Hello all, i'm working with mesenchymal stem cells and we're exploring the potential of MSCs in bone regeneration in a rabbit model via tissue engineering. So we have got chitosan powder but i dont know how to proceed with that. I've come across some articles where they have used freeze drying method for preparation of 3 D chitosan scaffolds.
I'm looking for a detailed protocol for proceeding with the same.
TIA
Ms Mahajan
I would not bother with chitosan...it is a non-mammalian polysaccharide which, though fun to play with, doesn't really do all that well in tissue regeneration. I presume you are looking at a rabbit model with the hopes of, eventually, reproducing good results in humans. So I suggest you start with demineralized rabbit bone. The Type I collagen in bone is an excellent scaffold for bone cell growth and, depending on how you create the demineralized bone, you can retain the bone morphogenic proteins (BMP-2 and others) that will help guide cell development. Let me know if you are interested in going that route and I can provide you with some detailed protocols.
Best,
JFK
Thankyou James kirk, I'm actually gonna be screening multiple scaffolds such as hydroxyapatite, beta tricalcium phosphate, chitosan , corals, platelet rich plasma gel and even demineralized bone matrix. I'd really appreciate the detailed protocols for preparation of DBM.
Thankyou Amin for sharing your protocol. No, we wont be working with the composites, I just want to test chitosan alone.
Ms Mahajan,
I just finish typing a response and hit send...at which point I was taken to the log in screen. If, by chance, it sent the previous response, I want to avoid duplicating my effort. If not, I will retype the protocol. Please let me know if you got the earlier post.
Thanks,
JFK
Okay, here goes...
1) Obtain fresh rabbit long bone (e.g. femur, tibia, humerus). Debride of soft tissue and marrow. Rinse with DI water.
2) Grind to 125 to 850 micron particle size. If using a powered mill, add ice or dry ice. If using a hand rasp, freeze the bone first.
3) Rinse the bone particles clean with chilled DI water (this and all following solution/solvent steps should be performed with chilled (e.g. 4 deg. C) solution.
4) Defat the bone particles with chilled ethanol or acetone. Place 10 volumes (e.g. if you have 50 grams of bone particles, use 500 mL of solvent) in a beaker with a stir bar; stir at low RPM for 10 minutes; decant. Repeat two more times.
5) Rinse defatted bone particles with 10 volumes of chilled DI water; stir at low RPM for 5 minutes; decant. Repeat two more times.
6) Demineralize bone particles with 10 volumes of 0.5 N HCl solution; stir at low RPM for 40 minutes; decant. Repeat two more times.
7) Repeat step 5).
8) If you are freeze-drying the DBM, freeze the damp DBM in a freezer first and then lyo. If you are air-drying the DBM, you will get faster results by dewatering the DBM by rinsing a couple of times with several volumes of ethanol (or acetone).
Let me know if you need any clarification on any of the steps. BTW, if you are looking for a source of hydroxyapatite, save the first two acid washes. Combine and filter this solution through a 0.22 micron filter to remove particulates. Place in a beaker with a stir bar and, while stirring on high RPM, add 0.5 N NaOH drop-wise. The solution will turn cloudy by the time you reach pH 6. Allow the precipitate to settle (or centrifuge); decant; and rinse several times with DI water. The recovered precipitate is bone hydroxyapatite and a little osteocalcin (the protein that caps hydroxyapatite crystal growth in native bone).
Best of luck,
JFK
Dear Aditi,
Maybe these papers help you.
Bone regeneration based on nano-hydroxyapatite and hydroxyapatite/chitosan nanocomposites: an in vitro and in vivo comparative study.
In vitro and in vivo investigations on bone regeneration potential of laminated hydroxyapatite/gelatin nanocomposite scaffold along with DBM.