In vitro hypertrophy and calcification of human fracture haematoma-derived cells in chondrogenic differentiation

Purpose

The haematoma at a fracture site plays an important role in fracture healing. Previously, we demonstrated that a fracture haematoma contains multilineage mesenchymal progenitor cells. We postulated that the haematoma provided a source of chondrogenic cells for endochondral ossification during fracture healing and preservation of the cells contributed to biological fracture healing. In this study, we investigated whether haematoma-derived cells (HCs) could differentiate into hypertrophic chondrocytes and finally induce calcification of the extracellular matrix in vitro.

Methods

Fracture haematomas were obtained from four patients. HCs were cultured for five weeks under conditions that induce chondrogenic differentiation, followed by two weeks of hypertrophic induction using a pellet culture system. The pellets were analysed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic, and angiogenic markers were measured by real-time PCR.

Results

The histological and immunohistochemical analyses revealed that HCs differentiated into chondrocytes and hypertrophic chondrocytes, followed by calcification of the extracellular matrix. This sequential differentiation was also reflected in the gene expression profiles. After chondrogenic induction, expression of osteogenic and angiogenic markers was not significantly upregulated. However, the expression of these markers was significantly upregulated following hypertrophic induction. These in vitro observations mimicked the process of endochondral ossification during fracture healing.

Conclusions

Our results suggest that the fracture haematoma may offer a source of cells with chondrogenic potential that play key roles in endochondral ossification during fracture healing. These findings support the opinion that the haematoma should be preserved for biological fracture healing.