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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