Peptide-Protein Coassemblies into Hierarchical and Bioactive Tubular Membranes.

Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.

Peptide Amphiphile Hydrogels Based on Homoternary Cucurbit[8]uril Host-Guest Complexes.

Supramolecular hydrogels based on peptide amphiphiles (PAs) are promising materials for tissue engineering and model extracellular matrixes for biological studies. While PA hydrogels are conventionally formed via electrostatic screening, new hydrogelation mechanisms might help to improve the design and functionality of these materials. Here, we present a host-guest-mediated PA hydrogelation method that relies on the formation of a host-guest homoternary complex with cucurbit[8]uril (CB[8]) and aromatic amino-acid-bearing PA nanofibers. As a result of the host-guest cross-linking between PA nanofibers, hierarchical morphologies and increased stiffness were found when host-guest-mediated PA hydrogels were compared to their ion-based equivalents. Additionally, both families of hydrogels exhibited similar biocompatibilities. These results demonstrate that CB[8]-mediated hydrogelation can be used as an alternative cross-linking method to upgrade the design of PA materials and extend their biomedical applications.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

Self-Assembling Hydrogels Based on a Complementary Host-Guest Peptide Amphiphile Pair.

Supramolecular polymer-based biomaterials play a significant role in current biomedical research. In particular, peptide amphiphiles (PAs) represent a promising material platform for biomedical applications given their modular assembly, tunability, and capacity to render materials with structural and molecular precision. However, the possibility to provide dynamic cues within PA-based materials would increase the capacity to modulate their mechanical and physical properties and, consequently, enhance their functionality and broader use. In this study, we report on the synthesis of a cationic PA pair bearing complementary adamantane and ß-cyclodextrin host-guest cues and their capacity to be further incorporated into self-assembled nanostructures. We demonstrate the possibility of these recognition motifs to selectively bind, enabling noncovalent cross-linking between PA nanofibers and endowing the resulting supramolecular hydrogels with enhanced mechanical properties, including stiffness and resistance to degradation, while retaining in vitro biocompatibility. The incorporation of the host-guest PA pairs in the resulting hydrogels allowed not only for macroscopic mechanical control from the molecular scale, but also for the possibility to engineer further spatiotemporal dynamic properties, opening opportunities for broader potential applications of PA-based materials.

Multicomponent self-assembly as a tool to harness new properties from peptides and proteins in material design.

Nature is enriched with a wide variety of complex, synergistic, and highly functional protein-based multicomponent assemblies. As such, nature has served as a source of inspiration for using multicomponent self-assembly as a platform to create highly ordered, complex, and dynamic protein and peptide-based nanostructures. Such an assembly system relies on the initial interaction of distinct individual building blocks leading to the formation of a complex that subsequently assembles into supramolecular architectures. This approach not only serves as a powerful platform for gaining insight into how proteins co-assemble in nature but also offers huge opportunities to harness new properties not inherent in the individual building blocks. In the past decades, various multicomponent self-assembly strategies have been used to extract synergistic properties from proteins and peptides. This review highlights the updates in the field of multicomponent self-assembly of proteins and peptides and summarizes various strategies, including covalent conjugation, ligand-receptor interactions, templated/directed assembly and non-specific co-assembly, for driving the self-assembly of multiple proteins and peptide-based building blocks into functional materials. In particular, we focus on peptide- or protein-containing multicomponent systems that, upon self-assembly, enable the emergence of new properties or phenomena. The ultimate goal of this review is to highlight the importance of multicomponent self-assembly in protein and peptide engineering, and to advocate its growth in the fields of materials science and nanotechnology.

Trauma induces overexpression of Cx43 in human fetal membrane defects.

OBJECTIVE: We developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects. METHOD: Term human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining. RESULTS: After trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture. CONCLUSION: Cx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Connexin 43 is overexpressed in human fetal membrane defects after fetoscopic surgery.

OBJECTIVE: We examined whether surgically induced membrane defects elevate connexin 43 (Cx43) expression in the wound edge of the amniotic membrane (AM) and drives structural changes in collagen that affects healing after fetoscopic surgery. METHOD: Cell morphology and collagen microstructure was investigated by scanning electron microscopy and second harmonic generation in fetal membranes taken from women who underwent fetal surgery. Immunofluoresence and real-time quantitative polymerase chain reaction was used to examine Cx43 expression in control and wound edge AM. RESULTS: Scanning electron microscopy showed dense, helical patterns of collagen fibrils in the wound edge of the fetal membrane. This arrangement changed in the fibroblast layer with evidence of collagen fibrils that were highly polarised along the wound edge but not in control membranes. Cx43 was increased by 112.9% in wound edge AM compared with controls (p < 0.001), with preferential distribution in the fibroblast layer compared with the epithelial layer (p < 0.01). In wound edge AM, mesenchymal cells had a flattened morphology, and there was evidence of poor epithelial migration across the defect. Cx43 and COX-2 expression was significantly increased in wound edge AM compared with controls (p < 0.001). CONCLUSION: Overexpression of Cx43 in the AM after fetal surgery induces morphological and structural changes in the collagenous matrix that may interfere with normal healing mechanisms. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Sparsely PEGylated poly(beta-amino ester) polyplexes enhance antigen specific T-cell response of a bivalent SARS-CoV-2 DNA vaccine.

DNA technology has emerged as a promising route to accelerated manufacture of sequence agnostic vaccines. For activity, DNA vaccines must be protected and delivered to the correct antigen presenting cells. However, the physicochemical properties of the vector must be carefully tuned to enhance interaction with immune cells and generate sufficient immune response for disease protection. In this study, we have engineered a range of polymer-based nanocarriers based on the poly(beta-amino ester) (PBAE) polycation platform to investigate the role that surface poly(ethylene glycol) (PEG) density has on pDNA encapsulation, formulation properties and gene transfectability both in vitro and in vivo. We achieved this by synthesising a non-PEGylated and PEGylated PBAE and produced formulations containing these PBAEs, and mixed polyplexes to tune surface PEG density. All polymers and co-formulations produced small polyplex nanoparticles with almost complete encapsulation of the cargo in all cases. Despite high gene transfection in HEK293T cells, only the fully PEGylated and mixed formulations displayed significantly higher expression of the reporter gene than the negative control in dendritic cells. Further in vivo studies with a bivalent SARS-CoV-2 pDNA vaccine revealed that only the mixed formulation led to strong antigen specific T-cell responses, however this did not translate into the presence of serum antibodies indicating the need for further studies into improving immunisation with polymer delivery systems.

Disassembly of self-assembling peptide hydrogels as a versatile method for cell extraction and manipulation.

Self-assembling peptide hydrogels (SAPHs) are increasingly being used as two-dimensional (2D) cell culture substrates and three-dimensional (3D) matrices due to their tunable properties and biomimicry of native tissues. Despite these advantages, SAPHs often represent an end-point in cell culture, as isolating cells from them leads to low yields and disruption of cells, limiting their use and post-culture analyses. Here, we report on a protocol designed to easily and effectively disassemble peptide amphiphile (PA) SAPHs to retrieve 3D encapsulated cells with high viability and minimal disruption. Due to the pivotal role played by salt ions in SAPH gelation, tetrasodium ethylenediaminetetraacetic acid (Na4EDTA) was used as metal chelator to sequester ions participating in PA self-assembly and induce a rapid, efficient, clean, and gentle gel-to-sol transition. We characterise PA disassembly from the nano- to the macro-scale, provide mechanistic and practical insights into the PA disassembly mechanism, and assess the potential use of the process. As proof-of-concept, we isolated different cell types from cell-laden PA hydrogels and demonstrated the possibility to perform downstream biological analyses including cell re-plating, gene analysis, and flow cytometry with high reproducibility and no material interference. Our work offers new opportunities for the use of SAPHs in cell culture and the potential use of cells cultured on SAPHs, in applications such as cell expansion, analysis of in vitro models, cell therapies, and regenerative medicine.

A Self-Assembled 3D Model Demonstrates How Stiffness Educates Tumor Cell Phenotypes and Therapy Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.

Considerations of growth factor and material use in bone tissue engineering using biodegradable scaffolds in vitro and in vivo.

Bone tissue engineering aims to harness materials to develop functional bone tissue to heal 'critical-sized' bone defects. This study examined a robust, coated poly(caprolactone) trimethacrylate (PCL-TMA) 3D-printable scaffold designed to augment bone formation. Following optimisation of the coatings, three bioactive coatings were examined, i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA), fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) applied sequentially (PEA/FN/BMP-2) and iii) both ELP and PEA/FN/BMP-2 coatings applied concurrently. The scaffold material was robust and showed biodegradability. The coatings demonstrated a significant (p < 0.05) osteogenic response in vitro in alkaline phosphatase gene upregulation and alkaline phosphatase production. The PCL-TMA scaffold and coatings supported angiogenesis and displayed excellent biocompatibility following evaluation on the chorioallantoic membrane assay. No significant (p < 0.05) heterotopic bone formed on the scaffolds within a murine subcutaneous implantation model, compared to the positive control of BMP-2 loaded collagen sponge following examination by micro-computed tomography or histology. The current studies demonstrate a range of innovative coated scaffold constructs with in vitro efficacy and clearly illustrate the importance of an appropriate in vivo environment to validate in vitro functionality prior to scale up and preclinical application.

Tissue engineering for articular cartilage repair--the state of the art.

Articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach - combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.

Synthetic extracellular matrices with function-encoding peptides.

The communication of cells with their surroundings is mostly encoded in the epitopes of structural and signalling proteins present in the extracellular matrix (ECM). These peptide epitopes can be incorporated in biomaterials to serve as function-encoding molecules to modulate cell-cell and cell-ECM interactions. In this Review, we discuss natural and synthetic peptide epitopes as molecular tools to bioengineer bioactive hydrogel materials. We present a library of functional peptide sequences that selectively communicate with cells and the ECM to coordinate biological processes, including epitopes that directly signal to cells, that bind ECM components that subsequently signal to cells, and that regulate ECM turnover. We highlight how these epitopes can be incorporated in different biomaterials as individual or multiple signals, working synergistically or additively. This molecular toolbox can be applied in the design of biomaterials aimed at regulating or controlling cellular and tissue function, repair and regeneration.

Intestinal Cell Differentiation and Phenotype in 2D and 3D Cell Culture Models.

Three-dimensional (3D) culture models are more physiologically relevant than two-dimensional (2D) cell culture models. 2D approaches cannot reproduce the complexity of the tumor microenvironment and are less able to translate biological insights; and drug response studies have many limitations to be extrapolated to the clinics. Here, we use the Caco-2 colon cancer cell line, which is an immortalized human epithelial cell line that under specific conditions can polarize and differentiate into a villus-like phenotype. We describe cell differentiation and cell growth in both 2D and 3D culture conditions, concluding that cell morphology, polarity, proliferation and differentiation are highly dependent on the type of cell culture system.

Predictors and efficacy of surgical treatment for consecutive exotropia.

PURPOSE: To study the surgical outcomes of consecutive exotropia and predictive factors, and to compare the medial rectus (MR) advancement, lateral rectus (LR) recession, or a combination of both procedures. METHODS: A retrospective study of patients diagnosed with consecutive exotropia that were operated on (2000-2020) were included. The convergence was classified from 0 to +++, with good: ++/+++ and poor: 0/+. A good outcome was considered when the final horizontal deviation was < 10 prism diopters (pd). Follow-up since the surgery and the number of reoperations were registered. RESULTS: A total of 88 cases were analyzed, mean age: 33.98 ± 17.68 years (57.95%: women). The near and distance horizontal deviation mean (±SD) was 34.3 pd (±16.45) and 34.36 pd (±16.33), respectively. MR advancement was performed in 36.36%, LR recession in 27.27%, and a combination of both in 36.36%. Surgery was unilateral in 65.91% (bilateral in 34.09%). A good outcome was obtained in 69.32% and reoperations in 11.36%. The insufficiency convergence was associated with a bad outcome. The near horizontal deviation (P = .006), the vertical deviation (VD) association (P = .036) and the combination of both MR advancement and LR recession (P = .017) were predictors of a bad result. The mean follow-up was 56.5 months ± 57.65. CONCLUSION: A long-term good surgical result was obtained in most patients. The greatest near deviation, the VD association, and the combination of MR advancement and the LR recession were predictive factors for bad results.

Elastin-like recombinamer-mediated hierarchical mineralization coatings on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces improve biocompatibility.

The biocompatibility of biomedical materials is vital to their applicability and functionality. However, modifying surfaces for enhanced biocompatibility using traditional surface treatment techniques is challenging. We employed a mineralizing elastin-like recombinamer (ELR) self-assembling platform to mediate mineralization on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces, resulting in the modification of surface morphology and bioactivity while improving the biocompatibility of the material. We modulated the level of nanocrystal organization by adjusting the cross-linker ratio. Nanoindentation tests revealed that the mineralized configuration had nonuniformity with respect to Young's modulus and hardness, with the center areas having higher values (5.626 ± 0.109 GPa and 0.264 ± 0.022 GPa) compared to the edges (4.282 ± 0.327 GPa and 0.143 ± 0.023 GPa). The Scratch test results indicated high bonding strength (2.668 ± 0.117 N) between the mineralized coating and the substrate. Mineralized Zr-16Nb-xTi (x = 4,16 wt%) alloys had higher viability compared to untreated alloys, which exhibited high cell viability (>100 %) after 5 days and high alkaline phosphatase activity after 7 days. Cell proliferation assays indicated that MG 63 cells grew faster on mineralized surfaces than on untreated surfaces. Scanning electron microscopy imaging confirmed that the cells adhered and spread well on mineralized surfaces. Furthermore, hemocompatibility test results revealed that all mineralized samples were non-hemolytic. Our results demonstrate the viability of employing the ELR mineralizing platform to improve alloy biocompatibility.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Integrating top-down and self-assembly in the fabrication of peptide and protein-based biomedical materials.

The capacity to create an increasing variety of bioactive molecules that are designed to assemble in specific configurations has opened up tremendous possibilities in the design of materials with an unprecedented level of control and functionality. A particular challenge involves guiding such self-assembling interactions across scales, thus precisely positioning individual molecules within well-organized, highly-ordered structures. Such hierarchical control is essential if peptides and proteins are to serve as both structural and functional building blocks of biomedical materials. To achieve this goal, top-down techniques are increasingly being used in combination with self-assembling systems to reproducibly manipulate, localize, orient and assemble peptides and proteins to form organized structures. In this tutorial review we provide insight into how both standard and novel top-down techniques are being used in combination with peptide or protein self-assembly to create a new generation of functional materials.

Embracing complexity in biomaterials design.

Animate materials, man-made materials behaving like living systems, are attracting enormous interest across a range of sectors, from construction and transport industry to medicine. In this leading opinion article, we propose that embracing complexity in biomaterials design offers untapped opportunities to create biomaterials with innovative life-like properties that extend their capabilities and unleash new paradigms in medical treatment.

Exploiting the fundamentals of biological organization for the advancement of biofabrication.

The field of biofabrication continues to progress, offering higher levels of spatial control, reproducibility, and functionality. However, we remain far from recapitulating what nature has achieved. Biological systems such as tissues and organs are assembled from the bottom-up through coordinated supramolecular and cellular processes that result in their remarkable structures and functionalities. In this perspective, we propose that incorporating such biological assembling mechanisms within fabrication techniques, offers an opportunity to push the boundaries of biofabrication. We dissect these mechanisms into distinct biological organization principles (BOPs) including self-assembly, compartmentalization, diffusion-reaction, disorder-to-order transitions, and out-of-equilibrium processes. We highlight recent work demonstrating the viability and potential of these approaches to enhance scalability, reproducibility, vascularization, and biomimicry; as well as current challenges to overcome.