MAGNETICALLY ACTUATED THERAPY FOR BONE REGENERATION

This research explores heterodimeric gold/iron nanoparticles as targeted radiofrequency (RF)-activated oligonucleotide delivery vectors as a means to provide spatiotemporal control of stem cell differentiation for the repair of segmental bone defects. This proposal addresses the Nanomaterials for Bone Regeneration focus area described in the Peer Reviewed Medical Research Program Discovery Award program announcement and addresses the areas of encouragement for (1) technologies addressing segmental/large bone defects in craniomaxillofacial and/or load-bearing regions and (2) research on nanomaterials-based methods to facilitate recruitment of endogenous cell populations for enhanced bone regeneration and osseointegration.Increasing survival of blast-injured patients has left many Service members with segmental bone defects and amputations. In total, 27% of battlefield injuries include injuries of the head and neck, 22% include of the upper extremity, and 32% involve the lower extremity. The lack of success with current methodologies of vascularized bone transport (limited donor sites), bone grafting (commonly resorbs), distraction osteogenesis (impractical for craniofacial), and stem cell-based therapies (lacking reliable directed differentiation in vivo) are the major technical challenges of segmental bone reconstruction.To address this challenge, we propose the development of an improved RF-modulated Targeted Inducible Gene Regulation System (TIGeRS), composed of a magnetic, targeted heterodimeric gold and iron nanoparticle system delivering miRNA mimics that have been demonstrated to drive de novo osteogenesis and endotheliogenesis in stem/progenitor cells. The heterodimeric composition of the nanoparticle (i.e., having two distinct faces) provides two independent surface chemistries for the attachment of antibody targeting moieties and miRNA mimics through independent chemistries. The TIGeRS vector is targeted to endogenous mesenchymal stem cell (MSC) cell population in the defect via the antibody where it is internalized by endocytosis. After localization in the defect site, the TIGeRS are activated by RF induction, releasing the miRNA mimics, resulting in improved bone regeneration. The antibody targeting and RF-modulated activation provide multiple pathways for increasing spatial and temporal selectivity, key features of clinically relevant gene regulation strategies. The proposed methodology, utilizing endogenous derived MSC and progenitors, is attractive clinically as it requires no manipulation or delivery of exogenous MSC, substantially simplifying the clinical and regulatory paradigm.The specific aims are design to test the hypothesis that spatiotemporally modulating the cell proliferation and osteogenic pathways of endogneous stem cells by RF-stimulated post-transcriptional gene regulation, we can augment the osteogenic potential of endogenous stem/progenitor cells resulting in rapid bone regeneration and complete closure of critical sized bone defects. The specific aims are three-fold. Aim 1: Determine the impact of combination treatments of miR-148b mimics and miR-21on MSC proliferation and differentiation. Aim 2: Explore the impact of RF heating and particle targeting/transfection on MSC viability and differentiation. Aim 3: A limited scope in vivo trial will be conducted to determine basic TIGeRS pharmacokinetics and in vivo dose-response.The combination of antibody targeting and RF induction activation of TIGeRS provides a minimally invasive method to spatially and temporally control of gene regulation in complex, deep tissue wound environments. Additionally, the further development of this technique will provide a unique tool to study the impacts of gene regulation in localized tissue in vivo. The capability to spatiotemporally modulate stromal cell function in deep tissue provides clinicians a new, minimally invasive tool guide and enhance musculoskeletal tissue regeneration. The development of this technology represents a paradigm shift compared to current autograft or allograft reconstruction methods, which often fail as a result of poor vascularization of the graft or the use of bone morphogenic protein (BMP) containing hydrogels wherein the implantation often results in heterotopic ossification as a result of BMP diffusion into surrounding tissue.This research will provide critical foundation for understanding; (1) the spatiotemporal interplay of proliferative and osteogenic miRNA cues in the optimal formation of bone tissue and (2) the basic pharmacokinetics and dynamics of the TIGeRS vector in small animal model. Future research will build on these findings to develop a minimally invasive therapeutic to accelerate the repair of non-healing deep tissue bone defects.