Full length articleMineralization pathways in the active murine epiphyseal growth plate
Introduction
The epiphyseal growth plate in the long bones of young animals is the focus of intensive dynamic mineralization activity. Within a short time, in a limited region, cartilage is deposited, mineralized and resorbed, and bone takes its place. These processes are intimately associated with vascular activity. The primary cartilage mineralization, the secondary bone mineralization and the vasculature are interwoven in space and time to form extremely complex structures. These mineralization and vascularization processes have been the focus of extensive research for decades [[2], [3], [4], [5]].
Long bones elongate by the process of endochondral ossification [6]. Endochondral ossification occurs at the ends of the long bones in the growth plates, which are located between the two contiguous mineralized regions; the distal epiphysis and the more centrally located metaphysis. The distal side of the growth plate, the proliferative zone, comprises columns of differentiating chondrocytes. The chondrocytes produce an extracellular matrix composed mainly of fine fibrillar collagens of types II, XI and IX [7] and proteoglycans [8]. The cartilage matrix is disordered close to the chondrocytes, but the collagen fibrils are more aligned between the columns of chondrocytes [2]. In the final differentiation stage that occurs in the so-called hypertrophic zone, the chondrocytes increase in size and the matrix becomes mineralized.
The hypertrophic chondrocytes secrete angiogenic factors such as VEGF-A [9], which induce the invasion of blood vessels from the metaphysis [10,11]. The diaphysis and metaphysis display two different endothelial cell populations, with distinctive vessel morphologies and functions [12]. Type L is most abundant in the diaphysis, while type H primarily occupies the metaphysis and exhibits a distinctive, narrow columnar structure [12,13]. Type H blood vessels are characterized by endothelial bud structure protrusions at the extremity of the vessel. The budding ends enable them to invade the avascular cartilage by continuous new bud extensions into the growth plate [12]. The bud protrusions lack basement membrane [14], and are delimited by endothelial cells surrounded by an extracellular matrix of type I collagen [15]. Type H vessels thus play a key role in supporting osteogenesis [5].
Osteoblasts are responsible for the type I collagen matrix formation surrounding the vasculature buds [15]. Osteoblasts and osteoclasts are introduced into the growth plate area through the blood vessels, and mediate the transformation of mineralized cartilage into bone. Thus the vascular patterning plays a key role in defining the morphogenesis of the forming bone trabeculae [15]. Following Brighton [16] we refer to the region of the growth plate that comprises the hypertrophic chondrocytes, the invading blood vessels and the newly formed bones, as the provisional ossification zone. This zone is also known as the interface zone [17].
It is widely accepted that the ions necessary for mineral formation are supplied to the forming bone as ions in solution in the blood serum [18]. In the rapidly forming bones of the chick embryo, vesicles containing mineral particles were observed in the serum, showing that a second pathway for supplying mineral in a solid phase exists [19]. Membrane-bound disordered mineral-containing vesicles have also been observed in cells responsible for bone formation in rapidly forming zebrafish tail bones and in embryonic mice [20,21] and chick bones [19]. These mineral bodies are translocated from inside the cells to the extracellular matrix [22] where they either penetrate the type I collagen fibrils and form intrafibrillar crystals, or form crystals between fibrils or both [21]. These precursor mineral bodies are highly disordered at the atomic level and only after secretion into the extracellular matrix do they lose their membranes and crystallize. It has also been observed in the rapidly forming zebrafish tail bones, that there is a direct pathway from the vasculature to the forming bone between cells, raising the possibility that an additional or alternative intercellular pathway for supplying ions/mineral to forming bones exists [23]. We emphasize that all these observations pertain to rapidly forming bone processes, and the presumably slower mineralization of mature bone may involve different pathways.
Much less is known about the mineralization of cartilage in the growth plate. The source of the ions for cartilage matrix mineralization is unlikely to be exclusively from the hypertrophic chondrocytes as the calcium concentrations in these cells is low when mineral is forming in the surrounding matrix [24,25]. Hypertrophic cartilage cells have been reported to release vesicles [4], which then concentrate ions in the extracellular environment and induce the formation of crystalline carbonated hydroxyapatite [26]. These matrix vesicles are thought to be an integral part of skeletal mineralization, including growth plate cartilage mineralization [27]. Other studies, however, have searched for and not found matrix vesicles in mineralizing cartilage [3].
Following the pioneering studies in which the growth plate was studied on frozen samples [24,25,28], we also use imaging technology that enables the investigation of the growth plate structures under cryogenic or fully hydrated conditions in 3D and at high resolution. We use these imaging technologies to shed light on mineral traffic and deposition pathways in the growth plate tissue of 9 week old mice, and especially the structures of the provisional ossification zone under close to physiological conditions. We show that in these mice the growth plate is still actively depositing and resorbing mineralized tissues.
Section snippets
Animal model
Nine week old BALB/c WT female mice were obtained from Envigo RMS (Israel). Tibiae were dissected immediately after sacrifice that followed the guidelines of the Weizmann Institute Animal Care and Use Committee (IACUC). The fresh tibiae were processed immediately after dissection.
Cryo-SEM
Tibiae were fixed by incubating while rotating in 4% paraformaldehyde (PFA) and 2% glutaraldehyde in 0.1 M cacodylate buffer at room temperature for two hours, and then 48 h at 4 °C. The samples were then washed three
Results
We analyzed the proximal growth plates in the tibiae of 9 week old BALB/c female mice. Mice of this age are just after the period of massive growth [31]. In order to determine whether the growth plate is still active and elongating, we carried out a pulse-chase experiment using calcein. One calcein injection was administered, and the animals were sacrificed after 24, 48 or 96 h post-injection (Fig. 1A, B and C, respectively). Ten-micron thick longitudinal cryo-sections of the proximal tibia (see
Discussion
The mineralization processes in the provisional ossification zone are extremely complex and occur within a volume about 100 μm thick. Mineralized cartilage and bone are deposited within this zone, but there are no defined sub-zones separating mineralized cartilage from bone. We did not observe any mineral located inside hypertrophic chondrocytes, but we did observe such intracellular mineral particles in cells immediately juxtaposed to the blood vessels, presumably osteoblasts. Furthermore, the
Conclusions
The 2D and 3D characterization of the provisional ossification zone of the mouse growth plate using samples preserved under close to physiological conditions, shows a complex and intimate distribution of hypertrophic chondrocytes and their associated mineralized matrix, bone trabeculae and blood vessel extremities. We show that membrane-bound mineral-containing vesicles are present in the blood serum, and mineral particles without membranes are associated with the blood vessel walls. We
Acknowledgements
We thank Keren Kahil Guterman, Ifat Kaplan-Ashiri and the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging (Weizmann Institute of Science) for their assistance in the electron microscopy studies. We thank Eli Zelzer, Sefi Addadi, Dana Hirsch and Yuri Kuznetsov for help with the mice and light microscopy experiments. We thank Maria Pierantoni and Andreas Roschger for help with the micro-CT and fruitful discussions. Financial support was provided by the Human Science Frontiers
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