Comparison of the potential for bioprinting of different 3D printing technologies

Abstract

26Additive manufacturing technologies offer a multitude of medical applications due to the advances in the development of the materials used to reproduce customized model products. The main problem with these technologies is obtaining the correct cell viability values, and it is where three-dimensional (3D) bioprinting emerges as a very interesting tool that should be studied extensively, as it has significant disadvantages with respect to printability. In this work, the comparison of 3D bioprinting technology in hydrogels and thermoplastics for the development of biomimetic parts is proposed. To this end, the study of the printability of different materials widely used in the literature is proposed, to subsequently test and analyze the parameters that indicate whether these materials could be used to obtain a biomimetic structure with structural guarantees. In order to analyze the materials studied, different tools have been designed to facilitate the quantitative characterization of their printability using 3D printing. For this purpose, different structures have been developed and a characterization methodology has been followed to quantify the printability value of each material in each test to subsequently discard the materials that do not obtain a minimum value in the result. After the study, it was found that only gelatin methacryloyl (GelMA) 5% could generate biomimetic structures faithful to the designed 3D model. Furthermore, by comparing the printing results of the different materials used in 3D bioprinting and consequently establishing the approach of different strategies, it is shown that hydrogels need to be further developed to match the results achieved by thermoplastic materials used for bioprinting.

1. Introduction

Humans are in a constant race to push the boundaries of science and knowledge in order to obtain tools that enable them to overcome adversity more effectively. For this reason, and due to a breakthrough in manufacturing technologies, three-dimensional (3D) printing has emerged. This printing is based on additive manufacturing technology, which makes it possible to create solid 3D objects from a digital model^[1].

27Today’s 3D printing methods can produce parts in a single operation, producing them fully assembled or, alternatively, facilitating the assembly process^[2]. In addition, objects are modeled and printed with a high degree of structural and spatial control, creating and optimizing objects that cannot be built with traditional processes.

This technology has wide applications in different fields^[3]: automotive (producing spare parts, creating production mechanisms); aerospace (creating complex parts); healthcare (operations planning, implant and prosthetic development, tissue bioprinting, medical training); retail (customized toys, use in simple repairs); etc.

3D printing is expanding its use in the field of health thanks to the manufacture of medical prostheses due to the high adaptability of each part created to the exact characteristics of the patient^[4]. The integration of 3D printing and biomimicry promotes improvements in the manufacture of functional materials and structures, which will lead to advances in various applications in the biomedical industry^[5,6]. Tissue engineering also offers very interesting solutions for regenerative medicine by combining cells, growth factors, biomaterials, and 3D printing technology to produce biological constructs in the desired shape, thus giving rise to 3D bioprinting.

3D bioprinting is a process of manufacturing functional tissues and organs from biomaterials by means of computer software that generates a 3D model. The process involves the addition of successive layers of biomaterial, with the added difficulty that, being living material, it must be carried out under conditions that ensure the survival and proliferation of the cells^[7-9].

To do this, first, the tissue or organ is digitized using some image processing technology (magnetic resonance, ultrasound...) in order to generate a 3D model. Subsequently, this digital model is converted into a Standard Triangle Language (STL) format file or, failing that, the type of file read by the bioprinter we are going to use. Finally, the biological part consists of obtaining and cultivating the cells. This step is key, as it requires choosing the right bioinks to emulate the tissue to be manufactured. Once the tissue or organ has been printed, it is kept in the bioprinter for maturation before it can be used or studied^[10].

The application of these additive printing technologies has, for example, exploded in popularity in the dental sector, which has started to use them in recent years. The World Dental Federation (FDI) states that oral health is paramount to the maintenance of general health and well-being. A healthy and functional dentition is a key indicator of quality of life.

The aim of this work is, first, to study the printability of different materials with potential for use in 3D bioprinting. To this end, tools will be designed using 3D printing to allow the complete characterization of different biocompatible materials using a 3D bioprinter. This will make it possible to quantify and standardize the results obtained by being able to control different variables, such as environmental humidity, printing temperature, or amount of material printed. The second objective is to compare the results obtained and study the uses of hydrogel and thermoplastic structures obtained using additive manufacturing technologies (3D printing and 3D bioprinting).

These two lines of study are becoming increasingly important in the field of medicine. In order to carry them out, the combination of 3D bioprinting with specialized computer-aided design (CAD) software will be considered, making it possible to manufacture biomimetic 3D structures.

2. Materials and methods

2.1. Starting materials

The following source materials were used in this study:

2.1.1. ColMA Lyophilizate

Methacrylated type 1 collagen (ColMA), supplied by CELLINK, is a hybrid hydrogel, which is obtained by the addition of photoactive methacrylate groups, allowing it to be cross-linked by the activation of a photoinitiator to provide the hydrogel with improved structural properties^[11].

2.1.2. LAP photoinitiator

The lithium salt LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is a free radical photoinitiator used to initiate the chain polymerization reaction after exposure to light and is combined with the different methacrylated bioinks, such as ColMA or gelatin methacryloyl (GelMA), to produce a photopolymer used in bioprinting^[12]. The LAP photoinitiator was supplied by CELLINK.

2.1.3. Reconstitution Agents A and P

Reconstitution Agent A is an acetic acid solution for dissolving and diluting freeze-dried CELLINK bioinks that include collagen in their composition, such as Coll1 Lyophilizate and ColMA Lyophilizate. The reconstituted collagen solutions, in combination with the collagen buffer, make the bioink isotonic and the pH easier to neutralize.

Reconstitution Agent P is ideal for dissolving and diluting GelMA Lyophilizate and hyaluronic acid methacrylate (HAMA) Lyophilizate for bioink formulation. Having physiological pH and isotonicity due 28to its composition based on phosphate-buffered saline (PBS) and HEPES buffer, Reconstitution Agent P makes the bioink suitable for cell culture applications.

Reconstitution Agents A and P were supplied by CELLINK^[13,14].

2.1.4. Collagen buffer

Collagen buffer allows both ColMA and Coll1 to have the right pH conditions for cell culture.

The usefulness of buffers, both in the regulation of acid–base balance in living organisms and when working in laboratories, lies in the possibility of keeping the concentration of hydrogen ions (H⁺) within very narrow limits. Thanks to the phenol red in their composition, they allow the pH to be adjusted so that it is suitable both for the viscosity of the bioink and for cell survival.

Collagen buffer was supplied by CELLINK^[15].

2.1.5. Sodium hydroxide (NaOH)

Sodium hydroxide (NaOH) in lentils was supplied by PanReac AppliChem^[16]. Both NaOH and HCl were used to adjust the solution to an appropriate pH^[17].

2.1.6. GelMA Lyophilizate

GelMA Lyophilizate is a porcine gelatin-based hybrid hydrogel that provides cells with an optimal medium for cell growth. The bonds of this bioink are modified with methacrylate groups to crosslink with a photoinitiator when exposed to ultraviolet light, conferring enhanced structural capacity.

GelMA Lyophilizate was supplied by CELLINK^[18].

2.1.7. CELLINK Start

CELLINK Start is a water-soluble gel used as a support material for complex structures, constructions with porosity along all three axes, and cell-loaded constructions. This gel is printed at room temperature^[19].

2.1.8. HAMA Lyophilizate

HAMA Lyophilizate is a hybrid hydrogel based on hyaluronic acid with methacrylate that can be cross-linked in the presence of a photoinitiator.

HAMA Lyophilizate was supplied by CELLINK^[20].

2.1.9. PCL

Ester-terminated polycaprolactone (PCL) is a biodegradable, high molecular weight, linear thermoplastic polyester with a melting point of 60°C and provides a reinforcing structure for tissue-loading constructs^[21].

2.1.10. Matrigel

Matrigel is an Engelbreth–Holm–Swarm (EHS) murine tumor-derived extract^[22]. It contains all known major components of many tissue basement membranes. It is a very useful substrate for 3D cell culture and in vivo studies to analyze differentiation, developmental, and pathological pathways, as well as for testing inhibitors/stimulators of these processes and for drug screening, toxicology testing, or disease modeling^[23]. This material converts to solid state at 37°C.

2.2. Equipment

The following equipment was used:

• (i) Ender 3 Pro 3D printer^[24], which allows the printing of numerous types of filaments, such as PLA, acrylonitrile butadiene styrene (ABS), polyethylene terephthalate glycol (PETG), among others.

• (ii) BIO X Bioprinter^[25], which is equipped with three interchangeable heads allowing the use of up to three different materials or tools for tissue bioprinting.

• (iii) Einscan-SE 3D scan^[26], which allows dual scanning, i.e., automatic scanning and fixed scanning.

• (iv) Autograph AG IS, which is a universal testing machine for compression and tensile studies.

2.3. Methods

2.3.1. ColMa Lyophilizate reconstitution

The desired volume of Reconstitution Agent A was added to the ColMA vial to achieve the target concentration of the stock solution (CS). Sterile stir bar was added for mixing gently overnight at 4°C without generating air bubbles. The appropriate amount of collagen buffer and photoinitiator was mixed. The mixture was filtered through 0.22-µm syringe filters. The collagen solution was neutralized on ice by adding the mixture to the appropriate amount of stock solution to obtain the desired final concentration. The pH was adjusted by adding 1M NaOH until a color indicative of pH 6.9–7.3 and PBS was reached, and the mixture was homogenized to obtain the neutralized ColMa hydrogel. The contents were transferred into a syringe.

2.3.2. GelMA Lyophilizate reconstitution

The desired amount of photoinitiator was dissolved in the corresponding volume of Reconstitution Agent P. The solution was filtered through 0.22-µm syringe filters. The filtered mixture was added to the vial of GelMA Lyophilizate, and the mixture was heated at 50°C for 60 min. The pH was adjusted by the use of NaOH or HCl to the optimum range of 7.0–7.4. The GelMA solution was transferred into a syringe.

2.3.3. HAMA Lyophilizate

The desired amount of photoinitiator was dissolved in the corresponding volume of Reconstitution Agent P. The solution was filtered through 0.22-µm syringe filters. The 29filtered mixture was added to the vial of GelMA Lyophilizate and the mixture was heated at room temperature for 60 min. The pH was adjusted by the use of NaOH or HCl to the optimum range of 6.5–7.4. The HAMA solution was transferred into a syringe.

3. Results

3.1. Starting materials and hydrogel formation

The hydrogels used in this study were GelMA, ColMA, HAMA, and Matrigel since they are able to obtain higher cell viability or proliferation compared to others. In addition, as a reference, a standard water-soluble hydrogel called CELLINK Start was used, which is often used as a sacrificial material for constructs due to its ease of use and printability^[27].

In addition to hydrogels, PCL, which is a biodegradable and hydrophobic thermoplastic composed of ester terminated polycaprolactone, was used as a scaffolding material. Five percent and 10% GelMA, ColMA with a target concentration of 5 mg/mL and 10 mg/mL, and finally 5% HAMA were reconstituted following the protocols provided by CELLINK.

3.2. Characterization of the used hydrogels

In order to know which of the hydrogels used in this work is the most suitable for printing a biomimetic structure, it is necessary to study their printability and some of their characteristics^[28]. In this way, we will be able to compare the hydrogel that offers the best guarantees with PCL. In other words, we can compare between 3D printing using fused deposition modeling (FDM) technology and 3D bioprinting using FDM technology.

To characterize the hydrogels, a 3D BIO X bioprinter was used in a chamber that allows the control of different key variables in the bioprinting process^[29], such as ambient humidity, printing temperature, temperature of the bed on which the printed material is deposited, and applied pressure.

Taking into account one of the main lines of research within bioprinting is the optimization of 3D printing technology for introducing cells into hydrogels, each hydrogel was bioprinted under specific conditions that allowed maximum cell viability, with the aim of comparing each hydrogel at its optimum printability point while ensuring the cell viability of the hydrogel.

3.2.1. Sessile drop method

The contact angle of a hydrogel droplet is a measure of the ability of a liquid to wet the surface of a solid. Angle values between 0° and 90° indicate a wettable, hydrophilic surface, while an angle between 90° and 180° indicates a non-wettable, hydrophobic surface^[9,30].

To make a correct measurement of the contact angle of the hydrogel drop, a cube was designed and manufactured with one of its sides open so that a glass plate or other material that does not generate absorption of the hydrogels could be placed at its base. This design prevents the generation of shadows caused by the lighting in the room. Also, at the top of the cube, a light-emitting diode (LED) is placed to generate a vertical illumination on the hydrogel drop to be analyzed (Figure 1).

Figure 1.

Chamber generated to isolate the droplets from the hydrogels and allow a good analysis by the sessile drop method, using a non-absorbent bed. Source: own elaboration.

The tests were performed at the solid–air interface and the contact angle images were taken perpendicular to the hydrogel droplet support plate and then processed using CAD software. Four measurements were taken with each hydrogel to obtain an accurate and real measurement (Figure 2).

Figure 2.

Obtaining the contact angle at the solid–air interface. Source: own elaboration.

Too hydrophilic surfaces (<35°) prevent interactions with cells, and too hydrophobic surfaces (>80°) cause protein denaturation, so the ideal contact angles for biocompatibility are approximately in the 35°–80° range, known as moderate wettability^[31].

30Although this test is not decisive to know if the hydrogel is going to present a correct cell viability, it does show us, at a low economic cost, other interesting characteristics:

• (i) Pressure that needs to be applied for a drop of hydrogel to be released. The greater the amount of pressure generated, the greater the original forces inside the syringe, so cell survival may be reduced.

• (ii) Viscosity of the hydrogel. Although this test does not allow us to quantify the viscosity of the hydrogel or its gelation, it does allow us to visually predict which hydrogels are going to present a greater state of gelation. This is a very important characteristic to obtain a correct printability.

• (iii) Hydrogel wettability. It is related to cell adhesion and spreading^[32].

Table 1 shows the results obtained.

Table 1. Contact angle results for the hydrogels studied.

Material	Temperature (°C)	Contact angle (°)
GelMA 5%	Ambient	116
	30	54
	40	51
GelMA 10%	Ambient	120
	30	109
	40	37
ColMA (Cs = 5)	9	32
	15	31
ColMA (Cs = 10)	9	45
	15	46
HAMA 5%	9	46
	Ambient	42
	30	40
	40	40
Matrigel	9	45
	15	48

Both Matrigel and ColMA with different concentrations could not be analyzed at the temperature of 25°C in the room where the experiment was carried out, as both crosslink at a temperature above 15°C. Ten percent GelMA at a temperature of 40°C showed an interesting contact angle, but a printing temperature above 37°C would subject the cells to high stress leading to cell death. Collagen-based materials had very low viscosity above 30°C, which made them difficult to bioprint. Therefore, their combination with other materials should be considered.

In view of the results in Table 1, the most interesting hydrogels for obtaining good viability and cell proliferation are 5% GelMA, ColMA with a target concentration of 10 mg/mL, 5% HAMA, and Matrigel, so the rest of the hydrogels, because they presented worse characteristics in this first test, were discarded in this study.

Matrigel is a material that requires low temperature for its maintenance, as it polymerizes at room temperature. Therefore, its temperature must be low both when performing the sessile drop method and when using it as a bioink in the bioprinter. Due to its low bioprinting temperature, this hydrogel is not suitable for cell-loaded bioprinting, but the good results obtained in the sessile drop method indicate that it is suitable for subsequent loading of cells into the bioprinter-generated structure, because the contact angles are between 35° and 80°.

3.2.2. Filament collapse test

The aim of this test is to analyze the deflection of the hydrogel filament to be analyzed for different distances. For this purpose, a platform has been developed with the same dimensions as a Petri dish, made up of pillars that are progressively spaced one unit apart (Figure 3).

Figure 3.

Design of the platform for the collapse test using Inventor. Source: own elaboration.

This platform was designed using Inventor, printed with PLA, and coated with resin. This platform, designed 31to be placed and adjusted to the printing bed of the BIO X bioprinter, allows us to control, for each hydrogel analyzed in this study, the conditions under which the study is carried out, as the bioprinter allows us to adjust the amount of extruded material, the extrusion speed and placement on the platform, the temperature and the pressure exerted on the syringe containing the bioink. In this way, this study can be easily reproduced by any research center with access to a bioprinter to compare the results (Figure 4).

Figure 4.

Final model of the Petri dish-shaped platform to be used together with a bioprinter to perform the collapse test by controlling the different influential parameters in 3D bioprinting. Image obtained using Inventor software. Source: own elaboration.

To determine the collapsed area, a hydrogel filament was deposited over all pillars, and it was determined when the theoretical area and the actual area differed greatly. In this way, the collapse rate can be obtained^[33]:

$$C_f=\frac{A_t^c-A_a^c}{A_t^c}•100\%$$ (I)

Where $A_t^c$ is the total area and y $A_a^c$ is the area generated after depositing the filament.

Figures 5 and 6 show an example of how the test is carried out and a sample of how it would look, respectively. If the filament does not collapse, coinciding the real area and the total area, the collapse coefficient is 0%. Table 2 shows the results of the test.

Figure 5.

Measuring the collapsed area. Source: own elaboration.

Figure 6.

Filament collapse test. The image on the left shows the standard hydrogel and the image on the right shows the GelMA (5%). Source: own elaboration.

Table 2. Filament collapse test tested on different hydrogels.

Hydrogel	Cf 1	Cf 2	Cf 3	Cf 4	Cf 5	Cf 6	Cf 7	Cf 8
CELLINK Start	0	0	0	0	2.43	5.64	9.83	10.302
ColMA (Cs = 10)	0	100	100	100	100	100	100	100
HaMA 5%	0	0	100	100	100	100	100	100
GelMA 5%	0	0	0	0	0	2.49	3.59	7.33
Matrigel	0	0	100	100	100	100	100	100

For this study, and based on the results of the sessile drop method shown in Table 1, 10% GelMA and ColMA with a target concentration of 5 mg/mL were discarded, as they were not within the 35°–80° range of the sessile drop method.

The use of this test, together with the proposed platform, allows us to quantify the deflection of the hydrogels as well as analyze their capacity to generate bridges and what would be the largest bridge they could support, thus obtaining an inferred result.

Table 2 shows that the only hydrogels that ensure collapse resistance conditions are the standard hydrogel and 5% GelMA, and that the hydrogel with the worst collapse result was ColMA (Cs = 10).

3.2.3. Quantitative assessment of the gelation state and printing grid test

This test aims to characterize extrusion hydrogels by performing two tests in one run. A filament with a good gel state is one that has a smooth surface and a constant width in all three dimensions, which facilitates bioprinting of regular dies with square holes^[34] (Figure 7).

Figure 7.

Comparative study of printability. Source: own elaboration.

32By analyzing the state of gelation, we can obtain the printability (Pr), which is defined as:

$$Pr=\frac{\pi }{4}\cdot \frac{1}{C}$$ (II)

$$\mathit{\text{If the figure is square }}\to C=\frac{4\pi }{16}\to Pr=\frac{\pi }{4}\cdot \frac{16}{4\pi }=1$$ (III)

This test also determines whether the hydrogel has sufficient mechanical properties to generate a “grid” or matrix of squares or rectangles of different dimensions, similar to the one shown in Figure 8^[35].

Figure 8.

Matrix formed by squares and rectangles of different dimensions. Image obtained using Inventor software. Source: own elaboration.

To perform this test, first, the area in mm² of the grid was obtained/designed, which will be referred to as the theoretical value. Subsequently, the actual bioprinted area is measured, which will be referred to as the true value, and both values are compared using the statistical tool of the standard deviation. In parallel, the value of Pr can be determined.

Once the dimensions of the designed matrix (theoretical value) are known, an image processing program is used to obtain the real dimensions (real value) generated by the hydrogel when creating the matrix. For this study, the ColMA (Cs = 10) was eliminated, as it was the one that gave the worst results in the filament collapse test (Table 3). From the results in Table 3, it can be seen that neither 5% HAMA nor Matrigel gel properly, i.e., they do not have a Pr equal to 1 or close to 1 to obtain a complex biomimetic structure.

Table 3. Results of the printability study.

The 5% GelMA was able to print a square grid with angles close to 90°C, although the gelation state was a little high since small hydrogel clots were visible. In view of the results, only the “grid” of the 5% GelMA was quantified, since, on the one hand, the standard hydrogel is soluble in water and therefore not useful to make a biomimetic structure that could be usable, and on the other hand, the rest of the materials did not show an acceptable behavior in the previous tests. Regarding the 5% GelMA, the quantification of the “grid” helps us to determine whether its gelation state, which is somewhat high, could be a problem to generate biomimetic structures.

The squares were measured from left to right and from bottom to top, starting with the square in the lower left corner, in row 1, and ending with the square in the upper right corner, in row 5. The measurements made are tabulated in Table 4, in which there is no great difference between the real value and the theoretical value.

Table 4. Summary table of the difference of actual and target values when performing the grid test.

	Real value (mm2)	Theoretical value (mm2)	Standard deviation
Row 1	0.42	2.55	1.51
	4.26	5.10	0.59
	8.05	10.20	1.52
	17.33	20.41	2.18
Row 2	4.48	5.10	0.44
	9.98	10.21	0.16
	17.52	20.41	2.04
	36.33	40.83	3.18
Row 3	6.50	7.65	0.81
	14.66	15.31	0.46
	25.78	30.62	3.42
	55.08	61.24	4.35
Row 4	8.96	10.20	0.88
	19.45	20.41	0.68
	36.23	40.83	3.26
	74.40	81.66	5.13
Row 5	10.36	12.76	1.69
	23.71	25.52	1.28
	48.80	51.03	1.58
	98.68	102.06	2.39

It should be noted that when printing the 5% GelMA with the bioprinter, the smaller grid of theoretical area of 0.42 mm² generated overlapping layers and bioprinting 33could not be correctly performed. The difference between the theoretical value and the real value in the area measurements is due, in most cases, to the fact that the hydrogel did not achieve perfect 90° angles and that at some points several layers of hydrogel overlapped, generating small overflows.

3.3. Study of printability of thermoplastics

In this study, we used PCL as a non-toxic thermoplastic because of its good biocompatibility and slow biodegradation characteristics. This last characteristic is very interesting for the objectives proposed, as it would allow our biomimetic scaffold to biodegrade as bone regeneration takes place. In addition, PCL has been shown to enhance and promote bone regeneration^[36].

In order to check the suitability of PCL for bioprinting and to see the significant differences with respect to hydrogels, the above-mentioned tests were used. The only test that was not used is the sessile drop method, which does not provide any relevant information on thermoplastics.

Table 5 shows the results of the PCL printing grid test, where it can be stated that the previously designed structure was reproduced with practically no difference between the theoretical area and the real area. The filament collapse test shown in Table 6 demonstrates that PCL has 34a great capacity to generate bridges with a high collapse resistance. In addition, it showed a very good printability with no difference between the theoretical value and the real value.

Table 5. Summary table of the difference of actual and target values when performing the grid test.

	Real value (mm2)	Theoretical value (mm2)	Standard deviation
Row 1	2.53	2.55	0.01
	5	5.10	0.07
	10.19	10.20	0.01
	20	20.41	0.29
Row 2	4.9	5.10	0.14
	10.12	10.21	0.06
	20.14	20.41	0.19
	40.9	40.83	0.05
Row 3	7.7	7.65	0.04
	15.25	15.31	0.04
	31	30.62	0.27
	62.2	62.24	0.03
Row 4	10	10.20	0.14
	20.44	20.41	0.02
	40.82	40.83	0.01
	81.62	81.66	0.03
Row 5	12.71	12.76	0.04
	25.55	25.56	0.01
	51	51.03	0.02
	102.02	102.06	0.03

Table 6. Filament collapse test tested on the different hydrogels.

Material	Cf 1	Cf 2	Cf 3	Cf 4	Cf 5	Cf 6	Cf 7	Cf 8
PCL	0	0	0	0	0	0	0	0

3.3.1. Results of printing a biomimetic structure

Good results, which were only obtained in the bioprinting of the gelatin-based hydrogel (GelMA) after following the printability methodology, show that the bioprinted structure had sufficiently acceptable structural characteristics for a complex structure. Before attempting to bioprint a biomimetic structure, the speed, temperature, and pressure parameters were adjusted to ensure the correct bioprinting of a simple scaffold with the different hydrogels created. Subsequently, using the Einscan-SE 3D scanner, a digital model of an example of a biomimetic structure, in this case a human tooth, was developed (Figure 9).

Figure 9.

Bioprinting using 5% GelMA hydrogel. Source: own elaboration.

As shown in Figure 9, the 5% GelMA hydrogel forms a tooth with a reliable shape with respect to the real tooth. After printing, a 405-nm ultraviolet light was applied for 30 s to harden the hydrogel. The hardness of the generated structure was very low, as it was not possible to apply any pressure without causing a loss of its shape.

35PCL, on the other hand, shows very acceptable compression results, as shown in Figure 10. Thus, hardness characteristics are not a problem of this material, which has negligible deformation when a range of pressures were applied.

Figure 10.

Deformation of the PCL under different applied forces.

Figure 11 shows the PCL-printed tooth after compression study.

Figure 11.

Tooth printed with PCL.

4. Conclusions

In conclusion, of all the materials used in this study, PCL and 5% GelMA are valid for the proposed methodology and for the manufacture of a scaffold with a biomimetic shape. Although the hydrogels studied are better than thermoplastics in terms of cell viability, which indicates the ability to introduce cells inside them during the bioprinting process, they have low mechanical properties for generating biomimetic structures, either because of poor gelation that leads to low printability or because they are unable to generate bridges without collapsing.

Only 5% GelMa allowed the realization of complex structures to be considered, as it passed all the tests proposed in this study. After bioprinting a tooth-shaped structure, it was found that it has high capacity to reproduce structures without causing problems during printing, but the 3D shape of the structure generated would change when forces are applied on it.

PCL, due to its high printability, its high strength, its ability to produce bridges without collapsing, and its ideal biological characteristics, is the most interesting of all the materials studied. On the other hand, it should be noted that 3D bioprinting, in combination with hybrid hydrogels, presents a greater capacity for evolution and a high potential in tissue engineering, and should therefore be the focus of our efforts to develop this methodology. Furthermore, the use of hydrogels provides good cell viability properties by better mimicking the extracellular matrix of the different tissues of the body, so their improvement and development should be a priority in the study of bioprinting.

Funding

This research was financed by the European Regional Development Fund (ERDF) within the framework of the Interreg VA Spain – Portugal (POCTEP) 2014–2020 program, project number 0633_BIOIMPACE_4_A.

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

Investigation: Jesús Manuel Rodríguez Rego, Laura Mendoza Cerezo, Antonio Macías García, Methodology, Writing-original draft

Methodology: Jesús Manuel Rodríguez Rego, Laura Mendoza Cerezo

Writing – original draft: Jesús Manuel Rodríguez Rego, Laura Mendoza Cerezo, Antonio Macías García

Writing – review & editing: Antonio Macías García, Juan Pablo Carrasco Amador

Funding acquisition: Alfonso Carlos Marcos Romero

Project administration: Alfonso Carlos Marcos Romero

Supervision: Alfonso Carlos Marcos Romero

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data

Not applicable.