227
Biomimetic Mineralization of Apatite on Bacterial Cellulose
of.the.dense.surface.layer,.middle.compartment,.and.lower.layer.as.well.as.
variations. of. their. thickness. reflect. the. activity. of. viable. cells,. which. was.
influenced.by.the.absence.or.presence.of.different.GlcNAc.concentrations.
in.the.medium.
Biomimetic Mineralization of Apatite on BC Templates
Soaking BC Templates in an SBF (1.5 SBF)
The.biomimetic.mineralization.of.a.Ca-P.coating.was.performed.in.a.. simple.
supersaturated. ion. solution,. in. which. ion. concentrations. were. 1.5. times.
those.of.the.SBF.developed.by.Kokubo.et.al..(1990)..The.1.5.SBF.was.prepared.
by. dissolving. sequentially. reagent-grade. sodium. chloride. (NaCl),. sodium.
hydrogen. carbonate. (NaHCO 3 ),. potassium. chloride. (KCl),. dipotassium.
hydrogen. phosphate. (K 2 HPO 4 ·3H 2 O),. magnesium. chloride. hexahydrate.
(MgCl 2 ·6H 2 O),.calcium.chloride.(CaCl 2 ),.and.sodium.sulfate.(Na 2 SO 4 ).(Wako.
Pure.Chemical,.Osaka,.Japan).in.distilled.water..The.pH.was.adjusted.to.pH.
7.4.±.0.01.with.tris-hydroxymethyl.aminomethane.(NH 2 C(CH 2 OH) 3 ).and.1.M.
HCl.while.maintaining.the.solution.temperature.at.36.5°C..The.stock.solution.was.filtered.through.a.0.2.μm.cellulose.acetate.filter.
Freeze-dried.BC.templates,.namely.native.BC,.BC-TEMPO,.and.BC-TEMPO-Ca.
with.a.size.of.10.mm.×.10.mm.×.5.mm.and.known.weight.were.soaked.in.10 ml.
of.1.5.SBF.at.37°C.for.3.weeks..The.SBF.solution.was.renewed.every.7 days..
Samples. were. taken. out. at. 2,. 7,. 14,. and. 21. days,. washed. thoroughly. with.
distilled.water,.and.freeze-dried..The.freeze-dried.samples.were.weighed.to.
determine.the.extent.of.mineral.deposition..Four.to.five.pieces.were.used.for.
each.BC.sample.for.each.designated.soaking.time.(Nge.and.Sugiyama.2007).
Soaking BC Templates in Calcium and Phosphate Solutions
The.calcium.solution.(0.05.M.CaCl 2 .in.50.mM.Tris-HCl.buffer.at.pH.7.4).and.
the.phosphate.solution.(0.03.M.NaH 2 PO 4 ).were.prepared.to.obtain.Ca:P.ratio.
of.1.67.similar.to.the.Ca:P.ratio.of.synthetic.hydroxyapatite.(Ca 10 (PO 4 ) 6 (OH) 2 )..
Freeze-dried. BC. samples. of. native. BC,. BC-TEMPO,. BC-TEMPO-Ca,. and.
BC-GlcNAc.were.alternatively.soaked.in.the.calcium.and.phosphate.solutions.
at.37°C.for.30.min.each.in.a.reciprocal.shaking.incubator.(50.strokes/min)..
The. samples. were. rinsed. thoroughly. with. distilled. water. between. each.
soaking. solution.. A. total. of. five. soaking. cycles. were. performed.. Samples.
were.withdrawn.from.each.alternate.soaking.cycle,.gently.and.thoroughly.
washed. with. distilled. water,. and. freeze-dried. to. determine. the. weight. of.
mineralized.Ca-P.that.deposited.on.different.BC.templates.(Nge.et.al..2010a)
Biomimetic Mineralization of Apatite on Bacterial Cellulose
of.the.dense.surface.layer,.middle.compartment,.and.lower.layer.as.well.as.
variations. of. their. thickness. reflect. the. activity. of. viable. cells,. which. was.
influenced.by.the.absence.or.presence.of.different.GlcNAc.concentrations.
in.the.medium.
Biomimetic Mineralization of Apatite on BC Templates
Soaking BC Templates in an SBF (1.5 SBF)
The.biomimetic.mineralization.of.a.Ca-P.coating.was.performed.in.a.. simple.
supersaturated. ion. solution,. in. which. ion. concentrations. were. 1.5. times.
those.of.the.SBF.developed.by.Kokubo.et.al..(1990)..The.1.5.SBF.was.prepared.
by. dissolving. sequentially. reagent-grade. sodium. chloride. (NaCl),. sodium.
hydrogen. carbonate. (NaHCO 3 ),. potassium. chloride. (KCl),. dipotassium.
hydrogen. phosphate. (K 2 HPO 4 ·3H 2 O),. magnesium. chloride. hexahydrate.
(MgCl 2 ·6H 2 O),.calcium.chloride.(CaCl 2 ),.and.sodium.sulfate.(Na 2 SO 4 ).(Wako.
Pure.Chemical,.Osaka,.Japan).in.distilled.water..The.pH.was.adjusted.to.pH.
7.4.±.0.01.with.tris-hydroxymethyl.aminomethane.(NH 2 C(CH 2 OH) 3 ).and.1.M.
HCl.while.maintaining.the.solution.temperature.at.36.5°C..The.stock.solution.was.filtered.through.a.0.2.μm.cellulose.acetate.filter.
Freeze-dried.BC.templates,.namely.native.BC,.BC-TEMPO,.and.BC-TEMPO-Ca.
with.a.size.of.10.mm.×.10.mm.×.5.mm.and.known.weight.were.soaked.in.10 ml.
of.1.5.SBF.at.37°C.for.3.weeks..The.SBF.solution.was.renewed.every.7 days..
Samples. were. taken. out. at. 2,. 7,. 14,. and. 21. days,. washed. thoroughly. with.
distilled.water,.and.freeze-dried..The.freeze-dried.samples.were.weighed.to.
determine.the.extent.of.mineral.deposition..Four.to.five.pieces.were.used.for.
each.BC.sample.for.each.designated.soaking.time.(Nge.and.Sugiyama.2007).
Soaking BC Templates in Calcium and Phosphate Solutions
The.calcium.solution.(0.05.M.CaCl 2 .in.50.mM.Tris-HCl.buffer.at.pH.7.4).and.
the.phosphate.solution.(0.03.M.NaH 2 PO 4 ).were.prepared.to.obtain.Ca:P.ratio.
of.1.67.similar.to.the.Ca:P.ratio.of.synthetic.hydroxyapatite.(Ca 10 (PO 4 ) 6 (OH) 2 )..
Freeze-dried. BC. samples. of. native. BC,. BC-TEMPO,. BC-TEMPO-Ca,. and.
BC-GlcNAc.were.alternatively.soaked.in.the.calcium.and.phosphate.solutions.
at.37°C.for.30.min.each.in.a.reciprocal.shaking.incubator.(50.strokes/min)..
The. samples. were. rinsed. thoroughly. with. distilled. water. between. each.
soaking. solution.. A. total. of. five. soaking. cycles. were. performed.. Samples.
were.withdrawn.from.each.alternate.soaking.cycle,.gently.and.thoroughly.
washed. with. distilled. water,. and. freeze-dried. to. determine. the. weight. of.
mineralized.Ca-P.that.deposited.on.different.BC.templates.(Nge.et.al..2010a)