*3.1. Biomineralization, 3D Printing and 3D Bio-Printing*

Biomineralization has been proven to be an incredible versatile process, exploitable for multiple purposes in different fields. The same could be stated for 3D printing, that with its simple basic principle paved the way for realization of incredibly ambitious and elaborated projects. The combination of these two elements have been giving rise, in the last decades, to incredible results.

The importance of scaffolds geometry at multiple level, from nano- to macro- scale, for cells attachment, spreading and viability has been so far well assessed and recognized. At the macro-scale, it is desirable for the scaffold to assume the defective part shape of the tissue or organ meant to be replaced or repaired, in order to help the neo tissue to organize into the required three-dimensional structure [74].

From the macro- point of view, especially for pursuing highly challenging goals, as it can be the regeneration of long bone defects, additive manufacturing has been attracting great interest. This, as it allows both a precise and controlled spatial-deposition of materials as well as their easy combination in complex multi-material structure, in addition to the possibility of creating shape-customized scaffold based on the anatomical site targeted [75].

If the scaffold shape and geometry play a crucial role at the macroscale, the same can be said for the composition of the material used as ink for the realization of the three-dimensional structure at the micro- and nanoscale

Speaking of bone regeneration, the key element lies in the mineral phase, in nature commonly produced by cells through the calcium-based biomineralization process.

Many approaches have been used through the years to mimic, reproduce, or induce this process in order to exploit its unique ability within the creation of new materials

suitable for bone tissue regeneration. 3D printing tries to combine the ultimate technology with the ancient principle of biomimesis. One of the most used approaches has been the use of different mineral phases, such as hydroxyapatite (HA), within the composition of the ink or as post-process modification. This, with the intention to not only to emulate, but also to stimulate the biomineralization, triggers the process by making the cells perceive a suitably functionalized environment and transforming the material from just osteoconductive to osteoinductive (Figure 2) [20,76]. In order to stimulate bone regeneration, other biomineralized polymers, including silk, a well-known high strength polymer, were exploited as biocompatible filler for 3D printing [77].

An alternative approach was devised by Romanazzo and collaborators. According to this strategy, it is possible to promote the formation of mineralized constructs in a support bath containing live cells and microgels, mimicking the complex and hierarchical structure of native bone. In these conditions, cells were able to differentiate at the interface of the constructs, while remaining multipotent in the intervening spaces, opening the potential for fabricating gradient tissue structures and for future in situ fabrication of bone-like tissues [78].

In this context, it is easy to understand how coatings to improve prosthetic implants biomimetic features have been the first to arise, but in recent years this trend has reached upgraded levels. For instance, Park and co-workers reported an osteoinductive coating of polydopamine, biomineralized HA, and bone morphogenetic protein-2 of a 3D printed polycaprolactone (PCL) structure elegantly exploits the advantages of three different strategies to reach noteworthy results. A different approach, which departs from post-process modifications and moves to the inclusion of biomineralization boosting elements into the ink formulation, is proposed by Hernandez and collaborators. A complex 3D printed multimaterial system made of PCL and HA-loaded hydrogel for long bone defect regeneration was developed. In this work, the PCL provided the appropriate mechanical support, while the hydrogel composition supports cells, promoting biomineralization [75]. Recently, more unconventional mineral phases, such as graphene oxide (GO) and black phosphorus (BP) have been used with the same purpose, with promising results [79].

Indeed, Yang and co-workers used an innovative combination of bio-glass and black phosphorus to devise three-dimensional therapeutic structures for localized photothermal osteosarcoma treatment and subsequent bone regeneration (Figure 3), profit from black phosphorus degradation to induce a phosphorus-driven in situ biomineralization [80].

A similar approach, but with different applications was exploited by Lin et al. They used a biomineralization inspired process to create a synthetic graphene composite with reshaping and self-healing features which can be used in a large variety of applications, including energy storage to actuators [81].

**Figure 2.** (**A**) Schematic illustration of the 3D printing of hydrogels based on sodium alginate and hydroxyapatite (SA/HAP). In this case, alginate and hydroxyapatite were solubilized in water and extensively mixed together. Glucono-delta-lactone (GDL), a controlled acidifying agent able to promote partial calcium release from hydroxyapatite, was then added to the alginate mixture (precrosslinking). The resulting mixture was then extruded. The progressive calcium release was able to promote the gelation of alginate, and thus the formation of hybrid hydrogels based on alginate and hydroxyapatite. These hydrogels were freeze-dried obtaining scaffolds. (**B**) Confocal Laser Scanning Microscopy (CLSM) micrographs of fluorophore labelled Bone Marrow Stem Cells (BMSCs) after 5 days of culture on the same scaffolds; (**C**) BMSCs proliferation at different timeframes (1, 3, and 5 days of culture) on the porous scaffolds with different HAP amount. Reproduced with permission from *Bioactive and Biocompatible Macroporous Scaffolds with Tunable Performances Prepared Based on 3D Printing of the Pre-Crosslinked Sodium Alginate/Hydroxyapatite Hydrogel Ink* by S. Liu et al., 2019, Macromolecular Materials and Engineering, 304 (4), 11 (doi:10.1002/mame.201800698). Copyright 2019 by John Wiley and Sons.

**Figure 3.** Schematic illustration of scaffolds fabrication based on black phosphorous/bio-glass (BP-BG) and devised for the elimination of osteosarcoma and the subsequent osteogenesis. These scaffolds were produced by 3D printing a black phosphorus and bio-glass mixture (BP-BG). These scaffolds can be exploited to promote osteosarcoma cells ablation upon light exposure. Subsequently, the scaffolds, due to their osteoconductive and osteoinductive properties, can be degraded into the main components of bone, promoting biomineralization and formation of new bone. Reproduced with permission from *2D-Black-Phosphorus-Reinforced 3D-Printed Scaffolds: A Stepwise Countermeasure for Osteosarcoma* by Shi et al., 2018, Advanced Materials, 30 (10), 12 [80]. Copyright 2018 by John Wiley and Sons.
