thermoresponsive macroporous scaffolds prepared by emulsion templating

CommunicationMacromolecular

Rapid Communications

Thermoresponsive Macroporous Scaffolds Prepared by Emulsion Templating

Shengzhong Zhou , Alexander Bismarck , * Joachim H. G. Steinke *

A versatile method to prepare non-covalently crosslinked polyHIPEs hydrogels from oil-in-water high internal phase emulsions (HIPEs) whose aqueous phase contained thermo-responsive linear polymers is described. The interconnected pore structure of the polyHIPEs is maintained by reversible physical aggregation of thermo-responsive polymer chains in an aqueous environment. This method to prepare intercon-nected porous hydrogels using a thermal trigger in the guise of thermo-responsive polymers by emulsion templating requires no chemical reac-tion during solidifi cation of the template. This particular feature could provide a safer route to injectable scaffolds as issues of polymerisation/crosslinking chemistry and residual initiator frag-ments or monomers do not arise

1. Introduction

Tissue engineering aims to create living three-dimensional (3D) tissues to regenerate, repair, or replace biological func-tions. Scaffolds, either preformed or injectable, offer phys-ical support as well as a 3D environment to facilitate the formation of new tissue through cell growth. [ 1 , 2 ] Compared with preformed scaffolds, injectable scaffolds offer many advantages such as reduced scar formation, patient dis-comfort, and risk of infection. [ 3 , 4 ] Cells, drugs, and bioactive molecules (e.g., growth factors) can be incorporated into the scaffold through mixing prior to injection [ 3 ] and irregular

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Dr. S. Zhou, Dr. J. H. G. Steinke Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK E-mail: [email protected] Prof. A. Bismarck Department of Chemical Engineering, Polymer & Composite Engineering (PaCE) Group, Imperial College London, South Kensington Campus, London SW7 2AZ, UK E-mail: [email protected]

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defects can be fi lled, which is not possible with preformed scaffolds. [ 4 ] Recently HIPEs have been used as a precursor to prepare highly interconnected porous scaffolds for tissue engineering. Covalent crosslinking was employed at physiological temperature to set in place and maintain the desired pore structure and with it impart mechanical prop-erties. [ 5–10 ] However, covalent crosslinking is also the pri-mary reason that limits the application of HIPE templates for injectable scaffolds. The crosslinking chemistry can lead to irritation of the surrounding tissue and may be diffi cult to control (in the case of radical [ 8 , 11 , 12 ] or sol–gel chemis-tries [ 13 ] ) or is too slow (when using enzymes) [ 14 ] to obtain the desired porous network structure. Here we describe the sole use of non-covalent interactions to crosslink injectable HIPEs using the phase transition of a thermoresponsive polymer with a lower critical solution temperature (LCST). The LCST of the polymer is close to body temperature so that a crosslinked macroporous hydrogel with an intercon-nected pore structure is created when it solidifi es once the HIPE has reached body temperature.

Beyond the realm of tissue engineering, HIPEs and poly(merised)HIPEs are also of considerable interest with potential applications in areas such as oil recovery, [ 15 ]

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water fi ltration membranes, [ 16 ] and food engineering (e.g., mayonnaise [ 17 ] ). HIPEs are defi ned as emulsions with an internal (or dispersed) phase volume ratio of 0.74 or greater. [ 11 , 12 ] When monomers, contained in the contin-uous phase of a HIPE, are polymerised, a polyHIPE is pro-duced with emulsion droplets embedded in the resulting polymer matrix. PolyHIPEs are characterised by high porosity and an interconnected pore structure [ 12 ] from which the internal phase can be removed (usually through evaporation or extraction). [ 12 , 18–22 ] PolyHIPEs commonly contain regular interconnected pores with pore sizes ranging from around 1 to more than 100 μ m. [ 11 ]

As interconnectivity, a hallmark of polyHIPEs, is con-sidered to be a necessary requirement for nutrient/waste diffusion to support cell growth and migration, HIPE-tem-plated materials have been actively investigated for tissue engineering although the focus has been on preformed scaffolds, [ 5–9 ] rather than injectable ones onto which cells are seeded and then implanted into a patient through invasive surgery.

An injectable polyHIPE scaffold requires the HIPE precursor to solidify (crosslink), only once injected. A non-covalent crosslinking approach, as introduced here, should ensure that an in vivo polyHIPE scaffold formation gives no rise to any irritation of the surrounding tissue, as no covalent bond forming reaction is necessary. Most HIPEs are crosslinked by radical polymerisation, [ 8 , 11 , 12 ] sol–gel reaction, [ 13 ] or enzymatic crosslinking. [ 14 ] As our crosslinking process is triggered by a temperature change, it also makes it easier to control the time point at which crosslinking occurs and avoid premature crosslinking, which otherwise can lead to clogging during the injection through, e.g., hypodermic needles. Thus a polyHIPE scaf-fold produced through a phase change of a thermosensi-tive polymer may be able to address the current limita-tions of available crosslinking chemistries.

Poly( N -isopropylacrylamide) (polyNIPAAm) is a ther-mosensitive polymer with a LCST in water of about 32 ° C. Below its LCST, polyNIPAAm chains form random coil structures in water but at temperatures above its LCST, polyNIPAAm chains expel water and collapse through a process of conformational reorganisation. [ 23 , 24 ] With the ability to reversibly phase separate and dissolve close to body temperature, polyNIPAAm and its biocom-patible biodegradable copolymers [ 25 ] continue to be inves-tigated widely for tissue engineering and drug delivery application. [ 4 ]

Covalently crosslinked polyNIPAAm derived polyHIPEs have been reported already [ 17 , 18 , 26 , 27 ] but we speculated that the physical aggregation between thermo-reversible polymer chains by itself, such as during the LCST transi-tion in polyNIPAAm, may provide enough non-covalent interpolymer chain interactions to stabilise a typical

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polyHIPE porous structure. Therefore the moulding and shaping of a scaffold could become possible below the LCST of whichever thermoresponsive polymer may have been selected for a particular application.

In our design we also took account of the fact that the wettability of a scaffold for tissue engineering is very important for cell seeding and proliferation in three dimensions. [ 28 ] Cell adhesion on synthetic polymer sur-faces is generally poor due to their low hydrophilicity and lack of surface cell recognition sites. [ 29–31 ] In order to improve the biocompatibility of our scaffolds, poly-NIPAAm was grafted to dextran. Dextran is a hydrophilic naturally occurring polysaccharide, which is better toler-ated by the human body than most synthetic polymers and exhibits good biocompatibility. [ 32 ] However, for in-situ applications one will have to consider that poly-NIPAAm fractions will be released after degradation of dextran, which results in signifi cant regulatory barriers to medical use. [ 25 ] However, polyNIPAAm based hydro-gels having the same thermal response as conventional polyNIPAAm but a much lower toxicity have already been reported. [ 25 ]

Following the strategy outlined above, we describe a versatile method to prepare oil-in-water (o/w) HIPEs and the corresponding non-covalently crosslinked poly-HIPEs [ 33 ] from water-soluble thermo-responsive linear polymers, with potential use as novel injectable scaffolds for tissue engineering (Figure 1 ).

2. Experimental Section

2.1. Synthesis of Dextran- b -PolyNIPAAm

Dextran (5.00 g) was dissolved in distilled water (50 mL) in a three necked fl ask which was placed in an oil bath at 27 ° C and purged with nitrogen. The nitrogen atmosphere was maintained throughout the polymerisation reaction. Cerium(IV) ammonium nitrate (0.01 mol, 5.50 g), nitric acid (69%, 6.10 g), and NIPAAm (5.00 g) were added to the reaction system. After 4 h the reaction was stopped by addition of 1 N sodium hydroxide solution. The reaction mixture was then transferred to a dialysis tube (MWCO = 12 000 Da) and dialyzed against distilled water for 3 d. The dis-tilled water was changed twice a day. A white fl uffy product was obtained after drying. The yield was 44%, which was low, how-ever, this was attributed to the removal of low-molecular-weight polymers during dialysis. The ratio of dextran glucose repeat units to polyNIPAAm repeat units was determined by comparing the 1 H NMR spectra of dextran, NIPAAm, and dextran- b -polyNIPAAm copolymer which were recorded in D 2 O using a Bruker DRX 400 (400 MHz) at room temperature. The spectra were processed using the software MestRe-C (version 4.8.1.1). The NMR analysis was carried out according to Wang et al. [ 34 ] Specifi cally, signals corre-sponding to the anomeric proton of the glucose ring of dextran at 4.9 ppm and the proton at the anomeric carbon of the α -1,3

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Figure 1 . Illustration of the formation of crosslinked o/w thermo-responsive polyHIPEs employing the temperature-triggered LCST phase transition of polyNIPAAm chain segments as a means of non-covalently crosslinking injectable HIPEs.

linkages at 5.1 ppm were clearly observed both in the 1 H NMR spectra of dextran and the dextran- b -polyNIPAAm copolymer. The proton signals of NIPAAm, such as the two methyl groups (1.1 ppm), the isopropyl methine group (3.8 ppm), and the back-bone CH and CH 2 groups (1.8 and 1.5 ppm) were identifi ed in the 1 H NMR spectra of NIPAAm and the dextran-b-polyNIPAAm block copolymer. The ratio of glucose units of dextran to NIPAAm units was 1:0.67.

2.2. Preparation of Dextran and Dextran- b -PolyNIPAAm PolyHIPEs

General procedure for the preparation of thermoresponsive poly-HIPEs exemplifi ed for polyHIPE DN1 . DN1 was prepared by dis-solving dextran- b -polyNIPAAm (0.050 g) in 2.5 mL of distilled water together with Triton X-405 (0.21 g) at room temperature. The solution was placed into a reaction vessel. The dispersed phase ( p -xylene, 22.5 mL) was added dropwise under stirring (450 rpm) at room temperature. The shear force was provided by an overhead stirrer with a D-shaped paddle. The HIPE preparation was complete once all the p -xylene was added. Approximately 2.5 mL of a p -xylene-containing HIPE was taken up in a 5 mL syringe and gently passed through a hypodermic needle (ID = 1.1 mm) into a round bottom fl ask. The emulsion was heated to 38 ° C for 30 min in an oil bath and the resulting material lyophilised for 2 d. The fi nal product was a soft white solid. The yield was 73% (attributed to the loss caused by incomplete transfer of the very viscous HIPE from the reaction vessel to freeze dryer). The com-position of dextran and dextran- b -polyNIPAAm HIPEs are listed in Table 1.


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2.3. Preparation of a PolyNIPAAm Solid Above the LCST

PolyNIPAAm N1 (0.30 g) was dissolved in 1.5 mL of distilled water and water was removed from the solution using a rotary evap-orator with the water bath set at 40 ° C to afford a transparent solid which was freeze-dried prior to use.

2.4. Solubility Tests of Thermoresponsive PolyHIPEs and Thermo-responsive PolyNIPAAm

Thermoresponsive polyHIPE DN1 or polyNIPAAm N1 (∼0.09 g) was placed into a glass beaker containing distilled water (200 mL) at either 24 or 38 ° C. The time of dissolution was defi ned as from when the solid came in contact with the water until the visual disappearance of all solids. (Dissolution times of thermo-responsive polyHIPE DN1 are listed in Table 3. Dissolution time of polyNIPAAm N1 at 24 ° C was around 12 min.)

3. Results and Discussion

We synthesised polyNIPAAm-grafted dextran using cerium( IV ) ammonium nitrate as a radical redox initi-ator, as described by Wang et al. [ 34 ] According to detailed investigations by Chauvierre et al. [ 35 ] and Bertholon et al., [ 36 ] the product of the radical polymerisation between dextran and water-soluble vinyl monomers ini-tiated by the redox system dextran and cerium( IV ) salts at low pH, leads to linear block copolymers. A dextran poly NIPAAm block copolymer (dextran- b -polyNIPAAm) was synthesised in this way with molecular weight

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change for the thermoresponsive polyHIPEs above their

Table 1. Composition of dextran and dextran- b -polyNIPAAm copolymer HIPE formulations.

Sample Code Aqueous phase/oil phase (v/v)

Polymer in aqueous phase

Aqueous phase composition: a) polymer/Triton X-405 [w/v%]

Organic Phase

DN0 1/9 dextran 20/8.5 p -xylene

DN1 1/9 dextran- b -polyNIPAAm

20/8.5 p -xylene


20/8.5 p -xylene


25/8.5 p -xylene

a) Concentration of dextran or dextran- b -polyNIPAAm copolymers and Triton X-405 in distilled water.

averages of M w = 28 000 Da and M n = 13 600 Da deter-mined by aqueous gel permeation chromatography (GPC). The LCST of this block copolymer was determined to be 34 ° C through turbidimetry of a 0.2% w/v aqueous solu-tion heated from 24 to 38 ° C. This LCST is suffi ciently removed from room temperature not to cause any pre-mature phase transition before and during injection and suffi ciently below body temperature to ensure a phase change under physiological conditions.

Several thermoresponsive polyHIPEs were prepared by o/w HIPE templating with dextran- b -polyNIPAAm copolymer as the constituent of the continuous aqueous phase. Triton X-405 (hydrophilic–lipophilic balance, HLB ≈ 18 [ 37 ] ) was chosen as the emulsifying surfactant stabiliser. For our initial investigation p -xylene was chosen as a model oil phase. It allows us to observe the pore morphology of the thermo-responsive polyHIPEs by SEM analysis as p -xylene can be removed directly through freeze drying unlike other water-immiscible organic solvents (e.g., toluene), biocom-patible squalene or even degradable oils, such as herring or soybean oil. As the freeze-drying step is only introduced to facilitate the characterisation of the scaffold morphology and not required when injecting the thermo-responsive polyHIPE, p -xylene therefore can be substituted appropri-ately with biocompatible oils. Emulsifi cation was carried out with stirring at 450 rpm and the investigated HIPE formulations are listed in Table 1 . In order to simulate the shear experienced by the HIPE when injected, a syringe was fi lled with the HIPE formulation and squeezed through a hypodermic needle into a fl ask. The fl ask was heated to 38 ° C for 30 min to trigger the phase transition of the dextran- b -polyNIPAAm copolymer. This was followed by lyophilisa-tion of the polyHIPE, producing a white fl uffy solid with a physical strength similar to popcorn. Serving as a control, a pure dextran polyHIPE ( DN0 ) was also prepared.

Scanning electron microscopy (SEM) analysis revealed that both the dextran polyHIPEs and dextran- b -polyNI-PAAm polyHIPEs clearly possess an open-porous structure (Figure 2 A, B, D and E). The pore diameters range from 4

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to 7 μ m (Table 2 ). Compared to the thermoresponsive dex-tran- b -polyNIPAAm polyHIPEs ( DN1 , DN2 , and DN3 ), DN0 possesses no clear pore throats (Figure 2 C) and its pore size is considerably smaller ( ≈ 2 μ m).

The pore morphology changed with increasing concen-tration of dextran- b -polyNIPAAm in the aqueous phase from 20% (w/v) (Figure 2 B; DN1 ) to 25% (w/v) (Figure 2 E; DN3 ), the latter exhibiting slightly thicker pore walls. DN3 with 25% (w/v) polymer concentration exhibited more uniform spherical pores. Reducing the volume per-centage of the dispersed phase also led to thicker pore walls (Figure 2 D).

The LCST of polyNIPAAm is reported to be 32 ° C, [ 38 ] and increased by 2 ° C in the case of the dextran- b -polyNI-PAAm copolymer to 34 ° C as determined by turbidimetry. Several experiments were conducted in order to investi-gate and compare the dissolution behaviour of our ther-moresponsive polyHIPEs in water below and above their LCST (Table 3 ). First DN0 was placed in distilled water and dissolved instantly at 24 and at 38 ° C as was expected for a pure dextran polyHIPE scaffold. Thermorespon-sive dextran- b -polyNIPAAm polyHIPE DN1 was treated in the same way. It dissolved completely at 24 ° C (below the LCST) but rather than in seconds in the case of DN0 the process took about 10 min to reach completion. We compared the dissolution rate with a freeze-dried sample of a synthesised polyNIPAAm homopolymer ( N1 ) ( M n = 37 600 Da and M w = 96 200 Da) conditioned by lyophili-sation which was carried out above its LCST. The polyNI-PAAm homopolymer required a similar period of time (12 instead of 10 min) to dissolve at 24 ° C. In comparison cova-lently crosslinked polyNIPAAm hydrogels with relatively similar pore size and/or porosity to our polyHIPEs, are reported to take about 20 s [ 39 ] in one example (for 10–50 μ m pore sizes) and in another one 1 min [ 40 ] (10 μ m pore size, 80% porosity) to start from their fully shrunken state (above LCST) and reach their fully swollen state (below LCST). In stark contrast there was no detectable volume


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Figure 2 . SEM images of polyHIPEs DN0–DN3 : A & B) Thermoresponsive dextran- b -polyNIPAAm polyHIPE with A) 90% (v/v) nominal pore volume and B) 20% (w/v) pol-ymer concentration ( DN1 ). C) Dextran polyHIPE with 90% (v/v) nominal pore volume and 20% (w/v) polymer concentration ( DN0 ). D) Thermoresponsive dextran- b -polyNIPAAm polyHIPE with 80% (v/v) nominal pore volume and 20% (w/v) polymer concentration ( DN2 ). E) Thermoresponsive dextran- b -polyNIPAAm polyHIPE with 90% (v/v) nominal pore volume and 25% (w/v) polymer concentration ( DN3 ). F) DN1 soaked in 38 ° C water for 14 d.

LCST (38 ° C) even after having left the polyHIPEs to fl oat on water for 14 d. This behaviour can be explained by assuming that the polyNIPAAm block segments phase inverted above the LCST of the block copolymer while simultaneously producing suffi ciently robust physical


Table 2. Densities, porosity, pore volume, and pore characteristics of dextran HIPE and d

polyHIPE Absolute density a) [g cm − 3 ]

Envelope density b)

[g cm − 3 ]

Porosity b) [%]

Pore volume b)

[cm 3 g − 1 ]

Po

DN0 1.490 0.245 83.6 3.416 1

DN1 1.438 0.131 90.0 6.928 3

DN2 1.500 0.145 90.4 6.243 4

DN3 1.466 0.123 91.6 7.448 4

a) Determined using GeoPyc 1360; b) Determined using AccuPyc 1330; c) Determined by SE

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(non-covalent) crosslinks through aggre-gation with neighbouring polyNIPAAm block segments. After 14 d the thermore-sponsive dextran- b -polyNIPAAm poly-HIPEs were freeze dried and again charac-terised by SEM to establish any changes in their morphology. Indeed two dif-ferent morphologies, globular and fi brous (Figure 2 F), were observed. The globular morphology (diameter ≈ 0.5–0.9 μ m) may be caused by the reorganisation/reshaping of the polymer walls around the pore throats into aggregates of globules in the continuous phase. The fi brous morphology (width ≈ 0.2–0.9 μ m) can be rationalised by plasticisation of the polymer through water, driven by minimisation of interfacial energy (Figure 2 F).

To summarize, our experiments with thermoresponsive dextran- b -polyNI-PAAm polyHIPEs have demonstrated that it is possible to prepare very porous (porosity > 90%) solid (elastic gel) macro-porous hydrogels by purely non-covalent interactions. The interconnected porous structure forms through aggregation of the polyNIPAAm blocks within the block copolymer. We have shown that the porous structure is maintained in an aqueous environment above 37 ° C. This is a new and versatile method to prepare o/w HIPEs and fabricate interconnected porous solid scaffolds using a thermal trigger in the guise of a thermorespon-sive polymer. No covalent bond forming

reaction is required during the preparation of the poly-HIPEs and their thermoresponsive nature allows the dissolution of the polyHIPE below its LCST into its com-ponents which could be an interesting approach for recy-cling as the LCST is tuneable. In particular, the absence

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extran- b -polyNIPAAm polyHIPEs.

re size c) [ μ m]

Pore throat size c) [ μ m]

Pore wall thickness c)

[ μ m]

.4 – 2.4 — 0.3 – 1.4

.6 – 7.5 0.4 – 1.3 0.2 – 1.6

.0 – 7.0 0.5 – 1.8 0.5 – 3.4

.7 – 7.3 1.0 – 2.7 0.5 – 2.5

M image analysis.

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Table 3. Observations made during solubility assessments of dextran polyHIPE and thermoresponsive dextran- b -polyNIPAAm polyHIPE in water, above and below the LCST.

Polymer Water

24 ° C 38 ° C

Dextran polyHIPE ( HIPE DN0 ) Dissolved instantly Dissolved instantly

Dextran- b -polyNIPAAm thermo-responsive polyHIPE ( HIPE DN1 )

Broke up into small pieces which sank to the bottom of container and dissolved

completely within 10 min

Floated in water, no apparent volume loss was observed visually during 14 d

of reactive chemistry otherwise required to solidify the HIPEs should provide a safer route to injectable scaf-folds. Issues of polymerisation/crosslinking chemistry or the biocompatibility of residual initiator fragments or monomers do not arise in our case. For the purpose of in vivo use, the internal oil phase can be easily substituted by other water-immiscible non-cytotoxic liquids, such as squalene or herring oil. [ 36 , 37 ] The potential health risk arising from the use of amphiphilic surfactants can be addressed by substituting them for non-toxic nanoparti-cles as emulsion stabiliser. [ 38 , 41 ]

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements : This work was supported by the Challenging Engineering Programme ( EP/E007538/1 ) of the UK Engineering and Physical Science Council and a China-UK Scholarships for Excellence award from China Scholarship Council for Shengzhong Zhou.

Received: May 15, 2012; Revised: June 22, 2012; Published online: ; DOI: 10.1002/marc.201200336

Keywords: emulsions ; polyNIPAAm ; porous materials ; thermo-responsive polymer

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