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Sandra Sánchez-Salcedo, Montserrat Colilla, Isabel Izquierdo-Barba, et al.

decrease in wettability due to a ‘‘lotus leaf effect’’ on Since the emergence of TE in the mid-1980s, a wide
the material surface [56] . To estimate the wettability of variety of shaping methodologies for manufacturing
the different surface samples, contact angles mea- 3D porous scaffolds have been developed. There are
surements were measured. The contact angle for the many manufacturing methods ranging from the more
initial Ti6Al4V substrate was 56º whilst that of the conventional ones, which lead to randomly intercon-
Nano-Ti6Al4V was 102º showing a drastic increase in nected porous scaffolds and that which are principally
the hydrophobicity for the nanostructured surfaces. based on the incorporation of porogen particles [66] , use
The antibacterial effect of the Nano-Ti6Al4V surfaces of foam replica technique [67] , gel-casting of foams [68] ,
was evaluated by means of bacterial adhesion experi- cold isostatic pressing [69] , deproteinization of bovine
ments and compared with those on medical grade bone [70] , particulate leaching [71] , freeze-drying [72] , gas
Ti6Al4V substrates. Different S. aureus strains from a foaming [73] , and a combination of the methods [74] ; to
collection strain and six clinical strains isolated from more sophisticated technologies based on solid free
different patients were used. Results showed that Na- form (SFF) fabrication such as rapid prototyping (RP).
no-Ti6Al4V exhibited a notable decrease in S. aureus RP techniques allowed accurate control in the ma-
adhesion for both the collection and clinical strains cro-microporosity scales and fabricating custom-made
(around 70%) with respect to the untreated Ti6Al4V implants, which allowed the fabrication of scaffolds
surfaces. both of bioceramic and metallic nature [75] . These tech-
Concerning biofilm formation, confocal microsco- niques constituted a general strategy in which 3D
py was used to characterize sequential biofilm forma- parts are printed layer-by-layer based on a comput-
tion after different periods (Figure 3B). The presence er-aided-design (CAD) to fabricate 3D interconnected
of a few scattered bacteria on the Nano-Ti6Al4V sur- porous scaffolds at a large scale [76,77] . Thus, the scaf-
face was noted, as well as the absence of biofilm after fold architecture can be adjusted and optimized to at-
24 hours of incubation. Additional confocal micro- tain the adequate mechanical response, accelerate bone
scopy experiments were performed using calcofluor regeneration process, and guide bone formation with
fluorescent stains to stain the extracellular matrix of the anatomic cortical-trabecular structure [78] . Several
biofilms after 48 hours (Figure 3B). The confocal 3D RP techniques have been used for scaffolds prepara-
images corresponding to biofilm formation demon- tion, such as robocasting (RC), selective laser sinter-
strated that non-coated Ti6Al4V substrates clearly ing (SLS), selective laser melting (SLM), stereolitho-
show biofilm formation from the blue staining of typ- graphy (SLA) [79–82] , 3D printing (3DP) [83–85] and fused
ical extracellular matrix covering the bacterial colo- deposition modeling (FDM) [86,87] . Herein we reviewed
nies. In contrast, blue staining was absent in Na- the two main RP techniques, namely robocasting (RC)
no-Ti6Al4V. and selective laser based techniques as SLS and SLM,
In vitro biocompatibility was assessed by culturing used for the manufacture of bioceramic and metallic
the HOS cell line on the Nano-Ti6Al4V. Results have scaffolds by itself or in combination with polymers.
indicated similar behavior regarding the initial os-
teoblast adhesion and mitochondrial activity between 3.1 Robocasting (RC)
both surfaces, indicating a good biocompatibility. SEM RC technology also known as direct-printing assembly,
micrographs after 1 day of culture confirmed that Na- is distinctive among these processes because it allo-
no-Ti6Al4V behaved as well as pristine Ti6Al4V with wed the building of ceramic scaffolds using water-
respect to human osteoblasts (Figure 3C). The surfac- based inks with minimal organic content (<1wt.%) [88] .
es in both cases appeared fully covered by cells, exhi- Slurries developed from RC must fulfill two important
biting good adhesion, proliferation and degree of ex- criteria [89] . Firstly, its viscoelastic properties must al-
tension. Higher magnification images showed the low it to flow through a deposition nozzle and then
anchoring elements spread by the cells. “set” instantaneously so that its shape is preserved as

3. Scaffolds for Bone Tissue Engineering additional layers are deposited or when it span gaps in
the underlying structure. Secondly, the suspension
The application of the above-mentioned antibacterial must have a high solid volume concentration to reduce
strategies to implants manufactured as 3D scaffolds shrinkage [90] . The stability of these slurries demands
would represent a step forward in bone tissue engi- high dispersive forces between particles, where the
neering (BTE). role of the dispersant is critical [90,91] . The resulting

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