Abstract
Introduction
The proteoglycan aggrecan is a major component of the cartilaginous matrices which provides resistance against compressive forces. Spontaneously occurring functional null mutations in the aggrecan gene (Acan) in various species lead to perinatal chondrodysplasia. The aim of the present study was to investigate the cellular and biomechanical properties of the cartilaginous growth plate, and the development of intervertebral disc in a novel, experimentally induced aggrecan mutant mouse strain carrying an insertion in exon 5 of the Acan gene.
Methods
The novel aggrecan mutant mice were generated by inserting a loxP site into exon 5 (E5i) by homologous recombination in ES cells. Wild type and homozygous mutant (Acan-E5i/E5i) mice were analyzed by skeletal staining, histology and immunohistochemistry. Proliferation and survival were assessed by phosphorylated histone H3 immunostaining and TUNEL assay, respectively. Shape index (SI) in the proliferative zone (PZ) of the growth plate (GP) was calculated as a ratio of the long and short axes of the cells. Orientation of the PZ chondrocytes was characterized by the angle between the cell long axis and longitudinal direction of the bone growth. Imaging and stiffness measurements were performed by atomic force microscopy (AFM).
Results
Acan-E5i/E5i mice are characterized by severe dwarfism, short snout, protruding tongue, cleft palate, and die at birth due to respiratory failure. On sections the cartilage of mutant mice appeared as tightly packed chondrocytes surrounded by a compressed matrix. At E18.5 and E14.5, the mutant PZ consisted of rounded (SI=1.71 at E18.5; SI=1.72 at E14.5) non-oriented chondrocytes, compared to the wild type PZ with flattened (SI=3.92 at E18.5; SI=3.90 at E14.5), columnar cells oriented with right angle to the longitudinal axis of the growth. At E13, the shape and orientation of mutant chondrocytes were similar to control. AFM at E14.5 and E18.5 demonstrated a stiffer matrix with denser collagen network in the mutant compared to wild type. The mutant cartilage had increased apoptosis and reduced proliferation rate at E18.5. The IVDs development appeared normal at E13.5-E14.5, however, the IVD was severely malformed at E18.5.
Discussion
We have shown that aggrecan deficiency impairs cartilage biomechanics and results in a stiffer matrix. The altered mechanical properties might be responsible for the disorganization of mutant GP and compression of the IVD at around birth. Interestingly, the altered matrix mechanics is dispensable for early flattening and orientation of GP proliferative chondrocytes. In summary, aggrecan is essential for proper cartilage cytoarchitecture and morphogenesis by ensuring the suitable mechanical environment.
