2.5.3. Lactate Dehydrogenase (LDH) Assay

The scaffolds for use in the lactate dehydrogenase LDH experiment were seeded in three 12-well plates. Each experiment assessed three scaffolds from each size, three Thermanox coverslips each as controls, a positive control, a negative control, and a blank to account for background absorbance in the ELISA reader. The experiments were repeated at least three times. A 200 μL cell solution containing 50,000 cells was seeded onto each scaffold, and a 100 μL cell solution containing 50,000 cells was seeded onto the Thermanox coverslips and additionally into two empty wells to act as the positive and negative controls, respectively. One well was left empty for use as a blank. The well plate was placed in an incubator at 37 ◦C with 5% CO2 for 2 h. Following incubation, 2.5 mL of DMEM-F12 phenol red free with the 1% P/S and 1% FBS additives was added into the samples wells and negative control wells. Since FBS itself contains LDH, a concentration of 10% in the medium might have triggered background absorption. Therefore, only a concentration

of 1% FBS was added to the medium. For the positive controls, 1% Triton X 100 (Art. No. X100, Sigma Aldrich, Saint Louis, MO, USA) was added to the DMEM-F12 medium with 1% P/S and 1% FBS to 100% to kill the cells. The LDH experiments were carried out at 24, 48 and 72 h following seeding, and the same procedure was repeated at each interval. Three 100 μL samples were taken from each well into a 96-well plate. An LDH reagent (100 μL) was added to each well in use, and the plate was incubated in darkness at room temperature for 30 min. Following incubation, the plate was placed in a Spectrostar Nano microplate reader, and absorbance was measured at a λ of 490 nm with a reference λ of 600 nm.

#### 2.5.4. GIEMSA Staining

MG-63 cells were seeded onto the samples analogous to Sections 2.5.1–2.5.3 and stained after 3, 7 and 10 days with GIEMSA solution (GIEMSA Azure Eosin Methylene Blue, Merck). For this purpose, the samples were washed with PBS and incubated with 1 mL GIEMSA solution (diluted 1:10 with deionized water) for 10 min at room temperature. Samples were subsequently rinsed with deionized water. Microscopy was performed on an OLYMPUS SZ-61 stereo microscope.

#### *2.6. In Vivo Preparations*

A total of 30 female Wistar rats aged between 12 and 15 weeks were used to test the TCP implants. They were divided in six groups of 5 rats each. One group was a negative control (SHAM-surgery without any implant), and the zirconium oxide group served as a positive control along with the four experimental groups. Before we started, the animals were housed in the IVC cages, with two daily feedings and water *ad libitum*, in the Animal Testing Center of the Fraunhofer IZI. Animals were examined for release 5 days before surgery. On surgery day, the rats were anesthetized with a fully antagonizable cocktail containing Medetomidin (0.15 mg/kg), Midazolam (2.0 mg/kg) and Fentanyl (0.005 mg/kg) i.m. The artificial bones were implanted into a prepared subdermal pocket of the rat's flanks and closed with clips. The sham was treated the same way but was put into the subderm of the flank. The antagonizing was done with a cocktail containing Atipamezol (0.75 mg/kg), Flumazenil (0.2 mg/kg) and Naloxon (0.12 mg/kg) i.m. On the surgery day, and up 2 days after this intervention, the rats were treated with meloxicam 0.2–0.5 mg/kg s.c. During the controls on day 2, 7 and 14, the rats were anesthetized in a box with 2.0–3.0% isoflurane (0.8–1.5 L/min oxygen) and kept in this unconscious status with 2.0% isoflurane (0.4–0.8 L/min oxygen). On surgery day (day 0) and every control day, 500 μL of blood was taken to test the liver (ALT, AST, GGT) and kidney values (Urea, Creatinine). The test was performed by the Clinic for Ungulates of the University of Leipzig after centrifugation of the blood at 10,000× *g* for 5 min at room temperature. The serum was then stored at −20 ◦C. On days 7 and 14, we also took a fine needle biopsy of the implant location averted from the incision and fixed it with 4% PFA. On day 14, the animal tests were finalized using deep isoflurane anesthesia. In the following necropsy, tissue was removed for further investigation from the liver, kidney, spleen implant location and local lymph nodes of the implant location, which was preserved in 4% PFA. Implants were transferred into a 15 mL BlueCup with saline, photographed with a Leica camera 2.0 after 1 to 3 h and evaluated semi-objectively according to vascularization/tissue ingrowth, removability of the tissue and loss of substance/tendency for break using a numerical system. In the case of an indifferent vascularization/tissue ingrowth, we evaluated two halves of each of the bones and took the average value.


This semi-objective data and the serum parameters were collected for each group in an Excel table and evaluated via box-plot. To assess the anticipated differences of tissue attachment to the porous foam section and the denser, rather smooth CerAM VPPmanufactured surface, whole artificial rat bones were cut in half. Each half was further analyzed.

#### *2.7. Statistics*

The collected data were analyzed descriptively using SPSS statistics software (Version 25, IBM, Armonk, NY, USA). Based on the raw data, the mean value and the standard deviation were calculated. The Mann-Whitney U-Test was used to evaluate the differences between experimental and control samples. *p*-values < 0.05 were considered to indicate statistical significance.

#### **3. Results**

The subsequent figure shows one exemplary manufactured near-net shaped hybrid foam (Figure 2a, right side) consisting of the foamed-in additively manufactured support structures (Figure 2a, left side) and the porous Freeze Foam, which together make up the support structure case. Figure 2b illustrates the Curasan control. Figure 3 displays the workflow from the rat bone to the reverse-engineered CAD file to the manufactured single Freeze Foams and bioceramic artificial *corticalis* (i.e., *corticalis* case).

**Figure 2.** Overview samples vs. Curasan control: (**a**) VPP-manufactured column support structure (left) and hybrid with Freeze Foam enclosing the VPP support (same figure, right); (**b**) Curasan control.

**Figure 3.** Reverse-engineering workflow from rat bone to a CAD file to the manufactured Freeze Foams and hybrid foams (artificial *corticalis* case).
