Nanocrystalline Cellulose as a Versatile Engineering Material for Extrusion-Based Bioprinting.

Naturally derived polysaccharide-based hydrogels, such as alginate, are frequently used in the design of bioinks for 3D bioprinting. Traditionally, the formulation of such bioinks requires the use of pre-reticulated materials with low viscosities, which favour cell viability but can negatively influence the resolution and shape fidelity of the printed constructs. In this work, we propose the use of cellulose nanocrystals (CNCs) as a rheological modifier to improve the printability of alginate-based bioinks whilst ensuring a high viability of encapsulated cells. Through rheological analysis, we demonstrate that the addition of CNCs (1% and 2% (w/v)) to alginate hydrogels (1% (w/v)) improves shear-thinning behaviour and mechanical stability, resulting in the high-fidelity printing of constructs with superior resolution. Importantly, LIVE/DEAD results confirm that the presence of CNCs does not seem to affect the health of immortalised chondrocytes (TC28a2) that remain viable over a period of seven days post-encapsulation. Taken together, our results indicate a favourable effect of the CNCs on the rheological and biocompatibility properties of alginate hydrogels, opening up new perspectives for the application of CNCs in the formulation of bioinks for extrusion-based bioprinting.

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.

Collagen-like Osteoclast-Associated Receptor (OSCAR)-Binding Motifs Show a Co-Stimulatory Effect on Osteoclastogenesis in a Peptide Hydrogel System.

Osteoclastogenesis, one of the dynamic pathways underlying bone remodelling, is a complex process that includes many stages. This complexity, while offering a wealth of therapeutic opportunities, represents a substantial challenge in unravelling the underlying mechanisms. As such, there is a high demand for robust model systems to understand osteoclastogenesis. Hydrogels seeded with osteoclast precursors and decorated with peptides or proteins mimicking bone's extracellular matrix could provide a useful synthetic tool to study pre-osteoclast-matrix interactions and their effect on osteoclastogenesis. For instance, fibrillar collagens have been shown to provide a co-stimulatory pathway for osteoclastogenesis through interaction with the osteoclast-associated receptor (OSCAR), a regulator of osteoclastogenesis expressed on the surface of pre-osteoclast cells. Based on this rationale, here we design two OSCAR-binding peptides and one recombinant OSCAR-binding protein, and we combine them with peptide-based hydrogels to study their effect on osteoclastogenesis. The OSCAR-binding peptides adopt the collagen triple-helical conformation and interact with OSCAR, as shown by circular dichroism spectropolarimetry and surface plasmon resonance. Furthermore, they have a positive effect on osteoclastogenesis, as demonstrated by appropriate gene expression and tartrate-resistant acid phosphatase staining typical of osteoclast formation. Combination of the OSCAR-binding peptides or the OSCAR-binding recombinant protein with peptide-based hydrogels enhances osteoclast differentiation when compared to the non-modified hydrogels, as demonstrated by multi-nucleation and by F-actin staining showing a characteristic osteoclast-like morphology. We envisage that these hydrogels could be used as a platform to study osteoclastogenesis and, in particular, to investigate the effect of costimulatory pathways involving OSCAR.

Incorporation of Natural and Recombinant Collagen Proteins within Fmoc-Based Self-Assembling Peptide Hydrogels.

Hydrogel biomaterials mimic the natural extracellular matrix through their nanofibrous ultrastructure and composition and provide an appropriate environment for cell-matrix and cell-cell interactions within their polymeric network. Hydrogels can be modified with different proteins, cytokines, or cell-adhesion motifs to control cell behavior and cell differentiation. Collagens are desirable and versatile proteins for hydrogel modification due to their abundance in the vertebrate extracellular matrix and their interactions with cell-surface receptors. Here, we report a quick, inexpensive and effective protocol for incorporation of natural, synthetic and recombinant collagens into Fmoc-based self-assembling peptide hydrogels. The hydrogels are modified through a diffusion protocol in which collagen molecules of different molecular sizes are successfully incorporated and retained over time. Characterization studies show that these collagens interact with the hydrogel fibers without affecting the overall mechanical properties of the composite hydrogels. Furthermore, the collagen molecules incorporated into the hydrogels are still biologically active and provide sites for adhesion and spreading of human fibrosarcoma cells through interaction with the α2ß1 integrin. Our protocol can be used to incorporate different types of collagen molecules into peptide-based hydrogels without any prior chemical modification. These modified hydrogels could be used in studies where collagen-based substrates are required to differentiate and control the cell behavior. Our protocol can be easily adapted to the incorporation of other bioactive proteins and peptides into peptide-based hydrogels to modulate their characteristics and their interaction with different cell types.

Self-Assembling Peptide Hydrogels as Functional Tools to Tackle Intervertebral Disc Degeneration.

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration, is a major contributor to global disability. In its healthy state, the IVD is a tough and well-hydrated tissue, able to act as a shock absorber along the spine. During degeneration, the IVD is hit by a cell-driven cascade of events, which progressively lead to extracellular matrix (ECM) degradation, chronic inflammation, and pain. Current treatments are divided into palliative care (early stage degeneration) and surgical interventions (late-stage degeneration), which are invasive and poorly efficient in the long term. To overcome these limitations, alternative tissue engineering and regenerative medicine strategies, in which soft biomaterials are used as injectable carriers of cells and/or biomolecules to be delivered to the injury site and restore tissue function, are currently being explored. Self-assembling peptide hydrogels (SAPHs) represent a promising class of de novo synthetic biomaterials able to merge the strengths of both natural and synthetic hydrogels for biomedical applications. Inherent features, such as shear-thinning behaviour, high biocompatibility, ECM biomimicry, and tuneable physiochemical properties make these hydrogels appropriate and functional tools to tackle IVD degeneration. This review will describe the pathogenesis of IVD degeneration, list biomaterials requirements to attempt IVD repair, and focus on current peptide hydrogel materials exploited for this purpose.

Acidic and basic self-assembling peptide and peptide-graphene oxide hydrogels: characterisation and effect on encapsulated nucleus pulposus cells.

Extracellular pH can have a profound effect on cell metabolism, gene and protein expression. Nucleus pulposus (NP) cells, for example, under acidic conditions accelerate the production of degradative enzymes and pro-inflammatory cytokines, leading ultimately to intervertebral disc degeneration, a major cause of back pain. Self-assembling peptide hydrogels constitute a well-established class of biomaterials that could be exploited as pH-tunable platform to investigate cell behaviour under normal and non-physiological pH. In this paper we formulated acidic (pH = 4) and basic (pH = 9) hydrogels, from the same octapeptide FEFKFEFK (F8) (F = phenyalanine, E = glutamic acid, K = lysine), to test the effect of non-physiological pH on encapsulated NP cells. Similarly, graphene oxide-containing F8 hydrogels (GO-F8) were formulated as stiffer analogues. Acidic and basic hydrogels showed peculiar morphologies and rheological properties, with all systems able to buffer within 30 minutes of exposure to cell culture media. NP cells seeded in acidic F8 hydrogels showed a more catabolic phenotype compared to basic hydrogels, with increased gene expression of degradative enzymes (MMP-3, ADAMTS-4), neurotrophic factors (NGF and BDNF) and NF-κB p65 phosphorylation. Acidic GO-F8 hydrogels also induced a catabolic response, although milder than basic counterparts and with the highest gene expression of characteristic NP-matrix components, aggrecan and collagen II. In all systems, the cellular response had a peak within 3 days of encapsulation, thereafter decreasing over 7 days, suggesting a 'transitory' effect of hydrogel pH on encapsulated cells. This work gives an insight on the effect of pH (and pH buffering) on encapsulated NP cells and offers new designs of low and high pH peptide hydrogels for 3D cell culture studies. STATEMENT OF SIGNIFICANCE: We have recently shown the potential of graphene oxide - self-assembling peptide hybrid hydrogels for NP cell culture and regeneration. Alongside cell carrier, self-assembling peptide hydrogels actually provide a versatile pH-tunable platform for biological studies. In this work we decided to explore the effect of non-physiological pH (and pH buffering) on encapsulated NP cells. Our approach allows the formulation of both acidic and basic hydrogels, starting from the same peptide sequence. We showed that the initial pH of the scaffold does not affect significantly cell response to encapsulation, but the presence of GO results in lower inflammatory levels and higher NP matrix protein production. This platform could be exploited to study the effect of pH on different cell types whose behaviour can be pH-dependent.

Hydroxyapatite-decorated Fmoc-hydrogel as a bone-mimicking substrate for osteoclast differentiation and culture.

Hydrogels are water-swollen networks with great potential for tissue engineering applications. However, their use in bone regeneration is often hampered due to a lack of materials' mineralization and poor mechanical properties. Moreover, most studies are focused on osteoblasts (OBs) for bone formation, while osteoclasts (OCs), cells involved in bone resorption, are often overlooked. Yet, the role of OCs is pivotal for bone homeostasis and aberrant OC activity has been reported in several pathological diseases, such as osteoporosis and bone cancer. For these reasons, the aim of this work is to develop customised, reinforced hydrogels to be used as material platform to study cell function, cell-material interactions and ultimately to provide a substrate for OC differentiation and culture. Here, Fmoc-based RGD-functionalised peptide hydrogels have been modified with hydroxyapatite nanopowder (Hap) as nanofiller, to create nanocomposite hydrogels. Atomic force microscopy showed that Hap nanoparticles decorate the peptide nanofibres with a repeating pattern, resulting in stiffer hydrogels with improved mechanical properties compared to Hap- and RGD-free controls. Furthermore, these nanocomposites supported adhesion of Raw 264.7 macrophages and their differentiation in 2D to mature OCs, as defined by the adoption of a typical OC morphology (presence of an actin ring, multinucleation, and ruffled plasma membrane). Finally, after 7 days of culture OCs showed an increased expression of TRAP, a typical OC differentiation marker. Collectively, the results suggest that the Hap/Fmoc-RGD hydrogel has a potential for bone tissue engineering, as a 2D model to study impairment or upregulation of OC differentiation. STATEMENT OF SIGNIFICANCE: Altered osteoclasts (OC) function is one of the major cause of bone fracture in the most commonly skeletal disorders (e.g. osteoporosis). Peptide hydrogels can be used as a platform to mimic the bone microenvironment and provide a tool to assess OC differentiation and function. Moreover, hydrogels can incorporate different nanofillers to yield hybrid biomaterials with enhanced mechanical properties and improved cytocompatibility. Herein, Fmoc-based RGD-functionalised peptide hydrogels were decorated with hydroxyapatite (Hap) nanoparticles to generate a hydrogel with improved rheological properties. Furthermore, they are able to support osteoclastogenesis of Raw264.7 cells in vitro as confirmed by morphology changes and expression of OC-markers. Therefore, this Hap-decorated hydrogel can be used as a template to successfully differentiate OC and potentially study OC dysfunction.

TGF-ß3-loaded graphene oxide - self-assembling peptide hybrid hydrogels as functional 3D scaffolds for the regeneration of the nucleus pulposus.

Intervertebral disc (IVD) degeneration is a process that starts in the central nucleus pulposus (NP) and leads to inflammation, extracellular matrix (ECM) degradation, and progressive loss of disc height. Early treatment of IVD degeneration is critical to the reduction of low back pain and related disability. As such, minimally invasive therapeutic approaches that can halt and reverse NP degeneration at the early stages of the disease are needed. Recently, we developed an injectable graphene oxide (GO) - self-assembling peptide FEFKFEFK (F: phenylalanine; K: lysine; E: glutamic acid) hybrid hydrogels as potential delivery platform for cells and/or drugs in the NP. In this current study, we explored the possibility of using the GO present in these hybrid hydrogels as a vehicle for the sequestration and controlled delivery of transforming growth factor beta-3 (TGF-ß3), an anabolic growth factor (GF) known to direct NP cell fate and function. For this purpose, we first investigated the potential of GO to bind and sequestrate TGF-ß3. We then cultured bovine NP cells in the new functional scaffolds and investigated their response to the presence of GO and TGF-ß3. Our results clearly showed that GO flakes can sequestrate TGF-ß3 through strong binding interactions resulting in a slow and prolonged release, with the GF remaining active even when bound to the GO flakes. The adsorption of the GF on the GO flakes to create TGF-ß3-loaded GO flakes and their subsequent incorporation in the hydrogels through mixing, [(GO/TGF-ß3Ads)-F8] hydrogel, led to the upregulation of NP-specific genes, accompanied by the production and deposition of an NP-like ECM, rich in aggrecan and collagen II. NP cells actively interacted with TGF-ß3-loaded GO flakes and remodeled the scaffolds through endocytosis. This work highlights the potential of using GO as a nanocarrier for the design of functional hybrid peptide-based hydrogels. STATEMENT OF SIGNIFICANCE: Intervertebral disc (IVD) degeneration is a process that starts in the central nucleus pulposus (NP) and leads to inflammation, extracellular matrix (ECM) degradation, and progressive loss of disc height. As such, minimally invasive therapeutic approaches that can halt and reverse NP degeneration at the early stages of the disease are needed. In this current study, we explored the possibility of using peptide - GO hybrid hydrogels as a vehicle for the sequestration and controlled delivery of transforming growth factor beta-3 (TGF-ß3), an anabolic growth factor (GF) known to direct NP cell fate and function.

Engineering a cell-hydrogel-fibre composite to mimic the structure and function of the tendon synovial sheath.

The repair of tendon injuries is often compromised by post-operative peritendinous adhesions. Placing a physical barrier at the interface between the tendon and the surrounding tissue could potentially solve this problem by reducing adhesion formation. At present, no such system is available for routine use in clinical practice. Here, we propose the development of a bilayer membrane combining a nanofibrous poly(ε-caprolactone) (PCL) electrospun mesh with a layer of self-assembling peptide hydrogel (SAPH) laden with type-B synoviocytes. This bilayer membrane would act as an anti-adhesion system capable of restoring tendon lubrication, while assisting with synovial sheath regeneration. The PCL mesh showed adequate mechanical properties (Young's modulus=19±4 MPa, ultimate tensile stress=9.6±1.7 MPa, failure load=0.5±0.1 N), indicating that the membrane is easy to handle and capable to withstand the frictional forces generated on the tendon's surface during movement (~0.3 N). Morphological analysis confirmed the generation of a mesh with nanosized PCL fibres and small pores (< 3 µm), which prevented fibroblast infiltration to impede extrinsic healing but still allowing diffusion of nutrients and waste. Rheological tests showed that incorporation of SAPH layer allows good lubrication properties when the membrane is articulated against porcine tendon or hypodermis, suggesting that restoration of tendon gliding is possible upon implantation. Moreover, viability and metabolic activity tests indicated that the SAPH was conducive to rabbit synoviocyte growth and proliferation over 28 days of 3D culture, sustaining cell production of specific matrix components, particularly hyaluronic acid. Synoviocyte-laden peptide hydrogel promoted a sustained endogenous production of hyaluronic acid, providing an anti-friction layer that potentially restores the tendon gliding environment.

The effect of terminal globular domains on the response of recombinant mini-spidroins to fiber spinning triggers.

Spider silk spidroins consist of long repetitive protein strands, flanked by globular terminal domains. The globular domains are often omitted in recombinant spidroins, but are thought to be essential for the spiders' natural spinning process. Mimicking this spinning process could be an essential step towards producing strong synthetic spider silk. Here we describe the production of a range of mini-spidroins with both terminal domains, and characterize their response to a number of biomimetic spinning triggers. Our results suggest that mini-spidroins which are able to form protein micelles due to the addition of both terminal domains exhibit shear-thinning, a property which native spidroins also show. Furthermore, our data also suggest that a pH drop alone is insufficient to trigger assembly in a wet-spinning process, and must be combined with salting-out for effective fiber formation. With these insights, we applied these assembly triggers for relatively biomimetic wet spinning. This work adds to the foundation of literature for developing improved biomimetic spinning techniques, which ought to result in synthetic silk that more closely approximates the unique properties of native spider silk.

Role of Sheet-Edge Interactions in ß-sheet Self-Assembling Peptide Hydrogels.

Hydrogels' hydrated fibrillar nature makes them the material of choice for the design and engineering of 3D scaffolds for cell culture, tissue engineering, and drug-delivery applications. One particular class of hydrogels which has been the focus of significant research is self-assembling peptide hydrogels. In the present work, we were interested in exploring how fiber-fiber edge interactions affect the self-assembly and gelation properties of amphipathic peptides. For this purpose, we investigated two ß-sheet-forming peptides, FEFKFEFK (F8) and KFEFKFEFKK (KF8K), the latter one having the fiber edges covered by lysine residues. Our results showed that the addition of the two lysine residues did not affect the ability of the peptides to form ß-sheet-rich fibers, provided that the overall charge carried by the two peptides was kept constant. However, it did significantly reduce edge-driven hydrophobic fiber-fiber associative interactions, resulting in reduced tendency for KF8K fibers to associate/aggregate laterally and form large fiber bundles and consequently network cross-links. This effect resulted in the formation of hydrogels with lower moduli but faster dynamics. As a result, KF8K fibers could be aligned only under high shear and at high concentration while F8 hydrogel fibers were found to align readily at low shear and low concentration. In addition, F8 hydrogels were found to fragment at high concentration because of the high aggregation state stabilizing the fiber bundles, resulting in fiber breakage rather than disentanglement and alignment.

Methylcellulose Hydrogel with Melissa officinalis Essential Oil as a Potential Treatment for Oral Candidiasis.

Candida spp. are the most prevalent fungi of the human microbiota and are opportunistic pathogens that can cause oral candidiasis. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Therefore, much interest in the antimicrobial potential of natural compounds has recently been evident. The use of hydrogels in the delivery of biocides has been explored due to their biocompatibility, ease with drug encapsulation, and due to their potential to confer mechanical and structural properties similar to biological tissue. Methylcellulose hydrogels (10% (w/v)) with 1% (v/v) and 2% (v/v) Melissa officinalis oil were synthesised. The rheological properties and gelation time of the hydrogels were evaluated. Antimicrobial action, the antifungal potential and ability to displace Candida were determined. Rheological tests revealed that the hydrogel jellified in three minutes at 37 °C. Loaded hydrogels successfully inhibited Candida albicans growth as evident by zone of inhibition and time-kill assays. A significant reduction in retained C. albicans was demonstrated with the hydrogel at 2% Melissa officinalis concentration. This work demonstrated that an essential oil-loaded hydrogel had the potential to provide a novel antimicrobial therapy for the treatment of oral candidiasis.

Modeling the Three-Dimensional Bioprinting Process of ß-Sheet Self-Assembling Peptide Hydrogel Scaffolds.

Extrusion-based three-dimensional (3D) bioprinting is nowadays the most efficient additive manufacturing technology to fabricate well-defined and clinical-scale relevant 3D scaffolds, exploiting soft biomaterials. However, trial and error approaches are usually employed to achieve the desired structures, thus leading to a waste of time and material. In this work, we show the potential of finite element (FE) simulation in predicting the printability of a biomaterial, in terms of extrudability and scaffold mechanical stability over time. To this end, we firstly rheologically characterized a newly developed self-assembling peptide hydrogel (SAPH). Subsequently, we modeled both the extrusion process of the SAPHs and the stability over time of a 3D-bioprinted wood-pile scaffold. FE modeling revealed that the simulated SAPHs and printing setups led to a successful extrusion, within a range of shear stresses that are not detrimental for cells. Finally, we successfully 3D bioprinted human ear-shaped scaffolds with in vivo dimensions and several protrusion planes by bioplotting the SAPH into a poly(vinyl alcohol)-poly(vinyl pyrrolidone) copolymer, which was identified as a suitable bioprinting strategy by mechanical FE simulation.

Graphene oxide containing self-assembling peptide hybrid hydrogels as a potential 3D injectable cell delivery platform for intervertebral disc repair applications.

Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP). In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (ß-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. STATEMENT OF SIGNIFICANCE: Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells' microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.