**4. Emerging research theme: Addressing the need for vascularisation**

#### **4.1. Vascularisation as limiting bottleneck**

Much effort has been focused on generating tissue engineered bone grafts in vitro, and several attempts have been made to heal bone defects with engineered grafts in vivo. The achievements in bone tissue engineering led considerable progress in finding potent osteogenic cell sources and suitable biomaterials, as well as the development of scaffolds and the use of bioactive factors. Currently, the attention has gradually shifted to strategies to improve vascular formation in tissue engineered bones as it emerges as the most crucial factor in ensuring graft survival and hence bone repair.

Having a network of blood vessels within a tissue-engineered graft is important for maintain‐ ing cellular survival particularly within the core of large bone grafts [67]. It has been shown that after implantation, neo-bone tissues were found only at where a vascular network was present [68]. Poor angiogenesis has been identified as the main reason for implant failure and is currently acknowledged as a major challenge in tissue engineering [69-71].

Currently, there are various strategies that are under investigation for improving vasculari‐ zation in tissue engineered grafts. These include the induction of vascularization in vivo, the design of scaffolds specific to improving vascularization, and prevascularization techniques using coculture systems [72]. The prospect of functional vascularized bone graft for defect healing brings a bright future for clinical application.

#### **4.2. Induction of angiogenesis and vasculogenesis**

Angiogenesis and vasculogenesis are natural vascularization process that occurs in tissue development and wound healing. The endothelial cells function as the main mediator of neovascularization through forming new blood vessels by angiogenesis and they can be expanded by vasculogenesis. In some studies, endothelial cells were used to generate capillary-like structure and connect vessels in vitro [73]. However, it is unclear as to whether these vascular generation approaches are effective in inducing vascularization in engineered bone graft in vivo.

In addition to directly using endothelial cells to form vessel network, some growth factors related to angiogenesis are used as another method to improve vascularization in engineered tissue. These factors include vascular endothelium growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) [74]. A major advantage of utilizing growth factors instead of living cells is that the risk of host rejection can be excluded. However, it seems inefficient using growth factors alone, because of the random formation of new vessels within implanted bone [68]. More importantly, the inappropriate delivery of growth factors may induce excessive angiogenesis which could cause severe pathogenic process such as tumour development, atheroscleosis, and proliferative retinopathies [68, 71].

#### **4.3. Scaffold design to promote vascular formation**

Aside from the selection of molecular and cellular mediator for effective vascularization, the choice of material used is also closely related to the vascular formation ability of the engineered bone graft. The scaffolds have been designed to allow vascular in-growth through a macro‐ porous structure and incorporating vascular cues such as with the use of growth factors and/ or cells, rather than just serving as osteoconductive surfaces [75].

Over the past years, the selection of material candidates for bone tissue engineering scaffolds has been focused on the compatibility of bone cell attachment and growth. But now, much attention has also been switched to homing vascular formation [76]. The materials can impact the vascularization outcome of the bone graft from two aspects: 1) supporting the endothelial cells growth and forming vessels; 2) incorporating active molecules that help to introduce vascular formation. For example, the usage of silk fibroin and polycaprolactone (PCL) as components for 3D porous scaffolds exhibited a good support to endothelial cell growth and consequent vascularization [69, 77]; while poly lactic-co-glycolic acid (PLGA) and poly lactideco-glycolide (PLG) scaffolds showed the ability to incorporate VEGFs and release them locally so that angiogenesis was improved [78, 79].

Additionally, the structural properties, such as geometry and porosity can also affect the angiogenic ability of the scaffolds. Narayan and Venkatraman reported that the pore size of scaffolds have a profound influence on the growth of endothelial cells, with enhanced cell growth with smaller pore sizes and lower interpose distances [80]. Currently, the influence of scaffold design on its osteoconductivity and vascular conductivity is still unclear and deserves more investigations [81].

#### **4.4. The use of stem cells for neo-vascularisation**

Compared to mature endothelial cells, stem/progenitor cell candidates has been shown to exhibit higher proliferative capacity and differentiation potential [82]. Endothelial progenitor cells undergo vasculogenesis and have been shown to improve vascularization through the release of a milieu of angiogenic factors [83, 84].

In addition to using endothelial progenitor cells to induce vascularization, stem cell candidates are also needed for bone formation. Therefore, a co-culture system of the cells from different lineages has been proposed by various groups which reviewed comprehensively by Liu et all [72]. Several attempts have been made in generating vascularized bone graft for defect healing through co-culture systems, combine endothelial lineages with osteogenic cell types on different types of scaffolds [85-89].We have recently shown that co-cultured human fetal mesenchymal stem cells (hfMSC) and umbilical cord blood derived endothelial progenitor cells (UCB-EPC) seeded on PCL-TCP macroporous scaffolds induced more neo-vasculariza‐ tion and better bone formation, compared with the use of hfMSC alone [88].
