# Preclinical Performance of Customized Polycaprolactone-Based Hybrid Bone grafts
## 2 Materials and Methods
### Sample Preparation  
The experimental hybrid inks were manufactured by the Polymers Research Group (CSIC, Madrid, Spain), following the subsequent stages:  
The synthesis of strontium-free and substituted hydroxyapatite involved adding 500 mL of 0.24 M di-ammonium hydrogen phosphate solution and 500 mL of 0.40 M of analytical-grade calcium acetate or calcium acetate plus a strontium acetate solution simultaneously in a closed polypropylene reaction vessel. The solutions were added in 10 mL aliquots with a 700 Dosino autoburette (Metrohm, Herisau, Switzerland), at a rate of 10 mL/min, until total addition. Ca/Sr ratios and sample codes of the prepared samples can be found in Table 11, Appendix A. The pH of the mixed solution was set to a value of 9.40 and it was maintained at this level throughout the reaction with a pH stat (Titrino 736, Metrohm, Switzerland) with the addition of 3.3 vol% ammonia solution. The temperature was set at 40 \({}^{\circ}\)C. After finishing the addition, the reaction was aged for 17 h. The obtained powders were filtered, washed with deionized water, and dried at 60 \({}^{\circ}\)C. Powders were calcined at 600 \({}^{\circ}\)C for 5 h using a 10 \({}^{\circ}\)C/min heating rate. Some of the powders were also calcined at 900 \({}^{\circ}\)C for further characterization (Appendix A). The synthesis reaction is:  
\[(10-x)Ca(CH_{3}COO)_{2}+xSr(CH_{3}COO)_{2}+6(NH_{4})_{2}\;HPO_{4}+OH-\] \[\longrightarrow Ca_{(10-x)}Sr_{x}(PO_{4})_{6}(OH)_{2}\]  
where for Sr0, \(x=0\); for Sr10, \(x=1\)Characterization results can be found in Appendix A, and references are provided there.  
The polymer was mixed with ceramic fillers in different percentages until the maximum possible load was optimized. Once all polymer-ceramic mixtures were obtained, a micro-extruder (Thermo-Haake Minilab system, Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain a printable filament. Pore size values of 300 microns with 100% interconnectivity were used as reference values for the internal face of the graft (oriented towards the bone substrate). On the contrary, the external face had porosities <50 um to prevent soft tissue invasion. Parametric design software, OpenScad v2021.01, was used for the design of the scaffolds. A fuse filament printer based on a Hephestos 2 (Prusa i3-BQ, Madrid, Spain) was used for the printing of 3D models. The porosity was designed directly into the slicing program, Cura (UltiMaker, Utrecht, The Netherlands). Two types of experimental, easily printable, hybrid filaments were obtained, i.e., PCL90 and PCL95. The PCL90 was composed of 90% of PCL (Sigma-Aldrich, St. Louis, MO, USA, Mn 80,000 g/mol) and 10% of hydroxyapatite, whereas PCL95 was made of 95% of PCL and 5% of strontium-loaded hydroxyapatite. In the preclinical testing, a control polymer made of 100% PCL was used (3D4 makers.com, Haarlem, The Netherlands, Mw: 84,500). Synthesis reactions have been added to the Appendix A.  
The first material weighed 0.0837 g, with a pure PCL concentration of 100%; the second material had a weight of 0.3186 g and a PCL concentration of 95% of PCL and 5% of strontium-loaded hydroxyapatite; finally, the third PCL material had a PCL concentration of 90% and 10% hydroxyapatite and a weight of 0.2034 g (Figure 1).  
These samples were introduced into empty 5 mL tubes and sterilized twice for 30\({}^{\prime}\) in a laminar UV/C flow cabinet at 254 nm. Once sterilization was completed, different amounts of DMEM culture medium without phenol red were added to each tube following ISO 10993-12 standards [16] and according to the weight of the material, resulting in 1.1 mL in the first tube, 3.186 mL in the second, and 2.34 mL in the third. The samples were stored for 24 h in the CO\({}_{2}\) incubator at 7.5%, 37 \({}^{\circ}\)C, and a relative humidity of 85%. After 24 h, the liquid resulting from incubation was used for the cell viability assay.