**1. Introduction**

The increase of life expectancy and the enhanced frequency of injuries and diseases are considered the most important causes for the escalating demand for dental and orthopedic devices. In this respect, the surface functionalization of implants with highly performant bioactive materials is currently of interest and necessary both for the prevention of failure and the prolongation of the bone implants' life. Thus, good examples are calcium phosphates (CaPs) and bioglasses [1–4], which are the most used bioceramic materials in medicine, in particular in orthopedics and dentistry [5,6], as coatings for various metallic implants [7]. From the first class, a special focus was put on hydroxyapatite (HA), Ca10(PO4)6(OH)2 [3,4,7]. Due to their role as sca ffolds for osteogenic di fferentiation [8], the ability to form strong bonds with the host bone tissues [9], and their excellent capacity to stimulate and accelerate the formation of new bone tissue around implants [10–12], HA ceramics are frequently utilized in bone grafting and dental devices as bone substitutes, either in their simple form, or in conjunction with other di fferent biomaterials [13,14]. One should note that, if for the healthy patients, the osseointegration rate takes place at a reasonable speed, in the case of older patients, or those presenting critical medical conditions from the systemic point of view (in particular HIV positive patients that reported an increase in early failures of dental implants [15,16]), this process could be improved by applying HA-based coatings on to the implants' surfaces.

Despite its excellent bone-regeneration properties, HA is brittle in bulk [17] and characterized by poor mechanical properties. Usually, an implant can be manufactured from Ti or its medical-grade alloys. In this respect, additive manufacturing (AM) is a technology that allows for cost-e ffective and rapid production of complex three dimensional (3D) metallic parts and is gaining nowadays increased attention in the field of personalized medicine [18,19]. In contrast to their excellent mechanical properties, Ti implants elicit low osseointegration rates. To overpass this shortcoming, HA can be applied as a coating onto the surface of Ti implants, to significantly improve the overall performances of the structures, by successfully combining the excellent bioactivity of the ceramic with the mechanical advantages of the metallic substrates [17,20,21]. To enhance these properties even more, HA doping with various concentrations of therapeutic ions is envisaged [4,7,22,23].

Currently, huge interest is focused on finding appropriate deposition techniques to modify the surface of metallic implants, by functionalization with coatings, proteins and/or drugs that could favor the enhancement of cellular adhesion, thus leading to the acceleration of the osseointegration time [24–27]. In the field of thin-film growth, pulsed laser deposition (PLD) has been established in the past 15–20 years as one of the most popular and e fficient methods for the deposition of a wide spectrum of materials, especially onto substrates with complex geometrical shapes, including implants [28]. Moreover, when CaPs of animal origin are involved, the PLD technique could be one of the most suitable choices to synthesize this type of coating. If the synthetic HA has a complex stoichiometry, with a large number of atoms and functional groups di fficult to be transferred in the form of thin layers by physical vapor deposition techniques, biological apatites become even more di fficult to transfer due to the supplemental presence of functional groups and substitutional ions, which further complicate their stoichiometry and structure [29]. Nevertheless, the PLD method is well-known for its ability to stoichiometrically transfer very complex molecules (due to the high ablation rate which causes all elements to evaporate at the same time [30]) and this advantage should place it among the main candidates for the successful transfer of such complex materials. Moreover, with the major drawback of this deposition technique (i.e., being capable of coating only small-area substrates, as the plasma plume diameter is restricted by the low spot size of the laser beam) being overcome by the implementation of laser scanning units in commercially, widely available PLD equipment, the path towards large-scale application is thus opened [31,32].

It is a documented fact that in vitro tests can play an important role in the evaluation of a material by providing useful information about its potential behavior in a biological environment. To better understand the complex processes that may occur in a living system and to provide the most accurate data for the complete confirmation of the performance of a biomaterial designed for clinical trials, in vivo tests in animal models are extremely important and should, therefore, follow thorough in vitro confirmation. Accordingly, after previous validation of both in vitro cytocompatibility [33,34] and antimicrobial activity [35] of biological HA (BHA) doped with lithium carbonate (further denoted as Li-C) and phosphate (further denoted as Li-P) coatings, a leap forward in our research was performed by investigating their preliminary bone bonding and bone bonding strength characteristics in vivo in an animal model. Thus, the aim of this preliminary study was both to (i) demonstrate superior bone–implant interactions in the case of PLD functionalized implants as compared to simple, uncoated ones (Ti controls), and (ii) verify the hypothesis that higher values of the extraction force are obtained in the case of implants extracted after longer periods of implantation time. It should be stressed that there are reports in the literature on the in vivo testing of CaPs (especially HA), but, to the best of our knowledge, the novelty of the current work resides both in the additive manufacturing (AM) technique used to fabricate the implantable medical devices, and the fact that this is the first preliminary study to address the in vivo mechanical behavior of these 3D printed implants functionalized by PLD with biological HA, doped with Li-C and Li-P coatings.

### **2. Materials and Methods**

### *2.1. Printing of Metallic Implants*

The laser melting deposition (LMD) technique was used for the manufacturing of three-dimensional (3D) metallic implants. A Ti6Al4V powder (with the particle size of <90 μm), further denoted as control, was used as precursor material. Implants were in "T"-shape (Figure 1a,b) and their dimensions are displayed in Figure 1a. It should be mentioned that, by using the LMD technique, the fabrication costs and manufacturing time of 3D metallic implants were significantly reduced.

**Figure 1.** (**a**) The technical drawing of the 3D metallic implant; (**b**) the 3D drawing of the implant; (**c**) the TruTops Cell® graphical software which generates the movement codes for the robotic arm.

First, incipient bulk prisms of (10 × 5 × 1) cm<sup>3</sup> were 3D printed using several experimental conditions, to find the optimal parameters in view of obtaining defect-free samples. Thus, by optimization of the laser and scanning parameters, compact structures without cracks or pores and with excellent compositional uniformity were obtained. The laser beam (supplied through an optical fiber) used for melting the powder

was generated by an Yb:YAG disk source (λ = 1030 nm, continuous wave), model TruDisk 3001 (Trumpf, Ditzingen, Germany). The surface focused spot size had a diameter of ~0.6 mm.

An automated powder feeder (Trumpf, Ditzingen, Germany), connected through hoses (having 6 mm diameters) to a nozzle with three flow channels, that was mounted on a robotic system TruLaser Robot 5020 (Trumpf, Ditzingen, Germany), made of a Kr30HA robot (Kuka, Augsburg, Germany), with six movement axes, was used for the powder delivery. The powder was sent to the robot through a delivery system provided with turntables. A three-beam particle flow nozzle was used and aligned so that the beams could be coherent in a single point, congruen<sup>t</sup> with the laser beam spot. The Ti implants were fabricated under Ar ambient.

The 2D technical drawing of the 3D metallic implants (Figure 1a) was generated in the graphical engineering software Solid Works® (Dassault Systems, Vélizy-Villacoublay, France), then translated to the 3D space (Figure 1b), and subsequently imported in TruTops Cell® (Trumpf, Ditzingen, Germany) (Figure 1c), a movement code generator for the robotic arm.

The design of the trajectory followed by the laser beam for structure growth proved to be essential to obtain samples that respect both the appearance and dimensions of the technical drawings. The separation distance of contours on the *z*-axis, established by tracing a calibration curve, was of 0.5 mm. All trajectories executed by the robotic arm were extremely important to obtain a homogeneous structure, free of discontinuities.

Three-dimensional metallic implants were fabricated onto a Ti substrate with a thickness of 10 mm, after which they were removed from it by a disk cutting machine model Brillant 200 (ATM, Mammelzen, Germany).

### *2.2. Pulsed Laser Deposition (PLD) Experiments*
