Towards intra-articular delivery for cartilaginous tissue regeneration

Abstract

In osteoarthritis (OA) and degenerate intervertebral discs (IVDs), chondrocytes and nucleus pulposus (NP) cells are responsible for producing secreted destructive proteinases to the extracellular matrix including collagenases, aggrecanases and metalloproteinases (MMPs). In addition, increased level of pro-inflammatory cytokines including interleukin-1 and tumor necrosis factor alpha (TNFα) are involved in catabolic effects, stimulating the cells to secrete proteolytic enzymes.

In addition to conventional therapies aiming merely at symptom relief, research focus toward regenerative approaches has emerged. For example, application of RNAi may be of great potential in the treatment of degenerative diseases of joints by inhibiting the local degenerative processes along with regenerative approaches.

In this thesis, strategies to deliver therapeutic compounds or small RNA molecules, with the use of biomaterials, are studied to ultimately treat degenerate musculoskeletal tissues toward regeneration by means of minimal invasive administration of therapeutic molecules, over a prolonged period of time.

A new in vitro inflammatory model was used to measure the bioactivity of anti-inflammatory agents released from novel poly(thioester)-functionalized PLGA microspheres. In the bioactivity assay where OA chondrocytes were batch-wise thawed for new cell culture every 3 days, prostaglandin E2 (PGE2) levels induced by TNFα were reduced extensively when microspheres loaded with 1 nmol of celecoxib (CXB) or triamcinolone acetonide (TA) were added. Gradually resumed PGE2 levels were observed within 21 days if 0.1 nmol of CXB or TA was loaded into microspheres.

The potential application of RNAi for treating cartilaginous diseases was examined from the aspects of safety and delivery efficiency. Non-specific effects of siRNA transfection with the use of lipid-based or naturally-derived transfection reagents were studied in different types of mesenchymal cells. Variable viability, changes in DNA content and differential cell cycle regulations all together showed the complexity of siRNA transfection, in addition to affecting target gene expression. A novel injectable and thermoresponsive hydrogel based on poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) was used and shown for its use in supporting siRNA delivery in OA chondrocytes. In another approach pre-transfection of hsa-miR-148a for modulating chondrogenic phenotype in OA chondrocytes, prior to incorporation in hydrogel systems as a means of strategic delivery, was studied. Although high levels of hsa-miR-148a were found until 14 days, the effect was limited to a decreased MMP13 expression compared to other previously shown regenerative effects.

To translate the application of the thermoresponsive hydrogel into future clinical practice, the hydrogel incorporated with the anti-inflammatory drug CXB was studied for safety and efficacy in a canine model with spontaneously degenerated IVD. After the intradiscal injection, no clinical symptoms (i.e. of IVD herniation or progression of degeneration) were observed. Controlled release of CXB resulted in a non-significant inhibition of PGE2, possibly because of the absence of its target, cyclooxygenase-2, in the model.

The studies presented in this thesis provided new insights into future regenerative therapies for OA or degenerate IVD, in a scope investigating safety and efficacy from in vitro to in vivo situations. These strategies have shown encouraging research outcomes. Further studies could promote translation into in vivo application and ultimately clinical practice.