Self-assembled peptide gels for 3D cell culture

Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progress towards the establishment of design rules for the preparation of scaffolds with tuneable properties.First, Fmoc-dipeptides composed of a combination of hydrophobic aromatic residues phenylalanine (F) and glycine (G) were studied with a particular emphasis on the effect of pH variations. The systems were investigated in order to assess what influence the position of such residues in the peptide sequence had on the physical properties of the molecules, and what impact the chemical structure had on the self-assembly behaviour and the gelation properties of the materials. Subsequently, phenylalanine was replaced by leucine (L), a non-aromatic amino acid that had the same relative hydrophobicity in order to determine whether the self-assembly of such molecules is driven by aromatic interactions or hydrophobic effects.Using potentiometry, the behaviour of the systems in solution has been investigated, revealing that they were all characterised by pKa shifts of up to six units above the theoretical values. Fmoc-FF exhibited two transitions whereas the other Fmoc-dipeptides only displayed one. These transitions were found to coincide with the formation of distinct self-assembled structures with differing molecular conformations and properties that were characterised using transmission electron microscopy, infrared and fluorescence spectroscopy, X-ray scattering and shear rheometry.π-stacking of the aromatic moieties was thought to be the driving force of the self-assembly mechanism, generating dimers that corresponded to the building blocks of the supramolecular structures formed. On the other hand, the peptide components were stabilised via hydrogen bonding and could form antiparallel β-sheets depending on the amino acid sequence and the associated influence on the rigidity of the molecules. Below their (first) apparent pKa transition, Fmoc-FF, Fmoc-LL, Fmoc-FG, Fmoc-LG and Fmoc-GG formed hydrogels, with the mechanical properties and stability varying depending on the amino acid sequence. Fmoc-FF and Fmoc-LL exhibited the lowest storage modulus values (G′ ~ 0.5–5 Pa) of the studied systems while Fmoc-LG displayed the highest (G′ ~ 1000–2100 Pa). Fmoc-FG and Fmoc-LG had the peculiarity of being obtained upon heating and where found to be particularly stable, as opposed to Fmoc-GG gels which showed a tendency to crystallise. On the microscopic scale, these gels were all associated with the presence of entangled fibrillar networks of different size and morphology, which in some cases could self-assemble further through a lamellar organisation. Again, Fmoc-FG and Fmoc-LG distinguished from the other systems as they were the only Fmoc-dipeptides to show a supramolecular chirality in the form of twisted ribbons under specific pH conditions. In contrast, Fmoc-GF and Fmoc-GL did not form hydrogels below their apparent pKa due to the formation of sheet-like and spherical structures respectively.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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

Modification of β-Sheet Forming Peptide Hydrophobic Face: Effect on Self-Assembly and Gelation

β-Sheet forming peptides have attracted significant interest for the design of hydrogels for biomedical applications. One of the main challenges is the control and understanding of the correlations between peptide molecular structure, the morphology, and topology of the fiber and network formed as well as the macroscopic properties of the hydrogel obtained. In this work, we have investigated the effect that functionalizing these peptides through their hydrophobic face has on their self-assembly and gelation. Our results show that the modification of the hydrophobic face results in a partial loss of the extended β-sheet conformation of the peptide and a significant change in fiber morphology from straight to kinked. As a consequence, the ability of these fibers to associate along their length and form large bundles is reduced. These structural changes (fiber structure and network topology) significantly affect the mechanical properties of the hydrogels (shear modulus and elasticity)

Evaluation of a synthetic peptide-based bioink (PeptiInk Alpha 1) for in vitro 3D bioprinting of cartilage tissue models

Cartilage pathology in human disease is poorly understood and requires further research. Various attempts have been made to study cartilage pathologies using in vitro human cartilage models as an alternative for preclinical research. Three-dimensional (3D) bioprinting is a technique that has been used to 3D-bioprint cartilage tissue models in vitro using animal-derived materials such as gelatine or hyaluronan, which present challenges in terms of scalability, reproducibility, and ethical concerns. We present an assessment of synthetic self-assembling peptides as bioinks for bioprinted human in vitro cartilage models. Primary human chondrocytes were mixed with PeptiInk Alpha 1, 3D-bioprinted and cultured for 14 days, and compared with 3D chondrocyte pellet controls. Cell viability was assessed through LIVE/DEAD assays and DNA quantification. High cell viability was observed in the PeptiInk culture, while a fast decrease in DNA levels was observed in the 3D pellet control. Histological evaluation using hematoxylin and eosin staining and immunofluorescence labeling for SOX-9, collagen type II, and aggrecan showed a homogeneous cell distribution in the 3D-bioprinted PeptiInks as well as high expression of chondrogenic markers in both control and PeptiInk cultures. mRNA expression levels assessed by - qRT-PCR (quantitative real time-polymerase chain reaction) confirmed chondrogenic cell behavior. These data showed promise in the potential use of PeptiInk Alpha 1 as a bioprintable manufacturing material for human cartilage in vitro models

Effect of the molecular structure of TPU on the cellular structure of nanocellular polymers based on PMMA/TPU blends

Producción CientíficaIn this work, the effects of thermoplastic polyurethane (TPU) chemistry and concentration on the cellular structure of nanocellular polymers based on poly(methyl-methacrylate) (PMMA) are presented. Three grades of TPU with different fractions of hard segments (HS) (60%, 70%, and 80%) have been synthesized by the prepolymer method. Nanocellular polymers based on PMMA have been produced by gas dissolution foaming using TPU as a nucleating agent in different contents (0.5 wt%, 2 wt%, and 5 wt%). TPU characterization shows that as the content of HS increases, the density, hardness, and molecular weight of the TPU are higher. PMMA/TPU cellular materials show a gradient cell size distribution from the edge of the sample towards the nanocellular core. In the core region, the addition of TPU has a strong nucleating effect in PMMA. Core structure depends on the HS content and the TPU content. As the HS or TPU content increases, the cell nucleation density increases, and the cell size is reduced. Then, the use of TPUs with different characteristics allows controlling the cellular structure. Nanocellular polymers have been obtained with a core relative density between 0.15 and 0.20 and cell sizes between 220 and 640 nm.Ministerio de Ciencia, Innovación y Universidades (Proyects RTI2018-098749-B-I00 and PTQ2019-010560)Ente Regional de la Energía de Castilla y León (Proyecto EREN_2019_L4_UVA

Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation

Self-Assembling Polypeptide Hydrogels as a Platform to Recapitulate the Tumor Microenvironment

From MDPI via Jisc Publications RouterHistory: accepted 2021-06-25, pub-electronic 2021-06-30Publication status: PublishedFunder: Innovate UKRI Research Knowledge Transfer Partnership; Grant(s): KTP: Self-assembling peptide matrices as a platform for cell biology studies and drug deliveryThe tumor microenvironment plays a critical role in modulating cancer cell migration, metabolism, and malignancy, thus, highlighting the need to develop in vitro culture systems that can recapitulate its abnormal properties. While a variety of stiffness-tunable biomaterials, reviewed here, have been developed to mimic the rigidity of the tumor extracellular matrix, culture systems that can recapitulate the broader extracellular context of the tumor microenvironment (including pH and temperature) remain comparably unexplored, partially due to the difficulty in independently tuning these parameters. Here, we investigate a self-assembled polypeptide network hydrogel as a cell culture platform and demonstrate that the culture parameters, including the substrate stiffness, extracellular pH and temperature, can be independently controlled. We then use this biomaterial as a cell culture substrate to assess the effect of stiffness, pH and temperature on Suit2 cells, a pancreatic cancer cell line, and demonstrate that these microenvironmental factors can regulate two critical transcription factors in cancer: yes-associated protein 1 (YAP) and hypoxia inducible factor (HIF-1A)

Effect of the Molecular Structure of TPU on the Cellular Structure of Nanocellular Polymers Based on PMMA/TPU Blends

From MDPI via Jisc Publications RouterHistory: accepted 2021-09-07, pub-electronic 2021-09-10Publication status: PublishedFunder: Junta de Castilla y León; Grant(s): I.Sanchez-CalderonFunder: Ministerio de Ciencia, Innovación y Universidades; Grant(s): RTI2018-098749-B-I00, PTQ2019-010560 (Victoria Bernardo-García)Funder: EREN; Grant(s): Ente Regional de la Energía de Castilla y León EREN_2019_L4_UVAIn this work, the effects of thermoplastic polyurethane (TPU) chemistry and concentration on the cellular structure of nanocellular polymers based on poly(methyl-methacrylate) (PMMA) are presented. Three grades of TPU with different fractions of hard segments (HS) (60%, 70%, and 80%) have been synthesized by the prepolymer method. Nanocellular polymers based on PMMA have been produced by gas dissolution foaming using TPU as a nucleating agent in different contents (0.5 wt%, 2 wt%, and 5 wt%). TPU characterization shows that as the content of HS increases, the density, hardness, and molecular weight of the TPU are higher. PMMA/TPU cellular materials show a gradient cell size distribution from the edge of the sample towards the nanocellular core. In the core region, the addition of TPU has a strong nucleating effect in PMMA. Core structure depends on the HS content and the TPU content. As the HS or TPU content increases, the cell nucleation density increases, and the cell size is reduced. Then, the use of TPUs with different characteristics allows controlling the cellular structure. Nanocellular polymers have been obtained with a core relative density between 0.15 and 0.20 and cell sizes between 220 and 640 nm

Controlling Doxorubicin Release from a Peptide Hydrogel through Fine-Tuning of Drug–Peptide Fiber Interactions

open access articleHydrogels are versatile materials that have emerged in the last few decades as promising candidates for a range of applications in the biomedical field, from tissue engineering and regenerative medicine to controlled drug delivery. In the drug delivery field, in particular, they have been the subject of significant interest for the spatially and temporally controlled delivery of anticancer drugs and therapeutics. Self-assembling peptide-based hydrogels, in particular, have recently come to the fore as potential candidate vehicles for the delivery of a range of drugs. In order to explore how drug–peptide interactions influence doxorubicin (Dox) release, five β-sheet-forming self-assembling peptides with different physicochemical properties were used for the purpose of this study, namely: FEFKFEFK (F8), FKFEFKFK (FK), FEFEFKFE (FE), FEFKFEFKK (F8K), and KFEFKFEFKK (KF8K) (F: phenylalanine; E: glutamic acid; K: lysine). First, Dox-loaded hydrogels were characterized to ensure that the incorporation of the drug did not significantly affect the hydrogel properties. Subsequently, Dox diffusion out of the hydrogels was investigated using UV absorbance. The amount of drug retained in F8/FE composite hydrogels was found to be directly proportional to the amount of charge carried by the peptide fibers. When cation−π interactions were used, the position and number of end-lysine were found to play a key role in the retention of Dox. In this case, the amount of Dox retained in F8/KF8K composite hydrogels was linked to the amount of end-lysine introduced, and an end-lysine/Dox interaction stoichiometry of 3/1 was obtained. For pure FE and KF8K hydrogels, the maximum amount of Dox retained was also found to be related to the overall concentration of the hydrogels and, therefore, to the overall fiber surface area available for interaction with the drug. For 14 mM hydrogel, ∼170–200 μM Dox could be retained after 24 h. This set of peptides also showed a broad range of susceptibilities to enzymatic degradation opening the prospect of being able to control also the rate of degradation of these hydrogels. Finally, the Dox released from the hydrogel was shown to be active and affect 3T3 mouse fibroblasts viability in vitro. Our study clearly shows the potential of this peptide design as a platform for the formulation of injectable or sprayable hydrogels for controlled drug delivery

Analysis of the Foaming Window for Thermoplastic Polyurethane with Different Hard Segment Contents

From MDPI via Jisc Publications RouterHistory: accepted 2021-09-11, pub-electronic 2021-09-17Publication status: PublishedA series of thermoplastic polyurethanes (TPUs) with different amounts of hard segments (HS) (40, 50 and 60 wt.%) are synthesized by a pre-polymer method. These synthesized TPUs are characterized by Shore hardness, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle X-ray diffraction (WAXD), dynamic mechanical thermal analysis (DMTA), and rheology. Then, these materials are foamed by a one-step gas dissolution foaming process and the processing window that allows producing homogeneous foams is analyzed. The effect of foaming temperature from 140 to 180 °C on the cellular structure and on density is evaluated, fixing a saturation pressure of 20 MPa and a saturation time of 1 h. Among the TPUs studied, only that with 50 wt.% HS allows obtaining a stable foam, whose better features are reached after foaming at 170 °C. Finally, the foaming of TPU with 50 wt.% HS is optimized by varying the saturation pressure from 10 to 25 MPa at 170 °C. The optimum saturation and foaming conditions are 25 MPa and 170 °C for 1 h, which gives foams with the lowest relative density of 0.74, the smallest average cell size of 4 μm, and the higher cell nucleation density of 8.0 × 109 nuclei/cm3. As a final conclusion of this investigation, the TPU with 50 wt.% HS is the only one that can be foamed under the saturation and foaming conditions used in this study. TPU foams containing 50 wt.% HS with a cell size below 15 microns and porosity of 1.4–18.6% can be obtained using foaming temperatures from 140 to 180 °C, saturation pressure of 20 MPa, and saturation time of 1 h. Varying the saturation pressure from 10 to 25 MPa and fixing the foaming temperature of 170 °C and saturation pressure of 1 h results in TPU foams with a cell size of below 37 microns and porosity of 1.7–21.2%