Review: the potential impact of surface crystalline states of titanium for biomedical applications.

In many biomedical applications, titanium forms an interface with tissues, which is crucial to ensure its long-term stability and safety. In order to exert control over this process, titanium implants have been treated with various methods that induce physicochemical changes at nano and microscales. In the past 20 years, most of the studies have been conducted to see the effect of topographical and physicochemical changes of titanium surface after surface treatments on cells behavior and bacteria adhesion. In this review, we will first briefly present some of these surface treatments either chemical or physical and we explain the biological responses to titanium with a specific focus on adverse immune reactions. More recently, a new trend has emerged in titanium surface science with a focus on the crystalline phase of titanium dioxide and the associated biological responses. In these recent studies, rutile and anatase are the major two polymorphs used for biomedical applications. In the second part of this review, we consider this emerging topic of the control of the crystalline phase of titanium and discuss its potential biological impacts. More in-depth analysis of treatment-related surface crystalline changes can significantly improve the control over titanium/host tissue interface and can result in considerable decreases in implant-related complications, which is currently a big burden on the healthcare system.

Immunomodulation with Self-Crosslinked Polyelectrolyte Multilayer-Based Coatings.

This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1ß). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.

Cationic homopolypeptides: A versatile tool to design multifunctional antimicrobial nanocoatings.

Postoperative infections are the most common complications faced by surgeons after implant surgery. To address this issue, an emerging and promising approach is to develop antimicrobial coatings using antibiotic substitutes. We investigated the use of polycationic homopolypeptides in a layer-by-layer coating combined with hyaluronic acid (HA) to produce an effective antimicrobial shield. The three peptide-based polycations used to make the coatings, poly(l-arginine) (PAR), poly(l-lysine), and poly(l-ornithine), provided an efficient antibacterial barrier by a contact-killing mechanism against Gram-positive, Gram-negative, and antibiotic-resistant bacteria. Moreover, this activity was higher for homopolypeptides containing 30 amino-acid residues per polycation chain, emphasizing the impact of the polycation chain length and its mobility in the coatings to deploy its contact-killing antimicrobial properties. However, the PAR-containing coating emerged as the best candidate among the three selected polycations, as it promoted cell adhesion and epithelial monolayer formation. It also stimulated nitric oxide production in endothelial cells, thereby facilitating angiogenesis and subsequent tissue regeneration. More interestingly, bacteria did not develop a resistance to PAR and (PAR/HA) also inhibited the proliferation of eukaryotic pathogens, such as yeasts. Furthermore, in vivo investigations on a (PAR/HA)-coated hernia mesh implanted on a rabbit model confirmed that the coating had antibacterial properties without causing chronic inflammation. These impressive synergistic activities highlight the strong potential of PAR/HA coatings as a key tool in combating bacteria, including those resistant to conventional antibiotics and associated to medical devices.

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell-derived epithelial tissue patches.

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

Corrigendum: Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation.

[This corrects the article DOI: 10.3389/fbioe.2018.00108.].

Use of Nanoparticles in Tissue Engineering and Regenerative Medicine.

Advances in nanoparticle (NP) production and demand for control over nanoscale systems have had significant impact on tissue engineering and regenerative medicine (TERM). NPs with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli-response delivery potential, and precise control over behavior (via external stimuli such as magnetic fields) have made it possible their use for improving engineered tissues and overcoming obstacles in TERM. Functional tissue and organ replacements require a high degree of spatial and temporal control over the biological events and also their real-time monitoring. Presentation and local delivery of bioactive (growth factors, chemokines, inhibitors, cytokines, genes etc.) and contrast agents in a controlled manner are important implements to exert control over and monitor the engineered tissues. This need resulted in utilization of NP based systems in tissue engineering scaffolds for delivery of multiple growth factors, for providing contrast for imaging and also for controlling properties of the scaffolds. Depending on the application, materials, as polymers, metals, ceramics and their different composites can be utilized for production of NPs. In this review, we will cover the use of NP systems in TERM and also provide an outlook for future potential use of such systems.

Multifunctional polymeric implant coatings based on gelatin, hyaluronic acid derivative and chain length-controlled poly(arginine).

Surface of the implantable devices is the root cause of several complications such as infections, implant loosening and chronic inflammation. There is an urgent need for multifunctional coatings that can address these shortcomings simultaneously in a manner similar to the structures of extracellular matrix. Herein, we developed a coating system composed of ECM components and a naturally derived polypeptide. The interactions between the coating components create an environment that enables incorporation of an antimicrobial/angiogenic polypeptide. The film composition is based gelatin and hyaluronic acid modified with aldehyde groups (HA-Ald) that can react with poly (arginine) (PAR) through transient interactions. Nanoplasmon measurements demonstrated a significantly higher loading of PAR in films containing HA-Ald with longer retention of PAR in the structure. The presence of PAR not only provides to the film surface antimicrobial (contact-killing) properties but also increased endothelial cell-cell contacts (PECAM) and VEGFA gene expression and secretion by human vascular endothelial cells. This multifunctional coating can be easily applied to surface of implants where it can enact on several problems simultaneously.

Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation.

The function of soft tissues is intricately linked to their connections with the other systems of the body such as circulation, nervous system, and immune system. The presence of resident macrophages in tissues provides a means to control tissue homeostasis and also a way to react to the physical/biological insults and tissue damage. Thus, incorporation of resident macrophage like phenotype-controlled macrophages in engineered tissues can improve their fidelity as model tissues and also improve their rate of integration and facilitate the resolution of inflammation for regenerative medicine applications. Herein, we demonstrate two potential ways to immunoassist the remodeling process of engineered soft tissues in three-dimensional (3-D) gelatin based hydrogels containing fibroblasts and/or endothelial cells: (i) with supplementation of interleukin-4 (IL-4) in the presence of macrophages and (ii) in tri-culture via naive monocytes or differentiated macrophages. The presence of IL-4 had a proliferative effect on fibroblasts, with a significant boosting effect on proliferation and cytokine secretion in the presence of differentiated macrophages with an upregulation of activin, interleukin-1 receptor antagonist (IL-1RA), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß), creating a more stimulating microenvironment. The addition of IL-4 in endothelial cell/macrophage co-culture configuration improved the organization of the sprout-like structures, with a boost in proliferation at day 1 and with an upregulation of IL-6 and IL-1RA at the earliest stage in the presence of differentiated macrophages creating a favorable microenvironment for angiogenesis. In tri-culture conditions, the presence of monocytes or macrophages resulted in a denser tissue-like structure with highly remodeled hydrogels. The presence of differentiated macrophages had a boosting effect on the angiogenic secretory microenvironment, such as IL-6 and IL-8, without any additional cytokine supplementation. The presence of fibroblasts in combination with endothelial cells also had a significant effect on the secretion of angiopoietin. Our results demonstrate that incorporation of macrophages in a resident macrophage function and their phenotype control have significant effects on the maturation and cytokine microenvironment of 3-D multiple cell type-laden hydrogels, which can be harnessed for better integration of implantable systems and for more physiologically relevant in vitro tissue models with an immune component.

Incorporation of resident macrophages in engineered tissues: Multiple cell type response to microenvironment controlled macrophage-laden gelatine hydrogels.

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, an in-vitro model of interaction was developed between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, naive THP-1 monocytes, endothelial cells and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. To mimic resident macrophages, THP-1 cells were encapsulated in the presence or absence of IL-4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage-laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes-laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in more secretion of both pro- and anti-inflammatory cytokines compared to M2 stimulation. It was demonstrated that there is a distinct effect of encapsulated macrophages on the behaviour of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation.

Auxiliary Biomembranes as a Directional Delivery System To Control Biological Events in Cell-Laden Tissue-Engineering Scaffolds.

Delivery of growth factors is an indispensable part of tissue engineering. Here, we describe a detachable membrane-based release system composed of extracellular matrix components that can be attached to hydrogels to achieve directional release of bioactive molecules. This way, the release of cytokines/growth factors can be started at a desired point of tissue maturation or directly in vivo. As a model, we develop thin films of an interpenetrating network of double-cross-linked gelatin and hyaluronic acid derivatives. The use of the auxiliary release system with vascular endothelial growth factor results in extensive sprouting by encapsulated vascular endothelial cells. The presence of the release system with interleukin-4 results in clustering of encapsulated macrophages with a significant decrease in M1 macrophages (proinflammatory). This system can be used in conjunction with three-dimensional structures as an auxiliary system to control artificial tissue maturation and growth.

Generation of anti-inflammatory macrophages for implants and regenerative medicine using self-standing release systems with a phenotype-fixing cytokine cocktail formulation.

The immediate tissue microenvironment of implanted biomedical devices and engineered tissues is highly influential on their long term fate and efficacy. The creation of a long-term anti-inflammatory microenvironment around implants and artificial tissues can facilitate their integration. Macrophages are highly plastic cells that define the tissue reactions on the implanted material. Local control of macrophage phenotype by long-term fixation of their healing activities and suppression of inflammatory reactions are required to improve implant acceptance. Herein, we describe the development of a cytokine cocktail (M2Ct) that induces stable M2-like macrophage phenotype with significantly decreased pro-inflammatory cytokine and increased anti-inflammatory cytokine secretion profile. The positive effect of the M2Ct was shown in an in vitro wound healing model; where M2Ct facilitated wound closure by human fibroblasts in co-culture conditions. Using a model for induction of inflammation by LPS we have shown that the M2Ct phenotype is stable for 12days. However, in the absence of M2Ct in the medium macrophages underwent rapid pro-inflammatory re-programming upon IFNg stimulation. Therefore, loading and release of the cytokine cocktail from a self-standing, transferable gelatin/tyraminated hyaluronic acid based release system was developed to stabilize macrophage phenotype for in vivo applications in implantation and tissue engineering. The M2Ct cytokine cocktail retained its anti-inflammatory activity in controlled release conditions. Our data indicate that the direct application of a potent M2 inducing cytokine cocktail in a transferable release system can significantly improve the long term functionality of biomedical devices by decreasing pro-inflammatory cytokine secretion and increasing the rate of wound healing. STATEMENT OF SIGNIFICANCE: Uncontrollable activation of macrophages in the microenvironment of implants and engineered tissues is a significant problem leading to poor integration of implants and artificial tissues. In the current manuscript we demonstrate that self-standing, transferable gelatin/tyraminated hyaluronic acid based thin films are perspective tools for controlled release of anti-inflammatory cytokine combinations and can be used to down-modulate macrophage activation on implant surfaces. We also show that optimized cytokine cocktail consisting of IL4/IL10/TGFß1 (M2Ct) induces long-term anti-inflammatory and pro-healing phenotype in human primary monocyte-derived macrophages. This cocktail formulation could be loaded on gelatin/tyraminated films and promoted favorable M2-like macrophage phenotype with low responsiveness to pro-inflammatory stimuli. Such self-standing release systems can be used for prolonged local control of macrophage phenotype upon implantation.

Hyaluronic Acid and Its Derivatives in Coating and Delivery Systems: Applications in Tissue Engineering, Regenerative Medicine and Immunomodulation.

As an Extracellular Matrix (ECM) component, Hyaluronic acid (HA) plays a multi-faceted role in cell migration, proliferation and differentiation at micro level and system level events such as tissue water homeostasis. Among its biological functions, it is known to interact with cytokines and contribute to their retention in ECM microenvironment. In addition to its biological functions, it has advantageous physical properties which result in the industrial endeavors in the synthesis and extraction of HA for variety of applications ranging from medical to cosmetic. Recently, HA and its derivatives have been the focus of active research for applications in biomedical device coatings, drug delivery systems and in the form of scaffolds or cell-laden hydrogels for tissue engineering. A specific reason for the increase in use of HA based structures is their immunomodulatory and regeneration inducing capacities. In this context, this article reviews recent literature on modulation of the implantable biomaterial microenvironment by systems based on HA and its derivatives, particularly hydrogels and microscale coatings that are able to deliver cytokines in order to reduce the adverse immune reactions and promote tissue healing.