Main 3D Manufacturing Techniques for Customized Bone Substitutes. A Systematic Review

Clinicians should be aware of the main methods and materials to face the challenge of bone shortage by manufacturing customized grafts, in order to repair defects. This study aims to carry out a bibliographic review of the existing methods to manufacture customized bone scaffolds through 3D technology and to identify their current situation based on the published papers. A literature search was carried out using “3D scaffold”, “bone regeneration”, “robocasting” and “3D printing” as descriptors. This search strategy was performed on PubMed (MEDLINE), Scopus and Cochrane Library, but also by hand search in relevant journals and throughout the selected papers. All the papers focusing on techniques for manufacturing customized bone scaffolds were reviewed. The 62 articles identified described 14 techniques (4 subtraction + 10 addition techniques). Scaffold fabrication techniques can be also be classified according to the time at which they are developed, into Conventional techniques and Solid Freeform Fabrication techniques. The conventional techniques are unable to control the architecture of the pore and the pore interconnection. However, current Solid Freeform Fabrication techniques allow individualizing and generating complex geometries of porosity. To conclude, currently SLA (Stereolithography), Robocasting and FDM (Fused deposition modeling) are promising options in customized bone regeneration.


Introduction
The bone performs many key functions in the body in general and in the mouth in particular, and enables, among other things, the fixation of dental elements. The bone can regenerate spontaneously in healthy conditions, as long as there are walls that limit the defect (self-contained defect); however, the "restitutio ad integrum" is exceptional, and is always performed for small defects. In the mouth, the dimensional loss of the alveolar bone after a tooth extraction or a maxillary intervention is inevitable [1] even if filling biomaterials are used concomitantly [2]. Therefore, the loss of the maxillary bones over time and especially as a result of therapeutic interventions aimed at eliminating dental elements is unavoidable today. Bone shortage is therefore the main challenge that implant surgeons face on a daily basis, and finding a way or system to deal with mandibular atrophy is one of their major concerns [3].
Craniofacial bone reconstruction is extremely complex, given the anatomical singularity of each defect, the presence of adjacent neurovascular structures, the risk of infection, etc. Autologous bone grafts are considered the gold standard, as they provide osteoconduction, osteoinduction and certain osteogenesis, and are usually extracted from extraoral (iliac bone, tibia, cranial calotte, etc.) or intraoral (chin, tuberosity, mandibular branch, etc.) areas, depending on the amount required and the surgeon's preferences [4]. However, they offer certain complications such as poor availability, additional surgeries, morbidity in the donor area, high reabsorption, etc. [5,6]. With respect to the osteogenic capacity of the

Materials and Methods
In May 2020, a systematic review was carried out in PubMed, Embase, Scopus and Cochrane Library with the following Boolean descriptors and operators: scaffold (Title/Abstract) AND (bone regeneration OR tissue engineering) AND (3D printing OR robocasting). After removing duplicates, this resulted in 63 articles that were screened by reading the abstracts, and afterwards 12 articles were finally selected taking into account the following eligibility criteria: articles published in English, focusing on scaffolds fabricated by distinct manufacturing techniques with clinical approach; The exclusion criteria: articles about a single case, regeneration of tissues other than bone or cartilage, papers dealing with bioprinting. By means of a complementary search from those 12 publications, another 40 papers were obtained, making a total of 52 papers (Figure 1).  From the first search, the 14 most relevant techniques were selected, and with the intention of fulfilling the same objective, a search was carried out combining the term bone scaffold for each of the 14 techniques with the following descriptors: (1) Thermally induced phase separation; (2) Solvent Casting; (3) Polymer-Sponge; (4) Sol-Gel Technique; (5) Gas foaming OR supercritical fluid processing; (6) SLA OR stereolithography; (7) SLS OR selective laser sintering; (8) 3DP OR 3D printing; (9) Multi Jet Fusion OR MJF; (10) FFF OR FDM OR fused filament fabrication OR fused deposition modeling; (11) Multi Head Deposition System (12) DIW OR direct ink OR direct ink writing; (13) Low-temperature deposition manufacturing; (14) Pressure-assisted microsyringe.

Results
With the aim of carrying out a classification of scaffold fabrication techniques and the materials that can be used for this purpose, a total 52 papers were reviewed, being  15 reviews, 17 in vitro studies and 20 in vivo studies (i.e., 16 on animals-with the New Zealand rabbit as the predominant animal-and 4 on human patients). Some of these works focused on the study of biomaterials (Table 1) while others were devoted to an in-depth analysis of fabrication techniques (Table 2), their regenerative efficacy and/or their mechanical properties. Roh HS. 2016 [20] Composite material. PCL + HA + MgO The scaffolds were treated with oxygen and nitrogen plasma. They were analyzed in vitro with pre-osteoblastic cells.

FFF
The addition of HA and MgO facilitated the initial adhesion, proliferation and differentiation of the cells. The treatment with plasma increased hydrophilicity, enhancing the bioactivity of the scaffolds.
In vitro, the degree of dissolution and the calcification and mineralization were improved by Calcium phosphate glass. In vivo in rats and dogs, a significant improvement in bone and cement formations was observed with Calcium phosphate glass.    In vivo: Fifteen New Zealand rabbits with radial diaphysis defects. They were analyzed at 8 (n = 9), 12 (n = 3) and 24 (n = 3) weeks.

β-TCP
At 12 and 24 weeks, a large amount of bone was found which led to the regeneration of the marrow space. The amount of scaffold was much higher at 8 than at 12 and 24 weeks, between which there was not much difference.
Silva DN. 2008 [50] SLS and 3D printing In vitro: Dry human skulls were used to measure and compare the accuracy of the techniques.
Gypsum powder and water were used as a binder.
Polymers have also been used as a manufacturing material for 3D structures for bone regeneration. They were used individually, mainly in the early years of development of additive manufacturing technologies (1984) [15,17,52], and more recently associated with nanoparticles [45], antibiotics [39], and other substances such as peptides and dopamine [16]. Their efficacy and biocompatibility have been proven in in vivo studies with experimental animals such as Beagle dogs [17] and New Zealand rabbits [16].
Finally, the use of bioceramics combined with polymers (biocomposites)-since the properties of both types of materials are added together-has been shown to provide greater advantages for bone regeneration, from impression accuracy [18] to compressive strength [19,22] and new bone formation [19][20][21][22]31,32,44]. To date, the biocompatibility of these composite materials has been demonstrated in in vitro [18][19][20]44] and in vivo studies with experimental animals only [21,22,31,32,40]. Biocomposites can result from the polymer-ceramic bonding of calcium phosphate (HA or TCP) or bioactive polymerglass, facilitating cell adhesion and proliferation [20,32] and, as a result, bone regeneration/reparation [15]. In this regard, it has recently been reported that the incorporation of hydroxyapatite (HAP) into a biodegradable polymer (i.e., poly l-lactic acid) (PLLA) matrix exhibit bioactivity and osteoconductivity showing excellent bone defect repair capacity with the formation of abundant new bone tissue and blood vessel tissue [53]. Moreover, authors such as Gendviliene I [18] proved that biocomposites achieved higher printing accuracy than pure polymers. However, biocomposites reduce the mechanical resistance of pure polymers to some extent [21]. In contrast to the previous case, Lin YH [19] showed that adding CSi to the PCL scaffold increased mechanical resistance as well as osteo-regenerative capacity.
Some authors have compared the outcomes gathered by several techniques. Silva [50] showed that the precision of 3D printing and SLS was comparable and acceptable in both cases; Salmi [51] highlighted the PolyJet over the previous two in terms of precision, and Tagliaferri [52] indicated that, among the FFF, SLS and MJF, the FFF was the less convenient option due to the high printing time and environmental impact, which was minimal with MJF ( Table 2). Within the additive manufacturing methods, in this review of the literature we found that some of them, such as stereolithography [33,34] and selective laser sintering [35], have been shown to be successful with regard to bone regeneration through studies in humans, which are the most relevant in terms of practical clinical effects. On the other hand, the success of today's most promising technologies, such as direct ink writing or, more specifically, robocasting, 3D printing or filament extrusion, is based solely on the results from experimental animals.
Seven of the review papers studied in this article [54][55][56][57][58][59][60] focused on the analysis of the properties, advantages and disadvantages of the different materials that have been and are currently used for bone regeneration. On the other hand, five of them [61][62][63][64][65] were dedicated to classifying and studying the different methods for manufacturing customized structures for bone regeneration and how these techniques have evolved over the years. The effect of the pore size on both the biocompatibility and the mechanical strength of ceramic scaffolds needs to be checked in vivo [66,67].

Discussion
The techniques used for the fabrication of bone scaffolding can be divided, according to the fabrication method, into Subtraction and Addition ( Figure 2). Furthermore, depending on the degree of manual versus computer control in the design and manufacturing process, the techniques may also be classified as conventional (less computerized) or current techniques (more computerized). The conventional techniques had the common problem that the pore architecture cannot be customized, so it is very difficult to control the size of the pores as well as achieving their controlled interconnection. According to Thavornyutikarn [61], these conventional techniques are mostly incapable of producing fully continuous interconnectivity and uniform pore morphology within a scaffold. Most of the conventional techniques manufacture by subtraction. By contrast, the current additive manufacturing techniques, also called Solid Freeform Fabrication Techniques (SFF), offer the possibility of individualizing scaffolds and generating complex geometries with controlled porosity.

Subtraction Techniques
These include all the techniques in which the porous scaffold is obtained after the removal of part of the material from an initial solid or liquid uniform block. Within this group, only conventional techniques are found.

Solvent Casting
A mixture of polymer and ceramic particles is dissolved in an organic solvent and this solution is melted and put into a mold. Afterwards, the solvent is evaporated, leaving a porous scaffold [61]. A variant of this technique is solvent casting + particulate leaching [31], in which the solution mentioned above is used but, in addition, porogen particles are added. After the evaporation of the organic solvent, the scaffold is placed in water or another solvent capable of removing these particles, which generates a higher porosity, with interconnected pores and rough surfaces [31]. The main advantage of this method is that the preparation process is easy and does not require expensive equipment. However, this technique can only form scaffolds of simple shapes (flat sheets and tubes), and the residual solvents left in the scaffold material could be harmful to cells and tissues [61].

Subtraction Techniques
These include all the techniques in which the porous scaffold is obtained after the removal of part of the material from an initial solid or liquid uniform block. Within this group, only conventional techniques are found.

Solvent Casting
A mixture of polymer and ceramic particles is dissolved in an organic solvent and this solution is melted and put into a mold. Afterwards, the solvent is evaporated, leaving a porous scaffold [61]. A variant of this technique is solvent casting + particulate leaching [31], in which the solution mentioned above is used but, in addition, porogen particles are added. After the evaporation of the organic solvent, the scaffold is placed in water or another solvent capable of removing these particles, which generates a higher porosity, with interconnected pores and rough surfaces [31]. The main advantage of this method is that the preparation process is easy and does not require expensive equipment. However, this technique can only form scaffolds of simple shapes (flat sheets and tubes), and the residual solvents left in the scaffold material could be harmful to cells and tissues [61].

Thermally Induced Phase Separation (TIPS)
An organic solvent is used to create the polymer dissolution. In this case, the solution, once introduced into the mold, cools down causing the solvent to solidify and leave spaces among the polymers. The solvent is then evaporated by sublimation, and a porous scaffold is obtained [61]. By means of this technique, a great variety of scaffolds with high porosity can be generated by modifying variables such as the type of polymer and solvent, the polymer concentration, and the phase separation temperature [68,69].

Thermally Induced Phase Separation (TIPS)
An organic solvent is used to create the polymer dissolution. In this case, the solution, once introduced into the mold, cools down causing the solvent to solidify and leave spaces among the polymers. The solvent is then evaporated by sublimation, and a porous scaffold is obtained [61]. By means of this technique, a great variety of scaffolds with high porosity can be generated by modifying variables such as the type of polymer and solvent, the polymer concentration, and the phase separation temperature [68,69].
The disadvantages of the two techniques mentioned above (solvent casting and TIPS) [61] are that only simple-shaped scaffolds can be made and that the residual organic solvent could denature proteins and therefore be harmful to biological cells and tissues. In addition, only polymeric structures can be manufactured and are therefore affected by the characteristic shrinkage of these materials [61].

Polymer-Sponge
Starting from a ceramic solution in a suitable solvent (water or alcohol), charges of sucrose, gelatin or PMMA (polymethylmethacrylate) are added so that, as these compounds evaporate during sintering, they will create porosities forming so-called green bodies [70]. Furthermore, it has been described that the addition of polysaccharides increases the resistance of the scaffold [70]. The formation of green bodies can be classified according to the process, since different geometries and porosities are obtained with each one. The main advantage of this also named replication technique relies on the ability to form uniform dispersion of ceramic powder within a template, resulting in controllable pore size, high porosity and well-interconnected scaffolds. However, the equipment needed is quite expensive and the process is time consuming [61].

Sol-Gel Technique
Sol-gel is a chemical route that begins with the synthesis of a colloidal suspension of solid ceramic particles that is called sol. The sol is subjected to a hydrolysis and conden-sation process that results in the formation of a solid within the solvent, which is called gel [71]. The solvent is extracted from the gel by simply allowing it to rest at room temperature for a period of time, called ageing, during which the gel will shrink by expelling the residual solvent, resulting in a highly porous scaffold [71]. Regarding the main advantages and disadvantages, it should be mentioned that the biodegradability of the structures is satisfactory, and a great variability of forms can be obtained; however, they have low mechanical resistance [70,[72][73][74][75][76].

Addition Techniques
These include all those techniques in which the porous geometry of the scaffold is achieved by adding matter, usually layer by layer, without using organic solvent. This group includes both conventional and new techniques.

Gas Foaming/Supercritical Fluid Processing
Mooney developed this conventional technique in 1996 [77] with the aim of eliminating the need for organic solvents and their drawbacks. The polymer is introduced into a chamber and saturated with high pressure CO 2 . The pressure is then rapidly lowered, causing a situation of gas-polymer thermodynamic instability that ends with the formation of pores [77]. Parameters such as temperature, pressure, degree of saturation and speed of depressurization influence the morphology and size of the pores. This technique has the disadvantages of forming closed, noninterconnected pores and a smooth, nonporous surface layer of the scaffold [61]. In addition, it requires excessive heat for its realization [61].

SLA/Stereolithography
This was the first additive manufacturing technique to be introduced in dentistry. It was developed and patented by Chuck Hull in 1984 with the Stratasys company (Eden Prairie, MN, USA). This technology consists of a tank of photosensitive liquid resin, a moving platform and an ultraviolet laser which, when impacted on the resin, will create a solid layer of it. The scaffold is created layer by layer as follows: once the first layer has been made, the platform will descend leaving a new surface of liquid resin that will be polymerized by light creating a second layer, and so on until the scaffolding is complete. At that point, the uncured resin is removed, and the scaffold is subjected to UV light to complete the cure [78][79][80].
Elomaa et al. [81] used degradable polymers as a material and obtained structures with 70-90% interconnected pores. SLA technology can also be used with bioceramics and glass. It was Chu who first described its use with ceramics [82][83][84]. The suspension of ceramics and/or glass in resin has a high density, which makes the SLA process difficult, so some researchers [85][86][87] developed a process combining the SLA technique and casting. The composite fabrication process using SLA is difficult due to the high viscosity of the polymer/ceramic suspensions [88], so this technology has not been widely used with this material.
The SLA technology was the first to create reproducible scaffolds with high dimensional accuracy (up to 50 microns) and surface quality [88,89]; however, it has many drawbacks. It requires expensive machinery, support structure during manufacture, and scaffolding manufacturing time is slow, depending on the size and resolution required. An inherent problem in the process is also shrinkage during sintering. Added to this is the logistical hurdle: there are a small number of photosensitive resins on the market and many of them are toxic at a cellular level [61], although this is a point that can be overcome over time, for example, by using resins based on vinyl esters that have better biocompatibility [90].
Current SLA technologies based on the original conception concept of SLA, there are different techniques, based on SLA technologies that differ in the method of curing the resin. First, the Micro SLA uses a single photon beam for greater precision. Lee et al. [91] used this technique to make poly propylene fumarate scaffolds and Seol et al. [92] for HA and TCP scaffolds. Both studies, performed in vitro, obtained scaffolds with mechanical properties similar to those of human cancellous bone.
First, the so-called Two-Photon Polymerization uses an ultra-short pulse laser and makes it possible to manufacture scaffolds with nanometric resolution [88][89][90]. Second, the Digital Light Processing uses visible light and creates an entire layer at once. It offers a solution to several of the problems of SLA technology. Its main advantage is the speed of synthesis, in addition to the high lateral resolution (40 microns), the large proportion of solid particles it allows (40-60%), and the absence of expensive equipment such as lasers or a heating chamber [92]. Moreover, it allows the manufacture of ceramic and bioglass scaffolds.

Selective Laser Sintering (SLS)
This technology was developed in 1986 and first marketed in 1992. It consists of a CO 2 laser that acts on a bed of powder to sinter certain regions of the powder to form a solid first layer. The platform lowers the corresponding layer thickness, and a roller deposits a new layer of powder [78][79][80]. Eshraghi and Das [15] manufactured orthogonal pore PCL scaffolds designed for placement in loaded locations. These scaffolds were precise with respect to the digital design and showed acceptable compressive strength. Other authors, such as Pereira et al. [93], have also found great reliability between the virtual model and the manufactured structure. Manufacturing bioceramics using the SLS technique directly has proved difficult, mainly due to the high heating and cooling speeds associated with the high energy laser used [94][95][96]. However, it is currently in use; for example, Feng P. [97] used a bioceramic powder loaded with titanium nanoparticles to improve the mechanical properties of the scaffold, obtaining a compressive strength of 23 MPa with 58% porosity [97]. The SLS technique has also been used to manufacture composite scaffolds but finding the right process parameters is a challenge: powder composition, laser power, particle size, and temperature [61]. This technique has the great advantage of being the only one capable of manufacturing metal structures (such as titanium and cobalt chrome). For example, F. Mangano [35] made dental implants with a sharp edge to rehabilitate highly atrophic maxillae.
The main advantage of this method is that it makes it possible to create reproducible scaffolds, provides greater dimensional accuracy than the SLA technique (<50 microns) and does not require a support structure. However, as with all techniques, it has its drawbacks. Shrinkage during melting or sintering remains a problem as with SLA technology. In addition, the use of high temperature, which could cause the degradation of biodegradable dust, and the difficulty or impossibility of removing the dust once the scaffold has been manufactured, which could hinder cell proliferation and cause an inflammatory reaction, are the major drawbacks. It should also be noted that the resolution will be limited by the size, shape, and arrangement of the dust particles.
To solve the excess of temperature of SLS and to allow the manufacturing of scaffolds with bioactive and biodegradable materials, Popov et al. developed selective laser sintering by surface (SLSS) [98]. This is a variation of the SLS in which the polymer particles are coated with CO 2 , so the melting is limited to the surface layer, maintaining the nature of the particles inside the polymer during the scaffold manufacture [79,99,100].

3D Printing (3DP)
This was developed in 1989. In this variant of SLS technology, instead of using a laser, a liquid binder is used on the bed of powder to solidify what would be the first layer of the scaffold. Similarly, once the scaffold has been built, any remaining dust must be removed [78][79][80]. This is the only SFF technique that can use hydrogels for the manufacture of scaffolds. The problem with hydrogels is the poor mechanical properties, which force the structure to be processed later to incorporate monomers or polymers so as to increase the mechanical resistance [37,38,101]. Some authors [38,102] have verified the validity of this technique in vivo for the manufacture of scaffolding, especially with calcium phosphate ceramics. However, the finished pieces require subsequent thermal treatment to improve the mechanical properties [30,36,61]. The manufacture of scaffolds from composite materials is also possible, e.g., Sherwood et al. [103] manufactured PLGA/TCP structures that showed a compressive and tensile strength similar to that of cancellous bone.
As it is a technique that does not require high temperature and works with hydrogels, it allows the incorporation of biologically active molecules or even cells. It allows the manufacture of high consistency scaffolds, without support structures and at high speed, which makes mass production feasible. Despite the high consistency, the bonds formed between particles are weak, so scaffolds have poor mechanical properties, as Jason found in a study showing that calcium phosphate scaffolds made with this technology had significantly lower torsional resistance than allografts [37]. Furthermore, it requires a large particle size, which reduces precision and resolution [61] and, as with the SLS techniques, it has the disadvantage of difficult or impossible removal of uncured dust.
When comparing the printing accuracy of SLS and 3DP technology, Silva et al. [50] found that it was acceptable in both cases, with dimensional errors of 2.1% and 2.67%, respectively, slightly higher in the 3DP technique.
A similar technique called PolyJet consists of the extrusion of liquid resin through multiple nozzles, which as soon as it is deposited on the platform, is cured by ultraviolet light. This technology stands out for its high manufacturing speed and printing precision. In the study by Salmi M [51], where the PolyJet is compared in terms of accuracy to 3D printing and SLS, the PolyJet technique showed significantly more accurate results.

Multi Jet Fusion (MJF)
This is a very new and promising technology developed by HP (Hewlett-Packard) [52]. It is based on numerous nozzles capable of releasing different liquid agents onto the printing surface [52]. On the one hand, they release a liquid binder and, on the other hand, a detailing agent to improve resolution. A lamp then runs over the surface, polymerizing and distributing the heat. Finally, the excess dust is removed by blasting [52]. The use of this technique in the field of dentistry has yet to be developed, but the results found in other materials are promising. One of the major advantages of this technology is that it allows mass production due to its speed of processing: it is capable of manufacturing as many parts as fit into the powder hopper at once. The powder used is very fine, so high density, resolution and precision structures are achieved. The uncured powder is reused for the next print, so the waste of material is minimal. This technique also offers the possibility of using different materials.

Fused Filament Fabrication (FFF)
This has also been referred to as Fused Deposition Modeling (FDM), and it was developed in 1992. This technology synthesizes scaffolds by casting material. The system consists of a substrate platform on which there is a mobile nozzle with a small hole. A filament with the corresponding material is introduced into this nozzle, where it melts and is deposited on the platform, giving rise to a first layer. The platform descends, leaving space for the second layer [77,78]. The first scaffolds created by FDM were made of PCL and showed great biocompatibility with human fibroblasts [104]. A filament composed of a thermoplastic polymer, ceramic powder and a binding agent is used to create bioceramic structures through FDM. The polymer and binder are removed during further processing [105,106]. Finished ceramic parts are sintered to improve their mechanical properties [107][108][109][110]. FDM technology has also been used to manufacture composite scaffolds. The research group of Hutmacher et al. [53,110,111] manufactured scaffolds based on various polymers and calcium phosphates that showed favorable mechanical properties, bioactivity, resorption and increased cell colonization and incorporation of growth factors. The study by Gendviliene I [18] showed that the PLA/10% HA filament printed with a 3D FFF printer produced scaffolds with equal or even better accuracy than those printed with pure PLA filament [18].
This technique has numerous advantages, such as its low cost and the achievement of scaffolds of good structural integrity with minimum material waste. In the X and Y axes, it has a high precision and versatility in the direction of the materials within each layer (0.5 microns [39]); however, the direction of the Z axis is not easily controlled (5 microns) [24,39]. As for the disadvantages, this technique requires high temperature, the scaffold manufacturing process is slow and requires support structures, so it does not allow mass production. For successful printing, the viscosity properties of the materials must be considered when casting.

Multi Head Deposition System (MHDS)
This new FFF-based technology, the MHDS (multi head deposition system) consists of using more than one extrusion head to create a composition from several materials, which can be laid out in the same layer [61]. It requires high temperature; however, Kundu J [112] was successful in manufacturing PCL cartilage regeneration scaffolds + alginate hydrogel with encapsulated chondrocytes by adapting the parameters to maintain cell viability [113]. Another variant of the FDP is the Precision Extruding Deposition. The difference between this technique and conventional FDM is that it employs material in the form of granules which is subsequently melted in a chamber, thus avoiding the need to use filament-shaped materials [114].

Direct Ink Writing (DIW)
This arises from the concept of filament extrusion. Here, instead of starting from a material in the form of a yarn, the starting point is a solution of material, which is extruded through a nozzle, so that scaffolds are manufactured layer by layer. Several different techniques can be identified within this group. The advantages are the same as with the FFF techniques, but some of the disadvantages are overcome: it does not require high temperatures to melt the filament and the properties of the material do not have to be considered when melting it.
Robocasting is probably the most promising of the techniques included in DIW technology. It was developed in 1998 by Cesarano et al. [115]. It allows a highly concentrated suspension to be deposited through a small channel on a nonwetting oil bath. The suspension becomes solid when the water evaporates [116]. This technique has been widely used to manufacture bioceramic structures. For example, Pedro Miranda [23] recommends the use of small dust particles and low-specificity surface area, in addition to using Ca-deficient powders to avoid the transition of TCP from beta to alpha. On the other hand, J. Franco [46] describes the preparation of ceramic-based inks (HA, b-TCP and BCP) using Pluronic F-127 as a hydrogel, which is fluid at 0 • C and gel at room temperature. One of its greatest advantages is that scaffolds made by this technique are more resistant than those made by other methods using the same materials. Many authors have supported this statement after finding satisfactory results in their studies [26,42,43,47] In this technique, instead of departing from a filament as in the other FFF techniques, it starts from a solution of the material to be used in a low melting point solvent. The deposition of material must be at very low temperatures to allow the material to solidify when deposited on the platform to form layers [117]. The solvent will then be removed by freeze-drying. Almeida et al. [81] developed scaffolds with this technique, which presented porosity greater than 90%, mechanical properties similar to cancellous bone and good biocompatibility and conductivity.

Pressure-Assisted Microsyringe (PAM)
It was developed in 2002 by Vozzi et al. [118]. In this technique, instead of using heat for extrusion, constant pressure is applied, and instead of using a filament, a solution is used. When the solvent evaporates due to pressure, the material solidifies. The higher the viscosity, the higher the resolution [118,119]. This technique has been widely used for the manufacture of drugs [120][121][122][123] and, to a lesser extent, to make polymer scaffolds [118,119]. The same research group [124][125][126] developed the so-called PAM 2, in which they replaced constant pressure with a mechanical piston as the driving force. As it does not require heat, it allows the incorporation of living cells, which is a great advantage, while its major limitation is the need to use low concentration solutions.

Scientific Support of the Techniques
The scientific literature supports the 14 techniques to different extents. In Figure 3, it is shown that 3DP, SLS, SLA and FFF are the most studied techniques, being supported by 6043 papers, 5135 papers, 3961 papers and 2368 papers, respectively. However, focusing on the percentage of articles that contained "bone scaffold" within those published papers, the Multi Head Deposition System (57%), the Low-Temperature Deposition Manufacturing (50%) and TIPS (28.2%) stand out as the main techniques applied in bone scaffold manufacturing. However, the Robocasting technique (a variant of DIW) may be considered a promising technique, with 49 out of 70 papers focusing on customized bone scaffolds. Robocasting biocomposites for bone regeneration is increasingly studied in recent years. In this regard, some authors studied the effect of different polymeric coatings (both natural and synthetic), on the mechanical performance of bioceramic robocast scaffolds [126,127], while others focused on the osteostimulative capability of the robocasted biocomposite in animal models [128]. Future experiences will clarify the best choice for customizing bone grafts with the available techniques. robocast scaffolds [126,127], while others focused on the osteostimulative capability of the robocasted biocomposite in animal models [128]. Future experiences will clarify the best choice for customizing bone grafts with the available techniques. Finally, patents may support most techniques and materials reported in this review; however, we did not search within patent databases and therefore our review is not exhaustive. Consequently, readers should be aware that several current promising techniques or materials could not be retrieved with the search strategy used in the present Finally, patents may support most techniques and materials reported in this review; however, we did not search within patent databases and therefore our review is not exhaustive. Consequently, readers should be aware that several current promising techniques or materials could not be retrieved with the search strategy used in the present work, as the patent procedure needs to check the innovativeness/originality of the material/method candidate for patenting.

Conclusions
There are many techniques for the manufacture of 3D scaffolds. Among them, we can differentiate traditional techniques, which are nowadays practically in disuse in the field of regenerative dentistry, because of the lack of mechanical integrity, as well as the limited capacity to control the internal and external architecture of scaffolds (i.e., pore morphology, pore size, pore interconnectivity and overall porosity). By contrast, the so-called solid freeform fabrication techniques, encompassed under additive manufacturing techniques, overcome the above-mentioned disadvantages. In this regard, SLA, Robocasting and FDM are promising options in customized bone regeneration that enable good mechanical and biological properties throughout the entire scaffold.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments:
The study was conducted within the Research Group "Avances en Salud Oral" (Advances in Oral Health) of the University of Salamanca, led by the first author. https: //avancessaludoral.usal.es.

Conflicts of Interest:
The authors declare no conflict of interest.