The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.
This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 µm). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 °C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.
Cellulose is a polysaccharide consisting of linear chains of β (1-4) linked D-glucose units. It is the most abundant natural polymer on Earth and is extracted from a variety of sources, including marine animals (e.g., tunicates), plants (e.g., wood, cotton, wheat straw), and bacterial sources, such as algae (e.g., Valonia), fungi, and even amoeba (protozoa)1,2. Cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) with at least one dimension on nanoscale are obtained through mechanical treatments and acid hydrolysis from cellulose. They not only possess the properties of cellulose, such as potential for chemical modification, low toxicity, biocompatibility, biodegradable and renewable, but it also has nanoscale characteristics like high specific surface area, high mechanical properties, rheological and optical properties. These attractive properties have made CNFs and CNCs suitable for biomedical applications, mainly in the form of 3-dimensional (3D) hydrogel scaffolds3. These scaffolds require customized dimensions with controlled pore structure and interconnected porosity. Our group and others have reported 3D porous cellulose nanocomposites prepared through casting, electrospinning and freeze-drying4,5,6,7,8. However, control on the pore structure and fabrication of complex geometry is not achieved through these traditional techniques.
3D printing is an additive manufacturing technique, in which 3D objects are created layer by layer through the computer-controlled deposition of the ink9. The advantages of 3D printing over traditional techniques includes design freedom, controlled macro and micro dimensions, fabrication of complex architectures, customization and reproducibility. In addition, 3D printing of CNFs and CNCs also offers shear-induced alignments of nanoparticles, preferred directionality, gradient porosity and can easily be extended to 3D bioprinting10,11,12,13,14,15. Recently, the dynamics of CNCs alignment during 3D printing has been reported16,17. Advances in the field of bioprinting have enable 3D printed tissues and organs despite the involved challenge such as choice and concentration of living cells and growth factors, composition of the carrier ink, printing pressures and nozzle diameters18,19,20.
The porosity and compressive strength of cartilage regenerative scaffolds are important properties that dictates its efficiency and performance. Pore size plays an important role for the adhesion, differentiation, and proliferation of cells as well as for the exchange of nutrients and metabolic waste21. However, there is no definite pore size that can be considered as an ideal value, some studies showed higher bioactivity with smaller pores while others showed better cartilage regeneration with larger pores. Macropores (<500 µm) facilitate tissue mineralization, nutrient supply and waste removal while micropores (150-250 µm) facilitate cell attachment and better mechanical properties22,23. The implanted scaffold must have sufficient mechanical integrity from the time of handling, implantation and until the completion of its desired purpose. The aggregate compressive modulus for natural articular cartilage is reported to be in the range of 0.1-2 MPa depending on age, sex and tested location4,24,25,26,27,28,29.
In our previous work11, 3D printing was used to fabricate porous bioscaffolds of a double crosslinked interpenetrating polymer network (IPN) from a hydrogel ink containing reinforced CNCs in a matrix of sodium alginate and gelatin. The 3D printing pathway was optimized to achieve 3D scaffolds with uniform and gradient pore structures (80-2,125 µm) where nanocrystals orient preferably in the printing direction (degree of orientation between 61-76%). Here, we present the continuation of this work and demonstrates the effect of porosity on the mechanical properties of 3D printed hydrogel scaffolds in simulated body conditions. CNCs used here, were earlier reported by us to be cytocompatible and non-toxic (i.e., cell growth after 15 days of incubation was confirmed30). Moreover, scaffolds prepared via freeze-drying using the same CNCs, sodium alginate and gelatin showed high porosity, high uptake of phosphate buffer saline and cytocompatibility toward mesenchymal stem cells5. The goal of this work is to demonstrate the hydrogel ink processing, 3D printing of porous scaffolds and the compression testing. Schematics of the processing route is shown in Figure 1.
1. Preparation of precursors
2. Preparation of hydrogel ink
3. Measurement of rheological properties of hydrogel
NTE: Perform the rheological properties by using a smooth cone-on-plate geometry, CP25-2-SN7617, diameter 25 mm, 2° nominal angle and gap height 0.05 mm at 25 °C.
4. File preparation for 3D Printing
NOTE: Cura 2.4.0 software is used for designing 3D scaffolds (20 mm3) having three types of pores. 1- Uniform pores of 0.6 mm, 2- uniform pores of 1.0 mm and 3- gradient pores of range 0.5-1 mm.
5. 3D printing porous scaffolds
6. Crosslinking of 3D printed scaffolds
7. Compression testing
NOTE: Perform compression tests with 100 N load cell in water at 37 °C.
CNCs based nanocomposite hydrogel ink shows a strong non-Newtonian shear thinning behavior (Figure 2a). The apparent viscosity of 1.55 × 105 Pa.s at a low shear rate (0.001 s-1) drops by five orders of magnitude to a value of 22.60 Pa.s at a shear rate of 50 s-1 (≈50 s-1 being a typical shear rate experienced during 3D printing)31. The hydrogel ink exhibits a viscoelastic solid behavior, as the storage modulus G’ (4.42 × 107 Pa) is an order of magnitude greater than the loss modulus G’’ (8.26 × 106 Pa) at low shear stress, with a well-defined dynamic yield stress value (G’=G’’) of 5.59 × 104 Pa (Figure 2b). The 3D printed porous nanocomposite hydrogel scaffolds are in shown in Figure 3. For all the printed scaffolds, the shape and dimensions are very well retained after printing as well as after double crosslinking. The pore sizes of the scaffolds, 110-1,100 µm, are in the range of 100-400 µm that is considered a benchmark for cartilage regeneration32.
The 3D printed scaffolds were tested in compression mode. This is the preferred mode of mechanical testing for cartilage materials because the role of natural cartilage is to bear loads in compression. To mimic the in vivo conditions, scaffolds were tested in water at 37 °C. Table 1 and Figure 4a represents the compressive data obtained for different porous nanocomposite hydrogel scaffolds at a strain rate of 2 mm/min. At low strain rates (1-5%), the compressive modulus (~ 0.17 MPa) is more or less similar for all types of porous scaffolds. This shows that the elastic nature of the hydrogel ink is preserved even in the presences of the macropores. However, at high strain rates (25-30%), the highest modulus of 0.45 MPa is obtained for reference scaffold with no porosity. However, as soon as the pore size increases, the modulus decreases, due to the decrease in density indicating the expected relationship between porosity of the scaffolds and the corresponding mechanical properties. In case of the gradient porous scaffolds, the modulus is higher (0.34 MPa) as compared to uniform porous scaffolds (0.20 and 0.26 MPa) because of the presence of smaller pore sizes and more solid walls. Furthermore, the compressive modulus of the 3D hydrogel scaffolds increases as the compression rate increases (Figure 4b), exhibiting and mimicking the viscoelasticity of natural cartilage tissues that is considered favorable for load bearing scaffolds33. The compressive modulus of 0.20 MPa at strain rate of 2 mm/min increases to 0.35 MPa at 5 mm/min and further increases to 0.47 MPa at 120 mm/min and is in the range reported for natural cartilage (i.e., compressive modulus of 0.1-2 MPa).
Figure 1. Schematics of the processing route. (a) Preparation of the nanocomposite hydrogel ink. (b) 3D printing porous scaffolds. (c) Double crosslinking of 3D printed scaffolds. (d) Compression testing of 3D porous scaffolds in water at 37 °C. Please click here to view a larger version of this figure.
Figure 2. Log–log plots of nanocomposite hydrogel ink. (a) Viscosity vs. shear rate and (b) G’ and G’’ vs. shear stress. Please click here to view a larger version of this figure.
Figure 3. 3D printed porous scaffolds. Scale: 500 µm. (a) Reference with no holes. (b) 1 mm pore size. (c) 0.60 mm pore size. (d) Gradient porosity 110-800 µm. Please click here to view a larger version of this figure.
Figure 4. Representative stress-strain curves for 3D printed porous nanocomposite hydrogel scaffolds. (a) At constant strain rate of 2 mm/s. (b) At different strain rates for 1 mm pore size scaffold. Please click here to view a larger version of this figure.
Target pore size (µm) | Average pore size (µm) | Compressive modulus at 1-5 % strain (MPa) | Compressive modulus at 25-30 % strain (MPa) |
Reference | 0 | 0.19 ± 0.04 | 0.45 ± 0.03 |
1,000 | 850-1,100 | 0.17 ± 0.02 | 0.2 ± 0.01 |
600 | 480-650 | 0.16 ± 0.01 | 0.26 ± 0.05 |
Gradient | 110-800 | 0.16 ± 0.01 | 0.34 ± 0.04 |
Table 1. Compression data for 3D printed nanocomposite hydrogel scaffolds.
3D printing requires suitable rheological properties of the hydrogel ink. The high viscosity ink will require extreme pressures for its extrusion while low viscosity ink will not maintain its shape after extrusion. The viscosity of the hydrogel ink can be controlled through the concentration of the ingredients. As compared to our previous work11, the solid content of the hydrogel ink is increased from 5.4 to 9.9 wt% resulting in concentrated hydrogel ink which helps to improve the resolution of the printed scaffold. It may be noted that, unlike long flexible CNFs, rigid rod like CNCs can produce inks with higher solid contents at a given viscosity due to the absences of physical entanglements14. Another important aspect that affect printability is the homogeneity of the ink. It was noted that heating the hydrogel ink at a temperature of 40 °C promotes the homogeneous mixing of CNCs with the matrix phase. To further ensure the smoothness of the hydrogel ink, it was passed through a series of nozzles, starting with the biggest diameter of 800 µm, then 600 µm and finally 400 µm. During these passes, the nozzle can be clogged which indicates the presences of big lumps but after these passes the hydrogel ink extruded effortlessly in the form of a continuous filament. The nozzle movement to obtain 3D printed constructs is also of great importance as indicated by our previous work11. The nozzle pathway should avoid repetitive movements and excess depositions of the hydrogel ink so that the resolution of the 3D print is preserved.
The porosity obtained in the 3D printed hydrogel scaffolds is in the acceptable range as compared to the targeted porosity (Table 1). An exact match cannot be expected because of the swelling nature of the hydrogel ink. The consistency of the hydrogel ink is an important factor especially when ex-situ crosslinking has to be done, i.e. crosslinking after the printing of the 3D construct. It was noted that the hydrogel ink was concentrated enough (solid content of 9.9 wt%) to maintain its shape, structure and dimensions during and after the printing process.
The pore size of the scaffold plays an essential role in cell interactions, oxygen diffusion and waste removal together with its mechanical properties to perform and support the desired functionality. Scaffolds with gradient porosity have the ability to better represent the actual in vivo conditions where cells are exposed to layers of different tissues with varying structural properties22,23,34. The porosity and mechanical properties are inversely related but the composition of the hydrogel scaffold can play an important role. CNCs has been selected as the main ingredient of the hydrogel ink because of its well-known mechanical properties2,35,36. The hydrogel ink fabricated here, possess its elasticity even in the presences of the pores, has an optimal pore size (110-1,100 µm) and a suitable compressive modulus (0.20-0.45 MPa) required for cartilage regeneration applications.
Compression testing was done in water and at body temperature to mimic the in vivo conditions as much as possible. There was no drying step involved between 3D printing and mechanical testing. In natural tissues, a porosity gradient is observed rather than one uniform pore size. The same is true for compression values for load bearing natural tissues, as the compressive modulus depends on the age, sex and on the tested location.
The advantage with the study presented here is that the final porosity and compressive modulus values of 3D porous scaffold can be controlled and customized through hydrogel ink composition and 3D printing process. This protocol is flexible and can be modified according to the specific requirements. The 3D printing is a powerful technique and can be explored in future to develop scaffolds with complex structural and compositional features. Multi material dispensing can introduce revolution by controlling the composition of the scaffolds, concentration of cells or growth factors, structural features such as directionality or porosity, mechanical properties and degradation rate in different parts of the 3D constructs.
The authors have nothing to disclose.
This study is financially supported by Knut and Alice Wallenberg Foundation (Wallenberg Wood Science Center), Swedish Research Council,VR (Bioheal, DNR 2016-05709 and DNR 2017-04254).
60 mL syringe | Structur3D Printing | ||
Alginic acid sodium salt | Sigma-Aldrich | 9005-38-3 | |
Anhydrous calcium chloride | Sigma-Aldrich | 10043-52-4 | |
Clamps, three pronged, Talon | VWR | 241-0404 | 102 mm, Dual adjustment clamp, large, clamp extension 127 mm |
Cura 2.4.0 | Ultimaker | Free slicing software | |
Discov3ry Complete | Structur3D Printing | Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder | |
Gelatin from bovine skin | Sigma-Aldrich | 9000-70-8 | |
Glutaraldehyde solution 50 wt. % in H2O | Sigma-Aldrich | 111-30-8 | |
homogenizer | SPX | APV-2000 | |
Instron 5960 | Instron | Instron 5960, Biopuls Bath, 100 N load cell, 37 °C, | |
Physica MCR 301 rheometer | Anton Paar | CP25-2-SN7617, gap height 0.05 mm, 25 °C | |
Sorvall Lynx 6000 centrifuge | AB Ninolab | s/n 41881692 | F12-rotor (6×500 ml) |
stainless steel nozzle | Structur3D Printing | 800, 600 and 400 µm | |
thingsinverse | MakerBot's | sharing and downloading 3D printable things in form of stl files | |
ultra sonication | Qsonica, LLC | Q500 | |
Unbarked wood chips | Norway spruce(Picea abies) | dry matter content of 50–55% |