This publication by K.B. Chien was summarized by Renee Cosme, with edits by Danielle Torrise. The original paper can be accessed here.
Fabricating three-dimensional (3D) porous scaffolds with controlled structure and geometry is crucial for tissue regeneration. To date, exploration in printing 3D natural protein scaffolds is limited. In this study, soy protein slurry was successfully printed using the 3D Bioplotter to form scaffolds. Various versions of the 3D scaffold were tested in terms of their ability to support the growth of human mesenchymal stem cells (hMSC). Seeding efficiency of human mesenchymal stem cells (hMSC) was highest for nontreated and thermally treated scaffolds, and all scaffolds supported hMSC viability over time.
Developing 3D scaffolds using bioplotting is a technique that involves continuously extruding, or forcing out, soft–or ‘slurry’–material unto a solid surface or liquid medium to form a structure.
According to Chien et al. (2013), our knowledge of bioplotting soft biomaterial sourced from animal and/or plant-based proteins remains limited, and there are challenges involved in developing scaffolds using these soft materials. For instance, because these biomaterials have diverse properties (i.e. viscosity, moisture content), fabrication of scaffolds/structures using these materials may require different conditions. There is also a challenge of finding an appropriate process to maintain a rigid scaffold after bioplotting.
To address these challenges, this research paper features Chien et al.’s (2013) study of assessing the efficacy of 3D scaffold printing using soy protein biomaterial for tissue regeneration applications.
Materials & Methods
1. Preparing a ‘soy protein slurry’
- Soy protein slurry was produced by mixing proportions of soy protein isolate with glycerol (among other compounds), which was sieved through 2 autoclaves of various pore sizes (297 + 105 μm respectively).
- Dithiothreitol (DTT) was added to the resulting soy protein slurry to observe the effects of disulfide bond development during solid structure formation (its importance will be discussed below).
2. Making the soy protein scaffolds (determining extrusion mass flow rate).
- To make the scaffolds, Chien et al. (2013) subjected the slurry batches into a bioplotter, measuring each batch’s mass flow rate, or mass that passes per unit of time, upon changing temperature (between 22 to 70C) during the extrusion process into a Petri dish.
- The slurry was extruded in a way that upon creation of a scaffold, each of its layer was arranged in a 45 or 90 degree orientation (Figure 1b).
- The resulting scaffolds were dehydrated using 95% ethanol.
3. Post-treatment of the scaffolds.
- All scaffolds (whether arranged in a 45 or 90o geometry) were subjected to one of four treatments:
- Non-treated (NT group) scaffolds were rinsed 3 times with a phosphate buffer solution (PBS) containing calcium + magnesium.
- Dehydrothermal treated (DHT group) scaffolds were rinsed 3x with PBS, dried with filter paper, and were subjected in a vacuum oven for 24 hr at 105cC.
- Freeze-dried and dehydrothermal treated (FD-DHT group) scaffolds were rinsed 3x with PBS, dried with filter paper, frozen at -80oC, and underwent DHT procedure (as outlined above).
- Chemically crosslinked (via carbodiimide crosslinking; EDC group) scaffolds were immersed in a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution, dissolved in 95% ethanol.
To assess the effects of post-treatments on the scaffolds, their morphologies were observed with a scanning electron microscope. Additionally, all scaffolds were subjected to a compression test to determine their mechanical properties (i.e. compressive modulus).
4. Determining cell seeding efficiency.
- ~10 000 to 100 000 prepared human mesenchymal cells (hMSCs)–a type of stem cell that can differentiate into bone, fat, muscle, or cartilage cells–were ‘seeded’ (inserted) on the surface of scaffolds with a 45o geometry and left to culture for 1-7 days. After this period, the scaffolds were harvested to determine cell seeding efficiency, proliferation, and morphology.
Results & Discussion
A Specific Mass Flow Rate Produces ‘Structurally-Defined’ 3D Scaffolds
- Chien et al. (2013) calculated that, in order to develop a 3D scaffold using soy protein (that is comprised of 20 wt.% of soy protein and 4 wt.% glycerol) with ideal pore structure, a mass flow rate of 0.0072 + 0.0002 g/s must be achieved.
- In this experiment, the bioplotter constructed structured scaffolds when it was subjected to temperatures between 22o and 37oC, because the mass flow rate did not change at these conditions (Figure 2)
- The researchers explained that a mass flow rate higher and/or lower than the determined value would create ‘less-than-ideal’ scaffolds, which may be influenced by changes in bioplotter temperature, extrusion pressure, and composition of soy protein slurry (Figure 2).
- As seen in Figure 4, low mass flow rates (and low pressures) created scaffolds with large uneven pores (Fig. 4a), while high flow rates (and high pressures) caused overflowing of material into the pores. Both conditions produce non-ideal scaffolds.
Addition of DTT to Soy Protein Slurry May Help Create More Robust Scaffolds
- Chien et al. (2013) observed that dithiothreitol-treated scaffolds are more robust and ‘smoother’ compared to scaffolds without the DTT (Figure 3B, C, D, E).
- DTT encourages disulfide bond formation within the soy protein slurry, thus decreasing the mass flow rate during bioplotting.
EDC Post-Treatments Improve Structural Integrity of Scaffolds
- EDC-treated scaffolds are the most ‘robust’ compared to other post-treated scaffolds due to carbodiimide crosslinking that promotes peptide bond formation. However, it was also observed that to a lesser degree, DHT scaffolds were also quite robust.
- DHT and EDC-treated scaffolds with 90o orientation were the most robust compared to other scaffolds.
hMSCs Characteristics on Soy Protein Scaffolds
- hMSC seeding efficiency was highest in NT + DHT scaffolds, with the highest cell count contained within NT scaffolds (Figure A, B), while such trends were lowest in the EDC and DHT scaffolds. Nonetheless, all post-treated scaffolds were capable of cell proliferation (growth).
Chien et al.’s (2013) study demonstrates that robust, 3D scaffold printing of soy protein slurry via bioplotting is possible, as long as certain parameters–such as mass flow rate and post-treatments–are considered, ultimately serving applications related to medicine and tissue regeneration.