An increase in Wharton's jelly membrane osteocompatibility by a genipin-cross-link.

Wharton's Jelly (WJ) has attracted significant interest in the field of tissue healing thanks to its biological properties, including antibacterial activity and immunomodulation. However, due to the fast degradation and poor mechanical behavior in biological environment, its application in bone regeneration is compromised. Here, we proposed to use genipin as an efficient cross-linking agent to significantly improve the elasticity and the enzymatical stability of the WJ matrix. The degree of cross-linking, linear elastic moduli, and collagenase resistance varied over a wide range depending on genipin concentration. Furthermore, our results highlighted that an increase in genipin concentration led to a decreased surface wettability, therefore impairing cell attachment and proliferation. The genipin cross-linking prevented rapid in vitro and in vivo degradation, but led to an adverse host reaction and calcification. When implanted in the parietal bone defect, a limited parietal bone regeneration to the dura was observed. We conclude that genipin-cross-linked WJ is a versatile medical device however, a careful selection is required with regards to the genipin concentration.

Recent Developments in Layer-by-Layer Assembly for Drug Delivery and Tissue Engineering Applications.

Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.

Preclinical in vitro evaluation of implantable materials: conventional approaches, new models and future directions.

Nowadays, implants and prostheses are widely used to repair damaged tissues or to treat different diseases, but their use is associated with the risk of infection, inflammation and finally rejection. To address these issues, new antimicrobial and anti-inflammatory materials are being developed. Aforementioned materials require their thorough preclinical testing before clinical applications can be envisaged. Although many researchers are currently working on new in vitro tissues for drug screening and tissue replacement, in vitro models for evaluation of new biomaterials are just emerging and are extremely rare. In this context, there is an increased need for advanced in vitro models, which would best recapitulate the in vivo environment, limiting animal experimentation and adapted to the multitude of these materials. Here, we overview currently available preclinical methods and models for biological in vitro evaluation of new biomaterials. We describe several biological tests used in biocompatibility assessment, which is a primordial step in new material's development, and discuss existing challenges in this field. In the second part, the emphasis is made on the development of new 3D models and approaches for preclinical evaluation of biomaterials. The third part focuses on the main parameters to consider to achieve the optimal conditions for evaluating biocompatibility; we also overview differences in regulations across different geographical regions and regulatory systems. Finally, we discuss future directions for the development of innovative biomaterial-related assays: in silico models, dynamic testing models, complex multicellular and multiple organ systems, as well as patient-specific personalized testing approaches.

A miniaturized genotoxicity evaluation system for fast biomaterial-related risk assessment.

Implants and prostheses are widely used to either repair damaged tissues or treat different diseases. Before an implant reaches the market, multiple preclinical and clinical tests must be performed. Along with cytotoxicity or hemocompatibility preclinical tests, genotoxicity is an essential feature to investigate. Indeed, the materials used for implantation should be non-genotoxic, i.e. they should not promote mutations that can potentially lead to tumour formation. However, given the complexity level of genotoxicity tests, such tests are not readily available to biomaterials researchers, which is the reason why this aspect is severely underreported in the literature. To solve this problem, we developed a simplified genotoxicity test that can be further adapted by standard biomaterials laboratories. We started by simplifying the classic Ames test in Petri dishes, after which we developed a miniaturized test in a microfluidic chip, which takes only 24 hours, requiring significantly less material and space. An automatization option with a customized testing chamber architecture and microfluidics-based control system has been designed as well. This optimized microfluidic chip system can significantly improve the availability of genotoxicity tests for biomaterials developers, with the additional benefit of more in-depth observation and quantitative comparison due to the availability of processable image components.

The Effect of Machine Learning Algorithms on the Prediction of Layer-by-Layer Coating Properties.

Layer-by-layer film (LbL) coatings made of polyelectrolytes are a powerful tool for surface modification, including the applications in the biomedical field, for food packaging, and in many electrochemical systems. However, despite the number of publications related to LbL assembly, predicting LbL coating properties represents quite a challenge, can take a long time, and be very costly. Machine learning (ML) methodologies that are now emerging can accelerate and improve new coating development and potentially revolutionize the field. Recently, we have demonstrated a preliminary ML-based model for coating thickness prediction. In this paper, we compared several ML algorithms for optimizing a methodology for coating thickness prediction, namely, linear regression, Support Vector Regressor, Random Forest Regressor, and Extra Tree Regressor. The current research has shown that learning algorithms are effective in predicting the coating output value, with the Extra Tree Regressor algorithm demonstrating superior predictive performance, when used in combination with optimized hyperparameters and with missing data imputation. The best predictors of the coating thickness were determined, and they can be later used to accurately predict coating thickness, avoiding measurement of multiple parameters. The development of optimized methodologies will ensure different reliable predictive models for coating property/function relations. As a continuation, the methodology can be adapted and used for predicting the outputs connected to antimicrobial, anti-inflammatory, and antiviral properties in order to be able to respond to actual biomedical problems such as antibiotic resistance, implant rejection, or COVID-19 outbreak.

Biomimetic Bilayered Scaffolds for Tissue Engineering: From Current Design Strategies to Medical Applications.

Tissue damage due to cancer, congenital anomalies, and injuries needs new efficient treatments that allow tissue regeneration. In this context, tissue engineering shows a great potential to restore the native architecture and function of damaged tissues, by combining cells with specific scaffolds. Scaffolds made of natural and/or synthetic polymers and sometimes ceramics play a key role in guiding cell growth and formation of the new tissues. Monolayered scaffolds, which consist of uniform material structure, are reported as not being sufficient to mimic complex biological environment of the tissues. Osteochondral, cutaneous, vascular, and many other tissues all have multilayered structures, therefore multilayered scaffolds seem more advantageous to regenerate these tissues. In this review, recent advances in bilayered scaffolds design applied to regeneration of vascular, bone, cartilage, skin, periodontal, urinary bladder, and tracheal tissues are focused on. After a short introduction on tissue anatomy, composition and fabrication techniques of bilayered scaffolds are explained. Then, experimental results obtained in vitro and in vivo are described, and their limitations are given. Finally, difficulties in scaling up production of bilayer scaffolds and reaching the stage of clinical studies are discussed when multiple scaffold components are used.

A Coculture Based, 3D Bioprinted Ovarian Tumor Model Combining Cancer Cells and Cancer Associated Fibroblasts.

Ovarian cancer remains a major public health issue due to its poor prognosis. To develop more effective therapies, it is crucial to set-up reliable models that closely mimic the complexity of the ovarian tumor's microenvironment. 3D bioprinting is currently a promising approach to build heterogenous and reproducible cancer models with controlled shape and architecture. However, this technology is still poorly investigated to model ovarian tumors. In this study, a 3D bioprinted ovarian tumor model combining cancer cells (SKOV-3) and cancer associated fibroblasts (CAFs) are described. The resulting tumor models show their ability to maintain cell viability and proliferation. Cells are observed to self-assemble in heterotypic aggregates. Moreover, CAFs are observed to be recruited and to circle cancer cells reproducing an in vivo process taking place in the tumor microenvironment. Interestingly, this approach also shows its ability to rapidly generate a high number of reproducible tumor models that can be subjected to usual characterizations (cell viability and metabolic activity; histology and immunological studies; and real-time imaging). Therefore, these ovarian tumor models can be an interesting tool for high throughput drug screening applications.

Escherichia coli Biofilm Formation, Motion and Protein Patterns on Hyaluronic Acid and Polydimethylsiloxane Depend on Surface Stiffness.

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9-574 kPa) and HA (hyaluronic acid gels, 44 Pa-2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth.

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers.

The engineering of skeletal muscle tissue, a highly organized structure of myotubes, is promising for the treatment of muscle injuries and muscle diseases, for replacement, or for pharmacology research. Muscle tissue development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers, which is challenging to achieve on flat surfaces. In this work, we designed hydrogen-bonded tannic acid/collagen layer-by-layer (TA/COL LbL) nanofilms using a simple brushing method to address this issue. In comparison to films obtained by dipping, brushed TA/COL films showed oriented COL fibers of 60 nm diameter along the brushing direction. Built at acidic pH due to COL solubility, TA/COL films released TA in physiological conditions with a minor loss of thickness. After characterization of COL fibers' orientation, human myoblasts (C25CL48) were seeded on the oriented TA/COL film, ended by COL. After 12 days in a differentiation medium without any other supplement, human myoblasts were able to align on brushed TA/COL films and to differentiate into long aligned myotubes (from hundreds of µm up to 1.7 mm length) thanks to two distinct properties: (i) the orientation of COL fibers guiding myoblasts' alignment and (ii) the TA release favoring the differentiation. This simple and potent brushing process allows the development of anisotropic tissues in vitro which can be used for studies of drug discovery and screening or the replacement of damaged tissue.

Polyarginine as a Simultaneous Antimicrobial, Immunomodulatory, and miRNA Delivery Agent within Polyanionic Hydrogel.

Implantation of biomedical devices is followed by immune response to the implant, as well as occasionally bacterial, yeast, and/or fungal infections. In this context, new implant materials and coatings that deal with medical device-associated complications are required. Antibacterial and anti-inflammatory materials are also required for wound healing applications, especially in diabetic patients with chronic wounds. In this work, hyaluronic acid (HA) hydrogels with triple activity: antimicrobial, immunomodulatory, and miRNA delivery agent, are presented. It is demonstrated that polyarginine with a degree of polymerization of 30 (PAR30), which is previously shown to have a prolonged antibacterial activity, decreases inflammatory response of lipopolysaccharide-stimulated macrophages. In addition, PAR30 accelerates fibroblast migration in macrophage/fibroblast coculture system, suggesting a positive effect on wound healing. Furthermore, PAR30 allows to load miRNA into HA hydrogels, and then to deliver them into the cells. To the authors knowledge, this study is the first describing miRNA-loaded hydrogels with antibacterial effect and anti-inflammatory features. Such system can become a tool for the treatment of infected wounds, e.g., diabetic ulcers, as well as for foreign body response modulation.

Prediction of coating thickness for polyelectrolyte multilayers via machine learning.

Layer-by-layer (LbL) deposition method of polyelectrolytes is a versatile way of developing functional nanoscale coatings. Even though the mechanisms of LbL film development are well-established, currently there are no predictive models that can link film components with their final properties. The current health crisis has shown the importance of accelerated development of biomedical solutions such as antiviral coatings, and the implementation of machine learning methodologies for coating development can enable achieving this. In this work, using literature data and newly generated experimental results, we first analyzed the relative impact of 23 coating parameters on the coating thickness. Next, a predictive model has been developed using aforementioned parameters and molecular descriptors of polymers from the DeepChem library. Model performance was limited because of insufficient number of data points in the training set, due to the scarce availability of data in the literature. Despite this limitation, we demonstrate, for the first time, utilization of machine learning for prediction of LbL coating properties. It can decrease the time necessary to obtain functional coating with desired properties, as well as decrease experimental costs and enable the fast first response to crisis situations (such as pandemics) where coatings can positively contribute. Besides coating thickness, which was selected as an output value in this study, machine learning approach can be potentially used to predict functional properties of multilayer coatings, e.g. biocompatibility, cell adhesive, antibacterial, antiviral or anti-inflammatory properties.

Recent Advances in Antiinflammatory Material Design.

Implants and prostheses are widely used to replace damaged tissues or to treat various diseases. However, besides the risk of bacterial or fungal infection, an inflammatory response usually occurs. Here, recent progress in the field of anti-inflammatory biomaterials is described. Different materials and approaches are used to decrease the inflammatory response, including hydrogels, nanoparticles, implant surface coating by polymers, and a variety of systems for anti-inflammatory drug delivery. Complex multifunctional systems dealing with inflammation, microbial infection, bone regeneration, or angiogenesis are also described. New promising stimuli-responsive systems, such as pH- and temperature-responsive materials, are also being developed that would enable an "intelligent" antiinflammatory response when the inflammation occurs. Together, different approaches hold promise for creation of novel multifunctional smart materials allowing better implant integration and tissue regeneration.

New Smart Antimicrobial Hydrogels, Nanomaterials, and Coatings: Earlier Action, More Specific, Better Dosing?

To fight against antibiotic-resistant bacteria adhering and developing on medical devices, which is a growing problem worldwide, researchers are currently developing new "smart" materials and coatings. They consist in delivery of antimicrobial agents in an intelligent way, i.e., only when bacteria are present. This requires the use of new and sophisticated tools combining antimicrobial agents with lipids or polymers, synthetic and/or natural. In this review, three classes of innovative materials are described: hydrogels, nanomaterials, and thin films. Moreover, smart antibacterial materials can be classified into two groups depending on the origin of the stimulus used: those that respond to a nonbiological stimulus (light, temperature, electric and magnetic fields) and those that respond to a biological stimulus related to the presence of bacteria, such as changes in pH or bacterial enzyme secretion. The bacteria presence can induce a pH change that constitutes a first potential biological trigger allowing the system to become active. A second biological trigger signal consists in enzymes produced by bacteria themselves. A complete panel of recent studies will be given focusing on the design of such innovative smart materials that are sensitive to biological triggers.

Polyanionic Hydrogels as Reservoirs for Polycationic Antibiotic Substitutes Providing Prolonged Antibacterial Activity.

Implantation of biomedical devices is often followed by bacterial infections that may seriously affect implant functionalities and lead to their failure. In the context of bacterial resistance to antibiotics, which is a growing problem worldwide, new strategies that are able to overcome these problems are needed. In this work, we introduce a new formulation of hyaluronic acid (HA)-based antimicrobial material: HA hydrogels loaded with polyarginine (PAR), a polycationic antibiotic substitute. The loading is possible through electrostatic interactions between negatively charged HA and positively charged PAR. Such hydrogels absorb high quantities of PAR, which are then gradually released from the hydrogel. This original system provides a long-lasting antibacterial effect on an in vitro model of repetitive infection, thus demonstrating a strong potential to fight multiple rounds of infections that are resistant to antibiotic treatment. In addition, HA-PAR hydrogels could be deposited onto/into medical devices such as wound dressings and mesh prostheses used in clinical applications. Finally, we performed first in vivo tests of hydrogel-coated mesh materials to verify their biocompatibility in a rat model, which show no difference between control HA hydrogel and PAR-loaded hydrogel in terms of inflammation.

Construction and myogenic differentiation of 3D myoblast tissues fabricated by fibronectin-gelatin nanofilm coating.

In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ∼76 µm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models.

Control of the Proliferation/Differentiation Balance in Skeletal Myoblasts by Integrin and Syndecan Targeting Peptides.

Controlling the different steps of cell differentiation in vitro using bioactive surfaces may be useful in view of future cell therapies. Substrates presenting peptides, which are minimal fragments of extracellular matrix (ECM) proteins may be used for this purpose. In this work, we used polyelectrolyte multilayer films presenting two peptides derived from different muscle ECM proteins to target syndecan or/and integrin receptors. We showed that the presence of laminin-derived peptide to target syndecan-1 promotes lamellipodia formation, increases migration speed, directionality, and cell proliferation but impaired myotube formation. The cellular effects of L2synd are under the control of Rac1 and Cdc42 activities and involved ß1 integrin in contrast to RGD-containing peptide, which enabled adhesion via ß3 integrins and muscle cell differentiation. Our results show that peptides grafted onto multilayered films can guide the proliferation/differentiation balance and reveal crosstalk between different adhesion receptors.

Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation.

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(L-lysine) (PLL) and poly(L-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.

Polyelectrolyte Multilayer Assemblies on Materials Surfaces: From Cell Adhesion to Tissue Engineering.

Controlling the bulk and surface properties of materials is a real challenge for bioengineers working in the fields of biomaterials, tissue engineering and biophysics. The layer-by-layer (LbL) deposition method, introduced 20 years ago, consists in the alternate adsorption of polyelectrolytes that self-organize on the material's surface, leading to the formation of polyelectrolyte multilayer (PEM) films.1 Because of its simplicity and versatility, the procedure has led to considerable developments of biological applications within the past 5 years. In this review, we focus our attention on the design of PEM films as surface coatings for applications in the field of physical properties that have emerged as being key points in relation to biological processes. The numerous possibilities for adjusting the chemical, physical, and mechanical properties of PEM films have fostered studies on the influence of these parameters on cellular behaviors. Importantly, PEM have emerged as a powerful tool for the immobilization of biomolecules with preserved bioactivity.

A material's point of view on recent developments of polymeric biomaterials: control of mechanical and biochemical properties.

Cells respond to a variety of stimuli, including biochemical, topographical and mechanical signals originating from their micro-environment. Cell responses to the mechanical properties of their substrates have been increasingly studied for about 14 years. To this end, several types of materials based on synthetic and natural polymers have been developed. Presentation of biochemical ligands to the cells is also important to provide additional functionalities or more selectivity in the details of cell/material interaction. In this review article, we will emphasize the development of synthetic and natural polymeric materials with well-characterized and tunable mechanical properties. We will also highlight how biochemical signals can be presented to the cells by combining them with these biomaterials. Such developments in materials science are not only important for fundamental biophysical studies on cell/material interactions but also for the design of a new generation of advanced and highly functional biomaterials.