC1966) diagnosed with knee or hip OA between the ages of 31 and 46 were used as OA cases. Study subjects without knee and hip OA were used as the controls. Height and weight were measured in a clinical examination at the age of 31 (baseline). Mean heights for the OA cases and the controls were compared by an independent samples t-test. Cox regression analysis was performed to calculate the risk for OA for different height quartiles. The results were adjusted for body mass index/weight, education, smoking and leisure-time physical activity at baseline. Additionally, a Kaplan-Meier analysis was performed. Results Men with knee OA were 2.6 cm taller (P < 0.001) and women with knee OA 1.2 cm taller (P = 0.048) than the controls. Hip OA cases were found to be slightly shorter than the controls, but no statistically significant differences were observed. The adjusted hazard ratios (HRs) for knee OA and hip OA in the highest quartile were 2.5 (95% CI 1.4–4.5) and 1.0 (95% CI 0.3–3.4) for men and 1.8 (95% CI 1.0–3.1) and 0.7 (95% CI 0.2–2.3) for women. Conclusions Height at the age of 31 was associated with incidence of early knee OA, diagnosed prior to age 46. However, the low incidence of hip OA made our results for hip OA inconclusive.

// Heikki Huhta 1, 2, 3, 4, * , Olli Helminen 1, 2, 3, 4, * , Sami Palomaki 3, 4, * , Joonas H. Kauppila 1, 2, 3, 4, 5 , Juha Saarnio 2, 3, 4 , Petri P. Lehenkari 2, 3, 4 , Tuomo J. Karttunen 1, 3, 4 1 Departments of Pathology, University of Oulu, Oulu, Finland 2 Departments of Surgery, University of Oulu, Oulu, Finland 3 Medical Research Center Oulu, Oulu, Finland 4 Oulu University Hospital, Oulu, Finland 5 Upper Gastrointestinal Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden * These authors contributed equally to these work Correspondence to: Heikki Huhta, email: heikki.huhta@oulu.fi Keywords: MCT1, MCT4, MTCO1, esophageal adenocarcinoma, Barrett’s esophagus Received: October 20, 2016 Accepted: January 27, 2017 Published: February 11, 2017 ABSTRACT Background: Monocarboxylate transporters (MCTs) are cell membrane proteins which transport pyruvate, lactate and ketone bodies across the plasma membrane. MCTs are activated in various cancers, but their expression in esophageal adenocarcinoma is not known. The present study was conducted to elucidate the expression of MCTs in esophageal adenocarcinoma and its precursor lesions. Results: Cytoplasmic MCT1, MCT4 and MTCO1 expression linearly increased from normal epithelium to Barrett's mucosa to dysplasia and cancer. Low cytoplasmic MCT1 expression associated with high T-class ( P < 0.01), positive lymph node metastases ( P < 0.05), positive distant metastases ( P < 0.01) and high tumor stage ( P < 0.01). High cytoplasmic MCT4 expression correlated significantly with positive distant metastases ( P < 0.05). Both low MCT1 and high MCT4 histoscore predicted survival in univariate analysis ( P < 0.01). MCT4 histoscore predicted survival in multivariate analysis ( P = 0.043; HR 1.8 95%CI 1.0–3.1). MTCO1 expression was not correlated to clinicopathological variables or survival. Materials and Methods: MCT1, MCT4 and mitochondrial cytochrome c oxidase (MTCO1) expression were determined with immunohistochemistry in esophageal specimens from 129 patients with columnar dysplasia or adenocarcinoma. Specimens including normal esophagus ( n = 88), gastric ( n = 67) or intestinal metaplasia ( n = 51), low-grade ( n = 42), high-grade dysplasia ( n = 37) and esophageal adenocarcinoma ( n = 99) were evaluated. Conclusions: Major increase in markers of tumor metabolism occurs during carcinogenesis and progression of esophageal adenocarcinoma. MCT1 and MCT4 are prognostic factors in esophageal adenocarcinoma.

Background:Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. Methods: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. TenascinC and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. Results: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. Conclusions: Stromal TNC and, especially, FN expressions differentiate patients into low-and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.

Nucleic acid-sensing toll-like receptors (TLRs) (3, 7, 8, 9) have a role both in antiviral innate immunity and in autoimmune disorders. We assessed the expression of TLR3, 7, 8 and 9 in human and mouse pancreas focusing on the subpopulations of cells in the Langerhans islets. We studied eight human samples with normal pancreatic islets and two samples from patients with type 1 diabetes. Additionally, 10 CD-1 mouse pancreases were analysed. Immunohistochemical double-stainings for the TLRs and insulin, glucagon or somatostatin, respectively, were performed along with appropriate controls. In human pancreas, strong immunoreaction of TLR7 and TLR8 was observed in the insulin-positive beta cells, whereas glucagon- or somatostatin-expressing cells of the islets were weakly stained or negative. In type 1 diabetes, the expression in islets was weak or lost (TLR7: p = 0.014, TLR8: p = 0.053), correlating with loss of beta cells. TLR3 and 9 were expressed only weakly with no correlation with specific cell types. In mouse pancreas, only TLR9 was detected. Intra-pancreatic nerve ganglia strongly expressed TLR7. The strong expression of TLR7 and TLR8 in the beta cells of normal human islets could be an important piece in the puzzle of type 1 diabetes pathogenesis, and be linked with destruction of this particular subpopulation of the islet cells. In normal mice, only TLR9 can be constantly detected in the islets, highlighting differences between the species.

Summary Objective To determine the associations between multi-feature structural pathology assessed using magnetic resonance imaging (MRI) and the presence of knee pain, and to determine the associations between the locations of structural changes and different knee pain patterns. Method Eighty symptomatic subjects with knee pain and suspicion or diagnosis of knee OA and 63 asymptomatic subjects underwent knee MRI. Severity of structural changes was graded by MRI Osteoarthritis Knee Score (MOAKS) in separate knee locations. The associations between cartilage damage, bone marrow lesions (BMLs), osteophytes, Hoffa's synovitis, effusion-synovitis, meniscal damage and structural pathologies in ligaments, tendons and bursas and both the presence of pain and the knee pain patterns were assessed. Results The presence of Hoffa's synovitis (adjusted RR 1.6, 95% CI 1.2–1.3) and osteophytes in any region (2.07, 1.19–3.60) was significantly associated with the presence of pain. Any Hoffa's synovitis was associated with patellar pain (adjusted RR 4.70, 95% CI 1.19–3.60) and moderate-to-severe Hoffa's synovitis with diffuse pain (2.25, 1.13–4.50). Medial knee pain was associated with cartilage loss in the medial tibia (adjusted RR 2.66, 95% CI 1.22–5.80), osteophytes in the medial tibia (2.66, 1.17–6.07) and medial femur (2.55, 1.07–6.09), medial meniscal maceration (2.20, 1.01–4.79) and anterior meniscal extrusions (2.78, 1.14–6.75). Conclusions Hoffa's synovitis and osteophytes were strongly associated with the presence of knee pain. Medial pain was associated most often with medially located structural pathologies.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin. Increased cytoplasmic and nuclear expression of some TLRs has been previously reported in dysplasia in Barrett's esophagus and various malignant lesions in association with survival and metastasis. We assessed nuclear expression of TLR5 in Barrett's esophagus and esophageal adenocarcinoma (n = 94) by immunohistochemistry and immunofluorescent staining. Nuclear expression was observed in the majority of studied lesions with high variation in the proportion of positive nuclei. Expression in lymph node metastases was significantly higher than in esophageal lesions (p

Despite increasing evidence that subchondral bone contributes to osteoarthritis (OA) pathogenesis, little is known about local changes in bone structure compared to cartilage degeneration. This study linked structural adaptation of subchondral bone with histological OA grade. Twenty-five osteochondral samples of macroscopically different degeneration were prepared from tibiae of 14 patients. Samples were scanned with micro-computed tomography (μCT) and both conventional structural parameters and novel 3D parameters based on local patterns were analyzed from the subchondral plate and trabecular bone. Subsequently, samples were processed for histology and evaluated for OARSI grade. Each bone parameter and OARSI grade was compared to assess structural adaptation of bone with OA severity. In addition, thicknesses of cartilage, calcified cartilage, and subchondral plate were analyzed from histological sections and compared with subchondral bone plate thickness from μCT. With increasing OARSI grade, the subchondral plate became thicker along with decreased specific bone surface, while there was no change in tissue mineral density. Histological analysis showed that subchondral plate thickness from μCT also includes calcified cartilage. Entropy of local patterns increased with OA severity, reflecting higher tissue heterogeneity. In the trabecular compartment, bone volume fraction and both trabecular thickness and number increased with OARSI grade while trabecular separation and structure model index decreased. Also, elevation of local patterns became longitudinal in the subchondral plate and axial transverse in trabecular bone with increasing OARSI grade. This study demonstrates the possibility of radiological assessment of OA severity by structural analysis of bone. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 35:785-792, 2017.

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p

Stem/progenitor cells (SPCs), such as the mesenchymal, hematopoietic, or neural SPCs, can be isolated and propagated from adult tissues for therapy purposes. Novel data show that the SPC lipid species composition is modified by the fatty acids provided by the culture medium or tissue niche, and that the fatty acid composition of the cells regulate their functions. The ratio of n-3 and n-6 polyunsaturated fatty acids (PUFAs) in cellular lipids determines the quality and quantity of signaling molecules synthesized from the PUFAs by SPCs. The produced lipid mediators control the metabolism, differentiation and fate of the SPCs and proximate cells in concert with other local or systemic effectors. For example, high content of arachidonic acid, 20:4n-6, may promote differentiation and loss of the stemness traits. The hereby-reviewed special requirements for the SPC lipids will help us to develop culture procedures that maintain cell functionality during the in vitro cell expansion, which is essential for effective and safe cell therapy.

Three-dimensional (3D) semi-quantitative grading of pathological features in articular cartilage (AC) offers significant improvements in basic research of osteoarthritis (OA). We have earlier developed the 3D protocol for imaging of AC and its structures which includes staining of the sample with a contrast agent (phosphotungstic acid, PTA) and a consequent scanning with micro-computed tomography. Such a protocol was designed to provide X-ray attenuation contrast to visualize AC structure. However, at the same time, this protocol has one major disadvantage: the loss of contrast at the tidemark (calcified cartilage interface, CCI). An accurate segmentation of CCI can be very important for understanding the etiology of OA and ex-vivo evaluation of tidemark condition at early OA stages. In this paper, we present the first application of Deep Learning to PTA-stained osteochondral samples that allows to perform tidemark segmentation in a fully-automatic manner. Our method is based on U-Net trained using a combination of binary cross-entropy and soft-Jaccard loss. On cross-validation, this approach yielded intersection over the union of 0.59, 0.70, 0.79, 0.83 and 0.86 within 15 \(\upmu \)m, 30 \(\upmu \)m, 45 \(\upmu \)m, 60 \(\upmu \)m. and 75 \(\upmu \)m padded zones around the tidemark, respectively. Our codes and the dataset that consisted of 35 PTA-stained human AC samples are made publicly available together with the segmentation masks to facilitate the development of biomedical image segmentation methods.

Fiber-based scaffolds produced by textile manufacturing technology offer versatile materials for tissue engineering applications since a wide range of crucial scaffold parameters, including porosity, pore size and interconnectivity, can be accurately controlled using 3D weaving. In this study, we developed a weavable, bioactive biodegradable composite fiber from poly (lactic acid) (PLA) and hydroxyapatite powder by melt spinning. Subsequently, scaffolds of these fibers were fabricated by 3D weaving. The differentiation of human mesenchymal stem cells (hMSCs) in vitro was studied on the 3D scaffolds and compared with differentiation on 2D substrates having the same material composition. Our data showed that the 3D woven scaffolds have a major impact on hMSCs proliferation and activation. The 3D architecture supports the differentiation of the hMSCs into osteoblast cells and enhances the production of mineralized bone matrix. The present study further confirms that a 3D scaffold promotes hMSCs differentiation into the osteoblast–lineage and bone mineralization.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC–MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Bone marrow (BM) and peripheral blood (PB) derived mononuclear cells are precursors of in vitro osteoclast differentiation. However, few studies have compared the phenotypic and functional properties of osteoclasts generated from these sources and the effects of different growth factors on osteoclastogenesis. Both cell types differentiated into functional osteoclasts, but culturing the cells with or without transforming growth factor beta (TGF-β) and dexamethasone revealed differences in their osteoclastogenic capacity. When receptor activator for nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were used for differentiation, we did not observe differences in bone resorption activity or expression of osteoclastogenic genes calcitonin receptor (CR) and nuclear factor of activated T-cells (NFATc1) between the osteoclasts formed from the two sources. Addition of TGF-β and dexamethasone led to higher number of nuclei in multinuclear cells and increased expression of tartrate resistant acid phosphatase (TRACP) 5a and 5b, CR and NFATc1 in PB- derived osteoclasts depicting the higher osteoclastogenic potential and responsiveness to TGF-β and dexamethasone in PB monocytes. These results conclude that the choice of the osteoclast precursor source as well as the choice of osteoclastogenic growth factors are essential matters in determining the phenotypic characteristics of heterogeneous osteoclast populations.

Aims The aim of the study was to compare the influence of gap junctional communication (GJC) in osteoclastogenesis from bone marrow (BM) and peripheral blood (PB) monocytes. These widely used sources differ in purity, since BM cultures contain a significant number of stromal cells. We studied whether stimulation of GJC in BM monocyte/stromal cell cultures differs from the effect in pure PB monocyte cultures. We compared the differentiation also in acidosis, which is a known inducer of bone resorption. Main methods Human BM and PB monocytes were isolated from BM aspirates or whole blood samples. The cells were cultured on human bone slices with osteoclastogenic growth factors and a GJC modulator, antiarrhythmic peptide AAP10, at physiological and acidic pH. Key findings Both BM and PB monocytes differentiated into osteoclasts. Acidosis increased resorption in both cultures but stimulated cell fusion only in BM cultures, which demonstrates the role of stromal cells in osteoclastogenesis. At physiological pH, AAP10 increased the number of multinuclear cells and bone resorption in both BM and PB cultures indicating that GJC is involved in differentiation in both of these osteoclastogenesis assays. Interestingly, in PB cultures at pH 6.5 the stimulation of GJC with AAP10 inhibited both osteoclastogenesis and bone resorption suggesting a different role of GJC in BM and PB monocytes at stressed environment. Significance The study is conducted with primary human tissue samples and adds new knowledge on factors affecting osteoclastogenesis from different monocyte sources.

Pancreatic cancer arises from precursor lesions called pancreatic intraepithelial neoplasia (PanIN) characterized by inflammatory microenvironment. In pancreatic cancer, strong innate immunity and hypoxia responses are typical. Occurrence and relationship of these responses in human PanINs is unknown. We have studied the expression of toll-like receptors (TLR) TLR2, TLR4 and TLR9, and hypoxia markers HIF-1alpha and Carbonic anhydrase IX (CAIX) in normal and inflamed pancreatic ducts, in PanINs and in cancers. The samples of 69 surgically resected pancreatic ductal adenocarcinoma patients were stained using immunohistochemistry. We found TLR2, TLR9, HIF-1alpha and CAIX to be prominently expressed in pancreatic intraepithelial neoplasia. Expression of TLR2 showed a linear increase from PanIN1 to PanIN3, while the highest TLR4 expression was detected in inflamed ducts, and TLR9 expression in PanIN1 lesions. Within the PanIN1-group, nuclear HIF-1alpha correlated with membranous and cytoplasmic TLR2 expression (ρ = 0.982 and 0.815; p < 0.001 and p = 0.025, respectively), and in the PanIN2-group nuclear HIF-1alpha correlated with nuclear TLR9 expression 0.636, p = 0.026). Our findings show that the expression of TLRs 2, 4 and 9, and hypoxia markers HIF-1alpha and CAIX is abnormal in pancreatic intraepithelial neoplasia suggesting that both the innate immunity activation and hypoxia response are involved in early pancreatic carcinogenesis. However, these processes might be independent.

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5′-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5′-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs.

Osteogenesis imperfecta (OI) is a group of genetic disorders, of which Type III is the most severe among survivors. The disease is characterised in particular by bone fragility, decreased bone mass and increased incidence of fractures. Other usual findings are muscle hypotonia, joint hypermobility and short stature. Fractures and weak bones may consequently cause limb and spinal deformity and chronic physical disability. Bisphosphonates have revolutionised the treatment of newborn children with severe OI type III. Surgery is still needed in most patients due to high frequency of the fractures. In this systematic review we describe the present state-of-art in treating the most severe type of OI in newborn and preschool children with their bone fractures.

The purpose of this study was to evaluate the usefulness of MRI-guidance for core decompression of avascular necrosis of the femoral head. Twelve MRI-guided core decompressions were performed on patients with different stages of avascular necrosis of the femoral head. The patients were asked to evaluate their pain and their ability to function before and after the procedure and imaging findings were reviewed respectively. Technical success in reaching the target was 100 % without complications. Mean duration of the procedure itself was 54 min. All patients with ARCO stage 1 osteonecrosis experienced clinical benefit and pathological MRI findings were seen to diminish. Patients with more advanced disease gained less, if any, benefit and total hip arthroplasty was eventually performed on four patients. MRI-guidance seems technically feasible, accurate and safe for core decompression of avascular necrosis of the femoral head. Patients with early stage osteonecrosis may benefit from the procedure. • MRI is a useful guidance method for minimally invasive musculoskeletal interventions. • Bone drilling seems beneficial at early stages of avascular necrosis. • MRI-guidance is safe and accurate for bone drilling.

Background aims Bone marrow mononuclear cells (BM-MNCs) and bone marrow–derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. Cells transplanted intravascularly may become entrapped in the lungs, which potentially decreases their therapeutic effect and increases the risk for embolism. Methods Twelve pigs were divided into groups of 3 and received 99m Tc- hydroxymethyl-propylene-amine-oxime–labeled autologous BM-MNCs or allogeneic BM-MSCs by either intravenous (IV) or intra-arterial (IA) transplantation. A whole body scan and single photon emission computed tomography/computed tomography (SPECT/CT) were performed 8 h later, and tissue biopsies were collected for gamma counting. A helical CT scan was also performed on 4 pigs to detect possible pulmonary embolism, 2 after IV BM-MSC injection and 2 after saline injection. Results The transplantation route had a greater impact on the biodistribution of the BM-MSCs than the BM-MNCs. The BM-MNCs accumulated in the spleen and bones, irrespective of the administration route. The BM-MSCs had relatively higher uptake in the kidneys. The IA transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. Lung atelectases were frequent due to mechanical ventilation and attracted transplanted cells. CT did not reveal any pulmonary embolism. Conclusions Both administration routes were found to be safe, but iatrogenic atelectasis might be an issue when cells accumulate in the lungs. The IA administration is effective in avoiding pulmonary entrapment of BM-MSCs. The cell type and administration method both have a major impact on the acute homing.

The clinical data considering the bone marrow mononuclear cell (BMMNC) therapy in treatment for acute myocardial infarction (AMI) are controversial and the mechanisms remain unknown. Our objective was to study the cardiac function and changes in cytokine levels after administration of BMMNC in experimental AMI model.Unlabeled or Super-Paramagnetic-Iron-Oxide-labeled BMMNCs or saline was injected into myocardium of 31 pigs after circumflex artery occlusion. Ejection fraction (EF) was measured preoperatively, postoperatively and at 21 days by echocardiography. Cardiac MRI was performed postoperatively and after 21 days in 7 BMMNC animals. Serum cytokine levels were measured at baseline, 24 h and 21 days. Cellular homing was evaluated comparing MRI and histology.From baseline to 21 days EF decreased less in BMMNC group (EF mean control -19 SD 12 vs. BMMNC -4 SD 15 percentage points p = 0.02). Cytokine concentrations showed high variability between the animals. MRI correlated with histology in cell detection and revealed BMMNCs in the infarction area. By MRI, EF improved 11 percentage points. The improvement in EF was associated with the number of transplanted BMMNCs detected in the myocardium.BMMNC injection after AMI improved cardiac function. Quantity of transplanted BMMNCs correlated with the improvement in cardiac function after AMI.

Magnetic resonance (MR) imaging, with its inherent good spatial resolution and without tissue penetration depth limitations associated with other cell tracking techniques, is considered a valuable tool to assess the effects of cellular therapy. However, in order to allow in vivo tracking of transplanted cells with MRI, the cells must be labeled with contrast agents, usually in the form of magnetic or paramagnetic nanoparticles. Typically these are iron oxides, which are associated with a number of drawbacks related e.g. to the generation of hypointensities of the MR image due to signal loss. In this study, two chemically distinct manganese oxide-based nanostructures were developed and their feasibility as labels for human mesenchymal stem cells (hMSCs) was investigated. The ability to monitor the produced particles alone or within the labeled cells in vitro using MR imaging was further evaluated. Two novel synthetic approaches, the polyol process and microwave digestion, were combined to yield a “green”, and extremely rapid means of producing water-dispersible crystalline (MnO) and amorphous (MnOx) manganese oxides. To increase their water dispersability, capping agents in the form of organic polymers, poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid) (PAA), were added to the synthesis process. Crystalline MnO was not formed when PAA was used as the capping agent, since Mn ions (Mn2+) cannot be hydrolyzed to Mn(OH)2, which is an intermediate step in the formation of MnO, under the acidic conditions provided by PAA. PVP, on the other hand, served to induce a spherical shape to the formed nanocrystals. Remarkably, the relaxation times of the as-prepared amorphous MnOx were significantly shorter than those of their crystalline counterparts, and the biocompatibility was also higher for MnOx. To the best of our knowledge, this is the first report which describes the use of an amorphous MnOx as a cell label for MR imaging.

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP‐linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti‐inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease‐linked defect occurs in this non‐neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α‐tubulin post‐translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non‐neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.

OBJECTIVEAutologous bone cranioplasty after decompressive craniectomy entails a notable burden of difficult postoperative complications, such as infection and bone flap resorption (BFR), leading to mechanical failure. The prevalence and significance of asymptomatic BFR is currently unclear. The aim of this study was to radiologically monitor the long-term bone flap survival and bone quality change in patients undergoing autologous cranioplasty.METHODSThe authors identified all 45 patients who underwent autologous cranioplasty at Oulu University Hospital, Finland, between January 2004 and December 2014. Using perioperative and follow-up CT scans, the volumes and radiodensities of the intact bone flap prior to surgery and at follow-up were calculated. Relative changes in bone flap volume and radiodensity were then determined to assess cranioplasty survival. Sufficient CT scans were obtainable from 41 (91.1%) of the 45 patients.RESULTSThe 41 patients were followed up for a median duration of 3.79 years (25th and 75th percentiles = 1.55 and 6.66). Thirty-seven (90.2%) of the 41 patients had some degree of BFR and 13 (31.7%) had a remaining bone flap volume of less than 80%. Patients younger than 30 years of age had a mean decrease of 15.8% in bone flap volume compared with the rest of the cohort. Bone flap volume was not found to decrease linearly with the passing of time, however. The effects of lifestyle factors and comorbidities on BFR were nonsignificant.CONCLUSIONSIn this study BFR was a very common phenomenon, occurring at least to some degree in 90% of the patients. Decreases in bone volume were especially prominent in patients younger than 30 years of age. Because the progression of resorption during follow-up was nonlinear, routine follow-up CT scans appear unnecessary in monitoring the progression of BFR; instead, clinical follow-up with mechanical stability assessment is advised. Partial resorption is most likely a normal physiological phenomenon during the bone revitalization process.

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.

The dependence of the proton NMR relaxation times on field strength and on location within the tissue has been determined for a number of bovine and human articular cartilage samples. While the strong variation of T2 across the triple-layered cartilage structure as well as its orientation dependence are well known from clinical and laboratory high-field studies, T1 shows similar behavior only in low magnetic fields. At 0.27 T, the ratio of longest to shortest T1 has been found to cover a ratio of about 3-5 in healthy tissue. At the same time, the average T1 was found to be strongly field dependent in the range down to 0.25 mT, but no spatially resolved data are available under these conditions. By correlating the spatially resolved T1 distribution obtained at field strengths of 0.27 T with mathematical decompositions of the signal recovery function into multiexponential components, an attempt is made to quantify the width of P(T1) for variable field strengths, and to identify the field value where this distribution is widest, being optimally situated as a biomarker for laboratory studies or preclinical low-field investigations where spatial resolution is absent or insufficient to resolve the cartilage layer structure.

Anatomy and Cell biology, University of Oulu, Oulu, FinlandBACKGROUND: Toll-like receptor 9 (TLR9) is a cellularreceptor, which recognizes bacterial and host-derivedDNA. Stimulation of TLR9 induces cellular invasion viamatrix metalloproteinase 13 (MMP-13). The aim of thisstudy was to evaluate the role of TLR9 in invasion of oraltongue squamous cell carcinoma (OTSCC).METHODS: The effects of TLR9 ligands on oral squa-mous cell carcinoma cell lines were studied with invasionand migration assays, as well as in a myoma organotypicmodel.RESULTS: The TLR9 ligand, CpG-ODN, increased inva-sion and migration in OTSCC lines. These effects werereduced by TLR9 siRNA or inhibition with TLR9antibodies. Immunohistochemical analysis of tissuesfrom 195 patients with OTSCC revealed that TLR9was expressed in 181/195 carcinomas. The expression ofTLR9 was higher in the malignant cells than in thenormal epithelium. High TLR9 expression was associ-ated with high MMP-13 expression and poor differenti-ation. High TLR9 expression was also identified as anindependent predictor of poor prognosis (HR 1.810,95% CI [1.053–3.112]).CONCLUSION: Toll-like receptor 9 mediates OTSCCinvasion and migration in vitro and is an independentprognostic factor of OTSCC. Inhibition of TLR9 may be anovel therapeutic opportunity in oral cancer.J Oral Pathol Med (2014)Keywords: invasion; matrix metalloproteinase 13; migration;Toll-like receptor 9; tongue cancer

To investigate the association of quantitative magnetic resonance imaging (qMRI) parameters with arthroscopic grading of cartilage degeneration. Arthroscopy of the knee is considered to be the gold standard of osteoarthritis diagnostics; however, it is operator-dependent and limited to the evaluation of the articular surface. qMRI provides information on the quality of articular cartilage and its changes even at early stages of a disease. qMRI techniques included T 1 relaxation time, T 2 relaxation time, and delayed gadolinium-enhanced MRI of cartilage mapping at 3 T in ten patients. Due to a lack of generally accepted semiquantitative scoring systems for evaluating severity of cartilage degeneration during arthroscopy, the International Cartilage Repair Society (ICRS) classification system was used to grade the severity of cartilage lesions. qMRI parameters were statistically compared to arthroscopic grading conducted with the ICRS classification system. qMRI parameters were not linearly related to arthroscopic grading. Spearman’s correlation coefficients between qMRI and arthroscopic grading were not significant. The relative differences in qMRI parameters of superficial and deep cartilage varied with degeneration, suggesting different macromolecular alterations in different cartilage zones. Results suggest that loss of cartilage and the quality of remaining tissue in the lesion site may not be directly associated with each other. The severity of cartilage degeneration may not be revealed solely by diagnostic arthroscopy, and thus, qMRI can have a role in the investigation of cartilage degeneration.

Purpose To evaluate the sensitivity of quantitative MRI techniques (T1, T1,Gd, T2, continous wave (CW) T1ρ dispersion, adiabatic T1ρ, adiabatic T2ρ, RAFF and inversion-prepared magnetization transfer (MT)) for assessment of human articular cartilage with varying degrees of natural degeneration. Methods Osteochondral samples (n = 14) were obtained from the tibial plateaus of patients undergoing total knee replacement. MRI of the specimens was performed at 9.4T and the relaxation time maps were evaluated in the cartilage zones. For reference, quantitative histology, OARSI grading and biomechanical measurements were performed and correlated with MRI findings. Results All MRI parameters, except T1,Gd, showed statistically significant differences in tangential and full-thickness regions of interest (ROIs) between early and advanced osteoarthritis (OA) groups, as classified by OARSI grading. CW-T1ρ showed significant dispersion in all ROIs and featured classical laminar structure of cartilage with spin-lock powers below 1000 Hz. Adiabatic T1ρ, T2ρ, CW-T1ρ, MT, and RAFF correlated strongly with OARSI grade and biomechanical parameters. Conclusion MRI parameters were able to differentiate between early and advanced OA. Furthermore, rotating frame methods, namely adiabatic T1ρ, adiabatic T2ρ, CW-T1ρ, and RAFF, as well as MT experiment correlated strongly with biomechanical parameters and OARSI grade, suggesting high sensitivity of the parameters for cartilage degeneration. Magn Reson Med 74:249–259, 2015. © 2014 Wiley Periodicals, Inc.

Toll-like receptor-9 (TLR9) is an intracellular DNA receptor that is widely expressed in breast and other cancers. We previously demonstrated that low tumor TLR9 expression upon diagnosis is associated with significantly shortened disease-specific survival times in patients with triple-negative breast cancer (TNBC). There are no targeted therapies for this subgroup of patients whose prognosis is among the worst in breast cancer. Due to the previously detected in vitro anti-invasive effects of chloroquine in these cell lines, the present study aimed to investigate the in vivo effects of chloroquine against two clinical subtypes of TNBC that differ in TLR9 expression. Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro. Daily treatment of mice with intraperitoneal (i.p.) chloroquine (80 mg/kg/day) for 22 days, did not inhibit the growth of control siRNA or TLR9 siRNA MDA-MB-231 breast cancer cells. In conclusion, despite the favorable in vitro effects on TNBC invasion and viability, particularly in hypoxic conditions, chloroquine does not prevent the growth of the triple-negative MDA-MB-231 cells with high or low TLR9 expression levels in vivo. This may be explained by the activating effects of chloroquine on MMP-13 expression or by the fact that chloroquine, by suppressing TLR9 expression, permits the activation of currently unknown molecular pathways, which allow the aggressive behavior of TNBC cells with low TLR9 expression in hypoxia.

Human mesenchymal stem cells (hMSCs) are multipotent cells that have aroused great expectations in regenerative medicine. They are assumed to originate from hypoxic stem cell niches, especially in the bone marrow. This suggests that O2 is of importance in their regulation. In order to characterize regulation of the oxygen sensing pathway in these cells, we studied hMSCs isolated from three origins, adult and pediatric bone marrow and umbilical cord blood (UCB). Surprisingly, pediatric bone marrow and UCB MSCs showed normoxic stabilization of hypoxia-inducible factor-1α (HIF-1α) that is normally degraded completely by HIF prolyl 4-hydroxylases in the presence of oxygen. This was due to a high expression level of HIF-1α mRNA rather than inappropriate post-translational degradation of HIF-1α protein. HIF-1α mRNA was also induced in normoxic adult bone marrow MSCs, but 40% less than in the pediatric cells, and this was apparently not enough to stabilize the protein. The high normoxic HIF expression in all the hMSCs studied was accompanied by increased expression of a large number of glycolytic HIF target genes and increased glycolysis. Osteogenic differentiation of bone marrow-derived hMSCs reduced HIF-1α mRNA and protein expression and the expression of glycolytic mRNAs, resulting in decreased glycolysis and induction of oxidative metabolism. Induced mitochondrial biogenesis, changes in mitochondrial morphology and size indicative of increased oxidative phosphorylation, and induction of extracellular matrix synthesis were observed following osteogenic differentiation. Altogether, these data suggest that HIF-1α is a general regulator controlling the metabolic fate and multipotency of the hMSCs.