Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair.

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like' printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2.

Mesenchymal stem cells (MSCs) are being exploited in regenerative medicine due to their tri-lineage differentiation and immunomodulation activity. Currently, there are two major challenges when directing the differentiation of MSCs for therapeutic applications. First, chemical and growth factor strategies to direct osteogenesis in vivo lack specificity for targeted delivery with desired effects. Second, MSC differentiation by gene therapy is difficult as transfection with existing approaches is clinically impractical (viral transfection) or have low efficacy (lipid-mediated transfection). These challenges can be avoided by directly delivering nonvirally derived recombinant protein transcription factors with the glycosaminoglycan-binding enhanced transduction (GET) delivery system (P21 and 8R peptides). We used the osteogenic master regulator, RUNX2 as a programming factor due to its stage-specific role in osteochondral differentiation pathways. Herein, we engineered GET-fusion proteins and compared sequential osteogenic changes in MSCs, induced by exposure to GET fusion proteins or conventional stimulation methods (dexamethasone and Bone morphogenetic protein 2). By assessing loss of stem cell-surface markers, upregulation of osteogenic genes and matrix mineralization, we demonstrate that GET-RUNX2 efficiently transduces MSCs and triggers osteogenesis by enhancing target gene expression directly. The high transduction efficiency of GET system holds great promise for stem cell therapies by allowing reproducible transcriptional control in stem cells, potentially bypassing problems observed with high-concentration growth-factor or pleiotropic steroid therapies. Stem Cells Translational Medicine 2017;6:2146-2159.

Controlled release of GAG-binding enhanced transduction (GET) peptides for sustained and highly efficient intracellular delivery.

Controlled release systems for therapeutic molecules are vital to allow the sustained local delivery of their activities which direct cell behaviour and enable novel regenerative strategies. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor signalling but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding domains which promote cell targeting, and cell penetrating peptides (CPPs) which allow cell entry. Herein we demonstrate that GET system can be used in controlled release systems to mediate sustained intracellular transduction over one week. We assessed the stability and activity of GET peptides in poly(dl-lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) prepared using a S/O/W double emulsion method. Efficient encapsulation (∼65%) and tailored protein release profiles could be achieved, however intracellular transduction was significantly inhibited post-release. To retain GET peptide activity we optimized a strategy of co-encapsulation of l-Histidine, which may form a complex with the PLGA degradation products under acidic conditions. Simulations of the polymer microclimate showed that hydrolytic acidic PLGA degradation products directly inhibited GET peptide transduction activity, and use of l-Histidine significantly enhanced released protein delivery. The ability to control the intracellular transduction of functional proteins into cells will facilitate new localized delivery methods and allow approaches to direct cellular behaviour for many regenerative medicine applications. STATEMENT OF SIGNIFICANCE: The goal for regenerative medicine is to restore functional biological tissue by controlling and augmenting cellular behaviour. Either Transcription (TFs) or growth factors (GFs) can be presented to cells in spatio-temporal gradients for programming cell fate and gene expression. Here, we have created a sustained and controlled release system for GET (Glycosaminoglycan-enhanced transducing)-tagged proteins using S/O/W PLGA microparticle fabrication. We demonstrated that PLGA and its acidic degradants inhibit GET-mediated transduction, which can be overcome by using pH-activated l-Histidine. l-Histidine inhibits the electrostatic interaction of GET/PLGA and allows enhanced intracellular transduction. GET could provide a powerful tool to program cell behaviour either in gradients or with sustained delivery. We believe that our controlled release systems will allow application of GET for tissue regeneration directly by TF cellular programming.