**4. Discussion**

The rabbit's bones manifest faster changes and turnover in comparison to larger animal models [47]. Taking into consideration the difficulty to extrapolate the results obtained on rabbit bones to the human ones, the current study represents a screening for implant design and a validation of the used biomaterials, before their testing on larger animal models.

Due to its attractive biological properties and resemblance to the mineral part of the bone, HA currently represents one of the most used CaPs. To overcome the drawback that synthetic HA does not completely match the chemical composition of bone mineral component [48], continuous efforts have been dedicated to find alternative, low-cost methods to produce HA, such as obtaining it from sustainable, biological (BioHA) resources (biogenic, mammalian, and/or natural fish bones). One should note that BioHA is a carbonated, non-stoichiometric Ca-deficient material, which is different from synthetic HA in terms of composition, stoichiometry, crystal size/morphology, crystallinity degree, degradation rate, and overall biological performance [29]. Another important aspect of the growing interest of using BioHA materials is related to the presence of trace elements and functional groups, which modify the chemical formula of the natural HA in bone. While the chemically-synthesized HA lacks these elements, mammalian bones contain a higher source of ions and trace elements [49–51], with Na<sup>+</sup> and Mg<sup>2</sup>+ as the most frequently found ones [29]. One notes that, the presence of these ions alongside HA plays an important role in the development of teeth and bone, whereas their absence could cause fragility or bone loss [6].

The three main steps for bone formation are (i) cellular osteogenic differentiation, (ii) matrix maturation and (iii) matrix mineralization. The initial adhesion of cells to an implant represents a key parameter for their proliferation and differentiation onto the surface of a biomaterial. The surface functionalization of implants by CaP coatings can ensure new bone formation, which might occur rapidly along the entire surface of the coating, as compared to the simple Ti implant (control) [52]. In this respect, in our previous studies [33,34], the effect of lithium addition (Li2CO3 and/or Li3PO4) in BHA coatings synthesized by PLD was thoroughly examined from the physical-chemical, mechanical and cytocompatibility points of view. In comparison to bare Ti implants and simple (undoped) BHA coatings, Li-C and Li-P structures elicited improved wettability properties, which could further induce improved bone regeneration [33]. These characteristics seemed to have a beneficial influence on the overall cytotoxicity of the materials, the proliferation of human mesenchymal stem cells (hMSC) reaching its highest values in the case of Li-C coatings, followed by Li-P and simple (undoped) BHA ones. After 72 h, the cellular viability of hMSC was superior for BHA-doped structures (Li-P and Li-C) as compared to simple (undoped) BHA ones. One should note that, the bonding strength values were two times (in the case of Li-P coatings) and up to more than three times (in the case of Li-C coatings) higher than the threshold (>15 MPa) imposed by the ISO standard regulating the load-bearing implant coatings [33]. The evaluation of simple (undoped) and doped (Li-P) BHA coatings to induce osteoblast (OB) cells di fferentiation was also performed [34]. The level of mineralization in the case of Li-P coatings was found to be higher and significantly higher as compared to simple (undoped) BHA ones and bare Ti, respectively. In addition, when referring to the in situ level of alkaline phosphatase (which is an early marker of osteogenic di fferentiation), in hMSC and di fferentiated OB cells, the slightly improved results in the case of doped BHA (Li-P) structures in comparison to simple (undoped) BHA ones, could be explained by the characteristics of the coatings induced by the lithium doping agen<sup>t</sup> (low roughness values and a hydrophilic behavior) [34]. Moreover, the comparative morphological investigations evidenced the presence of numerous nodules of mineralization originating from OB cells grown on the surface of Li-P coatings, as well as a network-like organization of OBs interconnected into the mineralized extracellular matrix. Therefore, it was suggested that this network of OBs could constitute the premise forin vitro early di fferentiation of cells and, consequently, for an improved osseointegration in vivo [34]. Analyzing the overall demonstrated improved characteristics of lithium-doped BHA structures in comparison to bare Ti implants and simple (undoped) BHA coatings, it was concluded that the incorporation of lithium might prove to be a delivery vehicle for bioactive agents to promote and even accelerate osseointegration in close relation with an improved anchorage of bone metallic implants. It should be emphasized here that, the current EU strategies applied for the optimization of in vivo experiments indicate that the maximum amount of relevant, high-quality data should be generated strictly in accordance with the 3 *R*s philosophy, i.e., reduction, refinement and replacement of animal use, as defined by Russell and Burch in 1959. Taking into consideration all these aspects, along with the fact that the aim of this pilot study was not to investigate the lithium e ffectiveness in in vivo experiments, but to demonstrate the superiority of lithium-doped BHA coatings over commercial Ti implants (which represent the "gold standard" for implantological applications), the use of simple (undoped) BHA coatings as a second control (next to bare Ti implants) was not considered necessary at this stage of research.

We have shown in the current study that, after implantation of the 3D printed coated and uncoated Ti implants, the bone in the vicinity of the devices started to react to the foreign body. In all cases, between 4 and 9 weeks the bone density increased. There was an obvious di fference between the uncoated (control) 3D Ti implants and those functionalized with Li-C and Li-P coatings, in terms of bone density. The coated implants were always surrounded by a denser bone as compared to the uncoated ones, after 4 and 9 weeks, respectively. The denser bone around Li-C and Li-P coatings was expected, as the synthesized structures are based on natural HA, which is very similar to the mineral part of the bone. The material is biomimetic and bioactive, shielding the metallic implant and further stimulating the bone growth.

Apparently, between the Li-C and Li-P coated implants there were no obvious di fferences in terms of bone density. Therefore, to elucidate which of the two type of coatings could be more beneficial for the bone growth, in vivo mechanical tests were performed. It should be noted that, the optimal functioning of an endosseous implant depends on the mechanical stability of the biomaterial, which can be evaluated by extraction tests. To obtain information regarding the force that occurs between the bone tissue and implanted materials, various experimental study models have been developed, each of these approaches having its own particularities [44,47,53]. Most animal models currently used to study the retention of implants in bone are influenced by shear forces introduced during the retention tests. Push-out and/or torsion tests are generally used to analyze these forces. Push-out tests have some limitations regarding the evaluation of the bone–implant force. Therefore, the implant's shape, the degree of surface roughness and the good alignment between the implant clamping system and the pushing device can significantly influence test results. The use of the torsion test has the theoretical advantage of achieving an equal distribution of the force field at the bone–implant interface. On the other hand, these experiments are far more complicated and time-consuming as compared to the push-out ones. At the same time, the torsion test does not provide a clear distinction between the chemical bone–implant interaction and the mechanical retention because of the implant's surface roughness. Therefore, some studies, including the current one, have been concentrated on tensile strength measurements which, very important, can be influenced only by the chemical links between the implant and the bone [54–56]. Therefore, it is considered that by tensile tests the strength of the chemical bond can be measured directly. Thus, after 4 weeks of implantation, the value of the detachment force was higher in the case of 3D Ti implants functionalized with Li-C coatings in comparison to uncoated (control) ones. For the case of Li-P structures, this trend was similar, but there were particular instances when the results for coated and uncoated implants were very similar. The trend of higher detachment force was maintained for the case of 3D Ti implants functionalized with Li-P coatings as compared to the uncoated implants, however the e ffect seemed to be attenuated in respect to the Li-C coatings. After 9 weeks of implantation, it was observed that the detachment force was clearly superior (~1.3 times) for the 3D Ti implants functionalized with Li-C coatings as compared to the ones functionalized with Li-P (106.6 ± 2.9 N vs. 83.7 ± 2.1 N). A possible explanation for the higher values of the bone strength in the first case (Li-C coatings), could be connected to an increased crystallinity of the structures [57]. In this respect, it was shown that the Li-C coatings displayed a higher degree of crystallinity as compared to Li-P ones [33].

In the case of biological HA materials, as compared to synthetic HA, the osseoconduction rate is faster because the latter (i) has a higher solubility (which increases if its chemical structure contains carbonate groups), and (ii) contains Mg<sup>2</sup>+ and Na<sup>+</sup> ions, which are two elements associated to bone remodeling [58]. This might represent one of the possible mechanisms through which the Li-C and Li-P coatings can improve the process of osseoconduction. One should therefore emphasize on the increased values of the detachment force obtained at 9 weeks, which demonstrate the capacity for long-term attachment to bone of our functionalized 3D metallic implants.

Usually, lithium is incorporated into CaPs in form of Li2O [59,60]. In the current study, we selected to mix it with HA in form of Li2CO3 and Li3PO4. The reason was that in an initial parametric study (data not shown here), Li2O mixed with natural origin HA displayed inferior results in vitro as compared to the Li2CO3 and Li3PO4 mixes. There were studies in the literature devoted to lithium relation to the human bone, some associating it with osteoporosis, while others signaling bone enhancement. In a study of Zamani et al. [61], it was shown that the treatment of patients with Li2CO3 preserved and even enhanced bone mass. Moreover, the results reported by Cohen et al. [62], demonstrated that Li2CO3 incorporated in drugs does not induce a risk of osteoporosis. One should note that, Li-P was tested in the current in vivo preliminary study because there are no reports in the dedicated literature related to its action on the bone. The results of the current work demonstrated that it possesses bioactive characteristics in conjunction with biological origin HA, however it was inferior to its Li-C counterpart.

All in all, the results of this preliminary in vivo assessment of the pulsed laser deposited BioHA doped with Li-C and Li-P coatings hold promise. Further and more insightful documentation on both the ingrowth characteristics' influence on the mechanical stability over various implantation time periods, and biomolecular analyses (i.e., related to gene expression of osteoblasts in contact with bone substitutes and/or HA [63]), will be considered. In addition, detailed in vivo studies, in which simple (undoped) BHA coatings will be used as controls, will be imagined not only to assess the lithium effectiveness in in vivo experiments, but to demonstrate their superiority also over the commercial Ti implants. All these investigations are necessary and should stand as the subject of a dedicated study which should start, however, from the results of this preliminary work.
