*Article* **Biomimetic Hierarchical Structuring of PLA by Ultra-Short Laser Pulses for Processing of Tissue Engineered Matrices: Study of Cellular and Antibacterial Behavior**

**Albena Daskalova 1,\* , Liliya Angelova <sup>1</sup> , Emil Filipov <sup>1</sup> , Dante Aceti <sup>1</sup> , Rosica Mincheva <sup>2</sup> , Xavier Carrete <sup>2</sup> , Halima Kerdjoudj 3,4 , Marie Dubus 3,4 , Julie Chevrier 3,4, Anton Trifonov <sup>5</sup> and Ivan Buchvarov <sup>5</sup>**


**Abstract:** The influence of ultra-short laser modification on the surface morphology and possible chemical alteration of poly-lactic acid (PLA) matrix in respect to the optimization of cellular and antibacterial behavior were investigated in this study. Scanning electron microscopy (SEM) morphological examination of the processed PLA surface showed the formation of diverse hierarchical surface microstructures, generated by irradiation with a range of laser fluences (F) and scanning velocities (V) values. By controlling the laser parameters, diverse surface roughness can be achieved, thus influencing cellular dynamics. This surface feedback can be applied to finely tune and control diverse biomaterial surface properties like wettability, reflectivity, and biomimetics. The triggering of thermal effects, leading to the ejection of material with subsequent solidification and formation of raised rims and 3D-like hollow structures along the processed zones, demonstrated a direct correlation to the wettability of the PLA. A transition from superhydrophobic (θ > 150◦ ) to super hydrophilic (θ < 20◦ ) surfaces can be achieved by the creation of grooves with V = 0.6 mm/s, F = 1.7 J/cm<sup>2</sup> . The achieved hierarchical architecture affected morphology and thickness of the processed samples which were linked to the nature of ultra-short laser-material interaction effects, namely the precipitation of temperature distribution during material processing can be strongly minimized with ultrashort pulses leading to non-thermal and spatially localized effects that can facilitate volume ablation without collateral thermal damage The obtained modification zones were analyzed employing Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), Energy dispersive X-ray analysis (EDX), and optical profilometer. The modification of the PLA surface resulted in an increased roughness value for treatment with lower velocities (V = 0.6 mm/s). Thus, the substrate gains a 3D-like architecture and forms a natural matrix by microprocessing with V = 0.6 mm/s, F = 1.7 J/cm<sup>2</sup> , and V = 3.8 mm/s, F = 0.8 J/cm<sup>2</sup> . The tests performed with Mesenchymal stem cells (MSCs) demonstrated that the ultra-short laser surface modification altered the cell orientation and promoted cell growth. The topographical design was tested also for the effectiveness of bacterial attachment concerning chosen parameters for the creation of an array with defined geometrical patterns.

**Keywords:** cell matrices; Fs bioscaffolds structuring; ultra-short functionalization of cell matrices; tissue engineering; temporal scaffolds; PLA texturing

**Citation:** Daskalova, A.; Angelova, L.; Filipov, E.; Aceti, D.; Mincheva, R.; Carrete, X.; Kerdjoudj, H.; Dubus, M.; Chevrier, J.; Trifonov, A.; et al. Biomimetic Hierarchical Structuring of PLA by Ultra-Short Laser Pulses for Processing of Tissue Engineered Matrices: Study of Cellular and Antibacterial Behavior. *Polymers* **2021**, *13*, 2577. https://doi.org/10.3390/ polym13152577

Academic Editor: Evgenia G. Korzhikova-Vlakh

Received: 9 July 2021 Accepted: 1 August 2021 Published: 3 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

According to data from World Population Prospects: the 2019 Revision, (reference date: 01.07 2020), 24.2% of all the people in the world are over the age of 50 [1]. This fact determines the recrudescence of diseases such as bone fragilities, osteoarthritis, cartilage, and cardiovascular pathologies as one of the major problems of modern societies. Tissue engineering, as an innovative field in biotechnology, deals with the creation, regeneration, and improvement of the function of injured or missing tissues. The design of smart porous biocompatible and biodegradable scaffolds, in association with specific tissue cells and growth factors, plays a crucial role in the regeneration of tissues and recovery of their functionality [2]. The main purpose of the engineered scaffold is to create a biomimetic environment that stimulates cell adhesion, proliferation, and differentiation; as a result, the ingrowing cells are reorganized into new three-dimensional tissue [3]. During this gradual biodegradation of the scaffold, a new tissue with the desired properties is naturally formed. The selection of appropriate biocompatible source materials is crucial for successful scaffold fabrication [4,5]. Polylactic acid (PLA) has excellent mechanical properties (stability, toughness, elasticity, and strength), biocompatibility, biodegradability, ease of processing, and thermal stability [6–8]. The up-listed properties are the main features for the successful application of PLA-engineered constructs in the regeneration of tissues, undergoing mechanical stress under normal physiological conditions [3,9]. PLA is an aliphatic polyester with backbone formula (C3H4O2)<sup>n</sup> or [–C(CH3)HC(=O)O–]n, approved by the U.S. Food and Drug Administration (FDA) for different biomedical applications, [10] for which the synthetic biopolymer is in direct contact with biological fluids [6,11]. An important advantage of this polymer is that it is naturally resorbed as the physiological repair process takes over the main product of PLA metabolism in mammals is lactic acid [12]. It is incorporated in the carboxylic acid cycle and completely excreted by natural degradation pathways [13]. The biocompatibility of the scaffold generally refers to inflammatory response, as well as to the intrinsic micro and nanoscale structure of the biosurfaces. On the other hand, PLA is a hydrophobic polymer, which lowers its bioactivity, regarding the interaction of the scaffolds with cells, tissue, and fluids [14–22]. Additional surface functionalization is needed for the creation of effective bio-interfaces between the cells seeded and the PLA-based scaffold [23]. This could be achieved by different chemical methods, most of which, however, use organic compounds in order to control surface properties of the tissue scaffolds (by modifying surface chemistry), leaving residual toxic components in the cells environment and thus may lead to risks of cell toxicity and carcinogenicity [24]. As an alternative, the properties of various cell matrices could also be controlled by the means of different physical methods (by controlled removal, addition, or deformation of the biomaterial surface in order to increase its roughness) [25,26]. The most innovative and bio-friendly approach are the laser-based techniques due to their non-contact nature of interaction with the material and thus the absence of chemicals used for additional treatment of the created cell/tissue matrices. In the literature, there is scarce information concerning femtosecond laser modification of PLA. Irradiation with ultra-short laser pulses has been extensively studied in the last years, especially for the functionalization of tissue-engineered scaffolds, based on various biomaterials [27,28]. Femtosecond (Fs) laser-based processing is an alternative approach, characterized by extremely high processing accuracy and the absence of thermal deformations on the processed material due to its extremely high-peak power and pulse duration which is shorter than material thermal relaxation time. Ultra-short pulses lead to non-thermal and spatially localized effects, a result of which volume ablation occurs without collateral thermal damage to the surrounding zones. The effects of temperature distribution during the interaction with extremely short pulses is strongly minimized. The creation of micro and nanostructures on the surface of the biomaterial can strongly affect cell behavior, as diverse surface roughness (in macro, micro, and nanoscale) can be achieved by controlling the applied laser parameters [29]. Such surface feedback can be used to finely tune and control diverse properties of the processed material like wettability and biomimetics. Wettability is an important factor in cell adhesion, and the fs laser surface modification

has the ability to aid in tuning it in respect to the effect of interest—namely achieving cell adhesive/antiadhesive scaffold surfaces, needed for diverse tissue regenerative applications [30–33] or even the creation of enhanced antibacterial cell surface environment [34]. Yada and Terakawa even achieved the generation of laser-induced periodic surface structures (LIPSS) on a highly biocompatible poly-L-lactic acid scaffold by femtosecond laser processing [35]. These structures are examined for cell behavior control. In the current study, PLA polymer surfaces were modified by Ti: sapphire femtosecond laser at diverse laser energies and scanning velocities were applied. Different surface treatments were used to obtain structures with hierarchical geometries for future preparation and design of porous PLA-based cellularized scaffolds and bio-interfaces between the tissues of the recipient and the foreign implant. Laser microstructured PLA scaffolds were investigated by SEM, EDX, XPS, optical profilometer studies, wettability, and FTIR. The thickness of the samples, before and after ultra-fast laser modification, was compared. In vitro degradation tests and preliminary cellular studies were accomplished. Mesenchymal stem cells from Wharton Jelly (MSCs) were cultured on PLA scaffolds for evaluating the effect of the patterned polymer surface on cell proliferation and cell morphology from day 1 to 14, compared to control PLA matrix. The adhesion of *Staphylococcus aureus* was also evaluated for the potential antibacterial effect which laser treatment could achieve. Thus, the aim of this examination will be directed towards achievement of conditions, associated with regime above modification threshold, and which gives a feedback concerning to which limit the PLA material could be processed without triggering chemical state alterations. This would potentially lead to the optimization of laser patterning conditions in order to achieve desired improved morphologies for cellular adhesion and bacterial rejection. The results obtained demonstrate that the precise control of the laser parameters applied to the PLA scaffolds could generate bioactive surfaces by means of the nondestructive Fs laser modification technique.
