Development of aptamer-enabled strategies for cartilage and bone regeneration applications

Abstract

Cartilage and bone defects are common skeletal diseases with a significant impact on health. People with cartilage lesions or bone damages have low quality of life due to continuous skeletal pain or stiffness, chronic inflammation at wounds, and emotional depression. Unattended skeletal impairment can progress to severe disability or even immobility. Although progress has been made in regenerative medicine, there still remains no effective treatments enabling fast and safe regeneration of damaged cartilage and bone tissues. The next generation of regenerative medicine aims to evolve tissue engineering strategies functionalized with advanced biomaterials to achieve improved tissue regeneration effects. Aptamers are remarkable biomolecules that enable specific and strong molecular interactions, making them attractive candidates for biomedical applications. For therapeutic applications, aptamers could serve as either drugs or drug carriers. Given the powerful properties of aptamers, this project aimed to apply DNA aptamers to develop advanced regenerative medicine for more efficient therapy of cartilage and bone impairments. This research includes three interlinked studies relating to aptamer application in cartilage and bone disease. First, two DNA aptamer inhibitor candidates for the osteoarthritis-associated biomarker, ADAMTS-5, were evaluated by determining their inhibitory effects on the aggrecanase activity of ADAMTS-5. Second, DNA aptamers against E.coli-expressed recombinant human bone morphogenetic protein 2 (rhBMP-2) were evolved using a bead-based systematic evolution of ligands by exponential enrichment (SELEX) strategy. Third, the selected BMP-2-binding aptamers were integrated with collagen to assemble fibrous scaffolds to promote the osteogenesis potential of BMP-2 for bone regeneration. In the ADAMTS-5 phase of this research, two G-rich DNA aptamers developed for ADAMTS-5 showed specific inhibition of aggrecanase activity of ADAMTS-5, suggesting they hold potential to be developed as therapeutics for osteoarthritis linked to ADAMTS-5. Regarding rhBMP-2 aptamers, the evolution of aptamers only succeeded using E.coli-expressed non-glycosylated rhBMP-2 but not on CHO-derived glycosylated targets. The identified DNA aptamer was an adenine-rich sequence that exhibited strong binding affinity and specificity to the target BMP-2. Serum stability results revealed that the BMP-2-binding aptamer was susceptible to nuclease degradation even when it was modified with 3’-end inverted thymidine, phosphorothioate substitutions, and circularization. However, the aptamer was significantly stabilized when incorporated into collagen scaffolds. Molecular interaction model of the aptamer-BMP-2 complex implied that the aptamer targeted BMP-2 mainly at heparin-binding domains, which was verified with empirical experiments using heparin as binding competitors. The BMP-2-binding aptamer was applied to assemble an extracellular matrix (ECM)-mimic scaffold with type I collagen. The assembly was spontaneous and yielded fibrous structures sized in micrometers. Aptamer-functionalized collagen scaffolds showed increased binding to BMP-2 and could significantly promote alkaline phosphatase (ALP) expression, cell adhesion, and wound healing process induced by BMP-2 in C2C12 cells. Such fibrous scaffolds hold promise to enhance the osteoinductive functionality of BMP-2 to achieve more efficient bone regeneration. This work envisions the potential of developing aptamer-mediated or aptamer-functionalized therapeutic strategies for cartilage and bone regeneration with enhanced efficacy and reduced side effects. This research forms a foundation for a plethora of applications of aptamers applied for cartilage and bone regeneration in regenerative medicine.