Synthetic polymers used as prosthetic materials induce significant clinical complications after the post oncologic intervention. Considering this, designing novel biomaterials able to overcome these disadvantages have become an important goal for the scientific community. The proposed project aims to develop new biocomposite systems with tailored antiadherent and antibacterial functions in the form of a membrane with multilayer structure (BIOMULTIPOL), intended for pelvic prosthetic meshes. The objectives of this project will be achieved through the use of polymers with improved biocompatibility: nanocellulose (NC), poly-L-lactic acid (PLLA) and poly(3-hydroxybutyrate) (PHB), these materials exhibiting an increased resistance to bacterial infection compared to non-biodegradable synthetic materials.

The project approach is new and is based on the association of different biocomposite layers, each having a well defined function, thus mimicking the natural structures. Another innovative aspect is related to the multiple role of NC, which enhances the mechanical properties and improves the biocompatibility of the BIOMULTIPOL membrane, eliminating the mesh-related complications. The proposed BIOMULTIPOL membrane will assure the needed mechanical support (Lm layer), antiadherent (La layer) and antibacterial (Lb layer) properties. Various highly hydrophobic compounds and different antibacterial agents will be screened for achieving the desired antiadherent and antibacterial properties.

BIOMULTIPOL membrane will consist of different biomaterials in the form of multiple distinct layers each performing a clearly defined function.

The specific objectives of the project are the following:

v    Development of new PLLA/PHB/NC biomaterials system with improved mechanical properties, antiadherent and antibacterial capabilities and controlled biocompatibility; Studying the correlations between composition, processing methodology (in meshes, fibers and films) and the properties (functions) of the system;

v    Correlations with the results of biocompatibility and cytotoxicity tests on the resulted biomaterials system (on both sides); Optimization of the multilayer membrane to provide the desired functions;

v    Technological conditions for the development of BIOMULTIPOL model and validation.

ü    The BIOMULTIPOL membrane will contribute to the improvement of the quality of life of patients because this multilayer membrane will reduce clinical complications in surgical oncology for the repair of pelvic floor;

ü    The project will have a big scientific impact due to both the complexity of the technological process of obtaining multilayered membranes and due to detailed mechanical and biological assessment (biocompatibility, antibacterial and biodegradability tests);

ü    BIOMULTIPOL membranes will also have an important impact in the medical field as they will be a new medical device based on the association of different layers of biocomposites, each with a well-defined function, mimicking natural structures.

ü    Social effects will consist in involving young researchers and PhD students together with experienced researchers in a cutting-edge research. Moreover, the development of such membranes will contribute to the creation of synergies between research institutes, industry and doctors.

Phase I

Title: Setting up the conditions for obtaining PLLA-PHB biomaterials for mechanical support (Lm) and complex characterization

Period: 02.05.2018-30.12.2018

Summary of the activity report

In this phase it was developed the first layer of the BIOMULTIPOL membrane, namely Lm layer, which has been designed as an outer layer capable of providing high mechanical strength.

In this respect, the biocompatible polymers proposed for the development of the Lm layer, polylactic acid and polyhydroxybutyrate were modified with several types of cellulosic nanofillers. In order to provide superior mechanical properties, the proposed compositional systems have been crosslinked in the presence of medical approved crosslinkers.

The obtained bionanocomposites have undergone a thorough characterization in order to assess their high-strength performance, thermal stability and the adhesion between components. By applying different melt processing techniques the Lm model was obtained in the form of filaments, films and meshes.

Phase II

Title: Designing the antiadherent (La) and antibacterial (Lb) layers; characterization of resulted biomaterials

Period: 30.12.2018 - 31.12.2019

Summary of the activity report

Within this phase three types of different polyhedral oligomeric silsesquioxanes (POSS) and two types of SiO₂, were used for the obtaining of the antiadherent layer (La). The morphological studies showed a good dispersion of both POSS and SiO₂ within the PLLA matrix, only few aggregates being noticed when using 7wt.% nanoparticles. The contact angle results confirmed the increased the hidrophobicity of resulted La biocomposites while TGA, DSC and DMTA analyses highlighting the increased thermal stability, crystallinity and thermo-mechanical properties. MTT and LDH assay were performed in order to assess the biocompatibility of La biocomposites. Heparin and bovin serum albumin were used in a polyester matrix in order to obtain biocomposites (Lb, Lb’) with antibacterial activity. Two approaches were employed for the obtaining of these biocomposites: modification of NC and direct mixing with polymeric matrix. FTIR results confirmed the presence of heparin and albumin in the NC structure and in structure of the Lb and Lb’ resulted biocomposites. The results obtained from the antibacterial tests indicated the biocomposite sample consisting of PLLA and cellulose grafted with heparin as having the best efficiency in reducing the colonies of Staphylococcus aureus and Escherichia coli. Thus, the antimicrobial activity of this sample contributed to the reduction of Staphylococcus aureus colonies by 14.22% and by 15.09% in the case of Escherichia coli, compared to the control sample and the sample containing albumin. Due to the satisfactory antibacterial effect as well as the higher thermal stability, this compositional system was selected as suitable to form the Lb layer in the BIOMULTIPOL membrane model. The multilayered membrane model was developed using the optimum formulations for the three layers. Compression molding was applied for the obtaining BIOMULTIPOL model. The obtained model showed good adhesion between layers and a satisfactory flexibility for the final application. Biodegradability tests on the BIOMULTIPOL model were performed in Phosphate Buffered Saline (PBS) for 208 days. TGA, FTIR and contact angle analysis were carried out on the samples after incubation in PBS and their results showed negligible biodegradability.

Phase III

Title: Technology for the production of BIOMULTIPOL membrane – model

Period: 31.12.2019 - 31.10.2020

Summary of the activity report

During this stage, it has been established the laboratory technology for the development of the BIOMULTIPOL model membrane and also the membrane model was validated. The validation of the membrane model involved a complex analysis of thermal stability and mechanical characteristics as well as morphological and biological properties.

The results of the biodegradability tests showed that the model membranes mentained there morphological properties and thermal stability thus being suitable for use as support membrane for post-oncological pelvic surgery. Moreover, in vitro biocompatibility and cell viability assays (MTT and LDH) indicated that the model membranes allowed the attachment of the tested cells (L929) in a percentage higher than 85%, as well as their lack of toxicity.

Also during this project phase, a patent application was prepared, the object of the invention being represented by the obtaining of polymeric biocomposites with antibacterial effect and a paper was published in Polymers.

Articles

1. Frone, A.N., Panaitescu, D.M., Chiulan, I., Gabor, A.R., Nicolae, C.A., Oprea, M., Gavrilescu, D., Puitel, D. Thermal and mechanical behavior of biodegradable polyester films containing cellulose nanofibers. J Therm Anal Calorim (2019) 138: 2387. https://doi.org/10.1007/s10973-019-08218-4

2. Frone, A.N., Batalu, D., Chiulan, I., Oprea, M., Gabor, A.R., Nicolae, C.-A., Raditoiu, V., Trusca, R., Panaitescu, D.M. Morpho-structural, thermal and mechanical properties of PLA/PHB/Cellulose biodegradable nanocomposites obtained by compression molding, extrusion, and 3D printing. (2020) Nanomaterials, 10 (1), art. no. 51. https://www.mdpi.com/2079-4991/10/1/51

3. Frone AN, Nicolae CA, Eremia MC, Tofan V, Ghiurea M, Chiulan I, Radu E, Damian CM, Panaitescu DM. Low Molecular Weight and Polymeric Modifiers as Toughening Agents in Poly(3-Hydroxybutyrate) Films. (2020) Polymers, 12 (11):2446. https://doi.org/10.3390/polym12112446

Conferences

1. Adriana N. Frone, Ioana Chiulan, Madalina Oprea, Alexandru S. Stoian, Denis M. Panaitescu, Augusta R. Gabor, Cristian A. Nicolae. Effect of different POSS structures on thermal and morphological properties of biodegradable polyester. The International Symposium "PRIOrities of CHEMistry for a sustainable development" PRIOCHEM XV-th Edition, ICECHIM - Bucharest – Romania October 30 - November 01, 2019, vol. 15/2019, ISSN 2504-3900
2. Adriana N. Frone,, Alexandru S. Stoian,Valentin Raditoiu, Cristian Andi Nicolae, Denis Mihaela Panaitescu, Augusta Raluca Gabor. Thermal and morphological characteristics of poly(l-lactic acid)/organic–inorganic hybrid nanocomposites. 5th Central and Eastern European Conference on Thermal Analysis and Calorimetry (CEEC-TAC5), 27-30 August 2019, Roma, Italy
3. A. N. Frone, D. Batalu, D. M. Panaitescu, I. Chiulan, A. R. Gabor, C. A. Nicolae. Filament extrusion and 3D printing of polyester based biocomposites. 7th International Conference on Biobased and Biodegradable Polymers (BIOPOL-2019), Stockholm (Sweden), 17-19 June 2019
4. Adriana Nicoleta Frone, D. M. Panaitescu, I. Chiulan, A. R. Gabor, C. A. Nicolae, M. Oprea, S. Sergiu, C.M. Damian. Effect of different cellulose nanofillers on the structural, thermal and mechanical properties of PLLA/PHB biocomposites. 2^nd International Conference on Advanced Structural and Functional Materials (ICAS 2019), 19 - 23, August  2019 Barcelona, Spain
5. Madalina Oprea, Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Raluca Augusta Gabor, Ioana Chiulan, Cristian Andi Nicolae, Sergiu Alexandru Stoian.The influence of silica based nanofillers on thermal and dynamic mechanical properties of polylactic acid. 21^st Romanian International Conference on Chemistry and Chemical Engineering, Constanta- Mamaia, ROMANIA - September 4 – 7, 2019
6. Adriana Nicoleta FRONE, Denis Mihaela PANAITESCU, Ioana CHIULAN, Raluca Augusta GABOR, Cristian Andi NICOLAE, Thermal behavior of biodegradable polyesters based membranes. 12th European Symposium on Thermal Analysis and Calorimetry, 27-30 August 2018, Brasov, Romania
7. Adriana Nicoleta FRONE, Denis Mihaela PANAITESCU, Raluca Augusta GABOR, Ioana CHIULAN, Cristian-Andi NICOLAE. PLA/PHB biomaterials modified by cellulose fibers, PRIOCHEM XIV – 2018.