The multifaceted role of the vasculature in endochondral fracture repair

Abstract

Fracture healing is critically dependent upon an adequate vascular supply. The normal rate for fracture delayed or non-union is estimated to be between 10 and 15%, and annual fracture numbers are approximately 15 million cases per year. However, when there is decreased vascular perfusion to the fracture, incidence of impaired healing rises dramatically to 46%. Reduction in the blood supply to the fracture can be the result of traumatic injuries that physically disrupt the vasculature and damage supportive soft tissue, the result of anatomical location (i.e., distal tibia), or attributed to physiological conditions such as age, diabetes, or smoking. The role of the vasculature during repair is multifaceted and changes during the course of healing. In this article, we review recent insights into the role of the vasculature during fracture repair. Taken together these data highlight the need for an updated model for endochondral repair to facilitate improved therapeutic approaches to promote bone healing.

Keywords: angiogenesis; bone biology; cartilage transformation; endochondral ossification; fracture repair.

Figure 1

Endochondral Fracture Schematic: A fracture that is not rigidly stabilized will heal through a combination of intramembranous and endochondral ossification. Fracture healing progression: bone is depicted in red and cartilage in blue, time course corresponds to healing in a mouse tibia. (A) The first stage of fracture healing is formation of a hematoma to initiate an inflammatory cascade characterized in part by an abundance of pro-inflammatory M1 macrophages, low pH, and elevated lactate level. (B) Next, local osteochondral progenitors differentiate into bone and cartilage to initiate healing. Intramembranous ossification occurs primarily at the periosteum and endosteum of the fracture callus, while the soft cartilage callus forms in the central portion of the fracture where there is maximal mobility. For healing to progress normally, inflammation must be down-regulated and there is a shift in the macrophage population from a pro-inflammatory M1 state to an anti-inflammatory M2 state. (B’) Within the cartilage callus chondrocytes differentiate and mature in a parallel fashion to the growth plate. (C) Conversion of cartilage to bone during endochondral ossification occurs concomitantly with the invasion of blood vessels. (C’) Blood vessels lead to mineralization of the cartilage matrix and new data suggest that these cells transform directly into osteoblasts. Chondrocytes do not undergo significant apoptosis and may re-enter the cell cycle. (D) The cartilage callus becomes fully converted to a trabeculated bone that will bridge the full fracture defect. This trabeculated structure will be remodeled into a cortical bone that is almost indistinguishable in form and function from the native bone.

Figure 2

Vasculature in the fracture callus transition zone: (A,B) Histology of fracture callus 10 days following injury stained with Hall’s and Brudt’s Quadruple (HBQ) stain that indicates the cartilage matrix in blue through alcian blue and the bone matrix in red through direct red dye. We have defined the region where the cartilage becomes bone in the fracture callus as the transition zone (“TZ”). In this region, the cartilage (“C”) matures through hypertrophy (“HC”) and junctions into bone in an area that is clearly marked by the blue to red matrix transition and the invasion of blood vessels (“BV”). Within the red bone matrix (“B”) cells with the large round hypertrophic chondrocyte morphology and the smaller elongated osteoblast morphology can be clearly identified. (C–F) Immunohistochemistry to VEGF protein. (C,D) The transition from normal to hypertrophic cartilage demonstrates that only hypertrophic chondrocytes make VEGF. (E,F) At the hypertrophic cartilage to bone transition this VEGF is responsible for recruiting the vasculature. Osteocytes no longer make VEGF protein but the protein binds to the extracellular matrix in this transition zone. (G,H) Immunohistochemistry to PECAM/CD31 protein marks the vasculature invading in the transition zone. Scale bar = 100 μm; B = bone, BV = blood vessel, C = cartilage, HC = hypertrophic cartilage, TZ = transition zone.

Figure 3

Cell death is not pervasive at the transition zone: the established model of endochondral ossification includes cell death of the hypertrophic chondrocytes. Here, we show TUNEL staining in the transition zone of the fracture callus to demonstrate that there is minimal cell death seen in these hypertrophic chondrocytes. (A) Bone marrow, as a positive control; (B) non-hypertrophic cartilage at tip of fractured bone; (C,D) transition zone with red dotted line indicating the demarcation between cartilage (“C”) and bone (“B”) that can be easily distinguished by cell morphology and bright field microscopy. Scale bar = 100 μm.

Figure 4

A new model for endochondral fracture repair: local osteochondral progenitors from the periosteum and endosteum are the stem cells that differentiate to form bone and cartilage in the fracture site. To generate the cartilage callus, osteochondral progenitors differentiate into chondrocytes (blue) that proliferate to generate the early soft callus. Chondrocytes within the callus mature into hypertrophic chondrocytes. Expression of angiogenic factors by these cells results in vascular invasion into the previously avascular soft callus. Mineralization of the hypertrophic cartilage occurs at this transition zone where blood vessels are invading. Hypertrophic chondrocytes begin to express many of the canonical markers of the osteoblast (red), including osteocalcin, osteopontin, and alkaline phosphatase. The fate of these mineralized hypertrophic chondrocytes remains unclear. Apoptosis is the classical fate ascribed to these cells. According to this model, new bone is formed by osteoblasts that arise from osteoprogenitor cells brought in through the invading vasculature. In addition, new reports indicate that at least a part of the newly formed bone in the fracture callus is chondrocyte derived. The mechanisms that enable this phenotypic conversion of cartilage to bone remain unclear. Here, we depict two proposed pathways – chondrocyte de-differentiation and direct maturation – that have been suggested in the literature, but without fully substantiated details as suggested by dotted lines.