**3. Bones that ossify via intramembranous ossification**

## **3.1. Directly ossifying bones**

**Figure 1.** Schematic showing the complex cross talk between angiogenesis and skeletogenesis. Mesenchymal cells aggregate in an avascular (hypoxic) zone to form skeletogenic condensations. Differences in the molecular characteristics of these cells determine the fate of the condensation. Cells within this condensation produce VEGF and other proangiogenic factors, and this induces angiogenesis. Left side: Cells within the chondrogenic condensation continue to produce these angiogenic factors, and ultimately, the condensation differentiates into a cartilage template, which is still avascular. Following angiogenesis surrounding this template, blood vessels invade the cartilage perichondrium layer, and this triggers osteoblast differentiation, cartilage matrix degradation, bone matrix production and the further release of more pro-angiogenic factors. Ossification begins in the diaphysis (shaft) of long bones and spreads to the epiphyses (ends of the bone). Right side: As cells within the skeletogenic condensation differentiate, they continue to produce angiogenic factors. These factors induce angiogenesis and subsequent blood vessel invasion into the outer edges of the condensation. As more cells within the condensation differentiate, the wave of bone matrix deposition and blood vessel

50 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

invasion spreads outwards.

Intramembranously ossifying bones form without a cartilage template. Motile mesenchymal cells fated to differentiate into osteoblasts aggregate to from skeletogenic condensations at the site of the future bone. As these osteogenic aggregations enlarge and reach a critical size, the central cells begin to differentiate into osteoblasts, lose their mobility and deposit bone matrix. This process results in osteoblasts becoming embedded or trapped in bone matrix, forcing them to differentiate into osteocytes [8]. The majority of the craniofacial skeleton forms via intramembranous ossification [9]. A common example is the skull vault (the calvariae). Less common examples are the scleral ossicles (in reptiles), parts of the clavicles and scapula (in mammals), the cleithra and opercula (in fish) and sesamoid bones (e.g., the patella in humans) [9]. Although vascularization has been extensively studied in endochondral ossification as discussed above, comparatively little research has been conducted to understand the relationship between angiogenesis and intramembranous ossification.

## **3.2. Angiogenesis during formation of the initial phase of directly ossifying bones**

The most studied intramembranous bones are the calvariae (or skull vault). Cells in the osteogenic condensations proliferate resulting in growth of the condensations until a critical size is reached. Ossification begins at the center of the condensation and expanding outwards toward the apex of the head [10]. Once this has occurred, cells at the osteogenic front proliferate, and the bones grow toward one another [11]. Areas that ossified first form a trabecular bone structure that later becomes woven bone [10]. Interestingly, the frontal and parietal bones in humans each develop two condensations, each with their own ossification centers; these centers then fuse as ossification progresses [12].

There is a significant difference in the gene expression patterns in prechondrogenic and preosteogenic condensations [1]. Avascularity within condensations may be necessary for the formation of the condensations themselves and/or to provide positional cues [10, 13]. Indeed, in scleral ossicles, an avascular zone develops surrounding the condensation [14, 15]. Manipulating the size of this avascular zone has a direct effect on subsequent bone development [15]. Although not much is known about the process of vascularization during intramembranous ossification, it is thought that similar to endochondral bones, hypoxic conditions are important to induce angiogenesis. Avascular zones likely surround all preosteogenic condensations in mammals and avians, however, the mechanism by which these zones are established is not known [10]. Percival and colleagues [10] postulate that this avascularity may be important for condensation growth, and subsequent intramembranous ossification (as in the case for prechondrogenic condensations of endochondrally ossifying bones, **Figure 1**).

A single study describes in detail, the association of angiogenesis and intramembranous bones [16]. This study of the development of the chick frontal bone showed that small capillaries invade the thin avascular layer of loose mesenchymal cells of the condensation prior to 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 external vasculature is remodeled.

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.

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

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

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., endochondral ossification) and well-organized blood vessels.

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 neovascularization after trauma and has been used *in vitro* to promote the organization of blood vessels in artificial tissue grafts similarly to VEGF [22, 23]. Furthermore, when these grafts were implanted subcutaneously in mice, there was increased bone formation of both directly and indirectly ossifying bone types [22]. For large defects, supplementing the graft with platelet-rich plasma results in increased bone formation [24].
