22 23 24 25 26 2 27Abstract 28 Joint morphogenesis is the process during which distinct and functional joint shapes emerge during pre-and 29 post-natal joint development. In this study, a repeatable semi-automatic protocol capable of providing a 3D 30 realistic developmental map of the prenatal mouse knee joint was designed by combining Optical Projection 31 Tomography imaging (OPT) and a deformable registration algorithm (Sheffield Image Registration toolkit, 32 ShIRT). Eleven left limbs of healthy murine embryos were scanned with OPT (voxel size: 14.63µm) at two 33 different stages of development: Theiler stage (TS) 23 (approximately 14.5 embryonic days) and 24 34 (approximately 15.5 embryonic days). One TS23 limb was used to evaluate the precision of the displacement 35 predictions for this specific case. The remaining limbs were then used to estimate Developmental Tibia and 36 Femur Maps. Acceptable uncertainties of the displacement predictions were found for both epiphyses 37 (between 0.7 and 1.4 µm, along all directions and anatomical sites) for nodal spacing of 1 voxel. The 38 protocol was found to be reproducible with maximum Modified Housdorff Distance differences equal to 1.9 39 µm and 1.5 µm for the tibial and femoral epiphyses respectively. The effect of the initial shape of the 40 rudiment affected the developmental maps by 21.7 µm and 21.9 µm for the tibial and femoral epiphyses 41 respectively, which correspond to 1.4 and 1.5 times the voxel size. To conclude, this study proposes a 42 repeatable semi-automatic protocol capable of providing mean 3D realistic developmental map of a 43 developing rudiment allowing researchers to study how growth and adaptation are directed by biological and 44 mechanobiological factors.3 46 Introduction 47 Growth and morphogenesis are two fundamental processes which every living system undergo during both 48 the prenatal and juvenile phase. If growth is more related to an increase in size and mass of an organism over 49 a period of time, morphogenesis is the biological process responsible for any organism to develop its shape. 50 Joint morphogenesis is the key process through which the two opposing cartilaginous rudiments of a joint 51 develop their reciprocal and fully functional shapes, and which starts during prenatal joint development. This 52 process is described in details by Pacifici et al., [1] and subsequently updated by Nowlan and Sharpe [2]. The 53 consequences of incomplete or abnormal joint morphogenesis can be very debilitating and may lead to 54 musculoskeletal diseases such as osteoarthritis (OA) [3, 4]. The formation of a skeletal joint is a highly 55 regulated process which is controlled by several biochemical factors (e.g. growth factors, Hox genes) [5].56 Moreover, several studies have experimentally found a relationship between prenatal joint motion and 57 physiological joint morphogenesis in mice [6, 7] and chicks [8][9][10][11], demonstrating the importance of 58 mechanical loads in the morphogenic process. For example, 2D histological assessm...