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Self-assembling processes are ubiquitous phenomena that drive the organization and the hierarchical formation of complex molecular systems. The investigation of assembling dynamics, emerging from the interactions among biomolecules like amino-acids and polypeptides, is fundamental to determine how a mixture of simple objects can yield a complex structure at the nano-scale level. In this paper we present HyperBeta, a novel open-source software that exploits an innovative algorithm based on hyper-graphs to efficiently identify and graphically represent the dynamics of $$\beta$$ β -sheets formation. Differently from the existing tools, HyperBeta directly manipulates data generated by means of coarse-grained molecular dynamics simulation tools (GROMACS), performed using the MARTINI force field. Coarse-grained molecular structures are visualized using HyperBeta ’s proprietary real-time high-quality 3D engine, which provides a plethora of analysis tools and statistical information, controlled by means of an intuitive event-based graphical user interface. The high-quality renderer relies on a variety of visual cues to improve the readability and interpretability of distance and depth relationships between peptides. We show that HyperBeta is able to track the $$\beta$$ β -sheets formation in coarse-grained molecular dynamics simulations, and provides a completely new and efficient mean for the investigation of the kinetics of these nano-structures. HyperBeta will therefore facilitate biotechnological and medical research where these structural elements play a crucial role, such as the development of novel high-performance biomaterials in tissue engineering, or a better comprehension of the molecular mechanisms at the basis of complex pathologies like Alzheimer’s disease.

Self-assembly peptides (SAPs) are an important class of hydrogels used in nanomedicine for tissue repair and neural regeneration. Due to their unique properties, SAPs may be used in a wide range of applications but some limitations, such as low bioavailability and rapid hydrolysis degradation, need to be overcome. Here, we describe the synthesis and characterization of two novel cyclic SAPs without the use of D/L-alternating amino acids, showing a reversible transition of their supramolecular nanostructures, from nanotubes/nanofibers into nanovesicles/nanospheres. The investigation, characterization and optimization are performed using atomic force microscopy (AFM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, Raman analysis, circular dichroism (CD), and rheology measurements. Also, in vitro cell viability assays show negligible toxicity of the representative optimized cyclic SAP towards human neural stem cells (hNSCs). Our results suggest that linear SAP theoretical background can be applied to develop cyclic SAPs, with important implications in the scalable fabrication of inter-changeable nanostructures, as well as for biomedical applications, including tissue regeneration, drug-delivery, drug-design, sensing, imaging, and size selectivity.

Activated microglia/macrophages infiltration, astrocyte migration, and increased production of inhibitory chondroitin sulfate proteoglycans (CSPGs) are standard harmful events taking place after the spinal cord injuries (SCI). The gliotic scar, viz. the outcome of chronic SCI, constitutes a long-lasting physical and chemical barrier to axonal regrowth. In the past two decades, various research groups targeted the hostile host microenvironments of the gliotic scar at the injury site. To this purpose, biomaterial scaffolds demonstrate to provide a promising potential for nervous cell restoration. We here focused our efforts on two self-assembling peptides (SAPs), featuring different self-assembled nanostructures, and on different methods of drug loading to exploit the neuroregenerative potential of Chondroitinase ABC (ChABC), a thermolabile pro-plastic agent attenuating the inhibitory action of CSPGs. Enzymatic activity of ChABC (usually lasting less than 72 hours in vitro) released from SAPs was remarkably detected up to 42 days in vitro. ChABC was continuously released in vitro from a few days to 42 days as well. Also, injections of ChABC loaded SAP hydrogels favored host neural regeneration and behavioral recovery in chronic SCI in rats. Hence, SAP hydrogels showed great promise for the delivery of Chondroitinase ABC in future therapies targeting chronic SCI.

Editorial of a Research Topic

Self-assembling peptides (SAPs) are a promising class of biomaterials amenable to easy molecular design and functionalization. Despite their increasing usage in regenerative medicine, a detailed analysis of their biomechanics at the nanoscale level is still missing. In this work, we propose and validate, in all-atom dynamics, a coarse-grained model to elucidate strain distribution, failure mechanisms and biomechanical effects of functionalization of two SAPs when subjected to both axial stretching and bending forces. We highlight different failure mechanisms for fibril seeds and fibrils, as well as the negligible contribution of the chosen functional motif to the overall system rupture. This approach could lay the basis for the development of "more" coarse-grained models in the long pathway connecting SAP sequences and hydrogel mechanical properties.

Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials.

Significance Cells reside in 3D microenvironments in living tissues; consequently, 3D cultures gained great interest because they better mimic the natural conditions of cells. Self-assembling peptides (SAPs) are synthetic bioabsorbable biomaterials that can provide customized 3D microenvironments regulating cell functionalities and tissue repair. Here we introduce a SAP-hydrogel designed to support human neural stem cell (hNSC) differentiation in 3D serum-free conditions, generating mature and active human neurons in vitro. We also demonstrate its functional neurorigenerative potential in rat spinal cord injuries, peaking when seeded with hNSCs progeny predifferentiated in vitro for 6 weeks. Despite these promising results, this approach should be confirmed in the future with medium-size animal models and with additional and refined behavioral tests before entering a clinical trial., Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the “engineered tissue” was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Self-assembling peptides are an exemplary class of supramolecular biomaterials of broad biomedical utility. Mechanistic studies on the peptide self-assembly demonstrated the importance of the oligomeric intermediates towards the properties of the supramolecular biomaterials being formed. In this study, we demonstrate how the overall yield of the supramolecular assemblies are moderated through subtle molecular changes in the peptide monomers. This strategy is exemplified with a set of surfactant-like peptides (SLPs) with different β-sheet propensities and charged residues flanking the aggregation domains. By integrating different techniques, we show that these molecular changes can alter both the nucleation propensity of the oligomeric intermediates and the thermodynamic stability of the fibril structures. We demonstrate that the amount of assembled nanofibers are critically defined by the oligomeric nucleation propensities. Our findings offer guidance on designing self-assembling peptides for different biomedical applications, as well as insights into the role of protein gatekeeper sequences in preventing amyloidosis.

Self‐assembling and molecular folding are ubiquitous in Nature: they drive the organization of systems ranging from living creatures to DNA molecules. Elucidating the complex dynamics underlying these phenomena is of crucial importance. However, a tool for the analysis of the various phenomena involved in protein/peptide aggregation is still missing. Here, an innovative software is developed and validated for the identification and visualization of b‐structuring and b‐sheet formation in both simulated systems and crystal structures of proteins and peptides. The novel software suite, dubbed Morphoscanner, is designed to identify and intuitively represent b‐structuring and b‐sheet formation during molecular dynamics trajectories, paying attention to temporary strand‐strand alignment, suboligomer formation and evolution of local order. Self‐assembling peptides (SAPs) constitute a promising class of biomaterials and an interesting model to study the spontaneous assembly of molecular systems in vitro. With the help of coarse‐grained molecular dynamics the self‐assembling of diverse SAPs is simulated into molten aggregates. When applied to these systems, Morphoscanner highlights different b‐structuring schemes and kinetics related to SAP sequences. It is demonstrated that Morphoscanner is a novel versatile tool designed to probe the aggregation dynamics of self‐assembling systems, adaptable to the analysis of differently coarsened simulations of a variety of biomolecules.

Self-assembling peptides (SAP) have drawn an increasing interest in the tissue engineering community. They display unquestionable biomimetic properties, tailorability and promising biocompatibility. However their use has been hampered by poor mechanical properties making them fragile soft scaffolds. To increase SAP hydrogel stiffness we introduced a novel strategy based on multiple ramifications of (LDLK)3, a well-known linear SAP, connected with one or multiple “lysine knots”. Differently branched SAPs were tested by increasing the number of (LDLK)3-like branches and by adding the neuro-regenerative functional motif BMHP1 as a single branch. While pure branched peptides did not have appealing self-assembling propensity, when mixed with the corresponding linear SAP they increased the stiffness of the overall hydrogel of multiple times. Notably, optimal results (or peak) were obtained 1) at similar molar ratio (between linear and branched peptides) for all tested sequences and 2) for the branched SAPs featuring the highest number of branches made of (LDLK)3. The functional motif BMHP1, as expected, seemed not to contribute to the increase of the storage modulus as efficiently as (LDLK)3. Interestingly, branched SAPs improved the β-sheet self-arrangement of (LDLK)3 and allowed for the formation of assembled nanofibers. Indeed in coarse-grained molecular dynamics we showed they readily integrate in the assembled aggregates providing “molecular connections” among otherwise weakly paired β-structures. Lastly, branched SAPs did not affect the usual response of human neural stem cells cultured on (LDLK)3-like scaffolds in vitro. Hence, branched SAPs may be a valuable new tool to enhance mechanical properties of self-assembling peptide biomaterials harmlessly; as neither chemical nor enzymatic cross-linking reactions are involved. As a consequence, branched SAPs may enlarge the field of application of SAPs in tissue engineering and beyond. Statement of Significance Self-assembling peptides stand at the forefront of regenerative medicine because they feature biomimetic nano-architectures that mimic the complexity of natural peptide-based extracellular matrices of living tissues. Their superior biocompatibility and ease of scale-up production are hampered by weak mechanical properties due to transient non-covalent interactions among and within the self-assembled peptide chains, thus limiting their potential applications. We introduced new branched self-assembling peptides to be used as “molecular connectors” among self-assembled nanostructures made of linear SAPs. Branched SAPs could be mixed with linear SAPs before self-assembling in order to have them intermingled with different β-sheets of linear SAPs after gelation. This strategy caused a manifold increase of the stiffness of the assembled hydrogels (proportional to the number of self-assembling branches), did not affect SAP propensity to form β-sheet but, instead, further stimulated their secondary structure rearrangements. It is now possible to modularly improve SAP scaffold mechanical properties without using harmful chemical reactions. Therefore, branched SAPs represent an additional tool to be adopted for efficient and harmless SAP scaffold customization in tissue engineering.

Self-assembling (SAPs) and co-assembling peptides (CAPs) are driving increasing enthusiasm as synthetic but biologically inspired biomaterials amenable of easy functionalization for regenerative medicine. On the other hand, electrospinning (ES) is a versatile technique useful for tailoring the nanostructures of various biomaterials into scaffolds resembling the extracellular matrices found in organs and tissues. The synergistic merging of these two approaches is a long-awaited advance in nanomedicine that has not been deeply documented so far. In the present work, we describe the successful ES of a library of diverse SAPs and CAPs into biomimetic nanofibrous mats. Our results suggest that suitable ES solutions are characterized by high concentrations of peptides, providing backbone physical chain entanglements, and by random coil/α-helical conformations while β-sheet aggregation may be detrimental to spinnability. The resulting peptide fibers feature interconnected seamless mats with nanofibers average diameters ranging from ∼100 nm to ∼400 nm. Also, peptide chemical nature and ES set up parameters play pivotal roles in determining the conformational transitions and morphological properties of the produced nanofibers. Far from being an exhaustive description of the just-opened novel field of ES-assembled peptides, this seminal work aims at shining a light on a still missing general theory for the production of electrospun peptidic biomaterials bringing together the spatial, biochemical and biomimetic of these two techniques into unique scaffolds for tissue engineering. Statement of Significance Construction of peptide hydrogels has received considerable attention due to their potential as nanostructures amenable of easy functionalization and capable of creating microenvironments suited for culturing cells and triggering tissue regeneration. They display a superior biocompatibility unmatched by other known synthetic biomaterials so far. However, their applications are confined to body fillers because most of them do spontaneously form hydrogels, while effective tissue regeneration often requires well-defined fibrous scaffolds. In this work, we developed electrospun fibers of various peptides (cross-beta self-assembling, hierarchically assembling, functionalized, co-assembling) and we provided a deep understanding of the crucial phenomena to be taken into account when peptides fibers fabrication. These results open new venues for exploring novel regenerative applications of peptide nanofibrous scaffolds.

Stem‐cell behavior is regulated by the material properties of the surrounding extracellular matrix, which has important implications for the design of tissue‐engineering scaffolds. However, our understanding of the material properties of stem‐cell scaffolds is limited to nanoscopic‐to‐macroscopic length scales. Herein, a solid‐state NMR approach is presented that provides atomic‐scale information on complex stem‐cell substrates at near physiological conditions and at natural isotope abundance. Using self‐assembled peptidic scaffolds designed for nervous‐tissue regeneration, we show at atomic scale how scaffold‐assembly degree, mechanics, and homogeneity correlate with favorable stem cell behavior. Integration of solid‐state NMR data with molecular dynamics simulations reveals a highly ordered fibrillar structure as the most favorable stem‐cell scaffold. This could improve the design of tissue‐engineering scaffolds and other self‐assembled biomaterials., In good molecular order: Solid‐state NMR spectroscopy demonstrates that atomic‐scale properties, including molecular order and mechanics, of tissue‐engineering scaffolds correlate with the viability of stem cell cultures. This insight could improve the design of tissue‐engineering scaffolds for regenerative medicine applications.

The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π–π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ∼8 nm to ∼70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G′) –from ∼1000 to ∼27 000 Pa – exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches.

The design of biomimetic scaffolds suitable for cell-based therapies is a fundamental step for the regeneration of the damaged nervous system; indeed growing interest is focusing on the discovery of peptide sequences to modulate the fate of transplanted cells and, in particular, the differentiation outcome of multipotent neural stem cells. By applying the Phage Display technique to murine neural stem cells we isolated a peptide, KLPGWSG, present in proteins involved in both stem cell maintenance and differentiation. We show that KLPGWSG binds molecules expressed on the cell surface of murine adult neural stem cells, thus may potentially be involved in stem cell fate determination. Indeed we demonstrated that this peptide in solution enhances per se cell differentiation toward the neuronal phenotype. Hence, we synthesized two LDLK-12-based self-assembling peptides functionalized with KLPGWSG peptide (KLP and Ac-KLP) and characterized them via atomic force microscopy, rheometry and circular dichroism, obtaining nanostructured hydrogels supporting murine neural stem cells differentiation in vitro. Interestingly, we demonstrated that, when scaffold stiffness is comparable to that of the brain in vivo, the Ac-KLP SAP-based scaffold enhances the neuronal differentiation of neural stem cells. These evidences place both KLPGWSG and the functionalized self-assembling peptide Ac-KLP as promising candidates for, respectively, biomimetic studies and stem cell therapies for nervous regeneration. © 2013 Elsevier B.V.