220
Bacterial Cellulose
physiological. conditions.. In. this. work,. chemical. modification. of. BC. by.
2,2,6,6-tetramethylpyperidine-1-oxyl.(TEMPO)-mediated.oxidation.to.introduce. carboxyl. functional. groups,. and. modification. of. the. culture. medium.
G.  xylinus. by. addition. of. amino. sugar. (N-acetylglucosamine). were. performed.to.render.the.BC.materials.biomimetic..TEMPO,.a.water-soluble.and.
stable.nitroxyl.radical,.is.well.known.for.its.catalytic.and.selective.oxidation.
of. primary. hydroxyl. groups. of. polysaccharides. under. aqueous. conditions.
(Montanari.et.al..2005;.Saito.and.Isogai.2004)..On.the.other.hand,.the.amino.
sugar. N-acetylglucosamine. (GlcNAc). is. the. monomer. unit. of. chitin,. the.
second. .most.abundant.natural.biopolymer,.which.is.commonly.found.in.the.
shells.of.marine.crustaceans.and.cell.walls.of.fungi.(Roberts.1992)..GlcNAc.
also.shares.the.structural.feature.of.glycosaminoglycan.(GAGs),.which.are.
unbranched.heteropolysaccharides.consisting.of.the.repeating.unit.[uronic.
acid-amino. sugar] n .. GAGs. are. extracellular. matrix. (ECM). constituents. of.
skeletal.tissues.and.are.considered.to.play.an.important.role.in.stimulating.
chondrogenesis. by. modulating. chondrocyte. morphology,. differentiation,.
and.function.(Di.Martino.et.al..2005;.Suh.and.Matthew.2000)..Considering.
the. function. of. GAGs. in. the. ECM. of. skeletal. tissues,. our. strategy. was. to.
attempt. incorporating. an. amino. sugar. in. the. BC. to. prepare. a. biomimetic.
ECM..The.overall.objective.is.to.fabricate.a.new.generation.of.BC-based.biomimetic.composites.for.potential.osteological.applications.
Preparation of BC Templates
Microbial Synthesis of BC from Standard Culture Medium (Native BC)
The.bacterial.strain.of.cellulose-producing.species,.Acetobacter aceti.(AJ12368),.
closely.related.to.G. xylinus,.and.the.Schramm–Hestrin.(SH).medium.(Hestrin.
and.Schramm.1954).were.used.for.BC.production.(Nge.and.Sugiyama.2007)..
The.pH.of.the.medium.was.adjusted.to.5.with.1N.hydrochloric.acid.(HCl).
and. autoclaved. at. 120°C. for. 20. min.. The. cells. used. as. the. inoculum. were.
statically.precultured.in.test.tubes.at.27°C.for.2.weeks..The.thick.gelatinous.
membrane. was. squeezed. aseptically. to. remove. cells. embedded. inside. the.
pellicle..A.25.mL.cell.suspension.(1.3.×.10 7 .cells/ml).was.then.transferred.to.
500.ml.fresh.medium.and.successively.distributed.(9.ml/well).into.six-well.
mately.35.mm.Φ).recovered.after.static.culture.at.27°C.for.9.days.were.purified.in.two.steps..First,.the.pellicles.were.placed.in.boiling.distilled.water.for.
1.h,.followed.by.incubation.in.0.1.M.sodium.hydroxide.(NaOH).at.80°C.for.
2h..The.pellicles.were.then.washed.with.distilled.water.until.neutral.pH.was.
reached..Some.of.the.purified.pellicles.were.subjected.to.freeze-drying.after.