Personalized Scaffolds for Diabetic Foot Ulcer Healing Using Extracellular Matrix from Induced Pluripotent Stem-Reprogrammed Patient Cells.

Diabetic foot ulcers (DFU) are chronic wounds sustained by pathological fibroblasts and aberrant extracellular matrix (ECM). Porous collagen-based scaffolds (CS) have shown clinical promise for treating DFUs but may benefit from functional enhancements. Our previous work showed fibroblasts differentiated from induced pluripotent stem cells are an effective source of new ECM mimicking fetal matrix, which notably promotes scar-free healing. Likewise, functionalizing CS with this rejuvenated ECM showed potential for DFU healing. Here, we demonstrate for the first time an approach to DFU healing using biopsied cells from DFU patients, reprogramming those cells, and functionalizing CS with patient-specific ECM as a personalized acellular tissue engineered scaffold. We took a two-pronged approach: 1) direct ECM blending into scaffold fabrication; and 2) seeding scaffolds with reprogrammed fibroblasts for ECM deposition followed by decellularization. The decellularization approach reduced cell number requirements and maintained naturally deposited ECM proteins. Both approaches showed enhanced ECM deposition from DFU fibroblasts. Decellularized scaffolds additionally enhanced glycosaminoglycan deposition and subsequent vascularization. Finally, reprogrammed ECM scaffolds from patient-matched DFU fibroblasts outperformed those from healthy fibroblasts in several metrics, suggesting ECM is in fact able to redirect resident pathological fibroblasts in DFUs towards healing, and a patient-specific ECM signature may be beneficial.

Mechanosignalling in cartilage: an emerging target for the treatment of osteoarthritis.

Mechanical stimuli have fundamental roles in articular cartilage during health and disease. Chondrocytes respond to the physical properties of the cartilage extracellular matrix (ECM) and the mechanical forces exerted on them during joint loading. In osteoarthritis (OA), catabolic processes degrade the functional ECM and the composition and viscoelastic properties of the ECM produced by chondrocytes are altered. The abnormal loading environment created by these alterations propagates cell dysfunction and inflammation. Chondrocytes sense their physical environment via an array of mechanosensitive receptors and channels that activate a complex network of downstream signalling pathways to regulate several cell processes central to OA pathology. Advances in understanding the complex roles of specific mechanosignalling mechanisms in healthy and OA cartilage have highlighted molecular processes that can be therapeutically targeted to interrupt pathological feedback loops. The potential for combining these mechanosignalling targets with the rapidly expanding field of smart mechanoresponsive biomaterials and delivery systems is an emerging paradigm in OA treatment. The continued advances in this field have the potential to enable restoration of healthy mechanical microenvironments and signalling through the development of precision therapeutics, mechanoregulated biomaterials and drug systems in the near future.

Layered Double Hydroxide as a Potent Non-viral Vector for Nucleic Acid Delivery Using Gene-Activated Scaffolds for Tissue Regeneration Applications.

Nonviral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit typically exhibiting lower transfection efficiencies. As a result, there remains a significant need for the development of a nonviral delivery system with low cytotoxicity and high transfection efficacy as a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide (LDH) as a nonviral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor physicochemical properties of the LDH-pDNA nanoparticles. However, the lower mass ratios required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to MSCs. Furthermore, incorporation of LDH-miRNA nanoparticles into collagen-nanohydroxyapatite scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.

Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing.

Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.

Scaffold-Based Delivery of Nucleic Acid Therapeutics for Enhanced Bone and Cartilage Repair.

Recent advances in tissue engineering have made progress toward the development of biomaterials capable of the delivery of growth factors, such as bone morphogenetic proteins, in order to promote enhanced tissue repair. However, controlling the release of these growth factors on demand and within the desired localized area is a significant challenge and the associated high costs and side effects of uncontrolled delivery have proven increasingly problematic in clinical orthopedics. Gene therapy may be a valuable tool to avoid the limitations of local delivery of growth factors. Following a series of setbacks in the 1990s, the field of gene therapy is now seeing improvements in safety and efficacy resulting in substantial clinical progress and a resurgence in confidence. Biomaterial scaffold-mediated gene therapy provides a template for cell infiltration and tissue formation while promoting transfection of cells to engineer therapeutic proteins in a sustained but ultimately transient fashion. Additionally, scaffold-mediated delivery of RNA-based therapeutics can silence specific genes associated with orthopedic pathological states. This review will provide an overview of the current state-of-the-art in the field of gene-activated scaffolds and their use within orthopedic tissue engineering applications. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1671-1680, 2019.