porous gelatine-GAG scaffolds
porous gelatine-GAG scaffolds
– Prepare porous gelatine-GAG scaffolds using freeze-drying fabrication and
determine the effect of cross-linking density on (i) the mechanical properties
and (ii) the viability of seeded MC3T3-E1 osteoblast cells.
– Establish whether Gelatin-GAG scaffolds might be suitable in a bone tissue
engineering strategy
Abstract should be approximately 150-200 words and should state:
– the problem investigated
– purpose and outline of the research carried out
– the principal results and major conclusions
• An abstract is often presented separately from the article, and
therefore must be able to stand-alone
• Abstract should not contain
– Figures or images,
– References to other literature
– Abbreviations.
Introduction
• One of the primary functions of the introduction is to establish
why the study is being carried out
• Introduction should address a number of important points
i. Establishing the existing state of knowledge in the field by citing
important and relevant literature
ii. Identifying a gap in the current literature, thus outlining a clear
need/motivation for the study undertaken
iii. Identify the specific focus of your work by outlining specific
objectives/hypotheses under investigation.
• Approximate length 500-800 wordsIntroduction
• Structure of Introduction
– First paragraph
• Broad information on the topic and some important/relevant background
information and literature.
– Second/Third paragraphs
• Progressively narrower the focus of the study, and begin to identify a
potential gap in the literature
Begin to outline the need/motivation for the study (“However, it is not
known…/However, the precise mechanism…”)
– Final paragraph
• Clearly state either objectives/hypothesis under investigation
• Briefly outline of how these are going to be investigated
• Common Mistakes
– Too much or not enough information
– Confusing structure (the introduction should flow – begins by talking broadly
about the topic and progressively narrows its scope leading up to the objectives)
– Unclear purpose
BME405 Tissue Engineering Laboratory Coursework
Week 4 – Preparation of Freeze-dried Gelatin-GAG scaffolds
Total number of students: 32 (approximately)
Students should split into 4 groups of 8 people
Background
Gelatin-GAG scaffolds are manufactured using a freeze-drying (or lyophilisation) process. Prior to
freeze-drying, Gelatin and GAG are mixed together with acetic acid to produce a Gelatin-GAG
suspension. This suspension is then placed into a freeze-dryer, which cools it in a controlled manner.
As the slurry freezes the Gelatin-GAG solute is localised between the growing ice crystals, forming a
continuous, interpenetrating network of ice and solute. Sublimation of the ice crystals during the
drying phase leads to the formation of a highly porous scaffold that can be used in a wide range of
Tissue Engineering applications.
Laboratory Description
The objective of this laboratory session is to prepare a Gelatin-GAG solution that will be freeze-dried
to create highly porous scaffolds that will be used in subsequent laboratory sessions.
Each group of students will prepare a single tray of Gelatin-GAG solution that will undergo a
freeze-drying cycle (see student procedure below).
When solutions are prepared, there will be a brief demonstration of how the freeze-drier
operates and a freeze-drying cycle will be initiated (Total cycle time=26 hours).
Finally, there will be a brief demonstration on how Gelatin-GAG scaffolds are chemically
crosslinking using EDAC chemical treatment.
Supplies
Gelatin (Sigma, G2500)
Deionised water
Acetic Acid (Sigma 695092)
Chondroitin-4-sulphate C-4-S (Sigma 27042-10G-F)
Equipment
Magnetic stirrer
Magnetic stirrer bars
Weighing scales
Beaker (200ml)
Stainless steel trays for freeze-dryer
Pipette gun and 25mL pipettes
Weigh boat trays
Spatula
Lab shaker
Timers
Personal Protective Equipment
Lab Coat
Safety Glasses
Gloves (provided)
Student Protocol
This protocol prepares a 50ml solution of Gelatin-GAG that consists of 1% Vol. Gelatin and 0.1% Vol.
Chondroitin-4-sulphate (GAG). Due to time constraints, a 37.5ml solution of Gelatin has been
premade for this lab session and will be distributed to each group of students (see Appendix for the
protocol used to prepare this 1% solution). This will be combined with a Chondroitin-4-sulphate/Acetic Acid solution to make up the final 50ml Gelatin-GAG solution.
Figure 1: Procedure to prepare Gelatin-GAG solution
1. Prepare a 25 mL solution of 0.05M Acetic Acid/Deionised water in a 50mL falcon tube.
2. Remove Chondroitin-4-sulphate (C-S-4) from fridge and allow to sit for 10 minutes.
3. Add 0.1g of Chondroitin-4-sulphate to the 25 mL tube of 0.05M Acetic Acid. (Note that this
measure is equivalent to a 0.1% Vol. of C-S-4 in the final 50ml Gelatin-GAG solution, i.e.
0.05g in 50mL)
4. Use the lab shaker to dissolve the C-4-S throughout the Acetic acid, until solution becomes
clear.
5. Add 12.5 mL of acetic acid/C-4-S solution to the 37.5mL Gelatin solution (premade and given
to each group at beginning of lab) in steps, by adding 2mL of acetic acid/C-4-S solution at 1
minute intervals.
6. Stir with the magnetic stirrer for at least 10 minutes until the solution appears
homogenously mixed.
7. Place beaker of Gelatin-GAG solution in the freeze dryer and turn on the vacuum by pressing
the vacuum switch. Turn off vacuum before the solution starts to boil (45-50 torr) by
pressing the same button. Release the vacuum at the vacuum release button.
8. Pipette 32.5 mL of Gelatin-GAG solution into each 90x90mm tray to get 4mm high solution
(Note: 88x88mm tray only needs 31ml of solution), taking care not to create bubbles or to
introduce lumped particles (Pipette a total of 35.5 mL into pipette and leave 3 mL in pipette
while filling the trays).
9. Place trays in the freeze dryer.
10. Initiate Freeze-drying cycle (total cycle-time 26 hours)
Freezing Step Temperature, ºC Time, min R/H
Pressure,
mTorr
1 (start) 20 15 H Atm
2 (ramping) -40 65 R Atm
3 (ramping) -40 60 H Atm
4 (drying) -40 5 H 200
5 (drying) 0 320 R 200
6 (drying) 0 1020 H 200
7 (drying) 20 40 R 200
8 (drying) 20 30 H 200
8 (drying) 20 30 H 200
9 (2
nd
drying) 20 70 H 200
Appendix A
Protocol used prepare Gelatin solution (pre-made)
A 1% solution of Gelatin (2 g in 200 mL) will be premade and ready for use in subsequent steps of
the protocol.
1. Prepare 200 mL of deionised water in a 400 mL beaker using a graduated cylinder.
2. Added 0.05 M acetic acid to dh2
O (2.32 mL acetic acid in 800 mL dh2
O) (0.58 mL in 200 mL).
3. Remove 50 mL of acetic acid solution from the beaker and place into a 50 mL tube and leave
the rest to the side.
4. Prepare a 1% solution of Gelatin (2 g in 200 mL). Add 2g Gelatin to the beaker with a 150 mL
of acetic acid. Stir at speed that creates a vortex. Maintain at 50ºC for a least 2-3 hrs until
solution becomes clear. (Try not to let the solution foam)
5. This will be aliquoted into 4 beakers (37.5ml each) on magnetic stirrers for each group.
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