Poppy O. Smith

Peripheral nerve injuries impose a substantial strain on the lives of millions of people worldwide. Engineered neural tissues provide a promising avenue to improve peripheral nerve repair strategies, potentially allowing the precise seeding of cells and materials that support regenerative processes. Current optimization of cell-seeding strategies relies on testing the impact of relevant parameters in vivo, requiring considerable time and resources. We propose an alternative approach to the design of cellular hydrogels based on a mathematical cell–solute model, informed via in vitro experiment, to identify promising cell-seeding strategies in silico at a limited cost. These designs are manufactured using 3D-printed moulds and validated in vivo. We evaluate the regenerative potential of these designs by focusing on the impact of different cell-seeding strategies on vascular endothelial growth factor secretion and gradient generation, both crucial elements of regenerative angiogenesis in early nerve repair. In this way, we provide a first proof-of-concept of a digital twin for nerve tissue engineering, which uses in silico, in vitro and in vivo repair models.

Arteriovenous grafts are widely used for hemodialysis access, yet high failure rates remain a major clinical challenge. Decellularized blood vessels offer promising alternatives, but effective protocols must remove cellular and antigenic components while preserving extracellular matrix (ECM) integrity. This is particularly challenging in elastic arteries, where smooth muscle cells are anchored by dense actin filaments that are difficult to remove without ECM damage from harsh treatments. Here, we present a decellularization strategy based on actin-disrupting agents, specifically Latrunculin B, to facilitate removal of these dense cytoskeletal structures. The protocol integrates osmotic shock, high-ionic-strength salts, and Triton X-100 within a shortened processing time. Full-length porcine carotid arteries were decellularized using a dual-flow perfusion system, and five protocols were evaluated for removal of nuclear and immunogenic material, ECM preservation, mechanical properties, and cytocompatibility with human endothelial cells (HUVECs). All protocols substantially reduced nuclear material while preserving elastin, collagen, and mechanical integrity. Complete removal of immunogenic proteins was achieved with a final alkaline Triton X-100 wash (pH 8–14), highlighting the effectiveness of alkaline treatment in solubilizing membrane-bound antigens. All scaffolds supported HUVEC adhesion and formed a confluent endothelial monolayer within 8 days. Overall, integrating actin disruption via Latrunculin B with dual-salt–Triton processing, alkaline washing, and perfusion enhances decellularization efficiency, preserves ECM structure, reduces processing time and cost, and yields cytocompatible small-diameter vascular scaffolds with strong translational potential.

Glial cell line-derived neurotrophic factor (GDNF) is a neuroprotective protein with widespread applications in regenerative medicine, though its clinical translation has been limited. Peptide mimetics have the potential to overcome the limitations associated with the translation of GDNF. Herein, peptides targeting the GDNF receptor GFRα1 were identified by screening a 12-mer linear peptide phage display library against GFRα1. Peptide hits were rationalized by computational alanine scanning mutagenesis studies to identify residues contributing to GDNF and GFRα1 binding. Four hit peptides were synthesized and tested for GFRα1 binding affinity. All peptides activated the downstream phosphatidylinositol 3 kinase signaling pathway in SH-SY5Y cells and increased cellular proliferation. The biological activity of two peptides matched that of recombinant GDNF, as measured by neurite outgrowth of primary dorsal root ganglion neurons. For the first time, we report monomeric GFRα1-targeting peptides that mimic the biological effects of GDNF, with potential applications in regenerative medicine.

Background/Objectives: Endothelial cells play a key role in peripheral nerve regeneration, forming aligned vasculature which bridges the gap in the injured nerve tissue and guides the regrowing tissue. This work aimed to mimic key features of this aligned vasculature by differentiating endothelial cells from human induced pluripotent stem cells (hiPSCs) and incorporating them into engineered neural tissue (EngNT). Methods: hiPSCs were differentiated into endothelial cells with the temporal addition of growth factors and biomolecules. These hiPSC-derived endothelial cells (hiPSC-ECs) were incorporated into EngNT fabricated from collagen hydrogels using the gel aspiration-ejection (GAE) technique and maintained in vitro to allow endothelial network formation. Results: At the mRNA and protein level, pluripotency marker expression decreased and endothelial cell marker expression increased over the course of hiPSC differentiation to endothelial cells. The derived endothelial cells expressed CD31, CD144, ENG, VEGFR2, and VWF, and formed network structures in the matrix tubulogenesis assay. hiPSC-ECs incorporated into EngNT were viable and aligned. They formed highly aligned tube-like structures containing lumens after four days in culture and the EngNT constructs supported neurite growth in vitro when co-cultured with rat dorsal root ganglion (DRG) neurons. Conclusions: This work rapidly generated engineered nerve tissue containing highly aligned endothelial tube-like structures, resembling key features of the early nerve regeneration bridge. Therefore, this 3D engineered tissue provides a platform to study the effects of endothelial cell structures in nerve repair treatment and translational development.

During heart disease, the cardiac extracellular matrix (ECM) undergoes a structural and mechanical transformation. Cardiomyocytes sense the mechanical properties of their environment, leading to phenotypic remodeling. A critical component of the ECM mechanosensing machinery, including the protein talin, is organized at the cardiomyocyte costamere. Our previous work indicated a different talin tension, depending on the ECM stiffness, but the effects on downstream signaling remained elusive. Here, we identify that the talin interacting proteins DLC1 (deleted in liver cancer 1), RIAM (Rap1-interacting adaptor molecule), and paxillin each preferentially bind to talin at a specific ECM stiffness, this interaction is preserved in the absence of tension, and the interaction is regulated through focal adhesion kinase signaling. Moreover, DLC1 regulates cardiomyocyte RhoA activity in a stiffness-dependent way, whereby the loss of DLC1 results in myofibrillar disarray. Together, this study demonstrates a mechanism of imprinting mechanical information into the talin interactome to fine-tune RhoA activity, with impacts on cardiac health and disease.

Tissue engineering has the potential to overcome the limitations of using autografts in nerve gap repair, using cellular biomaterials to bridge the gap and support neuronal regeneration. Various types of therapeutic cells could be considered for use in aligned collagen-based engineered neural tissue (EngNT), including Schwann cells and their precursors, which can be derived from human induced pluripotent stem cells (hiPSCs). Using Schwann cell precursors may have practical advantages over mature Schwann cells as they expand readily in vitro and involve a shorter differentiation period. However, the performance of each cell type needs to be tested in EngNT. By adapting established protocols, hiPSCs were differentiated into Schwann cell precursors and Schwann cells, with distinctive molecular profiles confirmed using immunocytochemistry and RT-qPCR. For the first time, both cell types were incorporated into EngNT using gel aspiration–ejection, a technique used to align and simultaneously stabilise the cellular hydrogels. Both types of cellular constructs supported and guided aligned neurite outgrowth from adult rat dorsal root ganglion neurons in vitro. Initial experiments in a rat model of nerve gap injury demonstrated the extent to which the engrafted cells survived after 2 weeks and indicated that both types of hiPSC-derived cells supported the infiltration of host neurons, Schwann cells and endothelial cells. In summary, we show that human Schwann cell precursors promote infiltrating endogenous axons in a model of peripheral nerve injury to a greater degree than their terminally differentiated Schwann cell counterparts.

Engineered neural tissue (EngNT) is a stabilised aligned cellular hydrogel that offers a potential alternative to the nerve autograft for the treatment of severe peripheral nerve injury. This work aimed to automate the production of EngNT, to improve the feasibility of scalable manufacture for clinical translation. Endothelial cells were used as the cellular component of the EngNT, with the formation of endothelial cell tube-like structures mimicking the polarised vascular structures formed early on in the natural regenerative process. Gel aspiration-ejection for the production of EngNT was automated by integrating a syringe pump with a robotic positioning system, using software coded in Python to control both devices. Having established the production method and tested mechanical properties, the EngNT containing human umbilical vein endothelial cells (EngNT-HUVEC) was characterised in terms of viability and alignment, compatibility with neurite outgrowth from rat dorsal root ganglion neurons and formation of endothelial cell networks in vitro. EngNT-HUVEC manufactured using the automated system contained viable and aligned endothelial cells, which developed into a network of multinucleated endothelial cell tube-like structures inside the constructs and an outer layer of endothelialisation. The EngNT-HUVEC constructs were made in various sizes within minutes. Constructs provided support and guidance to regenerating neurites in vitro. This work automated the formation of EngNT, facilitating high throughput manufacture at scale. The formation of endothelial cell tube-like structures within stabilised hydrogels provides an engineered tissue with potential for use in nerve repair.

摘要: 随着我们从第四次工业革命向第五次工业革命的过渡，生物制造领域的兴起正在改变生命科学和医疗保健。3D生物打印、组织工程、定向组装和自组装等先进技术在创建仿生支架、组织类器官、医疗设备和生物系统方面发挥着重要作用。英国和爱尔兰的生物制造领域成为生物科学和医疗保健的关键力量，得益于尖端的研究和发展。这些地区的研究专注于药物输送、体外模型, 类器官,和组织工程的生物功能产品，致力于创新符合伦理标准的医疗保健解决方案，同时支持环保和有效的医疗保健系统。