Validation and reproducibility of computerised cell-viability analysis of tissue slices

BACKGROUND: The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. METHODS: To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes. RESULTS: Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77). CONCLUSIONS: This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages

Survival of Chondrocytes in Rabbit Septal Cartilage After Electromechanical Reshaping

Electromechanical reshaping (EMR) has been recently described as an alternative method for reshaping facial cartilage without the need for incisions or sutures. This study focuses on determining the short- and long-term viability of chondrocytes following EMR in cartilage grafts maintained in tissue culture. Flat rabbit nasal septal cartilage specimens were bent into semi-cylindrical shapes by an aluminum jig while a constant electric voltage was applied across the concave and convex surfaces. After EMR, specimens were maintained in culture media for 64 days. Over this time period, specimens were serially biopsied and then stained with a fluorescent live–dead assay system and imaged using laser scanning confocal microscopy. In addition, the fraction of viable chondrocytes was measured, correlated with voltage, voltage application time, electric field configuration, and examined serially. The fraction of viable chondrocytes decreased with voltage and application time. High local electric field intensity and proximity to the positive electrode also focally reduced chondrocyte viability. The density of viable chondrocytes decreased over time and reached a steady state after 2–4 weeks. Viable cells were concentrated within the central region of the specimen. Approximately 20% of original chondrocytes remained viable after reshaping with optimal voltage and application time parameters and compared favorably with conventional surgical shape change techniques such as morselization

Clinical application of scaffolds for cartilage tissue engineering

The purpose of this paper is to review the basic science and clinical literature on scaffolds clinically available for the treatment of articular cartilage injuries. The use of tissue-engineered grafts based on scaffolds seems to be as effective as conventional ACI clinically. However, there is limited evidence that scaffold techniques result in homogeneous distribution of cells. Similarly, few studies exist on the maintenance of the chondrocyte phenotype in scaffolds. Both of which would be potential advantages over the first generation ACI. The mean clinical score in all of the clinical literature on scaffold techniques significantly improved compared with preoperative values. More than 80% of patients had an excellent or good outcome. None of the short- or mid-term clinical and histological results of these tissue-engineering techniques with scaffolds were reported to be better than conventional ACI. However, some studies suggest that these methods may reduce surgical time, morbidity, and risks of periosteal hypertrophy and post-operative adhesions. Based on the available literature, we were not able to rank the scaffolds available for clinical use. Firm recommendations on which cartilage repair procedure is to be preferred is currently not known on the basis of these studies. Randomized clinical trials and longer follow-up periods are needed for more widespread information regarding the clinical effectiveness of scaffold-based, tissue-engineered cartilage repair

Tissue engineering of functional articular cartilage: the current status

Osteoarthritis is a degenerative joint disease characterized by pain and disability. It involves all ages and 70% of people aged >65 have some degree of osteoarthritis. Natural cartilage repair is limited because chondrocyte density and metabolism are low and cartilage has no blood supply. The results of joint-preserving treatment protocols such as debridement, mosaicplasty, perichondrium transplantation and autologous chondrocyte implantation vary largely and the average long-term result is unsatisfactory. One reason for limited clinical success is that most treatments require new cartilage to be formed at the site of a defect. However, the mechanical conditions at such sites are unfavorable for repair of the original damaged cartilage. Therefore, it is unlikely that healthy cartilage would form at these locations. The most promising method to circumvent this problem is to engineer mechanically stable cartilage ex vivo and to implant that into the damaged tissue area. This review outlines the issues related to the composition and functionality of tissue-engineered cartilage. In particular, the focus will be on the parameters cell source, signaling molecules, scaffolds and mechanical stimulation. In addition, the current status of tissue engineering of cartilage will be discussed, with the focus on extracellular matrix content, structure and its functionality

Monoclonal antibodies against human chondrocytes

Bujia J, Sittinger M, Sudhoff H, et al. Monoclonal antibodies against human chondrocytes. In Vitro Cellular & Developmental Biology - Animal. 1996;32(6):366-371