GAMBA - Gene Activated Matrices for Bone and Cartilage Regeneration in Arthritis

Introduction: Mesenchymal stem cells (MSCs) are considered a promising cell source for cartilage repair strategies due to their chondrogenic differentiation potential. However, their in-vitro tendency to progress towards hypertrophy limits their clinical use. This unfavourable result may be due to the fact that MSCs used in tissue engineering approaches are all at the same developmental stage, and have lost crucial spatial and temporal signalling cues. In this study, we sought to investigate the effect of a spatial parathyroid hormone-related protein (PTHrP) signalling gradient on the chondrogenic differentiation of MSCs and progression to hypertrophy. Methods: Human bone marrow-derived MSCs were transduced with adenoviral vectors overexpressing PTHrP and seeded into fibrin-poly(ester-urethane) scaffolds. To investigate the effect of a spatial PTHrP signalling gradient, scaffolds were seeded with PTHrP-overexpressing MSCs positioned on top of the scaffold, with untransduced MSCs seeded evenly within. Scaffolds were cultured with or without 2 ng/ml TGF-β1 for 28 days. Results: PTHrP overexpression increased GAG production by MSCs irrespective of TGF-β1 treatment, and exerted differential effects on chondrogenic and hypetrophic gene expression when MSCs were cultured in the presence of a PTHrP signalling gradient. Furthermore, PTHrP-overexpressing MSCs were associated with an increase of endogenous TGF-β1 production and reduced total MMP-13 secretion compared to controls. Conclusion: The presence of a spatial PTHrP signalling gradient may support chondrogenic differentiation of MSCs and promote the formation of a more stable cartilage phenotype in tissue engineering applications.

Background: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. Results: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. Conclusion: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

The GAMBA Consortium is developing a novel gene-activated matrix platform for bone and cartilage repair with a focus on osteoarthritis-related tissue damage. The scientific and technological objectives of this project are complemented with an innovative program of public outreach, actively linking patients and society to the evolvement of this project. The GAMBA platform will implement a concept of spatiotemporal control of regenerative bioactivity on command and demand. A gene activated matrix is a biomaterial with embedded gene vectors that will genetically modify cells embedded in or colonising the matrix. The platform comprises modules that self-adapt to the biological environment and that can be independently addressed with endogenous biological and exogenous physical or pharmacological stimuli, resulting in a temporally and spatially coordinated growth factor gene expression pattern. This reproduces, within the matrix, key elements of natural tissue formation. The modules are a biomimetic hyaluronan gel, a ceramic matrix, growth factor-encoding gene vector nanoparticles, magnetic nanoparticles and mesenchymal stem cells. Anatomical adaptivity is achieved with engineered thermal properties of the polymer matrix, which embeds other modules, selected according to functional requirements. Mechanical support is provided by Micro Macroporous Biphasic Calcium Phosphate (MBCP™), a resorbable material approved for clinical use. Spatiotemporal control of bioactivity and responsiveness to physiological conditions is represented, firstly, in the spatial distribution and release profiles of gene vectors within the composite matrix and, secondly, by letting local and external biological or physical stimuli activate the promoters driving the expression of vector-encoded growth factor transgenes. This concept is implemented by a multidisciplinary team from leading European institutions. Here, we report on the concepts, objectives and some preliminary results of the GAMBA project which is funded in 7th Framework Programme of the European Union THEME [NMP-2009-2.3-1], Biomimetic gels and polymers for tissue repair.