Transverse Relaxation Anisotropy of the Achilles and Patellar Tendon Studied by MR Microscopy.

BACKGROUND: T2 * anisotropy affects the clinical assessment of tendons (magic-angle artifact) and may be a source of T2 *-misinterpretation. PURPOSE: To analyze T2 *-anisotropy and T2 *-decay of Achilles and patellar tendons in vitro at microscopic resolution using a variable-echo-time (vTE) sequence. STUDY TYPE: Prospective. SPECIMEN: Four human Achilles and four patellar tendons. FIELD STRENGTH/SEQUENCE: A 7 T MR-microscopy; 3D-vTE spoiled-gradient-echo-sequence (T2 *-mapping). ASSESSMENT: All tendons were measured at 0° and 55° relative to B0 . Additional angles were measured for one Achilles and one patellar tendon for a total of 11 angles ranging from 0° to 90°. T2 *-decay was analyzed with mono- and bi-exponential signal fitting. Mono-exponential T2 *-values (T2 *m ), short and long T2 *-components (T2 *s , T2 *l ), and the fraction of the short component Fs of the bi-exponential T2 *-fit were calculated. T2 *-decay characteristics were compared with morphological MRI and histologic findings based on a region-of-interest analysis. STATISTICAL TESTS: Akaike information criterion (AICC ), F-test, and paired t-test. A P value smaller than the α-level of 0.05 was considered statistically significant. RESULTS: T2 *m -values between fiber-to-field angles of 0° and 55° were increased on average from T2 *m (0°) = 1.92 msec to T2 *m (55°) = 29.86 msec (15.5-fold) in the Achilles and T2 *m (0°) = 1.46 msec to T2 *m (55°) = 23.33 msec (16.0-fold) in the patellar tendons. The changes in T2 *m -values were statistically significant. For the whole tendon, according to F-test and AICC , a bi-exponential model was preferred for angles close to 0°, while the mono-exponential model tended to be preferred at angles close to 55°. CONCLUSION: MR-microscopy provides a deeper insight into the relationship between T2 *-decay (mono- vs. bi-exponential model) and tendon heterogeneity. Changes in fiber-to-field angle result in significant changes in T2 *-values. Thus, we conclude that awareness of T2 *-anisotropy should be noted in quantitative T2 *-mapping of tendons to avoid T2 *-misinterpretation such as a false positive detection of degeneration due to large fiber-to-field angles. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

A 3-Dimensional In Vitro Model of Zonally Organized Extracellular Matrix.

OBJECTIVE: Functional cartilage repair requires the new formation of organized hyaline cartilaginous matrix to avoid the generation of fibrous repair tissue. The potential of mesenchymal progenitors was used to assemble a 3-dimensional structure in vitro, reflecting the zonation of collagen matrix in hyaline articular cartilage. DESIGN: The 3-dimensional architecture of collagen alignment in pellet cultures of chondroprogenitors (CPs) was assessed with Picrosirius red staining analyzed under polarized light. In parallel assays, the trilineage capability was confirmed by calcium deposition during osteogenesis by alizarin S staining and alkaline phosphatase staining. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), mRNA levels of ALP, RUNX2, and BGLAP were assessed after 21 days of osteoinduction. Lipid droplets were stained with oil red O and adipogenic differentiation was confirmed by RT-qPCR analysis of PPARG and LPL gene expression. RESULTS: Under conditions promoting the chondrogenic signature in self-assembling constructs, CPs formed an aligned extracellular matrix, positive for glycosaminoglycans and collagen type II, showing developing zonation of birefringent collagen fibers along the cross section of pellets, which reflect the distribution of collagen fibers in hyaline cartilage. Induced osteogenic and adipogenic differentiation confirmed the trilineage potential of CPs. CONCLUSION: This model promotes the differentiation and self-organization of postnatal chondroprogenitors, resulting in the formation of zonally organized engineered hyaline cartilage comparable to the 3 zones of native cartilage.

Analysis of Running-Related Injuries: The Vienna Study.

BACKGROUND: This study aimed to provide an extensive and up-to-date analysis of running-related injuries (RRI) and analyze a broad range of contributing factors for a large heterogeneous and non-selected running population from Central Europe. METHODS: Anthropometric, training, footwear, anatomic malalignment, and injury data from 196 injured runners were assessed case-controlled and retrospectively. Univariate and multivariate regression models were developed to identify associated factors for specific injury locations and diagnoses. RESULTS: The majority of patients were female (56%). Three most frequently observed malalignments included varus knee alignment, pelvic obliquity, and patellar squinting. The most common injuries were the patellofemoral pain syndrome (PFPS), the iliotibial band friction syndrome (ITBFS), patellar tendinopathy, spinal overload, and ankle instability. A number of contributing factors were identified. Previous injury history was a contributing factor for knee injuries and ITBFS. Lower training load was reported with a higher incidence of PFPS, while a higher training load was positively associated with injuries of the lower leg. Runners with a higher body mass index (BMI) were at a significantly higher risk for lower back injuries. CONCLUSIONS: Running-related injuries are multifactorial associated with a combination of variables including personal data, training load, anatomic malalignments, and injury history. They can furthermore result from a lack of experience/training as well as from overuse. Suffering a specific RRI of high risk could be defined based on individual predispositions and help to induce appropriate training balance.

Galectins-1 and -3 in Human Intervertebral Disc Degeneration: Non-Uniform Distribution Profiles and Activation of Disease Markers Involving NF-κB by Galectin-1.

Degeneration of the human intervertebral disc (IVD) is assumed to underlie severe clinical symptoms, in particular chronic back pain. Since adhesion/growth-regulatory galectins are linked to arthritis/osteoarthritis pathogenesis by activating a pro-degradative/-inflammatory gene expression signature, we hypothesized a similar functional involvement of galectins in IVD degeneration. Immunohistochemical evidence for the presence of galectins-1 and -3 in IVD is provided comparatively for specimens of spondylochondrosis, spondylolisthesis, and spinal deformity. Immunopositivity was detected in sections of fixed IVD specimens in each cellular compartment with age-, disease-, and galectin-type-related differences. Of note, presence of both galectins correlated with IVD degeneration, whereas correlation with age was seen only for galectin-3. In addition, staining profiles for these two galectins showed different distribution patterns in serial sections, an indication for non-redundant functionalities. In vitro, both galectins bound to IVD cells in a glycan-dependent manner. However, exclusively galectin-1 binding triggered a significant induction of functional disease markers (i.e., IL6, CXCL8, and MMP1/3/13) with involvement of the nuclear factor-kB pathway. This study thus gives direction to further network analyses and functional studies on galectins in IVD degeneration. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2204-2216, 2019.

Orientation dependence and decay characteristics of T2 * relaxation in the human meniscus studied with 7 Tesla MR microscopy and compared to histology.

PURPOSE: To evaluate: (1) the feasibility of MR microscopy T2 * mapping by performing a zonal analysis of spatially matched T2 * maps and histological images using microscopic in-plane pixel resolution; (2) the orientational dependence of T2 * relaxation of the meniscus; and (3) the T2 * decay characteristics of the meniscus by statistically evaluating the quality of mono- and biexponential model. METHODS: Ultrahigh resolution T2 * mapping was performed with ultrashort echo time using a 7 Tesla MR microscopy system. Measurement of one meniscus was performed at three orientations to the main magnetic field (0, 55, and 90°). Histological assessment was performed with picrosirius red staining and polarized light microscopy. Quality of mono- and biexponential model fitting was tested using Akaike Information Criteria and F-test. RESULTS: (1) The outer laminar layer, connective tissue fibers from the joint capsule, and the highly organized tendon-like structures were identified using ultra-highly resolved MRI. (2) Highly organized structures of the meniscus showed considerable changes in T2 * values with orientation. (3) No significant biexponential decay was found on a voxel-by-voxel-based evaluation. On a region-of-interest-averaged basis, significant biexponential decay was found for the tendon-like region in a fiber-to-field angle of 0°. CONCLUSION: The MR microscopy approach used in this study allows the identification of meniscus substructures and to quantify T2 * with a voxel resolution approximately 100 times higher than previously reported. T2 * decay showed a strong fiber-to-field angle dependence reflecting the anisotropic properties of the meniscal collagen fibers. No clear biexponential decay behavior was found for the meniscus substructures.

Galectin-8 induces functional disease markers in human osteoarthritis and cooperates with galectins-1 and -3.

The reading of glycan-encoded signals by tissue lectins is considered a major route of the flow of biological information in many (patho)physiological processes. The arising challenge for current research is to proceed from work on a distinct protein to family-wide testing of lectin function. Having previously identified homodimeric galectin-1 and chimera-type galectin-3 as molecular switches in osteoarthritis progression, we here provide proof-of-principle evidence for an intra-network cooperation of galectins with three types of modular architecture. We show that the presence of tandem-repeat-type galectin-8 significantly correlated with cartilage degeneration and that it is secreted by osteoarthritic chondrocytes. Glycan-inhibitable surface binding of galectin-8 to these cells increased gene transcription and the secretion of functional disease markers. The natural variant galectin-8 (F19Y) was less active than the prevalent form. Genome-wide array analysis revealed induction of a pro-degradative/inflammatory gene signature, largely under control of NF-κB signaling. This signature overlapped with respective gene-expression patterns elicited by galectins-1 and -3, but also presented supplementary features. Functional assays with mixtures of galectins that mimic the pathophysiological status unveiled cooperation between the three galectins. Our findings shape the novel concept to consider individual galectins as part of a so far not realized teamwork in osteoarthritis pathogenesis, with relevance beyond this disease.

Brazilin blocks catabolic processes in human osteoarthritic chondrocytes via inhibition of NFKB1/p50.

This study aimed to evaluate the chondroprotective and anti-inflammatory activity of brazilin in human osteoarthritic (OA) cartilage and chondrocytes with particular focus on the nuclear factor-kappa B (NF-κB) pathway. Therefore, brazilin was isolated from Caesalpinia sappan and identified using high performance liquid chromatography (HPLC). The effect of brazilin was assessed in cartilage explants treated with 10 ng/ml interleukin (IL)-1ß and 10 ng/ml tumor necrosis factor (TNF)-α using histological and biochemical glycosaminoglycan (GAG) analyses and in primary chondrocytes treated with 10 ng/ml IL-1ß using RT-qPCR, ELISA, and Western blot. The involvement of NF-κB signaling was examined using a human NF-κB signaling array and in silico pathway analysis. Brazilin was found to reduce the GAG loss from cartilage explants stimulated with IL-1ß and TNF-α. NF-κB pathway analysis in chondrocytes revealed NFKB1/p50 as a central player regulating the anti-inflammatory activities of brazilin. Brazilin suppressed the IL-1ß-mediated up-regulation of OA markers and the induction of NFKB1/p50 in chondrocytes. In conclusion, brazilin effectively attenuates catabolic processes in human OA cartilage and chondrocytes-at least in part due to the inhibition of NFKB1/p50-which indicates a chondroprotective potential of brazilin in OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2431-2438, 2018.

Galectin-3 Induces a Pro-degradative/inflammatory Gene Signature in Human Chondrocytes, Teaming Up with Galectin-1 in Osteoarthritis Pathogenesis.

Inflammatory chemo- and cytokines and matrix-degrading proteases underlie the progression of osteoarthritis (OA). Aiming to define upstream regulators for these disease markers, we pursued initial evidence for an upregulation of members of the adhesion/growth-regulatory galectin family. Immunohistochemical localization of galectin-3 (Gal-3) in sections of human cartilage with increasing levels of degeneration revealed a linear correlation reaching a chondrocyte positivity of 60%. Presence in situ was cytoplasmic, the lectin was secreted from OA chondrocytes in culture and binding of Gal-3 yielded lactose-inhibitable surface staining. Exposure of cells to the lectin led to enhanced gene expression and secretion of functional disease markers. Genome-wide transcriptomic analysis broadened this result to reveal a pro-degradative/inflammatory gene signature under the control of NF-κB. Fittingly, targeting this route of activation by inhibitors impaired the unfavourable response to Gal-3 binding, as also seen by shortening the lectin's collagen-like repeat region. Gal-3's activation profile overlaps with that of homodimeric galectin-1 (Gal-1) and also has distinctive (supplementing) features. Tested at subsaturating concentrations in a mixture, we found cooperation between the two galectins, apparently able to team up to promote OA pathogenesis. In summary, our results suggest that a network of endogenous lectins is relevant for initiating this process cascade.

Galectin-1 Couples Glycobiology to Inflammation in Osteoarthritis through the Activation of an NF-κB-Regulated Gene Network.

Osteoarthritis is a degenerative joint disease that ranks among the leading causes of adult disability. Mechanisms underlying osteoarthritis pathogenesis are not yet fully elucidated, putting limits to current disease management and treatment. Based on the phenomenological evidence for dysregulation within the glycome of chondrocytes and the network of a family of adhesion/growth-regulatory lectins, that is, galectins, we tested the hypothesis that Galectin-1 is relevant for causing degeneration. Immunohistochemical analysis substantiated that Galectin-1 upregulation is associated with osteoarthritic cartilage and subchondral bone histopathology and severity of degeneration (p < 0.0001, n = 29 patients). In vitro, the lectin was secreted and it bound to osteoarthritic chondrocytes inhibitable by cognate sugar. Glycan-dependent Galectin-1 binding induced a set of disease markers, including matrix metalloproteinases and activated NF-κB, hereby switching on an inflammatory gene signature (p < 10(-16)). Inhibition of distinct components of the NF-κB pathway using dedicated inhibitors led to dose-dependent impairment of Galectin-1-mediated transcriptional activation. Enhanced secretion of effectors of degeneration such as three matrix metalloproteinases underscores the data's pathophysiological relevance. This study thus identifies Galectin-1 as a master regulator of clinically relevant inflammatory-response genes, working via NF-κB. Because inflammation is critical to cartilage degeneration in osteoarthritis, this report reveals an intimate relation of glycobiology to osteoarthritic cartilage degeneration.

Medical Plant Extracts for Treating Knee Osteoarthritis: a Snapshot of Recent Clinical Trials and Their Biological Background.

In light of the growing global health problem associated with osteoarthritis, herbal remedies have become an important research focus in the scientific and medical community, and numerous studies have been published to identify their biological effects and mechanisms in vitro and in vivo. This review is a snapshot of the most recent clinical trials on the efficacy of medical plant extracts in knee osteoarthritis patients, and provides relevant background information on the biological mechanisms that may underlie the clinical observations. Therefore, we performed a PubMed literature survey and discussed a selection of clinical trials in the field, with special attention being drawn to the design and outcome measures of the studies. We further spotlighted on issues relating to the efficacy and safety of the plant extracts and discussed major challenges for upcoming studies in the field, which include the need for rigorously designed in vivo and in vitro studies, as well as the elucidation of potential additive effects and structure-modifying activities beyond symptom relief.

Sodium magnetic resonance imaging of ankle joint in cadaver specimens, volunteers, and patients after different cartilage repair techniques at 7 T: initial results.

OBJECTIVES: The goal of cartilage repair techniques such as microfracture (MFX) or matrix-associated autologous chondrocyte transplantation (MACT) is to produce repair tissue (RT) with sufficient glycosaminoglycan (GAG) content. Sodium magnetic resonance imaging (MRI) offers a direct and noninvasive evaluation of the GAG content in native cartilage and RT. In the femoral cartilage, this method was able to distinguish between RTs produced by MFX and MACT having different GAG contents. However, it needs to be clarified whether sodium MRI can be useful for evaluating RT in thin ankle cartilage. Thus, the aims of this 7-T study were (1) to validate our sodium MRI protocol in cadaver ankle samples, (2) to evaluate the sodium corrected signal intensities (cSI) in cartilage of volunteers, (3) and to compare sodium values in RT between patients after MFX and MACT treatment. MATERIALS AND METHODS: Five human cadaver ankle samples as well as ankles of 9 asymptomatic volunteers, 6 MFX patients and 6 MACT patients were measured in this 7-T study. Sodium values from the ankle samples were compared with histochemically evaluated GAG content. In the volunteers, sodium cSI values were calculated in the cartilages of ankle and subtalar joint. In the patients, sodium cSI in RT and reference cartilage were measured, morphological appearance of RT was evaluated using the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system, and clinical outcome before and after surgery was assessed using the American Orthopaedic Foot and Ankle Society score and Modified Cincinnati Knee Scale. All regions of interest were defined on morphological images and subsequently transferred to the corresponding sodium images. Analysis of variance, t tests, and Pearson correlation coefficients were evaluated. RESULTS: In the patients, significantly lower sodium cSI values were found in RT than in reference cartilage for the MFX (P = 0.007) and MACT patients (P = 0.008). Sodium cSI and MOCART scores in RT did not differ between the MFX and MACT patients (P = 0.185). No significant difference in sodium cSI was found between reference cartilage of the volunteers and the patients (P = 0.355). The patients showed significantly higher American Orthopaedic Foot and Ankle Society and Modified Cincinnati scores after treatment than they did before treatment. In the volunteers, sodium cSI was significantly higher in the tibial cartilage than in the talar cartilage of ankle joint (P = 0.002) and in the talar cartilage than in the calcaneal cartilage of subtalar joint (P < 0.001). Data from the cadaver ankle samples showed a strong linear relationship between the sodium values and the histochemically determined GAG content (r = 0.800; P < 0.001; R = 0.639). CONCLUSIONS: This study demonstrates the feasibility of in vivo quantification of sodium cSI, which can be used for GAG content evaluation in thin cartilages of ankle and subtalar joints at 7 T. A strong correlation observed between the histochemically evaluated GAG content and the sodium values proved the sufficient sensitivity of sodium MRI to changes in the GAG content of cartilages in the ankle. Both MFX and MACT produced RT with lower sodium cSI and, thus, of lower quality compared with reference cartilage in the patients or in the volunteers. Our results suggest that MFX and MACT produce RT with similar GAG content and similar morphological appearance in patients with similar surgery outcome. Sodium MRI at 7 T allows a quantitative evaluation of RT quality in the ankle and may thus be useful in the noninvasive assessment of new cartilage repair procedures.

Vascularization of primary and secondary ossification centres in the human growth plate.

BACKGROUND: The switch from cartilage template to bone during endochondral ossification of the growth plate requires a dynamic and close interaction between cartilage and the developing vasculature. Vascular invasion of the primarily avascular hypertrophic chondrocyte zone brings chondroclasts, osteoblast- and endothelial precursor cells into future centres of ossification.Vascularization of human growth plates of polydactylic digits was studied by immunohistochemistry, confocal-laser-scanning-microscopy and RT-qPCR using markers specific for endothelial cells CD34 and CD31, smooth muscle cells α-SMA, endothelial progenitor cells CD133, CXCR4, VEGFR-2 and mesenchymal progenitor cells CD90 and CD105. In addition, morphometric analysis was performed to quantify RUNX2+ and DLX5+ hypertrophic chondrocytes, RANK+ chondro- and osteoclasts, and CD133+ progenitors in different zones of the growth plate. RESULTS: New vessels in ossification centres were formed by sprouting of CD34+ endothelial cells that did not co-express the mature endothelial cell marker CD31. These immature vessels in the growth plate showed no abluminal coverage with α-SMA+ smooth muscle cells, but in their close proximity single CD133+ precursor cells were found that did not express VEGFR-2, a marker for endothelial lineage commitment. In periosteum and in the perichondrial groove of Ranvier that harboured CD90+/CD105+ chondro-progenitors, in contrast, mature vessels were found stabilized by α-SMA+ smooth muscle cells. CONCLUSION: Vascularization of ossification centres of the growth plate was mediated by sprouting of capillaries coming from the bone collar or by intussusception rather than by de-novo vessel formation involving endothelial progenitor cells. Vascular invasion of the joint anlage was temporally delayed compared to the surrounding joint tissue.

Human osteoarthritic knee cartilage: fingerprinting of adhesion/growth-regulatory galectins in vitro and in situ indicates differential upregulation in severe degeneration.

The apparent connection of galectin-3 to chondrocyte survival and osteoarthritis-like cartilage modifications in animal models provided incentive for the mapping of seven members of this family of adhesion/growth-regulatory proteins in human cartilage specimens. Starting with work in vitro, RT-qPCR analyses and immunocytochemistry revealed gene transcription and protein presence in cultured OA chondrocytes, especially for galectin-1, galectin-3 and galectin-8. Immunohistochemistry in clinical specimens with mild and severe cartilage degeneration detected galectins in chondrocytes-with upregulation, especially of galectin-1 in areas of severe degeneration-accompanied by α2,6-sialylation in the pericellular matrix. Given the possibility for additive/antagonistic activities between galectins, these results direct further research toward examining cellular effects of (1) these proteins (alone or in combination) on chondrocytes and (2) remodeling of the chondrocyte glycophenotype.

Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein.

INTRODUCTION: This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes. METHODS: Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤ 4 or ≥ 9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing ß-galactoside-specific endogenous lectins. RESULTS: We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration. CONCLUSIONS: In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA.

Aldehyde dehydrogenase 1, a potential marker for cancer stem cells in human sarcoma.

Tumors contain a small population of cancer stem cells (CSC) proposed to be responsible for tumor maintenance and relapse. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in different cancer types. This study used the Aldefluor® assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1(high) cells from five human sarcoma cell lines and one primary chordoma cell line. ALDH1(high) cells range from 0.3% (MUG-Chor1) to 4.1% (SW-1353) of gated cells. Immunohistochemical staining, analysis of the clone formation efficiency, and xCELLigence microelectronic sensor technology revealed that ALDH1(high) cells from all sarcoma cell lines have an increased proliferation rate compared to ALDH1(low) cells. By investigating of important regulators of stem cell biology, real-time RT-PCR data showed an increased expression of c-Myc, ß-catenin, and SOX-2 in the ALDH1(high) population and a significant higher level of ABCG2. Statistical analysis of data demonstrated that ALDH1(high) cells of SW-982 and SW-1353 showed higher resistance to commonly used chemotherapeutic agents like doxorubicin, epirubicin, and cisplatin than ALDH1(low) cells. This study demonstrates that in different sarcoma cell lines, high ALDH1 activity can be used to identify a subpopulation of cells characterized by a significantly higher proliferation rate, increased colony forming, increased expression of ABC transporter genes and stemness markers compared to control cells. In addition, enhanced drug resistance was demonstrated.