Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological, Molecular and Computational Analysis of Immobilised Embryos

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG’s), Verhoeff-Van Gieson (elastic fibers), Masson’s trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated