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

Reconstruction versus conservative treatment after rupture of the anterior cruciate ligament: cost effectiveness analysis

BACKGROUND: The decision whether to treat conservatively or reconstruct surgically a torn anterior cruciate ligament (ACL) is an ongoing subject of debate. The high prevalence and associated public health burden of torn ACL has led to continuous efforts to determine the best therapeutic approach. A critical evaluation of benefits and expenditures of both treatment options as in a cost effectiveness analysis seems well-suited to provide valuable information for treating physicians and healthcare policymakers. METHODS: A literature review identified four of 7410 searched articles providing sufficient outcome probabilities for the two treatment options for modeling. A transformation key based on the expert opinions of 25 orthopedic surgeons was used to derive utilities from available evidence. The cost data for both treatment strategies were based on average figures compiled by Orthopaedic University Hospital Balgrist and reinforced by Swiss national statistics. A decision tree was constructed to derive the cost-effectiveness of each strategy, which was then tested for robustness using Monte Carlo simulation. RESULTS: Decision tree analysis revealed a cost effectiveness of 16,038 USD/0.78 QALY for ACL reconstruction and 15,466 USD/0.66 QALY for conservative treatment, implying an incremental cost effectiveness of 4,890 USD/QALY for ACL reconstruction. Sensitivity analysis of utilities did not change the trend. CONCLUSION: ACL reconstruction for reestablishment of knee stability seems cost effective in the Swiss setting based on currently available evidence. This, however, should be reinforced with randomized controlled trials comparing the two treatment strategies

Role of biomechanics in the understanding of normal, injured, and healing ligaments and tendons

Ligaments and tendons are soft connective tissues which serve essential roles for biomechanical function of the musculoskeletal system by stabilizing and guiding the motion of diarthrodial joints. Nevertheless, these tissues are frequently injured due to repetition and overuse as well as quick cutting motions that involve acceleration and deceleration. These injuries often upset this balance between mobility and stability of the joint which causes damage to other soft tissues manifested as pain and other morbidity, such as osteoarthritis

Localized articular cartilage defects: a review on current modes of treatment and how nanotechnology can play a role

Current operative and non-operative treatments for articular cartilage (AC) defect repair still fail to meet clinical expectations. These treatment options and challenges will be reviewed from a clinical perspective. Various polymeric and naturally occurring materials serving as scaffolds have shown promising neocartilage formation, but few studies are able to draw good clinical correlations. While tissue and organ engineering have generated public demand and expectations that engineered tissues will soon be available, there are still several critical hurdles that need to be overcome. There is a general preference for (1) avoiding the harvesting of normal tissues, (2) a single minimally invasive operative procedure for material insertion, and (3) a durable material that reproduces normal hyaline cartilage and will provide a good lifetime warranty. To avoid harvesting normal tissues, alternative cell sourcing is considered. On the materials front, there is a demand for molecular diversity and synthetic flexibility. For minimally invasive surgery, injectable materials have been actively researched. While initial studies are promising, there still remain a few challenges to overcome before injectable scaffolds will become clinically relevant. Key considerations are reviewed in this article. Advances in nanotechnology have enabled us to employ bottom-up approaches to scaffold design, fabrication, and characterization to better mimic the biological dimensions of matter. One approach involves self-assembly of small DNA-like molecules into larger superaggregates with nanoscale dimensions. One such self-assembling organic system is the rosette nanotubes. The design and properties are highlighted as they are related to solving orthopedic problems.NRC publication: Ye

Correction: Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

[This corrects the article DOI: 10.1371/journal.pone.0248292.]

Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

Low oxygen and mechanical loading may play roles in regulating the fibrocartilaginous phenotype of the human inner meniscus, but their combination in engineered tissues remains unstudied. Here, we investigated how continuous low oxygen ("hypoxia") combined with dynamic compression would affect the fibrocartilaginous "inner meniscus-like" matrix-forming phenotype of human meniscus fibrochondrocytes (MFCs) in a porous type I collagen scaffold. Freshly-seeded MFC scaffolds were cultured for 4 weeks in either 3 or 20% O2 or pre-cultured for 2 weeks in 3% O2 and then dynamically compressed for 2 weeks (10% strain, 1 Hz, 1 h/day, 5 days/week), all with or without TGF-β3 supplementation. TGF-β3 supplementation was found necessary to induce matrix formation by MFCs in the collagen scaffold regardless of oxygen tension and application of the dynamic compression loading regime. Neither hypoxia under static culture nor hypoxia combined with dynamic compression had significant effects on expression of specific protein and mRNA markers for the fibrocartilaginous matrix-forming phenotype. Mechanical properties significantly increased over the two-week loading period but were not different between static and dynamic-loaded tissues after the loading period. These findings indicate that 3% O2 applied immediately after scaffold seeding and dynamic compression to 10% strain do not affect the fibrocartilaginous matrix-forming phenotype of human MFCs in this type I collagen scaffold. It is possible that a delayed hypoxia treatment and an optimized pre-culture period and loading regime combination would have led to different outcomes

Meniscal and articular cartilage lesions in the anterior cruciate ligament-deficient knee: correlation between time from injury and knee scores

Anterior cruciate ligament (ACL) rupture is associated with meniscal tears and/or articular cartilage damage. The aim of this study was twofold: (a) to report and correlate the incidence of meniscal and cartilage lesions in ACL-deficient knees with time from injury and (b) to correlate lesions of menisci and cartilage with widely used knee scores. Data were analysed from 109 consecutive patients with ACL rupture. Meniscal and articular cartilage lesions were documented during the arthroscopic reconstruction of the ACL. Patients were distributed into 3 groups according to time from injury; group A: 0-3 months (35 patients), group B: 3-12 months (39 patients) and group C: more than 12 months (35 patients). Lysholm, KOOS and IKDC rating scales were recorded preoperatively. Logistic regression analyses were applied to correlate the concomitant intra-articular pathologies with the time from injury and knee-rating scales. Of 109 patients, 32 (29 %) had a medial meniscus tear, 20 (19 %) had a lateral meniscus tear, 17 (15 %) had both menisci torn and 40 (37 %) had no meniscal tear. Analysis revealed that time from injury was not a significant factor for the presence of a meniscal lesion. The odds of development of a high-grade cartilage lesion in an ACL-deficient knee reconstructed more than 12 months from time from injury are 5.5 and 12.5 times higher when compared with knees that underwent ACL reconstruction less than 3 months and between 3 and 12 months after knee injury, respectively. No association was found between intra-articular pathology and the KOOS and Lysholm scores. A positive correlation between the IKDC score and patients without any intra-articular pathology was found. The presence of high-grade cartilage lesions is significantly increased in an ACL-deficient knee when reconstruction is performed more than 12 months after injury. However, the incidence of meniscal tears is not increased significantly. Correlation of intra-articular pathology in ACL-deficient knees with knee-rating scales is weak. Diagnostic study, Level II