Compromised autophagy precedes meniscus degeneration and cartilage damage in mice.

OBJECTIVE: Autophagy is a cellular homeostasis mechanism that facilitates normal cell function and survival. Objectives of this study were to determine associations between autophagic responses with meniscus injury, joint aging, and osteoarthritis (OA), and to establish the temporal relationship with structural changes in menisci and cartilage. METHODS: Constitutive activation of autophagy during aging was measured in GFP-LC3 transgenic reporter mice between 6 and 30 months. Meniscus injury was created by surgically destabilizing the medial meniscus (DMM) to induce posttraumatic OA in C57BL/6J mice. Levels of autophagy proteins and activation were analyzed by confocal microscopy and immunohistochemistry. Associated histopathological changes, such as cellularity, matrix staining, and structural damage, were graded in the meniscus and compared to changes in articular cartilage. RESULTS: In C57BL/6J mice, basal autophagy was lower in the meniscus than in articular cartilage. With increasing age, expression of the autophagy proteins ATG5 and LC3 was significantly reduced by 24 months. Age-related changes included abnormal Safranin-O staining and reduced cellularity, which preceded structural damage in the meniscus and articular cartilage. In mice with DMM, autophagy was induced in the meniscus while it was suppressed in cartilage. Articular cartilage exhibited the most profound changes in autophagy and structure that preceded meniscus degeneration. Systemic administration of rapamycin to mice with DMM induced autophagy activation in cartilage and reduced degenerative changes in both meniscus and cartilage. CONCLUSION: Autophagy is significantly affected in the meniscus during aging and injury and precedes structural damage. Maintenance of autophagic activity appears critical for meniscus and cartilage integrity.

Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis.

OBJECTIVE: To establish a standardized protocol for histopathological assessment of murine menisci that can be applied to evaluate transgenic, knock-out/in, and surgically induced OA models. METHODS: Knee joints from C57BL/6J mice (6-36 months) as well as from mice with surgically-induced OA were processed and cut into sagittal sections. All sections included the anterior and posterior horns of the menisci and were graded for (1) surface integrity, (2) cellularity, (3) Safranin-O staining distribution and intensity. Articular cartilage in the knee joints was also scored. RESULTS: The new histopathological grading system showed good inter- and intra-class correlation coefficients. The major age-related changes in murine menisci in the absence of OA included decreased Safranin O staining intensity, abnormal cell distribution and the appearance of acellular areas. Menisci from mice with surgically-induced OA showed severe fibrillations, partial/total loss of tissue, and calcifications. Abnormal cell arrangements included both regional hypercellularity and hypocellularity along with hypertrophy and cell clusters. In general, the posterior horns were less affected by age and OA. CONCLUSION: A new standardized protocol and histopathological grading system has been developed and validated to allow for a comprehensive, systematic evaluation of changes in aging and OA-affected murine menisci. This system was developed to serve as a standardized technique and tool for further studies in murine meniscal pathophysiology models.

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development.

OBJECTIVE: To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation. DESIGN: Human knees (n=125 from 65 donors; age range 23-92) were obtained from tissue banks. All cartilage surfaces were macroscopically graded. Osteochondral slabs representing the entire central regions of both femoral condyles, tibial plateaus, and the patella were processed for histology and Safranin O - Fast Green staining. Slides representing normal, aged, and osteoarthritis (OA) tissue were scanned and electronic images were scored online by five observers. Statistical analysis was performed for inter- and intra-observer variability, reproducibility and reliability. RESULTS: The inter-observer variability among five observers for the MANKIN system showed a similar good Intra-class correlation coefficient (ICC>0.81) as for the OARSI system (ICC>0.78). Repeat scoring by three of the five readers showed very good agreement (ICC>0.94). Both systems showed a high reproducibility among four of the five readers as indicated by the Spearman's rho value. For the MANKIN system, the surface represented by lesion depth was the parameter where all readers showed an excellent agreement. Other parameters such as cellularity, Safranin O staining intensity and tidemark had greater inter-reader disagreement. CONCLUSION: Both scoring systems were reliable but appeared too complex and time consuming for assessment of lesion severity, the major parameter determined in standardized scoring systems. To rapidly and reproducibly assess severity of cartilage degradation, we propose to develop a simplified system for lesion volume.

Macroscopic and histopathologic analysis of human knee menisci in aging and osteoarthritis.

OBJECTIVE: Meniscus lesions following trauma or associated with osteoarthritis (OA) have been described, yet meniscus aging has not been systematically analyzed. The objectives of this study were to (1) establish standardized protocols for representative macroscopic and microscopic analysis, (2) improve existing scoring systems, and (3) apply these techniques to a large number of human menisci. DESIGN: Medial and lateral menisci from 107 human knees were obtained and cut in two different planes (triangle/cross section and transverse/horizontal section as well) in three separate locations (middle portion, anterior and posterior horns). All sections included vascular and avascular regions and were graded for (1) surface integrity, (2) cellularity, (3) matrix/fiber organization and collagen alignment, and (4) Safranin-O staining intensity. The cartilage in all knee compartments was also scored. RESULTS: The new macroscopic and microscopic grading systems showed high inter-reader and intra-reader intraclass correlation coefficients. The major age-related changes in menisci in joints with no or minimal OA included increased Safranin-O staining intensity, decreased cell density, the appearance of acellular zones, and evidence of mucoid degeneration with some loss of collagen fiber organization. The earliest meniscus changes occurred predominantly along the inner rim. Menisci from OA joints showed severe fibrocartilaginous separation of the matrix, extensive fraying, tears and calcification. Abnormal cell arrangements included decreased cellularity, diffuse hypercellularity along with cellular hypertrophy and abnormal cell clusters. In general, the anterior horns of both medial and lateral menisci were less affected by age and OA. CONCLUSIONS: New standardized protocols and new validated grading systems allowed us to conduct a more systematic evaluation of changes in aging and OA menisci at a macroscopic and microscopic level. Several meniscus abnormalities appear to be specific to aging in the absence of significant OA. With aging the meniscal surface can be intact but abnormal matrix organization and cellularity were observed within the meniscal substance. The increased Safranin-O staining appears to represent a shift from fibroblastic to chondrocytic phenotype during aging and early degeneration.

DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes.

OBJECTIVE: In this study, we were interested in the overall methylation level in aged and degenerated cartilage. Also, we looked at one gene which might be involved in the re-initiation of replicative activity in osteoarthritis (OA) chondrocytes, p21(WAF1/CIP1). p21(WAF1/CIP1) was previously suggested to be down-regulated in OA chondrocytes and is known to be regulated by epigenetic modulation. METHODS: Total methylation levels were analyzed by high pressure liquid chromatography (HPLC), mRNA expression of p21(WAF1/CIP1) and DNMT enzymes by real-time polymerase chain reaction. The methylation status of the p21(WAF1/CIP1)- promotor using bisulfite genomic sequencing was evaluated. RESULTS: General methylation analysis of genomic DNA showed no difference in between normal and aged/OA chondrocytes. Also no difference in methylation of the promotor of the p21(WAF1/CIP1) gene was detectable, which was significantly down-regulated in OA chondrocytes. DNMT1 and DNMT3a were expressed with no significant changes of expression levels found in OA chondrocytes. CONCLUSION: Cell cycle progression inhibitor p21(WAF1/CIP1) is expressed in normal and significantly down-regulated in OA articular chondrocytes, which may mediate the re-initiation of cell proliferation in OA cartilage. However, the suppression of p21(WAF1/CIP1) mRNA expression is not due to hypermethylation of its promotor. No overall changes in genome methylation levels were found in aged or OA cartilage. Interestingly, significant expression of DNA methyltransferases was found in articular chondrocytes, which supports that DNA methylation could still be a relevant mechanism of gene regulation in (osteoarthritic) chondrocytes, though not on an overall genomic level nor specifically for the regulation of the p21(WAF1/CIP1) gene.

A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro.

OBJECTIVE: To characterise in vitro engineered cartilaginous constructs made employing a novel static, scaffold-free and closed chamber system. DESIGN: Chondrocytes derived from slaughter age pigs (3-6 months) were seeded at high density (20 x 10(6)) into cylindrical chambers (1.0 x 0.5cm) and were maintained between an upper and a lower membrane (100 kDa) for 21 days and subsequently cultured in open culture for 7 additional days. RESULTS: Viable constructs produced were approximately 10 mmx2mm in size and were stable enough to be handled by surgical pincers. Histology and electron microscopy evaluations revealed a neo-cartilage structure of high cell density with a comprehensive extracellular matrix. Predominately collagen type II and negligible amounts of collagen types I and X were detected using RT-PCR and SDS-PAGE analyses. CONCLUSIONS: In this study, we provide evidence of a scaffold-free system that can produce immature hyaline-like cartilaginous constructs suitable for in vivo implantation, or that may be useful for in vitro studies of events related to the process of chondrogenesis.