Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors

Abstract

Rapid vascularisation of tissue-engineered osteogenic grafts is a major obstacle in the development of regenerative medicine approaches for bone repair. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth. We investigated a cell-based gene therapy approach to generate osteogenic grafts with an increased vascularization potential in an ectopic nude rat model in vivo, by genetically modifying human bone marrow-derived stromal/stem cells (BMSC) to express rat VEGF. BMSC were loaded onto silicate-substituted apatite granules, which are a clinically established osteo-conductive material. Eight weeks after implantation, the vascular density of constructs seeded with VEGF-BMSC was 3-fold greater than with control cells, consisting of physiologically structured vascular networks with both conductance vessels and capillaries. However, VEGF specifically caused a global reduction in bone quantity, which consisted of thin trabeculae of immature matrix. VEGF did not impair BMSC engraftment in vivo, but strongly increased the recruitment of TRAP- and Cathepsin K-positive osteoclasts. These data suggest that VEGF over-expression is effective to improve the vascularization of osteogenic grafts, but also has the potential to disrupt bone homoeostasis towards excessive degradation, posing a challenge to its clinical application in bone tissue engineering.

Introduction

Tissue engineering is a promising strategy for the repair of large bone defects. For this purpose, bone marrow-derived mesenchymal stromal/stem cells (BMSC) constitute a rich source of osteoprogenitors [1]. However, one of the major limiting factors towards the clinical implementation of BMSC-based tissue-engineered osteogenic grafts is the need to rapidly provide a sufficient blood supply after implantation to ensure their engraftment. In fact, the lack of vascularization in the centre of large cell-loaded constructs invariably leads to ischaemia followed by necrosis [2], [3]. Apart from managing the supply of nutrients and oxygen to the cells and the removal of metabolic products, necessary for the survival, growth and differentiation of the engrafted cells, blood vessels also allow the recruitment of highly-specialised cells, like circulating osteoprogenitors, haematopoietic stem cells or monocytes, which can contribute to tissue regeneration and remodelling [4], [5], [6].

Several approaches to accelerate vascularization of tissue-engineered bone grafts upon implantation are currently being investigated [7]. These include surgical techniques, like flap- or arteriovenous loop-fabrication [8], [9], [10], biomaterial-based methods, like nano-/micro-fibre combined scaffolds or scaffold microfabrication designed to facilitate vascular in-growth [10], [11], as well as the co-culture of osteogenic and vasculogenic precursor cells inside the grafts [12], [13], [14]. Another strategy is the supply of pro-angiogenic factors [11]. Among these, Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth both in normal and pathological angiogenesis and is therefore the most attractive and well-characterized factor for inducing the therapeutic growth of new blood vessels [15]. On one hand, VEGF has a very short half-life in vivo, but on the other hand its expression needs to be sustained for approximately 4 weeks in order to allow the stabilization and persistence of newly formed vessels [16], [17], [18]. Therefore, the use of recombinant protein is challenging, since a continuous delivery of the factor is required. A stable, long-term production directly inside the tissue is more practically achieved by gene therapy, i.e. the delivery of the genetic sequence to allow a continuous secretion of the factor by the target cells [19].

Therefore, here we tested the hypothesis that sustained expression of VEGF by genetically modified human BMSC could generate osteogenic grafts with an increased pro-angiogenic potential and efficient bone formation in vivo.