Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)

8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)

httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 113

Chemical characterisation and fabrication ofchitosanndashsilica hybrid scaff olds with3-glycidoxypropyl trimethoxysilanedagger

Louise S Connella Frederik Romerb Marta Suarezcd Esther M Vallianta Ziyu Zhanga

Peter D Leee Mark E Smithbf John V Hannab and Julian R Jonesa

Chitosan has been explored as a potential component of biomaterials and scaffolds for many tissue

engineering applications Hybrid materials where organic and inorganic networks interpenetrate at the

molecular level have been a particular focus of interest using 3-glycidoxypropyl trimethoxysilane

(GPTMS) as a covalent crosslinker between the networks in a solndashgel process GPTMS contains both an

epoxide ring that can undergo a ring opening reaction with the primary amine of chitosan and a

trimethoxysilane group that can co-condense with silica precursors to form a silica network While manyresearchers have exploited this ring-opening reaction it is not yet fully understood and thus the 1047297nal

product is still a matter of some dispute Here a detailed study of the reaction of GPTMS with chitosan

under different pH conditions was carried out using a combination of solution state and solid state MAS

NMR techniques The reaction of GPTMS with chitosan at the primary amine to form a secondary amine

was con1047297rmed and the rate was found to increase at lower pH However a side-reaction was identi 1047297ed

between GPTMS and water producing a diol species The relative amounts of diol and chitosanndashGPTMS

species were 80 and 20 respectively and this ratio did not vary with pH The functionalisation pH had

an effect on the mechanical properties of 65 wt organic monoliths where the properties of the organic

component became more dominant Scaffolds were fabricated by freeze drying and had pore diameters

in excess of 140 mm and tailorable by altering freezing temperature which were suitable for tissue

engineering applications In both monoliths and scaffolds increasing the organic content disrupted the

inorganic network leading to an increase in silica dissolution in SBF However the dissolution of silica

and chitosan was congruent up to 4 weeks in SBF illustrating the true hybrid nature resulting from

covalent bonding between the networks

Introduction

As the worlds aging population increases the number of indi-

viduals aff ected by bone and cartilage disorders is also

increasing1 To treat these debilitating conditions and address

the scarcity of natural gra material it is necessary for synthetic

materials to be developed that mimic the physical properties of

natural tissue while also stimulating regeneration and remod-

elling by the body2ndash4 Bioactive glass scaff olds have many of the

properties required for tissue engineering scaff olds but they

are too brittle to be used in cyclically loaded applications5 A

so er tougher material is required67 Solndashgel derived hybrid

materials where interpenetrating networks (IPNs) of organic

polymers and inorganic components are covalently bonded8

have shown promise as biodegradable tissue regeneration

scaff olds Natural polymers such as chitosan9ndash14 poly-glutamic

acid15ndash18 and gelatin1920 are covalently bonded to a silica

network via gra ing a silane containing coupling agent to

functional groups along the polymer These hybrid materials

have been formed into scaff olds by various methods including

foaming20 freeze-drying1119 and electrospinning21 The poten-

tial benets of hybrids over conventional composites is that

mechanical properties and dissolution rates can be varied by

a Department of Materials Imperial College London South Kensington Campus SW7

2AZ UK E-mail j ulianrjonesimperialacuk Tel +44 (0) 2075946749b Department of Physics University of Warwick Gibbet Hill Rd Coventry CV4 7AL UK c Fundaci on ITMA Parque Technol ogico de Asturias 33428 Llanera Asturias Spaind Department of Nanostructured Material Centro de Investigaci on en Nanomateriales y

Nanotecnolog ıa Principado de Asturias Parque Tecnol ogico de Asturias 33428

Llanera SpaineSchool of Materials The University of Manchester Oxford Rd M13 9PL UK f The Vice-Chancellors O ffice University House Lancaster University Lancaster LA1

4YW UK

dagger Electronic supplementary information (ESI) available 1H NMR of chitosan and

GPTMS in D2ODCl at pH 4 over time 1Hndash

13C HSQC spectra of fully hydrolysed

GPTMS a er 72 h in D2ODCl at pH 2 29Si MAS NMR spectra of functionalised

chitosan at pH 2 and 4 with table of quantication and thermogravimetric

analysis of 50 wt organic chitosanndashsilica scaff olds a er 0 72 168 and 672 h

in SBF See DOI 101039c3tb21507e

Cite this J Mater Chem B 2014 2668

Received 25th October 2013Accepted 2nd December 2013

DOI 101039c3tb21507e

wwwrscorgMaterialsB

668 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014

Journal ofMaterials Chemistry B

PAPER View Article OnlineView Journal | View Issue









short time-points indicating that the hydrolysis of the silane

groups was rapid However at 5 min in solution at pH 4 and 6

there were multiple peaks around d1H 053 ppm due to

incomplete hydrolysis of the silane groups (ESI Fig S1dagger)

At pH 2 the hydrolysis was so rapid that no evidence of

partial hydrolysis was observed at 5 min This is in agreement

with Gabrielli et al who observed a pH dependence of the rate of

silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2

protons of the epoxide ring at d1

H 281 ppm and d1

H 261 ppmreduced in intensity over time however this occurred at a much

slower rate than silane hydrolysis New peaks were observed in

the d1H 390ndash330 ppm region although there was considerable

overlap in the 1H NMR spectra making it hard to distinguish

the peaks Using a combination of 13C and HSQC (showing 1H

and 13C coupling through one bond) experiments allowed the

diff erent species to be identied This was conrmed by

repeating the HSQC experiment for GPTMS alone in D2ODCl

a er 72 h at pH 2 where the epoxide ring was fully opened (ESI

Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3

Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C

5939 ppm) and (d1

H 341 ppm d13

C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by

water in solution43 At longer time points but at all pH values

other signals were observed at (d1H 357 ppm d13C 5096 ppm)

and (d1H 357 ppm d13C 5096 ppm) which were attributed to

the reaction of epoxide ring with the primary amine of chitosan

(ndashNH2) to form a secondary amine No other reactions were

identied suggesting that the only covalent coupling reaction

occurring between chitosan and GPTMS occurred at the primary

amine

The use of quantitative HSQC experiments showed that the

extent of epoxide opening a er 24 h decreased as pH increased

9 68 and 98 mol epoxide ring remained at pH 2 4 and 6

respectively (Fig 4a) This supports the observations of Gabrielli

et al that the opening of the epoxide ring of GPTMS in water is

acid catalysed and hence slightly acidic conditions are required

for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent

nucleophilic attack In contrast with the prediction of Gabrielli

et al altering the pH did not aff ect the relative numbers of diol

and secondary amine species formed the percentage of primary

amines that formed secondary amines remained constant at

around 20 (Fig 4b)

Analysis of 15N MAS NMR of chitosan dissolved at pH 4

quenched in liquid nitrogen and freeze dried showed clearly

that in pure chitosan there were two signals due to acetylated

and deacetylated forms of the chitosan monomer (Fig 5a) A er

24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm

split into two indicating a third nitrogen species is present (Fig 5b)

This is unequivocal evidence that there was a reaction

between chitosan and GPTMS at the primary amine It also

shows that the nucleophilic addition between the amine and

Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions

Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h


Journal of Materials Chemistry B Paper

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the epoxide ring is the only covalent bonding which occurs

between the amine and GPTMS However it should be noted

that this does not rule out the possibility that hydrogen bonding

may occur between amine amide or hydroxyl species or that all

of the epoxide groups will react

FTIR spectra of the chitosan functionalised with GPTMS for

24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure

chitosan FTIR spectrum Minor diff erences arise at 1507 cm1

where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group

in chitosan which is more prominent at pH 4 because fewer of

the amine groups were converted to secondary amines There is

no evidence of the epoxide ring remaining at either pH 2 or pH 4

as the bands for CndashOndashC stretching of GPTMS would be expected

at 909 cm1 and 846 cm131 This is potentially due to the small

amount of GPTMS used relative to the amount of chitosan and

diol formation which reduces the relative amount of epoxide

ring further Mahony et al showed in a silicagelatin system that

the bands corresponding to unopened epoxide ring could not

be distinguished until a molar ratio of GPTMS to gelatin of 1500

was used (at pH 5)

20

Structural characterisation of hybrid monoliths

The chemical structure of the hybrids was characterised in

order to determine the eff ect of pH and organic content on the

monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-

cated by combining hydrolysed TEOS with the chitosanndashGPTMS

solution at pH 4 or pH 2 to give a composition of 65 wt

organic show a strong SindashOndashSi stretching band that appeared at

1020 cm1 The band at 934 cm1 was attributed to non-

bridging SindashOH bonds and appears moreintenseat pH 2 than at

pH 4 indicating a more condensed network at pH 4 The

primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-

tionalised chitosan at pH 4 the intensity of the secondary

amine reduced whereas little change was observed at pH 2

Again this may be attributed to more prominent hydrogen

bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a

silica network The nomenclature Qn is used to describe silica

species where the silicon is bonded by n bridging oxygens and 4

n non-bridging oxygens whereas Tn is used to describe a

silicon atom bonded to a carbon (as in GPTMS) with n bridging

oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra

showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which

correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n

respectively44

Peak tting of the one pulse MAS 29Si NMR spectra allowed

quantication of each of the silicon species present in 65 wt

organic hybrids (spectra shown in Fig 7 and calculated

percentage abundance of silicon species in Table 1) In agree-

ment with the FTIR results the hybrids synthesized at pH 4

were more highly condensed than at pH 2 as indicated by the

higher numbers of Q4 and T3 species present In fact at pH 4

there were no Q2 species present whereas there were 50 04

Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h

Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4

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Paper Journal of Materials Chemistry B

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present at pH 2 Calculation of the degree of condensation ( Dc)

gave values of 927 and 902 for pH 4 and 2 respectively The

more condensed network is due to the fact that at pH lt22 the

transition state of condensation is stabilised by the ethoxy and

methoxy groups of TEOS and GPTMS The partially hydrolysed

silica precursor condenses faster leading to chains of silica

network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors

condense fastest leading to highly condensed silica networks

with fewer non-bridging species45 Repeating 29Si MAS NMR for

the functionalised chitosan shows only Tn species as expected

as there was no TEOS present (ESI Fig S3dagger) However it was

observed that within 5 min condensation had occurred

between the GPTMS molecules so that at pH 2 up to 60 of the

GPTMS was present in a T3 form (ESI Table S1dagger) This would

render the molecule unable to condense further when TEOS is

introduced potentially leading to two distinct silica networks

that do not interpenetrate The signicance of this is unknown

and further investigation is required to establish the degree of

interaction between the two networks

SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that

no macroscale phase separation occurred during hybrid

synthesis at any composition (Fig 8) Agglomerated particle

morphologies typical of that formed by the solndashgel process46

were observed This is due to silica nanoparticles that agglom-

erate and fuse to form a mesoporous silica gel46 The apparent

particle diameters were similar for samples made at pH 2 and

pH 4 (compare Fig 8a with b and 8c with d) but larger particles

are observed as organic content increased The particle size of

the 35 wt organic hybrids was more typical for solndashgel silica

microstructures so the larger particle size is likely due to chi-

tosan polymer coating the surface of the silica particles

Mechanical and dissolution properties of monoliths

From compression tests hybrid monoliths containing 35 wt

organic exhibited brittle behaviour with a strain at fracture of 4

to 8 Increasing the chitosan content reduced the brittle

character as shown by the deformation prior to fracture for 65

wt organic monoliths whereas 35 wt organic monoliths

failed catastrophically (Fig 9) The increase in chitosan content

also increased the strain at fracture to around 48 This had the

eff ect of reducing the compressive modulus of the monoliths

Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2

pH Q4 Q3 Q2 T 3 T 2 Dc

4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902

Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species

Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks



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freeze-dried Chitosan has been chosen for scaff old synthesis by

freeze-drying as the polymer forms sheets between the ice

crystals as the sol is forced out of the solidifying pure water

where ultimately the ice crystals form the interconnected pore

structure of the scaff olds4950

Hybrid scaff old morphology

Investigation of the morphology of the scaff

olds by SEM(Fig 11) showed that reducing freezing temperature reduced

the pore diameters This can be attributed to the higher degree

of supercooling that occurs at lower freezing temperatures

hence increasing the nucleation rate of ice crystals Although

more ice crystals form the lower temperatures means that the

growth of the crystals is slower resulting in many small ice

crystals and hence smaller pores in the nal scaff old The pores

were elongated and angular with a certain degree of direction-

ality as the gels tended to freeze from the outside-in with a

protrusion forming in the centre where the ice forced the gel as

it expanded during freezing

Pore interconnectivity and interconnect size is o en more

important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-

sion as a function of pressure Analysis of the modal pore

interconnect diameters by mercury porosimetry conrmed that

the interconnect diameter reduced as the freezing temperature

reduced The scaff olds frozen at 20 C had modal pore

diameters of 178 47 mm and 156 7 mm 80 C were 150

39 mm and 140 15 mm and those quenched in liquid nitrogen

were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic

respectively (Fig 12)

A guide for a suitable interconnect diameter for bone tissue

engineering scaff olds is 100 mm51 At 20 C and 80 C the

interconnect diameters were well above 100 mm Quenching in

liquid nitrogen caused a signicant decrease in pore intercon-

nect diameter The interconnect diameters of 65 wt organic

and 50 wt organic scaff olds were similar at each freezing

temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and

65 wt organic respectively Table 3) This is due to the water

content of the gels prior to freeze-drying The scaff olds with

higher organic content contained relatively more chitosan

solution (17 mg mL1) and so also contain more water When

the water is frozen and removed during freeze-drying the ulti-

mate result is to increase the porosity of the scaff olds

mCT images of the 65 wt organic scaff olds frozen at 20 C

and 80 C shown in Fig 13 illustrate the angular and

Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry

Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature

Organic content (wt) Freezing temp (C) Porosity ()

65 20 975 0480 975 01196 975 02

50 20 969 0280 967 02196 964 01

(Mean SD n frac14 10)

Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying

Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly



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irregular pore morphologies that are characteristic of scaff olds

fabricated via freeze-drying Applying 3D image analysis tech-

niques the modal pore diameter of the 20 C 65 wt organic

scaff old was 313 mm and the modal interconnect diameter was

189 mm which is in good agreement with the mercury poros-

imetry data The images also showed that the scaff olds were well

interconnected important for tissue ingrowth and vasculariza-

tion The mean tortuosity of the scaff olds another property

which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165

024 and 137 031 for 20 C 80 C and 196 C scaff olds

respectively This is within the range reported for cancellous

bone by Pakula et al of 11 to 2852

Mechanical behaviour of the chitosanndashsilica hybrid scaff olds

The mechanical properties of the scaff olds were investigated

under compression and the data is presented in Table 4

A slight increase in the compressive modulus was observed

at 50 wt organic compared with 65 wt organic however due

to the highly porous nature of the scaff olds there was a large

degree of scatter within the data and the diff erence was not

statistically signicant The strain at failure did not vary with

freezing temperature although a small increase in compressive

modulus and compressive strengths was observed for samples

quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids

respectively and 808 620 and 1030 kPa for20 C80 C and

liquid nitrogen 65 wt organic hybrid scaff olds respectively

the compressive strengths are far too low for load sharing

applications for bone regeneration as originally intended This

is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity

Given the promising mechanical properties of the monolith

samples if the porosity were reduced then the compressive

strengths may be increased making them more suitable for

bone regeneration scaff olds Alternatively these scaff olds may

be used in non-load sharing applications such as cartilage

regeneration These scaff olds may be particularly attractive for

cartilage regeneration due to the elongated pore morphologies

and since chitosan has a similar structure to anionic glycos-

aminoglycans found in articular cartilage53

Dissolution behaviour of hybrid scaff olds

The silicon release in SBF as measured in triplicate by ICP-OES

(Fig 10b) was very rapid for both the 65 wt and 50 wt

organic scaff olds The fastest rate of silicon release was up to 8

h with the silicon concentration in solution plateauing at

around 80 g L1 and 90 g L1 for 50 and 65 wt organic

respectively a er 24 h As with the monolith hybrid samples

greater silicon release was observed for higher organic content

hybrids due to disruption of the silica network by the organic

component Phosphorus and calcium ion concentrations did

not vary over the timescale of the experiment (data not pre-

sented) and so it can be concluded that no apatite formed on

the sample surfaces as expected

FTIR analysis of the remaining solids a er 4 weeks in SBF

(Fig 14) showed that the amide I and II bands were retained

although there was a signicant reduction in the intensity of the

amide II band This indicates that there was still chitosan

remaining in the hybrid a er the dissolution study conrmed

by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight

loss by TGA between 200 C and 600 C of the 50 wt organic

scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this

increased to 40 wt and then remained constant at 1 w and 4 w

This suggests that there is rapid silica dissolution within the

rst 72 h as also indicated by the ICP-OES dissolution proles

Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds

Organiccontent (wt)

Freezing temp (C)

Compressmodulus (MPa)

Failurestress (kPa)

Strain at failure ()

65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75


Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion



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whereas chitosan dissolution was slower However a er the

rst 72 h the two components are released at the same rate so

that the relative composition remains constant up to 4 w in SBF

Congruent dissolution seen here a er 72 h is one of the

dening features of a successful hybrid material and so this is a

promising result for the long term mechanical and chemical

stability of the chitosanndashsilica hybrid

Although the assessment of biological activity is beyond the

scope of this article similar chitosanndash

GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-

eration of osteoblastic MG63 cell cultures on chitosanndashsilica

hybrid membranes and freeze dried scaff olds with varying

GPTMS and TEOS contents showed that the hybrid materials

were biocompatible101137 Compared with pure chitosan scaff olds

and membranes the hybrid materials showed better prolifera-

tion and multilayers of well spread MG63 cells a er 6 days in cell

culture10 however the type of silica species present aff ected the

behaviour of the cells with an increase in TEOS promoting

osteodiff erentiation rather than proliferation as seen in hybrids

with high GPTMS contents but no TEOS37 Scaff olds freeze dried

at

20

C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo

studies were carried out in adult female Wistar rats to determine

the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds

and membranes54 For each animal three 2 2 cm samples were

implanted into 3 cm long dorsal incisions and were recovered

a er 1 2 4 and 8 weeks From the results of these studies the

authors are condent that the chitosanndashsilica hybrid materials

presented here would be suitable for tissue regeneration appli-

cations particularly the highly porous freeze dried scaff olds

Conclusions

Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques

showed a reaction between the epoxide ring of GPTMS and

chitosan at the primary amine Following the reaction at three

diff erent pH values has shown that this reaction was acid

catalyzed with signicantly more epoxide ring opening at pH 2

than at pH 4 or 6 However it was also shown that an unwanted

side reaction occurred between water and the epoxide ring

resulting in diol formation and that this was the dominant

reaction at all pH values Hydrolysis of the methoxysilane

groups of GPTMS was rapid under acidic conditions however

condensation occurred simultaneously so that within 5 min T3

species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a

sol of hydrolysed TEOS The silica network of the monoliths was

less condensed when chitosan was functionalised at pH 2

compared with those functionalised at pH 4 This had the eff ect

of increasing the rate of silica dissolution in SBF for the pH 2

sample The eff ect of pH on mechanical properties was minimal

at 35 wt organic as the brittle nature of the silica phase

appeared to predominate However at 65 wt organic the

organic phase had a more signicant eff ect on the mechanical

properties as the elongation at failure was increased from 7 to

40 The samples fabricated at pH 2 which had a greater

degree of coupling between the chitosan and GPTMS showed a

slight increase in compressive modulus

Summary of the fabrication and characterisation of hybrid

scaff olds

Chitosanndashsilica hybrid scaff olds were fabricated by combining

the solndashgel process with a freeze-drying step Chitosan was

functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing

temperatures had a dramatic eff ect on the modal pore inter-

connect diameter Scaff olds fabricated by quenching in liquid

nitrogen had interconnect diameters of 20ndash23 mm which is too

small for tissue engineering applications Scaff olds frozen

at 20 and 80 C are suitable as they have pore interconnects

well in excess of 100 mm the critical value required for tissue

engineering scaff olds The compressive strengths of the scaf-

folds were too low to be used in load-sharing applications

primarily due to their high porosities of 96ndash97 Reducing the

porosity will increase the compressive strengths of the scaff olds

for alternative applications such as non-load bearing cartilage

regeneration may be more appropriate A 4 weeks dissolution

study in SBF showed that silicon release was rapid within the

rst 24 h but a er this time the chitosan and silica are released

at the same rate so that the relative composition of the hybrid

remains unchanged a er 72 h up to 4 weeks This is an

important result that points towards long term mechanical

stability and chemical activity of the scaff olds

Here for the rst time

A combination of solution and solid state NMR techniques

have been used to probe the functionalisation reaction between

chitosan and GPTMS

It has been shown that covalent bonding occurs between

the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between

chitosan and a silica network

The extent of reaction at diff erent pH values was quantied

to show that both the reactions of GPTMS with water and with

chitosan are acid catalyzed and that the relative amounts of

product and side-product does not depend on pH

That functionalisation pH was shown to have an impact on

the mechanical properties of hybrids at 65 wt where the

properties of the organic component become more dominant

That high organic content was shown to disrupt the silica

network speeding up the rate of silica dissolution in both

monolith and scaff old hybrids

The interconnect diameters were quantied for freeze-

dried chitosan scaff olds and conrmed that 20 and80 C are

appropriate freezing temperatures for fabricating tissue engi-

neering scaff olds

Chitosan and silicon were shown to be released congru-

ently when immersed in SBF for up to 4 w

Acknowledgements

The authors would like to thank Mr Peter Haycock Department

of Chemistry Imperial College London for carrying out the



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quantitative HSQC experiments This research has been funded

by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)

and the Department of Materials Imperial College London

EMV was a Natural Sciences and Engineering Research Council

of Canada (NSERC) Canadian Centennial Scholar MS was

supported by Ficyt under the Argo program JVH and MES

acknowledge support for the solid-state NMR facilities at War-

wick used in this research which were funded by EPSRC and the

University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by

Advantage West Midlands (AWM) and the European Regional

Development Fund (ERDF) JVH and MES acknowledge EPSRC

support for FR via project EPI0046881

Notes and references

1 R Burge B Dawson-Hughes D H Solomon J B Wong

A King and A Tosteson J Bone Miner Res 2007 22 465ndash

475

2 L L Hench and J M Polak Science 2002 295 1014ndash1017

3 R Langer and D A Tirrell Nature 2004 428 487ndash

4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281

5 M M Pereira J R Jones and L L Hench Adv Appl Ceram

2005 104 35ndash42

6 J R Jones Acta Biomater 2013 9 4457ndash4486

7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095

8 B M Novak Adv Mater 1993 5 422ndash433

9 Y Shirosaki C M Botelho M A Lopes and J D Santos J

Nanosci Nanotechnol 2009 9 3714ndash3719

10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes

J Santos M Costa and M Fernandes Acta Biomater

2009 5 346ndash355

11 Y Shirosaki T Okayama K Tsuru S Hayakawa and

A Osaka Chem Eng J 2008 137 122ndash

12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes

J D Santos and M H Fernandes Biomaterials 2005 26

485ndash493

13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa

P P Cortez F Gartner J D Santos M A Lopes

S Geuna A S Varejao and A C Mauricio Acta Med Port

2011 24 43ndash52

14 G Toskas C Cherif R-D Hund E Laourine B Mahltig

A Fahmi C Heinemann and T Hanke Carbohydr Polym

2013 94 713ndash722

15 E M Valliant F Romer D Wang D S McPhail

M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671

16 G Poologasundarampillai C Ionescu O Tsigkou

M Murugesan R G Hill M M Stevens J V Hanna

M E Smith and J R Jones J Mater Chem 2010 20 8952

17 G Poologasundarampillai B Yu O Tsigkou E Valliant

S Yue P D Lee R W Hamilton M M Stevens

T Kasuga and J R Jones So Matter 2012 8 4822ndash4832

18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl

2011 25 581ndash594

19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials

2002 23 4765ndash4773

20 O Mahony O Tsigkou C Ionescu C Minelli L Ling

R Hanly M E Smith M M Stevens and J R Jones Adv

Funct Mater 2010 20 3835ndash3845

21 C Gao Q Gao Y Li M N Rahaman A Teramoto and

K Abe J Appl Polym Sci 2013 127 2588ndash2599

22 S V Madihally and H W T Matthew Biomaterials 1999 20

1133ndash1142

23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40

7029ndash

703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal

Charact 1999 5 267ndash276

25 S Minami M Morimoto Y Okamoto H Saimoto and

Y Shigemasa in Materials Science of Chitin and Chitosan

ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006

ch 7 pp 191ndash217

26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23

4915ndash4921

27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and

Y Shirosaki J R Soc Interface 2005 2 335ndash340

28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837

29 A-C Chao J Membr Sci 2008 311 306ndash

31830 J G Varghese R S Karuppannan and M Y Kariduraganavar

J Chem Eng Data 2010 55 2084ndash2092

31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol

2005 35 225ndash235

32 S S Rashidova D S Shakarova O N Ruzimuradov

D T Satubaldieva S V Zalyalieva O A Shpigun

V P Varlamov and B D Kabulov J Chromatogr B Anal

Technol Biomed Life Sci 2004 800 49ndash53

33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8

34 S Prochazkova K M V arum and K Ostgaard Carbohydr

Polym 1999 38 115ndash122

35 L Gabrielli L S Connell L Russo J Jimenez-Barbero

F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848

36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa

Y Nakamura I R Gibson and A Osaka J Ceram Soc

Jpn 2010 118 989ndash992

37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura

I R Gibson and A Osaka in Bioceramics Development and

Applications ed S Kim The Korean Society for

Biomaterials 2009 vol 22 pp 217ndash220

38 S Heikkinen M M Toikka P T Karhunen and

I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367

39 J R Jones G Poologasundarampillai R C Atwood

D Bernard and P D Lee Biomaterials 2007 28 1404ndash

141340 R C Atwood J R Jones P D Lee and L L Hench Scr

Mater 2004 51 1029ndash1033

41 S Yue P D Lee G Poologasundarampillai and J R Jones

Acta Biomater 2011 7 2637ndash2643

42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915

43 L Gabrielli L Russo A Poveda J R Jones F Nicotra

J Jimenez-Barbero and L Cipolla Chemistry 2013 19

7856ndash7864

44 K J D MacKenzie and M E Smith Multinuclear Solid-State

Nuclear Magnetic Resonance of Inorganic Materials Elsevier

Science 2002



View Article Online



45 J D Wright and N A J M Sommerdijk Sol ndash gel materials

chemistry and applications Taylor amp Francis Ltd London 2000

46 S Lin C Ionescu K J Pike M E Smith and J R Jones J

Mater Chem 2009 19 1276

47 J Zhong and D C Greenspan J Biomed Mater Res 2000

53 694ndash701

48 K Tsuru C Ohtsuki A Osaka T Iwamoto and

J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161

49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518

50 S Deville Adv Eng Mater 2008 10 155ndash169

51 S F Hulbert S J Morrison and J J Klawitter J Biomed

Mater Res 1972 6 347ndash374

52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust

Soc Am 2008 123 2415ndash2423

53 A Di Martino M Sittinger and M V Risbud Biomaterials

2005 26 5983ndash5990

54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs

Y Shirosaki M A Lopes J D Santos F Fregnan

G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials

2008 29 4409ndash4419


View Article Online


















H 281 ppm and d1












5939 ppm) and (d1

H 341 ppm d13









amine












around 20 (Fig 4b)














View Article Online






















was used (at pH 5)

20























respectively44













View Article Online













































4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902





View Article Online



















































65 20 975 0480 975 01196 975 02

50 20 969 0280 967 02196 964 01






View Article Online

































































Organiccontent (wt)

Freezing temp (C)


Failurestress (kPa)


65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75





View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online






















was used (at pH 5)

20























respectively44













View Article Online













































4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902





View Article Online



















































65 20 975 0480 975 01196 975 02

50 20 969 0280 967 02196 964 01






View Article Online

































































Organiccontent (wt)

Freezing temp (C)


Failurestress (kPa)


65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75





View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online













































4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902





View Article Online



















































65 20 975 0480 975 01196 975 02

50 20 969 0280 967 02196 964 01






View Article Online

































































Organiccontent (wt)

Freezing temp (C)


Failurestress (kPa)


65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75





View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online



















































65 20 975 0480 975 01196 975 02

50 20 969 0280 967 02196 964 01






View Article Online

































































Organiccontent (wt)

Freezing temp (C)


Failurestress (kPa)


65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75





View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online

































































Organiccontent (wt)

Freezing temp (C)


Failurestress (kPa)


65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32

50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75





View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online























at

20










Conclusions


























scaff olds

























chitosan and GPTMS

















neering scaff olds



Acknowledgements





View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online


















475





2005 104 35ndash42








2009 5 346ndash355





485ndash493




2011 24 43ndash52



2013 94 713ndash722










2011 25 581ndash594


2002 23 4765ndash4773







1133ndash1142


7029ndash






ch 7 pp 191ndash217


4915ndash4921








2005 35 225ndash235












Jpn 2010 118 989ndash992










Mater 2004 51 1029ndash1033






7856ndash7864



Science 2002



View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online






Mater Chem 2009 19 1276


53 694ndash701








Soc Am 2008 123 2415ndash2423


2005 26 5983ndash5990




2008 29 4409ndash4419


View Article Online

Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)

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Transcript of Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)