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Project titleCreation of a new composite material based on polysaccharide and polyester for biomedical applications, study of its physicochemical characteristics, biocompatibility and biodegradability in vitro

AffiliationFederal State Institution "Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences",

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-302 - Structure and properties of polymers and multicomponent polymer systems

KeywordsBiopolymers, poly-3-hydroxybutyrate, chitosan, composite materials, biocompatibility, biodegradation, tissue engineering, films, scaffolds

Annotation
Biocompatible polymers of natural origin, such as chitosan, poly-3-hydroxybutyrate, are often used by researchers in the field of regenerative medicine to develop matrices and tissue-engineered scaffolds. The purpose of this project is aimed at solving the problem of creating composites from polymers of natural origin, which can be used to create biomedical products, namely for the regeneration of bone and cartilage tissue. The novelty of the presented study, first of all, is the use of a new method for the manufacture of mixed composites based on PHB and chitosan, using acetic acid as a common solvent. Establishing the mechanism of biodegradation of polymers for medical use and the fundamental patterns of changes in their physicochemical and properties in the process of biodegradation is an urgent scientific task. Solving this problem will reveal the mechanisms for controlling the biological properties of polymer products (biodegradation, biocompatibility, biomechanics) through the control of their physical and chemical properties, which is necessary to create biodegradable medical products with a given therapeutic activity. It is expected that the resulting polymer composite mixtures will be cytocompatible, non-toxic, more hydrophilic than pure poly-3-hydroxybutyrate, and have improved mechanical and rheological characteristics compared to pure chitosan. Structural parameters and main physicochemical properties of the films will be analyzed using the following methods: differential scanning calorimetry, IR spectroscopy, microscopy (AFM, SEM), nanoindentation, rheometry, contact angle measurements. Based on the developed composite mixtures, not only macrofilms, but also scaffolds, as well as ultrathin films will be formed, which will be used as model objects for studying the structure and interactions of mixture components at the molecular level. Thus, the results obtained can be used in biomedicine to develop new medical devices based on natural polymers.

Expected results
In the course of this work, it is planned to develop a composite mixed material based on chitosan and poly-3-hydroxybutyrate, which in the future will be able to find application in the engineering of bone and cartilage tissue. During the implementation of the project, samples of composite mixtures with different percentages of components based on chitosan and PHB will be obtained and characterized, and micro-, macroscopic films, scaffolds will be made. The properties of polymer blend samples will be compared with those of pure polymers. It is expected that the resulting polymer composite mixtures will be cytocompatible, non-toxic, more hydrophilic than pure PHB, have improved mechanical and rheological characteristics compared to pure chitosan. Structural parameters and main physicochemical properties of the films will be analyzed using the following methods: differential scanning calorimetry, IR spectroscopy, microscopy (AFM, SEM), nanoindentation, rheometry, contact angle measurements. In this work, we will search for relationships between the composition and formation conditions of composite films and their characteristics. For this, ultrathin films will be additionally formed and studied on the basis of polymer mixtures. Ultrathin films are a model object for studying the structure and interactions of mixture components at the molecular level. In addition, the results obtained will be applicable to macroscopic products, which will make it possible to predict their properties, including biological compatibility. Experiments to study the rate of biodegradation of films will make it possible to evaluate their ability to decompose in various media. In addition, the study of the degradation of mixtures will make it possible to suggest a degradation mechanism under conditions simulating physiological conditions, as well as to estimate the degradation rate, which is important for predicting their subsequent practical application, for example, faster degradation will be useful for products used in soft tissue engineering, and more long-term - in bone tissue engineering. During the degradation process, it is also possible to assess the presence of toxic substances in solution, thus making the selection of the most promising materials for subsequent biological tests. At the stage of biological testing, it will be established how the cytocompatibility of films from chitosan and poly-3-hydroxybutyrate correlates in in vitro tests. The result of this work will be the development of new composite materials with optimal characteristics for the creation of biomedical materials on their basis. The results obtained during the implementation of the project will correspond to the world level, thanks to a multilateral integrated approach to studying the properties of composite mixtures, a variety of research methods used, as well as additional study of materials at the nanolevel.

Annotation of the results obtained in 2022
The aim of this project is to solve the problem of creating composites from natural polymers that can be used to create products for biomedical applications. In the course of the work, a method of obtaining composite mixed films and scaffolds based on polyester poly-3-oxybutyrate (PНB) with the addition of polysaccharide chitosan (CTS) was proposed. Glacial acetic acid was used as a solvent for poly-3-oxybutyrate. At the first stage of the work, the optimal conditions for obtaining the composite blended materials were selected: temperature, mixing mode, polymer concentrations, and drying conditions. The preliminary resuspension of PHB in chloroform made it possible to achieve rapid dissolution of the polymer in acid and avoid a significant decrease in the molecular weight of PHB. Further addition of chitosan solution to the mixture made it possible to obtain homogeneous composites. Films with different ratios of both polymers (1:1, 1:2, 1:4, 1:10, 1:20 for CTS and PHB, respectively) were formed by molding on the Teflon surface and drying. The characteristics of the obtained composites were studied using modern methods of investigation. The physical properties and surface parameters were studied for the films of different compositions. DSC, TGA, AFM and FTIR spectroscopy data showed that both polymers did not bind, but the addition of chitosan had an effect on the composite structure. Increasing the amount of chitosan reduced the degree of crystallinity from 59.4% for PHB to 24.6% for PHB/CTS (1:1) and slightly reduced the hydrophobicity by decreasing the contact wetting angle from 84.5° for PHB to 74.3° for PHB/CTS (1:1). The TGA method revealed that chitosan had an effect on the thermal decomposition of the composites. Since chitosan decomposes at higher temperatures, the composites also had greater resistance to thermal degradation. There was also a change in decomposition stage: PHB decomposed in one stage, while the composite with chitosan had a three-stage decomposition behavior. There was also a dependence on the content of chitosan in the composite - with an increase in the amount of chitosan the percentage of weight loss at the beginning and end of the temperature range increases and decreases in the middle. Atomic force microscopy of the films showed an increase in the RMS surface roughness of the composites compared to that of the homopolymers. Roughness values for PHB in acetic acid and for CTS averaged 21.8 nm and 13.3 nm, and for all composites exceeded 60 nm, indicating a more developed surface topography of the composites, which may further affect cell attachment to them. The morphology of the composite films with a high percentage of chitosan incorporated differed from that of each of its components, and was first characterized by the presence of elongated fibrous areas, which significantly increased surface roughness. The presence of chitosan was not visually detectable on the surface of most composites. The nanomechanical characteristics of the films (nanoindentation) were investigated by force volume mapping, with force curves analyzed to extract the Young's modulus value using the Hertz model. The Young's modulus in compression was higher for composite films than for homopolymers. The lowest Young's modulus was recorded for chitosan films, 1.36 GPa. However, no specific regularities related to the composition of the composite materials were identified. 3D-structures (scaffolds) were also obtained from composite mixtures by freezing and drying and their rheological properties were studied. All the samples were viscoelastic objects, and the values of conservation and loss moduli increased with decreasing amounts of chitosan in the composition of the samples. All the above-mentioned results together open up the prospects for the development of composite materials made of PHB and CTS with adjustable characteristics, but revealing the regularities associated with the influence of composition on its properties requires additional studies on the physicochemical properties, biocompatibility, and biodegradability.

Publications

1. Zhuikova Y.V., Zhuikov V.A., Varlamov V.P. Biocomposite Materials Based on Poly(3-hydroxybutyrate) and Chitosan: A Review Polymers, Том 14, № 24, № публикации 5549 (year - 2022) https://doi.org/10.3390/polym14245549