**5. Autologous bone tissue engineering**

HIF activity is involved in angiogenesis required for cancer tumour growth, so HIF inhibitors

In addition, there have been observations that suggest that HIF pathway is not only a pivotal inductor of neo-angiogenesis but also is relevant in questions of bone regeneration for example

The mechanism behind this hypothesis postulates the ability of osteoblasts to instrumentalize HIF-1 alpha as oxygen sensor and the corresponding signalling cascade to improve angio- and osteogenesis concurrently; the molecular interconnection is not finally elucidated. A dynamic

Therefore the application of HIF activators might improve bone healing by optimizing the angiogenic properties of the wounded bone but more important by inducing bone regeneration itself. Encouraging observations have been made in mouse fracture models where an overexpression of HIF and VEGF in long bones of mice results in pronounced vascularisation. Even a separate cultivation of the special osteoblasts without the corre‐ sponding endothelial cells does not affect their proliferation and differentiation (Wan,

The statements and examples of the preceding sections underlined clearly the impact of

One well investigated area of research in this context in the regeneration of bone, e.g. in terms of skeletal development or fracture repair. The vasculatures´ job is to bring oxygen and nutrients to the metabolically active areas, but also to provide the bone with precursors or inflammatory cells. As far as the cytokines are concerned, there are many factors that act as key protagonists in angiogenesis as well as in bone regeneration and remodelling: VEGF, especially its isoforms VEGF120, 164 and 188 play a significant role. But there are other relevant

VEGF in its isoforms with the corresponding receptors have emerged as the decisive coupling factors between epi- and metaphyseal vascularisation and cartilage develop‐ ment and enchondral ossification. A block of VEGFR-1 and -2 with selective antibodies leads to a reduced VEGF signalling and consecutively to a reduced intramembraneous bone formation in distraction osteogenesis; VEGF in this setting is produced by local inflamma‐

The angiopoietins Ang-1 and Ang-2, hepatocyte growth factor HGF, platelet-derived growth factor PDGF, the IGF family and the neurotrophins NGFs also have angiogenic properties. The effect of HIF 1-alpha as stimulator of bone regeneration also has been observed: in a mouse model with increased HIF activity the animals showed significantly higher bone mass. The stimulated HIF activity led to enhanced intramembraneous bone regeneration

are under investigation for anti-cancer effects (Semenza, 2006).

crosstalk between osteoblasts and endothelial progenitors is assumed.

**4. Influence of angiogenesis during bone regeneration**

players: bFGF, TGFβ, HIF are among the most potential ones.

Gilbert et al., 2008, Wang, Wan et al, 2007).

angiogenic processes on any tissue regeneration.

tory cells (Jacobsen, Al-Aql et al. 2005).

in fracture repair.

460 Regenerative Medicine and Tissue Engineering

Established concepts in the management of bony segmental defects or non-union after fracture rest upon the surgical implantation of either autologous bone as free grafts or micro-surgically anastomosed or artificial substitutes.

The concept of autologous bone tissue engineering wants to make available an amount of bone chips or bars that the organism itself is not able to supply without severe consequences. The base of the ideal three dimensional vascularised bone scaffold comprises the presence of a mechanically stable scaffold, seeded with different autologous cell populations and precur‐ sors, loaded with growth factors, embedded from the moment of implantation in a functioning vasculature to provide oxygen and nutrients and to remove metabolic by-products. Whereas many demands of this regenerative model can be met during in vitro culture in bioreactors, the vascular continuity remains the big problem to be solved.

As the supply of nutrition and oxygen via diffusion in three-dimensional tissue formations is restricted to an area of 100 μm around the nutritive capillary, resorption and devitalisation in the centre of the implant lead to a loss of mechanical stability.

The improvement of vascularisation therefore is an important demand on bone tissue engi‐ neering concepts. As far as the scaffold itself is concerned, there are different aspects to be considered; one decisive factor is the porosity of the material. In in-vitro studies the scaffolds with smaller pores (5-20 μm) come with increased endothelial cell growth and enhanced osteogenesis (Narayan, Venkatraman et al., 2008). In vivo the opposed effect is observed: higher porosity leads to more efficient osteo- and angiogenesis (Santos, Reis et al., 2010).

The modern materials provide the base for successful vascularisation simply by their design. In the structure of biodegradable polymers the negative of a vascular network can be imprinted and thus provide the architectural structure of an efficient vasculature; the endothelial (progenitor) cells have to populate the form, the structure is pre-fabricated. The predefined geometry has to fulfil special demand to grant for optimal results, so the network should be designed in branches with defined numbers and localization of vertical nodes.

This concept of microfabrication has been upgraded: with CAD/CAM techniques threedimensional scaffolds can be designed (Ciocca, De Crescenzio et al., 2009). So far these techniques are mainly applied in soft tissue engineering.

The loading of the scaffolds with growth factors is an established concept. The systems of drug delivery and release have become more refined, due to a combination of advanced scaffold materials and bio molecular perception concerning the anigogenic and osteogenic character‐ istics of the applied factors, their interconnection and vice-versa regulation. Combined application of several interacting growth factors is regarded as one of the pivotal steps towards successful factor application: from a polymeric scaffold a combination of VEGF and PDGF is delivered with defined dose and release kinetics. The advantage is the interaction of VEGF as endothelial mitogen and initiator of angiogenesis whereas PDGF impact on muscle cells and pericytes leads to vessel maturation and stabilization (Richardson, Peters et al., 2001).

In vitro pre-vascularisation of the scaffold often requires the colonisation with a co-culture of osteoblasts and endothelial (progenitor) cells, the duration of the in vitro phase ranges from hours to weeks. Investigations with poly-lactides implanted with a co-culture of endothelial progenitors and osteoblasts resulted in improved osteogenesis and vascularisation. The ischemic necrosis that was observed in the center of a graft that has only been implanted with osteoblast was not shown in the co-cultured scaffold (Yu, Vandevord et al., 2008).

Finally the success of any implant relies on a quick and efficient perfusion. In this context microsurgical techniques are combined with tissue engineering concepts in hybrid approaches that combine the respective advantages (Santos, Reis et al, 2010).

Modern approaches aim to design a custom made scaffold, loaded with autologous cells and growth factors including autologous vessel loops to grant for a spontaneous microvascular supply to support the expanding tissue. The details of this technique will have to be refined, but the first results are promising (Locmic, Stillaert et al., 2007). In autologous bone tissue engineering the combination of different regenerative strategies including tissue support and angiogenesis on various levels of the implant design and prefabrication finally will lead to successful therapeutic concepts.
