VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IN HUMAN PERIOSTEUM – NORMAL EXPRESSION AND RESPONSE TO FRACTURE

Abstract

Vascular Endothelial Growth Factor (VEGF) has been shown to stimulate angiogenesis in a number of tissues and, in addition, to possess direct vasoactive properties. Stimulation of blood flow and angiogenesis are important features of the fracture healing process, particular in the early phases of healing. Inadequate vascularity has been associated with delayed union after fracture. The periosteum, and in particular its osteogenic cambial layer, has been shown to be very reactive to fracture and to contribute substantially to fracture healing. Fracture haematoma contains a considerable concentration of VEGF and enhanced plasma levels are observed in patients with multiple trauma. VEGF has been suggested to play a role during new bone formation possibly providing an important link between hypertrophic cartilage, angiogenesis and consequent ossification. However, the expression of VEGF in normal periosteum and in periosteum close to a fracture has not been previously reported. We hypothesise that the expression of VEGF in long bone periosteum will show a distinct response to fracture.

We investigated the expression of VEGF in vivo in human periosteum, using immunocytochemistry to detect the expression of Factor VIII and VEGF protein respectively. Under prior approval from the local Ethics Committee, biopsies of periosteal tissues were collected from two distinct groups (1) control and (2) following long bone fracture. Patient age range was 16 – 45 years for both groups. Group 1 consisted of patients (n = 5) who underwent an elective orthopaedic procedure during which periosteum was disrupted. Group 2 patients (n = 8) had long bone fractures from which periosteal tissue was harvested close to the fracture site during internal fixation at various time points following fracture (24 hours to nine days).

In Group 1 the periosteum showed abundant but delicate blood vessels staining throughout for VEGF but there was no other visible staining of other structures or cells. In Group 2 the vasculature in the periosteum close to the fracture site demonstrated a characteristic, time-dependent course of expression of VEGF. At 24 and 48h following fracture the vasculature showed a heterogenous picture. The vessels in periosteum showed signs of activation: thickened endothelia and dilated lumina, but did not express VEGF. At 60h the vessels began to show signs of the presence of VEGF protein and by 4 days most periosteal vessels expressed VEGF. Also at this time, VEGF staining was visible in some of the stromal cells of the periosteum that was not seen in any of the earlier times. At 9 days VEGF was visible not only in the omnipresent vasculature, but now consistently in spindle shaped cells of fibroblastic appearance and chondrocytes throughout the early callus.

This study, though limited by the number of patients, shows for the first time the expression of VEGF in normal periosteum as well as in periosteum during fracture healing. Interestingly, activated vessels in the early healing phase show little expression of VEGF; however it is known that the fracture haematoma contains VEGF in abundance. It is possible that the vasoactive role of VEGF prevails in these early days. There may be a critical time point at around 48h post fracture following which angiogenesis begins and VEGF is expressed in the endothelium throughout the vessel wall. The study suggests an important role for VEGF in the regulation of fracture healing. VEGF is not only expressed in endothelial cells within the periosteum but also in fibroblast-like stem cells and chondrocytes throughout the early callus suggesting it may play an important role in both osteo- and angiogenesis