Laser-Based Hybrid Manufacturing of Endosseous Implants

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Bio-interactions and Biocompatibility

Laser-based hybrid manufacturing of endosseous implants: optimized titanium surfaces for enhancing osteogenic differentiation of human mesenchymal stem cells Guenaelle Bouet, Frederic Cabanettes, Guillaume Bidron, Alain Guignandon, Sylvie Peyroche, Philippe Bertrand, Laurence Vico, and Virginie Dumas ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00769 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 16, 2019

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ACS Biomaterials Science & Engineering

Laser-based hybrid manufacturing of endosseous implants: optimized titanium surfaces for enhancing osteogenic differentiation of human mesenchymal stem cells

Guénaëlle Bouet1, Frédéric Cabanettes1, Guillaume Bidron2, Alain Guignandon3, Sylvie Peyroche3, Philippe Bertrand1, Laurence Vico3, Virginie Dumas*1 1. University of Lyon, Ecole Nationale d'Ingénieurs de Saint-Etienne, Laboratoire de Tribologie et Dynamique des Systèmes, UMR 5513 CNRS, 58, rue Jean Parot 42023 Saint-Etienne, France 2. GIE Manutech-USD (Ultrafast Surface Design), 20 Rue Professeur Benoît Lauras 42000 SaintEtienne, France 3. University of Lyon, INSERM U1059-SAINBIOSE, 42270 Saint-Priest-en-Jarez, France

* Corresponding author: [email protected]

Abstract Additive manufacturing (AM) is becoming increasingly important in the orthopedic and dental sectors thanks to two major advantages: the possibility of custom manufacturing and the integration of complex structures. However, at smaller scales, surface conditions of AM products are not mastered. Numerous non-fused powder particles give rise to roughness values greater than 10μm (Sa), thus limiting biomedical applications since the surface roughness of, e.g. metal implants plays a major role in the quality and rate of osseointegration. In this study, an innovative hybrid machine combining AM and a femtosecond laser (FS) was used to obtain Ti6Al4V parts with biofunctional surfaces. During the manufacturing process, the FS laser beam "neatly" ablates the surface, leaving in its path nanostructures created by the laser/matter interaction. This step decreases the Sa from 11μm to 4μm and increases the surface wettability.

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The behavior of human mesenchymal stem cells was evaluated on these new AM+FS surfaces and compared with AM surfaces and also polished surfaces. The number of cells atttached 24hours after plating is equivalent on all surfaces, but cell spreading is higher on AM+FS surfaces compared with their AM counterparts. In the longer term (days 7 and 14), fibronectin and collagen synthesis increase on AM+FS surfaces as opposed to AM alone. Alkaline phosphatase activity, osteocalcin production and mineralization, markers of osteogenic differentiation, are significantly lower on raw AM surfaces, whereas on the AM+FS specimens they display a level equivalent to that on the polished surface. Overall, these results indicate that using an FS laser beam during the fabrication of AM parts optimizes surface morphology to favor osteoblastic differentiation. This new hybrid machine could make it possible to produce AM implants with functional surfaces directly at the end of AM, thereby limiting their post treatments.

Keywords: additive manufacturing, femtosecond laser, titanium, nanotexturing, biofunctional surfaces, osteogenic differentiation


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1. Introduction Additive manufacturing (AM) is attractive for fabricating biomedical implants as it allows the creation of customized parts with complex shapes. Selective Laser Melting (SLM), also known as Laser Powder Bed Fusion (LPBF), is a metal-based AM technique that can produce 3D parts layer by layer1. In biomedical cases, this process can be used to manufacture implants whose shape will be customized based on CT (computed tomography) images of the patient’s anatomy2, thus opening an interesting field in dental or orthopedic applications as well as bucomaxillofacial reconstructions3-6. The technology allows manufacturing of the general shape of an implant and also a 3D internal structure controlled by computer-aided design (CAD). However, biomedical devices developed to replace and interact with the bone tissue must be properly designed down to both micro- and nano- scales in order to ensure suitable osteointegration. Micro- and nano-scales are all very important for early steps of osteointegration7. In this respect, a major limitation of the SLM process for biomedical application is the poor surface quality of fabricated parts8. The technique leaves a detrimental microroughness on the surface, with an Sa>10µm due only to partial fusion of the powder particles during manufacturing9. It is known that the biological performances of metallic implants is significantly determined by the micro-roughness of their surface1011.

Cells recognize and react to the micro- and nano-topographies of the implant

surface, which affect adhesion, proliferation, and differentiation, thus influencing osseointegration12-16. According to Tsukanaka and co-workers17, a moderately rough titanium implant surface (Ra from 1µm to 5µm) and in particular a multiscale topography combining nano- and micrometric roughnesses accelerates osteoblastic differentiation18-19. Thus, the nanostructural topography of a biomaterial has the


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capacity to direct the differentiation of mesenchymal stem cells toward an osteogenic lineage20-22. Conventional methods of post surface treatment of Ti6AV4V implants can be used on AM generated material. The chemical methods include acid etching, oxidation, anodization etc23 and the physical methods involve mechanical treatments such as machining, grinding and sandblasting24. However, such post-treatments have drawbacks in terms of accessibility of complex and/or inner (pore) surfaces and of chemical risks. For this study, a hybrid equipment was designed and built to associate a femtosecond laser source (FS) with the SLM laser source in order to improve the surface morphology and topography of the implant fabricated by SLM. This hybrid 3D manufacturing machine brings together both additive and subtractive functions by combining two lasers for the finishing and functionalization of 3D surfaces. The goal is to obtain a bio-functional surface directly at the output of 3D printing and thus reduce the otherwise required post-processing. The FS laser surface functionalization is applied after every layer deposited during the printing process. The FS laser acts as a substractive laser and removes the partly melted powder particles attached to the surface of each successive layer, therefore the surfaces manufactured with this new equipment have lower micro-roughnesses. Interestingly, a nanostructuration covers the majority of the surface that is the product of the FS laser/matter interaction, a consequence of the process. Irradiation of material surface with linearly polarized ultrashort laser pulses can generate nanostructures known as “Laser-Induced Periodic Surface Structures” (LIPSS)25, on numerous types of materials, including common implant materials such as titanium2627.

Previous study has shown that LIPSS promote osteoblastic differentiation28-30

while decreasing adipogenic differentiation28.


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This study describes the testing of the biological performances of the new surfaces obtained by the hybrid manufacturing process. In-vitro experiments were performed to evaluate the response of human mesenchymal stem cells (hMSCs) on these new designed titanium surfaces. We evaluated the morphology and spreading of hMSCs, and also their long-term activity when it comes to matrix deposition and osteoblast differentiation on surface manufactured by this combination of additive and subtractive laser-based technologies.

2. Materials and methods 2.1. Sample preparation Titanium alloy (Ti6Al4V) substrates of biomedical ASTM Grade 5, conventionally manufactured by casting (10×10×1 mm3, mirror polished to Ra

Laser-Based Hybrid Manufacturing of Endosseous Implants

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