Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors
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.
Section snippets
BMSC isolation and culture
Human primary BMSC were isolated from bone marrow aspirates. The aspirates were obtained from the iliac crest of healthy donors during routine orthopaedic surgical procedures according to established protocols, after informed consent by the patients and following protocol approval by the local ethical committee (EKBB, Ref. 78/07). Cells were isolated and cultured as described [20]. Briefly, after centrifugation, the pellet was washed in PBS (Invitrogen, Grand Island, NY, USA), resuspended in
Generation of VEGF-expressing BMSC
Human BMSC were transduced to express rat VEGF linked to a truncated form of CD8a as a FACS-detectable cell surface marker (VEGF-BMSC). Rat VEGF was used in order to avoid an antibody response against the secreted factor after in vivo implantation in nude rats. Naïve BMSC and BMSC transduced with a retroviral vector expressing only the truncated CD8a marker (CD8-BMSC) were used as controls. Flow cytometry analysis showed that transduction efficiencies of >90% were routinely achieved by the
Discussion
In this study we found that sustained over-expression of VEGF by genetically modified human BMSC was effective to improve vascularization of tissue-engineered bone grafts, leading to a 2- to 3-fold increase in vessel density compared to naïve cells and the generation of normal, mature and physiologically organized vascular networks. However, our data indicate that this approach also has the potential to substantially increase osteoclast recruitment and bone resorption, which led to a net
Conclusions
Our data indicate that VEGF over-expression from genetically modified human BMSC is an effective strategy to improve the vascularization of osteogenic grafts, but also has the potential to impair bone tissue formation by increasing osteoclast recruitment and bone resorption. Therefore, the equilibrium between VEGF-triggered angiogenesis, osteogenesis and bone resorption needs to be carefully investigated in controlled models in order to devise rational strategies that exploit the pro-angiogenic
Acknowledgements
The authors gratefully acknowledge Francine Wolf (Basel University Hospital) for technical help with in situ hybridization and Dr. Andreas Goessl for generously supplying Tisseel® fibrin glue. This work was supported by an Intramural Research Grant of the Department of Surgery (Basel University Hospital), by the European Union FP7 grant MAGISTER (CP-IP 214685) and by the Swiss National Science Foundation grants 127426 and 143898 to A.B. and grants 120432 and 138519 to A.S. and I.M.
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