**5. Pathological implications**

invading the condensation at/near the site of initial ossification [16]. This association between vascular invasion and ossification continues and cascades as the bone expands in all directions. As the bone mineralizes in the wake of this vascular invasion front, the internal and

52 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Based on studies of endochondral ossification and distraction osteogenesis, Percival and colleagues [10] recently developed a model of angiogenesis during intramembranous ossification. They propose that prior to the onset of mineralization, small capillaries begin to invade the surrounding avascular loose mesenchymal tissue due to the presence of pro-angiogenic factors. At around the time of mineralization onset, these capillaries invade the osteogenic condensations and branch outwards from the ossification center (**Figure 1**). These capillaries branch toward regions of pro-angiogenic factor expression and thus support the proliferating mesenchymal cells of the condensation. Mineralization thus first occurs at sites closest to the capillaries and then at sites progressively further away. Once the osteogenic condensation stops expanding, the capillaries along with the mineralization front continue to moves toward the presumptive

sutures (i.e., the edges of the future bone), thus allowing continued calvarial growth.

The cross talk between these two dynamic processes is summarized in **Figure 1**.

**4. Insights from cell culture and bone graft studies**

chondral ossification) and well-organized blood vessels.

Interestingly, while osteoblasts in endochondrally ossifying bones express both HIF1α and HIF2α, only HIF2α is detected in the osteoblasts of directly ossifying bones (i.e., those that undergo intramembranous ossification) [17]. This altered expression pattern of the HIFα subunits could suggest that alternative regulatory pathways trigger angiogenesis in these distinct types of ossification [17]. Percival and Richtsmeier [10] provide a comprehensive list of hypotheses relating to intramembranous osteogenesis and angiogenesis that require testing.

VEGF is a chemoattractant for primary osteoblasts and mesenchymal progenitor cells [18] and can directly promote differentiation of primary human osteoblasts in culture in a dosedependent manner [19]. Osteoblasts and mesenchymal stem cells are the two cell types most often used in bone tissue engineering. Interestingly, the type of cell used can influence the mode of ossification that occurs and the organization of blood vessels [20]. Implantation of osteoblasts leads to the formation of fibrous tissue and disorganized blood vessels. The osteoblasts become trapped in the secreted bone matrix (i.e., intramembranous ossification occurs). In contrast, implantation of stem cells leads to the formation of cartilaginous tissue (i.e., endo-

Basic fibroblast growth factor (bFGF) is another important pro-angiogenic factor. When bone mesenchymal stem cells were transfected with bFGF and then implanted into rats with calvarial defects, an increase in vascularization and osteogenesis was observed [21]. Similarly, the addition of sonic hedgehog (Shh) in engineered bone grafts *in vitro* also promotes vascularization of the grafts [22]. Shh is expressed during fracture healing and

external vasculature is remodeled.

The role of growth factors like VEGF, FGF, CTGFs and others in both bone growth and angiogenesis has been demonstrated in a number of recent studies investigating the growth and healing of bones. For example, FGF9+/- mice exhibit a reduction in the healing of bones accompanied by a lack of VEGF expression in the area of injury, suggesting that FGF9 is required for angiogenesis and for healing long bones [25]. Hypomorphic VEGF120/120 mice have reduced mineralization of the calvarial bones and consequently reduced bone thickness. This has been attributed to a reduction and delay in vascular invasion [26]. Additionally, conditional deletion of VEGFA in mice cranial neural crest cells causes cleft palate with reduced ossification of the mandibular bone due to reduced endothelial cell proliferation and decreased angiogenesis [27]. Mice with a VEGF-deficient osteoblastic lineage exhibit age-dependent loss of bone mass and increased bone marrow fat, similar to the changes associated with osteoporosis in humans [28].

## **6. Conclusions and summary**

VEGF mediates angiogenesis, chondrocyte differentiation, osteoblast differentiation and osteoclast recruitment [29], and thus, its role during osteogenesis is complex ([10], **Figure 1**). Yang et al. [30] provide a useful tabulation of the effects of VEGF on intramembranous and endochondral ossification. Importantly, this chapter highlights that the relationship between angiogenesis and intramembranous ossification is not well understood. For example, only one study describes the detailed relationship between these two dynamic systems [16], and a very recent study provides a model of this process [10]. This model should be examined in several directly ossifying bones in the skeleton in order to confirm whether all directly ossifying bones follow the model or whether subtle differences exist depending perhaps on the location of the bone or the origin of the bone cells. This lack of a fundamental understanding about the developmental interactions between angiogenesis and skeletogenic condensations (particularly with respect to directly ossifying bones) contributes to our inadequate understanding of skull formation [10].

It should be noted that bones that ossify from the perichondrium of a cartilage template can be considered endochondral or intramembranous (since the perichondrium is a membrane of the cartilage). An example is Meckel's cartilage. The relationship between angiogenesis and bones that develop via the perichondrium has not been studied.
