Proof of concept

Bioink and 3D printing preparation

Our prototype bioink composited by sodium alginate solution and Protein R. The 3D printing preaparation procedure is shown as below:

  1. Prepare 4% (W/V) sodium alginate solution,R solution buffer (50mM Tris + 0.2M NaCl + 8M Urea), Protein R and 1 mol/l calcium chloride solution respectively
  2. Add Protein R solution buffer and Protein R into the sodium alginate solution, mix them together to be the bioink
  3. For printing, mix calcium chloride solution into sodium alginate + Protein R solution (bioink) according to volume proportion 1:1, then the sodium alginate hydrogel (product) form

Bio-ink applied in 3D printing

To testify the feasibility of 3D printing by bio-ink, we came up with two solutions, one is DIY and the other is the professional solution.

Solution1:DIY

We have designed a simple bio-ink printing system consisting of two parts: an automatic plotter and a microsyringe pump. Microsyringe pumps are responsible for outputting bio-ink at the specified speed, and the automatic plotter enables the bio-ink to be sprayed and molded in the shape we want.

Fig.1 The automatic plotter (Photo offered by equipement supplier - Bofeng Tech)


Fig.2 The microsyringe pump (Photo offered by equipement supplier - Yuanhang Tech)

Bio-ink and calcium chloride solution were stored in the two syringes which were fixed on microsyringe pump. After starting the microsyringe pumps, they could accurately and equally pump the bioink and calcium chloride solution into the slender hose connected to the outside of the syringe cone head respectively and the other end of the hoses were connected to the automatic plotter where the pen was originally installed. (see Fig.3) Started the automatic plotter that drived the head, the bioink and calcium chloride solution mixed together then sprayed into the shape we wanted, and the material were formed.


Fig.3 The schematic diagram of DIY Bio-3D- Printer
(Green and pink color represented bioink and calcium chloride solution respectively)

The biggest advantage of our DIY solution is the low cost, the overall equipment cost is about 200 US dollars, but there are also disadvantages such as low material-forming accuracy, especially in terms of the thickness control of the material.

Solution2:Professional solution

We commissioned Regenovo Biotechnology Co., Ltd., which specializes in the development of biotechnology 3D printing technology,to conduct the prototype product printing experiments for us via its professional biotechnology 3D printer. Regenovo's professional engineers help us readjust the formation of our bio-ink, we successfully printed three 10mm x 10mm samples using the company's Bio-Architect® Pro high-performance bio-3D printer. Compared with DIY solutions, professional companies can use our bio-inks to achieve very high printing accuracy. However, the cost of professional companies is very expensive and the initial offer is almost 10 times than that of our DIY solution. What’s more, for reasons of technical confidentiality, the detailed formulation of the bioink improved by engineers cannot be disclosed to us. Due to the limited funds we raised, we were unable to work in depth with them, but it proved the practical feasibility of using our bioink and greatly encouraged us.

Fig.4 Bio-Architect® Pro high-performance bio-3D printer ( (Photo offered by Regenovo Biotech)


Fig.5 The 3 pieces of prototype samples printed by Regenovo Biotech

Compared with DIY solutions, professional companies could use our bio-inks to achieve very high printing accuracy. However, the cost of professional companies was very expensive, actually the initial offer of printing service only was almost 5 times higher than the cost of our whole DIY system. Moreover, for technical confidentiality reasons, the detailed formulation of the bioink modified by engineers did not be disclosed to us. Due to the limited funds we raised, we were unable to work in depth with the professional company, but it proved the practical feasibility of using our bioink and greatly encouraged us.

Mechanic properties test and Protein R function

In order to verify function of Protein R in bioink printed samples, we prepared a series of bioink with different composition (see Fig. 6) then tested printed samples mechanic properties.

Fig. 6：Samples with different compostion
C： 4% Sodium alginate without Protein R
T1：4% Sodium alginate + 0.5% Protein R
T2：4% Sodium alginate + 1% Protein R

With sodium alginate hydrogel without Protein R as the control, the samples exhibit different mechanical properties. As shown in Table 1, we tested the performance of the hydrogel from the stretching, compression and puncture to explore the mechanic function of Protein R in the mixed hydrogel. We run the experiments with three replications and the data were recorded accordingly.

Table 1： Mechanic function test results of samples with different compostion

We found that Protein R has an obvious effect on the resistance in stretching (T1 increased 41% and T2 increased 69% comparing with C) and puncture (T1 increased 43% and T2 increased 109% comparing with C), while its resistance to compression is relatively weaker (T1 increased 6% and T2 increased13% comparing with C). And the enhancement effect was positively correlated with the amount of Protein R added. The results proved spider silk Protein R could enhance mechanic properties of bio-3D printed material as we expected.