Histological analysis of second-look biopsy tissue from patients treated with BioSeed-C

<p><b>Copyright information:</b></p><p>Taken from "Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results"</p><p>http://arthritis-research.com/content/9/2/R41</p><p>Arthritis Research & Therapy 2007;9(2):R41-R41.</p><p>Published online 23 Apr 2007</p><p>PMCID:PMC1906819.</p><p></p> At 9 to 12 months after implantation, second-look biopsy tissue was stained for proteoglycans with alcian blue. One patient's biopsy tissue showed the formation of mixed repair tissue with areas of fibrocartilage ((c), black triangle) and hyaline-like cartilage ((c), white triangle) and a firm bonding to the subchondral bone that was undergoing remodeling ((c), asterisk). Biopsy tissue from three patients shows the formation of a hyaline-like cartilaginous repair tissue with intense staining of proteoglycans by alcian blue (e-h), good integration with the underlying bone tissue (f), viable, round cells within lacunae (g) and a smooth surface (h). Chondrocytes showed a columnar distribution and some clustering (g-j). Hematoxylin/eosin staining (i, j) of biopsy tissue of two patients confirmed the presence of viable chondrocytes and the absence of abnormal calcification, apoptosis, necrosis or formation of a fibrous repair tissue

Clinical outcome after 2 years evaluated by the Modified Cincinnati Knee Rating System

<p><b>Copyright information:</b></p><p>Taken from "Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results"</p><p>http://arthritis-research.com/content/9/2/R41</p><p>Arthritis Research & Therapy 2007;9(2):R41-R41.</p><p>Published online 23 Apr 2007</p><p>PMCID:PMC1906819.</p><p></p> The score from this system is shown for the entire patient cohort compared with patients with posttraumatic and mild degenerative defects (Jaeger-Wirth score < 3) and patients with osteoarthritic defects (Jaeger-Wirth score = 3). The preoperative and follow-up times are as indicated. Scores are presented as medians; the ends of the boxes define the 25th and 75th centiles, and error bars the 10th and 90th centiles. Where indicated (asterisks), differences were statistically significant (< 0.05) compared with the preoperative situation

Arthroscopic and magnetic resonance imaging evaluation of cartilage defects treated with autologous chondrocyte grafts (BioSeed-C)

<p><b>Copyright information:</b></p><p>Taken from "Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results"</p><p>http://arthritis-research.com/content/9/2/R41</p><p>Arthritis Research & Therapy 2007;9(2):R41-R41.</p><p>Published online 23 Apr 2007</p><p>PMCID:PMC1906819.</p><p></p> Intra-operative situation of a cartilage defect situated at the femoral condyle covered with transosseously fixed BioSeed-C (20 mm × 30 mm). Note that the healthy cartilage rim is partly intact. At 9 months after surgery, second-look arthroscopy showed the formation of a cartilage repair tissue of a tough condition (asterisk). Magnetic resonance imaging (MRI) at 6 months and 12 months after implantation of BioSeed-C shows transosseous drilling holes (white asterisks) due to fixation of the graft. The repair tissue covers the defect (white triangles) and gives a slightly altered MRI signal

Validation of gene expression of stimulated chondrocytes by real-time RT-PCR

Semi-quantitative real-time RT-PCR of selected genes that were differentially expressed in chondrocytes stimulated with supernatant of a synovial fibroblast cell line derived from a patient with rheumatoid arthritis (RASFsn) as determined by microarray analysis was performed. Real-time RT-PCR gene expression analysis determined that the expression of (), () and () was significantly induced during stimulation of cartilage-like cultures with RASFsn compared with stimulation with supernatant of a synovial fibroblast cell line derived from normal donor (NDSFsn). The gene expression of (), (), () and () was significantly repressed during stimulation with RASFsn. Consistent changes were observed between real-time RT-PCR and microarray analysis for all genes examined. The expression of selected genes was calculated as the percentage of () expression. The mean of each triplicate well of both experimental groups is plotted and the error bars represent SD. For statistical analysis, Students t-test was applied (*, ≤ 0.05; ***, ≤ 0.001).<p><b>Copyright information:</b></p><p>Taken from "Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an study"</p><p>http://arthritis-research.com/content/10/1/R9</p><p>Arthritis Research & Therapy 2008;10(1):R9-R9.</p><p>Published online 18 Jan 2008</p><p>PMCID:PMC2374452.</p><p></p

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (<i>r</i> = 0.90), human cartilage (<i>r</i> = 0.71), and human micromass culture (<i>r</i> = 0.75) were observed including 34 cartilage markers such as <i>COL2A1</i>, <i>COMP</i>, and <i>aggrecan</i>. TNF-α stimulation led to significant proteoglycan (−75%) and collagen depletion (−50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (<i>MMP1</i>, <i>-2</i>, -<i>13</i>, <i>ADAM10</i>), chemokines (<i>IL8</i>, <i>CCL2</i>,<i> CXCL2</i>, <i>CXCL12</i>, <i>CCXL14</i>), and genes associated with cell death (<i>TNFSF10</i>, <i>PMAIPI</i>, <i>AHR</i>) and skeletal development (<i>GPNMB</i>, <i>FRZB</i>) including transcription factors (<i>WIF1</i>,<i> DLX5</i>, <i>TWIST1</i>) and growth factors (<i>IGFBP1</i>, <i>-3</i>, <i>TGFB1</i>) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (<i>COL2A1</i>, <i>COL9A1</i>, <i>COMP</i>, <i>aggrecan</i>) as well as hypertrophic bone formation (<i>COL1A1</i>, <i>COL10A1</i>) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA

Hierarchical clustering and functional classification of differentially expressed genes

Genome-wide expression analysis was performed for two different experimental groups (G) of chondrocytes stimulated with supernatant of a synovial fibroblast cell line derived from a patient with rheumatoid arthritis (RASFsn) and chondrocytes stimulated with supernatant of a synovial fibroblast cell line derived from normal donor (NDSFsn) (= 2). Each experimental group was a pool of RNA isolated from stimulated chondrocytes that originated from three different donors; that is, group 1 (G1) consisted of equal amounts of RNA from stimulated chondrocytes of donors 1 to 3 and group 2 (G2) of donors 4 to 6. Genes that displayed ≥2-fold increase or ≤-2-fold decrease in RASFsn-stimulated compared with NDSFsn-stimulated chondrocytes determined by both analyses with GeneChip Operating Software and Robust Multi-array Analysis were hierarchically clustered and functionally classified into six groups. Colors represent relative levels of gene expression: bright red indicates the highest level of expression and bright green indicates the lowest level of expression. Expression data from the different experimental groups were compared and showed that the expression patterns were similar for the corresponding experimental groups of both RASFsn-stimulated and NDSFsn-stimulated chondrocytes because they clustered and were therefore most similar to each other, showing little variability.<p><b>Copyright information:</b></p><p>Taken from "Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an study"</p><p>http://arthritis-research.com/content/10/1/R9</p><p>Arthritis Research & Therapy 2008;10(1):R9-R9.</p><p>Published online 18 Jan 2008</p><p>PMCID:PMC2374452.</p><p></p