In Situ Transglutaminase Cross-Linking Improves Mechanical Properties of Self-Assembling Peptides for Biomedical Applications.

The development of three-dimensional (3D) biomaterials that mimic natural tissues is required for efficiently restoring physiological functions of injured tissues and organs. In the field of soft hydrogels, self-assembled peptides (SAPs) stand out as distinctive biomimetic scaffolds, offering tunable properties. They have garnered significant attention in nanomedicine due to their innate ability to self-assemble, resulting in the creation of fibrous nanostructures that closely mimic the microenvironment of the extracellular matrix (ECM). This unique feature ensures their biocompatibility and bioactivity, making them a compelling area of study over the past few decades. As they are soft hydrogels, approaches are necessary to enhance the stiffness and resilience of the SAP materials. This work shows an enzymatic strategy to selectively increase the stiffness and resiliency of functionalized SAPs using transglutaminase (TGase) type 2, an enzyme capable of triggering the formation of isopeptide bonds. To this aim, we synthesized a set of SAP sequences and characterized their cross-linking via rheological experiments, atomic force microscopy (AFM), thioflavin-T binding assay, and infrared spectroscopy (ATR-FTIR) tests. The results showed an improvement of the storage modulus of cross-linked SAPs at no cost of the maximum stress-at-failure. Further, in in vitro tests, we examined and validated the TGase capability to cross-link SAPs without hampering seeded neural stem cells (hNSCs) viability and differentiation, potentially leaving the door open for safe in situ cross-linking reactions in vivo.

Structural and Biomechanical Properties of Supramolecular Nanofiber-Based Hydrogels in Biomedicine.

The field of supramolecular nanofiber-based hydrogels in biomedicine has witnessed remarkable growth over the past two decades [...].

Dynamic surgical anatomy using 3D reconstruction technology in complex hepato-biliary surgery with vascular involvement. Results from an international multicentric survey.

INTRODUCTION: Three-dimensional liver modeling can lead to substantial changes in choosing the type and extension of liver resection. This study aimed to explore whether 3D reconstruction helps to better understand the relationship between liver tumors and neighboring vascular structures compared to standard 2D CT scan images. METHODS: Contrast-enhanced CT scan images of 11 patients suffering from primary and secondary hepatic tumors were selected. Twenty-three experienced HBP surgeons participated to the survey. A standardized questionnaire outlining 16 different vascular structures (items) having a potential relationship with the tumor was provided. Intraoperative and histopathological findings were used as the reference standard. The proper hypothesis was that 3D accuracy is greater than 2D. As a secondary endpoint, inter-raters' agreement was explored. RESULTS: The mean difference between 3D and 2D, was 2.6 points (SE: 0.40; 95 % CI: 1.7-3.5; p < 0.0001). After sensitivity analysis, the results favored 3D visualization as well (mean difference 1.7 points; SE: 0.32; 95 % CI: 1.0-2.5; p = 0.0004). The inter-raters' agreement was moderate for both methods (2D: W = 0.45; 3D: W = 0.44). CONCLUSION: 3D reconstruction may give a significant contribution to better understanding liver vascular anatomy and the precise relationship between the tumor and the neighboring structures.

Unleashing the Power of Artificial Intelligence in Materials Design.

The integration of artificial intelligence (AI) algorithms in materials design is revolutionizing the field of materials engineering thanks to their power to predict material properties, design de novo materials with enhanced features, and discover new mechanisms beyond intuition. In addition, they can be used to infer complex design principles and identify high-quality candidates more rapidly than trial-and-error experimentation. From this perspective, herein we describe how these tools can enable the acceleration and enrichment of each stage of the discovery cycle of novel materials with optimized properties. We begin by outlining the state-of-the-art AI models in materials design, including machine learning (ML), deep learning, and materials informatics tools. These methodologies enable the extraction of meaningful information from vast amounts of data, enabling researchers to uncover complex correlations and patterns within material properties, structures, and compositions. Next, a comprehensive overview of AI-driven materials design is provided and its potential future prospects are highlighted. By leveraging such AI algorithms, researchers can efficiently search and analyze databases containing a wide range of material properties, enabling the identification of promising candidates for specific applications. This capability has profound implications across various industries, from drug development to energy storage, where materials performance is crucial. Ultimately, AI-based approaches are poised to revolutionize our understanding and design of materials, ushering in a new era of accelerated innovation and advancement.

Computational Mechanics of Form-Fitting 3D-Printed Lattice-Based Wrist-Hand Orthosis for Motor Neuron Disease.

Motor neuron disease (MND) patients often experience hand-wrist muscle atrophy resulting in severe social consequences and hampering their daily activities. Although hand-wrist orthosis is commonly used to assist weakened muscles, its effectiveness is limited due to the rapid progression of the disease and the need for customization to suit individual patient requirements. To address these challenges, this study investigates the application of three-dimensional (3D) printing technology to design and fabricate two lattice structures inspired by silkworm cocoons, using poly-ε-caprolactone as feedstock material. Finite element method (FEM) analysis is employed to study the mechanical behavior, enabling control over the geometric configuration incorporated into the hand-wrist orthosis. Through tensile displacement and three-point bending simulations, the stress distribution is examined for both lattice geometries. Geometry-1 demonstrates anisotropic behavior, while geometry-2 exhibits no strict directional dependence due to its symmetry and uniform node positioning. Moreover, the biocompatibility of lattices with human skin fibroblasts is investigated, confirming excellent biocompatibility. Lastly, the study involves semi-structured interviews with MND patients to gather feedback and develop prototypes of form-fitting 3D-printed lattice-based hand-wrist orthosis. By utilizing 3D printing technology, this study aims to provide customized orthosis that can effectively support weakened muscles and reposition the hand for individuals with MND.

Biomimetic scaffolds using triply periodic minimal surface-based porous structures for biomedical applications.

The design of biomimetic porous scaffolds has been gaining attention in the biomedical sector lately. Shells, marine sponges, shark teeth, cancellous bone, sea urchin spine, and the armadillo armor structure are examples of biological systems that have already been studied to drive the design of innovative, porous, and multifunctional structures. Among these, triply periodic minimal surfaces (TPMSs) have attracted the attention of scientists for the fabrication of biomimetic porous scaffolds. The interest stems from their outstanding properties, which include mathematical controllable geometry features, highly interconnected porous architectures, high surface area to volume ratio, less stress concentration, tunable mechanical properties, and increased permeability. All these distinguishing features enable better cell adhesion, optimal integration to the surrounding tissue avoiding stress shieldings, a good permeability of fluid media and oxygen, and the possibility of vascularization. However, the sophisticated geometry of these TPMS-based structures has proven challenging to fabricate by conventional methods. The emergence of additive manufacturing (AM) and the enhanced manufacturing freedoms and flexibility it guarantees could solve some of the bottlenecks, thus leading to a surge of interest in designing and fabricating such structures in this field. Also, the feasibility of using AM technologies allows for obtaining size programmable TPMS printable in various materials, from polymers to metal alloys. Here, a comprehensive overview of 3D-printed TPMS porous structures is provided from a design for additive manufacturing (DfAM) and application perspective. First, design strategies, geometry design algorithms, and related topological optimization are introduced according to diverse requirements. Based on that, the performance control of TPMS and the pros and cons of the different AM processes for fabricating TPMS scaffolds are summarized. Lastly, practical applications of 3D-printed biomimetic TPMS porous structures for the biomedical field are presented to clarify the advantages and potential of such structures.

Human Neural Stem Cell-Based Drug Product: Clinical and Nonclinical Characterization.

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.

Quality Assessment of the Protein Ingredients Recovered by Ultrasound-Assisted Extraction from the Press Cakes of Coconut and Almond Beverage Preparation.

The manufacture of vegetal beverages has the drawback of producing large amounts of press cakes that are generally used as feed components. This work had the objective of valorizing the press cakes deriving from almond and coconut drinks production by using ultrasound-assisted extraction (UAE) to obtain protein ingredients for human use. Starting from coconut and almond press cakes, whose initial protein contents were 19.7% and 18.6%, respectively, the UAE treatment allowed liquid fractions to be obtained that were then freeze-dried: the extraction yields were 24.4 g dry extract/100 g press cake in case of coconut and 49.3 g dry extract/100 g press cake in case of almond. The protein contents of these dried materials were 30.10% and 22.88%, respectively. The quality of the extracted protein ingredients was assessed in term of phytic acid content, protein profile, techno-functional features, and antioxidant properties. The sonication had also a favorable effect on digestibility.

Exploitation of Olive (Olea europaea L.) Seed Proteins as Upgraded Source of Bioactive Peptides with Multifunctional Properties: Focus on Antioxidant and Dipeptidyl-Dipeptidase-IV Inhibitory Activities, and Glucagon-like Peptide 1 Improved Modulation.

Agri-food industry wastes and by-products include highly valuable components that can upgraded, providing low-cost bioactives or used as an alternative protein source. In this context, by-products from olive production and olive oil extraction process, i.e., seeds, can be fostered. In particular, this work was aimed at extracting and characterizing proteins for Olea europaea L. seeds and at producing two protein hydrolysates using alcalase and papain, respectively. Peptidomic analysis were performed, allowing to determine both medium- and short-sized peptides and to identify their potential biological activities. Moreover, an extensive characterization of the antioxidant properties of Olea europaea L. seed hydrolysates was carried out both in vitro by 2,2-diphenyl-1-picrylhydrazyl (DPPH), by ferric reducing antioxidant power (FRAP), and by 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, respectively, and at cellular level by measuring the ability of these hydrolysates to significant reduce the H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels in human intestinal Caco-2 cells. The results of the both hydrolysates showed significant antioxidant properties by reducing the free radical scavenging activities up to 65.0 ± 0.1% for the sample hydrolyzed with alcalase and up to 75.7 ± 0.4% for the papain hydrolysates tested at 5 mg/mL, respectively. Moreover, similar values were obtained by the ABTS assays, whereas the FRAP increased up to 13,025.0 ± 241.5% for the alcalase hydrolysates and up to 12,462.5 ± 311.9% for the papain hydrolysates, both tested at 1 mg/mL. According to the in vitro results, both papain and alcalase hydrolysates restore the cellular ROS levels up 130.4 ± 4.24% and 128.5 ± 3.60%, respectively, at 0.1 mg/mL and reduce the lipid peroxidation levels up to 109.2 ± 7.95% and 73.0 ± 7.64%, respectively, at 1.0 mg/mL. In addition, results underlined that the same hydrolysates reduced the activity of dipeptidyl peptidase-IV (DPP-IV) in vitro and at cellular levels up to 42.9 ± 6.5% and 38.7 ± 7.2% at 5.0 mg/mL for alcalase and papain hydrolysates, respectively. Interestingly, they stimulate the release and stability of glucagon-like peptide 1 (GLP-1) hormone through an increase of its levels up to 660.7 ± 21.9 pM and 613.4 ± 39.1 pM for alcalase and papain hydrolysates, respectively. Based on these results, olive seed hydrolysates may represent new ingredients with antioxidant and anti-diabetic properties for the development of nutraceuticals and functional foods for the prevention of metabolic syndrome onset.

Bioinspired photo-crosslinkable self-assembling peptides with pH-switchable "on-off" luminescence.

Significant progress has been made in peptide self-assembly over the past two decades; however, the in situ cross-linking of self-assembling peptides yielding better performing nanomaterials is still in its infancy. Indeed, self-assembling peptides (SAPs), relying only on non-covalent interactions, are mechanically unstable and susceptible to solvent erosion, greatly hindering their practical application. Herein, drawing inspiration from the biological functions of tyrosine, we present a photo-cross-linking approach for the in situ cross-linking of a tyrosine-containing LDLK12 SAP. This method is based on the ruthenium-complex-catalyzed conversion of tyrosine to dityrosine upon light irradiation. We observed a stable formation of dityrosine cross-linking starting from 5 minutes, with a maximum peak after 1 hour of UV irradiation. Furthermore, the presence of a ruthenium complex among the assembled peptide bundles bestows unusual fluorescence intensity stability up to as high as 42 °C, compared to the bare ruthenium complex. Also, due to a direct deprotonation-protonation process between the ruthenium complex and SAP molecules, the fluorescence of the photo-cross-linked SAP is capable of exhibiting "off-on-off-on" luminescence switchable from acid to basic pH. Lastly, we showed that the photo-cross-linked hydrogel exhibited enhanced mechanical stability with a storage modulus of ∼26 kPa, due to the formation of a densely entangled fibrous network of SAP molecules through dityrosine linkages. As such, this ruthenium-mediated photo-cross-linked SAP hydrogel could be useful in the design of novel tyrosine containing SAP materials with intriguing potential for biomedical imaging, pH sensing, photonics, soft electronics, and bioprinting.

Artificial Intelligence-Empowered 3D and 4D Printing Technologies toward Smarter Biomedical Materials and Approaches.

In the last decades, 3D printing has played a crucial role as an innovative technology for tissue and organ fabrication, patient-specific orthoses, drug delivery, and surgical planning. However, biomedical materials used for 3D printing are usually static and unable to dynamically respond or transform within the internal environment of the body. These materials are fabricated ex situ, which involves first printing on a planar substrate and then deploying it to the target surface, thus resulting in a possible mismatch between the printed part and the target surfaces. The emergence of 4D printing addresses some of these drawbacks, opening an attractive path for the biomedical sector. By preprogramming smart materials, 4D printing is able to manufacture structures that dynamically respond to external stimuli. Despite these potentials, 4D printed dynamic materials are still in their infancy of development. The rise of artificial intelligence (AI) could push these technologies forward enlarging their applicability, boosting the design space of smart materials by selecting promising ones with desired architectures, properties, and functions, reducing the time to manufacturing, and allowing the in situ printing directly on target surfaces achieving high-fidelity of human body micro-structures. In this review, an overview of 4D printing as a fascinating tool for designing advanced smart materials is provided. Then will be discussed the recent progress in AI-empowered 3D and 4D printing with open-loop and closed-loop methods, in particular regarding shape-morphing 4D-responsive materials, printing on moving targets, and surgical robots for in situ printing. Lastly, an outlook on 5D printing is given as an advanced future technique, in which AI will assume the role of the fifth dimension to empower the effectiveness of 3D and 4D printing for developing intelligent systems in the biomedical sector and beyond.

A New Polysaccharide Carrier Isolated from Camelina Cake: Structural Characterization, Rheological Behavior, and Its Influence on Purple Corn Cob Extract's Bioaccessibility.

A polysaccharide fraction obtained from camelina cake (CCP), selected as a carrier to encapsulate purple corn cob extract (MCE), was investigated. A wide population of carbohydrate polymers (with a polydispersivity index of 3.26 ± 0.07 and an average molecular weight of about 139.749 × 103 ± 4.392 × 103 g/mol) with a gel-like behavior and a thixotropic feature characterized the fraction. MCE-CCP combinations (50-50 and 25-75, w/w), selected based on CCP encapsulation efficiency, were tested for their stability and MCE polyphenols' bioaccessibility during digestion (monitored using an in vitro static procedure). During the oral and gastric phases of the digestion process, CCP gradually swelled and totally released MCE polyphenols. MCE-CCP50 had the fastest release. Moreover, anthocyanins were still detectable during the duodenal phase, in both MCE-CCP ingredients. Furthermore, CCP (5 mg/mL) exerted in vitro potential hypocholesterolemic activity via bile salts binding during digestion.

Selection and Optimization of an Innovative Polysaccharide-Based Carrier to Improve Anthocyanins Stability in Purple Corn Cob Extracts.

The extraction process of alcohol-insoluble polysaccharides from exhausted Moradyn cob (Zea mays L. cv. Moradyn) (EMCP), camelina cake (Camelina sativa L. Crantz) (CCP), and common bean seeds (Phaseolus vulgaris L.) (CBP) was investigated and optimized by Response Surface Methodology. Each fraction was tested at different core/carrier ratios in the encapsulation of Moradyn cob extract (MCE), a rich source of antioxidant anthocyanins, and the obtained ingredients were screened for their encapsulation efficiency (EE%) and extraction process sustainability. The ingredients containing 50% and 75% CCP had EE% higher than 60% and 80%, respectively, and were selected for further studies. Preliminary structural analysis indicated CCP was mostly composed of neutral polysaccharides and proteins in a random-coiled conformation, which was also unchanged in the ingredients. CCP-stabilizing properties were tested, applying an innovative stress testing protocol. CCP strongly improved MCE anthocyanins solid-state stability (25 °C, 30% RH), and therefore it could be an innovative anthocyanins carrier system.

Gel-Forming of Self-Assembling Peptides Functionalized with Food Bioactive Motifs Modulate DPP-IV and ACE Inhibitory Activity in Human Intestinal Caco-2 Cells.

Food bioactive peptides are increasingly used for formulating food products, nutraceuticals, and functional food, since they are generally considered safe for human consumption and metabolic syndrome prevention. They are also becoming popular as sustainable sources of novel functional biomaterials such as hydrogels, edible nanonutraceuticals, delivery systems, and packing materials. However, such food peptides are mostly unstable, and degrade during food processing, or in a gastrointestinal environment, thus resulting in low bioavailability precluding their practical applications. Here, we decided to functionalize the well-known and characterized self-assembling peptide RADA16 with two synthetic analogues of food bioactive peptides deriving from the hydrolysis of soybean glycinin and lupin ß-conglutin (namely IAVPTGVA and LTFPGSAED) for control of and improvement in their gel-forming nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), Thioflavin T (ThT) binding assay, rheological measurements, and Atomic Force Microscopy (AFM) analysis. Lastly, since self-assembling peptides (SAPs) can be co-assembled with diluent SAPs (without a bioactive epitope) as an approach to control the density of biological signals and therefore attain enhanced bioactivity, we investigated the effect of the co-assembly of RADA16 and functionalized food bioactive SAPs (dubbed cAP-Soy1 and cAP-Lup1) for the growth of Caco-2 human intestinal cells and contextually we characterized their biological activities as DPP-IV and ACE inhibitors, in order to demonstrate their potential use for the prevention of metabolic syndrome.

Boosted Cross-Linking and Characterization of High-Performing Self-Assembling Peptides.

Tissue engineering (TE) strategies require the design and characterization of novel biomaterials capable of mimicking the physiological microenvironments of the tissues to be regenerated. As such, implantable materials should be biomimetic, nanostructured and with mechanical properties approximating those of the target organ/tissue. Self-assembling peptides (SAPs) are biomimetic nanomaterials that can be readily synthesized and customized to match the requirements of some TE applications, but the weak interactions involved in the self-assembling phenomenon make them soft hydrogels unsuited for the regeneration of medium-to-hard tissues. In this work, we moved significant steps forward in the field of chemical cross-linked SAPs towards the goal of stiff peptidic materials suited for the regeneration of several tissues. Novel SAPs were designed and characterized to boost the 4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulpho-N-hydroxysuccinimide ester (Sulfo-SMCC) mediated cross-linking reaction, where they reached G' values of ~500 kPa. An additional orthogonal cross-linking was also effective and allowed to top remarkable G' values of 840 kPa. We demonstrated that cross-linking fastened the pre-existing self-aggregated nanostructures, and at the same time, a strong presence of ß-structures is necessary for an effective cross-linking of (LKLK)3-based SAPs. Combining strong SAP design and orthogonal cross-linking reactions, we brought SAP stiffness closer to the MPa threshold, and as such, we opened the door of the regeneration of skin, muscle and lung to biomimetic SAP technology.

Emerging technologies for management of patients with amyotrophic lateral sclerosis: from telehealth to assistive robotics and neural interfaces.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is characterized by the degeneration of both upper and lower motor neurons, which leads to muscle weakness and subsequently paralysis. It begins subtly with focal weakness but spreads relentlessly to involve most muscles, thus proving to be effectively incurable. Typically, death due to respiratory paralysis occurs in 3-5 years. To date, it has been shown that the management of ALS patients is best achieved with a multidisciplinary approach, and with the help of emerging technologies ranging from multidisciplinary teleconsults (for monitoring the dysphagia, respiratory function, and nutritional status) to brain-computer interfaces and eye tracking for alternative augmentative communication, until robotics, it may increase effectiveness. The COVID-19 pandemic created a spasmodic need to accelerate the development and implementation of such technologies in clinical practice, to improve the daily lives of both ALS patients and caregivers. However, despite the remarkable strides that have been made in the field, there are still issues to be addressed. This review will be discussed on the eureka moment of emerging technologies for ALS, used as a blueprint not only for neurodegenerative diseases, examining the current technologies already in place or being evaluated, highlighting the pros and cons for future clinical applications.

Nanostructure, Self-Assembly, Mechanical Properties, and Antioxidant Activity of a Lupin-Derived Peptide Hydrogel.

Naturally occurring food peptides are frequently used in the life sciences due to their beneficial effects through their impact on specific biochemical pathways. Furthermore, they are often leveraged for applications in areas as diverse as bioengineering, medicine, agriculture, and even fashion. However, progress toward understanding their self-assembling properties as functional materials are often hindered by their long aromatic and charged residue-enriched sequences encrypted in the parent protein sequence. In this study, we elucidate the nanostructure and the hierarchical self-assembly propensity of a lupin-derived peptide which belongs to the α-conglutin (11S globulin, legumin-like protein), with a straightforward N-terminal biotinylated oligoglycine tag-based methodology for controlling the nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), rheological measurements, and Atomic Force Microscopy (AFM) analyses. By using the biotin tag, we obtained a thixotropic lupin-derived peptide hydrogel (named BT13) with tunable mechanical properties (from 2 to 11 kPa), without impairing its spontaneous formation of ß-sheet secondary structures. Lastly, we demonstrated that this hydrogel has antioxidant activity. Altogether, our findings address multiple challenges associated with the development of naturally occurring food peptide-based hydrogels, offering a new tool to both fine tune the mechanical properties and tailor the antioxidant activities, providing new research directions across food chemistry, biochemistry, and bioengineering.

Assessment of the Physicochemical and Conformational Changes of Ultrasound-Driven Proteins Extracted from Soybean Okara Byproduct.

This study was aimed at the valorization of the okara byproduct deriving form soy food manufacturing, by using ultrasound at different temperatures for extracting the residual proteins. The physicochemical and conformational changes of the extracted proteins were investigated in order to optimize the procedure. Increasing the temperature from 20 up to 80 °C greatly enhanced the yields and the protein solubility without affecting the viscosity. The protein secondary and tertiary structures were also gradually modified in a significant way. After the ultrasonication at the highest temperature, a significant morphological transition from well-defined single round structures to highly aggregated ones was observed, which was confirmed by measuring the water contact angles and wettability. After the ultrasound process, the improvement of peptides generation and the different amino acid exposition within the protein led to an increase of the antioxidant properties. The integrated strategy applied in this study allows to foster the okara protein obtained after ultrasound extraction as valuable materials for new applications.

Self-assembling peptide hydrogels for the stabilization and sustained release of active Chondroitinase ABC in vitro and in spinal cord injuries.

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.

Cross-Linked Self-Assembling Peptides and Their Post-Assembly Functionalization via One-Pot and In Situ Gelation System.

Supramolecular nanostructures formed through peptide self-assembly can have a wide range of applications in the biomedical landscape. However, they often lose biomechanical properties at low mechanical stress due to the non-covalent interactions working in the self-assembling process. Herein, we report the design of cross-linked self-assembling peptide hydrogels using a one-pot in situ gelation system, based on 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide/N-hydroxysulfosuccinimide (EDC/sulfo-NHS) coupling, to tune its biomechanics. EDC/sulfo-NHS coupling led to limited changes in storage modulus (from 0.9 to 2 kPa), but it significantly increased both the strain (from 6% to 60%) and failure stress (from 19 to 35 Pa) of peptide hydrogel without impairing the spontaneous formation of ß-sheet-containing nano-filaments. Furthermore, EDC/sulfo-NHS cross-linking bestowed self-healing and thixotropic properties to the peptide hydrogel. Lastly, we demonstrated that this strategy can be used to incorporate bioactive functional motifs after self-assembly on pre-formed nanostructures by functionalizing an Ac-LDLKLDLKLDLK-CONH2 (LDLK12) self-assembling peptide with the phage display-derived KLPGWSG peptide involved in the modulation of neural stem cell proliferation and differentiation. The incorporation of a functional motif did not alter the peptide's secondary structure and its mechanical properties. The work reported here offers new tools to both fine tune the mechanical properties of and tailor the biomimetic properties of self-assembling peptide hydrogels while retaining their nanostructures, which is useful for tissue engineering and regenerative medicine applications.