Abstract
The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.
Highlights
The skeleton is a highly intricate, dynamic and complex organ that has a range of functions spanning from locomotion, to ion homeostasis
Assessment of the growth and mineralization potential of individually isolated and cultured embryonic day 15 (E15) 2nd, 3rd and 4th metatarsals revealed no significant differences in the appearance or extent of mineralization after 7 days of culture (Figure 2)
Studies have validated that E15 mouse metatarsals undergo a normal pattern of skeletal growth and differentiation
Summary
The skeleton is a highly intricate, dynamic and complex organ that has a range of functions spanning from locomotion, to ion homeostasis. Hypertrophic chondrocytes mineralize their surrounding ECM through the release of matrix vesicles, membrane bounded structures specialized for the production of amorphous calcium phosphate through their inclusion of phosphatases (tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1) and calcium channeling proteins (annexins) 9-11 This calcified cartilage is invaded by blood vessels from the underlying marrow resulting in osteoclast recruitment and matrix resorption. The metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cellmatrix interactions, providing conditions closer to the in vivo situation than cells in culture [19,20,21] This model allows for direct examination of linear bone growth which is not possible in primary chondrocyte cultures. Embryonic metatarsals from different mouse strains may be cultured in vitro, their growth and mineralization rates may differ to those detailed
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