This recent article describes the experimental procedures and results for a new mixture of biological gels which can be extruded to form structures of biological interest and serve as a skeletal framework on which to seed cultured animal cells for the purpose of growing proteins. This new mixture consists of 3 key components: sodium alginate, polyethylene glycol (PEG), and a nanoclay and goes by the acronym PEGDA.
This paper can be divided into three parts.
1) The first part describes experiments to demonstrate the mechanical properties of the mixture,
to guarantee it would make a suitable extrudate for 3D printers.
The role of the nanoclay was to increase viscosity and shear-thinning properties.
Color photographs are shown of the samples of PEG-alginate-nanoclay hydrogels, roughly 3 centimeters in diameter, stretched by a factor of three for one minute, which then restore their original structure upon relaxation. Similarly, compressive resilience is demonstrated: the hydrogel retains 97% of its original structure within 5 minutes after a compressive strain of 95%: being crushed to 1/20 of its original volume.
The experimenters also demonstrated that the addition of approximately 2.5% of 1 molar calcium sulfate increases the fracture energy from the unmanageable low level of 211 joules per square meter to 1500 joules per square meter. A fracture energy of 1500 joules per square meter means that 1500 joules of energy are required to pull apart a hypothetically rectangular object with a cross section of 1 square meter, thereby creating an additonal square meter of surface area by pulling one piece into two. The authors explained the mechanism for this lowered brittleness with a diagram (Figure 1): crosslinking of the Ca+2 cations with the alginate.
2) The second part describes tests of PEGDA to be an excellent support network for real animal cells.
The main real animal cells studied were human mesenchymal cells (hMSC). Mesenchymal cells are stem cells with high ability to differentiate into other cell types (multipotent). The hMSCs where soaked in 20% fetal bovine serum and 1% penicillin/streptomycin: at a concentration of 3 million per milliliter of gel for a 7-day culture. Viability of cells ranged as high as 86.0% with an error of 3.8% to 75.5% with an error of 11.6%.The researchers provided microphotographs of this cellular suspension (Figure 3).
3) The third part (p4037ff) describes research which combines the mechanical and cellular viability studies from the first two parts. This third part demonstrated the capability of constructing complex cellularized 3D structures with the 3D printers with an extrusion needle diameter of half a millimeter.
Human embryonic kidney cells (HEK): also at 3 million per milliliter of collagen gel. The authors encapsulated the HEK cells with rat tail collagen. The suspension was cultured for 7 days. The results were highly positive. 95% of the cells survived the 7-day culture. One photograph (Figure 4c) was provided of the surviving HEK cells.
This reviewer felt the paper could have done a better job of highlighting, by giving separate titles to separate sections, the fact that the researchers had three separate experimental goals: improving mechnical properties, confirming high cellular viability, and confirming complex 3D extrusion while maintaining high cellular viability. However, the significance of the researchers’ achievement simply overshadowed any minor complaints about the organization of their paper.
The reviewer also wishes to mention the following “backstory” about sodium alginate, which was not necessary for the usual audience of this journal but is good to include for a more general audience. Sodium alginate is the sodium salt of alginic acid: an anionic polysaccharide and alternating copolymer of M & G units, where M is (1,4)-linked beta-D-mannuronate and G its C-5 epimer alpha-L-guluronate residues. As its name suggests, alginic acid is found in algae: specifically, in the cell walls of brown algae.