Microvascular invasion during endochondral ossification in experimental fractures in rats

Abstract

In this study morphologic techniques have been used to detail the angiogenic response that accompanies endochondral fracture healing in a clinically relevant, reproducible rat model. In this displaced fracture, the gap fills with cartilage that later is replaced by bone, via endochondral ossification. A transient periosteal circulation, followed by a permanent medullary circulation accompany this progression. From 2 to 6 weeks, vessels grow out from the periosteal tissue and give rise to vascular buds, which abut directly onto the avascular zone corresponding to the fracture defect. From 3 weeks onwards, a second wave of vessels grows out from the marrow to the cartilage-filled fracture defect, terminating as vascular buds and loops lined by endothelial and perivascular cells. The loops and buds stain strongly for laminin but transmission electron microscopy does not demonstrate an identifiable basement membrane, pointing to a region of active extracellular matrix turnover. These vessels are intimately associated with osteoblasts and newly formed woven bone forming finger-like composite structures that protrude into the mineralized cartilage matrix with which they form a clearly demarcated interface. Invading vessels and woven bone successively replace the cartilage matrix to mediate repair. Both the vascular structures and progression of endochondral ossification observed, closely resemble those described in the normal epiphyseal growth plate, indicating that the fundamental processes are similar. However, there is a difference in the spatial orientation of cells such that the healing front in the fracture model is relatively disorganized, compared to the orderly linear array of cells at the epiphyseal growth plate.

Introduction

Neovascularization is essential for growth and healing of all tissue types [3]. With respect to bone, microangiographic investigations have elucidated both normal microvascular anatomy in bone and microvessel development in healing long bones [22]. A “centrifugal” microcirculation—from medullary vessels through anastomoses in the cortex to periosteal vessels—has been demonstrated in normal bone and in rigid undisplaced fractures [7], [22], [23]. However, in less rigid displaced fractures, the microcirculation is reversed during the early course of fracture healing, that is, periosteal vessels instead of medullary are the dominant source of blood supply. As new vessels and bone bridge the fracture zone, “centrifugal” microcirculation is re-established [22].

Fractures generally heal by intramembranous and/or endochondral ossification. Intramembranous ossification is considered to be a recapitulation of the bone formation that occurs during embryonic development of flat bones in which committed osteoprogenitor cells differentiate directly into bone-producing osteoblasts [7], [9]. Endochondral ossification is believed to be a recapitulation of the bone formation that occurs in the growth plate during embryonic development of long bones where avascular cartilage becomes hypertrophic and is followed by mineralization of the matrix [7], [9], [10]. Angiogenesis occurs in parallel with endochondral ossification, leading to erosion of the mineralized cartilage and deposition of bone. During endochondral ossification, the formation of new capillaries is considered to be indispensable for the deposition of bone [1], [7], [8], [14], [15].

Light- and scanning electron microscopy of corrosion casts from the growth plate in rats have shown that longitudinal vessels arise from the methaphysis and extend toward the epiphysis [1], [13], [14], [15]. These vessels form loops and sprouts in a border zone located beneath the last row of mineralized hypertrophic chondrocytes of the metaphyseal growth plate [1], [14], [15]. The same observations have been made in secondary ossification centers of femoral head bone in rats [12]. The hypertrophic chondrocytes and surrounding matrix form septae separated by cylindrical vascular compartments and terminal vascular loops and capillary sprouts have been identified in these compartments. The mineralized matrix septae are covered by osteoblasts sequentially replacing the matrix by bone [1].

The microvascular anatomy associated with endochondral ossification in fractures has not been clearly described [1], [12], [14], [15], [22]. In this report, we used a reproducible model of endochondral fracture healing [17], [18] to study the development of the accompanying neovascularization.