Developing reservoir systems for therapeutic delivery of biomolecules to a degenerated disc

Abstract

Low back pain has severe consequences for the quality of life of the affected patients and strong evidence suggests that this condition is caused by degeneration of the intervertebral disc (IVD). Disc degeneration coincides with alterations in structure and composition of the extracellular matrix (ECM) and a loss of anabolic potential by the disc cells. Unfortunately, current therapies can only provide symptomatic relief without addressing the underlying biological problem. The failure in regenerating a degenerated disc not only aggravates the patient¿s discomfort but also risks to impair the ability of the spine to absorb shocks, with consequent spreading of degeneration to the adjacent discs. The unique physiology of the IVD and the disruption of a vast array of intercellular signalling mechanisms in disc degeneration are posing serious challenges for the tissue engineering of the disc. In fact, unimodal strategies such as cell delivery or growth factor delivery alone had only moderate success. For this reason, this project synergistically combines cell and gene therapy approaches to trigger regeneration in a key region of the IVD, the nucleus pulposus (NP). The main objective is to develop an optimal microenvironment, tailored for the NP, in which stem cells can be programmed to secrete specific proteins of interest. Overall, the project has three phases. In the first phase, a three dimensional (3D) type II collagen/hyaluronan microgel platform was designed to deliver adipose-derived stem cells (ASCs) to the nucleus pulposus (NP). The macromolecular concentration of the microgel was tailored to prime desired cellular phenotype and ultimately to favour the process of stem cell differentiation towards a NP-like phenotype. The second phase consisted in the fabrication of a microsphere reservoir system for the delivery of non-viral gene vectors such as polyplexes. The key features of this reservoir system are high control over size and shape of the microspheres and also the use of type II collagen as a building block for their fabrication. In the last phase, the therapeutic potential of the platforms developed was characterized and 3D microgels were functionalized with polyplex-loaded microsphere reservoirs. These collagen reservoirs shielded the cells from the toxicity of the polyplexes, while allowing high levels of transfection. Although lower levels of transfection were observed in 3D microgels than in monolayer, ASCs embedded in 3D microgels could be transfected for a prolonged period and, as a result, functional transgenic proteins were released from the 3D microgel system. Hence, the functionalization of 3D microgels with polyplex-loaded microsphere reservoirs allows for the production of cell factories able to manufacture targeted therapeutic proteins. This offers great potential for regenerative therapies of the NP.