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Engineering Artificial Vascularized Bone Grafts for the Repair of Large Bone Defects
Liu Yuchun
National University of Singapore, Singapore

Clinical translation of bone tissue engineering (BTE) approaches for the repair of large bone defects is currently limited by poor vascularization, leading to consequential impaired bone repair. Despite bone having a natural healing ability, fractures beyond a certain size are incapable of spontaneous healing and hence, require surgical intervention for union. My PhD project focuses on the generation of vascularized bone grafts for the repair of large bone defects, using a co-culture of stem cells in conjunction with bioresorbable polymeric-ceramic scaffolds and bioreactor technologies. By leveraging on the osteogenic capacity of mesenchymal stem cells (MSC) and the vasculogenic potential of endothelial progenitor cells (EPC), it is hypothesized that the incorporation of EPC will improve osteogenicity and vascularity of the grafts, hence improving cell survival and aid in fracture repair.

Firstly, EPC and MSC were randomly mixed and cultured in a 2-dimensional monolayer culture. Direct contact of EPC/MSC demonstrated an increase in mineralization (Stage 1 bone formation) compared to MSC alone. This phenomenal observation was found to be mediated by the soluble factors secreted by EPC, where potent bone forming growth factors such as BMPs and TGF-ßs were identified in EPC conditioned media. Compared to the traditional function of EPC in augmenting vascularization, this research has identified a new role for EPC as an osteogenic regulator.

For the purpose of animal implantations, the co-culture was translated into a 3-dimensional scaffold. Fluorescently-labeled EPC (green) were observed to Liu YuchunNational University of Singapore, SingaporeEngineering Artificial Vascularized Bone Grafts for the Repair of Large Bone Defectsredistribute themselves amongst the MSC (red), forming a complex in vitro vessel-like network throughout the scaffold pores (Figure 1). The unique honeycomb-like architecture of the macro-porous polymeric/ceramic scaffold is believed to play an important role in providing a conducive support-frame for vessel development through its interconnected filament struts. Hypothesized that the action of biomechanical stimulation will aid in vessel development within the EPC/MSC-grafts, a biaxial bioreactor was used for culturing the cellular-grafts. Results however, showed that the dynamic bioreactor culture inhibited in vitro vessel formation. This finding is likely attributed to improved mass transport by the action of the bioreactor and hence, reduction in the hypoxic gradient generated within the sub-confluent cellular-scaffolds. This observation also unravels new insights on the scale-down culture models for optimizing bioreactor cultures for the facilitation of vessel and bone formation.

Next, subcutaneous mice implantations of cellular-grafts demonstrated improved vascularity, with the formation of a human-vessel network and greater infiltration of mouse-vessels in EPC/MSC-grafts compared to MSC alone. In a separate study, dynamically-cultured EPC/MSC-grafts were seen to exhibit higher levels of bone formation and earlier evidence of vasculogenesis in vivo compared to statically-cultured ones, suggesting the positive influence of biomechanical stimulation on graft maturation.

Additionally, it is well understood that hypoxia is the stimulus for angiogenesis, which triggers the entire cascade of signaling molecules necessary to induce vessel network formation. Experiments performed on endothelial cells under hypoxia showed the formation of an extensive in vitro vessel network with higher branch points as compared to under atmospheric oxygen tensions during differentiation on a Matrigel substrate. In relation to the osteogenicity of MSC, continuous hypoxic exposure of MSC monocultures demonstrated more robust osteogenic potential, suggesting the important role of hypoxia in retaining stem cell properties and improving therapeutic efficacy of the stem cells. Hence, the introduction of hypoxia is likely a critical parameter for consideration in cell culture systems.

Revisiting the natural physiology of bone, the reservoir of stem cells in the bone marrow niche resides in a low oxygen tension environment within the hollow interior of the trabecular bone. By leveraging on and understanding bone physiology and its properties, engineering solutions in this project have been designed to closely mimic the natural bone for efficacious tissue repair and regeneration.

In my opinion, the future of vascularized tissue engineering and regenerative medicine lies in an integrated approach of superior BTE components and the utility of co-culture sys-tems for accelerating vascularization. More investigations involving the optimization of the culture conditions of the bone graft, time of exposure of various biochemical, biome-chanical and micro-environmental cues are essential towards successful BTE strategies for clinical application. Additionally, the use of advanced imaging methodologies for real-time tracking of vascular development within osteogenic grafts will better aid in the understanding of these tissue-engineered co-cultures. Improvements in the spatial distribution of each cell type if seeded in a patterned manner would enable improved cell-cell interactions and formation of an organized vascular network throughout the large bone graft. It would be ideal if these bio-patterns could be imprinted onto a mechanically strong bioresorbable scaffold that is tailored to achieve similar strength and architecture as native tissues. Also, as opposed to current methods of in vitro graft maturation in bioreactors, continuous mechanical stimulation of the cellular-grafts in vivo could possibly play an important role in the long term success of the BTE-graft. Think about novel soft biodegradable micro-robots/actuators directly adhered to cell surfaces, providing a controlled and continuous mechanical stimulus to the cellular-graft in vivo until it fully degrades. It is envisioned to exert profound effects in accelerating and inducing more robust vessel networks, bone regeneration and improving functionality in the long term. This elegant micro-robot strategy could possibly replace current bioreactors, minimizing the time required for graft maturation and hence shortening the patient wait-time for bone graft implantation.

Now, imagine a large artificial bone graft that is well-infiltrated with an extensive network of vessels throughout the scaffold graft. The rapid reconnection and reperfusion with the patient’s blood vessel system is expected to accelerate bone repair and facilitate integration with host bone. Compared to the current bone graft strategies that are either invasive or having poor bone regeneration or remodeling efficacy, wouldn’t it be ideal to have such an efficacious off-the-shelf bone graft readily available for implantation?

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