A simplified index to quantify the irritation/corrosion potential of chemicals - Part II: Eye.

Eye irritation is a key human health endpoint assessed by in vitro and in vivo methods. One of the commonly used scoring methods to quantify the eye irritation potential of chemicals is the Modified Maximum Average Score (MMAS). It is dependent on the eye irritation effects (e.g. corneal opacity) originally proposed by Draize and then partially adopted by the OECD TG 405. These scores are not always fully reported in regulatory dossiers and lead to several drawbacks, 1) the difficulty to translate MMAS into a classification within the existing EU CLP/UN GHS criteria, 2) the absence of corrosion (serious eye damage), and 3) the dependency on input parameters which are usually not required under the OECD TGs (e.g. eye surface area). This study determined if classification can be driven by a maximum of two observed effects thereby simplifying the scoring calculation. The Simplified Irritation Index (SIIEYE), based only on corneal opacity and conjunctival redness, was developed using validated studies representing multiple chemical groups. A correlation was observed between the MMAS and the SIIEYE allowing harmonisation of the classification for the existing data. This index proved to be useful in the development of in silico model.

Assessment of method reliability and determination of reference intervals for rotational thromboelastometry in horses.

OBJECTIVES: To assess the measurement reliability of rotational thromboelastometry (ROTEM) measurements in horses, establish reference intervals for healthy horses, and evaluate the relationship between ROTEM variables, hematologic variables, and standard coagulation tests. DESIGN: Prospective observational study. SETTING: University teaching hospital. ANIMALS: Fifty healthy and 10 diseased adult horses. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was sampled from 10 healthy and 10 diseased horses and samples were repeatedly analyzed to evaluate measurement reliability of various ROTEM variables. Four different ROTEM assays (ie, EXTEM, INTEM, FIBTEM, and APTEM) were run simultaneously under standardized conditions. The device-related, operator-related, and day-to-day variability for the majority of ROTEM variables was very low to low, as indicated by a coefficient of variation (CV) of < 15%. Most of test-retest variability of ROTEM variables appeared to be device-related. Blood samples from 50 clinically healthy horses were used to establish reference intervals for ROTEM variables. Multiple stepwise regression analyses identified associations of different ROTEM variables with hematocrit, total protein concentration, fibrinogen concentration, platelet count, prothrombin time, activated partial thromboplastin time, and thrombin time. CONCLUSIONS: ROTEM is a feasible method to evaluate coagulation in horses. Its measurement reliability is acceptable, but device-related measurement variability has to be considered. Reference intervals are presented, but the influence of hematocrit, platelet count, and fibrinogen concentration may need to be taken into account when interpreting individual test results.

Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges.

Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.

A unique tool to selectively detect the chondrogenic IIB form of human type II procollagen protein.

Type II collagen, the major fibrillar collagen of cartilage, is synthesized as precursor forms (procollagens) containing N- and C-terminal propeptides. Three splice variants are thought to be translated to produce procollagen II isoforms (IIA/D and IIB) which differ in their amino propeptide parts. The IIA and IID are transient embryonic isoforms that include an additional cysteine-rich domain encoded by exon 2. The IIA and IID transcripts are co-expressed during chondrogenesis then decline and the IIB isoform is the only one expressed and synthesized in fully differentiated chondrocytes. Additionally, procollagens IIA/D can be re-expressed by dedifferentiating chondrocytes and in osteoarthritic cartilage. Therefore, it is an important point to determine which isoform(s) is (are) synthesized in vivo in normal and pathological situations and in vitro, to fully assess the phenotype of cells producing type II collagen protein. Antibodies directed against the cysteine-rich extra domain found in procollagens IIA and IID are already available but antibodies detecting only the chondrogenic IIB form of type II procollagen were missing so far. A synthetic peptide encompassing the junction between exon 1 and exon 3 of the human sequence was used as immunogen to produce rabbit polyclonal antibodies to procollagen IIB. After affinity purification on immobilized peptide their absence of crossreaction with procollagens IIA/D and with the fibrillar procollagens I, III and V was demonstrated by Western blotting. These antibodies were used to reveal at the protein level that the treatment of dedifferentiated human chondrocytes by bone morphogenic protein (BMP)-2 induces the synthesis of the IIB (chondrocytic) isoform of procollagen II. In addition, immunohistochemical staining of bovine cartilage demonstrates the potential of these antibodies in the analysis of the differential spatiotemporal distribution of N-propeptides of procollagens IIA/D and IIB during normal development and in pathological situations.

Cartilage-characteristic matrix reconstruction by sequential addition of soluble factors during expansion of human articular chondrocytes and their cultivation in collagen sponges.

OBJECTIVE: Articular cartilage has a poor capacity for spontaneous repair. Tissue engineering approaches using biomaterials and chondrocytes offer hope for treatments. Our goal was to test whether collagen sponges could be used as scaffolds for reconstruction of cartilage with human articular chondrocytes. We investigated the effects on the nature and abundance of cartilage matrix produced of sequential addition of chosen soluble factors during cell amplification on plastic and cultivation in collagen scaffolds. DESIGN: Isolated human articular chondrocytes were amplified for two passages with or without a cocktail of fibroblast growth factor (FGF)-2 and insulin (FI). The cells were then cultured in collagen sponges with or without a cocktail of bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine (BIT). The constructs were cultivated for 36 days in vitro or for another 6-week period in a nude mouse-based contained-defect organ culture model. Gene expression was analyzed using polymerase chain reaction, and protein production was analyzed using Western-blotting and immunohistochemistry. RESULTS: Dedifferentiation of chondrocytes occurred during cell expansion on plastic, and FI stimulated this dedifferentiation. We found that addition of BIT could trigger chondrocyte redifferentiation and cartilage-characteristic matrix production in the collagen sponges. The presence of FI during cell expansion increased the chondrocyte responsiveness to BIT.