Advanced Biomaterials Based On Biopolymers For Tissue Engineering Strategies

Growing interest has been devoted in the design and fabrication of novel biomimetic materials which has fasten the pace on the use of biopolymers as building blocks in tissue engineering approaches. Among of biopolymers systems those based on proteins are very promising ones due to their unique ability to control cell function and mimicking tissue properties. Proteins that show similarity to those found in the extracellular matrix (ECM) are excellent candidates to control and enhance cellular process including adhesion, spreading, migration, proliferation, and differentiation. For this reason, it is important to extend the knowledge on protein-based biomaterials, boosting their development beyond the state-of-the-art. Thereby, it is my plan to use different natural structural fibrous proteins such as keratin (from human or animals’ source); silk fibroin (from animals’ source derived from cocoons Bombyx mori) collagen (from animals’ source); and elastin (from animals’ source derived from bovine neck domain). These proteins are characterized by highly repetitive amino acid sequences that give rise to unique mechanical and architectural properties, as well as to notable biological functions.

These repetitive amino acid sequences usually result in the formation of relatively homogeneous secondary structures (e. g. β-pleated sheets, coiled coils, or triple helices), which in turn promote the spontaneous polymerization of protein monomers that self-assemble into hierarchical materials. It will be also my aim the use of peptides sequence that can be synthetically generated and are comparable to full-length proteins as they can be synthetized with a higher degree of specificity. Therefore, for the next years the work I am planning will focus on new and innovative ways to increase the knowledge in the use of ECM-like proteins as tools in tissue engineering strategies. To successfully achieve this goal, the scientific strategy will include different interrelated tasks, such as the development and optimization of the extraction procedures of proteins; the development of protein-based biomaterials with different morphologies and compositions; the effect of the enzymatic modification on their physicochemical properties, and also the validation of such hybrid biomaterials for tissue engineering approaches by multicellular studies in an attempt to recreate the complex in vivo microenvironment. These studies will complement the work that is being developed in University of Stuttgart, and at the same time differentiate me from several studies already published and/or in development in other universities.

My work will unravel the potential of ECM-like protein to create intelligent systems bringing new and disruptive insights in tissue engineering strategies, which is expected not only to have a major impact in rationally designing proteins-based materials, but also to apply them for therapeutic benefit. The successful of the current proposal will take advantage of my know-how in the field, as in last years I have been working with different proteins coming from different sources, such as bovine serum albumin (BSA), human serum albumin (HSA), soy protein isolate, elastin from bovine neck domain that are commercially available and keratin (from human hair and from wool), silk fibroin (from Bombyx mori) that need to be extracted. The extraction of proteins its an important and crucial step in order to achieve stable and functionalized platforms. In general, the classical extraction methods are widely established with possibility to obtain protein yields up to 70% in the case of keratin. Despite this being an important topic, it loses strength when reproducible amount of protein extracted is need, limiting our analysis when we want to build different biomaterials systems. Furthermore, the combination of denaturating agents with surfactants often present problems associated with non-uniform protein yields, protein hydrolysis, interference with chemical and physical analyses and difficulties related with their complete removal from solutions.

Therefore, different methodologies will be employed to extracted proteins and an extensive characterization will be also performed (e. g. , total protein content by Lowry method, SDS-PAGE electrophoresis and amino acid analysis) to successfully validate the adopted methodology. It is my vision and goal for the next years to refine these procedures to obtain extracts that are well characterized in terms of proteins’ composition. To achieve such goal, as primary objective I will adopt two-level factorial design to fully understand the effect of extraction variables (e. g. , concentrations of denaturating agents, time and temperature of extraction step and presence of surfactants) so that optimal conditions will be designed to obtain maximum protein yields in a reproducible way. The major advantage of studying the influence of several parameters by means of factorial design methodology is the possibility to distinguish possible interactions among factors, which would not be possible by the classical experimental methods. The remarkable properties of these protein extracts will lead to the development of a myriad of novel protein-based biomaterials for tissue engineering purposes. My know-how on the development and characterization of protein-based biomaterials over the last years its mainly in two areas: micro and nanoparticles for controlled release and hydrogels, films and capsules for different tissue engineering applications.

To achieved these structures, I have used the sonochemistry methodology, which, compared to traditional energy sources, it is a non-invasive technique that provides rather unusual reaction conditions (i. e. , a short duration of extremely high temperatures and pressures conditions in liquids). This is a very powerful and versatile technique to obtain reproducible soft materials with controlled size by adjusting several parameters, such as temperature, amplitude, and production of radicals. Till now, few biomaterials based on ultrasound techniques have been proposed, which open an avenue to use this technology to solve challenges that remain unmet for tissue engineering approaches. Therefore, I propose the use of these powerful technique for the creation of innovative hybrid biomaterials or to modified them creating unique functionalities for tissue engineering. The hydrogels fabrication will take advantage of the ultrasound technique to finely control the protein fiber microstructure during the fabrication of the three-dimensional network. With such approach it will be possible to spatial pattern various fibrous microstructures, enhancing the level of complexity of the engineered constructs to one that closely resemble the ECM microenvironment.

In order to obtain stable hydrogels structures several parameters will be controlled such as the protein concentration, sonication time and the use of additives (e. g. Ca2+). I also pretend to develop rather unique 3D hybrid system combining capsules and microparticles. The development of these hybrid systems will hence comprise the preparation of protein microparticles with or without bioactive agents (e. g. growth factors (GFs), by ultrasonication methodology and further encapsulated those particles into capsules. The capsules will be prepared using a pneumatic extrusion technique where different raw materials, concentration, crosslinkers, and air pressures will be tested. The capsules will be mainly obtained using either ECM-like proteins and a blend of proteins with alginate that is a polysaccharide extracted from algae. The blend of alginate capsules will be obtained by ionotropic gelation with calcium chloride, whereas fibrous protein capsules will be enzymatic crosslinked. The enzymatic crosslinked will be selected due to their environmentally friendly nature and also due to the fact that the majority of enzymes are involved in the catalyze of reactions naturally occurring in our body.

Few works up to now reported the use of transglutaminases (TGases) (EC2. 3. 1. 13) in the stabilization of protein-based biomaterials and there is a need to explore the use of these enzymes in order to finely tune the physicochemical properties of the final construct. My concern in this step would be the selection of commercial TGase enzymes from mammalian and microbial source follow by the assessment of optimal TGase enzyme and crosslinking conditions in order to get successful results. Thereby, in this research part I will start with enzymatic crosslinking using TGase but other enzymes may also be tested such as the horseradish peroxidase (HRP), where some studies already demonstrated its ability to crosslink the silk fibroin. The microparticles, previously produced by ultrasound technique, will be dispersed in the core of the capsules providing an additional solid 3D support for encapsulated cells, surpassing one of the main limitations of these constructs, which is the lack of adhesion sites that impair the cell proliferation and differentiation. At the same time, the loading of growth factors (GFs) in this microparticles will allow a tuned supply and elicit specific cellular actions. The GFs regulate several cellular activities by binding to specific cellular receptors present on the surface of target cells, which in turns initiate a cascade of biological events that stimulate the regenerative process. Among the plethora of GFs, I intend to use those with applicability in cartilage, bone and vascularization, such as transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).

For that, GFs can be directly embedded in protein solution prior to microparticles formation or immobilized on the surface of protein microparticles. The developed hydrogels and hybrid systems will be physicochemical characterized based on their: morphology (e. g. , topography, porosity and surface area); mechanical properties (e. g. compressive and tensile strength); hydrolytic stability (e. g. water-uptake and degradation) and surface properties (e. g. surface energy, chemistry, charge). The latter approach is particularly useful for additive manufacturing through bioplotting, which has the advantage of producing scaffolds with complex geometries, where microcapsules can be used to design a hierarchical scaffold containing different materials and different cell types. As with all the tissue engineering strategies, the possible therapeutic outcome is not solely linked with an optimal combination of biomaterials, and GFs, but it also relies on the cell performance. Thereby, I also pretend to evaluate the biological performance of the developed hydrogels and hybrid systems by multicellular studies in an attempt to recreate the complexity of the in vivo microenvironment. Co-cultures of heterogeneous cells can better mimic the in vivo microenvironment than monotypic cell cultures, as they allow to study the cellular crosstalk among different cell types.

Therefore, it is my aim to co-culture different cells sources, including osteoblasts, fibroblasts, smooth muscle cells, endothelial cells, bone marrow stromal cells (MSC), and adipose stem cells (ASC), etc. Naturally, this will depend on both the organ system as well as the biological/pathological process being studied. The proposed work intends several methodological linkages and is complementary to the work done in other groups, such as in the Institute of Biomaterials and Biomolecular Systems, Institute of Interfacial Process Engineering and Plasma Technology, Institute of Biomedicine Technik, Institute of Cell Biology and Immunology, Institute of Microbiology and, Institute of Chemical Process Engineering. All projects will address and develop the goals of the department and are in accordance with the structure and development plan of the Faculty 4: Energie-, Verfahrens- und Biotechnik and with the Institute of Biomaterials and Biomolecular Systems for the next years, especially in the field of development of protein-based materials. My plan is to involve master and doctoral students in my research work starting in the next years, or more depending on personal resources allocated to the group. Teaching conceptDespite a few modules during the bachelor and master courses in Technical Biology, the students have limited options to learn about the requirements in tissue engineering.

For this reason, I propose two modules, one on the bachelor level and another on the master level. These modules will improve the students’ knowledge and increase their interests in advanced biomaterials for tissue engineering.

Module: Principles in Tissue engineering – Bachelor studiesThe module aims to give bachelor students the fundamentals of the history and current state of art of the tissue engineering field. It will consist of introductory lectures to the research topics and practical applications. In the lectures the students are introduced to the concept of tissue engineering, the methodologies and essential elements. The module will cover the different types of devices used in tissue engineering, their main requirements, design tools (materials, processing methodologies) and characterization techniques. Moreover, it will give inputs in regards the basics on the general anatomy and composition/structure as well as regeneration ability of the main human tissue. Finally, the properties of stem cells, primary cells, and growth factors as well as their impact on the development of tissue-engineered devices will also be covered in this module. In the end of the module, the students should be capable of: a) Understand the core concepts of tissue engineering;b) Know about the strategies typically incorporated into tissue engineered devices or utilized during their development.

Students will be evaluated with a final exam. Module: Advanced Biomimetic Biomaterials – Master studiesThis module seeks on the one hand to show the latest trends in the development of materials for biomedical applications, including advanced therapies. On the other hand, it is intended that students seize the processes required to construct different biomaterials. This module it will consist of introductory lectures to the research topics and laboratory classes. During the introductory lectures it will be given the information regarding: Mechanical behavior of natural systems and biomaterials (including advanced biomaterials such as nano-composites and hierarchical systems); Systems that react to external stimulus; Biomaterials based on "soft matter", including for example, hydrogels; Biomaterials obtained by self-organization ("self-assembly"); and surfaces with specific surface properties, including biomimetic surfaces (extreme wettability, adhesive properties, etc. ).

The objectives of Advanced Biomimetic Biomaterials module, translate the specific skills that are expected to be developed by the students:a) Plan, execute and interpret experiments;b) Learn techniques to prepare and characterize different biomaterials (e. g. Hydrogels, films, scaffolds, etc. );d) Improve oral and written communication and Critical thinking. Students will be evaluated with a final exam and a paper presentation.