**6. Regenerative concepts**

The support of developing vasculature happens on different levels from the (systemic) application of growth factors to the local application of loaded implants.

Next to the selection of growth factors, the colonisation with prefabricated cell populations or the nano-structural design of the implants are decisive factors considering the implant integration and the development of a functional vessel network.

#### **6.1. Therapeutic angiogenesis**

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

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

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).

osteoblast was not shown in the co-cultured scaffold (Yu, Vandevord et al., 2008).

that combine the respective advantages (Santos, Reis et al, 2010).

successful therapeutic concepts.

**6. Regenerative concepts**

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

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

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

The support of developing vasculature happens on different levels from the (systemic)

Next to the selection of growth factors, the colonisation with prefabricated cell populations or the nano-structural design of the implants are decisive factors considering the implant

application of growth factors to the local application of loaded implants.

integration and the development of a functional vessel network.

designed in branches with defined numbers and localization of vertical nodes.

techniques are mainly applied in soft tissue engineering.

462 Regenerative Medicine and Tissue Engineering

In different clinical applications the angiogenic effect of different growth and transcription factors could be observed. In the context of angio- and osteogenesis, their coupling and the chance of therapeutic intervention, the administration of VEGF is the best investigated one. In fracture healing and bone regeneration therapeutic angiogenesis finds many points of attack. Beside the acute trauma the especially interesting indications considering bony regeneration are non-unions and distraction osteogenesis.

There are several approaches to stimulate angiogenesis and consecutively bone regeneration. The administration of angiogenic factors, VEGF or FGF, is supposed to effect a direct angio‐ genic up regulation. Another initiator of angiogenesis is HIF; its application or the inhibition of its degradation results in angiogenic effects. Generally these therapies aim to promote angiogenesis, to block anti-angiogenic processes and to bring endothelial progenitor cells to the wounded bone (Hankenson, Dishowitz et al., 2011).

The effects of VEGF as a promoter not only of angiogenesis but also of bone regeneration have been reported in a femur fracture model in mice and in a rabbit radial segmental defect; improved ossification and callus maturation where observed (Street et al, 2002).

Another growth factor with angiogenic and osteogenic characteristics is platelet derived growth factor (PDGF) that acts as mitogen for osteoblasts and up-regulates VEGF expression. In animal models the administration of PDGF came with increased mechanical stability and callus density (Hollinger, Onikepe et al., 2008). In human pilot projects these positive results of PDGF application in combination with fracture stabilization could be verified.

A modern area of research dealing with VEGF as a means of vascular protection and regen‐ eration aims to neuroprotection; VEGF has been reported to protect motor neurons in vitro from hypoxia induced toxicity, reactive oxygen and other degrading factors (Svensson, Peters et al., 2002). In addition, VEGF seems to be able to stimulate growth and development of neuronal stem cells as well as to recruit neuronal progenitor cells (Schaenzer, Wachs et al., 2004). In ALS rat models the protective effect of VEGF in the cerebrospinal fluid has recently been reported, showing a protracted course of disease with delayed paralysis and increased survival time (Storkebaum, Lambrechts et al., 2005).

The potential of angiogenesis as a pivotal factor in many areas of tissue maintenance and regeneration is obvious; in future regenerative medical concepts the manipulation of angio‐ genesis in parallel with tissue regeneration will be integral part and lead to successful strat‐ egies.

The most important growth and transcription factors enhancing vascularisation are summar‐ ized in table 3. The variety of different angiogenic factors with similar functions implies the idea of redundancy: the quick and undisturbed succession of events of angiogenesis is too important for the function of the whole organism to take the risk of relying on unique regulators or promoters.


**Table 3.** Growth and transcription factors stimulating angiogenesis (Losordo, Dimmeler et al., 2004)

#### **6.2. Nanotechnology in regenerative medicine**

Modern biomaterials have to meet many requirements: not only do they have to provide mechanic support and stability; they have also to enhance regenerative processes, to be antiinfective and non-inflammatory. The problems of bone implants so far were seen in the poor osseointegration and bone regeneration on the one hand and implant loosening and fracture on the other (Dhillon, Schwarz et al., 2011).

The relevance of nanotechnology in the area of tissue engineering research is reflected by the fact that it has become an independent field of interest in regenerative medicine: the nanome‐ dicine. Nanomedicine deals with implant structures that have (surface) dimensions of fewer than 100 nm. The used materials include fibres and particles, imitating natural bone structure to improve the mechanic and biological properties of the implants.

Figure 3 illustrates the dimension of nanostructures in the context of bone anatomy (Sato, Webster et al., 2004, Khang, Lu et al., 2008).

**Figure 3.** Nanostructure in bone structure (Sato, Webster et al., 2004)

**Growth factor Molecular target Effects on progenitor cells**

Mobilization of EPC Improves survival and differentiation of EPC

Mobilization of hematopoietic stem cells and EPC

Included in EPC culturing media

Mobilizes EPC and hematopoietic progenitor cells

Included in EPC culturing media

Mobilization of EPC

VEGF receptors expressed on endothelial cells, monocytes, hematopoietic stem cells; stimulates proliferation, migration, and tube formation

VEGF receptor 1 (cross talk with VEGF receptor 2)

FGF receptors expressed on endothelial cells, smooth muscle cells, and myoblasts; stimulates proliferation

Tie-2 receptor expressed on endothelial cells; enhances vessel maturation and stability

IGF receptor expressed on vascular cells and satellite cells; enhances skeletal muscle regeneration

Activates the Epo receptor, which is expressed on hematopoietic stem cells, EPC, endothelial cells, and cardiac myocytes; improves survival

Activation of gene expression (eg, VEGF, VEGF receptor 2, erythropoietin, IGF-2, and NO synthase)

Modern biomaterials have to meet many requirements: not only do they have to provide mechanic support and stability; they have also to enhance regenerative processes, to be antiinfective and non-inflammatory. The problems of bone implants so far were seen in the poor osseointegration and bone regeneration on the one hand and implant loosening and fracture

The relevance of nanotechnology in the area of tissue engineering research is reflected by the fact that it has become an independent field of interest in regenerative medicine: the nanome‐ dicine. Nanomedicine deals with implant structures that have (surface) dimensions of fewer than 100 nm. The used materials include fibres and particles, imitating natural bone structure

Figure 3 illustrates the dimension of nanostructures in the context of bone anatomy (Sato,

**Table 3.** Growth and transcription factors stimulating angiogenesis (Losordo, Dimmeler et al., 2004)

to improve the mechanic and biological properties of the implants.

VEGF

464 Regenerative Medicine and Tissue Engineering

Placenta-derived growth factor (PIGF)

Fibroblast growth factor (FGF)

Angiopoietin-1

Insulin-like growth factor (IGF)

Erythropietin (EPO)

Hypoxia inducible factor 1 (HIF-1)

**6.2. Nanotechnology in regenerative medicine**

on the other (Dhillon, Schwarz et al., 2011).

Webster et al., 2004, Khang, Lu et al., 2008).

One important task of nanomedicine is to come up with improved, probably intelligent biomaterial. In fact there are two different strategies to modify established materials. One deals with the materials chemistry, the other cares for the surface properties. By individually adapting chemical and physical characteristics the idea scaffold can be designed (Khang, Carpenter et al., 2010).

The established nano-materials in bone regeneration are nano-hydroyapatite, silk and nano structured titanium surfaces. To reach optimal cell adhesion and function there have been efforts to imitate the physiological anisotropy of natural bone. These modifications led to enhanced adhesion and mineralisation (Khang, Lu et al., 2008).

These nano-materials will find their way into clinical practice as far as orthopaedic indications or dental implants are concerned.

These developments are also realized in vascular tissue regeneration: the material surfaces are supposed to promote endothelial cell migration, adhesion and proliferation of vascular graft or stents. In an investigation of polylactide-co-clycolic acid (PLGA) surfaces with spherical surfaces features with ascending diameters a positive correlation of vertical surface feature dimension to cell adhesion and protein adsorption was measured; the optimal dimension was 20 nm (Carpenter, Khang et al., 2008). In special etching techniques, titanium inductively coupled plasma deep etching (TIDE), a linear nano-structured surface pattern is created that allows for increased endothelial cell proliferation compared to smooth titanium surfaces and even to randomly nano-structures titanium surfaces after five days of cultivation (Lu, Rao et al., 2008). Recent concepts deal with mechanical strain applying pulsed or sustained pressure to the implanted scaffolds to meet the demands of physiological vascular tension.

Nanotechnology in current tissue engineering concepts investigates the cell- biomaterial interaction and perfects the surface properties to achieve maximum regenerative support in combination with a prolonged implant lifetime. In the context of angiogenesis the development and integration of nano-materials will be of vital importance in regenerative strategies.

#### **6.3. Critical aspects**

Many promising investigations featuring growth factor therapy are performed in cell culture or healthy young animals. The therapeutic use of these developments especially addresses the ageing population suffering from ischemic diseases and vascular degeneration. Special attention in coming investigations has to be shifted to the functionality and regenerative demands of diseased of damages cells and tissues. Therapeutic angiogenic strategies have to be scrutinized under the focus of safety and effectiveness in systems with impaires endogenous endothelial function (Sun, Bai et al., 2009).

Beside the form of application, the definition of the ideal dose of angiogenic growth factor is one the most difficult questions to answer. When dealing with loaded scaffolds one has to define release kinetics. Additionally, in histological investigations, the vasculature that develops under the influence of high doses of VEGF repeatedly showed malformations and an insufficiency in the cell-cell junctions (Zisch, Lutolf 2003).

In most therapeutic concepts the desired effect of VEGF is a local one and aims to improve the formation of nutritive vasculature supporting tissue regeneration in a limited area of tissue as well as a in a limited period of time. VEGF application has to take place locally and only during defined period; considering the fact, the VEGF coming with increased angiogenesis is part of many pathologic processes, e.g. tumour vascularisation or proliferative retinopathy, the control of VEGF effect has to be granted. These demands require a lot of conceptional research considering the therapeutic application of VEGF. The angiogenic and osteogenic effects of VEGF delivered from poly-lactide scaffolds in irradiated osseous defects was illustrated impressively in increased vascularisation and bone formation, the application of a potent growth factor in tumour patients however bears many risks (Kaigler, Wang et al., 2006)

In every therapeutic concept the medical gain and the patients´ profit has to be weighed against the impending costs. Nowadays the production of recombinant VEGF in the desired doses is enormous. For routine clinical application the methods of generation, application and delivery have to be refined (Barralet, Gbureck et al., 2009).
