Mapping the microcarrier design pathway to modernise clinical mesenchymal stromal cell expansion.

Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs.

A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces.

Mesenchymal stem cells (MSCs) used for clinical applications require in vitro expansion to achieve therapeutically relevant numbers. However, conventional planar cell expansion approaches using tissue culture vessels are inefficient, costly, and can trigger MSC phenotypic and functional decline. Here we present a one-step dry plasma process to modify the internal surfaces of three-dimensional (3D) printed, high surface area to volume ratio (high-SA:V) porous scaffolds as platforms for stem cell expansion. To address the long-lasting challenge of uniform plasma treatment within the micrometre-sized pores of scaffolds, we developed a packed bed plasma immersion ion implantation (PBPI3) technology by which plasma is ignited inside porous materials for homogeneous surface activation. COMSOL Multiphysics simulations support our experimental data and provide insights into the role of electrical field and pressure distribution in plasma ignition. Spatial surface characterisation inside scaffolds demonstrates the homogeneity of PBPI3 activation. The PBPI3 treatment induces radical-containing chemical structures that enable the covalent attachment of biomolecules via a simple, non-toxic, single-step incubation process. We showed that PBPI3-treated scaffolds biofunctionalised with fibroblast growth factor 2 (FGF2) significantly promoted the expansion of MSCs, preserved cell phenotypic expression, and multipotency, while reducing the usage of costly growth factor supplements. This breakthrough PBPI3 technology can be applied to a wide range of 3D polymeric porous scaffolds, paving the way towards developing new biomimetic interfaces for tissue engineering and regenerative medicine.

Stress-induced senescence in mesenchymal stem cells: Triggers, hallmarks, and current rejuvenation approaches.

Mesenchymal stem cells (MSCs) have emerged as promising cell-based therapies in the treatment of degenerative and inflammatory conditions. However, despite accumulating evidence of the breadth of MSC functional potency, their broad clinical translation is hampered by inconsistencies in therapeutic efficacy, which is at least partly due to the phenotypic and functional heterogeneity of MSC populations as they progress towards senescence in vitro. MSC senescence, a natural response to aging and stress, gives rise to altered cellular responses and functional decline. This review describes the key regenerative properties of MSCs; summarises the main triggers, mechanisms, and consequences of MSC senescence; and discusses current cellular and extracellular strategies to delay the onset or progression of senescence, or to rejuvenate biological functions lost to senescence.

Cellular modifications and biomaterial design to improve mesenchymal stem cell transplantation.

Research has advanced considerably since the first clinical trial of human mesenchymal stem cells (MSCs) in the early 1990s. During this period, our understanding of MSC biology and our ability to expand and manipulate these cells have provided hope for the repair of damaged tissues due to illness or injury. MSCs have conventionally been injected systemically or locally into target tissue; however, inconsistent cell homing and engraftment efficiencies represent a major bottleneck that has led to mixed results in clinical studies. To overcome these issues, MSCs have been pre-conditioned with biomolecules, genetically altered, or surface engineered to enhance their homing and engraftment capabilities. In parallel, a variety of cell-encapsulating materials have been designed to improve cell delivery and post-transplantation survival and function. In this review, we discuss the current strategies that have been employed on cultured MSCs to improve targeted cell delivery and retention for tissue repair. We also discuss the advances in injectable and implantable biomaterial technologies that drive the success of MSC-based therapies in regenerative medicine. Multi-faceted approaches combining cellular modification and cell-instructive material design can pave the way for efficient and robust stem cell transplantation for superior therapeutic outcomes.

Biomimetic Culture Strategies for the Clinical Expansion of Mesenchymal Stromal Cells.

Mesenchymal stromal/stem cells (MSCs) typically require significant ex vivo expansion to achieve the high cell numbers required for research and clinical applications. However, conventional MSC culture on planar (2D) plastic surfaces has been shown to induce MSC senescence and decrease cell functionality over long-term proliferation, and usually, it has a high labor requirement, a high usage of reagents, and therefore, a high cost. In this Review, we describe current MSC-based therapeutic strategies and outline the important factors that need to be considered when developing next-generation cell expansion platforms. To retain the functional value of expanded MSCs, ex vivo culture systems should ideally recapitulate the components of the native stem cell microenvironment, which include soluble cues, resident cells, and the extracellular matrix substrate. We review the interplay between these stem cell niche components and their biological roles in governing MSC phenotype and functionality. We discuss current biomimetic strategies of incorporating biochemical and biophysical cues in MSC culture platforms to grow clinically relevant cell numbers while preserving cell potency and stemness. This Review summarizes the current state of MSC expansion technologies and the challenges that still need to be overcome for MSC clinical applications to be feasible and sustainable.

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses.

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1.

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin-cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5ß1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5ß1 integrin-promoted attachment and spreading on domains 12-16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

A New Vascular Engineering Strategy Using 3D Printed Ice.

Vascular engineering requires integrating dimensional flexibility, strength, and bioactivity to fabricate materials that enable diffusive exchange of oxygen and nutrients between cells and their environment. A recent publication (Biomaterials 2019;192:334-345) has described a new method of creating freestanding, tailorable, and biocompatible vascular constructs by coating ice scaffolds with natural or synthetic polymers.

Soluble matrix protein is a potent modulator of mesenchymal stem cell performance.

We challenge the conventional designation of structural matrix proteins primarily as supporting scaffolds for resident cells. The extracellular matrix protein tropoelastin is classically regarded as a structural component that confers mechanical strength and resilience to tissues subject to repetitive elastic deformation. Here we describe how tropoelastin inherently induces a range of biological responses, even in cells not typically associated with elastic tissues and in a manner unexpected of typical substrate-dependent matrix proteins. We show that tropoelastin alone drives mesenchymal stem cell (MSC) proliferation and phenotypic maintenance, akin to the synergistic effects of potent growth factors such as insulin-like growth factor 1 and basic fibroblast growth factor. In addition, tropoelastin functionally surpasses these growth factors, as well as fibronectin, in allowing substantial media serum reduction without loss of proliferative potential. We further demonstrate that tropoelastin elicits strong mitogenic and cell-attractive responses, both as an immobilized substrate and as a soluble additive, via direct interactions with cell surface integrins αvß3 and αvß5. This duality of action converges the long-held mechanistic dichotomy between adhesive matrix proteins and soluble growth factors and uncovers the powerful, untapped potential of tropoelastin for clinical MSC expansion and therapeutic MSC recruitment. We propose that the potent, growth factor-like mitogenic and motogenic abilities of tropoelastin are biologically rooted in the need for rapid stem cell homing and proliferation during early development and/or wound repair.

Plasma-Activated Substrate with a Tropoelastin Anchor for the Maintenance and Delivery of Multipotent Adult Progenitor Cells.

Conventional wound therapy utilizes wound coverage to prevent infection, trauma, and fluid and thermal loss. However, this approach is often inadequate for large and/or chronic wounds, which require active intervention via therapeutic cells to promote healing. To address this need, a patch which delivers multipotent adult progenitor cells (MAPCs) is developed. Medical-grade polyurethane (PU) films are modified using plasma immersion ion implantation (PIII), which creates a radical-rich layer capable of rapidly and covalently attaching biomolecules. It is demonstrated that a short treatment duration of 400 s maximizes surface activation and wettability, minimizes reduction in gas permeability, and preserves the hydrolytic resistance of the PU film. The reactivity of PIII-treated PU is utilized to immobilize the extracellular matrix protein tropoelastin in a functional conformation that stably withstands medical-grade ethylene oxide sterilization. The PIII-treated tropoelastin-functionalized patch significantly promotes MAPC adhesion and proliferation over standard PU, while fully maintaining cell phenotype. Topical application of the MAPC-seeded patch transfers cells to a human skin model, while undelivered MAPCs repopulate the patch surface for subsequent cell transfer. The potential of this new wound patch as a reservoir for the sustained delivery of therapeutic MAPCs and cell-secreted factors for large and/or non-healing wounds is indicated in the findings.

Tropoelastin is a Flexible Molecule that Retains its Canonical Shape.

Tropoelastin is the dominant building block of elastic fibers, which form a major component of the extracellular matrix, providing structural support to tissues and imbuing them with elasticity and resilience. Recently, the atomistic structure of human tropoelastin is described, obtained through accelerated sampling via replica exchange molecular dynamics simulations. Here, principal component analysis is used to consider the ensemble of structures accessible to tropoelastin at body temperature (37 °C) at which tropoelastin naturally self-assembles into aggregated coacervates. These coacervates are relevant because they are an essential intermediate assembly stage, where tropoelastin molecules are then cross-linked at lysine residues and integrated into growing elastic fibers. It is found that the ensemble preserves the canonical tropoelastin structure with an extended molecular body flanked by two protruding legs, and identifies variations in specific domain positioning within this global shape. Furthermore, it is found that lysine residues show a large variation in their location on the tropoelastin molecule compared with other residues. It is hypothesized that this perturbation of the lysines increases their accessibility and enhances cross-linking. Finally, the principal component modes are extracted to describe the range of tropoelastin's conformational fluctuation to validate tropoelastin's scissor-twist motion that was predicted earlier.

Molecular model of human tropoelastin and implications of associated mutations.

Protein folding poses unique challenges for large, disordered proteins due to the low resolution of structural data accessible in experiment and on the basis of short time scales and limited sampling attainable in computation. Such molecules are uniquely suited to accelerated-sampling molecular dynamics algorithms due to a flat-energy landscape. We apply these methods to report here the folded structure in water from a fully extended chain of tropoelastin, a 698-amino acid molecular precursor to elastic fibers that confer elasticity and recoil to tissues, finding good agreement with experimental data. We then study a series of artificial and disease-related mutations, yielding molecular mechanisms to explain structural differences and variation in hierarchical assembly observed in experiment. The present model builds a framework for studying assembly and disease and yields critical insight into molecular mechanisms behind these processes. These results suggest that proteins with disordered regions are suitable candidates for characterization by this approach.

Plasma ion implantation enabled bio-functionalization of PEEK improves osteoblastic activity.

Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK.

Targeted Modulation of Tropoelastin Structure and Assembly.

Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and functionally atypical hydrogels, and markedly impaired and abnormal elastic fiber formation. Our work indicates the crucial significance of both E345 and E414 residues in modulating specific local structure and higher-order assembly of human tropoelastin.

A cell adhesive peptide from tropoelastin promotes sequential cell attachment and spreading via distinct receptors.

Tropoelastin is the dominant monomer that assembles to form elastin, which confers elasticity to vertebrate elastic tissues including skin, arteries, and lungs. Tropoelastin interacts with cells through cell surface receptors including integrins and glycosaminoglycans (GAGs). As the region 17-18 is recognized as a key region in cell attachment and spreading, we utilized C-terminal truncated tropoelastin constructs containing dissected sections of domain 18. We mapped a cell-interactive sequence of tropoelastin to domain 17 and the first six amino acids (aa) of domain 18. Further delineation identified a 21-residue sequence (Peptide 302-322) which promoted cell attachment and spreading indistinguishable from that to N18, a construct encompassing the full domains 17-18. Alanine substitution of the lysines at positions 11 and 14 in Peptide 302-322 effectively abolished cell binding. This reliance on lysines pointed to a role for GAGs, which was assessed by heparan sulfate inhibition, leading to 85.9 ± 4.2% decreased cell binding, while inhibition of integrins using ethylenediaminetetraacetic acid did not affect attachment. In contrast, selective antibody blocking of the integrin αv family prevented cell spreading by 92.5 ± 8.9%. We propose a two-step mechanism by which cell interactions occur at this central region of tropoelastin: initially, cell adhesion is mediated by GAGs, which contact the lysine residues within the target sequence, and subsequently facilitate cell spreading modulated by integrins, specifically αv ß3 and αv ß5 . We conclude that this region comprises a tropoelastin-derived, cell-interactive sequence that independently mediates potent cell binding and spreading via sequential recognition of GAG and integrin cell surface receptors.

A sterilizable, biocompatible, tropoelastin surface coating immobilized by energetic ion activation.

Biomimetic materials which integrate with surrounding tissues and regulate new tissue formation are attractive for tissue engineering and regenerative medicine. Plasma immersion ion-implanted (PIII) polyethersulfone (PES) provides an excellent platform for the irreversible immobilization of bioactive proteins and peptides. PIII treatment significantly improves PES wettability and results in the formation of acidic groups on the PES surface, with the highest concentration observed at 40-80 s of PIII treatment. The elastomeric protein tropoelastin can be stably adhered to PIII-treated PES in a cell-interactive conformation by tailoring the pH and salt levels of the protein-surface association conditions. Tropoelastin-coated PIII-treated PES surfaces are resistant to molecular fouling, and actively promote high levels of fibroblast adhesion and proliferation while maintaining cell morphology. Tropoelastin, unlike other extracellular matrix proteins such as fibronectin, uniquely retains full bioactivity even after medical-grade ethylene oxide sterilization. This dual approach of PIII treatment and tropoelastin cloaking allows for the stable, robust functionalization of clinically used polymer materials for directed cellular interactions.

Tropoelastin coated PLLA-PLGA scaffolds promote vascular network formation.

The robust repair of large wounds and tissue defects relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable tissue constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with ECM proteins to create a natural environment for the tissue-specific cells. Tropoelastin, the precursor of the elastin, is the ECM protein responsible for elasticity in diverse tissues, including robust blood vessels. Here, we seeded endothelial cells with supporting cells on PLLA/PLGA scaffolds treated with tropoelastin, and examined the morphology, expansion and maturity of the newly formed vessels. Our results demonstrate that the treated scaffolds elicit a more expanded, complex and developed vascularization in comparison to the untreated group. Implantation of tropoelastin-treated scaffolds into mouse abdominal muscle resulted in enhanced perfusion of the penetrating vasculature and improved integration. This study points to the great potential of these combined materials in promoting the vascularization of implanted engineered constructs, which can be further exploited in the fabrication of clinically relevant engineered tissues.

Subtle balance of tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly.

The assembly of the tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial ß sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule's assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly.