Effect of Peptide-Polymer Host-Guest Electrostatic Interactions on Self-Assembling Peptide Hydrogels Structural and Mechanical Properties and Polymer Diffusivity.

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E(FKFE)2 and K(FEFK)2, that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

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

Hydrogels 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 ß-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.

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.

Sequence-Specific Detection of Unlabeled Nucleic Acid Biomarkers Using a "One-Pot" 3D Molecular Sensor.

DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide building blocks to provide sequence-specific, PCR-free fluorescent detection of unlabeled nucleic acid sequences. We discriminated at picomolar detection limits (<7 pM) "perfect-match" from mismatched sequences, down to a single nucleotide mutation, buried within longer lengths of the target. Detailed characterization by NMR, TEM, mass spectrometry, and rheology provided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sensitivity and detection limit. We discuss the generic design, which is based on a highly predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g., SNPs) and RNA (messenger, micro, and viral genomic RNA).

Designing Peptide/Graphene Hybrid Hydrogels through Fine-Tuning of Molecular Interactions.

A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling ß-sheet-forming, self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs were characterized; subsequently, the molecular interaction between peptides nanofibers and GDs were probed before formulating and mechanically characterizing the hybrid hydrogels. We show how the interplay between electrostatic interactions, which can be attractive or repulsive, and hydrophobic (and π-π in the case of peptide containing phenylalanine) interactions, which are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fiber adhesion to the flakes, the overall hydrophobicity of the peptides, as well as the type of fibrillar network formed. Finally, the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesemchymal stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels, allowing the incorporation of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications.

Controlling Self-Assembling Peptide Hydrogel Properties through Network Topology.

Self-assembling peptide-based hydrogels have encountered increasing interest in the recent years as scaffolds for 3D cell culture or for controlled drug delivery. One of the main challenges is the fine control of the mechanical properties of these materials. The bulk properties of hydrogels not only depend on the intrinsic properties of the fibers but also on the network topology formed. In this work we show how fiber-fiber interactions can be manipulated by design to control the final hydrogel network topology and therefore control the final properties of the material. This was achieved by exploiting the design features of ß-sheet forming peptides based on hydrophobic and hydrophilic residue alternation and exploiting the ability of the arginine's guanidine side group to interact with itself and with other amino acid side groups. By designing octa-peptides based on phenylalanine, glutamic acid, lysine, and arginine, we have investigated how fiber association and bundling affect the dynamic shear modulus of hydrogels and how it can be controlled by design. This work opens the possibility to fine-tune by design the bulk properties of peptide hydrogels.

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

ß-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 ß-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).

A modular self-assembly approach to functionalised ß-sheet peptide hydrogel biomaterials.

Two complementary ß-sheet-forming decapeptides have been created that form binary self-repairing hydrogels upon combination of the respective free-flowing peptide solutions at pH 7 and >0.28 wt%. The component peptides showed little structure separately but formed extended ß-sheet fibres upon mixing, which became entangled to produce stiff hydrogels. Microscopy revealed two major structures; thin fibrils with a twisted or helical appearance and with widths comparable to the predicted lengths of the peptides within a ß-sheet, and thicker, longer, interwoven fibres that appear to comprise laterally-packed fibrils. A range of gel stiffnesses (G' from 0.05 to 100 kPa) could be attained in this system by altering the assembly conditions, stiffnesses that cover the rheological properties desirable for cell culture scaffolds. Doping in a RGD-tagged component peptide at 5 mol% improved 3T3 fibroblast attachment and viability compared to hydrogel fibres without RGD functionalisation.

Controlling network topology and mechanical properties of co-assembling peptide hydrogels.

Oligopeptides are well-known to self-assemble into a wide array of nanostructures including ß-sheet-rich fibers that when present above a critical concentration become entangled and form self-supporting hydrogels. The length, quantity, and interactions between fibers influence the mechanical properties of the hydrogel formed and this is typically achieved by varying the peptide concentration, pH, ionic strength, or the addition of a second species or chemical cross-linking agent. Here, we outline an alternative, facile route to control the mechanical properties of the self-assembling octa-peptide, FEFEFKFK (FEKII); simply doping with controlled quantities of its double length peptide, FEFEFKFK-GG-FKFKFEFE (FEKII18). The structure and properties of a series of samples were studied here (0­100 M% of FEKII18) using Fourier transform infrared, small angle X-ray scattering, transmission electron microscopy, and oscillatory rheology. All samples were found to contain elongated, flexible fibers and all mixed samples contained Y-shaped branch points and parallel fibers which is attributed to the longer peptide self-assembling within two fibers, thus creating a cross-link in the network structure. Such behavior was reflected in an increase in the elasticity of the mixed samples with increasing quantity of double peptide. Interestingly the elastic modulus increased up to 30 times the pure FEKII value simply by adding 28 M% of FEKII18. These observations provide an easy, off-the-shelf method for an end-user to control the cross-linked network structure of the peptide hydrogel, and consequently strength of the hydrogel simply by physically mixing pre-determined quantities of two similar peptide molecules.

Enzymatically triggered peptide hydrogels for 3D cell encapsulation and culture.

We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.

Nanofibrillar Peptide hydrogels for the immobilization of biocatalysts for chemical transformations.

Enzymes are attractive, "green" alternatives to chemical catalysts within the industrial sector, but their robustness to environmental conditions needs optimizing. Here, an enzyme is tagged chemically and recombinantly with a self-assembling peptide that allows the conjugate to spontaneously assemble with pure peptide to form ß-sheet-rich nanofibers decorated with tethered enzyme. Above a critical concentration, these fibers entangle and form a 3D hydrogel. The immobilized enzyme catalyzes chemical transformations and critically its stability is increased significantly where it retains activity after exposure to high temperatures (90 °C) and long storage times (up to 12 months).

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Effect of enzyme concentration of the morphology and properties of enzymatically triggered peptide hydrogels.

We have recently shown that thermolysine, a protease enzyme obtained from Bacillus thermoproteolyticus rokko , can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by Cenz, where the shear moduli of the hydrogels were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the microscale are at the origin of their difference in mechanical properties. In this paper, we propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. These were confirmed by TEM measurements. The existence of these denser network regions will result in the reinforcement of the hydrogels, thus, explaining the high shear moduli obtained increasing Cenz.

Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool†Dedicated to the memory of Joachim H. G. Steinke.‡Electronic supplementary information (ESI) available: Further experimental data. See DOI: 10.1039/c3sm00131hClick here for additional data file.

The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheology studies and TEM of the corresponding hydrogels (including PNIPAAm-containing systems) showed no significant variation in properties and hydrogel morphology compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.

Novel hydrogels: are they poised to transform 3D cell-based assay systems in early drug discovery?

INTRODUCTION: Success in drug discovery remains unpredictable. However, more predictive and relevant disease models are becoming pivotal to demonstrating the clinical benefits of new drugs earlier in the lengthy drug discovery process. Novel hydrogel scaffolds are being developed to transform the relevance of such 3D cell-based in vitro assay systems. AREAS COVERED: Most traditional hydrogels are still of unknown composition and suffer significant batch-to-batch variations, which lead to technical constraints. This article looks at how a new generation of novel synthetic hydrogels that are based on self-assembling peptides are poised to transform 3D cell-based assay systems by improving their relevance, reproducibility and scalability. EXPERT OPINION: The emerging advantages of using these novel hydrogels for human 3D screening assays should enable the discovery of more cost-effective drugs, leading to improved patient benefits. Such a disruptive change could also reduce the considerable time lag from obtaining in vitro assay data to initiating clinical trials. There is now a sufficient body of data available in the literature to enable this ambition to become a reality by significantly improving the predictive validity of 3D cell-based assays in early drug discovery. Novel hydrogels are key to unlocking the full potential of these assay systems.

Growth and differentiation of human induced pluripotent stem cell (hiPSC)-derived kidney organoids using fully synthetic peptide hydrogels.

Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully-controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G'). The resulting organoids contained complex structures comparable to those differentiated within the animal-derived matrix, Matrigel. Single-cell RNA sequencing (scRNA-seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids, while SAPH-derived organoids were enriched for stromal-associated cell populations. Notably, differentiation within a higher G' SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide-based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids.

A Systematic Comparative Assessment of the Response of Ovarian Cancer Cells to the Chemotherapeutic Cisplatin in 3D Models of Various Structural and Biochemical Configurations-Does One Model Type Fit All?

Epithelial Ovarian Cancer (EOC) is a silent, deadly and aggressive gynaecological disease with a relatively low survival rate. This has been attributed, to some extent, to EOC's high recurrence rate and resistance to currently available platinum-based chemotherapeutic treatment methods. Multiple groups have studied and reported the effect of chemotherapeutic agents on various EOC 3D in vitro models. However, there are very few studies wherein a direct comparative study has been carried out between the different in vitro 3D models of EOC and the effect of chemotherapy within them. Herein, we report, for the first time, a direct comprehensive systematic comparative study of three different 3D in vitro platforms, namely (i) spheroids, (ii) synthetic PeptiGels/hydrogels of various chemical configurations and (iii) polymeric scaffolds with coatings of various extracellular matrices (ECMs) on the cell growth and response to the chemotherapeutic (Cisplatin) for ovary-derived (A2780) and metastatic (SK-OV-3) EOC cell lines. We report that all three 3D models are able to support the growth of EOC, but for different time periods (varying from 7 days to 4 weeks). We have also reported that chemoresistance to Cisplatin, in vitro, observed especially for metastatic EOC cells, is platform-dependent, in terms of both the structural and biochemical composition of the model/platform. Our study highlights the importance of selecting an appropriate 3D platform for in vitro tumour model development. We have demonstrated that the selection of the best platform for producing in vitro tumour models depends on the cancer/cell type, the experimental time period and the application for which the model is intended.

Biofabrication of the osteochondral unit and its applications: Current and future directions for 3D bioprinting.

Multiple prevalent diseases, such as osteoarthritis (OA), for which there is no cure or full understanding, affect the osteochondral unit; a complex interface tissue whose architecture, mechanical nature and physiological characteristics are still yet to be successfully reproduced in vitro. Although there have been multiple tissue engineering-based approaches to recapitulate the three dimensional (3D) structural complexity of the osteochondral unit, there are various aspects that still need to be improved. This review presents the different pre-requisites necessary to develop a human osteochondral unit construct and focuses on 3D bioprinting as a promising manufacturing technique. Examples of 3D bioprinted osteochondral tissues are reviewed, focusing on the most used bioinks, chosen cell types and growth factors. Further information regarding the applications of these 3D bioprinted tissues in the fields of disease modelling, drug testing and implantation is presented. Finally, special attention is given to the limitations that currently hold back these 3D bioprinted tissues from being used as models to investigate diseases such as OA. Information regarding improvements needed in bioink development, bioreactor use, vascularisation and inclusion of additional tissues to further complete an OA disease model, are presented. Overall, this review gives an overview of the evolution in 3D bioprinting of the osteochondral unit and its applications, as well as further illustrating limitations and improvements that could be performed explicitly for disease modelling.

Substrate Stiffness-Driven Membrane Tension Modulates Vesicular Trafficking via Caveolin-1.

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the ß1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.