3.4.4. Implant Parameters

As written above we judged the implants according to different semi-objective parameters. One of them was their ability to be vascularized or rather the adherence and ingrowth of tissue to the implants after 14 days. Vascularization is important for the supply of oxygen, nutrients, the transport of metabolic products and immune cells. As the goal is to help bone to grow into this pattern, a high vascularization results in a high metabolic rate and growth factors in the area. As shown in the following figure, the more porous the artificial bones the more tissue grows into them (84% TCP Freeze Foam > 81% TCP hybrid foam halves > 71% ZrO2 Freeze Foams). The zirconium oxide group showed less tissue adherence than the TCP specimen (Figure 19A). Additionally, we noted a non-adherence of host tissue to the part of the hybrid foam halves that were made by CerAM VPP for the artificial *corticalis*. As we do not only want vessels to grow into the implants but ultimately, host tissue to replace the implant and later giving stability, we evaluated the removability of tissue from the implants after 14 days. It is obvious that not only the porosity seems to be an important factor for the surrounding tissue but also the material. As expected, TCP was more integrative than zirconium oxide (Figure 19B). We also observed that parts of broken implants were held together by the immigrated tissue giving them additional stability. On the other hand, this adherent growing can be seen critical in case of a removal of an implant for instance due to incompatibility or failure. Though this growth was very invasive, we detected no signs of macroscopic fibrosis, capsular formation, inflammation, or calcification in the implantation area. An additional statement could be given after the evaluation of histological samples (fine needle aspiration and explanted implantation area). As the biodegradable implants are developed to replace bone in the short to midterm, they have to provide enough stability until the hosts own bone material is calcified. Therefore, we also looked at the scaffolds loss of stability and the tendency to break after 14 days (Figure 19C). In necropsy we did not find evidence of a broken implant in the ZrO2 group, likely due to a slightly decreased porosity as well as its general material properties), one broken implant in the TCP Freeze Foams and one nearly broken implant in the hybrid bone, whereby the fracture was located at the connection between porous and additively manufactured shell part. These tendencies likely reflect the material and porosity properties.

#### 3.4.5. In Vivo Conclusion

From the macroscopic and clinical point of view, and according to incompatibility and toxicity, we had no sign that any of the implants, independent of the material, the manufacturing or the handling before implantation, negatively influenced the results of this oriented and leveled study. This well-founded statement is based on proven literature references as discussed above and as demonstrated in [36,37]. In accordance, our in vivo results are unobtrusive. In that regard, we can make a recommendation that the scaffolds be developed further as a result of their vascularization/tissue ingrowth tendency, which is an important factor for an implant in the muscoskeletal system. The TCP Freeze Foams are the most promising scaffolds for a use in artificial trabecular bones according to the determined parameters in the study. The TCP hybrid foam halves (artificial *corticalis* case) showed insufficient connection of additively manufactured parts to the tissue. In accordance with the in vitro analyses, where the cells only sporadically colonized/attached to the CerAM VPP-manufactured columns, the tissue did not adhere to the CerAM VPP shell structure but only to the porous artificial *spongiosa* acting Freeze Foams (see Figure 15). Roughness measurements indicated that there were clear differences between the manufactured components, with the additively manufactured one likely being too smooth for cell attachment (see Figure 9). However, they still may be good candidates for further development, considering the fact that the shell part is very stable, likely for a long time, and thus this implant could potentially allow bridging of very big/long bone defects. However, a solution must be found to enhance cell attachment capability (e.g., chemical and/or physical surface modification and/or adding porosity). Alternatively, the support structure case might be chosen.

#### **4. Discussion**

An ideal engineered tissue scaffold for the regeneration of load-bearing bones should possess appropriate mechanical functions to provide structural support, share the biomechanical load, and distribute stress that stimulates bone growth and remodeling. Due to excellent biocompatibility, calcium phosphate scaffolds have been successfully used for non-load-bearing bone restoration in recent decades. Current bioceramic scaffolds cannot re-establish massive load-bearing bones. The mechanical properties of materials decrease with increasing porosity and pore size. For optimal new bone building, scaffolds normally

require an interconnected macroporous structure, with a high porosity of over 90% and a pore size ranging from 100 to 1000 μm. Such porous constructs typically have low mechanical properties. Therefore, in this study, porous bone-like foams were fitted around/in a customized additively manufactured support structure to manufacture bioceramic hybrid foams that are mechanically much more stable than single porous components. The scaffolds were made of β-TCP and were analyzed in terms of their biocompatibility and mechanical behavior. As a result, the authors postulate that these complex structural hybrids, due to the combination of load-bearing support and porous cell-ingrowth allowing interior growth, will eventually allow the manufacturing of bone-mimicking, mechanically stable implants.
