Final Report Summary - IMMODGEL (Local Immunomodulation around implants by innovative auxiliary hydrogel-based systems encapsulating autologous and phenotype controlled macrophages)
Executive Summary:
IMMODGEL aimed to identify adverse immune reactions to implantable biomaterials with a particular focus on titanium and silicone. By developing innovative immunomodulatory systems as novel therapeutic strategies, the project outcomes will significantly decrease the implant and medical device failure caused by such reactions.
Immune reactions to implants, biomedical devices, engineered tissues and transplants are a big obstacle in the biomedical field. For instance, electrodes can lose their functionality due to host immune responses, while the need to evade rejection of transplants narrows down the sources for transplantable tissues and contributes to the persistent donor tissue shortage.
The IMMODGEL project aimed at exploring the nature and the underlying mechanisms of adverse reactions with systems biology approaches. Such an understanding enabled the development of immunomodulatory therapeutic systems with biomaterials using tissue engineering and regenerative medicine methodologies. Our aim was to design immunomodulatory systems applicable to different situations (transplants, implants, biomedical devices, etc.). The design consists of an immunomodulatory hydrogel which contains encapsulated macrophages and is anchored to the implant surfaces and/or cytokine cocktail releasing adhesives/coatings. The system additionally presents antimicrobial properties via release of antimicrobial agents from the adhesive component.
The third axis of research in IMMODGEL (aside from systems biology level understanding of immune responses to titanium and therapeutic immunomodulation) consisted in the development of enabling technologies that can provide the tools for future research on immunomodulation, diagnostics related to immune reactions to implants and overall immunology research. For this end, founded on the data generated, IMMODGEL developed a patient-specific immunoprofiler in the form of an on-chip system that is currently at the stage of validation based on multiplex cytokine quantification and Mimotope Variance Analysis (MVA). Additionally, a "Foreign Body Response on-a-chip" has been designed and tested to predict patients´ specific responses to implant materials (tested with miniaturized titanium samples). The project also developed immunocompetent engineered tissues (respiratory epithelium and 3D hydrogel based model systems containing resident macrophages) to have in vitro models of immune reactions to biomaterials.
The key innovation was the development of IMMODGEL as an auxiliary system to improve the outcomes of implantation that has been validated in vitro and in vivo within the timeframe of the project. This innovation will help reducing the cost of implant complication and related medical costs in Europe. We have achieved to provide easily transferable research tools with value for diagnostic as well as therapeutic purposes together with a significant improvement in the understanding of the underlying causes of adverse reactions to biomaterials with methods to control them. The activities resulted in 41 scientific publications, 4 patent applications and more than 100 dissemination activities.
Project Context and Objectives:
Organ/tissue damage and loss are important clinical problems, where the current gold standard is transplantation. One of the main problems with transplants is the use immunosuppressants (IS) for preventing rejection. This results in several side effects such as increased tumor formation risk, higher susceptibility to infections and IS-related toxicity. According to Health Resources and Services Administration (HRSA) reports, depending on the target organ, the success rate of transplants can change between 50 to 90%, also many patients suffer while waiting for a suitable donor due to immunological concerns. Moreover, even though the survival rates are high, the deterioration of the transplant is not completely evitable. Therefore, there is an unmet need for locally applicable systems that can attenuate immune response towards transplants without having systemic effects. Beyond transplantation, implants, transplants and implantable biomedical devices have become mainstream solutions for a wide variety of health problems, and their use in medical practices either for therapeutic applications, prevention or diagnosis is increasing constantly. However, often adverse immune reactions against these foreign materials are observed in the host body. These can lead to dramatic immediate outcomes like immense pain, excessive inflammation or even rejection of the implanted material/tissue.
Overall these adverse immune reactions cause the following symptoms: i) delayed recovery, ii) deterioration of patients’ life quality following implantation iii) unresolved persistent chronic health problems, and iv) additional health problems due to side effects of rejection and/or inflammation. Beyond the obvious problems posed on the patient’s well-being, problems related to adverse immune reactions represent a massive burden on the healthcare system as they usually require additional surgeries, subsequent treatments and in turn long hospitalisation of the patients.
A series of cellular and molecular events following biomaterial implantation poses an important bottleneck for developing breakthrough implantable solutions. Moreover, chronic inflammation and also the local microenvironment created by it can be detrimental for the long term functionality of the implants, especially complex systems with several parts such as in vivo biosensors and artificial biopancreas systems. With inflammation increasingly recognized as a crucial component influencing regeneration, immunomodulation or immuno-engineering has emerged as a potential solution to overcome this key challenge in biomedical engineering. IMMODGEL proposed to develop a new therapeutic strategy based on the design and development of an auxiliary system which will be able to modulate the immune system response around implanted materials or tissues once attached on them. This immunomodulation will trigger remodelling around them and improve their integration in the host body.
This therapeutic strategy was based on harnessing the component of the immune response, namely inflammation that is an inevitable consequence of implantation/transplantation and is closely linked to the clinical outcomes. Upon implantation, immune cells migrate to the implantation site and initiate a localized inflammatory response. Although inflammation is an indispensable element in tissue regeneration, a chronic inflammatory response will significantly limit natural healing, which is quite common in the case of implants.
Among the variety of immune cells, monocytes and macrophages play a particularly critical role that determines successful tissue–implant integration or implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the balanced induction of different macrophage phenotypes. Classically, activated M1 macrophages are associated with a pro-inflammatory response. By contrast, alternatively activated M2 macrophages are associated with an anti-inflammatory and regenerative response which induces angiogenesis and proliferation. Therefore, harnessing macrophage polarity presents a unique opportunity to control inflammation, prevent rejection, and accelerate integration of biomaterials and medical devices. Different macrophage phenotypes with distinct functional properties have been identified. For instance, M1 macrophages are induced by interferon gamma (IFN-γ) from T helper 1 (TH1) cells, CD8+ cytotoxic T cells (CTLs) or natural killer (NK) cells in the presence of microbial products such as lipopolysaccharide (LPS). M1 macrophages have pro-inflammatory and anti-tumour functions and secrete high levels of pro-inflammatory cytokines such as interleukin 12 (IL-12) and IL-23. On the other hand, M2 macrophages are induced by IL-4 and/or IL-13, which are mainly secreted by TH2 cells or polymorphonuclear cells such as mast cells. M2 macrophages have anti-inflammatory and pro-wound healing activities and secrete large amounts of the anti-inflammatory cytokine IL-10. Currently, macrophage polarization is most commonly controlled via exposure to biochemical factors. Specifically, the M1 macrophage phenotype is typically induced through interferon gamma (IFNγ) or lipopolysaccharide (LPS) stimulation, while the M2 macrophage phenotype is typically induced through interleukin-4 (IL-4) or interleukin-13 (IL-13) stimulation. The M1/M2 polarization of macrophages has been described in a generalized manner and the optimum macrophage differentiation status for remodelling is not known, and thus one of the objectives was the determination of the optimum and a long-lasting phenotype based on the induction by cytokine cocktails.
Beyond cytokine based macrophage control, in IMMODGEL, one of the objectives was to determine whether physical stimuli such as encapsulation or presentation of micropatterns can control macrophage polarization. Recent advances in biomaterials science have identified that a biomaterial's design can be leveraged to instruct the host's immune system. For example, novel biomaterial surfaces, improved immune-instructive biomaterials and incorporating immune modulating cells could influence the wound healing process. In addition, several physical and biochemical factors have recently been reported to regulate macrophage polarization such as pore size, mechanical stimulation and extracellular matrix proteins (ECM) among others. However, the underlying mechanisms of how biomaterials steer macrophage polarity has remained poorly understood. The biomaterials in vivo will be in close contact with macrophages and characteristics such as surface chemistry and topography may have a critical role in initiating pro- or anti-inflammatory immune responses. Controlling biomaterial surface attributes provides a powerful tool for modulating the phenotype and function of immune cells with the aim of reducing detrimental pro-inflammatory responses and promoting beneficial healing responses. For this end, one of the objectives was to identify the specific surface topography features (surface micropatterns) that can induce M2 phenotype in incoming macrophages or induce M1 to M2 conversion.
The concept of IMMODGEL was that it is possible to design and engineer a stand-alone immunomodulatory system. This product can be attached to any implant to achieve local immunomodulation and thus protect the implant, transplant or tissue engineered product from the adverse effects of immune response while preventing systemic effects and subsequent infection risks. This is achieved via in vitro phenotype control of autologous macrophages which can be used to orchestrate the inflammation cascade. More precisely, the design of a cellular microenvironment that induces M2 phenotype was the main goal. Through this, it was possible to convert local reaction from an inflammatory route to a remodelling route. For achieving this, the objective in IMMODGEL was to develop a transferable, 3D hydrogel for macrophage (M2) encapsulation which can fix the encapsulated macrophage phenotype for 3 weeks with more than 90% viability.
One of the most common implant materials is titanium which is used in dentistry, as permanent tooth implants, and in orthopaedics as knee and hip replacement. Despite its biocompatibility, implants based on this metal commonly fail due to bad osseointegration, peri-implantitis, osteolysis etc. Depending on the target area, the rate of failure can be in the range of 2% to 10%. Currently there are only a few generic indicators (such as smoking, diabetes, factors that are known to affect healing overall) that are in use in order to indicate an elevated risk of implant failure. In an ageing society, the effects of implant failure have more reverberating effects as the problems related to implants mean additional health problems for ailing patients. One of the objectives of IMMODGEL was to determine a profile of specific phenotypic markers that describes the macrophage state around titanium implants.
In line with the silver economy initiative of the European Commission, it is important to provide tools to the implant practitioners to inform and guide elderly patients in order to minimize the adverse effect of such implantation procedures which otherwise are improving life quality. In IMMODGEL, another objective was to detect the patients prone to implant failure which affects a significant number of patients via development of personalized immunoprofiling tools such as a novel personalised multiplex diagnostic antibody-based array to identify personalized immune response to titanium implants.
One of the persistent problems around nondegradable metallic and polymeric implants is failure of macrophages to resolve the inflammation and their tendency to stay in a state, named “frustrated phagocytosis”. Implanted materials can induce a mixed pro/anti-inflammatory phenotype, supporting chronic inflammatory reactions accompanied by microbial contamination and resulting in implant failure. Several materials based on natural polymers for improved interaction with host tissue or surfaces that release anti-inflammatory drugs/bioactive agents have been developed for implant coating to reduce implant rejection. However, no definitive, long-term solution to avoid adverse immune responses to the implanted materials is available to date. The prevention of implant-associated infections or chronic inflammation by manipulating the macrophage phenotype is a promising strategy to improve implant acceptance. For this end, in IMMODGEL, the objective was to develop a polyelectrolyte multilayer based attachment system with cytokine release capacity for simultaneous anti-inflammatory and antimicrobial effects.
For the development of biomaterial based solutions, one potential route is the use of natural polymers with known anti-inflammatory functions. Hyaluronic acid (HA) plays a multi-faceted role in cell migration, proliferation and differentiation at micro level and system level events. 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. A specific reason for the increase in use of HA based structures is their immunomodulatory and regeneration inducing capacities. Thus, in IMMODGEL one of the objectives was to harness the advantageous properties of HA in both coating and hyrogel formulation.
The expected added value of IMMODGEL project was: i) Decreased risk of chronic inflammation, ii) Improved remodelling response by shortening of the inflammatory cascade, iii) A temporal control over the remodelling of the implant, iv) Applicability to different structures which can provide a comprehensive solution to immunological responses without the need to change the design of the original implants.
Additional expected outcomes of the project were: i) Elucidation of Macrophage behaviour within 3D hydrogels, ii) Determination of the synergy between physical control and cytokines to obtain long term control over cell phenotype while keeping product cost feasible, iii) Development of complex immuno-competent tissue models to better understand activities of immune cells in artificial tissue settings, iv) Development of benchmarks for trial of immunomodulating implants, v) Utilization of bioprinter technology to augment the capacities of other implants.
Project Results:
Scientific and Technical aspects of IMMODGEL project were implemented in 6 workpackages over the course of 48 months. The results and foreground generated in each workpackage was described below:
WP1: Systems immunology: identification of macrophage-mediated chronic inflammatory reactions to titanium implants (WP Leader: University of Heidelberg)
Parts of artificial larynx implants were put into contact with 1ary human macrophages and also tissue samples around titanium dental implants were collected. We detected specific markers that provide insight to the nature of the reaction to titanium in different patients. The results were used to establish the optimal biomaterial and cytokine cocktail delivery system for the immunomodulatory hydrogel design. The specific aim of the cocktail was to revert inflammatory reactions elicited by M1 macrophages to the anti-inflammatory phenotype (M2). The components of the cytokine cocktail have been determined. Its effect in the presence of biomaterials has been assessed within other WPs. To develop a diagnostic tool that predicts the immunological profile of the patient with respect to cytokine levels, target cytokines were selected. A lab-level prototype of the system was developed and tested for validation.
An additional part of the results of this workpackage form Foreground 1 (Titanum signature), which is planned to be published and is not described here to allow IP protection.
Foreground 2: Development of Cytokine Cocktail (A M2 macrophage phenotype fixing cytokine cocktail (long term stability) that can be used for immunomodulation):
We achieved to formulate 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 12 days. 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 (from Workpackage 3) 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. 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.
Foreground 3: Effect of Oxygen Plasma treatment on macrophage polarisation:
To assess the impact of different surface chemistries on macrophage polarisation, human monocytes were cultured for 6 days on untreated (hydrophobic) and O2 plasma-etched surfaces (hydrophilic). Our data clearly show that monocytes cultured on the O2 plasma-etched surface are polarised towards an M1-like phenotype, as evidenced by significantly higher expression of the pro-inflammatory transcription factors STAT1 and IRF5. By comparison, monocytes cultured on the non-etched surface exhibited an M2-like phenotype with high expression of mannose receptor (MR) and production of the anti-inflammatory cytokines IL-10 and CCL18. While the molecular basis of such different patterns of cell differentiation is yet to be fully elucidated, we hypothesise that it is due to the adsorption of different biomolecules on these surface chemistries. Indeed, our surface characterisation data show quantitative and qualitative differences between the protein layers on the O2 plasma-etched surface compared to non-etched surface which could be responsible for the observed differential macrophage polarisation on each surface. We showed that changes in surface chemistry resulting from O2 introduction by plasma treatment affect protein adsorption on the material. This in turn appears to influence monocyte polarisation towards macrophages with distinct phenotypes. Specifically, non-etched surfaces were shown to suppress expression of M1-associated markers and cytokines while promoting M2-associated markers. On the other hand, O2 plasma-etched surfaces had the opposite effect. Protein overlayer thickness and amino acid profiles were different on the surfaces, suggesting differences in protein adsorption.
WP2: Effect of topography on macrophage phenotype (WP Leader: University of Nottingham)
To determine the effect of surface micropatterns on macrophage phenotype and how this can be used to control it, microwells, micropillars and microgrooves with a wide range of dimensions were produced and screened with model monocytes and 1ary macrophages. 2 optimal types of surface micropatterns were selected. Further work has been undertaken to determine the synergistic effects of cytokine induction and surface micropatterns. The effect of optimized patterns on macrophage polarisation was quantified at gene level. The patterns can be introduced reproducibly to macrophage-laden hydrogels developed in WP3 and used in the final therapeutic system.
Foreground 4: Machine Learning based Image Analysis for simplified detection of macrophage phenotype:
Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time-consuming, require large numbers of cells and are resource intensive. Instead, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus, high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers.
Foreground 5: Effect of co-stimulation of macrophages with micropatterns and cytokines on their polarisation: As the surface microtopography is shown to exert significant effects on cell phenotype, we hypothesized that the presence of micropatterns on implant/medical device surfaces can attenuate the immune response. To this end, enzymatically crosslinked micropatterned gelatin films of varying groove widths (2, 5, 10, 20, and 40 µm) were tested for their effect on incoming monocyte behavior. In order to distinguish the effect of cytokine microenvironment on pattern presence, monocytes were seeded on micropatterned films in normal culture medium or M1/M2 inducing media and their morphology and cytokine secretions were observed for 6 d. The presence of the patterns induces microenvironment-specific changes on the secretions of the attached cells and also on their size. IL-1ß, IL-4, IL-12, TNF-α, and CCL-18 secretions were significantly affected particularly in M1 induction media by pattern presence. It was demonstrated for the first time that micropatterned surfaces can be used to control the initial attachment and cytokine secretion of incoming macrophages if they were linked with a polarization inducing cytokine microenvironment.
Foreground 6: Unbiased transcriptomics analysis of the effect of micropatterns on macrophage phenotype:
The activation state adopted by macrophages in response to biomaterials determines their own phenotype and function as well as those of other resident and infiltrating immune and nonimmune cells in the area. In biomaterials research, cell-instructive surfaces that favor or induce M2 macrophages have been considered as beneficial due to the anti-inflammatory and pro-regenerative properties of these cells. We used a gelatin methacryloyl (GelMA) hydrogel platform to determine whether micropatterned surfaces can modulate the phenotype and function of human macrophages. The effect of microgrooves/ridges and micropillars on macrophage phenotype, function, and gene expression profile were assessed using conventional methods (morphology, cytokine profile, surface marker expression, phagocytosis) and gene microarrays. Our results demonstrated that micropatterns did induce distinct gene expression profiles in human macrophages cultured on microgrooves/ridges and micropillars. Significant changes were observed in genes related to primary metabolic processes such as transcription, translation, protein trafficking, DNA repair, and cell survival. However, interestingly conventional phenotyping methods, relying on surface marker expression and cytokine profile, were not able to distinguish between the different conditions, and indicated no clear shift in cell activation towards M1 or M2 phenotypes. This highlighted the limitations of studying the effect of different physicochemical conditions on macrophages by solely relying on conventional markers that are primarily developed to differentiate between cytokine polarized M1 and M2 macrophages. We therefore propose the adoption of unbiased screening methods in determining macrophage responses to biomaterials. We showed that the exclusive use of conventional markers and methods for determining macrophage activation status could lead to missed opportunities for understanding and exploiting macrophage responses to biomaterials.
Foreground 7: Use of Electrohydrodynamic printing for implant surface micropatterning:
We developed a direct write printing method to modify metallic implant surfaces with biocompatible polymers with microscale precision particularly for controlling macrophage phenotype. Application of polymeric micropatterns on metallic implant surfaces can (i) improve their interaction with the host tissue, (ii) enable the delivery of growth factors, antibiotics, anti-inflammatory cytokines etc from the implant surface and (iii) can control the immune responses to the implant via controlling the attachment of immune cells, such as macrophages. Surface patterns with a resolution of less than 50 μm can be created using an electro hydrodynamic (EHD) printing, a template-free and single-step process. We devised a revised EHD printing method for the deposition of parallel strips of photocrosslinkable, cell adhesive polymeric composites with spacing of around 20 μm onto medical grade titanium substrates. Optimization of voltage, feeding rate and collection speed resulted in regular structures via very rapid movement of the grounded rotating collector driven to equivalent of the linear surface speed of above 100 cm s−1. A mixture of chemically modified PEG /gelatin was deposited onto a conductive titanium substrate with different surface pretreatments with an area of 400 mm2. Acid etched or UV treated titanium surfaces improved the stability of the printed structures. Polymeric lines induced temporary orientation of human monocytes and induced a thicker cell multilayer formation by fibroblasts. Staining of the monocytes for M1(CD80) and M2 (CD206) macrophage markers on the patterned surface showed mixed populations with higher anti-inflammatory cytokine secretion compared to tissue culture plastic control. The results demonstrated the suitability of this method for the preparation of biomaterials with structured surfaces on large areas and with desired chemical composition.
WP3: Development of physical constituents of IMMODGEL (Adhesive polyelectrolyte, cell encapsulating hydrogel) (WP Leader: Protip Medical)
The overall aim was to develop a gel formulation for macrophage encapsulation and also develop an adhesive (coating) to attach the macrophage encapsulated hydrogels to implants. For hydrogels, the goal was to develop a wide array of implantable, encapsulation systems for autologous macrophages which would enable their transfer and facilitate their phenotype control. For this end, biodegradable base materials such as gelatin, non-degradable polymers such as PEG and immunomodulatory molecules such as hyaluronic acid and polyarginine were utilized to achieve maximum viability and the highest M2 response. During optimization of encapsulation, an adhesive coating system based on gelatin/hyaluronic acid or polyarginine/hyaluronic acid was simultaneously developed to obtain a versatile adhesive structure. Four separate formulations have been successfully used for encapsulation of THP-1 cells and two formulations were also tested with 1ary macrophages. The physical and mechanical characterization of the hydrogels has been done and 4 formulations have been selected for further use in WP 4. Four working adhesive systems have been developed and tested with 1ary macrophages for their ability to release cytokines. Finally, the assembly of the optimal gel formulations together with the optimal adhesive has been established and as tested in contact with different biomaterials. An additional wet adhesive has been developed to ensure strong adhesion to the surfaces.
Foreground 8: Simultaneously anti-inflammatory/antimicrobial coatings:
Major problems with biomedical devices in particular implants located in nonsterile environments concern: (i) excessive immune response to the implant, (ii) development of bacterial biofilms, and (iii) yeast and fungi infections. We developed a multifunctional coating that addresses all these issues concomitantly. A new exponentially growing polyelectrolyte multilayer film based on polyarginine (PAR) and hyaluronic acid (HA) was first designed. The films had a strong inhibitory effect on the production of inflammatory cytokines released by human primary macrophage subpopulations. This could reduce potential chronic inflammatory reaction following implantation. Next, it was shown that PAR, due to its positive charges, has an antimicrobial activity in film format against Staphylococcus aureus for 24 h. In order to have a long-term antimicrobial activity, a precursor nanoscale silver coating was deposited on the surface before adding the PAR/HA films. Moreover, the PAR/HA films can be easily further functionalized by embedding antimicrobial peptides, like catestatin (CAT), a natural host defense peptide. The PAR/HA+CAT coatings were effective as an antimicrobial coating against yeast and fungi and cytocompatible. This all-in-one system constituted a new strategy to limit inflammation and prevents bacteria, yeast, and fungi infections.
Foreground 9: Development of novel release systems for delivery of anti-inflammatory cytokines:
We designed 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 adverse immune reactions. The chemical modification of the HA with aldehyde moieties allowed 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) stimulated the differentiation of primary human monocytes seeded on the films into pro-healing macrophage phenotype (M2). This induced the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrated that cross-linking PLL/HA film using HA-aldehyde was already effective by itself to limit inflammatory processes. This functionalized self-cross-linked PLL/HA-aldehyde films are an efficient candidate for immunomodulation of different kinds of implants of various architecture and properties.
Foreground 10: Discovery of the Chain-size dependent Antimicrobial activity of Polyarginine/Hyaluronic acid coatings (Part 1):
The number of nosocomial infections related to implants and medical devices increase alarmingly worldwide. As, we have observed in one of our anti-inflammatory coating formulations strong antimicrobial capacities, we stidied the underlying mechanism for such action. By investigating films built up with poly(arginine) (PAR) of various chain lengths (10, 30, 100, and 200 residues) and hyaluronic acid (HA), we demonstrated that exclusively films constructed with poly(arginine) composed of 30 residues (PAR30) acquired a strong antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria associated with infections of medical devices. This chain-size effect was extremely striking and was the first example reported where the length of the polyelectrolytes played a key-role in the functionality of the films. Moreover, this unexpected functionality of nanolayered polypeptide/polysaccharide PAR30/HA films occured without adding any specific antimicrobial agent, such as antibiotics or antimicrobial peptides. PAR30/HA film inhibited bacteria through a contact-killing mechanism due to the presence of mobile PAR30 chains. These chains were assumed to diffuse toward the interface, where they interacted with the bacteria with the consequence of killing them. This new coating with unique properties based on the association of a homopolypeptide of 30 residues with a polysaccharide is simple system to prevent implant-related infections with a reasonable production cost. (Patent application submitted)
Foreground 11: Discovery of the Hyaluronic acid dependent Antimicrobial activity of Polyarginine/Hyaluronic acid coatings (Part II):
As described above, first we showed that poly(arginine)/hyaluronic acid (PAR/HA) multilayers built with PAR chains constituted from 30 arginine residues (PAR30) have strong antimicrobial properties through a contact-killing mechanism. This property wass due to the ability of PAR30 chains, when associated with HA, to diffuse in the multilayer. Then, we investigated the effect of the nature of the polyanion on the antimicrobial activity of (PAR30/polyanion) multilayers. Four polysaccharides, one polypeptide, and one synthetic polyelectrolyte were investigated. Surprisingly, only HA leads to films with antimicrobial character. We related this property to the strong diffusion capacity of PAR30 chains in (PAR30/HA) multilayers compared to their diffusion ability in the other (PAR30/polyanion) films. Through isothermal microcalorimetry experiments, we also demonstrated that interactions in solutions of PAR30 chains with the different polyanions were characterized by a negative reaction enthalpy for all of the investigated polyanions except for HA, where the enthalpy of reaction was positive. Moreover, the molecular weight of HA was not a key parameter for the diffusion ability of PAR chains or for the bioactivity of the film. These results constituted an important step toward the establishment of rules to design contact-killing antimicrobial polyelectrolyte multilayers.
Foreground 12: Biomcompatible Adhesive development for establishing stable contact with implants and IMMODGEL system:
Modification of implant surfaces with an adhesive represents a promising strategy to promote the adhesion of cell-laden hydrogels on the implant. For this end, we developed a peptidic adhesive based on mussel foot protein (L-DOPA-L-Lysine)2-L-DOPA that can be applied directly on the surface of an implant. At physiological pH, unoxidized (L-DOPA-L-Lysine)2-L-DOPA expected to strongly adhere on metal/metal oxide surface formed only very thin coatings. Once oxidized at physiological pH, (L-DOPA-L-Lysine)2-L-DOPA formed an adhesive coating about 20 nanometers thick. In oxidized conditions, L-Lysine can adhere to metallic substrate via electrostatic interaction. Oxidized L-DOPA allows to form a coating through self-polymerization and can react with amines so this adhesive can be used to fix ECM based materials on implant surfaces through the reaction of quinones with amino groups. Hence, a stable interface between a soft gelatin hydrogel and metallic surfaces was achieved and the strength of adhesion was investigated. We have shown that the adhesive is non-cytotoxic to encapsulated cells and enabled the adhesion of gelatin soft hydrogels for 21 days on metallic substrates in liquid conditions. The adhesion properties of this anchoring peptide were quantified by a 180° peeling test.
WP4: Control of macrophage phenotype subsets in 3D hydrogel systems (WP Leader: Protobios)
In WP4, the aim was to quantify the effect of encapsulation and the physical properties of the hydrogel on the phenotype of the encapsulated macrophages. Several parameters such as stiffness, degradability, presence of cell adhesion sequences, hydrogel composition and crosslinking parameters have been varied and their effect on macrophage phenotype was tested in the presence and the absence of cytokine induction. This process fed into the selection process in WP3. Finally, the effect of adhesive and hydrogel composite on encapsulated macrophages in the presence of cytokine release was quantified. Macrophages can modulate tissue vascularisation and remodelling depending on their phenotype, this can be controlled using physical and chemical cues. Using cell-laden 3D structures the effect of phenotype controlled macrophages on primary fibroblasts and vascular endothelial cells was quantified.
Foreground 12: The Effect of Degradability of the Encapsulation Microenvironment on Macrophage Phenotype:
In order to see whether the nature of hydrogel had an effect on encapsulated macrophage phenotype, a biodegrable, natural polymer based hydrogel system (Methacrylated gelatin (GelMA)) and a synthetic, non-degradable hydrogel system (Poly(ethylene glycol) diacrylate (PEGDA)) were sued to encapsulate macrophages. We demonstrated that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and a collagen-derived matrix (gelatin). Surface marker, gene expression, biochemical, and cytokine profiling consistently indicated that THP-1 cells within a biomaterial lacking cell attachment motifs yielded pro-inflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induced an anti-inflammatory M2-like phenotype and propagated the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2β1 played a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity was further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively.
Foreground 13: Adhesive Film/Hydrogel composites with controlled cytokine release: Delivery of growth factors is an indispensable part of tissue engineering and immunomodulation. We used a detachable membrane-based release system (from WP3) 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 developed 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 resulted in extensive sprouting by encapsulated vascular endothelial cells. The presence of the release system with interleukin-4 resulted in clustering of encapsulated macrophages with a significant decrease in M1 macrophages (pro-inflammatory). This system was used in conjunction with three-dimensional structures as an auxiliary system to control artificial tissue maturation, growth and harnessing of immune response. (Patent in preparation for parts beyond the disclosed part)
WP5: Enabling Technologies: Foreign Body Response on-a-chip, immunocompetent artificial tissues, bioprinted immunomodulatory hydrogels
In WP5, we designed a prototype for Foreign Body Response on-a-chip system which was first used to observe the macrophage reaction to titanium microbeads. For the immunocompetent epithelial tissue development, we have developed artificial basement membrane mimics and tested them with respiratory epithelial cells. We also developed 3D sandwich-like structures for the connective part of the epithelial tissue and the methods of incorporating macrophages and macrophage-laden hydrogels in the model tissues. The application of bioprinting methodologies for printing of immunomodulatory hydrogels was also carried out.
Foreground 14: Resident Macrophage Model Development; Encapsulation of phenotype controlled macrophages and quantifying their interactions with incoming cells:
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.
Foreground 15: Sandwich-like multicellular organ models for respiratory epithelium development:
Many organs are multicellular and each cell type requires a different microenvironment. Thus, there is a need for modular structures where the microenvironment of each cell type can be tuned separately. We developed enzymatically crosslinked gelatin based double layered film structures where each layer can be loaded with growth factors separately. As a model, we have developed a bi-layer system to produce a respiratory epithelium. This system constitutes an in vitro “epithelial patch”. Crosslinking of the patches with transglutaminase resulted in 7 days of stability at 37 °C. The film layer was first used to release growth factors and it was shown that the release significantly improved the proliferation over 5 days. Human lung epithelial cells were used and under the release of an epithelial growth supplement mix, there was a significant improvement on the epithelial proliferation. The stability of the epithelial patch under in vitro conditions for 7 days without deterioration. Under co-culture conditions for three days both cell types were alive.
WP6: In vivo tests of IMMODGEL of the immunomodulatory system with titanium implants (WP Leader: University of Strasbourg/INSERM)
The implantation protocol for testing of immunomodulatory hydrogels was developed and implemented in a mouse model for the subcutaneous immunomodulation around implants with developed hydrogel and adhesive systems. The second part of the WP focused on immunomodulation in orthogonal positions particularly partial tracheal replacement with 3D printed structures in the presence or absence of immunomodulation where we have shown significant positive effects of immunomodulation at local and systemic levels.
Potential Impact:
Implant technology has become widespread and several types of implants, such as hip implants, knee implants are being implanted in more than a million cases worldwide. The decreasing cost of dental implants has increased their demand in Europe, and an additional several billions of Euros growth in this field is expected in the next 5 years. In a field (dental) which is valued at more than 2 billion Euros and where over 40% of global demand is in Europe, improvement of the current protocols via immune response control as developed in IMMODGEL will have great positive economic consequences. The removal of adverse immunological reactions, a very common problem in the field of biomedical engineering, can also augment the direct investment from public and private sector in areas where they would be normally considered too risky.
Prognostic or Susceptibility/Risk, Monitoring Biomarkers: The occurrence of mild and severe inflammatory processes of implants are on the rise. There are several contributing factors to this trend such as the overall ageing society, the availability of the implants to a wider range of demographics and a longer expected implant life time. The exact reasons in detail are still unknown, however, it is clear that the lack of diagnostic methods to actually determine high-risk patients and treat them accordingly is playing a big role in this problem. IMMODGEL aimed to fill this gap by studying in which form the generated scientific knowledge of the personalized set of candidate genes will be of the best use.
The immunomodulatory system targeted in this project consisted of 1) autologous macrophages with a controlled phenotype 2) a composite hydrogel for macrophage and cytokine encapsulation and 3) a polyelectrolyte coating which will allow adhesion of the encapsulated macrophages to any implant.
We have optimized several encapsulation systems and polyelectrolyte coatings that fit the requirements of the final therapeutic system. We have also developed the protocols for theassembly of the structures and their in vivo testing. Encapsulation of macrophages has not been undertaken before the IMMODGEL project in the literature; thus IMMODGEL has provided the initial conditions for future research in this area. Cell adhesion plays an integral role in enabling communication between cells and their microenvironment. However, their role in monocyte to macrophage differentiation and particularly macrophage polarization are yet to be fully understood. Here, we hypothesized that integrin-mediated cell–biomaterial interactions could play a key role in macrophage polarization. Given that in vivo these events take place in the context of ECM and in 3D, obtaining a clear understanding of the role of integrins in macrophage polarization in a 3D microenvironment will be more physiologically relevant than in a 2D environment. We have used two distinct hydrogel systems to probe the effect of cell–biomaterial interactions on macrophage polarization in a 3D environment. We investigated whether macrophage polarity can be controlled through integrin-mediated biomaterial-based programming. Our findings will pave the way for future endeavors in elucidating immune system/biomaterial interactions in 3D configurations.
We have devised specific cytokine cocktails for precise control of macrophage phenotype that have been tested in 3D conditions. Artificial tissue model development for the testing of immunomodulation has been done. We have provided the biomaterial community with new tools such as incoming macrophage models and a foreign body response on a chip system for incorporating the immunology aspect in their activities.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. We demonstrated that self-standing, transferable 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 cocktails induces long-term anti-inflammatory and pro-healing phenotype in human primary monocyte-derived macrophages. This cocktail formulation could be loaded on 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.
Via systems immunology, we were able to determine specific markers that can define the tissue response to medical grade titanium implants. This can be of significant prognostic value of the fate of such implants and will facilitate the clinicians and immunologists to holistically understand the adverse immune responses to implants, how these are controlled by macrophages and how to make judicious choices regarding host-implant compatibility. We also developed immunoprofiling methods based on cytokine measurements and epitope characterisation which can also be used for pre-implantation diagnostics applications.
A foreign body response (FBR) on-a-chip system has been developed that will significantly reduce the research cost and the need for animal tests by replacing the compulsory biocompatibility tests with a sophisticated in vitro test. The initial design of the FBR on-a-chip system is ready and the future iterations are currently underway within the framework of future projects.
Personalized Implants: Macrophages are master cells of the innate immune system that protect all tissues of our body from exogenous danger of different origins by induction of inflammation and elimination of foreign materials and organisms. Moreover, there are other cases of macrophage and metal based complications such as myofascitis due to the Aluminium content in vaccine adjuvants. As the immune system of each individual differs, it is important to have ways to evaluate the possible responses of the individual patients. The discoveries in IMMODGEL will allow the stratification of the population with regards to the response to Ti allowing to protect the patients from complications. It is also possible to provide suggestions for possible modifications of commercially available implants to make them more amenable for a given patient thus paving the way of personalized implants. Finally, it was our aim to utilize its predictions for the development of therapeutic solutions for implant related problems which can then be further extended to transplants and biomedical devices, particularly those of titanium and other metals/alloys, polymers, ceramics and eventually to engineered tissues. It can also open the door for looking beyond the innate immunity and into adaptive immunity via effects of lymphocytes.
The results obtained on model implants can provide a necessary framework for a wide range of biomedical materials. By developing a system that can be applied to any kind of implant, transplant or biomedical device, the project will expand the capabilities of immunomodulation. In this sense, IMMODGEL will have a widespread impact and can improve the chances and success of many other European healthcare products. Our results up to now are in line with this claim and we hope to deliver the proposed impact following the animal tests.
IMMODGEL will yield high socio-economic impacts by significantly improving the health and quality of life of patients in EU and world-wide, and by decreasing therapy costs due to adverse immunological reactions. This research will not only improve the outcomes of implants and biomedical devices, but will also provide meaningful improvement of non-communicable diseases (NCDs). In an ageing society, by improving the lifetime of implants and biomedical devices, immunomodulatory systems can provide a meaningful saving on healthcare costs. The project will also help the development of personalized medicine or stratified medicine approaches to implantology and development of necessary tools to achieve the personalization.
The proposed system can be used in conjunction with all types of implants, particularly with the implants such as dental, orthopaedic, spine or larynx implants. Applicability to different structures can provide a comprehensive solution to adverse immunological responses without the need to change the design of the original implants. This will also allow reducing the need of animal experimentation due to the elimination of immune system related failures which will not only have positive economic impacts but also ethical ones. Also, the development of a diagnostic tool to quantify immune response to implants will decrease health costs by predicting and preventing possible excessive immune reactions. Creation of new employment opportunities followed by the newly developed technologies and the know-how will also offer a positive economic impact on the society.
The development of this unique immunomodulatory system and relevant know-how is expected to provide significant opportunity for the industrial partners to both maintain and build further on their existing market positions. The overall implant industry is projected to increase between 5 and 10% per year (a growth of 20% is expected only in dental implant industry for the next 5 years), therefore a product that enhances the success of these surgeries and reduces several other associated health risks will fetch benefit of lower health sector costs and improved health conditions. For commercial exploitation and benefit to the industrial partners; Protip Medical has developed IP and new competencies thanks to the project. One of the technologies developed in IMMODGEL (patented) is under pre-clinical testing at the moment for eventual up-scaling and potential commercialisation. Protobios has entered into a potential new market (biomaterial diagnostics) and Contipro has found a new area for utilisation of their Hyaluronic acid based products as base material
System Immunology and Bioengineering have a continuously increasing importance due to the issues related to immune-competence, associated reliability and success issues, lack of broader industrial familiarity with wide-scale solutions and embedded commercial interests. The research undertaken in IMMODGEL project has generated academic and industrial interest. The development and application of an immunomodulatory system to control the adverse immune reactions in the host body will demonstrate their effectiveness for different types of surgical applications. The ability of implants to alleviate chronic disease related problems is significantly reduced by adverse immunological reactions to them, which makes immunomodulation a highly impactful area of technological development. This is expected to encourage further academic research and additional industry funding into academia to further lead in this technology.
Benefit to other academic research fields – Diagnostic and Immunological research is of great interest to researchers in many differing fields. Assessment of undiscovered fields of adverse immune reactions and host: implant interactions against specific cases will open up research options to academics which may not be immediately evident. For example, we have demonstrated a new means of classifying macrophage populations using image based machine learning. Given the heterogeneity of macrophage phenotype and current limitations of the machine learning approach it may be too early to suggest use of image analysis as an alternative to conventional cell phenotyping. However, our data provide strong evidence for the ability of high content and automated image analysis approaches for accurate, less resource intensive and fast phenotyping of functional diverse cell populations. Moreover, the data organized and information collected during the project will support the developement of other systemic researches.
Biomaterial-mediated immunomodulation by programming macrophage polarity is a promising tool in tissue engineering, regenerative medicine and implantology to decrease adverse immune reactions, accelerate implant integration, facilitate tissue regeneration and increase implant lifetime. Overall, biomaterial-based control over macrophages represents a novel technique to obtain a fundamental understanding of macrophage behavior and is a strong therapeutic tool for immunomodulation for implants, drug, and cell delivery systems.
Engineering a tissue requires the design of the optimal conditions for the cells in the target tissue involved. Additionally, it is also crucial to develop methods to recruit and direct the host cells that will take an active role in the integration of the implanted tissue. A modular ECM-based system such as IMMODGEL can be used as a component of engineered tissues for directional release of cytokines and growth factors to attain precise microenvironment control. Development of a system composed of two modular components decouples the release and cell loading, which allows a higher degree of control over the encapsulated cell behavior.
Silver Economy, welfare: An aging population brings new challenges to health professionals in general and dentists in particular. It is well known that dental implants are likely to become used within a larger population pool and that a given individual may have several implants in the mouth. IMMODGEL will make it possible to access to safer titanium dental implant procedures thus allowing the access to implants to a wider population range and improving its welfare.
Controlling Healthcare Cost: Inflammation around the implants can cause severe peri-implantitis. A study in 2015, states that the treatment of peri-implantitis can cost several thousand Euros depending on the treatment method. A better understanding of the patient’s innate response profile can result in a decrease of implant related complications. Moreover, this “complications” problem is not limited to dental implants as they are also known to exist in further implants and in various target organs. For example, implant related problems have been on the rise during the last few years (~5 000 complications out of 200 000 implantations for cardiovascular devices) which caused an increase in long term mortality (about 2%). In average, such complications cost about 15.000 dollars each. Similarly, in total hip replacement, a postsurgical treatment has a cost of 50.000 dollars (on average) with a rate of incidence of 2% (in the extreme cases the replacement of the implant or amputation of the limb could be necessary). By providing an innovative way to predict adverse immune reactions (reactions that in most cases are indirectly responsible for the infections as the implant is not integrated well), IMMODGEL will open the door to such a practice in other fields of implantology (hip, knee, cardiac, etc.) therefore contributing to a larger use of personalized medicine and a better control of healthcare costs.
HA is an important tool in biomaterial research as evidenced by its widespread use in surgical applications as a filler, as hydrogel material, cell carrier and drug delivery system. The ease of isolation/production of HA and its extensive biological activities makes it a very attractive target for therapeutic biomaterial based system development. The presence of several sites amenable to chemical modification on HA chains further improves its practical use as a wide array of derivatives can be developed. The current state of research has put emphasis on 3 directions in the use of HA in biomedical applications: i) injectable formulations that will improve the delivery and defect conformation for both drug and cell delivery applications ii) development of new derivatives of HA that enables further control over physicochemical and biological properties of the final structures iii) Harnessing the biological activities of HA for more controlled biological effects such as controlled retention of multiple growth factors or specific, directed differentiation of cells to desired phenotypes. Thus, in the upcoming years, it is reasonable to expect the development of more derivatives of HA for applications in regenerative medicine, particularly in immunomodulation, angiogenesis, nerve regeneration and hybrid materials containing HA. As a HA provider, IMMODGEL partner Contipro will benefit from the use of HA in immunomodulatory formulations.
As an outlook, the next in line in the sophistication of engineered tissues can be the inclusion of immune system components. Nearly all tissues have resident macrophage populations which has been shown to be an important factor in tissue homeostasis and healing upon injury. Recently, there has been a growing focus on the control over innate immune response in the microenvironment of implanted materials particularly through well-established macrophage polarization pathways that have been shown to have a crucial role in vascularization of implanted scaffolds. Immunoassisted tissue engineering approaches can harness the ability of innate immune cells to resolve inflammation and promote regeneration and healing. This can be achieved by exploiting the phenotypic plasticity of immune cells either via controlled delivery of specific phenotype inducing cytokines or direct co-delivery of phenotype controlled immune cells together with the cells relevant to the target organ function. A new focus on establishing a cross-talk with the host immune system, rather than trying to evade it, could pave the way for more functional and fast-integrating artificial tissues. Concomitant use of new developments in temporal control of multiple growth factor/cytokine delivery; advanced bottom-up assembly methods of engineered tissues such as robotic assembly; use of bioactive miRNAs within scaffolds; and micro/nanoscale topographical and chemical control of scaffold features for inducing anti- or proinflammatory immune cell phenotypes would provide the tools for engineering multicellular organs and establishing in vitro organoids that faithfully model physiological conditions with immune system components. These efforts would bring forth the aspects of ‘regenerative immunology’ in regenerative medicine.
IMMODGEL project provided a better understanding of the significant role of innate immune cells in tissue remodeling and regeneration for regenerative medicine and implants. A wide variety of strategies are available to orchestrate the initial immune response to implanted structures as detailed above. However, because the immune response is tightly regulated both spatially and temporally, more elaborate techniques will be necessary to attain optimal functional integration. Apart from control of the innate immune responses, the next line of control can be achieved at the level of adaptive immunity via B cell and T cell responses. Some of the most commonly used biomaterials, such as synthetic polymers and ceramics, can be spared of adaptive immunological responses owing to the lack of potential immunogenic components. However, new generations of biomaterials, where hybrids of organic and inorganic components, synthetic peptide structures, and cell-responsive polymeric components are increasingly used, necessitate the consideration of the adaptive immune responses to biomaterial structures. For a designed self-assembling polypeptide chain, for example, it is important to know whether the sequence and structure of the design resembles an antigen and can therefore be a functional epitope that triggers adaptive immunity. To optimize the adaptive immune response to biomaterials, a better understanding of the mechanism of events related to adaptive immunity, such as leukocyte attachment, is needed which can be outlook studies after IMMODGEL.
It is becoming increasingly important to ask fundamental immunological questions in the context of biomaterial development. Topographical and/or chemical design of biomaterial surfaces with respect to the APC responses can pave the way for a new generation of ‘cell instructive’ materials with immunomodulatory properties with a wide range of clinical applications. In future, high-throughput systems which will be key to better mimic the FBR in vitro will allow the elucidation of biomaterial-specific responses in real-time and will therefore substantially improve our ability to identify, predict, and control the immune response to implanted biomaterials. We believe, as the concepts surrounding the biocompatibility of biomaterials evolve, that the focus will shift from an evasion of the host immune system to an orchestrated interaction with it. This will also enable the discovery of new biomaterials.
List of Websites:
www.immodgel.eu
Scientific coordinator: Dr. Nihal Engin Vrana, e.vrana@protipmedical.com
Administrative coordinator: Dr. Mercedes Dragovits, dragovits@steinbeis-europa.de
IMMODGEL aimed to identify adverse immune reactions to implantable biomaterials with a particular focus on titanium and silicone. By developing innovative immunomodulatory systems as novel therapeutic strategies, the project outcomes will significantly decrease the implant and medical device failure caused by such reactions.
Immune reactions to implants, biomedical devices, engineered tissues and transplants are a big obstacle in the biomedical field. For instance, electrodes can lose their functionality due to host immune responses, while the need to evade rejection of transplants narrows down the sources for transplantable tissues and contributes to the persistent donor tissue shortage.
The IMMODGEL project aimed at exploring the nature and the underlying mechanisms of adverse reactions with systems biology approaches. Such an understanding enabled the development of immunomodulatory therapeutic systems with biomaterials using tissue engineering and regenerative medicine methodologies. Our aim was to design immunomodulatory systems applicable to different situations (transplants, implants, biomedical devices, etc.). The design consists of an immunomodulatory hydrogel which contains encapsulated macrophages and is anchored to the implant surfaces and/or cytokine cocktail releasing adhesives/coatings. The system additionally presents antimicrobial properties via release of antimicrobial agents from the adhesive component.
The third axis of research in IMMODGEL (aside from systems biology level understanding of immune responses to titanium and therapeutic immunomodulation) consisted in the development of enabling technologies that can provide the tools for future research on immunomodulation, diagnostics related to immune reactions to implants and overall immunology research. For this end, founded on the data generated, IMMODGEL developed a patient-specific immunoprofiler in the form of an on-chip system that is currently at the stage of validation based on multiplex cytokine quantification and Mimotope Variance Analysis (MVA). Additionally, a "Foreign Body Response on-a-chip" has been designed and tested to predict patients´ specific responses to implant materials (tested with miniaturized titanium samples). The project also developed immunocompetent engineered tissues (respiratory epithelium and 3D hydrogel based model systems containing resident macrophages) to have in vitro models of immune reactions to biomaterials.
The key innovation was the development of IMMODGEL as an auxiliary system to improve the outcomes of implantation that has been validated in vitro and in vivo within the timeframe of the project. This innovation will help reducing the cost of implant complication and related medical costs in Europe. We have achieved to provide easily transferable research tools with value for diagnostic as well as therapeutic purposes together with a significant improvement in the understanding of the underlying causes of adverse reactions to biomaterials with methods to control them. The activities resulted in 41 scientific publications, 4 patent applications and more than 100 dissemination activities.
Project Context and Objectives:
Organ/tissue damage and loss are important clinical problems, where the current gold standard is transplantation. One of the main problems with transplants is the use immunosuppressants (IS) for preventing rejection. This results in several side effects such as increased tumor formation risk, higher susceptibility to infections and IS-related toxicity. According to Health Resources and Services Administration (HRSA) reports, depending on the target organ, the success rate of transplants can change between 50 to 90%, also many patients suffer while waiting for a suitable donor due to immunological concerns. Moreover, even though the survival rates are high, the deterioration of the transplant is not completely evitable. Therefore, there is an unmet need for locally applicable systems that can attenuate immune response towards transplants without having systemic effects. Beyond transplantation, implants, transplants and implantable biomedical devices have become mainstream solutions for a wide variety of health problems, and their use in medical practices either for therapeutic applications, prevention or diagnosis is increasing constantly. However, often adverse immune reactions against these foreign materials are observed in the host body. These can lead to dramatic immediate outcomes like immense pain, excessive inflammation or even rejection of the implanted material/tissue.
Overall these adverse immune reactions cause the following symptoms: i) delayed recovery, ii) deterioration of patients’ life quality following implantation iii) unresolved persistent chronic health problems, and iv) additional health problems due to side effects of rejection and/or inflammation. Beyond the obvious problems posed on the patient’s well-being, problems related to adverse immune reactions represent a massive burden on the healthcare system as they usually require additional surgeries, subsequent treatments and in turn long hospitalisation of the patients.
A series of cellular and molecular events following biomaterial implantation poses an important bottleneck for developing breakthrough implantable solutions. Moreover, chronic inflammation and also the local microenvironment created by it can be detrimental for the long term functionality of the implants, especially complex systems with several parts such as in vivo biosensors and artificial biopancreas systems. With inflammation increasingly recognized as a crucial component influencing regeneration, immunomodulation or immuno-engineering has emerged as a potential solution to overcome this key challenge in biomedical engineering. IMMODGEL proposed to develop a new therapeutic strategy based on the design and development of an auxiliary system which will be able to modulate the immune system response around implanted materials or tissues once attached on them. This immunomodulation will trigger remodelling around them and improve their integration in the host body.
This therapeutic strategy was based on harnessing the component of the immune response, namely inflammation that is an inevitable consequence of implantation/transplantation and is closely linked to the clinical outcomes. Upon implantation, immune cells migrate to the implantation site and initiate a localized inflammatory response. Although inflammation is an indispensable element in tissue regeneration, a chronic inflammatory response will significantly limit natural healing, which is quite common in the case of implants.
Among the variety of immune cells, monocytes and macrophages play a particularly critical role that determines successful tissue–implant integration or implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the balanced induction of different macrophage phenotypes. Classically, activated M1 macrophages are associated with a pro-inflammatory response. By contrast, alternatively activated M2 macrophages are associated with an anti-inflammatory and regenerative response which induces angiogenesis and proliferation. Therefore, harnessing macrophage polarity presents a unique opportunity to control inflammation, prevent rejection, and accelerate integration of biomaterials and medical devices. Different macrophage phenotypes with distinct functional properties have been identified. For instance, M1 macrophages are induced by interferon gamma (IFN-γ) from T helper 1 (TH1) cells, CD8+ cytotoxic T cells (CTLs) or natural killer (NK) cells in the presence of microbial products such as lipopolysaccharide (LPS). M1 macrophages have pro-inflammatory and anti-tumour functions and secrete high levels of pro-inflammatory cytokines such as interleukin 12 (IL-12) and IL-23. On the other hand, M2 macrophages are induced by IL-4 and/or IL-13, which are mainly secreted by TH2 cells or polymorphonuclear cells such as mast cells. M2 macrophages have anti-inflammatory and pro-wound healing activities and secrete large amounts of the anti-inflammatory cytokine IL-10. Currently, macrophage polarization is most commonly controlled via exposure to biochemical factors. Specifically, the M1 macrophage phenotype is typically induced through interferon gamma (IFNγ) or lipopolysaccharide (LPS) stimulation, while the M2 macrophage phenotype is typically induced through interleukin-4 (IL-4) or interleukin-13 (IL-13) stimulation. The M1/M2 polarization of macrophages has been described in a generalized manner and the optimum macrophage differentiation status for remodelling is not known, and thus one of the objectives was the determination of the optimum and a long-lasting phenotype based on the induction by cytokine cocktails.
Beyond cytokine based macrophage control, in IMMODGEL, one of the objectives was to determine whether physical stimuli such as encapsulation or presentation of micropatterns can control macrophage polarization. Recent advances in biomaterials science have identified that a biomaterial's design can be leveraged to instruct the host's immune system. For example, novel biomaterial surfaces, improved immune-instructive biomaterials and incorporating immune modulating cells could influence the wound healing process. In addition, several physical and biochemical factors have recently been reported to regulate macrophage polarization such as pore size, mechanical stimulation and extracellular matrix proteins (ECM) among others. However, the underlying mechanisms of how biomaterials steer macrophage polarity has remained poorly understood. The biomaterials in vivo will be in close contact with macrophages and characteristics such as surface chemistry and topography may have a critical role in initiating pro- or anti-inflammatory immune responses. Controlling biomaterial surface attributes provides a powerful tool for modulating the phenotype and function of immune cells with the aim of reducing detrimental pro-inflammatory responses and promoting beneficial healing responses. For this end, one of the objectives was to identify the specific surface topography features (surface micropatterns) that can induce M2 phenotype in incoming macrophages or induce M1 to M2 conversion.
The concept of IMMODGEL was that it is possible to design and engineer a stand-alone immunomodulatory system. This product can be attached to any implant to achieve local immunomodulation and thus protect the implant, transplant or tissue engineered product from the adverse effects of immune response while preventing systemic effects and subsequent infection risks. This is achieved via in vitro phenotype control of autologous macrophages which can be used to orchestrate the inflammation cascade. More precisely, the design of a cellular microenvironment that induces M2 phenotype was the main goal. Through this, it was possible to convert local reaction from an inflammatory route to a remodelling route. For achieving this, the objective in IMMODGEL was to develop a transferable, 3D hydrogel for macrophage (M2) encapsulation which can fix the encapsulated macrophage phenotype for 3 weeks with more than 90% viability.
One of the most common implant materials is titanium which is used in dentistry, as permanent tooth implants, and in orthopaedics as knee and hip replacement. Despite its biocompatibility, implants based on this metal commonly fail due to bad osseointegration, peri-implantitis, osteolysis etc. Depending on the target area, the rate of failure can be in the range of 2% to 10%. Currently there are only a few generic indicators (such as smoking, diabetes, factors that are known to affect healing overall) that are in use in order to indicate an elevated risk of implant failure. In an ageing society, the effects of implant failure have more reverberating effects as the problems related to implants mean additional health problems for ailing patients. One of the objectives of IMMODGEL was to determine a profile of specific phenotypic markers that describes the macrophage state around titanium implants.
In line with the silver economy initiative of the European Commission, it is important to provide tools to the implant practitioners to inform and guide elderly patients in order to minimize the adverse effect of such implantation procedures which otherwise are improving life quality. In IMMODGEL, another objective was to detect the patients prone to implant failure which affects a significant number of patients via development of personalized immunoprofiling tools such as a novel personalised multiplex diagnostic antibody-based array to identify personalized immune response to titanium implants.
One of the persistent problems around nondegradable metallic and polymeric implants is failure of macrophages to resolve the inflammation and their tendency to stay in a state, named “frustrated phagocytosis”. Implanted materials can induce a mixed pro/anti-inflammatory phenotype, supporting chronic inflammatory reactions accompanied by microbial contamination and resulting in implant failure. Several materials based on natural polymers for improved interaction with host tissue or surfaces that release anti-inflammatory drugs/bioactive agents have been developed for implant coating to reduce implant rejection. However, no definitive, long-term solution to avoid adverse immune responses to the implanted materials is available to date. The prevention of implant-associated infections or chronic inflammation by manipulating the macrophage phenotype is a promising strategy to improve implant acceptance. For this end, in IMMODGEL, the objective was to develop a polyelectrolyte multilayer based attachment system with cytokine release capacity for simultaneous anti-inflammatory and antimicrobial effects.
For the development of biomaterial based solutions, one potential route is the use of natural polymers with known anti-inflammatory functions. Hyaluronic acid (HA) plays a multi-faceted role in cell migration, proliferation and differentiation at micro level and system level events. 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. A specific reason for the increase in use of HA based structures is their immunomodulatory and regeneration inducing capacities. Thus, in IMMODGEL one of the objectives was to harness the advantageous properties of HA in both coating and hyrogel formulation.
The expected added value of IMMODGEL project was: i) Decreased risk of chronic inflammation, ii) Improved remodelling response by shortening of the inflammatory cascade, iii) A temporal control over the remodelling of the implant, iv) Applicability to different structures which can provide a comprehensive solution to immunological responses without the need to change the design of the original implants.
Additional expected outcomes of the project were: i) Elucidation of Macrophage behaviour within 3D hydrogels, ii) Determination of the synergy between physical control and cytokines to obtain long term control over cell phenotype while keeping product cost feasible, iii) Development of complex immuno-competent tissue models to better understand activities of immune cells in artificial tissue settings, iv) Development of benchmarks for trial of immunomodulating implants, v) Utilization of bioprinter technology to augment the capacities of other implants.
Project Results:
Scientific and Technical aspects of IMMODGEL project were implemented in 6 workpackages over the course of 48 months. The results and foreground generated in each workpackage was described below:
WP1: Systems immunology: identification of macrophage-mediated chronic inflammatory reactions to titanium implants (WP Leader: University of Heidelberg)
Parts of artificial larynx implants were put into contact with 1ary human macrophages and also tissue samples around titanium dental implants were collected. We detected specific markers that provide insight to the nature of the reaction to titanium in different patients. The results were used to establish the optimal biomaterial and cytokine cocktail delivery system for the immunomodulatory hydrogel design. The specific aim of the cocktail was to revert inflammatory reactions elicited by M1 macrophages to the anti-inflammatory phenotype (M2). The components of the cytokine cocktail have been determined. Its effect in the presence of biomaterials has been assessed within other WPs. To develop a diagnostic tool that predicts the immunological profile of the patient with respect to cytokine levels, target cytokines were selected. A lab-level prototype of the system was developed and tested for validation.
An additional part of the results of this workpackage form Foreground 1 (Titanum signature), which is planned to be published and is not described here to allow IP protection.
Foreground 2: Development of Cytokine Cocktail (A M2 macrophage phenotype fixing cytokine cocktail (long term stability) that can be used for immunomodulation):
We achieved to formulate 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 12 days. 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 (from Workpackage 3) 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. 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.
Foreground 3: Effect of Oxygen Plasma treatment on macrophage polarisation:
To assess the impact of different surface chemistries on macrophage polarisation, human monocytes were cultured for 6 days on untreated (hydrophobic) and O2 plasma-etched surfaces (hydrophilic). Our data clearly show that monocytes cultured on the O2 plasma-etched surface are polarised towards an M1-like phenotype, as evidenced by significantly higher expression of the pro-inflammatory transcription factors STAT1 and IRF5. By comparison, monocytes cultured on the non-etched surface exhibited an M2-like phenotype with high expression of mannose receptor (MR) and production of the anti-inflammatory cytokines IL-10 and CCL18. While the molecular basis of such different patterns of cell differentiation is yet to be fully elucidated, we hypothesise that it is due to the adsorption of different biomolecules on these surface chemistries. Indeed, our surface characterisation data show quantitative and qualitative differences between the protein layers on the O2 plasma-etched surface compared to non-etched surface which could be responsible for the observed differential macrophage polarisation on each surface. We showed that changes in surface chemistry resulting from O2 introduction by plasma treatment affect protein adsorption on the material. This in turn appears to influence monocyte polarisation towards macrophages with distinct phenotypes. Specifically, non-etched surfaces were shown to suppress expression of M1-associated markers and cytokines while promoting M2-associated markers. On the other hand, O2 plasma-etched surfaces had the opposite effect. Protein overlayer thickness and amino acid profiles were different on the surfaces, suggesting differences in protein adsorption.
WP2: Effect of topography on macrophage phenotype (WP Leader: University of Nottingham)
To determine the effect of surface micropatterns on macrophage phenotype and how this can be used to control it, microwells, micropillars and microgrooves with a wide range of dimensions were produced and screened with model monocytes and 1ary macrophages. 2 optimal types of surface micropatterns were selected. Further work has been undertaken to determine the synergistic effects of cytokine induction and surface micropatterns. The effect of optimized patterns on macrophage polarisation was quantified at gene level. The patterns can be introduced reproducibly to macrophage-laden hydrogels developed in WP3 and used in the final therapeutic system.
Foreground 4: Machine Learning based Image Analysis for simplified detection of macrophage phenotype:
Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time-consuming, require large numbers of cells and are resource intensive. Instead, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus, high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers.
Foreground 5: Effect of co-stimulation of macrophages with micropatterns and cytokines on their polarisation: As the surface microtopography is shown to exert significant effects on cell phenotype, we hypothesized that the presence of micropatterns on implant/medical device surfaces can attenuate the immune response. To this end, enzymatically crosslinked micropatterned gelatin films of varying groove widths (2, 5, 10, 20, and 40 µm) were tested for their effect on incoming monocyte behavior. In order to distinguish the effect of cytokine microenvironment on pattern presence, monocytes were seeded on micropatterned films in normal culture medium or M1/M2 inducing media and their morphology and cytokine secretions were observed for 6 d. The presence of the patterns induces microenvironment-specific changes on the secretions of the attached cells and also on their size. IL-1ß, IL-4, IL-12, TNF-α, and CCL-18 secretions were significantly affected particularly in M1 induction media by pattern presence. It was demonstrated for the first time that micropatterned surfaces can be used to control the initial attachment and cytokine secretion of incoming macrophages if they were linked with a polarization inducing cytokine microenvironment.
Foreground 6: Unbiased transcriptomics analysis of the effect of micropatterns on macrophage phenotype:
The activation state adopted by macrophages in response to biomaterials determines their own phenotype and function as well as those of other resident and infiltrating immune and nonimmune cells in the area. In biomaterials research, cell-instructive surfaces that favor or induce M2 macrophages have been considered as beneficial due to the anti-inflammatory and pro-regenerative properties of these cells. We used a gelatin methacryloyl (GelMA) hydrogel platform to determine whether micropatterned surfaces can modulate the phenotype and function of human macrophages. The effect of microgrooves/ridges and micropillars on macrophage phenotype, function, and gene expression profile were assessed using conventional methods (morphology, cytokine profile, surface marker expression, phagocytosis) and gene microarrays. Our results demonstrated that micropatterns did induce distinct gene expression profiles in human macrophages cultured on microgrooves/ridges and micropillars. Significant changes were observed in genes related to primary metabolic processes such as transcription, translation, protein trafficking, DNA repair, and cell survival. However, interestingly conventional phenotyping methods, relying on surface marker expression and cytokine profile, were not able to distinguish between the different conditions, and indicated no clear shift in cell activation towards M1 or M2 phenotypes. This highlighted the limitations of studying the effect of different physicochemical conditions on macrophages by solely relying on conventional markers that are primarily developed to differentiate between cytokine polarized M1 and M2 macrophages. We therefore propose the adoption of unbiased screening methods in determining macrophage responses to biomaterials. We showed that the exclusive use of conventional markers and methods for determining macrophage activation status could lead to missed opportunities for understanding and exploiting macrophage responses to biomaterials.
Foreground 7: Use of Electrohydrodynamic printing for implant surface micropatterning:
We developed a direct write printing method to modify metallic implant surfaces with biocompatible polymers with microscale precision particularly for controlling macrophage phenotype. Application of polymeric micropatterns on metallic implant surfaces can (i) improve their interaction with the host tissue, (ii) enable the delivery of growth factors, antibiotics, anti-inflammatory cytokines etc from the implant surface and (iii) can control the immune responses to the implant via controlling the attachment of immune cells, such as macrophages. Surface patterns with a resolution of less than 50 μm can be created using an electro hydrodynamic (EHD) printing, a template-free and single-step process. We devised a revised EHD printing method for the deposition of parallel strips of photocrosslinkable, cell adhesive polymeric composites with spacing of around 20 μm onto medical grade titanium substrates. Optimization of voltage, feeding rate and collection speed resulted in regular structures via very rapid movement of the grounded rotating collector driven to equivalent of the linear surface speed of above 100 cm s−1. A mixture of chemically modified PEG /gelatin was deposited onto a conductive titanium substrate with different surface pretreatments with an area of 400 mm2. Acid etched or UV treated titanium surfaces improved the stability of the printed structures. Polymeric lines induced temporary orientation of human monocytes and induced a thicker cell multilayer formation by fibroblasts. Staining of the monocytes for M1(CD80) and M2 (CD206) macrophage markers on the patterned surface showed mixed populations with higher anti-inflammatory cytokine secretion compared to tissue culture plastic control. The results demonstrated the suitability of this method for the preparation of biomaterials with structured surfaces on large areas and with desired chemical composition.
WP3: Development of physical constituents of IMMODGEL (Adhesive polyelectrolyte, cell encapsulating hydrogel) (WP Leader: Protip Medical)
The overall aim was to develop a gel formulation for macrophage encapsulation and also develop an adhesive (coating) to attach the macrophage encapsulated hydrogels to implants. For hydrogels, the goal was to develop a wide array of implantable, encapsulation systems for autologous macrophages which would enable their transfer and facilitate their phenotype control. For this end, biodegradable base materials such as gelatin, non-degradable polymers such as PEG and immunomodulatory molecules such as hyaluronic acid and polyarginine were utilized to achieve maximum viability and the highest M2 response. During optimization of encapsulation, an adhesive coating system based on gelatin/hyaluronic acid or polyarginine/hyaluronic acid was simultaneously developed to obtain a versatile adhesive structure. Four separate formulations have been successfully used for encapsulation of THP-1 cells and two formulations were also tested with 1ary macrophages. The physical and mechanical characterization of the hydrogels has been done and 4 formulations have been selected for further use in WP 4. Four working adhesive systems have been developed and tested with 1ary macrophages for their ability to release cytokines. Finally, the assembly of the optimal gel formulations together with the optimal adhesive has been established and as tested in contact with different biomaterials. An additional wet adhesive has been developed to ensure strong adhesion to the surfaces.
Foreground 8: Simultaneously anti-inflammatory/antimicrobial coatings:
Major problems with biomedical devices in particular implants located in nonsterile environments concern: (i) excessive immune response to the implant, (ii) development of bacterial biofilms, and (iii) yeast and fungi infections. We developed a multifunctional coating that addresses all these issues concomitantly. A new exponentially growing polyelectrolyte multilayer film based on polyarginine (PAR) and hyaluronic acid (HA) was first designed. The films had a strong inhibitory effect on the production of inflammatory cytokines released by human primary macrophage subpopulations. This could reduce potential chronic inflammatory reaction following implantation. Next, it was shown that PAR, due to its positive charges, has an antimicrobial activity in film format against Staphylococcus aureus for 24 h. In order to have a long-term antimicrobial activity, a precursor nanoscale silver coating was deposited on the surface before adding the PAR/HA films. Moreover, the PAR/HA films can be easily further functionalized by embedding antimicrobial peptides, like catestatin (CAT), a natural host defense peptide. The PAR/HA+CAT coatings were effective as an antimicrobial coating against yeast and fungi and cytocompatible. This all-in-one system constituted a new strategy to limit inflammation and prevents bacteria, yeast, and fungi infections.
Foreground 9: Development of novel release systems for delivery of anti-inflammatory cytokines:
We designed 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 adverse immune reactions. The chemical modification of the HA with aldehyde moieties allowed 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) stimulated the differentiation of primary human monocytes seeded on the films into pro-healing macrophage phenotype (M2). This induced the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrated that cross-linking PLL/HA film using HA-aldehyde was already effective by itself to limit inflammatory processes. This functionalized self-cross-linked PLL/HA-aldehyde films are an efficient candidate for immunomodulation of different kinds of implants of various architecture and properties.
Foreground 10: Discovery of the Chain-size dependent Antimicrobial activity of Polyarginine/Hyaluronic acid coatings (Part 1):
The number of nosocomial infections related to implants and medical devices increase alarmingly worldwide. As, we have observed in one of our anti-inflammatory coating formulations strong antimicrobial capacities, we stidied the underlying mechanism for such action. By investigating films built up with poly(arginine) (PAR) of various chain lengths (10, 30, 100, and 200 residues) and hyaluronic acid (HA), we demonstrated that exclusively films constructed with poly(arginine) composed of 30 residues (PAR30) acquired a strong antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria associated with infections of medical devices. This chain-size effect was extremely striking and was the first example reported where the length of the polyelectrolytes played a key-role in the functionality of the films. Moreover, this unexpected functionality of nanolayered polypeptide/polysaccharide PAR30/HA films occured without adding any specific antimicrobial agent, such as antibiotics or antimicrobial peptides. PAR30/HA film inhibited bacteria through a contact-killing mechanism due to the presence of mobile PAR30 chains. These chains were assumed to diffuse toward the interface, where they interacted with the bacteria with the consequence of killing them. This new coating with unique properties based on the association of a homopolypeptide of 30 residues with a polysaccharide is simple system to prevent implant-related infections with a reasonable production cost. (Patent application submitted)
Foreground 11: Discovery of the Hyaluronic acid dependent Antimicrobial activity of Polyarginine/Hyaluronic acid coatings (Part II):
As described above, first we showed that poly(arginine)/hyaluronic acid (PAR/HA) multilayers built with PAR chains constituted from 30 arginine residues (PAR30) have strong antimicrobial properties through a contact-killing mechanism. This property wass due to the ability of PAR30 chains, when associated with HA, to diffuse in the multilayer. Then, we investigated the effect of the nature of the polyanion on the antimicrobial activity of (PAR30/polyanion) multilayers. Four polysaccharides, one polypeptide, and one synthetic polyelectrolyte were investigated. Surprisingly, only HA leads to films with antimicrobial character. We related this property to the strong diffusion capacity of PAR30 chains in (PAR30/HA) multilayers compared to their diffusion ability in the other (PAR30/polyanion) films. Through isothermal microcalorimetry experiments, we also demonstrated that interactions in solutions of PAR30 chains with the different polyanions were characterized by a negative reaction enthalpy for all of the investigated polyanions except for HA, where the enthalpy of reaction was positive. Moreover, the molecular weight of HA was not a key parameter for the diffusion ability of PAR chains or for the bioactivity of the film. These results constituted an important step toward the establishment of rules to design contact-killing antimicrobial polyelectrolyte multilayers.
Foreground 12: Biomcompatible Adhesive development for establishing stable contact with implants and IMMODGEL system:
Modification of implant surfaces with an adhesive represents a promising strategy to promote the adhesion of cell-laden hydrogels on the implant. For this end, we developed a peptidic adhesive based on mussel foot protein (L-DOPA-L-Lysine)2-L-DOPA that can be applied directly on the surface of an implant. At physiological pH, unoxidized (L-DOPA-L-Lysine)2-L-DOPA expected to strongly adhere on metal/metal oxide surface formed only very thin coatings. Once oxidized at physiological pH, (L-DOPA-L-Lysine)2-L-DOPA formed an adhesive coating about 20 nanometers thick. In oxidized conditions, L-Lysine can adhere to metallic substrate via electrostatic interaction. Oxidized L-DOPA allows to form a coating through self-polymerization and can react with amines so this adhesive can be used to fix ECM based materials on implant surfaces through the reaction of quinones with amino groups. Hence, a stable interface between a soft gelatin hydrogel and metallic surfaces was achieved and the strength of adhesion was investigated. We have shown that the adhesive is non-cytotoxic to encapsulated cells and enabled the adhesion of gelatin soft hydrogels for 21 days on metallic substrates in liquid conditions. The adhesion properties of this anchoring peptide were quantified by a 180° peeling test.
WP4: Control of macrophage phenotype subsets in 3D hydrogel systems (WP Leader: Protobios)
In WP4, the aim was to quantify the effect of encapsulation and the physical properties of the hydrogel on the phenotype of the encapsulated macrophages. Several parameters such as stiffness, degradability, presence of cell adhesion sequences, hydrogel composition and crosslinking parameters have been varied and their effect on macrophage phenotype was tested in the presence and the absence of cytokine induction. This process fed into the selection process in WP3. Finally, the effect of adhesive and hydrogel composite on encapsulated macrophages in the presence of cytokine release was quantified. Macrophages can modulate tissue vascularisation and remodelling depending on their phenotype, this can be controlled using physical and chemical cues. Using cell-laden 3D structures the effect of phenotype controlled macrophages on primary fibroblasts and vascular endothelial cells was quantified.
Foreground 12: The Effect of Degradability of the Encapsulation Microenvironment on Macrophage Phenotype:
In order to see whether the nature of hydrogel had an effect on encapsulated macrophage phenotype, a biodegrable, natural polymer based hydrogel system (Methacrylated gelatin (GelMA)) and a synthetic, non-degradable hydrogel system (Poly(ethylene glycol) diacrylate (PEGDA)) were sued to encapsulate macrophages. We demonstrated that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and a collagen-derived matrix (gelatin). Surface marker, gene expression, biochemical, and cytokine profiling consistently indicated that THP-1 cells within a biomaterial lacking cell attachment motifs yielded pro-inflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induced an anti-inflammatory M2-like phenotype and propagated the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2β1 played a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity was further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively.
Foreground 13: Adhesive Film/Hydrogel composites with controlled cytokine release: Delivery of growth factors is an indispensable part of tissue engineering and immunomodulation. We used a detachable membrane-based release system (from WP3) 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 developed 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 resulted in extensive sprouting by encapsulated vascular endothelial cells. The presence of the release system with interleukin-4 resulted in clustering of encapsulated macrophages with a significant decrease in M1 macrophages (pro-inflammatory). This system was used in conjunction with three-dimensional structures as an auxiliary system to control artificial tissue maturation, growth and harnessing of immune response. (Patent in preparation for parts beyond the disclosed part)
WP5: Enabling Technologies: Foreign Body Response on-a-chip, immunocompetent artificial tissues, bioprinted immunomodulatory hydrogels
In WP5, we designed a prototype for Foreign Body Response on-a-chip system which was first used to observe the macrophage reaction to titanium microbeads. For the immunocompetent epithelial tissue development, we have developed artificial basement membrane mimics and tested them with respiratory epithelial cells. We also developed 3D sandwich-like structures for the connective part of the epithelial tissue and the methods of incorporating macrophages and macrophage-laden hydrogels in the model tissues. The application of bioprinting methodologies for printing of immunomodulatory hydrogels was also carried out.
Foreground 14: Resident Macrophage Model Development; Encapsulation of phenotype controlled macrophages and quantifying their interactions with incoming cells:
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.
Foreground 15: Sandwich-like multicellular organ models for respiratory epithelium development:
Many organs are multicellular and each cell type requires a different microenvironment. Thus, there is a need for modular structures where the microenvironment of each cell type can be tuned separately. We developed enzymatically crosslinked gelatin based double layered film structures where each layer can be loaded with growth factors separately. As a model, we have developed a bi-layer system to produce a respiratory epithelium. This system constitutes an in vitro “epithelial patch”. Crosslinking of the patches with transglutaminase resulted in 7 days of stability at 37 °C. The film layer was first used to release growth factors and it was shown that the release significantly improved the proliferation over 5 days. Human lung epithelial cells were used and under the release of an epithelial growth supplement mix, there was a significant improvement on the epithelial proliferation. The stability of the epithelial patch under in vitro conditions for 7 days without deterioration. Under co-culture conditions for three days both cell types were alive.
WP6: In vivo tests of IMMODGEL of the immunomodulatory system with titanium implants (WP Leader: University of Strasbourg/INSERM)
The implantation protocol for testing of immunomodulatory hydrogels was developed and implemented in a mouse model for the subcutaneous immunomodulation around implants with developed hydrogel and adhesive systems. The second part of the WP focused on immunomodulation in orthogonal positions particularly partial tracheal replacement with 3D printed structures in the presence or absence of immunomodulation where we have shown significant positive effects of immunomodulation at local and systemic levels.
Potential Impact:
Implant technology has become widespread and several types of implants, such as hip implants, knee implants are being implanted in more than a million cases worldwide. The decreasing cost of dental implants has increased their demand in Europe, and an additional several billions of Euros growth in this field is expected in the next 5 years. In a field (dental) which is valued at more than 2 billion Euros and where over 40% of global demand is in Europe, improvement of the current protocols via immune response control as developed in IMMODGEL will have great positive economic consequences. The removal of adverse immunological reactions, a very common problem in the field of biomedical engineering, can also augment the direct investment from public and private sector in areas where they would be normally considered too risky.
Prognostic or Susceptibility/Risk, Monitoring Biomarkers: The occurrence of mild and severe inflammatory processes of implants are on the rise. There are several contributing factors to this trend such as the overall ageing society, the availability of the implants to a wider range of demographics and a longer expected implant life time. The exact reasons in detail are still unknown, however, it is clear that the lack of diagnostic methods to actually determine high-risk patients and treat them accordingly is playing a big role in this problem. IMMODGEL aimed to fill this gap by studying in which form the generated scientific knowledge of the personalized set of candidate genes will be of the best use.
The immunomodulatory system targeted in this project consisted of 1) autologous macrophages with a controlled phenotype 2) a composite hydrogel for macrophage and cytokine encapsulation and 3) a polyelectrolyte coating which will allow adhesion of the encapsulated macrophages to any implant.
We have optimized several encapsulation systems and polyelectrolyte coatings that fit the requirements of the final therapeutic system. We have also developed the protocols for theassembly of the structures and their in vivo testing. Encapsulation of macrophages has not been undertaken before the IMMODGEL project in the literature; thus IMMODGEL has provided the initial conditions for future research in this area. Cell adhesion plays an integral role in enabling communication between cells and their microenvironment. However, their role in monocyte to macrophage differentiation and particularly macrophage polarization are yet to be fully understood. Here, we hypothesized that integrin-mediated cell–biomaterial interactions could play a key role in macrophage polarization. Given that in vivo these events take place in the context of ECM and in 3D, obtaining a clear understanding of the role of integrins in macrophage polarization in a 3D microenvironment will be more physiologically relevant than in a 2D environment. We have used two distinct hydrogel systems to probe the effect of cell–biomaterial interactions on macrophage polarization in a 3D environment. We investigated whether macrophage polarity can be controlled through integrin-mediated biomaterial-based programming. Our findings will pave the way for future endeavors in elucidating immune system/biomaterial interactions in 3D configurations.
We have devised specific cytokine cocktails for precise control of macrophage phenotype that have been tested in 3D conditions. Artificial tissue model development for the testing of immunomodulation has been done. We have provided the biomaterial community with new tools such as incoming macrophage models and a foreign body response on a chip system for incorporating the immunology aspect in their activities.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. We demonstrated that self-standing, transferable 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 cocktails induces long-term anti-inflammatory and pro-healing phenotype in human primary monocyte-derived macrophages. This cocktail formulation could be loaded on 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.
Via systems immunology, we were able to determine specific markers that can define the tissue response to medical grade titanium implants. This can be of significant prognostic value of the fate of such implants and will facilitate the clinicians and immunologists to holistically understand the adverse immune responses to implants, how these are controlled by macrophages and how to make judicious choices regarding host-implant compatibility. We also developed immunoprofiling methods based on cytokine measurements and epitope characterisation which can also be used for pre-implantation diagnostics applications.
A foreign body response (FBR) on-a-chip system has been developed that will significantly reduce the research cost and the need for animal tests by replacing the compulsory biocompatibility tests with a sophisticated in vitro test. The initial design of the FBR on-a-chip system is ready and the future iterations are currently underway within the framework of future projects.
Personalized Implants: Macrophages are master cells of the innate immune system that protect all tissues of our body from exogenous danger of different origins by induction of inflammation and elimination of foreign materials and organisms. Moreover, there are other cases of macrophage and metal based complications such as myofascitis due to the Aluminium content in vaccine adjuvants. As the immune system of each individual differs, it is important to have ways to evaluate the possible responses of the individual patients. The discoveries in IMMODGEL will allow the stratification of the population with regards to the response to Ti allowing to protect the patients from complications. It is also possible to provide suggestions for possible modifications of commercially available implants to make them more amenable for a given patient thus paving the way of personalized implants. Finally, it was our aim to utilize its predictions for the development of therapeutic solutions for implant related problems which can then be further extended to transplants and biomedical devices, particularly those of titanium and other metals/alloys, polymers, ceramics and eventually to engineered tissues. It can also open the door for looking beyond the innate immunity and into adaptive immunity via effects of lymphocytes.
The results obtained on model implants can provide a necessary framework for a wide range of biomedical materials. By developing a system that can be applied to any kind of implant, transplant or biomedical device, the project will expand the capabilities of immunomodulation. In this sense, IMMODGEL will have a widespread impact and can improve the chances and success of many other European healthcare products. Our results up to now are in line with this claim and we hope to deliver the proposed impact following the animal tests.
IMMODGEL will yield high socio-economic impacts by significantly improving the health and quality of life of patients in EU and world-wide, and by decreasing therapy costs due to adverse immunological reactions. This research will not only improve the outcomes of implants and biomedical devices, but will also provide meaningful improvement of non-communicable diseases (NCDs). In an ageing society, by improving the lifetime of implants and biomedical devices, immunomodulatory systems can provide a meaningful saving on healthcare costs. The project will also help the development of personalized medicine or stratified medicine approaches to implantology and development of necessary tools to achieve the personalization.
The proposed system can be used in conjunction with all types of implants, particularly with the implants such as dental, orthopaedic, spine or larynx implants. Applicability to different structures can provide a comprehensive solution to adverse immunological responses without the need to change the design of the original implants. This will also allow reducing the need of animal experimentation due to the elimination of immune system related failures which will not only have positive economic impacts but also ethical ones. Also, the development of a diagnostic tool to quantify immune response to implants will decrease health costs by predicting and preventing possible excessive immune reactions. Creation of new employment opportunities followed by the newly developed technologies and the know-how will also offer a positive economic impact on the society.
The development of this unique immunomodulatory system and relevant know-how is expected to provide significant opportunity for the industrial partners to both maintain and build further on their existing market positions. The overall implant industry is projected to increase between 5 and 10% per year (a growth of 20% is expected only in dental implant industry for the next 5 years), therefore a product that enhances the success of these surgeries and reduces several other associated health risks will fetch benefit of lower health sector costs and improved health conditions. For commercial exploitation and benefit to the industrial partners; Protip Medical has developed IP and new competencies thanks to the project. One of the technologies developed in IMMODGEL (patented) is under pre-clinical testing at the moment for eventual up-scaling and potential commercialisation. Protobios has entered into a potential new market (biomaterial diagnostics) and Contipro has found a new area for utilisation of their Hyaluronic acid based products as base material
System Immunology and Bioengineering have a continuously increasing importance due to the issues related to immune-competence, associated reliability and success issues, lack of broader industrial familiarity with wide-scale solutions and embedded commercial interests. The research undertaken in IMMODGEL project has generated academic and industrial interest. The development and application of an immunomodulatory system to control the adverse immune reactions in the host body will demonstrate their effectiveness for different types of surgical applications. The ability of implants to alleviate chronic disease related problems is significantly reduced by adverse immunological reactions to them, which makes immunomodulation a highly impactful area of technological development. This is expected to encourage further academic research and additional industry funding into academia to further lead in this technology.
Benefit to other academic research fields – Diagnostic and Immunological research is of great interest to researchers in many differing fields. Assessment of undiscovered fields of adverse immune reactions and host: implant interactions against specific cases will open up research options to academics which may not be immediately evident. For example, we have demonstrated a new means of classifying macrophage populations using image based machine learning. Given the heterogeneity of macrophage phenotype and current limitations of the machine learning approach it may be too early to suggest use of image analysis as an alternative to conventional cell phenotyping. However, our data provide strong evidence for the ability of high content and automated image analysis approaches for accurate, less resource intensive and fast phenotyping of functional diverse cell populations. Moreover, the data organized and information collected during the project will support the developement of other systemic researches.
Biomaterial-mediated immunomodulation by programming macrophage polarity is a promising tool in tissue engineering, regenerative medicine and implantology to decrease adverse immune reactions, accelerate implant integration, facilitate tissue regeneration and increase implant lifetime. Overall, biomaterial-based control over macrophages represents a novel technique to obtain a fundamental understanding of macrophage behavior and is a strong therapeutic tool for immunomodulation for implants, drug, and cell delivery systems.
Engineering a tissue requires the design of the optimal conditions for the cells in the target tissue involved. Additionally, it is also crucial to develop methods to recruit and direct the host cells that will take an active role in the integration of the implanted tissue. A modular ECM-based system such as IMMODGEL can be used as a component of engineered tissues for directional release of cytokines and growth factors to attain precise microenvironment control. Development of a system composed of two modular components decouples the release and cell loading, which allows a higher degree of control over the encapsulated cell behavior.
Silver Economy, welfare: An aging population brings new challenges to health professionals in general and dentists in particular. It is well known that dental implants are likely to become used within a larger population pool and that a given individual may have several implants in the mouth. IMMODGEL will make it possible to access to safer titanium dental implant procedures thus allowing the access to implants to a wider population range and improving its welfare.
Controlling Healthcare Cost: Inflammation around the implants can cause severe peri-implantitis. A study in 2015, states that the treatment of peri-implantitis can cost several thousand Euros depending on the treatment method. A better understanding of the patient’s innate response profile can result in a decrease of implant related complications. Moreover, this “complications” problem is not limited to dental implants as they are also known to exist in further implants and in various target organs. For example, implant related problems have been on the rise during the last few years (~5 000 complications out of 200 000 implantations for cardiovascular devices) which caused an increase in long term mortality (about 2%). In average, such complications cost about 15.000 dollars each. Similarly, in total hip replacement, a postsurgical treatment has a cost of 50.000 dollars (on average) with a rate of incidence of 2% (in the extreme cases the replacement of the implant or amputation of the limb could be necessary). By providing an innovative way to predict adverse immune reactions (reactions that in most cases are indirectly responsible for the infections as the implant is not integrated well), IMMODGEL will open the door to such a practice in other fields of implantology (hip, knee, cardiac, etc.) therefore contributing to a larger use of personalized medicine and a better control of healthcare costs.
HA is an important tool in biomaterial research as evidenced by its widespread use in surgical applications as a filler, as hydrogel material, cell carrier and drug delivery system. The ease of isolation/production of HA and its extensive biological activities makes it a very attractive target for therapeutic biomaterial based system development. The presence of several sites amenable to chemical modification on HA chains further improves its practical use as a wide array of derivatives can be developed. The current state of research has put emphasis on 3 directions in the use of HA in biomedical applications: i) injectable formulations that will improve the delivery and defect conformation for both drug and cell delivery applications ii) development of new derivatives of HA that enables further control over physicochemical and biological properties of the final structures iii) Harnessing the biological activities of HA for more controlled biological effects such as controlled retention of multiple growth factors or specific, directed differentiation of cells to desired phenotypes. Thus, in the upcoming years, it is reasonable to expect the development of more derivatives of HA for applications in regenerative medicine, particularly in immunomodulation, angiogenesis, nerve regeneration and hybrid materials containing HA. As a HA provider, IMMODGEL partner Contipro will benefit from the use of HA in immunomodulatory formulations.
As an outlook, the next in line in the sophistication of engineered tissues can be the inclusion of immune system components. Nearly all tissues have resident macrophage populations which has been shown to be an important factor in tissue homeostasis and healing upon injury. Recently, there has been a growing focus on the control over innate immune response in the microenvironment of implanted materials particularly through well-established macrophage polarization pathways that have been shown to have a crucial role in vascularization of implanted scaffolds. Immunoassisted tissue engineering approaches can harness the ability of innate immune cells to resolve inflammation and promote regeneration and healing. This can be achieved by exploiting the phenotypic plasticity of immune cells either via controlled delivery of specific phenotype inducing cytokines or direct co-delivery of phenotype controlled immune cells together with the cells relevant to the target organ function. A new focus on establishing a cross-talk with the host immune system, rather than trying to evade it, could pave the way for more functional and fast-integrating artificial tissues. Concomitant use of new developments in temporal control of multiple growth factor/cytokine delivery; advanced bottom-up assembly methods of engineered tissues such as robotic assembly; use of bioactive miRNAs within scaffolds; and micro/nanoscale topographical and chemical control of scaffold features for inducing anti- or proinflammatory immune cell phenotypes would provide the tools for engineering multicellular organs and establishing in vitro organoids that faithfully model physiological conditions with immune system components. These efforts would bring forth the aspects of ‘regenerative immunology’ in regenerative medicine.
IMMODGEL project provided a better understanding of the significant role of innate immune cells in tissue remodeling and regeneration for regenerative medicine and implants. A wide variety of strategies are available to orchestrate the initial immune response to implanted structures as detailed above. However, because the immune response is tightly regulated both spatially and temporally, more elaborate techniques will be necessary to attain optimal functional integration. Apart from control of the innate immune responses, the next line of control can be achieved at the level of adaptive immunity via B cell and T cell responses. Some of the most commonly used biomaterials, such as synthetic polymers and ceramics, can be spared of adaptive immunological responses owing to the lack of potential immunogenic components. However, new generations of biomaterials, where hybrids of organic and inorganic components, synthetic peptide structures, and cell-responsive polymeric components are increasingly used, necessitate the consideration of the adaptive immune responses to biomaterial structures. For a designed self-assembling polypeptide chain, for example, it is important to know whether the sequence and structure of the design resembles an antigen and can therefore be a functional epitope that triggers adaptive immunity. To optimize the adaptive immune response to biomaterials, a better understanding of the mechanism of events related to adaptive immunity, such as leukocyte attachment, is needed which can be outlook studies after IMMODGEL.
It is becoming increasingly important to ask fundamental immunological questions in the context of biomaterial development. Topographical and/or chemical design of biomaterial surfaces with respect to the APC responses can pave the way for a new generation of ‘cell instructive’ materials with immunomodulatory properties with a wide range of clinical applications. In future, high-throughput systems which will be key to better mimic the FBR in vitro will allow the elucidation of biomaterial-specific responses in real-time and will therefore substantially improve our ability to identify, predict, and control the immune response to implanted biomaterials. We believe, as the concepts surrounding the biocompatibility of biomaterials evolve, that the focus will shift from an evasion of the host immune system to an orchestrated interaction with it. This will also enable the discovery of new biomaterials.
List of Websites:
www.immodgel.eu
Scientific coordinator: Dr. Nihal Engin Vrana, e.vrana@protipmedical.com
Administrative coordinator: Dr. Mercedes Dragovits, dragovits@steinbeis-europa.de
