The role of the TRAP enzyme in endochondral bone formation

Most parts of our skeleton develop from primordial cartilaginous anlagen whereby the long bones (e.g. femur, tibia) are formed via perichondral and endochondral ossification. The perichondral (cortical) bone develops within a soft connective tissue (perichondrium) at the outer surface of the cartilaginous model. The endochondral bone is formed within the anlagen and leads to the establishment of a diaphyseal primary (POC) as well as an epiphyseal secondary ossification centre (SOC). The result is the establishment of the growth plate, a structure critical for longitudinal bone growth. A first and an essential event for the formation of the SOC is the early generation of vascularized cartilage canals (lvarez et al. 2005A; Holmbeck and Szabova 2006; Blumer et al. 2007). This process requires the proteolytic cleavage of the cartilaginous matrix which in turn will allow the canals to grow into the epiphysis (Holmbeck et al. 1999, 2003; Zhou et al. 2000; Lee et al. 2001; Davoli et al. 2001; lvarez et al. 2005A; Holmbeck and Szabova 2006). To define the essential role of the proteolytic enzymes several knockout mice have been generated and investigated. In membrane-bound type-1 matrix metalloproteinase (MT1-MMP = MMP 14) deficient mice, for instance, the invasion of cartilage canals fails to occur. Consequently, these mice do not develop a SOC; they show distinct growth plate abnormalities and have a reduced longitudinal growth of their long bones (Holmbeck et al. 1999; Zhou et al. 2000). Similarly, in tartrate-resistant acid phosphatase (TRAP) knockout mice the long bones are shorter than normal, most likely affected by an aberrant ossification of the epiphysis leading to an altered structure of the growth plate (Hayman et al. 1996, 2000; Hollberg et al. 2002; Hayman and Cox 2003; Roberts et al. 2007). In TRAP -/- mice the tibial epiphysis ossifies (Hollberg et al. 2002), but it was not examined whether this process takes place via the formation of cartilage canals or an alternative mechanism. To understand the individual role of the TRAP enzyme and its interactions with other proteolytic enzymes and bone relevant molecules we now intend to examine TRAP -/- mice. Regarding the bone formation within the epiphysis we consider that two scenarios are possible: (1) Cartilage canals are present within the TRAP -/- mice: if so, we think it, however, very probable that the processes triggering the formation of the canals as well as the SOC are significantly altered. Therefore, we first intend to examine the machinery of the cells and proteins responsible for the formation of cartilage canals and their advancement. Second, we will examine the timing and the location of bone relevant proteins during the development of the epiphysis. (2) Cartilage canals are not present within the TRAP -/- mice: if so, the ossification of the epiphysis proceeds via an alternative mechanism which should be investigated in detail. Based on the above mentioned considerations, we will examine an age series of postnatal mice, and we will focus on the femur. We will compare the results obtained from TRAP -/- mice with those from wild type and heterozygote TRAP +/- mice. We will cooperate with two well-established international research groups, one of them providing the knockout mice. For the research project, duration of three years is planned and we would ask for a financial support of 266.903,7.

The human skeleton is composed of bones, the majority of which develop indirectly via a cartilaginous precursor. Only a few bones (e.g.: skull, clavicle) develop directly by membranous ossification. According to their external shape, bones can be classified as long, short, flat and irregular bones. In long bones (e.g.: femur, tibia), the perichondral (cortical) bone develops first on the outer surface of the cartilaginous anlagen. Once the osseous collar has evolved, the endochondral bone begins to form within the model and leads to the formation of a diaphyseal primary ossification centre followed by the establishment of the epiphyseal secondary ossification centre. The development of the primary and secondary sites of ossification leads to the establishment of the growth plate, a specialized structure essential for longitudinal bone growth. During long bone development the original cartilaginous model is continuously replaced by bone but at the long bone endings the articular cartilage remains. Angiogenesis is a key feature in endochondral ossification, and with the vessels bone forming cells migrate into the cartilaginous model. Their ingrowth and advancement requires a machinery of proteolytic enzymes that disintegrate the components of the cartilaginous matrix. Several studies in mice have indicated that deletion of distinct enzymes clearly impairs the ingrowth of the epiphyseal blood vessels. These mice show delayed formations of the secondary ossification centre, leading to an altered histology of the growth plate. Consequently, its function is affected and the longitudinal growth of the bone is reduced. Mice lacking tartrate resistant acid phosphatase (TRAP) develop normally, but adults reveal a malformation of the skeleton. Specifically, they display a mild osteopetrotic phenotype with increased bone tissue and mineral density. The bones are brittle and shortened due to a malfunction of the growth plate. It has been speculated that in TRAP knockout mice both the invasion of the epiphyseal blood vessels and the formation of the secondary ossification centre are delayed. Our data provide evidence that in TRAP knockout mice the mineralization of the epiphyseal cartilage, formation of the marrow cavity, and localization of bone relevant proteins occur prematurely. On examination of a longer postnatal period, however, it becomes evident that TRAP deficiency does not impair the vascularization and development of the secondary ossification centre, despite these early events. In knockout mice the histology of the growth plate is expanded throughout development. The osteopetrotic phenotype is only detectable in the metaphysis. It is, however, remarkable that these histological alterations are not seen in young animals but become manifest 3 weeks after birth. We conclude that TRAP is not essential for the epiphyseal vascularization and establishment of the SOC in long bones.