2.2.1. Powders

Biological-derived hydroxyapatite (BHA) powders were obtained from the cortical part of bovine femoral bones, according to the protocol described in [29]. Concisely, heads of femoral bones were cut-off and shafts were further processed. The bone marrows were then extracted and the unwanted soft tissue residues or macroscopic adhering impurities and substances were gently removed from shafts, which were sliced, cleaned and washed with distilled water and deproteinized for 14 days in an alkali media of 1% sodium hypochlorite. The elimination of any potential hazardous contaminants was achieved by a calcination process (at 850 ◦C, for 4 h, in air) of the resulting dry bone fragments [36,37]. Prior to undergoing ball-milling to fine powders, the calcined bone specimens were crushed with a mortar and pestle. It should be emphasized that, for the fabrication of BHA powders, European Union (EU) regulations [38] and ISO 22442-1 [39] were followed. Batches of BHA fine powders were admixed with 1 wt.% of Li-C and Li-P (Sigma-Aldrich GmbH, St. Louis, MO, USA).

### 2.2.2. Target Preparation

The as-obtained Li-C and Li-P mixed powders were pressed at ~6 MPa in a 20 mm diameter mold. The resulting pellets were thermally treated in air, using an oven, for 4 h, at 700 ◦C. A heating rate of 20 ◦C/min and a cooling ramp of 5 ◦C/min were applied. Following this protocol, the fabrication of hard and compact targets was carried out.

### 2.2.3. Coating Fabrication

PLD experiments were conducted inside a stainless-steel deposition chamber, in an ambient water vapor pressure of 50 Pa. The target-to-substrate separation distance was of 5 cm. Coatings were synthesized using a KrF\* excimer laser source (COMPexPro 205, Coherent, Santa Clara, CA, USA, λ = 248 nm, τFWHM ≤ 25 ns). The incident laser fluence was set at 3.5 J/cm<sup>2</sup> (with a corresponding pulse energy of 360 mJ). The laser beam was incident at 45◦ on the target surface. For the growth of one film, 15000 consecutive laser pulses were applied. During the multi-pulse laser irradiation, the target

was continuously rotated with 0.3 Hz and translated along two orthogonal axes, to avoid piercing and to obtain unidirectional plasma.

Prior to introduction into the deposition chamber, all substrates were successively cleaned following a three-step protocol [34]. In addition, to avoid any possible micro-contamination, targets were submitted to a "cleaning" process with 1000 laser pulses. To collect the flux of expulsed micro-impurities, a shutter was interposed between the target and the 3D metallic substrates.

To ensure the complete deposition of the 3D Ti implants, a rotating stainless-steel support flange and four clamping pins [40] were used. During depositions, the substrates' temperature was kept constant at (500 ± 5%) ◦C, using a heater connected to a PID-EXCEL temperature controller (EXCEL Instruments, Mumbai, India). A heating rate of 25 ◦C/min and a cooling ramp of 10 ◦C/min were applied.

### 2.2.4. Thermal Treatments

Both the temperature used for heating the substrates during experiments and post-deposition thermal treatments, were demonstrated to have an important influence on the coatings' characteristics and, in particular, on their composition and stoichiometry. Therefore, all synthesized structures were submitted to post-deposition thermal-treatments in water-vapors enriched atmosphere. A removable flange, positioned on the heater so as to ensure an optimal contact with the substrates, was used.

### *2.3. Animals and Surgical Experimental Protocol*

The animal spectrum generally used to test CaP coatings synthesized by the PLD technique is limited to rats, mini-pigs, dogs, goats, sheep and rabbits [17]. Both the advantages and disadvantages of using different animal models have been widely discussed in the study reported by Lu et al. [41]. It should be emphasized that, the rabbit represents one of the most used animals for medical studies [42], due to its size and ease of handling. Moreover, its skeleton reaches maturity in a rather short period of time (approximatively six months) [43].

For the in vivo experiments reported in this study, that took place in the biobase of the University of Medicine and Pharmacy (UMF), Craiova, Romania, a total of 26 skeletally-matured New Zealand White rabbits, aged six months and weighing between 3 and 3.5 kg were used. One should note that, animals were weighed at the beginning of the study and before sacrifice, and no significant weight changes were observed. All animals were free of disease. They were housed in individual plastic cages, in a climate-controlled environment at 22 ◦C, 45% humidity, and 12-h alternating light–dark cycles. During the experimental period, free access to a standard laboratory diet and tap water were provided. All animals were acclimatized for 10 days before use in this study.

Before surgery, the control and functionalized 3D Ti implants were sterilized by autoclaving (at 120 ◦C, for 1 h).

The 26 rabbits were randomly assigned to two groups (*n* = 13 each). The 3D metallic implants were introduced into the femoral condyles [44] (two implants in each rabbit), using the following sequence: at the level of the right femur, the 3D Ti implants functionalized with Li-C and/or Li-P coatings, and at the level of the left femur, the simple Ti implants (controls).

Antibiotic prophylaxis was performed pre-operatively, by intramuscular administration of a cephalosporin (sulfate diluted in physiological serum, at a dose of 40 mg/kg body). The antibiotic treatment continued for up to seven days (two doses/day).

All surgeries were performed under general anesthesia, following a three-step procedure: (i) sedation by subcutaneous administration of fentanyl (0.1 mL/Kg) and midazolam (2 mg/Kg), (ii) maintenance (during the surgical act) of the anesthesia by administration of diluted fentanyl with physiological serum (1 mL fentanyl to 9 mL physiological serum), and (iii) injection at the incision site (under sterile conditions), of 1% xylin (5 mL), as an adjuvant.

At the beginning of the surgical procedure, the incision site was shaved and washed well with water and soap, and disinfected with betadine solution, followed by covering the animal with sterile overlays. An incision of ~3 cm was performed on the lateral face of the distal femoral epiphysis. This interested the epidermis, the dermis, and the facial layers. The muscle present at this level was dissociated and the periosteum was incised longitudinally, exposing the bony lateral face of the distal femoral epiphysis.

Using a dental burr (Stryker Core Reamer), with adjustable rotational drill speed, under continuous saline irrigation, to which burr drills (Osstem surgical kit for dental implants, model TS III SA) with progressive diameter were attached, an opening hole was drilled through the cortical bone. Later, this hole was enlarged progressively (to avoid a possible cortical fracture), to a diameter slightly inferior to the implant's dimensions. Into the bone socket thus created, the 3D metallic implant was inserted in a press-fit manner, ensuring both complete coverage of its surface and stability. The implant was then carefully covered and protected by soft tissues and the muscle fascia and epidermis were closed with sutures (Figure 2).

**Figure 2.** Surgical steps for the correct positioning of the 3D Ti implant: (**a**) exposure of lateral side of the distal femur, with visualization of the cortical bone; (**b**) opening hole drilled through the cortical bone using a dental burr; (**c**) insertion of the implant into the bone socket in a press-fit manner; (**d**) cover and protection of the implant by soft tissues and closing of the wound.

Immediate postoperative radiological examinations by computed tomography (CT) were performed both to confirm the correct position of the implants and to rule out any bone fractures or other possible surgical complications.

After the surgery, all animals were allowed to move freely in their cages, being monitored for general health conditions, on a daily basis. The operative wound was carefully checked and bandaged until complete healing. At the end of the implantation procedures, all rabbits were permitted free access to normal pellet food and water. No post-operative complications were reported.

The surgical protocol used for introducing the 3D metallic implants into the rabbits' femoral condyles was approved by the "Committee of Ethics and Academic and Scientific Deontology" at the UMF in Craiova, Romania (document no. 135/20.12.2019), and the entire experimental process and the surgical technique complied with regulations and precautions of the EU Council Directive of 22 September 2010, regarding the care and use of laboratory animals for scientific purposes (2010/63/EU).

### *2.4. Characterization of Control and Functionalized Three-Dimensional (3D) Ti Implants*
