3D Bioprinting

Week 8

Introduction

This weeks class discussed on using naturally occuring biomaterials for building complex materials. The prime idea is to engineer structural bio-polymers into new materials applying principles of material science and advanced fabrication.
There are two main materials of focus in the class, namely silk and keratin. There materials where to known to mankind for 1000s years and now we are reengineering them to meet different needs of the mankind. These efforts will be a bridge between organic and inorganic worlds. The experiments of this week are focused on silk, which is a natural protein composed of fibrin. This material is widely and commercially produced from Bombyx mori silkwork.

The silk obtained from this organism has two parts namely the fibroin and sticky glue like protein called sericin. Fibrion is having excellent mechanical properties making it a good candidate for being used as a structural material.
The following is an intereseting TED talk about the possibilities of silk.

In the following experiments we try to isolate fibroin from the silk coccoon and use it for various applications.

Lab Task

A. Regeneration of silk fibroin into an aqueous suspension (please, refer to Step 1-23 in Rockwood et al. Nature Protocols, 6, 1612–1631 (2011) in background reading).

Silk Fibroin Extraction Protocol

1. Boil 2 liter of ultra pure water in a stainless steel vessel .
2. Use a scissor to cut the cocoons into small pieces. Measure 5g of cocoon pieces on a scale.
3. Measure 2.24g of sodium carbonate on a weighing scale.
4. Gradually add sodium carbonate weighted above to the water and let it completely dissolve.
5. Add the cocoon pieces once the water starts to boil and continue boiling for 30 min. Stir occasionally with a spoon.
6. Remove the silk fibroin and cool it by rinsing with ultra pure cold water. You will have to rince is 2-3 times.
7. Squeeze off excess water and spread it out on a piece of aluminum foil.
8. Let the silk dry overnight.
9. Once the silk is dry, keep it in a clean plastic bag.

Dissolve silk fibroin in LiBr

10. Calculate the amount of 9.3 M Lithium bromide needed to prepare a 20% (wt/vol) solution based on the amount of dried fibroin available. The ratio of silk to LiBr is 1:4. Thus multiply the amount of dried silk fibroin by a factor of 4 to obtain the volume of LiBr needed.
11. The volume of LiBr solution used for 3.5 g silk was 14ml.
12. Add the LiBr solution on top of the silk fibroin and make sure that the solution completly covers the fibrion.
13. Let the fibroin dissolve in an oven at 60 °C for 4 h. Once completely dissolved, it appear transparent with an amber color.

Dialysis and Centrifugation

14. Hydrate dialysis cassettes in water for 2 minutes.
15. With a 20ml syringe and an 18-gauge needle, insert 12ml of the silk-LiBr solution into the cassette.
16. Dialyze against 50ml of ultra pure water. Change the water every 12hs atleast.
17. Remove the silk from the cassettes after 72h with a syringe and needle. Split the total volume into two tubes.
18. Centrifuge to remove impurities. Spin at 9.000 r.p.m. at 4°C for 20 min.
19. Transfer the silk solution with a pipette into another centrifuge tube.
20. Repeat steps 18 and 19 twice.
21. To determine the concentration of the silk solution, measure the weigh boat. Thereafter, add 0,5 ml of the silk solution to the boat and allow to dry at 60°C. Once the silk is dry, determine the weight of the silk and divide it by 0,5 ml. This will yield the weight per volume percentage.
Lyophilization and concentration

22. The fibroin solution can either be used as is or it can be lyophilized (option A) or concentrated (option B). For storage for longer than 1 month the silk solution should be lyophilized. The concentrated solution can be used directly for preparing silk tubes.
23. This silk fibroin can be used to prepare a number of different materials

Due to unavailablity of dialysis casette, we are yet to complte this expriment.

B. Fabrication of an implantable, biodegradable diffraction grating through soft lithography (please, refer to Step 25H in Rockwood et al. Nature Protocols, 6, 1612–1631 (2011) in background reading).

Steps: Fabrication of an implantable

1. Prepare work area by placing a sheet of aluminum foil and collecting the following materials : pipette, kimwipes, a paper cup, a needle, a Sylgard base and curing agent, a bottle of 70% ethanol and diffraction grating).
2. Clean the diffraction grating by spraying ethanol, wiping with kim wipes and blow-dry with pressurized air.
3. Weigh out an appropriate amount of base in the paper cup and then add curing agent from the Sylgard kit. For one mold (5cmx5cm) use 4.5 of base and 0.5 of curing agent (9:1 ratio).
4. Mix well for around a min.
5. To remove air bubbles place PDMS in a vacuum oven for 30-40 min .
6. Slowly pour the PDMS over the diffraction grating without introducing bubbles after placing it on a pretri dish.
7. Once the grating is covered in pdms, remove any bubbles with a needle.
8. Leave the setup to dry at room temperature for over 24 hours.
10. Once dry, slowly pull out the PDMS from the diffraction grating. Be careful not no damage the PDMS mould.
11. Clean the mold with ethanol and wipe with kim wipes.
12. Punch out 14-mm disks from the PDMS.
13. Place the PDMS molds in a Petri dish.
14. Add 100 μl of 8% silk solution(already prepared) onto each 14-mm disk
15. Allow the films to dry overnight.
16. Allow the films to water anneal for 1 day.
17. Allow the films to dry for 10 min and gently peel films from the mold using tweezers.

Ethics/ safety considerations this week

Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.

This weeks experimentation does not rise any ethical concern but the in this experiment we use LIBr which is a carcinogen so it is essential to be very careful and take all safety precautions while using this compound.