Overview of the Results Obtained (Summary of Progress in Phases I-III)

During the first phase of project execution, various topographies influencing the type of cells in contact with the breast implant were designed to obtain molds, implicitly matrices for the preliminary production of textured surfaces through replication in PDMS. The characteristics of the matrices and corresponding PDMS replicas were studied through SEM, AFM, profilometry, and preliminary biological analyses. Geometries at the nano/micro-scale of matrix structures were designed and obtained. Using grayscale masks and an excimer laser, molds with defined shapes in polycarbonate were obtained. Preliminary replication stages resulted in micro and nanostructured surfaces in PDMS. Replicated surface characterization and preliminary in vitro testing provided initial indications of topographies beneficial for breast implants.

In the second phase, new nano-microstructured PDMS substrates were obtained and characterized through simple replication, along with micro and macro-textured structures using the salt leaching technique with crystals of varying sizes (from 20 mm to 100 mm). These multidimensional PDMS surfaces, replicated with or without the use of salt crystals and their melting (salt loss), were characterized through SEM, AFM, contact angle, and surface energy. Wear and friction studies of PDMS substrates revealed that those with the most significant defects, including mass loss after friction and immersion testing in basic and acidic pH solutions, were obtained using the salt loss technique.

In the third phase, new complex functionalized surfaces were obtained and characterized through the MAPLE method with zwitterionic polymers, antifibrotic, and anti-inflammatory compounds for applications in improving coatings of breast implant capsules. Multidimensional and functionalized surface analysis was correlated with the biological response of the obtained biointerfaces to choose optimal surfaces. In vitro biological studies were conducted on different textured surfaces using macrophage cells as an experimental model for inflammation. Regardless of surface topography—linear, honeycomb, with or without salt-based PDMS materials—facilitated different macrophage cell adhesion. For all types of texturing, the morphological appearance of macrophages adapted to the topography of PDMS material surfaces. Inflammatory response, specifically TNFa cytokine secretion, was quantified through ELISA tests, suggesting optimal structures were linear and hexagonal with depths of up to 5 microns. Interaction with PDMS functionalized with star and honeycomb topography (depth of 5µm), covering with superhydrophilic pMPC material and containing Lfcin-PFD, significantly reduced cell proliferation and adhesion, as well as the ability to induce a pro-inflammatory response. Additionally, fibroblast interaction with these types of structures inhibited collagen fiber alignment, directly impacting fibrosis development. The antibacterial capacity of optimized substrates was evaluated on both Gram-positive and Gram-negative microorganism lines. Both non-functionalized and pMPC film-functionalized structures showed reduced initial microorganism adhesion. However, in the long term, interrupted linear structures proved effective in inhibiting biofilm formation (tested at intervals of 1, 2, 9, and 21 days).

Results Recorded During the Project Implementation Period

The project results were valorized and disseminated nationally and internationally through articles (4 published, 2 submitted for evaluation in ISI journals), patents (3), invited presentations (3), oral presentations (4), posters, as well as participation in International Invention Salons (4 international awards: 1 gold medal Euroinvent, 2 gold medals PROINVENT, and Best Student Award at the EMRS conference in France for Dr. A. Bonciu). All reported results from funded activities mentioned the funder's name and the contract number.

The project's website was continually updated in both English and Romanian (https://ancabonciuub.wixsite.com/breaslife/).

 

 

 

Phase 1: "Design and production of masks, molds and preliminary production of textured surfaces by replication in PDMS; Study of the characteristics of molds and corresponding replicas in PDMS obtained by SEM, AFM, profilometry and preliminary biological analysis."

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The results that are being published will be included in the web page after the articles are published.

 

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Stage summary:

      During the first stage of the project, various topographies were designed that can influence the type of cells that come into contact with the breast implant, in order to obtain masks, implicitly molds for the preliminary production of textured surfaces by replication in PDMS. The study of the characteristics of the molds and of the corresponding replicas in PDMS obtained was done by SEM analyzes, AFM, profilometry and preliminary biological analyzes.

      Micrometric scale geometries of the mold structure architecture were designed and obtained. Using gray level masks and an excimer laser, molds with defined shapes in polycarbonate were obtained.

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                                                           Diagram of the laser micro-processing system of polycarbonate surfaces

 

            Obtaining micro and nanostructured surfaces in PDMS was done by replication-preliminary stage. The study of the characteristics of the molds and of the corresponding replicas in PDMS obtained was done by SEM analyzes, AFM, contact angle, surface energy, profilometry and were correlated, following preliminary biological analyzes with the cellular response.

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    The characterization of the replicated surfaces and the preliminary in vitro testing of the replicated surfaces gave the first indications on the types of topographies that can have a beneficial effect for breast implants, by testing with fibroblasts and macrophages. PDMS-based materials, regardless of surface topography - unstructured and structured are characterized by the fact that low adhesion of dermal fibroblasts is in the form of round cell agglomerations, suggesting a preference for cell-cell interaction over interaction with the surface of materials.

    Macrophage cells have a different behavior from fibroblasts, with a morphological appearance it is adapted to the surface topography of PDMS-based materials. It should be noted that the morphological phenotype of the adhered cells is not the one corresponding to macrophages stimulated with bacterial endotoxins (M1), which indicates the absence of an inflammatory potential.

 

 

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                                                        Cellular response to control surfaces and textured PDMS and PDMS.

 

The project website was also developed, and the dissemination included 1 patent application, 1 ISI article in a journal located in the red zone (In Vitro Effect of Replicated Porous Polymeric Nano-MicroStructured Biointerfaces Characteristics on Macrophages Behavior, NL Dumitrescu et al, Nanomaterials, Volume 11 Issue 8 10.3390 / nano11081913) and 5 conference presentations (a guest lecture, 3 oral presentations and a poster).

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The dissemination included:

• 1 patent application : Polymer films with zwitterionic characteristics obtained by laser evaporation for applications in the biomedical field -Anca Bonciu, Nicoleta Dumitrescu, Simona Nistorescu, Valentina Marascu, Laurentiu Rusen, Gratiela Gradisteanu si Valentina Dinca

• 1 ISI published article : In Vitro Effect of Replicated Porous Polymeric Nano-MicroStructured Biointerfaces Characteristics on Macrophages Behavior, Luminita Nicoleta Dumitrescu, Madalina Icriverzi, Anca Bonciu, Anca Roșeanu, Antoniu Moldovan, Valentina Dinca, Nanomaterials, Volume 11  Issue 8  10.3390/nano11081913

• 5  conference participations (1 invited lecture, 3 oral presentations and 1 poster presentation):

ICPAM 13, Sant Feliu de Guixol, Spain, 23sept-1 oct 2021-Invited presentation-V. Dinca: On the road to bioactive and multifunctional biointerfaces for implant research: a laser-based approach concept

HPLA 2021, 13 - 15 April 2021, online-Oral presentation  V.Dinca: Mesenchymal Stem Cells Interaction with Hierarchical Textured Surfaces Obtained by Laser Processing

Sesiunea de Comunicări Științifice a Studenților Facultății de Biologie – 2021, 28 mai, 2021, Modificări ale morfologiei celulare în urma interacției cu microtopografii de PDMS, Simona Stroescu (Nistorescu), Cristina Staicu, Anca Bonciu, Iuliana Urzică, Simona Brajnicov, Dincă Valentina, Anișoara Cîmpean

            EMRS Spring 2021 31Mai-4Iunie, online-Oral presentation: Pyramidal shaped ceria nano-biointerfaces for early bone cell response, A.F. Bonciu, S. Orobeti, L. E. Sima, M. Icriverzi, V. Dinca,, M. Filipescu, A. Moldovan, A.Popescu and M. Dinescu

EMRS Spring 2021 31Mai-4Iunie, online-Poster presentation : Influence of laser-designed microstructures density on interfaces characteristics and on preliminary responses of cells-Anca Bonciu, Alixandra Wagner, Nicoleta Dumitrescu, Valentina Marascu, Antoniu Moldovan, Cerasela Zoica Dinu, Valentina Dinca

 

International prizes: YOUNG SCIENTIST AWARD - ANCA BONCIU, Symposium H

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Phase NO.2 - The design, obtaining and characterization of new nano, micro and macro textured surfaces for applications in obtaining improved coatings of breast implant capsules. Study of the development and characterization of multidimensional surfaces in PDMS by replication and/or replication combined with the technique of using salt microcrystals (loss salt). Advanced analysis of the biological response of the obtained biointerfaces.

(January - December 2022)

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Stage Summary:

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    In the second stage, new nano-micro structured PDMS substrates were obtained and characterized by simple replication, and micro and macrotextured structures by combination with the salt loss technique, using salt crystals with different grain sizes (from 20 mm to at 100 mm). These multidimensional surfaces in PDMS by replication, with or without the combination with the technique of using salt microcrystals and their melting (salt loss) were characterized by SEM, AFM, contact angle and surface energy. The wear and friction studies of the PDMS substrates showed that the substrates that present the most significant defects, including mass loss, after testing by friction and immersion in solutions with basic and acidic pH are those obtained by the salt loss technique.

    The in vitro biological study was carried out on different textured surfaces using, as in the previous stage, macrophage cells as an experimental model for inflammation. It was observed that materials based on PDMS, regardless of the surface topography - unstructured and structured linear, honeycomb, with or without salt, favor the adhesion of macrophage cells in a different way. For all types of texturing, the morphological appearance of the macrophages is adapted to the type of topography of the surfaces of the PDMS-based materials. Cells spread between topographic features, adjusting the edge of the cell body to outline the side walls of the structures. At the same time, depending on the size of the structures and the distance between the structures, the cells make the connection between the topographic features; acquiring an elongated morphology as in the case of linear structures. Hexagonal surfaces of put type impose a restriction of the cells mainly inside the cavities. Unlike the other types of texturing, the micro- and macro-structured ones using salt microcrystals induce a much weaker response of the cells, as they generally adapt in the cavities created by the salt microcrystals.

For the collagen fiber formation studies, human dermal fibroblasts (CCD 1070SK) were used, and cell adhesion, as well as the formation of these collagen fibers, were investigated by SEM after a period of 7 days, being observed collagen fibers especially on the surfaces used as Control, but not the ones textured in PDMS. The inflammatory response, namely the secretion of TNFα cytokines, was quantified by ELISA tests, suggesting that linear and hexagonal structures with a maximum depth of 5 microns are optimal.

    The evaluation of the ability of the antibacterial capacity of the optimized substrates was carried out on 3 lines of microorganisms (E. Coli, S. Aureus and C. Albicans). Structured surfaces with high roughness and topographical structural units larger than 5 microns do not effectively inhibit bacteria, which led to the need to expand microbiological analyzes on other types of topographies. Multidimensional linear structures were proposed in interrupted steps with dimensions of the order of microorganisms that could segregate colonies and reduce the adhesion of microorganisms. The microbiological tests, as well as those on macrophage cells, indicated that, in addition to the linear radial and honeycomb structures found to be optimal, a new direction of interest is given by linear multidimensional structures.

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    Thus, we can say that the objectives of this stage were fully met, and the deliverables were achieved. The project web page was updated, the dissemination included 1 patent application (New microstructured polymer surfaces used as biointerfaces of silicone capsules in breast implants. (A 16704 from 25.11.2022), 1 ISI article published and one in the process of publication, 4 participation in conferences (2 invited lectures and 2 posters) and an international award (Euroinvent gold medal).