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. PLACE THIS ORDER OR A SIMILAR ORDER WITH US TODAY AND GET AN AMAZING DISCOUNT :)