An assessment of bone marrow mesenchymal stem cell and human articular cartilage derived chondroprogenitor cocultures vs. monocultures.

BACKGROUND: Cell based therapy in cartilage repair predominantly involves the use of chondrocytes and mesenchymal stromal cells (MSC). Co-culture systems, due to their probable synergistic effect on enhancement of functional chondrogenesis and reduction in terminal differentiation have also been attempted. Chondroprogenitors, derived from articular cartilage and regarded as MSCs, have recently garnered interest for consideration in cartilage regeneration to overcome limitations associated with use of conventional cell types. The aim of this study was to assess whetherco-culturing bone marrow (BM)-MSCs and chondroprogenitors at different ratios would yield superior results in terms of surface marker expression, gene expression and chondrogenic potential. METHODS: Human BM-MSCs and chondroprogenitors obtained from three osteoarthritic knee joints and subjected to monolayer expansion and pellet cultures (10,000 cells/cm2) as five test groups containing either monocultures or co-cultures (MSC: chondroprogenitors) at three different ratios (75:25, 50:50 and 25:75) were utilized. RESULTS: Data analysis revealed that all groups exhibited a high expression of CD166, CD29 and CD49e. With regard to gene expression, high expression of SOX9, Aggrecan and Collagen type I; a moderate expression of Collagen type X and RUNX2; with a low expression of Collagen type II was seen. Analysis of pellet culture revealed that chondroprogenitor monoculture and chondroprogenitor dominant coculture, exhibited a subjectively larger pellet size with higher deposition of Collagen type II and glycosaminoglycan. CONCLUSION: In conclusion, this study is suggestive of chondroprogenitor monoculture superiority over MSCs, either in isolation or in a coculture system and proposes further analysis of chondroprogenitors for cartilage repair.

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.

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.

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.

Characterization of regional meniscal cell and chondrocyte phenotypes and chondrogenic differentiation with histological analysis in osteoarthritic donor-matched tissues.

Meniscus degeneration is closely related to the progression of knee osteoarthritis (OA). However, there is currently a lack of quantitative and objective metrics to assess OA meniscal cell phenotypes. In this study we investigated the phenotypic markers and chondrogenic potency of avascular and vascular meniscal cells and chondrocytes from medial OA knee joints (n = 10). Flow cytometry results showed that a significantly greater percentage of meniscal cells were positive for CD49b, CD49c and CD166 compared to donor-matched chondrocytes after 14 days in monolayer culture. The integrins, CD49b and CD29, were expressed at a significantly higher level on avascular meniscal cells derived from tissues with a more degenerated inner border than non-degenerate menisci, suggesting that the integrin family may play an important role in meniscus OA pathology. Collagen fibres arranged in a "tree-like" formation within the meniscus appeared to have less blood vessels associated with them in the vascular region of the most degenerate menisci, which may indicate that such structures are involved in the pathological process. We have demonstrated that meniscal cells derived from the lateral meniscus in medial OA patients have chondrogenic capacity in vitro and hence could represent a potential cell source to consider for meniscus tissue engineering.

Changes in Composition of ADAMTS-5 and CD-68 in the Knee Joint Synovial Fluid Cells of Meniscal Tears Patients an Immunohistochemical Study / Cambios en la Composición de ADAMTS-5 y CD-68 en las Células del Líquido Sinovial de la Articulación de la Rodilla en Pacientes con Desgarro de Menisco. Un Estudio Inmunohistoquímico

SUMMARY: Meniscus tear is an important injury affecting the quality of life. This work is aimed to investigate the activity of CD68 and ADAMTS-5 in cells in synovial fluid in male and female patients with meniscal tear. In this study ,18 male and 22 female patients with meniscal tears were included. Local pain sensation during patients' physical examination, swelling, performing daily activities and difficulty in running-walking complaints were determined. 5 cc synovial fluids were aspirated from the lateral suprapatellar pouch part of the knees with meniscal pain. After routine histological follow-up of the samples, they were embedded in paraffin and sectioned with microtome and 5 micrometer thickness. CD68 and ADAMTS-5 primary antibodies were used for immunohistochemical analysis. Sections were taken and evaluated with a stylish microscope. The distribution of blood cells after meniscus tear was higher in female patients than in male patients. CD68 distribution in female patients appeared higher than in male patients. CD68 expression was high in macrophage cell cytoplasm. ADAMTS-5 expression was higher in female patients in degenerative cells and apoptotic cells. ADAMTS-5 is an important metallo-protein involved in the development of apoptotic signal and extracellular matrix synthesis in patients with ADAMTS-5 meniscus tear, and it may be an important criterion for the treatment developed after injury. CD68 and ADAMTS-5 activity was thought to be one of the important signal pathways that can be identified in the treatment of meniscus tear.

RESUMEN: La rotura del menisco es una lesión importante que afecta la calidad de vida. El objetivo fue investigar la actividad de CD68 y ADAMTS-5 en células del líquido sinovial en pacientes masculinos y femeninos con desgarro meniscal. Se incluyeron 18 pacientes masculinos y 22 femeninos con desgarros meniscales. Se determinó la sensación de dolor local durante el examen físico de los pacientes, la hinchazón, la realización de actividades diarias y la dificultad al correr y caminar. Se aspiraron 5 cc de líquido sinoviale de la parte de la bolsa suprapatelar lateral de las rodillas de los pacientes con dolor meniscal. Después del seguimiento histológico de rutina, las muestras se incluyeron en parafina y se seccionaron con un micrótomo de grosor de 5 micrómetros. Para el análisis inmunohistoquímico se usaron los anticuerpos primarios CD68 y ADAMTS-5. La distribución de las células sanguíneas después del desgarro del menisco fue mayor en pacientes femeninos que en pacientes masculinos. La distribución de CD68 en pacientes femeninos fue más alta que en pacientes masculinos. Además la expresión de CD68 fue alta en el citoplasma de los macrófagos. La expresión de ADAMTS-5 fue mayor en pacientes femeninos en las células degenerativas y células apoptóticas. ADAMTS-5 es una metaloproteína importante en el desarrollo de la señal apoptótica y la síntesis de matriz extracelular en pacientes con rotura de menisco ADAMTS-5, y puede ser un criterio importante para el tratamiento después de la lesión. La actividad de CD68 y ADAMTS-5 era una de las vías de señal importantes que se pueden identificar en el tratamiento de la rotura del menisco.

Acetaminophen, bupivacaine, Duramorph, and Toradol: A comparison of chondrocyte viability and gene expression changes in osteoarthritic human chondrocytes.

BACKGROUND: A multitude of chemical agents are currently used intra-articularly to decrease pain after orthopaedic procedures including total knee arthroplasty. However, the possible deleterious effects of these injectable chemicals on chondrocyte viability have not been weighed against their potential benefits. Using a human osteoarthritic chondrocyte model, the purpose of this study was to assess the potential for cartilage damage caused by bupivacaine, Toradol, Duramorph, and acetaminophen from surgical local anesthesia. METHODS: Human distal femur and proximal tibia cross sections were obtained during total knee arthroplasty and divided into control group and experimental groups treated by bupivacaine, Toradol, Duramorph, and acetaminophen respectively. Chondrocytes obtained from enzymatically digested cartilage were cultured using a 3D alginate bead culture method to ensure lower rates of dedifferentiation. Chondrocyte bead cultures were exposed to the study chemicals. The gene expression and chondrocyte viability were measured by RT-PCR and flow cytometry, respectively. RESULTS: Compared with untreated group bupivacaine treatment led to the greatest cellular apoptosis with 30.5 ± 11% dead cells (P = 0.000). Duramorph and acetaminophen did not result in a significant increase in cell death. Bupivacaine treatment led to an increase in Caspase 3 gene expression (P = 0.000) as well as the acetaminophen treatment (P = 0.001) when compared to control. CONCLUSION: Our data demonstrated that Duramorph and Toradol were not cytotoxic to human chondrocytes and may be better alternatives to the frequently used and more cytotoxic bupivacaine. Acetaminophen did not result in increased cell death; however, it did show increased caspase 3 gene expression and caution should be considered.

Compositional Variation and Functional Mechanism of Exosomes in the Articular Microenvironment in Knee Osteoarthritis.

Osteoarthritis (OA) is a major cause of disability worldwide with increasing age. Knee OA (KOA) is the most prevalent type of OA. Recently, it is considered that KOA is a whole joint disease, including articular cartilage, subchondral bone, synovium, ligaments, joint capsules, and muscles around the joint. Exosomes in knee joint are mainly secreted by articular chondrocytes and synoviocytes. They participate in cell and tissue cross-talk by carrying a complex cargo of proteins, lipids, nucleic acids, etc. Under normal conditions, exosomes maintain the microenvironmental homeostasis of the joint cavity. Under pathological conditions, the composition and function of exosomes changes, which in turn, disrupts the balance of anabolism and catabolism of articular chondrocyte and facilitates inflammatory responses, thus accelerating KOA progression. As a regenerative medicine, mesenchymal stem cells (MSCs) are promised to facilitate repair of degenerated cartilage and decelerate OA process. The therapeutic function of MSC mainly depends on MSC-derived exosomes, which can restore the homeostasis of the articular microenvironment. In the future, the specific mechanism of exosomes for OA treatment needs further elucidation, and the treatment effect of exosomes for long-term and/or severe OA needs further exploration.

Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage.

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.

Characterization of human articular chondrocytes and chondroprogenitors derived from non-diseased and osteoarthritic knee joints to assess superiority for cell-based therapy.

PURPOSE: Cell based therapy is constantly underway since regeneration of genuine hyaline cartilage is under par. Much attention has been afforded to chondroprogenitors recently, as an alternative cell substitute for cartilage repair. Although single source derivation of chondrocytes and chondroprogenitors is advantageous, lack of a characteristic differentiating marker obscures clear identification, which is essential to create a biological profile and is also required to assess cell type superiority for cartilage repair. METHODS: Cells obtained from three non-diseased/osteoarthritic human knee joints each, were expanded in culture up to passage 10. Characterization studies were performed using flow cytometry; gene expression was studied using RT-PCR; growth kinetics and tri-lineage differentiation was also studied to construct a better profile of chondroprogenitors as well as chondrocytes. RESULTS AND CONCLUSION: Our results showed that both cell populations exhibited similar cell surface characteristics except for non-diseased chondroprogenitors, which showed markedly low expression of CD34 and high expression of CD166. Trilineage data was suggestive of multilineage potential for both cell types with chondroprogenitors showing notably higher glycosaminoglycan and lower calcified matrix deposition. Data acquired from this study aided in describing cellular behavior of human articular cartilage derived chondroprogenitors in conditions not reported earlier. Our comparative analysis suggests that sorting based on a combination of markers (CD34- and CD166+) would yield a population of cells with minimal contamination by chondrocytes, which may provide translatable results in terms of enhanced chondrogenesis and reduced hypertrophy; both indispensable for the field of cartilage regeneration.

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.

A multiscale framework for evaluating three-dimensional cell mechanics in fibril-reinforced poroelastic tissues with anatomical cell distribution - Analysis of chondrocyte deformation behavior in mechanically loaded articular cartilage.

Characterization of the mechanical environment of cells in collagenous biological tissues during different daily activities is crucial for understanding the role of mechanics on cell biosynthesis and tissue health. However, current imaging methods are limited in characterizing very fast deformations of cells. This could be achieved with computational multiscale modeling, but current models accommodating collagen fibril networks and poroelastic ground matrix have included only a single cell. In this study, a workflow was developed for generating a three-dimensional multiscale model with imaging-based anatomical cell distributions and their micro-environment (pericellular and extracellular matrix). Fibril-reinforced poroelastic material models with (FRPES) and without (FRPE) swelling were implemented into the model and simulations were performed for evaluating cell deformations before and after experimental loading conducted for rabbit knee joint cartilage. We observed that the cells experienced considerably different deformation based on their location in all models. Both FRPE and FRPES models were able to predict the trends in the changes in cell deformations, although the average and median magnitudes differed between the model predictions and experiments. However, the FRPES model results were generally closer to the experimental results. Current findings suggest that morphological properties of cells are affected by the variations in the tissue properties between the samples and variations within the sample caused by the measurement geometry, local structure and composition. Thus, it would be important to consider the anatomical distribution and location of multiple cells along with the structure of fibril networks if cell deformation metrics are evaluated in collagenous tissues.

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.

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.

Timing of Intra-Articular Injection of Synovial Mesenchymal Stem Cells Affects Cartilage Restoration in a Partial Thickness Cartilage Defect Model in Rats.

OBJECTIVE: We investigated the effect of administration of intra-articular mesenchymal stem cells (MSCs) on cartilage repair at different timings, and the distribution of MSCs in the knee. DESIGN: A partial thickness cartilage defect (PTCD) was created on the medial femoral condyle in 14-week-old Sprague-Dawley rats. Intra-articular injection of 1 × 106 MSCs was performed at 3 time points, namely at the time of surgery (0w group), at 1 week after surgery (1w group), and at 2 weeks after surgery (2w group). For the control, 50 µL phosphate-buffered saline was injected at the time of surgery. The femoral condyles were collected at 6 weeks after creation of PTCD and assessed histologically. To investigate the distribution of MSCs, fluorescent-labeled MSCs were injected into the knee joint. RESULTS: In the control group, the cartilage lesion was distinguishable from surrounding cartilage. In the 0w group, hypocellularity and a slight decrease in safranin O stainability were observed around the injured area, but cartilage was restored to a nearly normal condition. In contrast, in the 1w and 2w groups, the cartilage surface was irregular and safranin O stainability in the injured and surrounding areas was poor. Histological score in the 0w group was significantly better than in the control, 1w, and 2w groups. At 1 day postinjection, fluorescent-labeled MSCs were mostly distributed in synovium. However, no migration into the PTCD was observed. CONCLUSIONS: Early intra-articular injection of MSCs was effective in enhancing cartilage healing in a rat PTCD model. Injected MSCs were distributed in synovium, not in cartilage surrounding the PTCD.

Comparison of Electrophysiological Properties and Gene Expression between Human Chondrocytes and Chondroprogenitors Derived from Normal and Osteoarthritic Cartilage.

OBJECTIVES: Bone-marrow mesenchymal stem cells (MSCs) and chondrocytes are currently used for cell-based therapy in cartilage repair. Chondroprogenitors (CPs), resident cells of articular cartilage, demonstrate likeness to stem cells. Reports suggest that chondrocytes phenotype is altered in culture, thus making differentiation between the two cell populations difficult. Our objectives were to electrophysiologically assess chondrocytes and CPs, compare their mRNA expression with that of ionic channels already reported in MSCs, and to observe the effect of time in culture and osteoarthritic damage on cells. DESIGN AND RESULTS: Chondrocytes and CPs at passages 0 (p0) and 5 (p5) derived from normal and osteoarthritic (OA) knee joints were used. Ionic currents were recorded by subjecting cells to depolarizing voltage pulses, and reverse transcriptase-polymerase chain reaction (RT-PCR) was used for studying ion channel expression. Our results demonstrated that both chondrocytes and CPs showed the presence of similar currents belonging to voltage-gated potassium channel subfamily, with RT-PCR confirming high mRNA expression of Maxi K, HKv1.1, HKv1.4, HKv4.2, and hEAG1 channels. Our finding also suggested that CPs were comparatively more sensitive to increased time in culture and inflammatory processes as observed in OA, as was evidenced by the significant decrease in mean current density (p0 normal CP: 183.171 ± 50.80 pA/pF; p5 normal CP: 50.225 ± 17.63 pA/pF; P = 0.0280) and significant increase in cellular size (p0 normal CP: 21.564 ± 2.98 pF; p0 OA CP: 37.939 ± 3.55 pF; P = 0.0057). CONCLUSION: Both cell types appear to be optimal candidates for cell-based therapy although initial seeding density, cell source (normal vs. OA), and time in culture are matters of concern, prior to cell-type selection.

Simvastatin induces differentiation in rabbit articular chondrocytes via Wnt/ß-catenin pathway.

Simvastatin is widely used as a specific inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase to reduce the levels of low-density lipoprotein cholesterol. However, its regulatory mechanism in chondrocyte differentiation is unclear. This study was conducted to evaluate the effects and signalling pathway of simvastatin on chondrocyte differentiation. We found that simvastatin induced chondrocyte differentiation, as confirmed by increased type II collagen expression and induced sulphated proteoglycan synthesis. Western blotting results showed that expression of type II collagen increased 6-fold in a dose-dependent manner compared with that in the control. Further, nuclear/cytosol fraction analysis revealed that simvastatin reduced the expression and translocation of ß-catenin into the nucleus from the cytoplasm by approximately 50% compared with that in the control. A luciferase assay using a T cell factor/lymphoid enhancer factor reporter construct was performed to test the transcriptional activity of ß-catenin. Simvastatin-induced differentiation was dependent on inactivation of ß-catenin, as simvastatin inhibited accumulation of ß-catenin, which was characterized by translocation of ß-catenin to the nucleus as shown by immunofluorescence staining and the luciferase assay. Prevention of ß-catenin degradation by inhibition of the proteasome with z-Leu-Leu-Leu-CHO blocked the increase in type II collagen expression. Simvastatin treatment reduced chondrocyte dedifferentiation induced by retinoic acid or serial monolayer culturing by 50% compared to that in the non-treated cells. Our findings demonstrate that simvastatin increases differentiation of rabbit articular chondrocytes via the ß-catenin pathway.

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.

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.

Mechanism of miR-15a regulating the growth and apoptosis of human knee joint chondrocytes by targeting SMAD2.

Objective: To investigate the effects of miR-15a on proliferation and apoptosis of human knee articular chondrocytes and explore its underlying mechanism. Methods: qRT-PCR was used to detect the expression of miR-15a in normal chondrocytes and knee arthritic chondrocytes; miR-con (transfected miR-con), miR-15a (transfected miR-15a mimics), anti-miR-con group (transfected anti-miR-con), anti-miR-15a group (transfected anti-miR-15a mimics), pcDNA group (transfected pcDNA), pcDNA-SMAD2 group (transfected pcDNA-SMAD2), the miR-15a + pcDNA group (co-transfected miR-15a and pcDNA), miR-15a + pcDNA-SMAD2 group (co-transfected miR-15a mimics and pcDNA-SMAD2), were transfected into knee articular chondrocytes by liposome method, respectively. The cell proliferation and apoptosis of each group were detected by MTT assay and flow cytometry. The protein expression of SMAD2 was detected by Western blot. The fluorescence activity of each group was detected by dual luciferase reporter gene assay. Results: The expression of miR-15a in knee arthritis chondrocytes was significantly increased (p < .05) compared with that in normal chondrocytes. Moreover, overexpression of miR-15a and silencing of SMAD2 inhibited proliferation and promoted apoptosis in knee arthritis chondrocyte. MiR-15a targeted SMAD2. Overexpression of SMAD2 reversed the inhibitory effects on proliferation and promotion effects on apoptosis induced by miR-15a in knee arthritis chondrocytes. Conclusion: miR-15a can inhibit the proliferation and promote apoptosis of knee arthritis chondrocytes. The mechanism may be related to SMAD2, which will provide a new target for the treatment of knee arthritis.