Abstract
PurposeHypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Materials and MethodsFetal mouse tibiae (E17.5) explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively). Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.ResultsThe oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.ConclusionsCollectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.
Highlights
Longitudinal growth of long bones is a tightly regulated process that is driven by hypertrophic differentiation and endochondral ossification of hyaline cartilage [1]
Normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL
Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes
Summary
Longitudinal growth of long bones is a tightly regulated process that is driven by hypertrophic differentiation and endochondral ossification of hyaline cartilage [1]. The cartilaginous ends of long bones can be divided into three general zones: the resting, proliferative and hypertrophic zone. At the end of the proliferative zone, chondrocytes start maturing into terminally differentiated enlarged chondrocytes, which are located in the hypertrophic zone. Hypertrophic chondrocytes produce large amounts of angiogenic factors, such as vascular endothelial growth factor (Vegf) that recruits invading blood vessels into the hypertrophic cartilage [2]. This allows for the infiltration of amongst others bone forming cells, and the alleviation of hypoxic stress (less than 5% oxygen) that occurs in most of the hyaline cartilage [3,4]
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