Enhancing the potential of aged human articular chondrocytes for high-quality cartilage regeneration.

Autologous chondrocyte implantation (ACI) is a regenerative procedure used to treat focal articular cartilage defects in knee joints. However, age has been considered as a limiting factor and ACI is not recommended for patients older than 40-50 years of age. One reason for this may be due to the reduced capacity of aged chondrocytes in generating new cartilage. Currently, the underlying mechanism contributing to aging-associated functional decline in chondrocytes is not clear and no proven approach exists to reverse chondrocyte aging. Given that chondrocytes in healthy hyaline cartilage typically display a spherical shape, believed to be essential for chondrocyte phenotype stability, we hypothesize that maintaining aged chondrocytes in a suspension culture that forces the cells to adopt a round morphology may help to "rejuvenate" them to a younger state, thus, leading to enhanced cartilage regeneration. Chondrocytes isolated from aged donors displayed reduced proliferation potential and impaired capacity in generating hyaline cartilage, compared to cells isolated from young donors, indicated by increased hypertrophy and cellular senescence. To test our hypothesis, the "old" chondrocytes were seeded as a suspension onto an agarose-based substratum, where they maintained a round morphology. After the 3-day suspension culture, aged chondrocytes displayed enhanced replicative capacity, compared to those grown adherent to tissue culture plastic. Moreover, chondrocytes subjected to suspension culture formed new cartilage in vitro with higher quality and quantity, with enhanced cartilage matrix deposition, concomitant with lower levels of hypertrophy and cellular senescence markers. Mechanistic analysis suggested the involvement of the RhoA and ERK1/2 signaling pathways in the "rejuvenation" process. In summary, our study presents a robust and straightforward method to enhance the function of aged human chondrocytes, which can be conveniently used to generate a large number of high-quality chondrocytes for ACI application in the elderly.

Methylprednisolone acetate mitigates IL1ß induced changes in matrix metalloproteinase gene expression in skeletally immature ovine explant knee tissues.

OBJECTIVE AND DESIGN: This study aimed at evaluating the effect of methylprednisolone (MPA) on messenger ribonucleic acid (mRNA) expression levels in immature ovine knee joint tissue explants following interleukin (IL)1ß induction and to assess responsiveness of the explants. MATERIAL OR SUBJECTS: Explants were harvested from the articular cartilage, synovium, and infrapatellar fat pad (IPFP) from immature female sheep. TREATMENT: Methylprednisolone. METHODS: The samples were allocated into six groups: (1) control, (2) MPA (10-3 M), (3) MPA (10-4 M), (4) IL1ß, (5) IL1ß + 10-3 M MPA, or (6) IL1ß + 10-4 M MPA. mRNA expression levels for molecules relevant to inflammation, cartilage degradation/anabolism, activation of innate immunity, and adipose tissue/hormones were quantified. Fold changes with MPA treatment were compared via the comparative CT method. RESULTS: Methylprednisolone treatment significantly suppressed MMPs consistently across the cartilage (MMP1, MMP3, and MMP13), synovium (MMP1 and MMP3), and IPFP (MMP13) (all p < 0.05). Other genes that were less consistently suppressed include endogenous IL1ß (cartilage) and IL6 (IPFP) (all p < 0.05), and others not affected either by IL-1 exposure or subsequent MPA include TGFß1, TLR4, and adipose-related molecules. CONCLUSIONS: Methylprednisolone significantly mitigated IL1ß induced mRNA expression for MMPs in the immature cartilage, synovium, and IPFP, but the extent of the responsiveness was tissue-, location-, and gene-specific.

Platelet-Rich Plasma Devices Can Be Used to Isolate Stem Cells From Synovial Fluid at the Point of Care.

PURPOSE: To assess whether point-of-care devices designed for collecting cellular components from blood or bone marrow could be used to isolate viable stem cells from synovial fluid. METHODS: Male and female patients older than 18 years old with either an acute, anterior cruciate ligament (ACL) injury or knee osteoarthritis (OA) with a minimum estimated 20 mL of knee effusion volunteered. Ten patients with an ACL injury and 10 patients with OA were enrolled. Two milliliters of collected synovial effusion were analyzed and cultured for cellular content. The remaining fluid was combined with whole blood and processed using a buffy-coat based platelet-rich plasma (PRP) processing system. Specimens were analyzed for cell counts, colony-forming unit (CFU) assays, differentiation assays, and flow cytometry. RESULTS: ACL effusion fluid contained 42.1 ± 20.7 CFU/mL and OA effusion fluid contained 65.4 ± 42.1 CFU/mL. After PRP processing, the counts in ACL-PRP were 101.6 ± 66.1 CFU/mL and 114.8 ± 73.4 CFU/mL in the OA-PRP. Cells showed tri-lineage differentiation potential when cultured under appropriate parameters. When analyzed with flow cytometry, >95% of cells produced with culturing expressed cell surface markers typically expressed by known stem cell populations, specifically CD45-, CD73+, CD29+, CD44+, CD105+, and CD90+. CONCLUSIONS: Multipotent viable stem cells can be harvested from knee synovial fluid, associated with an ACL injury or OA, and concentrated with a buffy coat-based PRP-processing device. CLINICAL RELEVANCE: PRP devices can be used to harvest stem cells from effusion fluids. Methods to use effusion fluid associated with an ACL injury and OA should be investigated further.

Cruciate ligament, patellar tendon, and patella formation involves differential cellular sources and dynamics as joint cavitation proceeds.

BACKGROUND: Cruciate ligament (CL) and patellar tendon (PT) are important elements of the knee joint, uniting femur, patella, and tibia into a single functional unit. So far, knowledge on the developmental mechanism of CL, PT, and patella falls far behind other skeletal tissues. RESULTS: Here, employing various lineage tracing strategies we investigate the cellular sources and dynamics that drive CL, PT, and patella formation during mouse embryonic development. We show that Gdf5 and Gli1 are generally expressed in the same cell population that only contributes to CL, but not PT or patella development. In addition, Col2 is expressed in two independent cell populations before and after joint cavitation, where the former contributes to the CL and the dorsal part of the PT and the latter contributes to the patella. Moreover, Prrx1 is always expressed in CL and PT progenitors, but not patella progenitors where it is switched off after joint cavitation. Finally, we reveal that patella development employs different cellular dynamics before and after joint cavitation. CONCLUSIONS: Our findings delineate the expression changes of several skeletogenesis-related genes before and after joint cavitation, and provide an indication on the cellular dynamics underlying ligament, tendon, and sesamoid bone formation during embryogenesis.

Using engineering models to shorten cryoprotectant loading time for the vitrification of articular cartilage.

Osteochondral allograft transplantation can treat full thickness cartilage and bone lesions in the knee and other joints, but the lack of widespread articular cartilage banking limits the quantity of cartilage available for size and contour matching. To address the limited availability of cartilage, vitrification can be used to store harvested joint tissues indefinitely. Our group's reported vitrification protocol [Biomaterials 33 (2012) 6061-6068] takes 9.5 h to load cryoprotectants into intact articular cartilage on bone and achieves high cell viability, but further optimization is needed to shorten this protocol for clinical use. Herein, we use engineering models to calculate the spatial and temporal distributions of cryoprotectant concentration, solution vitrifiability, and freezing point for each step of the 9.5-h protocol. We then incorporate the following major design choices for developing a new shorter protocol: (i) all cryoprotectant loading solution concentrations are reduced, (ii) glycerol is removed as a cryoprotectant, and (iii) an equilibration step is introduced to flatten the final cryoprotectant concentration profiles. We also use a new criterion-the spatially and temporally resolved prediction of solution vitrifiability-to assess whether a protocol will be successful instead of requiring that each cryoprotectant individually reaches a certain concentration. A total cryoprotectant loading time of 7 h is targeted, and our new 7-h protocol is predicted to achieve a level of vitrifiability comparable to the proven 9.5-h protocol throughout the cartilage thickness.

Platelet-Rich Fibrin Facilitates One-Stage Cartilage Repair by Promoting Chondrocytes Viability, Migration, and Matrix Synthesis.

The main aim of this study is to develop a one-stage method to combine platelet-rich fibrin (PRF) and autologous cartilage autografts for porcine articular cartilage repair. The porcine chondrocytes were treated with different concentrations of PRF-conditioned media and were evaluated for their cell viability and extracellular glycosaminoglycan (GAG) synthesis during six day cultivation. The chemotactic effects of PRF on chondrocytes on undigested cartilage autografts were revealed in explant cultures. For the in vivo part, porcine chondral defects were created at the medial femoral condyles of which were (1) left untreated, (2) implanted with PRF combined with hand-diced cartilage grafts, or (3) implanted with PRF combined with device-diced cartilage grafts. After six months, gross grades, histological, and immunohistochemical analyses were compared. The results showed that PRF promotes the viability and GAG expression of the cultured chondrocytes. Additionally, the PRF-conditioned media induce significant cellular migration and outgrowth of chondrocytes from undigested cartilage grafts. In the in vivo study, gross grading and histological scores showed significantly better outcomes in the treatment groups as compared with controls. Moreover, both treatment groups showed significantly more type II collagen staining and minimal type I collagen staining as compared with controls, indicating more hyaline-like cartilage and less fibrous tissue. In conclusion, PRF enhances the viability, differentiation, and migration of chondrocytes, thus, showing an appealing capacity for cartilage repair. The data altogether provide evidences to confirm the feasibility of a one-stage, culture-free method of combining PRF and cartilage autografts for repairing articular cartilage defects. From translational standpoints, these advantages benefit clinical applications by simplifying and potentiating the efficacy of cartilage autograft transplants.

Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.

Chondrocyte viability is lost during high-rate impact loading by transfer of amplified strain, but not stress, to pericellular and cellular regions.

OBJECTIVE: Deleterious impact loading to cartilage initiates post-traumatic osteoarthritis (OA). While cytokine and enzyme levels regulate disease progression, specific mechanical cues that elucidate cellular OA origins merit further investigation. We defined the dominant pericellular and cellular strain/stress transfer mechanisms following bulk-tissue injury associated with cell death. METHOD: Using an in vitro model, we investigated rate-dependent loading and spatial localization of cell viability in acute indentation and time-course studies. Atomic force microscopy (AFM) and magnetic resonance imaging (MRI) confirmed depth-wise changes in cartilage micro-/macro-mechanics and structure post-indentation. To understand the transfer of loading to cartilage domains, we computationally modeled full-field strain and stress measures in interstitial matrix, pericellular and cellular regions. RESULTS: Chondrocyte viability decreased following rapid impact (80%/s) vs slow loading (0.1%/s) or unloaded controls. Viability was lost immediately during impact within regions near the indenter-tissue contact but did not change over 7 days of tissue culture. AFM studies revealed a loss of stiffness following 80%/s loading, and MRI studies confirmed an increased tensile and shear strain, but not relaxometry. Image-based patterns of chondrocyte viability closely matched computational estimates of amplified maximum principal and shear strain in interstitial matrix, pericellular and cellular regions. CONCLUSION: Rapid indentation worsens chondrocyte death and degrades cartilage matrix stiffness in indentation regions. Cell death at high strain rates may be driven by elevated tensile strains, but not matrix stress. Strain amplification beyond critical thresholds in the pericellular matrix and cells may define a point of origin for early damage in post-traumatic OA.

Functional analysis of synovial fluid from osteoarthritic knee and carpometacarpal joints unravels different molecular profiles.

OBJECTIVE: In this work, we aimed to elucidate the molecular mechanisms driving primary OA. By studying the dynamics of protein expression in two different types of OA joints we searched for similarities and disparities to identify key molecular mechanisms driving OA. METHODS: For this purpose, human SF samples were obtained from CMC-I OA and knee joint of OA patients. SF samples were analysed by label-free quantitative liquid chromatography mass spectrometry. Disease-relevant proteins identified in proteomics studies, such as clusterin, paraoxonase/arylesterase 1 (PON1) and transthyretin were validated by enzyme-linked immunosorbent assays, and on the mRNA level by droplet digital PCR. Functional studies were performed in vitro using primary chondrocytes. RESULTS: Differential proteomic changes were observed in the concentration of 40 proteins including clusterin, PON1 and transthyretin. Immunoassay analyses of clusterin, PON1, transthyretin and other inflammatory cytokines confirmed significant differences in protein concentration in SF of CMC-I and knee OA patients, with primarily lower protein expression levels in CMC-I. Functional studies on chondrocytes unequivocally demonstrated that stimulation with SF obtained from knee OA, in contrast to CMC-I OA joint, caused a significant upregulation in pro-inflammatory response, cell death and hypertrophy. CONCLUSION: This study demonstrates that differential expression of molecular players in SF from different OA joints evokes diverse effects on primary chondrocytes. The pathomolecular mechanisms of OA may significantly differ in various joints, a finding that brings a new dimension into the pathogenesis of primary OA.

Cryopreservation in 95% serum with 5% DMSO maintains colony formation and chondrogenic abilities in human synovial mesenchymal stem cells.

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs. METHODS: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 105 cells) were suspended in 400 µL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 µL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 µL; 3 × 103 cells) were cultured in 60 cm2 dishes for 14 days for colony formation assays. Additional 62.5 µL samples of cell suspensions (1.25 × 105 cells) were added to tubes and cultured for 21 days for chondrogenesis assays. RESULTS: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group. CONCLUSION: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.

Expression of minor cartilage collagens and small leucine rich proteoglycans may be relatively reduced in osteoarthritic cartilage.

BACKGROUND: In osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix. METHODS: Cartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy. RESULTS: The result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression. CONCLUSIONS: This may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA.

Arthroscopic autologous chondrocyte implantation in the knee with an in situ crosslinking matrix: minimum 4-year clinical results of 15 cases and 1 histological evaluation.

PURPOSE: To clinically evaluate an arthroscopic autologous chondrocyte implantation (ACI) technique with an in situ crosslinking matrix for the treatment of full thickness cartilage defects of the knee and to present histological results of a graft cartilage biopsy obtained after 1.5 years. METHODS: Fifteen cases of arthroscopic autologous chondrocyte implantation in the knee performed between November 2011 and October 2012 were included in the study. Medical charts and operational reports were screened and the patients were contacted after 0.8 ± 0.3 years (0.4-1.3) and 4.3 ± 0.3 years (4.0-4.8) to asses subjective IKDC and re-operation. The Tegner activity scale was collected at the second follow-up time point. Subjective IKDC response rates were assessed at both follow-up time points. RESULTS: The first and second follow-up was completed by all 15 patients (100%). The subjective IKDC scores showed a significant improvement (pre-operative 44.5 ± 15.9, first follow-up 71.1 ± 15.9, p < 0.001, second follow-up 72.6 ± 17.3, p < 0.001). The overall response rate was 66.7% (n = 10) at follow-up one and two. There were no significant differences in pre-injury (4, range 1-9) and follow-up two (4, range 2-7) Tegner activity scales (p = n.s.). Two patients required re-operation in the index knee, not related to the ACI procedure. No complication related to the ACI or the implantation technique occurred. The histological results showed excellent cartilage regeneration. CONCLUSION: Arthroscopic ACI using an in situ crosslinking matrix is a safe and reliable treatment option for full-thickness cartilage defects of the knee.

Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.

An extended OpenSim knee model for analysis of strains of connective tissues.

BACKGROUND: OpenSim musculoskeletal models provide an accurate simulation environment that eases limitations of in vivo and in vitro studies. In this work, a biomechanical knee model was formulated with femoral articular cartilages and menisci along with 25 connective tissue bundles representing ligaments and capsules. The strain patterns of the connective tissues in the presence of femoral articular cartilage and menisci in the OpenSim knee model was probed in a first of its kind study. METHODS: The effect of knee flexion (0°-120°), knee rotation (- 40° to 30°) and knee adduction (- 15° to 15°) on the anterior cruciate, posterior cruciate, medial collateral, lateral collateral ligaments and other connective tissues were studied by passive simulation. Further, a new parameter for assessment of strain namely, the differential inter-bundle strain of the connective tissues were analyzed to provide new insights for injury kinematics. RESULTS: ACL, PCL, LCL and PL was observed to follow a parabolic strain pattern during flexion while MCL represented linear strain patterns. All connective tissues showed non-symmetric parabolic strain variation during rotation. During adduction, the strain variation was linear for the knee bundles except for FL, PFL and TL. CONCLUSIONS: Strains higher than 0.1 were observed in most of the bundles during lateral rotation followed by abduction, medial rotation and adduction. In the case of flexion, highest strains were observed in aACL and aPCL. A combination of strains at a flexion of 0° with medial rotation of 30° or a flexion of 80° with rotation of 30° are evaluated as rupture-prone kinematics.

Reliability of cartilage digestion and FDA-EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts.

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA-EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA-EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA-EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.

[Current Options and Importance of Imaging of the Hyaline Articular Cartilage of the Knee Prior to the Surgery - a Different Perspective of an Orthopaedic Surgeon and a Radiologist]. / Soucasné moznosti a význam zobrazování hyalinní chrupavky kolenního kloubu pred operacním zákrokem ­ rozdílný pohled ortopeda a radiologa.

The authors present an overview of the commonly used techniques and new trends of the cartilage imaging, especially postoperatively, and also discuss the potential of MRI imaging of the cartilage from the perspective of an experienced orthopaedic surgeon. In conclusion, the authors propose possible explanations for the potential discrepancies between the MRI and the arthroscopic findings. Hyaline cartilage damage and subsequent reparation of this tissue is one of the topical issues of orthopaedics and traumatology. Due to the expanding possibilities of treatment of this tissue and a relatively good effect of the surgery, the number of patients indicated for magnetic resonance imaging prior to the surgery has been on an increase. To make a decision concerning the subsequent type of treatment, it is necessary to get an idea of the cartilage cover condition, articular surfaces and also of the associated pathologies. The degree of cartilage damage can be assessed by arthroscopy or magnetic resonance imaging, which provides also the possibility of the subchondral lesion detection. Thanks to the noninvasive nature of the MRI examination, it has become the most important method in full imaging of the articular cartilage. The MRI of the cartilage has many options and at present the evaluation of the hyaline cartilage should be an integral part of each MRI examination of joints. For a more accurate assessment of the cartilage there are several advanced techniques available that can be used not only for preoperative diagnostics, but also for monitoring after the surgery. Key words: hyaline cartilage, magnetic resonance, arthroscopy.

Interleukin-1ß affects the phospholipid biosynthesis of fibroblast-like synoviocytes from human osteoarthritic knee joints.

OBJECTIVE: Phospholipids (PLs), together with hyaluronan and lubricin, are involved in boundary lubrication within human articular joints. Levels of lubricants in synovial fluid (SF) have been found to be associated with the health status of the joint. However, the biosynthesis and release of PLs within human joints remains poorly understood. This study contributes to our understanding of the effects of cytokines on the biosynthesis of PLs using cultured fibroblast-like synoviocytes (FLS) from human osteoarthritic knee joints. METHODS: Cultured FLS were stimulated with IL-1ß, TNFα, IL-6, or inhibitors of cell signaling pathways such as QNZ, SB203580 and SP600125 in the presence of stable isotope-labeled precursors of PLs. Lipids were extracted and quantified using electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Our analyses provide for the first time a detailed overview of PL species being synthesized by FLS. IL-1ß increased the biosynthesis of both phosphatidylethanolamine (PE) and PE-based plasmalogens. We show here that the NF-κB, p38 MAPK and JNK signaling pathways are all involved in IL-1ß-induced PL biosynthesis. IL-6 had no impact on PLs, whereas TNFα increased the biosynthesis of all PL classes. CONCLUSION: The biosynthesis of various PLs is controlled by IL-1ß and TNFα. Our detailed PL species analysis revealed that FLS can partly contribute to the elevated PL levels found in human osteoarthritis (OA) SF. IL-1ß in particular stimulates PE and PE-based plasmalogens which can act as cell-protective antioxidants. These results suggest that during OA progression, FLS undergo alterations in their PL composition to adapt to the new diseased environment.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an Anti-C5aR1 Antibody and C5 Small Interfering RNA.

Rheumatoid arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated Abs or small interfering RNAs (siRNAs) or small molecule inhibitors against C5 and C5aR1 (CD88) that have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. In this study, we test in collagen Ab-induced arthritis (CAIA) a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that although C5aR2 or C5L2 (GPR77) plays no role in CAIA, C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking C5, C5aR1, or the combination decreased clinical disease activity in mice with CAIA by 45%, 51%, and 58%, respectively. Anti-C5 Ab (BB5.1) has only limited efficacy, but significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1 Ab-protamine-C5 siRNA conjugate. To our knowledge, we show for the first time that whereas unconjugated Ab plus siRNAs reduce arthritis by 19%, our anti-C5aR1 Ab-protamine-C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils, and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Extracorporeal Shockwave Therapy Enhances Expression of Pdia-3 Which Is a Key Factor of the 1α,25-Dihydroxyvitamin D 3 Rapid Membrane Signaling Pathway in Treatment of Early Osteoarthritis of the Knee.

The goal of our research was demonstrated that multiple molecules in microenvironments of the early osteoarthritis (OA) joint tissue may be actively responded to extracorporeal shockwave therapy (ESWT) treatment, which potentially regulated biological function of chondrocytes and synovial cells in early OA knee. We demonstrated that shockwave treatment induced the expression of protein-disulfide isomerase-associated 3 (Pdia-3) which was a significant mediator of the 1α,25-Dihydroxyvitamin D 3 (1α,25(OH)2D3) rapid signaling pathway, using two-dimensional electrophoresis, histological analysis and quantitative polymerase chain reaction (qPCR). We observed that the expression of Pdia-3 at 2 weeks was significantly higher than that of other group at 4, 8, and 12 weeks post-shockwave treatment in early OA rat knee model. The other factors of the rapid membrane signaling pathway, including extracellular signal-regulated protein kinases 1 (ERK1), osteopontin (OPG), alkaline phosphatase (ALP), and matrix metallopeptidase 13 (MMP13) were examined and were found to be significantly increased at 2 weeks post-shockwave treatment by qPCR in early OA of the knee. Our proteomic data revealed significant Pdia-3 expression in microenvironments of OA joint tissue that could be actively responded to ESWT, which may potentially regulate the biological functions of chondrocytes and osteoblasts in the treatment of the early OA of the knee.

Tissue engineering-relevant characteristics of ex vivo and monolayer-expanded chondrocytes from the notch versus trochlea of human knee joints.

PURPOSE: The aim was to analyse the biological characteristics of chondrocytes from the two biopsy sites notch vs. trochlea of human knee joints. The question was whether tissue engineering-relevant characteristics such as viability and mRNA expression profile would be comparable ex vivo and after monolayer expansion, as these are parts of routine autologous chondrocyte implantation (ACI). METHODS: Biopsies from the intercondylar notch and the lateral aspect of the trochlea from 20 patients with ICRS grades 3 and 4 cartilage defects were harvested during arthroscopy. Collagen types 1, 2, and 10 mRNA were quantified by polymerase chain reaction. RESULTS: Compared with notch chondrocytes, ex vivo trochlea chondrocytes had comparable cell numbers, vitality and aggrecan, collagen types 1, -2 and -10 mRNA expression. After monolayer expansion both notch and trochlea chondrocyte characteristics were comparably altered, regardless of their biopsy origin, and no significant differences in viability and mRNA expression were noted. CONCLUSIONS: Collectively, these findings suggest that tissue engineering-relevant characteristics of notch and trochlea chondrocytes are comparable ex vivo and after monolayer expansion. Thus, trochlea chondrocytes promise clinical potential and chondrocytes for ACI could potentially be generated from both notch and trochlea biopsy sites.