Bioactive Motifs Research Articles

Effects of vegetative propagation on protein content and bioactivity of the red seaweed Palmaria palmata

Production of the red seaweed Palmaria palmata is currently hindered by a lack of standardised cultivation methods leading to uncertainties in yield and product quality. This study assessed vegetative propagation of meristematic fragments and the protein content and bioactivity potential of resulting plants. Growth was strong and sustained, averaging 5% day−1. Total protein contents initially decreased but recovered as the fragments grew larger and thicker. Samples displayed the highest antioxidant activity early in the experiment, suggesting that wounds may increase the secretion of antioxidant compounds. In silico analysis identified 762 potentially bioactive motifs, including 70 matching in vitro results. The newly discovered peptide SLLYSDITRPGGNMYTTR (SR18), linked to the pigment allophycocyanin, had very strong antioxidant properties and may drive the recorded in vitro activity. Vegetative propagation appears as a strong potential cultivation tool, and the utilised approach can be applied to assess the cultivation and nutritional potential of other seaweed species.

O10 Tuneable Caf1 protein polymers as an innovative and scalable solution for regenerative wound care

Abstract Introduction and aims Wound healing is a complex process essential for tissue restoration, where the extracellular matrix (ECM) plays a crucial role by providing structural and biochemical support to cells. Laminin 332, a key ECM component, is a promising target for protein-based therapeutics in view of its pathogenic role in junctional epidermolysis bullosa (JEB). Bioengineered capsular antigen F1 protein (Caf1) has emerged as a potential candidate molecule. We have developed the technology to enable bacteria to produce Caf1 polymers with multiple tailored bioactive motifs. Additionally, the unique properties of Caf1 minimize the potential for a host immune response. In this study, we engineered Caf1 proteins to express specific synthetic basement membrane components related to laminin 332 with the aim of restoring ECM equilibrium and achieving effective wound healing. Methods To evaluate Caf1 construct effects on human epidermal keratinocytes (HEKs) (CCD 1106) and human dermal fibroblasts (HDFs), scratch assays were performed under different treatment with Caf constructs. The Incucyte® ZOOM system (Sartorius AG, Göttingen, Germany) automated scratch wound and Live-Cell Analysis System screened constructs enhancing wound healing, capturing images every 2 h. Analysis involved quantifying the area under the curve of wound confluence (%) over 72 h to assess wound closure. Results The migration of HDFs was enhanced by 10.4% ± 0.95% (P < 0.05 Anova, n = 9 independent experiments) when there was interaction with Caf1-CTB4 (PMQKMRGDVFSP). The migration of CCDs was enhanced by 12.7% ± 1.03% when interacting with Caf1-J3 (KNSFMALYLSKGR) and by 14.4% ± 1.11% when interacting with Caf1-J4 (TALIRATYGEYSTGYI) (P < 0.05 Anova; n = 3 independent experiments). Combining Caf1-J3 and Caf1-J4 synergistically boosts CCD migration by 19.1% ± 1.42% (P < 0.05 Anova; n = 3 independent experiments), yielding better Results compared with individual treatments. Conclusions Caf1 exhibits promise as a platform for motif delivery, and the incorporation of cell migration motifs into Caf1 holds the potential to enhance wound healing.

Nickel-catalysed enantioselective alkene dicarbofunctionalization enabled by photochemical aliphatic C–H bond activation

The development of novel strategies to rapidly construct complex chiral molecules from readily available feedstocks is a long-term pursuit in the chemistry community. Radical-mediated alkene difunctionalizations represent an excellent platform towards this goal. However, asymmetric versions remain highly challenging, and more importantly, examples featuring simple hydrocarbons as reaction partners are elusive. Here we report an asymmetric three-component alkene dicarbofunctionalization capitalizing on the direct activation of C(sp3)–H bonds through the combination of photocatalysed hydrogen atom transfer and nickel catalysis. This protocol provides an efficient platform for installing two vicinal carbon–carbon bonds across alkenes in an atom-economic fashion, providing a wide array of high-value chiral α-aryl/alkenyl carbonyls and phosphonates, as well as 1,1-diarylalkanes from ubiquitous alkane, ether and alcohol feedstocks. This method exhibits operational simplicity, broad substrate scope and excellent regioselectivity, chemoselectivity and enantioselectivity. The compatibility with bioactive motifs and expedient synthesis of pharmaceutically relevant molecules highlight the synthetic potential of this protocol.

Molecularly Imprinted Macroporous Hydrogel Promotes Bone Regeneration via Osteogenic Induction and Osteoclastic Inhibition.

Macroporous hydrogels offer physical supportive spaces and bio-instructive environment for the seeded cells, where cell-scaffold interactions directly influence cell fates and subsequently affect tissue regeneration post-implantation. Effectively modifying bioactive motifs at the inner pore surface provides appropriate niches for cell-scaffold interactions. A molecular imprinting method and sacrificial templates are introduced to prepare inner pore surface modification in the macroporous hydrogels. In detail, acrylated bisphosphonates (Ac-BPs) chelating to templates (CaCO3 particles) are anchored on the inner pore surface of the methacrylated gelatin (GelMA)-methacrylated hyaluronic acid (HAMA)-poly (ethylene glycol) diacrylate (PEGDA) macroporous hydrogel (GHP) to form a functional hydrogel scaffold (GHP-int-BP). GHP-int-BP, but not GHP, effectively crafts artificial cell niches to substantially alter cell fates, including osteogenic induction and osteoclastic inhibition, and promote in situ bone regeneration. These findings highlight that molecular imprinting on the inner pore surface in the hydrogel efficiently creates orthogonally additive bio-instructive scaffolds for bone regeneration.

Synthesis of palladium complexes containing benzimidazole core and their catalytic activities in direct C[sbnd]H functionalization of five-membered heterocyclic bioactive motifs

Herein, we reported the synthesis of nine new PEPPSI-Pd(II)–N-heterocyclic carbene (NHC) complexes 3a-h containing benzimidazole core and examined their catalytic activities in the direct arylation of heteroaromatic species with aryl bromides and arylchlorides. Nine new Pd benzimidazole complexes were synthesized and their structures were elucidated by 1H and 13C NMR spectroscopy, FT-IR spectroscopy, and elemental analysis methods. In the 13C NMR spectra, NCHN peaks of Pd(II) complexes were observed between 159.7–161.5 ppm. Further, direct arylation reaction, using KOAc as base and DMAc as solvent under inert conditions, were carried out with the synthesized complexes in the presence of various aryl bromides and chlorides, and 2-acetyl furan, and furfuryl acetate 2-acetylthiophene and N-methylpyrrole-2- carboxaldehyde. Moreover, moderate to hig yields were obtained in the direct arylation of heteroarenes and aryl bromides under 1 mol% catalyst loading at 150 °C for 2 h loading conditions. The results showed that electron-donating and electron-withdrawing substituents on the aryl halides might produce coupling products in good yields in the presence of complexes 2a-d. Taken together, our findings are expected to contribute to the synthesis and design of palladium complexes containing benzimidazole core structures.

Digital Light Processing 3D Bioprinting of Gelatin-Norbornene Hydrogel for Enhanced Vascularization.

Digital light processing (DLP) bioprinting can be used to fabricate volumetric scaffolds with intricate internal structures, such as perfusable vascular channels. The successful implementation of DLP bioprinting in tissue fabrication requires using suitable photo-reactive bioinks. Norbornene-based bioinks have emerged as an attractive alternative to (meth)acrylated macromers in 3D bioprinting owing to their mild and rapid reaction kinetics, high cytocompatibility for in situ cell encapsulation, and adaptability for post-printing modification or conjugation of bioactive motifs. In this contribution, the development of gelatin-norbornene (GelNB) is reported as a photo-cross-linkable bioink for DLP 3D bioprinting. Low concentrations of GelNB (2-5wt.%) and poly(ethylene glycol)-tetra-thiol (PEG4SH) are DLP-printed with a wide range of stiffness (G' ≈120 to 4000Pa) and with perfusable channels. DLP-printed GelNB hydrogels are highly cytocompatible, as demonstrated by the high viability of the encapsulated human umbilical vein endothelial cells (HUVECs). The encapsulated HUVECs formed an interconnected microvascular network with lumen structures. Notably, the GelNB bioink permitted both in situ tethering and secondary conjugation of QK peptide, a vascular endothelial growth factor (VEGF)-mimetic peptide. Incorporation of QK peptide significantly improved endothelialization and vasculogenesis of the DLP-printed GelNB hydrogels, reinforcing the applicability of this bioink system in diverse biofabrication applications.

Recent Developments in Ultrasound-Promoted Aqueous-Mediated Greener Synthesis of Biologically Vital Indole Derivatives

AbstractThe extensive range of uses of N-heterocycles as potent bioactive motifs has attracted researchers to expand newer methods for their efficient synthesis. Particularly, indoles are widely known for their prevalent pharmacological properties. Green chemistry provides various synthetic tools viz. alternative energy resources, nonconventional solvents, nano-catalysts, etc. Modern strategies of using ultrasound as an alternative energy resource in organic synthesis has led to the development of environment friendly and cost-effective techniques. The chemical and mechanical effects of ultrasound waves impart significant enhancement in both stoichiometric and catalytic reactions. The exclusive physicochemical properties of water offer widespread utility for carrying out organic reactions in this medium. The aim of this review article is to provide an inclusive summary of the combined use of ultrasound and aqueous media for the facile synthesis of biologically vital indole derivatives.1 Introduction2 Synthesis of Biologically Vital Indoles2.1 Spirocyclic Indoles2.2 Non-spiro 3-Substituted Indoles2.3 Miscellaneous Indole Syntheses3 Conclusions

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Synthesis, characterization, and DFT study of the s-triazine analogues of medicinal interest incorporated with five- and six-membered bioactive heterocyclic scaffolds

The present paper reports the synthesis, NMR study, and detailed computational investigation of various novel s-triazine analogues 9–12 of medicinal interest embellished with bioactive heterocyclic scaffolds such as isoxazole, pyrazole, and pyrimidine, linked to triazine backbone through an oxyphenyl spacer. The synthesis of 9–12 was performed by exploiting the synthetic potential of the chalcone 4 which resulted from the reaction of benzaldehyde and s-triazine bound aryl ketone derivative 3. The later was in turn obtained by the reaction of p-hydroxyacetophenone with 6‑chloro-2,4-dicyclopropylamino-1,3,5-triazine(2). This strategy allowed the s-triazine to act as a three-pronged stencil to embrace three bioactive motifs jointly in the same molecular framework in 9–12.Detailed density function theory (DFT) study corroborated the experimental findings. It was shown that the molecules of the synthesized compounds would be quite uncrowded and therefore would be packed quite loosely in the crystalline phase. The molecular orbital (MOs) analysis showed that all the synthesized compounds should have quite pronounced thermodynamic stability and thatthe modification of the substituting group would allow to finely tune the reactivity of the s-triazine derivatives. Also, possible formation of various noncovalent interactions between the molecules of the studied compounds within their crystals or between the molecules of these compounds with various polar solvents and biologically important molecules was suggested. Moreover, potential reactivity of these compounds towards various electrophiles and nucleophiles suggesting possibility of their successful employment for medical purposes was proposed. Further, the global reactivity parameters (GRP) analysis suggested that the compound studied would be quite stable in redox processes, with the pyridine derivatives showing higher stability.

Poly(ethylene glycol)-Norbornene as a Photoclick Bioink for Digital Light Processing 3D Bioprinting.

Digital light processing (DLP) bioprinting is an emerging technology for three-dimensional bioprinting (3DBP) owing to its high printing fidelity, fast fabrication speed, and higher printing resolution. Low-viscosity bioinks such as poly(ethylene glycol) diacrylate (PEGDA) are commonly used for DLP-based bioprinting. However, the cross-linking of PEGDA proceeds via chain-growth photopolymerization that displays significant heterogeneity in cross-linking density. In contrast, step-growth thiol-norbornene photopolymerization is not oxygen inhibited and produces hydrogels with an ideal network structure. The high cytocompatibility and rapid gelation of thiol-norbornene photopolymerization have lent itself to the cross-linking of cell-laden hydrogels but have not been extensively used for DLP bioprinting. In this study, we explored eight-arm PEG-norbornene (PEG8NB) as a bioink/resin for visible light-initiated DLP-based 3DBP. The PEG8NB-based DLP resin showed high printing fidelity and cytocompatibility even without the use of any bioactive motifs and high initial stiffness. In addition, we demonstrated the versatility of the PEGNB resin by printing solid structures as cell culture devices, hollow channels for endothelialization, and microwells for generating cell spheroids. This work not only expands the selection of bioinks for DLP-based 3DBP but also provides a platform for dynamic modification of the bioprinted constructs.

Plausible PEPPSI catalysts for direct C–H functionalization of five-membered heterocyclic bioactive motifs: synthesis, spectral, X-ray crystallographic characterizations and catalytic activity

In this study, a series of benzimidazolium salts were synthesized as asymmetric N-heterocyclic carbene (NHC) precursors.

Self-Assembled Peptide Nanostructures for ECM Biomimicry.

Proteins are functional building blocks of living organisms that exert a wide variety of functions, but their synthesis and industrial production can be cumbersome and expensive. By contrast, short peptides are very convenient to prepare at a low cost on a large scale, and their self-assembly into nanostructures and gels is a popular avenue for protein biomimicry. In this Review, we will analyze the last 5-year progress on the incorporation of bioactive motifs into self-assembling peptides to mimic functional proteins of the extracellular matrix (ECM) and guide cell fate inside hydrogel scaffolds.

Self-assembling peptides-based nano-cargos for targeted chemotherapy and immunotherapy of tumors: recent developments, challenges, and future perspectives

Self-assembling peptides (SAPs) have enormous potential in medical and biological applications, particularly noninvasive tumor therapy. SAPs self-assembly is governed by multiple non-covalent interactions and results in the formation of a variety of morphological features. SAPs can be assembled in a variety of ways, including chemical conjugation and physical encapsulation, to incorporate multiple bioactive motifs. Peptide-based nanomaterials are used for chemotherapy, delivery vehicles, immunotherapy, and noninvasive tumor therapies (e.g. photodynamic therapy) by employing the self-assembling properties of peptides. The recent increase of SAPs is almost entirely due to their excellent biocompatibility, responsiveness toward tumor microenvironment, multivalency, and structural versatility. Synergistic therapy is a more effective and powerful approach to treat the tumor. Notably, SAPs can be used to subtly combine various treatments. Importantly, SAPs are capable of subtly making the combination of various treatments. This review describes mechanisms of peptides self-assemble into various structures and their biomedical applications with a focus on possible treatments.

Bioactive Film-Guided Soft-Hard Interface Design Technology for Multi-Tissue Integrative Regeneration.

Control over soft‐to‐hard tissue interfaces is attracting intensive worldwide research efforts. Herein, a bioactive film‐guided soft–hard interface design (SHID) for multi‐tissue integrative regeneration is shown. Briefly, a soft bioactive film with good elasticity matchable to native ligament tissue, is incorporated with bone‐mimic components (calcium phosphate cement, CPC) to partially endow the soft‐film with hard‐tissue mimicking feature. The hybrid film is elegantly compounded with a clinical artificial ligament to act as a buffer zone to bridge the soft (ligament) and hard tissues (bone). Moreover, the bioactive film‐decorated ligament can be rolled into a 3D bio‐instructive implant with spatial‐controllable distribution of CPC bioactive motifs. CPC then promotes the recruitment and differentiation of endogenous cells in to the implant inside part, which enables a vascularized bone growth into the implant, and forms a structure mimicking the biological ligament–bone interface, thereby significantly improving osteointegration and biomechanical property. Thus, this special design provides an effective SHID‐guided implant‐bioactivation strategy unreached by the traditional manufacturing methods, enlightening a promising technology to develop an ideal SHID for translational use in the future.

Supramolecular Peptide Nanofiber Hydrogels for Bone Tissue Engineering: From Multihierarchical Fabrications to Comprehensive Applications.

Bone tissue engineering is becoming an ideal strategy to replace autologous bone grafts for surgical bone repair, but the multihierarchical complexity of natural bone is still difficult to emulate due to the lack of suitable biomaterials. Supramolecular peptide nanofiber hydrogels (SPNHs) are emerging biomaterials because of their inherent biocompatibility, satisfied biodegradability, high purity, facile functionalization, and tunable mechanical properties. This review initially focuses on the multihierarchical fabrications by SPNHs to emulate natural bony extracellular matrix. Structurally, supramolecular peptides based on distinctive building blocks can assemble into nanofiber hydrogels, which can be used as nanomorphology‐mimetic scaffolds for tissue engineering. Biochemically, bioactive motifs and bioactive factors can be covalently tethered or physically absorbed to SPNHs to endow various functions depending on physiological and pharmacological requirements. Mechanically, four strategies are summarized to optimize the biophysical microenvironment of SPNHs for bone regeneration. Furthermore, comprehensive applications about SPNHs for bone tissue engineering are reviewed. The biomaterials can be directly used in the form of injectable hydrogels or composite nanoscaffolds, or they can be used to construct engineered bone grafts by bioprinting or bioreactors. Finally, continuing challenges and outlook are discussed.

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 β-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.

Amino acid containing amphiphilic hydrogelators with antibacterial and antiparasitic activities.

Nanoscale self-assembly of peptide constructs represents a promising means to present bioactive motifs to develop new functional materials. Here, we present a series of peptide amphiphiles which form hydrogels based on β-sheet nanofibril networks, several of which have very promising anti-microbial and anti-parasitic activities, in particular against multiple strains of Leishmania including drug-resistant ones. Aromatic amino acid based amphiphilic supramolecular gelators C14-Phe-CONH-(CH2)n-NH2 (n = 6 for P1 and n = 2 for P3) and C14-Trp-CONH-(CH2)n-NH2 (n = 6 for P2 and n = 2 for P4) have been synthesized and characterized, and their self-assembly and gelation behaviour have been investigated in the presence of ultrapure water (P1, P2, and P4) or 2% DMSO(v/v) in ultrapure water (P3). The rheological, morphological and structural properties of the gels have been comprehensively examined. The amphiphilic gelators (P1 and P3) were found to be active against both Gram-positive bacteria B. subtilis and Gram-negative bacteria E. coli and P. aeruginosa. Interestingly, amphiphiles P1 and P3 containing an L-phenylalanine residue show both antibacterial and antiparasitic activities. Herein, we report that synthetic amphiphiles with an amino acid residue exhibit a potent anti-protozoan activity and are cytotoxic towards a wide array of protozoal parasites, which includes Indian varieties of Leishmania donovani and also kill resistant parasitic strains including BHU-575, MILR and CPTR cells. These gelators are highly cytotoxic to promastigotes of Leishmania and trigger apoptotic-like events inside the parasite. The mechanism of killing the parasite is shown and these gelators are non-cytotoxic to host macrophage cells indicating the potential use of these gels as therapeutic agents against multiple forms of leishmaniasis in the near future.

Injectable self-assembling peptide nanofiber hydrogel as a bioactive 3D platform to promote chronic wound tissue regeneration

Chronic wounds remain a worldwide clinical challenge, and bioactive materials that can promote skin regeneration are required. Self-assembling peptide (SAP) hydrogels have shown great potential in tissue repair, but their regenerative efficacy and possible mechanism in chronic wound healing are unclear. Here, we report an SAP (KGH) that enhances extracellular matrix (ECM) remodeling and angiogenesis, thereby promoting chronic wound healing in diabetic mice. In vivo, the KGH hydrogel was retained in wounds up to 7 days after injection, and it was effective in speeding up wound closure by ∼20% compared to the control groups and enhancing angiogenesis (e.g., VEGFA, CD31+ capillaries), cell proliferation (e.g., PCNA+ cells), formation of granulation tissue (e.g., α-SMA), and ECM deposition/remodeling (e.g., collagen I, fibronectin). In vitro, the KGH hydrogel created a 3D microenvironment for skin cells, maintained the sustained growth of cell spheroids, and increased the secretion of ECM proteins (e.g., laminin) and growth factors (e.g., PDGFB, VEGFA, and TGF-β) in skin keratinocytes compared to the conventional 2D culture. Mechanistically, the KGH hydrogel might promote wound tissue regeneration by activating the Rho/ROCK and TGF-β/MEK/MAPK pathways. As a type of designed material, SAP can be further re-engineered with biological motifs, therapeutic reagents, or stem cells to enhance skin regeneration. This study highlights that SAP hydrogels are a promising material platform for advanced chronic wound healing and might have translational potential in future clinical applications. Statement of significanceChronic wounds are a common and serious health issue worldwide, and bioactive dressing materials are required to address this issue. SAP hydrogels have shown certain tissue repair potential, but their regenerative efficacy and underlying mechanism in chronic wound healing remain elusive. Herein, we report that SAP hydrogels create a native 3D microenvironment that can remarkably stimulate angiogenesis and ECM remodeling in diabetic wounds. Mechanistically, the SAP hydrogel promoted ECM proteins and GFs secretion in skin cells through the activation of the Rho/ROCK and TGF-ß/MEK/MAPK pathways. Additionally, SAP can be readily engineered with various bioactive motifs or therapeutic drugs/cells. This work highlights SAP hydrogels as a promising biomaterial platform for chronic wound healing and the regeneration of many other tissues.

Dual Functionalization of Gelatin for Orthogonal and Dynamic Hydrogel Cross-Linking.

Gelatin-based hydrogels are widely used in biomedical fields because of their abundance of bioactive motifs that support cell adhesion and matrix remodeling. Although inherently bioactive, unmodified gelatin exhibits temperature-dependent rheology and solubilizes at body temperature, making it unstable for three-dimensional (3D) cell culture. Therefore, the addition of chemically reactive motifs is required to render gelatin-based hydrogels with highly controllable cross-linking kinetics and tunable mechanical properties that are critical for 3D cell culture. This article provides a series of methods toward establishing orthogonally cross-linked gelatin-based hydrogels for dynamic 3D cell culture. In particular, we prepared dually functionalized gelatin macromers amenable for sequential, orthogonal covalent cross-linking. Central to this material platform is the synthesis of norbornene-functionalized gelatin (GelNB), which forms covalently cross-linked hydrogels via orthogonal thiol-norbornene click cross-linking. Using GelNB as the starting material, we further detail the methods for synthesizing gelatin macromers susceptible to hydroxyphenylacetic acid (HPA) dimerization (i.e., GelNB-HPA) and hydrazone bonding (i.e., GelNB-CH) for on-demand matrix stiffening. Finally, we outline the protocol for synthesizing a gelatin macromer capable of adjusting hydrogel stress relaxation via boronate ester bonding (i.e., GelNB-BA). The combination of these orthogonal chemistries affords a wide range of gelatin-based hydrogels as biomimetic matrices in tissue engineering and regenerative medicine applications.

Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.