Muscle induced mechanical forces have been implicated as an important regulator in the development of the skeleton. Immobilisation studies have demonstrated that in the absence of muscle contraction, skeletal elements are shorter, cartilage growth is reduced and cavity formation fails (Nowlan et ah, 2008b;​ Osborne et ah, 2002;​ Drachman and Sokoloff, 1966). The avian knee joint is a complex 3D structure and while we know much about the steps involved in the formation of joints in general and some of the molecules that regulate this process, it is not yet clear how this complex structure develops its characteristic shape and structure. The objective of this thesis was to investigate how morphogenesis is mechanically regulated during the emergence of joint shape and structure, particularly how cellular processes such as proliferation and molecular events such as the expression of regulatory genes in the joint can be modulated by local mechanical signals.

The development of the avian knee joint and its associated structures, including the joint capsule, muscles and tendons of the hind limb were captured using the 3D imaging technique OPT. Musculoskeletal development between stages HH28 and HH34 was captured using a combination of wholemount histological staining for cartilage using alcian blue, double immunolabelling of the muscles and tendons using antibodies against myosin and tenascin and in situ hybridisation for both Col XI, a marker of the joint capsule and Scleraxis another tendon marker. This data was used to create a detailed 3D dataset for the study of the tissues of the chick knee joint. This 3D dataset was used to construct a FE model of the developing knee joint at HH30, HH32 and HH34. This anatomically accurate model was used to simulate the patterns of biophysical stimuli experienced by the tissues of the embryonic joint during development. These realistic stimuli patterns were compared with patterns of cartilage growth and rates of proliferating cells in the distal femur;​ a correlation between the magnitude and location of these biophysical stimuli and the relative rates of cell proliferation and cartilage growth was found indicating that cartilage growth and chondrocyte proliferation in the epiphysis is modulated by local patterns of biophysical stimuli.

In ovo immobilisation was found to significantly affect the development of shape in the knee joint. Predicted patterns of biophysical stimuli indicated that the stimuli experienced by the immobilised animals were dramatically different from that experienced by control embryos. Cell proliferation was investigated in the distal femur of immobilised and control joints and it was determined that immobilisation reduced the proportion of proliferating cells in a location specific manner. This supported the hypothesis that regional patterns of biophysical stimuli regulate local patterns of cartilage growth and chondrocyte cell proliferation, ultimately modulating the process of joint morphogenesis.

The expression of key regulatory genes PTHrP, BMP 2, FGF 2, FGFr 2, CD44, HAS 2 and extracellular matrix markers collagen II and tenascin C were investigated by in situ hybridisation in normal and DMB immobilised knee joints. These molecules are known to play vital roles in regulating cartilage growth, cell proliferation and the emergence of the joint cavity (Edwards et al., 1994;​ Dowthwaite et ah, 2003;​ Kavanagh et ah, 2006;​ Minina et ah, 2001;​ 2002). The loss of BMP2, HAS 2 and CD44 expression in specific emerging tissues of the joints of immobilised animals was also associated with ectopic expression of PTRrP and the cartilage marker Gol Ha, normally excluded from the joint. Therefore immobilisation not only affected cell proliferation in the rudiment it also appeared to impact the process of cell differentiation in the interzone, as indicated by changes in characteristic gene expression patterns and the ectopic expression of the chondrocyte marker Gol H. This thesis demonstrates the integral role that muscle induced mechanical forces play in the development of both tissue shape and structure in the embryonic joint.