Engineering microporosity in bacterial cellulose scaffolds

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE R E S E A R C H A R T I C L EJ Tissue Eng Regen Med 2008; 2: 320–330.Published online 10 July 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.97

Engineering microporosity in bacterial cellulosescaffolds

Henrik Backdahl1,2, Maricris Esguerra2, Dick Delbro3,4, Bo Risberg2 and Paul Gatenholm1*1Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Goteborg,Sweden2Vascular Engineering Centre, Department of Surgery, Sahlgrenska University Hospital, SE-413 45 Goteborg, Sweden3School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82, Kalmar, Sweden4Department of Surgery, Sahlgrenska University Hospital, SE-413 45 Goteborg, Sweden

Abstract

The scaffold is an essential component in tissue engineering. A novel method to prepare three-dimensional (3D) nanofibril network scaffolds with controlled microporosity has been developed. Byplacing paraffin wax and starch particles of various sizes in a growing culture of Acetobacter xylinum,bacterial cellulose scaffolds of different morphologies and interconnectivity were prepared. Paraffinparticles were incorporated throughout the scaffold, while starch particles were found only in theoutermost area of the resulting scaffold. The porogens were successfully removed after culture withbacteria and no residues were detected with electron spectroscopy for chemical analysis (ESCA)or Fourier transform infra-red spectroscopy (FT–IR). Resulting scaffolds were seeded with smoothmuscle cells (SMCs) and investigated using histology and organ bath techniques. SMC were selectedas the cell type since the main purpose of the resulting scaffolds is for tissue engineered bloodvessels. SMCs attached to and proliferated on and partly into the scaffolds. Copyright 2008 JohnWiley & Sons, Ltd.

Received 8 January 2008; Revised 10 April 2008; Accepted 17 April 2008

Keywords microporous scaffold; bacterial cellulose; particle leaching; smooth muscle cells

1. Introduction

A scaffold seeded with cells is generally considered theparadigm of tissue engineering. Scaffolds with differentarchitectures and pore sizes can be manufactured fromsynthetic or naturally occurring materials. It has beenfound that when the scaffold has a nanoscale morphology,cell attachment, proliferation and expression of matrixcomponents is increased (Pattison et al., 2005). Ascompared with macroscale scaffolds, nanoscale scaffoldspossess larger surface areas for the adsorption of proteinsand present many more binding sites to cell membranereceptors (Stevens and George, 2005).

Fibrils in the nanoscale are produced by the majorityof biological systems. Collagen, for example, is the maincomponent of animal and human connective tissue and

*Correspondence to: Paul Gatenholm, Department of Chem-ical and Biological Engineering, Chalmers, SE-412 96Goteborg, Sweden. E-mail: [email protected]

is a widely used scaffold for tissue-engineering purposes.Blood vessels (Weinberg and Bell, 1986) and skin (Augeret al., 1998) are just a few examples of tissues which havebeen engineered using collagen scaffolds. The material isbiodegradable but also potentially immunogenic. Most ofthe collagen used is of bovine origin but attempts havebeen made to produce synthetic collagen (Paramonovand Hartgerink, 2005). Plants but also bacteria producenanofibrils composed not of collagen but based oncellulose. Bacterial cellulose fibrils are in the nanometerscale and are microscopically similar to collagen fibres(Backdahl et al., 2006). This makes them interesting touse as a collagen-mimicking component in scaffolds.

Cultured bacterial cellulose (BC) consists of anentangled nanofibrous network, which provides theBC network with strong mechanical properties. Severalresearch groups have tried to alter the porosity of BCnetworks. A very thin honeycomb-patterned BC networkcan be fabricated by culturing Gluconcetobacter xylinus ona patterned agarose film (Uraki et al., 2007). Attempts

Copyright 2008 John Wiley & Sons, Ltd.

Engineering microporosity in bacterial cellulose scaffolds 321

have been made to introduce foreign substances into aBC network. Solid particles (aluminium, iron, silica geland glass beads, among others) have been incorporatedwhile culture was taking place in a rotating disk reactor.No effect on the rate of BC formation was observed whenthis experimental approach was used. The particles usedwere not removed after culture but were instead allowedto remain in the structure in order to alter the propertiesof the resulting product (Serafica et al., 2002).

Porogen/particle-leaching techniques have been usedby several research groups for scaffold fabrication. Theporogens are usually placed in a mould and a polymeris cast in such a mould. This is followed by a leachingstep to remove the porogen particles from the system,leaving a scaffold of the cast polymer. Different porogenmaterials have been used, including salt (Mikos et al.,1994), paraffin (Ma and Choi, 2001), ice (Chen et al.,2001), gelatine (Zhou et al., 2005) and sugar (Capeset al., 2005), among others.

Smooth muscle cells (SMCs) are of particular interestin tissue engineering of blood vessels, since they make upa part of the native vessel wall. SMCs were cultured on BCin a previous study by the present team, and an ingrowthof up to 40 µm in width was detected (Backdahl et al.,2006). The material has also been tested in vivo withgood biocompatibility results (Helenius et al., 2006). Abacterial cellulose substrate has been shown to improvecell viability compared to cell culture plastics (Watanabeet al., 1993). The SMCs are able to contract and relaxin response to different types of stimuli in vivo. By theuse of organ bath methodology (Percy, 1996) and/or amyograph (Mulvany and Halpern, 1977), SMC reactivitycan be investigated in vitro. The former method wasutilized in the current study. This implies that mechanicalactivity (i.e. contractions or relaxations) of a piece oftissue may be investigated by suspending the tissue in anorgan chamber, in which it is bathed in a physiologicalsalt solution (Krebs buffer) at a constant 37 ◦C, which isgased for optimal oxygen tension and pH. One end of thetissue is attached to a force transducer for the monitoringof mechanical activity on-line by a pen recorder. Thetissue may be stimulated either electrically (by means of abipolar electrode, surrounding the tissue) or chemically,by adding a muscle-stimulating or -inhibiting compoundto the chamber. The action of such a compound may beterminated by repeated rinsing of the tissue with Krebsbuffer.

In the present study, the particle leaching technique wasmodified to be used in growing cultures of Acetobacterxylinum. This resulted in a novel method to introducemicroporosity in BC tubes intended as scaffolds fortissue-engineered blood vessels. Preliminary cell studieswere destined to investigate whether pore size is aregulator of cellular ingrowth. It was further exploredwhether the SMC ingrowth would alter the biomechanicalproperties of the scaffold, as studied by the organ bathtechnique.

2. Materials and methods

2.1. Particles preparation

Potato starch particles (Lyckeby Culinar AB, Fjalkinge,Sweden) with a size range of 5–100 µm diameter anda mean diameter of 43 µm were used with no fur-ther processing. Polyvinyl alcohol (PVA) powder (averagemolecular weight 30 000–70 000) (Sigma-Aldrich, Stein-heim, Germany) was allowed to dissolve in deionizedwater, 0.5% (g/ml). The solution was heated to 70 ◦Cand stirred at 800 r.p.m. Paraffin wax (Joel SvenssonsVaxfabrik, Ljungby, Sweden) was allowed to melt on ahot plate. The melted paraffin was then poured slowly intothe heated PVA solution to form particles, as describedelsewhere (Ma and Choi, 2001). The particles were thenthoroughly rinsed in deionized water and sieved to collectparticles in the range 90–500 µm diameter.

2.2. Sterilization of particles

Starch and wax particles were placed in separate beakersand an ethanol : water mixture (70 : 30) was added sothat all particles were immersed. Gentle stirring ensuredthat all particles came into contact with the solution. Thebeaker with particles was then placed in a freezer for 24 hprior to freeze-drying (PowerDry PL3000, Heto).

2.3. Fermentation of porous bacterial cellulosescaffolds

Corn steep liquid media, described elsewhere (Matsuokaet al., 1996), was used as a culture medium for both pre-cultures and scaffold fermentation. The strain used wasAcetobacter xylinum subsp. sucrofermentas BPR2001, tradenumber 700 178, purchased from the American TypeCulture Collection (LGC Promochem AB, Boras, Sweden).Pre-cultures of BC were made as described elsewhere(Bodin et al., 2007). Fermentation of the scaffolds wasperformed submerged in 70 ml glass tubes, using asilicone tube (outer diameter 6 mm, thickness 0.5 mm;Lebo Production AB, Skogas, Sweden) as an oxygen-permeable material. The same set-up as described inearlier work for producing BC tubes was utilized here(Bodin et al., 2007). An illustration of the set-up can befound in Figure 1.

Four types of culture were produced: normal BC tubeculture as reference and using three different porogens,viz. potato starch, paraffin wax and fused paraffinparticles. Porogen particles and culture medium weregently stirred together in a beaker to moisten all particlesbefore filling the culture glass tubes. The paraffin particleswere fused by placing the particle-filled glass tubes ina 40 ◦C water bath for 30 min. After pre-culture thebacteria were liberated from the resulting BC hydrogelby vigorous shaking and 2.5 ml bacteria suspensionwere added to each fermentation vessel, yielding a cell

Copyright 2008 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2008; 2: 320–330.DOI: 10.1002/term

322 H. Backdahl et al.

Figure 1. Schematic picture of the fermentation set-up. Paraffin particles float and starch particles sink in the culture medium. Thebacteria produce an entangled bacterial cellulose network on and between the particles close to the silicone tubing

concentration of 3.7 × 106 cfu/ml. Oxygen (100%) wasblown into the silicone tube during fermentation. Thescaffold fermentations were completed after 7 days.

2.4. Particle leaching, cleaning and sterilizationof porous scaffolds

Excess particles were rinsed away by washing the scaffoldsin Millipore water. These were then boiled in 0.1 M

NaOH at 60 ◦C for 4 h and then repeatedly washed inMillipore water at 60 ◦C. Different cleaning procedureswere then developed and used, depending on the porogenchosen, all carried out in a shaking water bath.

2.4.1. Paraffin particles

Scaffolds were placed in a 1 vol% solution of the surfactantBerol 543 (Akzo Nobel, Stenungsund, Sweden) for 24 hat 75 ◦C, followed by a cleaning step in ethanol at 75 ◦Cfor 12 h. These steps were repeated until no signs ofparticles were seen under a light microscope. The scaffoldswere then cleaned by repeated washing in Millipore

water (60 ◦C) prior to electron spectroscopy for chemicalanalysis (ESCA).

2.4.2. Starch particles

Scaffolds were boiled repeatedly in water for 30 min todissolve the starch particles; this step was completedbefore the NaOH treatment described above. The pH ofthe water was adjusted to 4.4 with acetic acid beforestarting the enzymatic cleaning step. 200 µl of the starchdegrading enzyme Termamyl Ultra 300L (Novozymes,

Bagsvaed, Denmark) were added and kept at 90 ◦C for1 h. The temperature was then lowered to 65 ◦C and200 µl of another enzyme, AMG 300L (Novozymes), wereadded and the reaction was allowed to continue for 12 h.The scaffolds were placed in a 1 vol% solution of Berol543 for 24 h at 75 ◦C followed by a cleaning step inethanol at 75 ◦C for 12 h. Repeated cleaning steps withMillipore water were then carried out to finish thecleaning procedure.

Cleaning cycles were undertaken until ESCA showedthat the material was free from porogen residues. Thescaffolds were then steam sterilized (1 bar, 120 ◦C) for20 min and kept refrigerated until use.

2.5. Electron spectroscopy for chemical analysis(ESCA)

Samples were allowed to dry at 30 ◦C in an oven for 24 h toform films for analysis. The ESCA analysis was conductedon a Quantum 2000 scanning ESCA microprobe (PhysicalElectronics, Chanhassen, MN, USA). An Al Kα (1486.6 eV)X-ray source was used, and the beam size was 100 µm.Five areas of each sample were analysed, 500 × 500 µmeach, with a take-off angle of 45◦ with respect to thesample surface. The depth was approximately 4–5 nm.

2.6. Culture of smooth muscle cells (SMCs)on/in bacterial cellulose scaffolds

2.6.1. Cell isolation and culture

Human SMCs were prepared from macroscopicallyhealthy spare parts of the great saphenous veins obtained



from by-pass surgery donors. The study was approvedby the ethics committee at Gothenburg University.The cells were isolated from the medial layer by anexplant technique previously described (Heydarkhan-Hagvall et al., 2003). The medial layer was separatedfrom the intima and the adventitia and cut into smallpieces (1 mm2), which were placed in six-well polystyrenetissue culture plates. The pieces of tissue were coveredwith cover slips and the wells filled with 2 ml Dulbecco’sModified Eagle Medium (DMEM) supplemented with 20%fetal calf serum (FCS), penicillin/streptomycin (5 U/ml),Na-pyruvate (1 mM), non-essential amino acids (0.1 mM,Invitrogen, Paisley, UK) and L-glutamine (2 mM). Theculture medium was changed once a week for the first2 weeks and then every 72 h. The cells were grown untilconfluence and were then detached with trypsin/EDTA(PAA, Linz, Austria) for expansion by seeding the cellsfrom one confluent culture plate into three new. Afterthe first passage the amount of FCS was changed to10% in the culture medium, and cultures were allowedto proceed until passage 5, when the cells were usedfor experimentation. The SMCs tested negative formycoplasma contamination.

2.6.2. Cell culture on/in bacterial cellulosescaffolds

Cells were cultured in a migration chamber, as describedin an earlier report (Backdahl et al., 2006). The tubularporous scaffolds were cut open with scissors and laid flatwith the inner side down in Transwell culture plateinserts (Corning, NY, USA). SMCs were seeded ontoeach piece of BC (about 150 000 cells/piece BC) andthe inserts filled with 150 µl DMEM 10% FCS. The wellswere filled with 1 ml DMEM, 20% FCS with an additionof 10 ng/ml platelet derived growth factor (PDGF; AMSBiotechnology, Oxon, UK). The cells were cultured for 1or 2 weeks; three samples of each material and time pointwere used.

2.7. Scanning electron microscope (SEM)analysis

The porous BC scaffolds were visualized using an SEM(LEO 982 Gemini field emission SEM). BC samples werequenched in liquid nitrogen and freeze dried. The sampleswere then mounted on SEM Specimen Pin Al-stubs (AgarScientific Ltd, Stansted, UK) with the help of Carbon Tabs(Agar Scientific Ltd) and were sputtered for 2 min usingpalladium in a Emitech K550X Sputter Coater (Emitech,Ashford, UK).

2.8. Histochemical analysis

The samples were fixed in 4% phosphate bufferedformaldehyde at room temperature overnight anddehydrated in a series of ethanols in the range 70–99.5%,

followed by a xylene step. The specimens were thenembedded in paraffin and routinely processed as 5 µmsections for light microscopy. The sections were dewaxedin xylene and rehydrated in a series of 99.5% and95% ethanols, then stained with haematoxylin and eosin(H&E) in order to be able to detect the SMCs with thehelp of a light microscope (Axiophot, Zeiss, Germany).Briefly, the sections were rinsed in water and then stainedwith Mayer’s haematoxylin for 6 min, rinsed again in tapwater followed by rinsing with 1% HCl in 70% ethanolfor 30 s and finally rinsed in tap water. The sectionswere then stained with eosin for 1 min and dehydratedin a series of 95% and 99.5% ethanols and xylene.Finally, the sections were mounted with Pertex mountingmedium (Histolab AB, Goteborg, Sweden) and a coverslip. The Alcian blue and van Geison stains were made byHistolab AB (Histolab AB, Goteborg, Sweden). Collagenwas visualized by UV light in a fluorescence microscope(Imager M1, Zeiss). Collagen is autofluorescent, sono staining was needed. The same microscope wasused for visualization of bacterial cellulose nanofibrilsafter staining with Fungifluor, solution A, for 1 min(Polysciences, Eppelheim, Germany).

2.9. Analysis of mechanical activity

The set-up of organ bath methodology can be foundelsewhere (Percy, 1996). A strip of the cellulose (5–8 ×2–3 mm), with or without SMCs, was mounted verticallyin the organ chamber under an initial load of either 5 or2 mN. One end of the strip was attached to a Grass FT03 transducer for the monitoring of mechanical activityon a Grass polygraph (Grass Instruments Co., Quincy,MA, USA). The samples tested were SMCs cultured onporous BC scaffolds for 2 weeks (three samples, 5 mNinitial load), and BC reference material without cells (11samples, 2 and 5 mN initial load). For comparison, afemale weight-stable (approximately 20 g) mouse of theC57 bl/6 strain (Scanbur, Sollentuna, Sweden) was sac-rificed by cervical dislocation and the gallbladder wasdissected and mounted in the organ bath chamber underan initial load of 2 mN. The preparations were bathedin continuously oxygenated (95% O2, 5% CO2) Krebssolution, containing (mM): NaCl 115.5, KCl 4.6, KH2PO4

1.2, NaHCO3 21.9, MgSO4 1.2, glucose 11.5 and CaCl22.5 at 37 ◦C. After an equilibration period of 60 min witha wash-out at 15 min intervals, various contractile orrelaxatory chemical compounds were added to the prepa-rations for 5 min, followed by washout for 30 min. Thecompounds tested were papaverine sulphate (a smoothmuscle relaxant; Recip AB, Arsta, Sweden), KCl (whichcauses cell depolarization and thereby muscle contraction;Merck, Darmstadt, Germany), noradrenaline (a contrac-tile agent via α-adrenoceptors; APL, Goteborg, Sweden),adrenaline (a relaxatory agent via β-adrenoceptors; MerckNM AB, Stockholm, Sweden) and acetylcholine iodide(a contractile agent via cholinoceptors; Sigma-Aldrich,St. Louis, MO, USA). The porous BC scaffold with cells



and gallbladder were only exposed to KCl and papaver-ine, while the other tests on BC reference material wereperformed to further analyse its characteristics.

2.10. Tensile testing

An Instron Corp 1122 tensile tester (Instron Corp.,Norwood, MA, USA) with the computer softwareTestWorks 4 (MTS Systems Corp, Eden Prairie, MN,USA) was used. Each sample’s width and thickness wasmeasured and used as input data. The cross-head speedwas set to 50 mm/min.

2.11. Fourier transform infra-red spectroscopy(FT–IR)

The spectroscopy was performed using a Perkin-ElmerFTIR sytem 2000 (Perkin-Elmer, Waltham, MA, USA).Small fractions of the dried scaffold samples were mixedwith KBr (Merck, Darmstadt, Germany). The mixturewas then pressed into disks with 10 tons pressure for1 min. 20 scans were made in the wavelength interval of370–4000 cm−1. As a pure cellulose reference material,a filter paper was used (Munktell Filter AB, Grycksbo,Sweden).

3. Results

3.1. Fermentation of microporous BC scaffolds

The porogen materials behaved differently in thefermentor because of differences in density. Paraffin

particles floated in the culture medium and were packedat the top, while starch particles sank and were packed atthe bottom of the fermentation vessel. This resulted in asmall area with no porogen particles in all fermentations;this area was removed after culture. When both particletypes were transferred to the same bioreactor, a tube-like scaffold displaying three distinct regions was formed:porous from starch at the bottom, a non-porous regionin the middle and porous from paraffin in the upper part(Figure 1).

The outer part of the resulting tubular scaffolds changedwith the type of porogen used. Potato starch particles(5–100 µm) were smaller than the paraffin particles(90–500 µm), which influenced the resulting pore size(Figure 2).

Cross-sections of the different scaffolds showed thatall porogen materials were incorporated but that thisoccurred to different extents. Starch resulted in onlya modification in the outermost layer of the structure.Paraffin originated pores through half of the scaffold,although with the exception of a few areas pores werefound throughout the entire scaffold. The swelling ofthe potato starch particles during the cleaning procedurecaused many of the pores to break, leaving only a smallarea with pores close to the outer surface. This explainsthe differences in scaffold thickness seen in the pictures(Figure 3).

By fusing the paraffin particles together, connectionsbetween the particles could be made so that the resultingscaffold material had pores throughout the entire wall, onthe inner side (Figures 2–4); a comparison of Figures 2Dand 4B shows that the wall of the pores is denser onthe inner side than on the outer (see the square marked

(A)

(C) (D)

(B)

Figure 2. SEM images of the outer sides of bacterial cellulose (BC) tubes cultured without porogens (A), BC cultured with potatostarch (B), BC cultured with paraffin (C) and BC cultured with fused paraffin particles (D). The outer side of the BC network seenin (A) is the densest, followed by potato starch-cultured BC (B). The two paraffin-cultured scaffolds are similar to each other, buta difference in the degree of packing can be seen. Fusion of the paraffin particles seems to affect how tightly the paraffin particlesare packed together, the interconnectivity of the pores and space between the pores. The circle in (C) marks an area between pores



(A)

(C)

(B)

(D)

Figure 3. Cross-sections of BC tubes without porogens (A), BC cultured with potato starch (B), paraffin (C) and fused paraffin (D).The inner side is located at the bottom of the pictures (even though the border is not visible). BC has a layered structure towardsthe inner side and a more homogenous BC network toward the outer side (A). Potato starch particles were only integrated inthe outermost part of the tube (B). Paraffin particles became integrated about halfway through the BC tube wall (C), while fusedparaffin cultures had particles throughout the wall (D)

(A) (B)

Figure 4. SEM images taken on the inner side of paraffin-cultured BC, with (B) and without (A) fused particles. A few pores wereseen on the inner side of paraffin-cultured BC, indicating that pores were distributed throughout the wall, but only in a limitednumber of places. The fused paraffin culture had pores that were more evenly distributed on the inner side of the scaffold. Thesquare highlights an area with dense pore walls

area in Figure 4B in comparison with a similar area inFigure 2D).

3.2. Purification of scaffolds

In order to assure complete removal of porogen materials,purification procedures were developed and the scaffoldswere analysed using ESCA and FT–IR. The scaffolds weredried before analysis, resulting in thin films about 5 µmin thickness. The chemical composition of the surface wasinvestigated using ESCA, while the entire cross-sectionwas analysed using FT–IR. The scaffolds were successfully

purified and no trace of residues could be detected, eithervisually (Figure 7) or chemically (Table 1, Figure 6). Theamount of porogen material influences the number ofpurification steps needed. An example of the cleaningprocedure can be seen in Table 1.

3.3. Tensile test

The incorporation of pores into the scaffolds resulted in alower stress at break and modulus compared to a normalBC tube (Table 2).



Table 1. ESCA results of a BC scaffold produced with paraffinand one made with potato starch as porogen

No. of cleaningcycles

C–C(%) O : C

N(%)

0 Paraffin 43 0.44 7.292 Paraffin 64 0.31 0.424 Paraffin 10 0.78 0.267 Paraffin 4 0.83 00 Potato 22 0.55 5.781 Potato 27 0.58 1.872 Potato 6 0.79 0

All analyses were made after the ethanol cleaning step, with theexception of ‘1 Potato’, which was taken after enzymatic treatment.C–C, (contribution to C(1s)peak); O : C, (O : C atomic ratio). After culture,the paraffin BC scaffold had a C–C contribution of ∼42% and an O : Cratio of 0.4, while this was lowered to only C–C ∼ 4% and O : C = 0.8after all cleaning steps. The nitrogen content (N) also decreases withincreasing number of purification sequences. For scaffolds made withpotato starch, a lower number of cleaning cycles was needed. All valuesare the mean of the five areas analysed from each sample.

Table 2. Tensile test results from tests on normal BC tube,scaffold made porous with paraffin and fused paraffin

Sample nameStress at break

(MPa)Modulus

(MPa)

BC ref 0.33 (0.10) 8.25 (1.14)Paraffin 0.37 (0.16) 5.97 (1.9)Fused paraffin 0.27 (0.12) 5.73 (2.14)

BC ref, BC tube: Paraffin, scaffold made porous with paraffin; Fusedparaffin, scaffold made porous with fused paraffin.Values are presented as mean (SD). By fusing the paraffin, pores areobtained throughout the entire scaffold wall, resulting in a lower stressat break and modulus.

3.4. Smooth muscle cell culture on the scaffolds

SMCs are one of the cell types found in some blood vessels;they have the ability to contract and relax in their nativestate and thus were used in these experiments. SMCs werecultured on the materials and samples were harvestedafter 1 and 2 weeks of culture. The SMCs grew on andpartly into the material. The surface seeded with cellscontained varying amounts of cells; in some cases therewas a single cell layer, while in others there was a layeredstructure of about 200 µm. On scaffolds made with potatostarch only, a single cell layer was observed. SMCs couldbe found throughout the BC scaffolds that had beenconstructed of paraffin particles. Collagen productioncould be seen microscopically after 2 weeks of culture(Figure 5).

3.5. Analysis of mechanical activity

The tests showed that the SMC-seeded scaffolds didnot contract when exposed to KCl, neither did theyrelax when papaverine was used. Unexpectedly, in 4/11experiments, the BC reference relaxed when challengedwith the smooth muscle relaxant papaverine. The BCreference at best relaxed 1.25 mN after 2 min, while thegall bladder showed a relaxation of 1.5 mN after the same

time. No response was observed for the other relaxatory(i.e. noradrenaline or adrenaline) or contractile (i.e. KClor acetylcholine) compounds under investigation.

4. Discussion

Acetobacter xylinum was able to produce bacterialcellulose (BC) when paraffin particles and starch particleswere present in the culture medium. The bacteria grew onand between the porogen particles, creating a BC networkaround them (Figure 1). Previous studies on BC tubeproduction have shown that BC production increased withhigher oxygen ratio (Bodin et al., 2007). When the samebacteria are cultured statically, a pellicle is formed thatpropagates downwards (Borzani and de Souza, 1995).Comparison between the inner and the outer side of thefused paraffin-cultured scaffold shows a difference in porewall structure. The walls of the pores are denser on theinner side than on the outer side (Figures 2D, 4B). Oneexplanation for this discrepancy could be that, on theinner side, more bacteria are active and are localizedcloser to the oxygen support, resulting in denser porewalls.

A difference in BC network density was observedwhen comparing the pore walls with their surroundings.Cellulose synthesis was observed to occur more rapidlywhen the Acetobacter cells were attached to the static partsinside an air-lift fermentor (Vandamme et al., 1998). Ifparticles were placed in the same fermentor, the cellsattached as a biofilm on those particles (Vandamme et al.,1998), similarly to the particles used in this study. Thevelocity of movement of A. xylinum on a nematic orderedcellulose surface is ∼4.5 µm/min at 24 ◦C (Kondo et al.,2002), while movement in culture medium is about2 µm/min at 25 ◦C (Brown et al., 1976). The velocityof movement of A. xylinum on a paraffin surface was notmeasured, but an increase in movement velocity couldbe one explanation for the BC nanofibril network beingdenser around the porogen particle than in the poresurroundings (Figure 7A). Another explanation relies onthe fact that bacteria proliferate more abundantly whenattached to a surface than when moving through themedia. In that case the particles would have a greaternumber of bacteria attached to them, resulting in a denserBC network.

The scaffolds were kept in a wet state throughoutthe fermentation and cleaning processes, since drying orusing many solvents influences the structure. Removalof porogens that had been trapped in the bacterialcellulose network was a real challenge. Scaffold thicknessinfluences the time needed for purification and thenumber of purification steps required. A long purificationtime is required for paraffin, since the wax material mustbe given time to diffuse out of the central regions of thescaffold. Initially, an attempt was made to melt out theparaffin, but a substantial amount of paraffin remainedin the scaffold. The use of the surfactant Berol 543 made



(A) (B)

(C)

(E) (F)

(D)

Figure 5. H&E staining of SMC after 2 weeks of culture on a porous BC scaffold made with paraffin particles as porogens (A) andpotato starch particles as porogens (B). Alcian blue and van Gieson staining of scaffold made with paraffin (C) and fused paraffinscaffold (D). Fluorescent microscope images of collagen visualized with UV light (D) and together with fungiflour staining of the BCscaffold (F). The same porous BC scaffold made with paraffin as porogen material is seen (A, C, E, F). All scaffolds have the seededside to the upper left in the pictures. SMCs formed a thick cell layer on the seeded outer surface, ranging from ∼200 µm (A) to asingle cell layer (B). Scaffolds made with potato starch only had a single cell layer on them (B). The scaffold is easier to distinguishwith the Alcian blue and van Gieson stain than with the H&E stain. A small number of SMCs can be found in the scaffolds madewith paraffin, up to half-way into the scaffold (C). SMCs were found to have migrated all the way to the inner side on some placesof the scaffolds made with fused paraffin particles (D). SMCs have produced collagen in the scaffold made with paraffin as porogen(E, F). The BC scaffold wall is clearly distinguished with the fungiflour stain (F). The space indicated by a black bar in the figures iscaused by sample preparation. All pictures were taken after 2 weeks of SMCs culture

it possible to overcome this problem. Berol 543 has anoptimal temperature range around the melting point ofthe wax used, ∼75 ◦C. This non-ionic surfactant formsmicelles around paraffin residues that allow them todiffuse out of the material. The size of the micelles wasnot controlled and it is possible that some of them becameso large that they were entrapped in the scaffold. This was

the reason for using ethanol, which is a suitable solventfor non-ionic surfactants. The starch particles swelledwhen placed into the culture medium and kept at 30 ◦C;the extent of swelling was found to be approximately13% over an observation period of 6 days. One of thecleaning steps for removing starch involves boiling, whichbreaks up the starch granules, since they swell more than



200%. This swelling was higher than the capacity of theBC network to withstand it and, accordingly, some ofthe pores broke. This finding may explain why starchparticles were only incorporated close to the outer side ofthe scaffolds.

To investigate whether the samples were clean,several areas were studied with ESCA, since the samplematerial was greater in size than the scanning areaof 500 × 500 µm. The depth of analysis was 4–5 nm,while the dried scaffold was about 5 µm thick. The longpurification times allowed paraffin residues to diffuse outof the scaffold. Thus, some eventual residues should bedetectable with ESCA, despite the differences in samplethickness and depth of analysis. Great differences could beobserved with increased cleaning steps (Table 1). FT–IRwas used to investigate cross-sections of the scaffoldsto be sure that all paraffin residues had been removed(Figure 6). The scaffolds were considered clean, sincealmost no variations were found between them and a purecellulose reference material (Figure 6). A comparison scanof potato starch and cellulose was also made, and distinctpeaks at ∼924, ∼857 and ∼761 were found on the starchsample but not in cellulose. There is no peak indicating agreater absorbance in the cleaned scaffold at that range

(should be a downwards peak) (Figure 6B). No signs ofeventual paraffin residues could be visualized with SEMeither (Figure 7B).

A cell layer was observed on all seeded scaffold sides(seeded side is upper left in the figures), ranging fromone cell layer to layers of up to 200 µm in thickness(Figure 5A). For scaffolds made with potato starch, acell layer of only a few cells was seen (Figure 5B).The SMCs grew mostly on and partly into porous BCscaffolds made with paraffin. A small number of SMCscould be found throughout the scaffolds but they werenot evenly distributed. Scaffolds pre-treated with a fusiontime of 30 min (Figure 6D) had cell ingrowth to agreater depth than on scaffolds with no pre-treatment(Figure 6C). This was most likely due to the betterinterconnectivity between the pores, as seen on the SEMpictures (Figure 3C, D). These were far better results thanfor a BC pellicles, where an ingrowth of only ∼40 µm wasseen (Backdahl et al., 2006). The SMCs had producedsome collagen fibres both in the surface cell layer andfurther into scaffolds made with paraffin (Figure 6E).Fungifluor stains both cellulose and collagen, which madeit possible to clearly visualize the structure of the BCscaffold (Figure 6F). It was not clear whether the cell

Figure 6. FT-IR spectra illustrating absorbance (A) to wave length (X-axis). Comparison between pure cellulose (A and B, dottedline) and cleaned BC scaffolds made with the porogen materials paraffin (A, black) and potato starch (B, black). The lower curvecorresponds pure cellulose subtracting the scaffold. Both scaffolds are very similar to pure cellulose; no residues of potato starchor paraffin can be seen. When considering the peaks of the comparison curve a difference at ∼3500 (A and B) and ∼1600 (B) canbe noted, due to changes in water content between the samples. The peak at ∼2350 corresponds to CO2 in the test chamber. Thescaffold is considered clean



(A) (B)

Figure 7. SEM image of a dense pore (right) and surrounding BC network (A) and a higher magnification of the pore wall (B). Asseen in (A), some of the pores had a denser BC network than the fibrils connecting the pores. The dense pore wall is composed ofsmall fibrils in the nanometer scale and has no visual signs of eventual paraffin residues (B)

layers had incorporated BC nanofibrils. The scaffolds arenot completely flat, and cells could therefore have beenclumped together during the seeding, giving a tentativeexplanation for the changes in thickness. The thick celllayers could have incorporated BC nanofibrils; however,this is unlikely, since denser unpopulated areas shouldthen have been observed in the cell layers, similarly to thevisible scaffold areas (Figure 6F).

No mechanical response was registered for the SMC-seeded scaffolds and there could be several explanationsfor this. In order to obtain a mechanical response by aSMC-seeded scaffold when challenged with appropriatepharmacological probes, it is a prerequisite that the SMCsbe distributed throughout the scaffold, with intercellularjunctions formed so that the cells can contract or relax.Otherwise, it is not likely that any mechanical responsecan be recorded. The SMCs also need to be contractile.SMCs can be guided towards a proliferative and syntheticphenotype by the use, for example, of platelet-derivedgrowth factor (PDGF). This event is probably beneficialfor cell migration, but it makes the cells lose theircontractile phenotype (Thyberg, 1996; Stegemann andNerem, 2003). This notion, when taken together withthe discussion on PDGF (above), may explain whyno mechanical responses of the seeded scaffolds wereobserved. Surprisingly, relaxatory responses of the BCnetwork reference material were sometimes found in theorgan baths when exposed to papaverine. A previous studyhas shown that a cellulose film could show a large bendingdisplacement with low actuation voltage and low powerconsumption (Kim et al., 2006). This phenomenon is theresult of a combination of two mechanisms: ion migrationand dipolar orientation (Kim et al., 2006). It was noticedthat papaverine seemingly reacted with some componentof the Krebs solution, resulting in a precipitate when thecompound was added to the organ bath chambers. Theother substances tested did not behave in such a way.The formation of a solid material has probably influencedthe ion balance in the system. Papaverine is likely tohave reacted with some of the ions of the Krebs solutionrather than with those found within the BC nanofibrilnetwork, since the former were more easily accessible.This would result in a higher concentration of those ions

inside the BC network than in the surrounding Krebssolution. Accordingly, those ions would diffuse out of theBC network, bringing their associated water with them.This, in turn, could cause a volumetric change similar towhat was described by Kim et al. (2006), resulting in therelaxation observed. This hypothesis will, however, needa more thorough investigation to be verified.

Cellulose is degradable in nature by fungal and micro-bial enzymes, and such enzymes are, to our knowledge,absent in the human body. Degradation occurs by hydro-lase attack on the β(1–4) linkages. Degradation studies ofcellulose sponge (Martson et al., 1999), regenerated cel-luloses and cellulose derivatives (Miyamoto et al., 1989)have been carried out. Cellulose sponges were regardedas a slowly degradable implantation material; it was notfully degraded after 60 weeks. A study of BC degradationby our group showed no direct sign of degradation after12 weeks of implantation (Helenius et al., 2006). Basedon this knowledge the scaffolds described in this paperwould not degrade in a normal cell culture, giving thempromising properties as scaffolds for long term 3D cell cul-tures. This would, however, require a degradation studyon BC to be verified.

There are several advantages of the scaffolds presentedin this work compared to many of the synthetic scaffoldsused today. They are built up of nanofibres that arevisually similar to collagen. The water-holding capacity ishigh and there is no observed degradation of the scaffolds.Since the scaffolds are built up by an entangled nanofibrenetwork, with fibres of great length, the mechanicalproperties are likely to be stronger than other scaffoldswith the same solid material content.

5. Conclusions

It is possible to construct porous scaffolds by culturingAcetobacter xylinum together with porogen materials ofstarch and paraffin. A microporous BC scaffold wasconstructed with the help of porogens when placedin bacterial cellulose tube fermentation. The scaffoldspresented in this paper are built up of bacterialcellulose fibrils in the nanometer scale. A toolbox was



developed, allowing alteration of the porosity, thicknessand interconnectivity of the scaffolds by varying theporogen sizes and fermentation conditions. The porogenscould be removed with several cleaning steps. Smoothmuscle cells could migrate into the material to a greaterextent than in a pellicle produced in a static culture. Thesescaffolds may be suitable for 3D cell cultures, but futurework will show whether it is possible to make such seededscaffolds respond in a way similar to native blood vessels.

Acknowledgements

The authors wish to thank Professor Ulf Nannmark for his kindand helpful advice, especially regarding SEM analysis; ProfessorKrister Holmberg for consultation regarding surfactant selection;Lena Hallsberg and the University of Kalmar for help with theanalysis of mechanical activity; Anne Wendel for assistancewith ESCA analysis; and Anders Martensson for assistancewith FT–IR measurements. The authors acknowledge fundingfrom the Swedish Research Council (Grant No. 621-2004-3824),Biosynthetic Blood Vessels (Grant No. 311-2006-7570) and EUExpertissues (Grant No. NMP3-CT-2004-500283).

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Engineering microporosity in bacterial cellulose scaffolds

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