Three-Dimensional Printing Using a Maize Protein: Zein-Based Inks in Biomedical Applications


The use of three-dimensional (3D) printing for biomedical applications has expanded exponentially in recent years. However, the current portfolio of 3D printable inks is still limited. For instance, only few protein matrices have been explored as printing/bioprinting materials. Here, they introduce the use of zein, the primary constitutive protein in maize seeds, as a 3D printable material. Zein-based inks were prepared by dissolving commercial zein powder in ethanol with or without polyethylene glycol (PEG400) as a plasticizer. The rheological characteristics of theirmaterials, studied during 21 days of aging/maturation, showed an increase in the apparent viscosity as a function of time in all formulations. The addition of PEG400 decreased the apparent viscosity. Inks with and without PEG400 and at different maturation times were tested for printability in a BioX bioprinter. They optimized the 3D printing parameters for each ink formulation in terms of extrusion pressure and linear printing velocity. Higher fidelity structures were obtained with inks that had maturation times of 10 to 14 days. They present different proof-of-concept experiments to demonstrate the versatility of the engineered zein inks for diverse biomedical applications. These include printing of complex and/or free-standing 3D structures, tablets for controlled drug release, and scaffolds for cell culture.

Preparation and characterization of zein inks for 3D printing. Schematic representation of the protocol for preparation of zein-based inks. Zein powder was added to an aqueous ethanol solution and gradually incorporated by vortexing to generate a suspension, which was subsequently heated to produce a homogeneous solution. These formulations were placed in tightly closed syringes and stored for up to 21 days.

Engineers and scientists are actively seeking to harness the advantages of three-dimensional (3D) printing to provide improved solutions to current challenges associated with bioengineering (i.e., the development of tissues and organs, the smart design of implants or wearables for real-time physiological monitoring,the production of cultured meat, and the engineering of ecofriendly, and cost-effective products,among others). The advantages of traditional extrusion-based 3D printing include layer-by-layer fabrication of almost any 3D form at relatively high resolution, down to the microscale.

Rheological characterization of zein (Z) and zein + PEG400 (ZP) inks was conducted at day 0 and 21: (A) storage (G′) and loss modulus (G″) and (B) apparent viscosity of zein formulations at room temperature as a function of the shear rate (n = 3). (C) Shear stress vs storage modulus for different Z and ZP inks (aged at day 1 or 21) as calculated at room temperature. The yield points (or yield stresses), calculated from a graphic analysis, are indicated with vertical lines.

For instance, 3D printing has enabled the fabrication of free-form products, such as personalized dosed pills, temporary stents, tissue-engineered heart valves, or full-sized bladders.However, the diversity of biomedical devices that can be 3D printed is severely limited by the present portfolio of available printable materials or inks capable of satisfying the demanding requirements of biomedical applications. Printing will be further enabled by expanding the spectrum of extrudable and biofriendly materials (i.e., biocompatible, resorbable, amenable to biological functionalization, etc.).Some materials frequently used as inks in extrusion 3D bioprinting are biocompatible and even safe to eat (collagen, gelatin, silk, alginate, agarose, and glycosaminoglycans, among others).

Effect of maturation time on the printability of Z and ZP inks. Different maturation times (i.e., 4, 7, 10, and 14 days), linear printing velocities (i.e., 3, 5, and 7 mm/s), and extrusion pressures (i.e., 65, 75, and 85 kPa) were explored (n = 3). Scale bar: 1 cm. All printings were conducted at room temperature (∼25 °C). Blue stars highlight the best printings.

In addition to these, several food-based matrices have been explored recently for use in extrusion 3D printing.Some examples are carbohydrate-rich foods (e.g., baking dough, glucose and sucrose,starch,cellulose, lignin,and plant-based powders), fat-rich materials (such as chocolate), and protein-rich foods (e.g., milk protein, meat, insect powder,cheese, egg protein, and vegetable proteins). In the near future, some of these materials could be engineered (in terms of rheology, stability, biocompatibility, etc.) for use in the biomedical field.

Analysis of the fidelity and quality of our printed zein structures. (A) STL grid design. The theoretical dimensions of vertical (V) and horizontal (H) lines are shown (i.e., V and H = 0.627 cm). (B) Actual printed grid; vertical and horizontal lines are indicated. Scale bar: 1 cm. (C) Equation used to calculate the ISL. (D) ISL values as determined for different printing conditions. Grids printed with zein (Z) and zein–PEG formulations (ZP) were analyzed. ISL values for (i) Z (blue bars), and (ii) ZP (red bars) grids printed at different pressure and speed conditions. Each triplet of bars shows variations depending on linear speed. Analysis is conducted for inks aged 7 (D7), 10 (D10), and 14 (D14) days. Cases in which printing parameters were inadequate to generate continuous and defined structures are marked with a circle (o). When the linear velocity of the printing head was too slow, or the pressure was higher than needed, a cross (+) was assigned. ISL values for all the pressure and linear speed tested.

Among the many possibilities, protein-based materials are particularly attractive. Proteins are constitutive blocks in the human body; therefore, their use may lead to the design of biocompatible, resorbable, cell-friendly, and biosmart inks for biomedical applications. Today, only a limited number of protein matrices have been explored as printing/bioprinting materials. Relevant examples are mammal-derived proteins, such as collagen,fibrin, or gelatin.Some of these materials (i.e., collagen and gelatin) are extensively used in bioprinting applications because of their cell-adhesive and bioactive properties that allow cell attachment or interaction. Their mechanical properties are also similar to those of natural tissues, making them suitable host environments for cultured cells. However, these proteins have limited availability and typically lack the physical stability needed for 3D printing of precise and large structures (centimeter range). Furthermore, their use often requires tight control over the processing and printing temperature, chemical prefunctionalization such as methacrylation, and postprinting cross-linking steps. These processability challenges are further complicated by the limited availability of mammal-derived materials and, thus, their high cost.

Temperature effect on zein ink printability. (A) Complex viscosity vs temperature. (B) Images of grids 3D printed at different temperatures (i.e., 10, 25, and 45 °C) using Z and ZP inks 14 day old. These grids were printed at 75 kPa and 3 mm/s. Scale bar: 1 cm.

Fortunately, alternative 3D printing materials can be found outside the mammal group. For instance, silks from arthropods and insects exhibit excellent characteristics of biocompatibility and stability under physiological conditions and have been confirmed as 3D printable, thereby greatly enhancing their potential in biofabrication.

3D printing of multilayered constructs. (A) “Tecnológico de Monterrey” logo in STL format. (B) 3D printed with high resolution. (C) Close-up of the logo showing the resolution of the printed structure. (D) Self-standing hollow box printed with 15 layers. The printing parameters were 85 kPa, speed 33 mm/s, and room temperature. We used a Z ink with 30 days of aging. (E) Strain vs stress plots derived from a compression test conducted with 3D printed cylinders of Z (blue curves; N = 5) and ZP (red curves; N = 5). (F) Young modulus of 3D printed cylinders of Z and ZP, as determined from compression tests.

In the present study, they explore the use of zein, the most abundant constitutive protein in maize seeds, as a suitable material for developing 3D printing materials. Zein, as a byproduct of corn syrup or cornstarch production, is a widely abundant and cost-effective plant-derived biopolymer. It is a prolamine, a protein rich in the amino acid proline, and occurs as aggregates linked by disulfide bonds. It has a molecular weight around 25,000 to 35,000 kDa and is poorly soluble in water due to its relatively high content of hydrophobic amino acids (proline, leucine, and alanine); however, it also exhibits amphiphilic behavior due to its high glutamine content (21–26%).

Here, they explore the use of concentrated zein solutions in ethanol/water as inks for 3D printing, aiming to expand the portfolio of proteins used in 3D printing. Zein exhibits several attractive attributes as a printable material. Zein is biodegradable, edible, has been classified as “generally recognized as safe” by the U.S. Food and Drug Administration (FDA), and has been used in biomedical applications, such as coatings for pharmaceutical capsules, fabrication of scaffolds for cell culture, and synthesis of nanoparticles, fibers, and membranes for controlled drug release.

Stability of zein-printed constructs in aqueous environments. (A) Z and ZP construct integrity under aqueous conditions over time. (B) FTIR analysis of Z and ZP inks at two different aging times (days 7 [Z7, ZP7] and 14 [Z14, ZP14]) and after immersion in water. FTIR of samples after water immersion is indicated with the suffix H2O. (C) Effect of wetting freshly printed or desiccated zein constructs on their surface topography. (D) Schematic representation of the effect of swelling in zein constructs.

Zein and poly ε-caprolactone blendsand zein–lignin blends have also been used before in 3D printing applications. Furthermore, previous studies have shown that concentrated zein solutions in ethanol/water exhibit shear-thinning properties required characteristic of inks used for extrusion-based 3D printing. To their knowledge, this is the first report of the use of zein as a 3D printing material.

Drug release. (A) Cumulative release of NA from Z (blue line) and ZP (red line) for 48 h (chemical structure of NA is shown on the bottom right). (B) Bacterial inhibition by 3D printed tablets of Z-NA and ZP-NA against suspension cultures of S. aureus. (C,D) Bacterial inhibition caused by 3D printed tablets of Z-NA and ZP-NA against suspension cultures of S. aureus in plate cultures. (C) Images of two independent replicas are presented. The imbibition halos induced by a disc of NA (as a positive control) (+), 3D printed tablets of Z-NA (1, 2), and ZP-NA (3, 4) are shown, and (D) their area was quantified by image analysis.

Zein-based formulations, optionally supplemented with polyethylene glycol (PEG400) as a plasticizer,were characterized in terms of their rheology, printability, and stability. They also report the effect of ink aging time on the rheological properties and printability. The shape fidelity and self-standing behavior of zein-based inks was successfully demonstrated by the 3D printing of complex multilayer constructs. Furthermore, they show proof-of-concept experiments that demonstrate potential applications of their formulations. For instance, they 3D printed zein (Z) and zein–PEG (ZP) antibiotic-loaded tablets and studied their release kinetics and bacterial inhibition properties. Moreover, 3D printed zein scaffolds were seeded with C2C12 myoblasts to assess cytocompatibility.

Zein-based 3D printed constructs as cellular scaffolds. (A) Actin (red)-stained C2C12 agglomerate formed on the surface of a grid printed with Z at day 3 of culture; scale bar: 200 μm. (B) Actin (red)/nucleic (blue)/staining of C2C12 cells at day 7 of culture on a ZP grid; scale bar: 200 μm. (C) Normalized C2C12 myoblast PrestoBlue proliferation assay on the 3D printed grids of Z (blue) and ZP (red) over 7 days of culture. (D) Actin (red)/nucleic (blue) staining of C2C12 cells at day 7 of culture on a ZP grid; scale bar: 2000 μm. (E) Actin (red)/nucleic (blue) staining of C2C12 cells at day 7 of culture on ZP grids covered with fibronectin; scale bar: 2000 μm. (F) Closeup of cells spreading at day 7 of culture on ZP grids covered with fibronectin, scale bar: 200 μm.

1. Three-Dimensional Printing Using a Maize Protein: Zein-Based Inks in Biomedical Applications

Jorge Alfonso Tavares-Negrete, Alberto Emanuel Aceves-Colin, Delia Cristal Rivera-Flores, Gladys Guadalupe Díaz-Armas, Anne-Sophie Mertgen, Plinio Alejandro Trinidad-Calderón, Jorge Miguel Olmos-Cordero, Elda Graciela Gómez-López, Esther Pérez-Carrillo, Zamantha Judith Escobedo-Avellaneda, Ali Tamayol, Mario Moisés Alvarez, and Grissel Trujillo-de Santiago

ACS Biomaterials Science & Engineering Article ASAP

DOI: 10.1021/acsbiomaterials.1c0054