Citation: Touya, N.; Al-Bourgol, S.; Désigaux, T.; Kérourédan, O.; Gemini, L.; Kling, R.; Devillard, R. Bone Laser Patterning to Decipher Cell Organization. Bioengineering 2023, 10, 155. https://doi.org/10.3390/ bioengineering10020155 Academic Editors: Min Lee and Elena A. Jones Received: 12 January 2023 Revised: 18 January 2023 Accepted: 19 January 2023 Published: 24 January 2023 Copyright: © 2023 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/). bioengineering Article Bone Laser Patterning to Decipher Cell Organization Nicolas Touya 1, * , Samy Al-Bourgol 2 , ThéoDésigaux 1 , Olivia Kérourédan 1,3,4 , Laura Gemini 2 , Rainer Kling 2 and Raphaël Devillard 1,3,4 1 Univ. Bordeaux, INSERM, BIOTIS, U1026, F-33000 Bordeaux, France 2 ALPhANOV, Rue François Mitterrand, 33400 Talence, France 3 Faculty of Dentistry, University of Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France 4 Pôle de Médecine et Chirurgie Bucco-Dentaire, CHU de Bordeaux, Place Amélie Raba Léon, 33076 Bordeaux, France * Correspondence: nicolas.touya@u-bordeaux.fr Abstract: The laser patterning of implant materials for bone tissue engineering purposes has proven to be a promising technique for controlling cell properties such as adhesion or differentiation, resulting in enhanced osteointegration. However, the possibility of patterning the bone tissue side interface to generate microstructure effects has never been investigated. In the present study, three different laser-generated patterns were machined on the bone surface with the aim of identifying the best surface morphology compatible with osteogenic-related cell recolonization. The laser-patterned bone tissue was characterized by scanning electron microscopy and confocal microscopy in order to obtain a comprehensive picture of the bone surface morphology. The cortical bone patterning impact on cell compatibility and cytoskeleton rearrangement on the patterned surfaces was assessed using Stromal Cells from the Apical Papilla (SCAPs). The results indicated that laser machining had no detrimental effect on consecutively seeded cell metabolism. Orientation assays revealed that patterns with larger hatch distances were correlated with higher cell cytoskeletal conformation to the laser-machined patterns. To the best of our knowledge, this study is the first to consider and evaluate bone as a biological interface that can be engineered for improvement. Further investigations should focus on the in vivo implications of this direct patterning. Keywords: tissue engineering; bone; laser; femtosecond; patterning; direct 1. Introduction In recent years, surface modification has become a critical aspect of tissue engineer- ing [1,2] to improve the osteointegration of implants and influence cell fate [3]. Investi- gations have been conducted on various synthetic materials designed to substitute bone tissue as prostheses, such as ceramics [4–6], metals [7–10], and polymers [11]. An in vivo study outlined the strategic importance of modifying the topography of implants with lasers by showing a stronger bone-to-implant bond [12]. Femtosecond (fs) lasers allow for the biological response tailoring of surface morphology by controlling the surface pattern geometry down to a 100 s-nm scale [13–15] thanks to their unique characteristics. Indeed, Ultra Short Pulses (USP) allow for a quasi-non-thermal interaction with materials and the generation of interference-based physical phenomena [16]. For example, Carvalho et al. [13] reported the increased metabolic activity of MC3T3 (osteoblastic-related cells) cultured on Alumina-Toughened Zirconia (ATZ), where grid-structures or groove-like structures were generated via fs laser machining. Gnilitskyi et al. [14] demonstrated the higher ratio of HDFa (fibroblast cells) growth on both laser-nanostructured titanium alloy and zirconium, showing the predominant role of surface morphology over the material type for these types of biological responses. A similar result was achieved by Lee et al. [15], where a variation in the Saos-2 (osteoblastic-related cells) response and surface biocompatibility was observed down to a few 100 s-nm pattern size differences. Bioengineering 2023, 10, 155. https://doi.org/10.3390/bioengineering10020155 https://www.mdpi.com/journal/bioengineering