Introduction: The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate this to the mechanical fixation stability during the course of healing.

Materials and Methods: A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization.

Results: Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks) and decreased over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point.

Discussion: The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

The human cystein-rich protein 61 (hCYR61) belongs to an emerging family of genes which modulate growth and differentiation. Previously, hCYR61 was identified by us as a fast and transiently 1,25(OH)2-vitamin D3 responsive gene product in human osteoblasts by differential display PCR. Here, we further studied the role of the protein in human osteoblasts.

Using the human hFOB cell line hCYR61 mRNA was analysed by northern hybridisation. Protein levels were detected using western blotting. Intracellular localisation of the hCYR61 protein was determined using the expression as a fusion protein with green fluorescent protein. Immunohistology was performed in hFOB cells as well as primary human osteoblasts and human bone samples.

From northern analyses the hCYR61 mRNA was regulated by 1,25(OH)2-vitamin D3 as well as the growth factors TNFa, EGF, bFGF and IL1b 5-10-fold within 1 hour in the hFOB cell line. Here we show that the same factors markedly upregulated the hCYR61 protein within 24 to 48 hours in hFOB cells as has been analysed by western blotting. From cellular supernatants a highly upregulation of the hCYR61 protein by the growth factors was observed. A full length hCYR61 protein fused to the green fluorescent protein localised to the Golgi-apparatus. From immunohistology proliferating hFOB cells and primary osteoblasts express significant hCYR61 protein, whereas differentiated osteoblasts display a marked downregulation of hCYR61. In human bone high levels of hCYR61 were observed at the human growth plate as well as on surfaces of mineralised structures.

In summary, hCYR61 in human bone represents an immediate early regulated gene. The secreted protein plays a role as an extracellular matrix signaling protein which could play an important role in cell-cell communication within the bone microenvironment. The high expression level and regulation patterns observed in our studies suggest an important role in situations of bone repair and remodeling.