Endochondral ossification: A delicate balance between growth and mineralisation

Abstract

The rapid proliferation of chondrocytes within the constraints of the cartilaginous matrix leads to formation of the characteristic orderly columns of chondrocytes seen in the growth plate. It is this stage of chondrocyte differentiation which facilitates longitudinal bone growth. Both autocrine and paracrine factors play roles in controlling the rate of chondrocyte proliferation. Growth hormone, produced in the pituitary, induces the local production of insulin-like growth factors which promote proliferation. Chondrocyte proliferation is balanced by negative regulation, the best characterised pathway being via FGF receptor 3 (FGFR3). Mutations in FGFR3, which is expressed in the resting zone, cause the most common form of dwarfism, achondroplasia, the milder hypochondroplasia and the lethal thanatophoric dysplasia. These mutations lead to constitutive activation of the receptor resulting in reduced chondrocyte proliferation while knocking out the receptor in mice leads to skeletal overgrowth. Thus activation of FGFR3 appears to stop cells entering the proliferative phase, most likely via the up regulation of cell cycle inhibitors.The maturation zone between proliferation and hypertrophy represents the site of active control over chondrocyte exit from the cell cycle and entry into hypertrophy which sets the pace of differentiation. Regulation of chondrocyte exit from the cell cycle is poorly understood whereas the regulation of entry into hypertophy is one of the better understood areas of chondrocyte biology. In this latter process, parathyroid hormone related peptide, PTHrP, and the secreted factor Ihh play crucial roles. Ihh is expressed by chondrocytes in the lower part of the maturation zone, which are already committed to hypertrophy. Ihh induces PTHrP expression in the perichondrium. PTHrP diffuses into the maturation zone where its receptor is expressed, and prevents further chondrocyte differentiation. Once Ihh expressing cells have undergone hypertrophy they stop producing Ihh. Thus PTHrP and Ihh form a negative feedback loop to regulate entry into hypertrophy (Fig. 2Fig. 2). Ihh is also a positive regulator of chondrocyte proliferation and osteoblast differentiation and is thus critical for many aspects of endochondrial ossification.Fig. 2Key regulators of chondrocyte differentiation within the growth plate.View Large Image | View Hi-Res Image | Download PowerPoint SlideDuring hypertrophy, the chondrocytes increase in size by five to ten-fold and thereby contribute to long bone growth. They also prepare the cartilage matrix for vascular invasion and bone deposition. Intrinsic to both of these functions is the need for matrix remodelling, which consists both of matrix degradation and deposition of new matrix components. Degradation of the cartilage matrix involves cleavage of collagen by matrix metalloproteinases (MMPs) such as MMP-13 and MMP-2 from the collagenase and gelatinase subgroups respectively; proteoglycans are largely degraded by stromelysins, such as MMP-10, and the aggrecanases. MMPs can also remove anti-angiogenic molecules and generate angiogenic molecules either by releasing them from a matrix bound state or by forming angiogenic fragments from larger molecules.Hypertrophic cells secrete a unique matrix component, the short chain type X collagen. This collagen is thought to form a hexagonal lattice ‘basket’ around the expanding chondrocytes, which may provide structural support as they expand and degrade other matrix molecules. Recent studies of a strain of mice lacking type X collagen show that they have defective haematopoiesis, suggesting that the remaining spicules of hypertrophic chondrocyte matrix may have an essential role in directing the development of the haematopoietic cells of the bone marrow.In addition to type X collagen, hypertrophic chondrocytes also secrete matrix molecules such as osteopontin, osteonectin and bone sialoprotein which were once thought to be uniquely produced by osteoblasts. These molecules create a scaffold for the attachment and differentiation of the osteoblast and osteoclast precursor cells. The production of these proteins is under the control of the master regulator of the osteoblast phenotype, the transcription factor Cbfa-1, which is also required for the terminal differentiation of hypertrophic chondrocytes. Expression of bone markers by chondrocytes has led to the controversial opinion that they are transdifferentiating. However there is now overwhelming evidence that the fate of hypertrophic chondrocyte is apoptosis, mediated by the downregulation of the anti-apoptotic Bcl-2, leading to an imbalance with the levels of its pro-apoptotic partner Bax. The final trigger for apoptosis appears to depend on signals from the invading vasculature. This has been highlighted by analysis of the MMP-9 knockout mouse. MMP-9 is produced by osteoclasts at the chondro–osseous junction, and its absence delays vascular invasion, probably through decreased production of an angiogenic factor, and also delays hypertrophic chondrocyte apoptosis. This signalling interplay highlights the complex reciprocal regulation that exists between the chondrocytes and the invading vasculature.Endochondral ossification represents a fascinating combination of virtually every known biological pathway. Precisely how these pathways are integrated in the formation of a skeleton with elements of the appropriate size, shape and structure remains however a major challenge.