The research aims at developing implants (hydrogels, polymeric scaffolds) delivering growth factors, drugs and cells that provide sustained delivery of bioactive molecules, support survival, infiltration and proliferation of cells for tissue engineering, and in particular spinal cord injury (Figure 1).
Our group has gained expertise in drug delivery to the spinal cord that we would like to combine with transplantation of adult mesenchymal stem cells; more particularly human dental stem cells. Indeed, human dental stem cells display superior neural stem cell properties than bone marrow-derived mesenchymal stem cells since they originate from the neural crest. We have evaluated the impact of hypoxia, an important pathological cue in the injured spinal cord, on differentiation of dental cells derived from the human apical papilla (SCAPs).
In our most recent works, we have incorporated SCAPs in different hydrogels that are now ready to be tested for their potential in spinal cord repair strategies.
We evaluated the effect of VEGF and GDNF delivery, free or encapsulated, from an alginate: fibrinogen hydrogel injected in a rat spinal cord hemisection model.
Local VEGF delivery from alginate: fibrinogen hydrogel gelifying in situ induced angiogenesis and neurite growth but no functional improvement. The animals treated with free GDNF-loaded hydrogel experienced superior functional recovery compared to the animals treated with GDNF microsphere-loaded hydrogels and non-treated animals (in collaboration with Prof. Blanco-Prieto, Navarra University, Spain, Drs Schakman and Deumens, UCL, IoNS)
When stem cells are implanted in vivo as part of strategies for regenerative strategies, it is not unusal for the cells to spend at least a week in hypoxia before a new vasculature develops. Our objective was to assess the impact of hypoxia on SCAP proliferation, differentiation and growth factor expression/production. We worked with a well-characterized primary cell line (RP89 cells) provided by our collaborator, Pr. Anibal Diogenes, from University of Texas at San Antonio (USA). We have grown SCAPs in hypoxia (1%O2) and in normoxia and compared growth rate and expression of stemness genes (SURVIVIN, CD90, and CD105), osteogenic (RUNX2 and ALP), adipogenic (ALBP), neurogenic (CNP, NSE, and SNAIL) lineages genes and growth factor genes (VEGFA, VEGFB, BFGF, TGF-b1, GDNF, and NT3). Impact of hypoxia on neuro-, osteo- and adipo-differentiation of SCAPs was also determined. We showed a clear impact of hypoxic culture conditions on the differentiation potential of SCAPs. In particular, the up-regulation of neuro- and osteospecific genes and the pro-angiogenic factor in SCAPs cultured in basal medium supports the potential of SCAPs to promote tissue regeneration. Hypoxia was also particularly favorable for neurodifferentiation, which is promising for neuroregenerative events. We are now looking in more details at the potential of hypoxia cultured SCAP for spinal cord repair.
Cell-based therapies are the most common approaches in regenerative medicine. One important characteristic for the ideal scaffold to be used in regenerative procedures is to provide appropriate conditions to ensure cell attachment, growth and differentiation. Despite significant advances in the use of dental stem cells in regenerative procedures, no study has previously evaluated the effect of SCAP encapsulation in alginate, Corgel®, and fibrin hydrogels on cellular survival, proliferation and neurodifferentiation. We studied the impact of SCAP encapsulation in different hydrogels. Incorporation in fibrin hydrogels of medium and high fibrinogen concentrations maintained SCAP viability, supported SCAP proliferation and neuro-differentiation in vitro but also allowed SCAP proliferation, collagen secretion and angiogenesis in vivo. Depending on the objective (SCAP viability, proliferation, growth factors secretion or neuro-differentiation) but also taking into account specific constrains (residence time, injectability, gelification time), a different fibrin formulation can be selected among the tested combinations. Fibrin hydrogels prepared with 30 mg/ml or 50 mg/ml of fibrinogen could be efficient and suitable SCAP scaffolds for CNS regeneration. Regarding SCAP incorporation in different alginates and Corgel®, our results demonstrated the important role of hydrogel properties on SCAP viability. This study highlights that not a single property, but the appropriate combination of surface (presence of adhesion sites and hydro-phobicity) and mechanical characteristics dictate SCAP fate.
Another promising type of hydrogel is extra cellular matrix (ECM)-based hydrogels. This type of hydrogel has been developed by Pr. Badylak (University of Pittsburgh, USA) and Pr. Shakesheff (University of Nottingham, UK) with whom we collaborate. ECM-based hydrogels present the advantages of being thermosensitive, thus injectable, and to share the same origin (tissue-wise) than the tissue to repair. ECM hydrogels have been obtained from various tissues like bone, bladder, brain and spinal cord. In the scope of an Erasmus Mundus program (NanoFar), a joint PhD thesis between the University of Nottingham and the Université catholique de Louvain is currently ongoing and deals with the potential of porcine spinal cord ECM hydrogel for spinal cord repair. Up to know we have studied the impact of SCAP culture in bone ECM and spinal cord hydrogel in vitro.
- In order to evaluate the impact of SCAP on spinal cord repair, we have delivered SCAP in fibrin hydrogel or a whole apical papilla in a spinal cord hemisection model and observed a significant functional recovery of the rats after 6 weeks when they were treated with apical papilla.
Figure 5: Drug and cell delivery in tissue engineering