biocompatibility and in vivo evaluation of oligochitosans as cationic modifiers of alginate/ca...

Biocompatibility and in vivo evaluation of oligochitosansas cationic modifiers of alginate/Ca microcapsules

Maria De Castro,1 Gorka Orive,1 Rosa M. Hernandez,1 Artur Bartkowiak,2 W. Brylak,3 Jose L. Pedraz11Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country,Vitoria-Gasteiz, Spain2Department of Packaging and Polymers, University of Agriculture, Szczecin, Poland3Polymer Institute, University of Technology, Szczecin, Poland

Received 9 July 2007; revised 14 March 2008; accepted 26 March 2008Published online 14 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32270

Abstract: The present article investigates the substitutionof poly-L-lysine by two different oligochitosans as mem-brane-coating capsule, and its effect on different function-ality parameters of cell microencapsulation. To addressthis issue, initially the biocompatibility of the two types ofoligochitosan solutions was evaluated using erythropoietinsecreting C2C12 myoblast cell line as model. In a secondstep, we encapsulated the cells within alginate microcap-sules coated with each oligochitosan and a complete mor-phological and mechanical evaluation was performed.Finally, the in vivo functionality of the enclosed cells in thealginate-oligochitosan microcapsules was studied in Balb/cmice. Results show that both oligochitosans were biocom-patible, not detecting statistical differences between them.

The alginate-oligochitosan microcapsules were totallyspherical and uniform and resulted in high hematocrit lev-els after subcutaneous implantation in mice. In fact, signifi-cantly higher hematocrit levels were found in the animalstransplanted with the encapsulated cells compared withthe control group. Finally, the histological analysis showeda mild fibroblastic reaction around the capsule membrane.These results suggest that alginate-oligochitosan capsulesmay be an interesting alternative to conventional alginate-poly-L-lysine capsules. � 2009 Wiley Periodicals, Inc.J Biomed Mater Res 91A: 1119–1130, 2009

Key words: encapsulation; oligochitosan; alginate; C2C12cells; erythropoietin; mechanical properties

INTRODUCTION

Cell microencapsulation has been widely appliedin various therapeutic approaches including oraldelivery of live bacterial cells,1,2 production of mono-clonal antibodies by immobilized hybridoma cells,3

the development of bioartificial pancreas,4,5 bioartifi-cial kidney,6 and the entrapment of cells geneticallymodified to secrete the desired protein in differentdiseases.7–10 All of these therapeutic approachesrequire the use of biocompatible materials andimmobilization technology to avoid cell rejection.

In general in bioencapsulation the microcapsuleshave to fulfill not only biological but also technologi-cal requirements. Thus, in the development of anyimmobilization system some parameters must becarefully evaluated such as permeability, mechanicalstability, biocompatibility, immunoisolation, the ad-aptation of the system to the nature of the cell andthe presence of vascularization close to the trans-plant.11

Revising the literature the overwhelming majorityof the cell encapsulation groups have used themicrocapsules based on the initial model of Lim andSum,12 only with slight modifications in the elabora-tion procedure. Thus, firstly alginate matrix isformed and secondly the beads are coated with apolycation membrane which consists of poly-L-lysine(PLL) and alginate. However, some reportsaddressed that the biocompatibility of this type ofcapsule could be limited by the immunogenicity andcytotoxicity of PLL.13 Therefore some other polyca-tions such as poly-L-ornithine,14 chitosan,15,16 and oli-gochitosan17 have been recently evaluated as possi-ble alternatives to the classical PLL.

Correspondence to: J. L. Pedraz; e-mail: [email protected] grant sponsor: University of the Basque Coun-

try (UPV/EHU); contract grant number: 9/UPV 00101.125-13496/2001Contract grant sponsor: Basque Government; contract

grant number: ETORTEK 02, IE019

� 2009 Wiley Periodicals, Inc.

Over the last decade, one of the biopolymers thathas gained attention of scientists as an immobiliza-tion matrix material and as coating agent is chito-san.18 Thu et al. 19 reached to the conclusion that thepolyelectrolyte complex formed by the interpolymerionic interaction between chitosan and alginateunder certain conditions can be considered mechani-cally more stable than the complex formed betweenPLL and alginate. The average molar mass (MM)and the fraction of N-acetylation (FA) of chitosanwere considered to be essential variables. Chitosanswith MM below 10,000 g/mol could bind to alginatebeads at higher rates and to larger extent than chito-sans with higher MM. And that occurs becauseshorter chains of chitosans could diffuse fasterthrough cell membranes. On the other hand, thebinding of chitosans was found to increase withdecreasing fractions of FA.15 However, the completesolubility of chitosans (with a MM above 10 kDa)implies the use of a pH value below 6.6 and suchacidic conditions are not well-tolerated by mamma-lian cells.15,16 Recently, some reports have shownthat by adjusting the MM of the oligochitosan in therange of 1000–10,000 g/mol,20 it is possible to obtain,under physiological conditions, biocompatible andmechanically stable capsules.17

The main difference between unmodified oligochi-tosan and the quaternary ammonium derivative oli-gochitosan (modified oligochitosan) is the side chainpresented in the modified form, which results of thereaction between an oligochitosan of defined MM(unmodified oligochitosan) and the 3-chloro-2-hy-droxypropyltrimethylammonium chloride (CHPTMAC).Thus, in unmodified oligochitosan there are only pri-mary amino groups which at a pH value of 7.0–7.4only 10% of these groups are in chemical active/pro-tonated form. Therefore the complex between algi-nate and unmodified oligochitosan is weak ormechanically instable. In contrast, in the modifiedoligochitosan there are quaternary ammoniumgroups which are permanently charged withoutinfluence of pH (at a pH range 2–8). Therefore thepolyelectrolyte complex formed between alginateand modified oligochitosan is less porous and has ahigher mechanical stability.

In the present article a comparative study betweenthese two different oligochitosans as coating agentswas carried out. Thus we have evaluated the bio-compatibility and citotoxicity of the oligochitosans incell culture. Furthermore, microcapsules with eachpolymer have been fabricated and technologicallyevaluated. Finally, to test the long-term functionalityof the different encapsulation systems, we have im-mobilized erythropoietin (EPO)-secreting cells andimplant the capsules with any immunosuppresion inthe subcutaneous tissue of inmmunocompetent allo-geneic Balb/c mice.

MATERIALS AND METHODS

All cell culture media and serum were obtained fromGibco BRL/Invitrogen Life Technologies (Barcelona,Spain). C2C12 myoblasts genetically engineered to secreteEPO were kindly provided by the the Laboratoire D’etudede la Neurodegenerescence of Institut des Neurosciences(Ecole Polytechnique Federale de Lausanne, Lausanne,Switzerland). Low viscosity and high guluronic (LVG) al-ginate (250 mPa s at 2% solution and 258C) was purchasedfrom NovaMatrix/FMC (Oslo, Norway). The remainingchemicals were obtained from Sigma Chemicals (St. Louis,MO) unless otherwise indicated.

Oligochitosans preparation and characterization

Unmodified oligochitosan sample with MM 5 4300 g/mol was prepared by controlled radical degradation viacontinuous addition of hydrogen peroxide to 2.5% chitosansolution of pH 3.5–4.0 at 808C. Chitosan with a MM of1200 kDa and a degree of deacetylation 85% was used asthe starting material (Sigma-Aldrich). The oligochitosansample, after thermal degradation with hydrogen peroxideas chloride salts had relatively low polydispersity around2.0.

Quaternary ammonium derivatives of chitosan (modi-fied oligochitosan) were obtained in reaction between oli-gochitosan of defined MM 4300 g/mol) and CHPTMAC,21

which reacts with the amino or hydroxyl groups of poly-saccharides in neutral or basic pH, respectively.

Oligochitosan (5 g) was dissolved in distilled water(50 cm3) and heated to reaction temperature, then 20 cm3

solution of etherifying agent suspended in aqueous so-dium hydroxide solution (1/1/1 molar equivalent) wasadded. All reactions were carried out in aqueous mediumat 608C for 6 h. The modified oligochitosans were precipi-tated and washed 3–4 times with acetone before drying inoven at 408C. Such products were used without any fur-ther purification. FTIR spectra of oligosaccharide sampleswere recorded on a Nicolet 640 spectrometer using aGolden-Gate ATR system. The gel permeation chromatog-raphy analysis indicates that there is no significant changein MM of both ologochitosan samples, before and aftermodification.

Cell culture and MTT assay

In relation with the cell culture 10,000 C2C12 myoblastcells were seeded into each well of a 96-well cell cultureplate. One hundred microliters of Dulbecco’s modifiedeagle medium (DMEM) with high glucose (4500 mg/L)supplemented with 10% FBS and 1% penicillin/streptomy-cin were added to each well and afterwards the plate wasmaintained at 378C in a humidified 5% CO2/95% airatmosphere. Once the cells were attached to culture clus-ters, culture medium was replaced by 100 lL of one of thetwo oligochitosans in assay (unmodified and modified oli-gochitosans) at one of the two concentrations in assay

1120 DE CASTRO ET AL.

Journal of Biomedical Materials Research Part A

(0.05% or 0.5%). Afterwards the plate was incubated for3 h. The oligochitosans solutions of each concentrationwere prepared in DMEM and they were sterilized by fil-tration (0.2 lm, Iwaki, Japan). The pH of all the solutionswere controlled so that the assay was carried out at physi-ological conditions (pH 5 7.4). Related to the positive con-trol (100% viability) of the assay complete DMEM withoutany oligochitosans was used and to set the negative con-trol (0% viability) a solution of 0.1% (w/v) Triton-100 wasemployed. Mitochondrial activity as an indicator of cellu-lar activity was determined by the tetrazolium assay (theMTT assay) described by Fischer et al.22 The relative cellviability related to the control wells was according to thefollowing formula: [test viability]/[control viability] 3 100.Results for three replicates are expressed as mean 6 stand-ard deviation (SD).

Lactatate dehydrogenase assay

The lactatate dehydrogenase (LDH) assay quantitativelymeasures the cytosolic enzyme LDH concentrations, whichis released upon cell lysis, in the cell culture media after3 h of incubation, using a commercial kit (DG 1340,Sigma). The LDH concentration was determined using themethod based on reduction of nicotinamide adenine dinu-cleotide absorbance at 492 nm with a reference to 690 nmcorresponding to lactate and LDH according to the manu-facture’s protocol. Complete DMEM without any oligochi-tosan was used as negative control (0% LDH release) whileto set the positive control 0.1% (w/v) Triton-100 was used(100% LDH release). The relative LDH release of any sam-ple was determined by [(LDH release test)2(0% LDHrelease)]/[100% LDH release 2 0% LDH release)] 3 100.All results have been expressed as mean 6 SD for threereplicates.

Microscopic observation

After 3 h of incubation with the different oligochitosans,morphology of the C2C12 cells was observed using aninverted optical microscope (Nikon TMS) equipped with acamera (Sony CCD-Iris). After microcapsule preparation, amicroscopic observation was carried out to check the gen-eral morphology all the microcapsules.

Finally, at day 80 microcapsules maintained in vitrowere observed using a confocal scanning laser microscope(Olympus FluoView FV500), applying tetramethylrhod-amine and fluorescein isothiocyanate filter optics. Fluo-resce images were obtained applying an assay calledlive/dead viability/cytotoxicity assay (Molecular Probes,Invitrogen, Barcelona, Spain). Samples were processedaccording to the protocol provided by the manufacturer.Briefly, 20–40 capsules were washed with Dulbecco’sphosphate-buffered saline in order to remove all thegrowth media to avoid the false results due to too highesterase activity. Afterwards the ethidium–calceim mix-ture was added and maintained in the incubator for45 min at 378C. Only live cells with intracellular esteraseactivity could digest non-fluorescent calcein-AM into flu-

orescent calcein, producing an intense uniform green flu-orescence. In contrast, dead or dying cells containingdamaged membranes allowed the entrance of ethidiumhomodimer-1 to stain the nuclei, thereby producing abright red fluorescence. Therefore, this assay represents aqualitative way of evaluate the viability of the immobi-lized cells.

Microcapsule elaboration

All solutions for capsule preparation were sterile filteredand all equipments were autoclaved. Two types of micro-capsules were prepared at room temperature and underaseptic conditions using an electrostatic droplet generator.Both types of capsules differ in the first coating whereunmodified and modified oligochitosans have beenapplied. EPO-secreting C2C12 cells were suspended in1.5% (w/v) solution of LVG alginate to a final concentra-tion of 5 3 106 cells per 1 mL of alginate. The suspensionwas extruded into a 55 mM CaCl2 solution. The alginatedroplets were coated with 1% (w/v) solution of any of theoligochitosans for 20 min. Subsequently, a second coatwith alginate LVG 0.1% during 5 min was applied. Finallythe A-O-A microcapsules were maintained in completeDMEM during the period of the assays at 378C in a 5%CO2 humidified atmosphere and the medium was replacedevery 2–3 days.

Mechanical resistance study

The mechanical characterization of capsules was per-formed using a standard texture analyzer (TA-XT2i, StableMicrosystems, Surrey, UK) at a mobile probe constantspeed of 0.1 mm/s until 85% deformation was reached.The uniaxial compression experiment is used to study thedeformation behavior of the microcapsules.23 The forceexerted by the probe on the capsule was recorded as afunction of the compression distance leading to a force-versus-strain relation. Twenty capsules per batch were an-alyzed to obtain statistically relevant data.

Microcapsule implantation

Female 6 week-old Balb/c mice (n 5 5) have beenselected as allogeneic transplant model. Before implanta-tion capsules were washed several times in Hank’s bal-anced salt solution (HBSS). Under ether anesthesia, 0.2 mLof each type of microcapsule loaded with EPO-secretingC2C12 myoblasts (5 3 106 cells/mL) suspended in 0.5 mLof HBSS were implanted subcutaneously using a catheter(Nipro 18 gauge-, Nisho). Control mice received 1 mL ofHBSS by subcutaneously route. Upon recovery, animalshad access to food and water ad libitum.

Hematocrit measurement

Blood was collected by retro-orbital bleeding on anes-thetized animals using heparinized capillary tubes. During

BIOCOMPATIBILITY AND IN VIVO EVALUATION OF OLIGOCHITOSANS 1121


the first month after transplantation blood samples wereobtained weekly, afterwards the hematocrit was measuredeach 20 days. The hematocrit was measured by a standardmicrohematocrit method.24

In vitro/in vivo characterization of microcapsules

Before transplantation microcapsules were character-ized in term of morphology, and their functionality. Themorphology was evaluated by microscopic observationusing an inverted optical microscope (Nikon eclipseTE2000-S) equipped with a digital camera (Nikon DS-U1).Both, the cell viability and the EPO production rate wereused as capsule functionality indicators. The cell viabilitywas determined by the MTT assay and the EPO produc-tion was measured using an ELISA kit for human EPO.Cross-reaction of the kit allowed the detection of mouseEPO in culture supernatants. EPO secretion from 0.2 mLof A-O-A microcapsules in 24 h was determined. Aftermicrocapsules were explanted the MTT assay and theELISA kit were used to compare the in vitro/in vivoresults.

Histological analysis

At day 82 after implantation, two animals were sacri-ficed and microcapsules were retrieved and fixed in a4% paraformaldehyde solution in 0.1M sodium phos-phate, pH 7.2 for a minimum of 24 h at 48C. Afterwards,paraformaldehyde solution was replaced by 70% (v/v)ethanol solution. Serial horizontal cryostat sections (14lm) were processed for hematoxylin–eosin (H & E)staining.

Statistical analysis

The different parameters under study were comparedby the Student’s t-test or the Mann–Whitney test accordingto the result of the Levene test of homogeneity of varian-ces. When more than two groups were compared one-wayANOVA followed by Scheffe or Tamhane post-hoc testdepending on the homogeneity of variances was applied.SPSS 14.0 software (SPSS, Chicago, IL) was used for thestatistical analysis. Significance was set at p < 0.05 for allanalyses.

RESULTS

Oligochitosan characterization

The degree of chitosan derivatization was monitoredby FTIR spectroscopy (Fig. 1). Peak at 1540 cm21 (A)in unmodified oligochitosan spectrum corresponds toadsorption of amino groups. In the spectrum ofmodified oligochitosan the same peak has consider-ably decreased in intensity due to the reaction ofamine group with epoxide end of quaternary ammo-nium derivative compound. In IR spectra of modifiedoligochitosans one can observe appearance of newpeaks in wave range 1450–1500 cm1 (B) which corre-sponds to specific functional groups of substitutedside chain (methyl groups of CHPTMAC).21

On the basis of relative comparison of characteris-tic peaks at 1200–1250 cm21 and 1H nuclear mag-netic resonance analysis we could determine thedegree of substitution of modified products. Onecould observe that the prepared sample has lower

Figure 1. Comparison of FTIR spectra of unmodified and modified oligochitosan with their characteristic peaks, (A) and(B), respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



than theoretical degree of substitution; however itwas in the range 0.45–0.5.

In vitro cytotoxicity effect of the oligochitosans

Morphological characterization of C2C12 cells

EPO-secreting C2C12 myoblasts were incubated attwo oligochitosan concentrations (0.05% and 0.5%)for 3 h. In all cases the cells appeared as an adherentconfluent monolayer (Fig. 2). There were no signifi-cant changes in cell morphology neither in function

of polymer type, nor in concentration. In fact, thecharacteristic spindle shape of the myoblasts wasmaintained during all the study. No apparent celllysis or cells detached from the culture clusters werefound. On the contrary, treatment with Triton-100,induced an important alteration in cell morphologyand shape (Fig. 2 Cþ).

Metabolic activity of the C2C12 cells

The metabolic activity of the C2C12 cells culturedwith the two types of oligochitosans at two different

Figure 2. Morphological evaluation of the EPO-secreting myoblast cells incubated during 3 h with the two oligochitosans(modified and unmodified) at two concentrations selected (0.05% and 0.5%). The symbol C2 corresponds to the negativecontrol (complete culture medium), whereas Cþ corresponds to the positive control (Triton-100). The photomicrographswere taken with an inverted optical microscope equipped with a camera. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]



concentrations (0.05% and 0.5%) after an incubationtime of 3 h was evaluated using the MTT assay. Ingeneral, cells maintained at least 85–90% of their via-bility at the end of the study compared with the con-trol cells [Fig. 3(A)]. This fact indicates that both oli-gochitosans presented a very reduce cytotoxicity forC2C12 myoblasts. Contrary, cells treated with Tri-ton-100 presented a completely loss of viability.

Lactate dehydrogenase assay

The possible cell damage caused by the two typesof oligochitosans after 3 h of incubation was eval-uated measuring the activity of LDH [Fig. 3(B)].None of the oligochitosans induced a relevant LDHrelease (<5%). These results are complementary tothose obtained in the MTT assay after 3 h of incuba-tion. From a statistically point of view there was nodifference of LDH release for microcapsules coatedwith both types of oligochitosans.

Mechanical resistance study

The mechanical resistance of capsules (mNw/cap-sule) was determined as the force presented by themicrocapsules against compression exerted by themobile probe of the texturometer. This is the so-called bursting force. During the compression of thetwo types of coated capsules one could observe twobursting points. The small first one corresponds tothe outer oligochitosan/alginate Ca complex mem-brane, whereas the second is higher and correspondsto the inner core of alginate/Ca complex. After thefirst point there are many small peaks which corre-spond to micro-cracks due to the rigid structure ofmicrocapsule core25 [Fig. 4(A)].

The mechanical resistance of the both types of oli-gochitosans was studied during a time period of 21days in vitro. Both microcapsules presented a similarcompressive behavior along the time. The burstingforce decreased from 277 6 14 mNw/capsule formodified oligochitosan capsules and 345 6 56mNw/capsule for unmodified oligochitosan capsulesat day 1 to 188 6 12 mNw/capsule and 143 6 13Figure 3. A: Metabolic activity of the EPO-secreting

C2C12 myoblast cells cultured with the two oligochitosansat the two selected concentrations and at an incubationtime of 3 h using the MTT assay. The relative viability ofthe cells was calculated by [test viability]/[control viabil-ity] 3 100. Results are expressed as mean 6 S.D. for threereplicates. B: Lactate dehydrogenase release from the EPO-secreting C2C12 myoblast cells cultured with the two oli-gochitosans at the two selected concentrations and at anincubation time of 3 h. The relative LDH release was cal-culated by [LDH release-low control]/[high control-lowcontrol] 3 100. Results are expressed as mean 6 S.D. forthree replicates.

Figure 4. A: Force versus deformation for alginate-oligo-chitosan-alginate. B: Compression resistance of alginate-microcapsules coated with modified or unmodified oligo-chitosan at different time points. Results are expressed asmean 6 SD for three replicates. (*p < 0.05).



mNw/capsule, respectively, at day 21 [Fig. 4(B)].Therefore for modified oligochitosan the rate ofdecrease in the bursting force is lower that forunmodified oligochitosan treated microcapsules.However, none of the two immobilization systemscould be considered as mechanically instable.

Microencapsulation of C2C12 myoblasts and EPOsecretion from immobilized cells

We measured EPO production from the EPO-secreting myoblast cell line using an ELISA specificfor the measurement of human EPO. However, thecross-reactivity of the assay allowed the measure-ment of murine EPO levels. The selected cell linesecreted 46,264 6 1451 UI EPO/106 cells/24 h. EPO-secreting myoblasts were immobilized in two differ-ent types of alginate-oligochitosan-alginate (A-O-A)microcapsules using purified alginate and an electro-static droplet generator, which enabled the produc-tion of uniform shape microcapsules of small sizeand very narrow dispersion. Microcapsules coatedwith modified oligochitosan presented a diameter of519.9 6 14 lm [Fig. 5(A)] whereas those microcap-sules coated with unmodified oligochitosan showeda significantly smaller diameter of 488.0 6 33 lm(p < 0.05) [Fig. 5(B)].

Assuming previous studies,26 a final dose of 0.2mL of cell-loaded capsule was selected for implanta-tion. The latter produced a therapeutic dose of14,389 6 0.547 UI EPO/24 h and 16,889 6 0.478 UIEPO/24 h, for the modified and the unmodifiedmicrocapsules, respectively.

Hematocrit measurement

0.2 mL of modified and unmodified oligochitosanmicrocapsules loaded with 5 3 106 cells/mL have

been implanted in Balb/c mice by subcutaneousroute (n 5 5). The level of hematocrit in mice receiv-ing the microcapsules coated with modified oligochi-tosan was significantly higher than the one from thecontrol group (p < 0.05). In the first 2 weeks, itincreased from a basal value of (50.5 6 2)% to avalue of (73 6 2)%, reaching a value of (78.2 68.9)% at day 28. Thereafter, two of the mice receivingmodified capsules suffered a graft failure. However,the rest of the transplanted mice (n 5 3) maintaineda constant level of hematocrit between 81 and 86%until day 82 post-transplantation (Fig. 6).

On the other hand, the hematocrit of the micereceiving unmodified oligochitosan microcapsulesrose rapidly from a basal level of (52.3 6 2)% to(74.6 6 2)% as early as 14 days after implantation.From day 7 the mice receiving capsules coated withunmodified oligochitosan increased their hematocritlevel compared with the control group (p < 0.05),and these levels were significantly higher until the

Figure 5. Photomicrographs of the alginate-microcapsules coated with (A) modified oligochitosan and (B) unmodified oligo-chitosan. Photographs were taken using an inverted optical microscope equipped with a camera. Original magnification:3100. Scale Bar 5 250 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6. Hematocrit levels of Balb/c mice implantedsubcutaneously with EPO-secreting C2C12 myoblast cellsimmobilized in alginate-microcapsules (5 3 106 cells/mL)coated with unmodified or modified oligochitosan. Resultsare expressed as mean 6 S.D. (*p < 0.05).



last day of the assay (86.6% 6 3%). No statistical dif-ferences were observed between hematocrit profilesof the two groups of mice transplanted with bothtypes of microcapsules during the entire assay.Besides control mice presented a stable baseline (50–53%) during the study (Fig. 6).

Microscopic observation post-transplantation

During the assay two sets of A-O-A microcapsuleswere maintained in vitro in order to check the cellgrowth profile. At day 82 post-implantation, the via-bility of enclosed cells was measured qualitatively

Figure 7. Photomicrographs of alginate-microcapsules coated with (A,C,E) modified oligochitosan and (B,D,F) unmodi-fied oligochitosan. The first pair of photographs corresponds to in vitro microcapsules maintained in culture and theywere taken using a confocal scanning laser microscope (Olympus FluoView FV500). The second pair of photographs corre-sponds to explanted microcapsules and they were taken with an optical microscope equipped with a camera. Finally, thelast pair of photographs corresponds to the histological analysis of the explanted cell-loaded microcapsules implanted inBal/c mice. (Arrows, indicate the presence of vessels surrounding the microcapsules).



by the live/dead viability Assay using a confocalscanning laser microscope [Fig. 7(A,B)]. As itobserved, the living cells were distinguished by anintense uniform green fluorescence produce by theenzymatic conversion of non-fluorescent calcein-AMto fluorescent calcein. There were not differences inthe level of fluorescence between both of the photo-micrographs. The presence of yellow points in theimages was due to the superposing of green and redfluorescent corresponding to live and dead cells,respectively.

Additionally, modified and unmodified oligochito-san-based capsules were explanted at day 82 post-transplantation and analyzed by optical microscopyusing an inverted optical microscope (Nikon eclipseTE2000-S). After the transplantation, most of themicrocapsules maintained their spherical shape andwere free of any deformation [Fig. 7(C,D)].

The histological analysis of the capsules revealed aminimal inflammatory reaction around the microcap-sule [Fig. 7(E,F)]. This reaction consists of a fibroblasticlayer surrounding the membrane of the microcapsule.Comparing the histological analysis of the two typesof microcapsules no major differences were observed.

In vitro/in vivo correlation

To evaluate the viability and functionality of theenclosed cells both in vitro and after explantation,cell viability and EPO production were measuredand compared. No statistical differences wereobserved between the viability of the enclosed cellsat day 1 and at day 82 for both types of cell-loadedmicrocapsules [Fig. 8(A)].

The EPO (mUI/mL) production from 0.2 mL cap-sules in 24 h in vitro at day 82 and from theexplanted microcapsules at day 82 are shown in Fig-ure 8(B). It is possible to observe a reduction in thesecretion of the protein probably due to implant andexplantation process. On the other hand, there werenot statistical differences (p < 0.05) between the levelof EPO secretion from unmodified oligochitosan-coated microcapsules in vitro at day 82 and explantedat day 82. However, there were statistical differencesbetween modified oligochitosan microcapsules in vitroat day 82 and explanted at day 82 (p < 0.05). Wespeculate that this decrease in the level of EPO pro-duction could be related with the decrease in the he-matocrit level for this group of mice.

DISCUSSION

In cell microencapsulation technology the mostwidely used system is the so-called APA (alginate-PLL-alginate) microcapsule, which was initially

introduced by Lim and Sum.12 Nowadays, this typeof microcapsule has been applied for immobilizationa wide range of cells.27–30 It has been proven thatthere are some problems related to the use of PLL asa polycation in the preparation of capsule membranewhich has provoked the search of new polycations.Some of these drawbacks include the toxic manage-ment, immunogenicity, and cytotoxicity of PLL. Inthe search of substitutes of PLL, we address thepotential advantages of oligochitosans.17 In generaloligochitosans with different MM are obtained byradical degradation, using chitosans as the startingmaterial.

The aim of this article was to evaluate new algi-nate-oligochitosan systems for the long-term encap-sulation of mammalian cells. Thus, in vitro cell cyto-toxicity of both oligochitosans has been indicated bymorphological appearance, the metabolic activity,and the cell damage as function of the LDH release.Several studies have proven the high biocompatibil-ity of the chitosan molecule31–34 and also of the oli-gochitosan.17,35 In our experiments, no morphologi-cal changes were observed when cells were incu-bated with oligochitosan solutions independently ofthe polymer type or concentration. The latter indi-

Figure 8. (A) Metabolic activity in vitro at day 1 and atday 82 of cells immobilized in alginate-microcapsulescoated with modified or unmodified oligochitosan. (B)EPO secretion from microcapsules maintained in vitro atday 82 and from capsules explanted from mice at day 82for both types of microcapsules.



cates the high biocompatibility of the two polyca-tions studied.

Additionally, EPO-secreting C2C12 myoblast cellspresented a high level of cell viability (close to100%) after a 3 h incubation period with the two dif-ferent oligochitosans at the two concentrationsassayed. The other parameter under evaluation wascell damage measured by the LDH release. None ofthe oligochitosans induced a LDH release higherthan 30%, which is the limit to consider a materialcytotoxic. For example, other polycations used in cellmicroencapsulation such as PLL and (poly)ethyleni-mine provoke a LDH release of 70 and 80%, respec-tively, after only 1 h of incubation.

The following step was to prepare A-O-A micro-capsules using the two-step procedure. There aretwo different ways of elaborating alginate-chitosanmicrocapsules: a one-stage and a two-stage proce-dure.16,17 The former consists of dropping directlythe alginate solution into a solution of chitosanwhereas the latter comprises the preparation of algi-nate beads followed the formation of outer mem-brane by the suspension of the beads in the chitosansolution. The quantitative binding of chitosandepends strongly on selected preparation procedure.Therefore, in this work we decided to use the two-step procedure because microcapsules elaborated bythe two-step procedure bind approximately 100times more chitosan than capsules prepared with theone-step procedure when the chitosan has a MM ofless than 20 kDa.16 In relation with microcapsuleelaboration there is another problem that is solubilityof the chitosans. Chitosans with a MM higher than10 kDa need a pH below 6.6 to assure their solubil-ity. Although this problem can be solved by applica-tion of oligochitosan of MM lower than 10 kDa. As aresult higher pH between 6.8 and 7.4 can be appliedthat does not affect to the functionality of mamma-lian cells.

As our main goal was to design an efficient A-O-A system for the long-term encapsulation of mam-malian cells, different parameters that affect theinfluence encapsulation procedure were evaluated.Over the last few years various parameters affectingcapsule’s long-term stability including permeabil-ity18,22 and mechanical resistance23,27 have beenstudied. The mechanical strength of the microcap-sule is a crucial parameter when considering A-O-Asystem as a therapeutic approach for its use in vivo.The capsule mechanical resistance depends on struc-ture of polyelectrolyte complex formed between algi-nate and oligochitosan as well as on the type of oli-gochitosan and origin of immobilized cells. Based onour results it could be concluded that microcapsulescoated with modified oligochitosan offer more resist-ance against compression than microcapsules coatedwith unmodified oligochitosan (p < 0.05). In our pre-

vious paper17 the same unmodified oligochitosanswere used to coat G-alginate matrices and theobtained results are in agreement with the presentstudy. The statistical differences (p < 0.05) in thebursting force for coated microcapsule with modifiedand unmodified oligochitosan could be explain tak-ing into consideration the difference in their chemi-cal structure and MM. Thus the unmodified oligochi-tosan with a slighter lower MM and a less proto-nated structure could penetrate easier the alginate/Ca network and develop thicker and less densemembranes.25

In relation with the in vivo part of study there aretwo important facts to take into account. Firstly,there were not statistical differences (p < 0.05) in thehematocrit level of the two mice groups along theentire assay. Secondly, in the group of mice trans-planted with microcapsules coated with modifiedoligochitosan, there were two mice that from the be-ginning of the assay maintained lower hematocritlevels than the rest of the same group. When werecovered the capsules from these two mentionedmice and measured the EPO release from explantedmicrocapsules it resulted that 0.2 mL capsulessecreted 11 mUI EPO/24 h so the level of EPO secre-tion was too low in comparison with the rest EPOrelease values obtained from the other three respon-siveness mice. This could be the reason for obtaininga variable hematocrit response in the mice trans-planted with modified oligochitosan microcapsules.

The photomicrograph and histological analysis ofthe in vitro maintained capsules and of the explantedmicrocapsules at day 82 post-transplantationrevealed some differences between the two groupsof mice. First, the photomicrographs obtained withthe confocal scanning laser microscope [Fig. 7(A,B)]showed the life and dead cells. It could be observedin these photomicrographs that microcapsules coatedwith the unmodified oligochitosan presented ahigher density of green points that is a higher num-ber of life cells. However, these data were not inagreement with cell viability [Fig. 8(A)] and EPOproduction values obtained in vitro at day 82 [Fig.8(B)] for immobilized cells as these values werehigher for modified microcapsules than for unmodi-fied ones.

In relation with the explanted microcapsules fromthe mice, in both cases they maintained a sphericalshape without surface irregularities of the mem-brane. This fact proofs a high degree of biocompati-bility of the mentioned microcapsules. The histologi-cal analysis revealed the presence of vessels sur-rounding the microcapsules. Such angiogenicfunction of EPO has been widely studied not only invitro36 but also in vivo.37 In this study we could notdistinguish any difference in vascularization betweenboth types of explanted microcapsules.



There were no differences between both groups ofmice in terms of inflammatory reaction. Robitailleet al.38 had discovered that only the implantationprocess of the microcapsules in the peritoneal spaceincrement the number of neutrophlis and the levelsof some citoquines as the interleukin-1 beta (IL-1b)and the IL-6. Whereas the increments of some othercitoquines such as transforming growth factor (TGF-b1) and IL-1b indicate that the process is chronic. Inrecent years, it has been proven that EPO apart fromits hematopoietic effect has extra-hematopoieticeffects such as antiapoptotic and anti-inflammatoryeffects, especially in the central nervous system andthe cardiac level. For instance in the central nervoussystem EPO exerts its neuroprotector effect maintain-ing the cellular integrity and the cellular homeosta-sis, and also modulating the activity of the microgliaand the release of some pre-inflammatory citoquinessuch as IL-6, tumoral necrosis factor TNF-a andmonocyte chemotactic protein MCP-1.

In conclusion, results presented in this manuscriptsuggest that alginate-oligochitosan capsules may bean interesting alternative to conventional alginate-PLL capsules for long-term EPO release.

The authors thank the Basque Government’s grant ofresearch given to Maria De Castro. Confocal microscopyimages were taken at the ‘‘Servicio General de MicroscopiaAnalıtica y de Alta Resolucion en Biomedicina’’ at the Uni-versity of the Basque Country.

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