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Gelatin crosslinked with dehydroascorbic acid as anovel scaffold for tissue regeneration withsimultaneous antitumor activityTo cite this article M Falconi et al 2013 Biomed Mater 8 035011

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OPEN ACCESSIOP PUBLISHING BIOMEDICAL MATERIALS

Biomed Mater 8 (2013) 035011 (8pp) doi1010881748-604183035011

Gelatin crosslinked with dehydroascorbicacid as a novel scaffold for tissueregeneration with simultaneousantitumor activityM Falconi14 V Salvatore1 G Teti1 S Focaroli1 S Durante1 B Nicolini1A Mazzotti12 and I Orienti3

1 DIBINEMmdashDepartment of Biomedical and Neuromotor Sciences University of BolognaVia Irnerio 48 40126 Bologna Italy2 DIBINEM and Rizzoli Orthopaedic Institute IRCCS Via di Barbiano 110 40136 Bologna Italy3 FaBiTndashDepartment of Pharmacy and Biotechnologies University of Bologna Via San Donato 19240127 Bologna Italy

E-mail mirellafalconiuniboit vivianasalvatore2uniboit gabriellateti2uniboitstefanofocaroliuniboit sandradurante3uniboit benedettanicolini3uniboitantoniomazzotti2studiouniboit and isabellaorientiuniboit

Received 14 December 2012Accepted for publication 11 March 2013Published 26 April 2013Online at stacksioporgBMM8035011

AbstractA porous scaffold was developed to support normal tissue regeneration in the presence ofresidual tumor disease It was prepared by gelatin crosslinked with dehydroascorbic acid(DHA) A physicochemical characterization of the scaffold was carried out SEM and mercuryporosimetry revealed a high porosity and interconnection of pores in the scaffold Enzymaticdegradation provided 56 weight loss in ten days The scaffold was also evaluated in vitro forits ability to support the growth of normal cells while hindering tumor cell development Forthis purpose primary human fibroblasts and osteosarcoma tumor cells (MG-63) were seededon the scaffold Fibroblasts attached the scaffold and proliferated while the tumor cells afteran initial attachment and growth failed to proliferate and progressively underwent cell deathThis was attributed to the progressive release of DHA during the scaffold degradation and itscytotoxic activity towards tumor cells

(Some figures may appear in colour only in the online journal)

1 Introduction

Tissue regeneration represents an important issue mainly withregard to tumor disease where tissue damage may be due tothe diseasersquos progression or may be caused by chemotherapy

4 Authors to whom any correspondence should be addressed

Content from this work may be used under the terms ofthe Creative Commons Attribution 30 licence Any further

distribution of this work must maintain attribution to the author(s) and thetitle of the work journal citation and DOI

radiotherapy or chirurgical resections To this purpose porousscaffolds have been used either of natural [1 2] or syntheticorigin to favour cell attachment cell growth and differentiation[3 4] It has been highlighted that in order to promote tissueregeneration the scaffold needs to hold suitable porosity andpore interconnectivity [5] in order to maintain mechanicalintegrity while undergoing biodegradation At the same timeit also needs to sustain cell proliferation and new extracellularmatrix deposition [6ndash8] Several authors have investigatedthe use of different porous scaffolds especially for boneregeneration purposes [9ndash12] They mainly focused on the

1748-604113035011+08$3300 1 copy 2013 IOP Publishing Ltd Printed in the UK amp the USA

Biomed Mater 8 (2013) 035011 M Falconi et al

scaffoldrsquos ability to promote the regeneration of healthy tissuesbut have rarely addressed the possibility that prevention of theattachment and proliferation of tumor cells is possible at thesame time [13] Indeed it is well known that tumor cells maypersist in the body after antitumor therapy and may cause overtime a relapse of the disease

In this work we prepared and evaluated a novel porousscaffold based on gelatin crosslinked with dehydroascorbicacid (DHA) where the antitumor activity relied on the DHAreleased following scaffold degradation Gelatin was chosenas it is one of the most regularly used biomaterials for thepreparation of porous scaffolds due to its innocuous natureand its bioadhesive properties ascribable to both its biologicalcharacteristics and the presence of the RGD sequence involvedin cell attachment [14] Usually in order to obtain stable gelatinscaffolds at physiological temperatures chemical crosslinkingagents such as butadiene or glutaraldehyde are typically usedwhose toxicities are well known [14] In this study DHA wasused as the gelatin crosslinking agent due to the presenceof two ketone groups on its molecule linking the aminegroups of gelatin Besides its ability to crosslink gelatinthe DHA released due to scaffold degradation also providesantitumor activity Indeed the antitumor activity of DHA hasbeen reported either due to its reduction to ascorbic acid(AA) once it has entered the cells by the glucose transportmechanism or due to its intrinsic activity as an inhibitor of thekinases IKKβ and IKKα [15] Moreover the DHA conversionto AA is expected to promote collagen deposition and thusconnective tissue regeneration [16] In particular this workaims to demonstrate that this novel gelatinndashDHA scaffoldmay be suitable to induce healthy cell adhesion and growthwith simultaneous antitumor activity Regeneration shouldbe favoured by the bioadhesive properties of gelatin whichsupport cell attachment and the formation of extracellularmatrixes [16] The antitumor activity should correlate withthe DHA release following scaffold degradation

2 Materials and methods

21 Scaffold preparation

Gelatin type III from bovine skin 225 Bloom (Sigma Aldrich)was solubilised in water (20 wv) at 40 C 200 ml of thissolution was added to 100 ml of a DHA solution (10 wv)in water After stirring for 6 h at 80 C a gel was obtainedwhich was collected on a filter and then washed thoroughlywith water To remove unreacted DHA the gel was placedin a beaker with 200 ml of water at 37 C Water wasreplaced every hour after being analysed by HPLC for itsDHA content until no presence of DHA could be detected TheHPLC analysis was performedusing a Phenomenex Luna C18analytical column (100 mm times 46 mm id 3 μm) fitted witha Brownlee RP-18 precolumn (15 mm times 32 mm id 7 μm)The mobile phase consisted of 005 M sodium phosphate005 M sodium acetate 189 μM dodecyltrimethylammoniumchloride and 366 μM tetraoctylammonium bromide in 2575methanolwater (volvol) pH 4 [17] The elution rate was05 ml minminus1 UV-detection was carried out at 264 nm [18 19]

22 Characterisation of the scaffold

Total porosity of the freeze dried scaffold was measuredby mercury pycnometry (MP) [28] The morphology ofthe scaffold without cells was processed by high resolutionscanning electron microscopy (HRSEM) and coupled to anelectron dispersive spectroscopy (EDS Oxford System) ForHRSEM analysis scaffold samples of 5 times 1 times 3 mm werefixed with a solution of 25 glutaraldehyde in a 01 Mphosphate buffer (Sigma Aldrich St Louis USA) for 2 hat 4 C and subsequently post-fixed with 1 OsO4 in a 01 Mphosphate buffer (Societa Italiana Chimici Roma Italy) for 1 hat room temperature (RT) After several washes in the 015 Mphosphate buffer the samples were dehydrated in an ascendingalcohol series and critical point dried (CPD 030 Balzers LeicaMicrosystems GmbH Wetzlar Germany) The samples werethen metal coated with a thin layer of carbonplatinum (CPD030 Balzers Leica Microsystems GmbH Wetzlar Germany)and observed under field emission in-lens scanning electronmicroscopy (FEISEMJSM 890 Jeol Company Tokio Japan)with 7 kV accelerate voltage and 1 times 10ndash11 mA HRSEMimages were analyzed by Image pro plus software (MediaCybernetics MD USA)

23 Water uptake

The water uptake of the scaffold was determined by placinga known amount of scaffold in phosphate buffer saline (PBS)pH 74 at 37 C for 10 days Every day the scaffold weight wasdetermined and the medium changed The water uptake at eachtime point was expressed as WUPt = (Wt-W0)W0 whereW0 was the initial weight of the dry scaffold and Wt the weightof the soaked scaffold at each time point The experiment wasrepeated several times and the values of water uptake wereexpressed as the mean plusmn standard deviation (n = 10)

24 Degradation of the scaffold

To gain information on the scaffoldrsquos ability to undergodegradation in body fluids its weight loss in MEM mediumcontaining lysozyme was measured over time Lysozyme isknown as the major enzyme in human serum responsible forenzymatic degradation of biodegradable materials [20] Theexperiments were carried out by using 1 ml MEM medium(Invitrogen San Giuliano Milanese Italia) supplemented with10 FCS 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycinand 16 μg mlminus1 lysozyme which corresponded to thelysozyme concentration in human serum [21] The scaffoldsamples (5 times 1 times 5 mm) were placed individually incapped vials and after recording their initial weight (W0)1 mL of MEM containing lysozyme was added to each vialand maintained at 37 C in static incubation The MEMndashlysozyme solution was refreshed daily to ensure continuousenzyme activity Each day the samples were taken fromthe medium rinsed with deionized water freeze dried andweighed The weight loss was expressed as the percentageof the scaffold weight remaining after lysozyme treatmentWr = (WtW0) times 100 W0 denoted the initial weight of thescaffold and Wt the remaining weight of the scaffold at time (t)

2

Biomed Mater 8 (2013) 035011 M Falconi et al

The values were expressed as the mean plusmn standard deviation(n = 3)

25 Release of DHA from the scaffold

The release of DHA from the scaffold was evaluated bothin the presence of cells and without cells Scaffold samples(5 times 1 times 5 mm 59 mg plusmn 004) were placed in 96 well plateseither without cells or in the presence of cells which wereseeded upon their surfaces (30000 cellswell) They wereincubated for 1 2 5 7 or 10 days in the presence of 100 μl ofMEM supplemented with 10 FCS 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin At each time point the medium wascollected 200 μl of acetonitrile was added and the medium wascentrifuged at 5000 rpm for 10 min to separate proteins Thesupernatant was collected and analysed by high performanceliquid chromatography-mass spectrometry (HPLC) for itsDHA content using the same conditions reported in section 21Scaffolds without cells and cells seeded on the plates withoutscaffolds were used as blanks at the experimental time points

26 Primary culture of human fibroblasts

Dental pulps were obtained from human third molarsextracted for orthodontic reasons Informed consent wasobtained from patients Pulp explants were dissected insmall pieces (3ndash4 mm3) and cultured in MEM mediumsupplemented with 10 FCS and 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin The human pulp fibroblasts (HPFs)obtained were cultured at 37 C in a humidified atmosphereof 5 CO2 Cells from passages 3 to 10 were utilized for thefollowing experiments

27 Cell viability in the presence of the scaffold

HPFs and MG-63 cells were seeded onto the surface of scaffoldsamples (5 times 1 times 5 mm 59 mg plusmn 004) contained in 96-wellplates The cells were seeded at a density of 30 000 cellswelland cultured in MEM medium supplemented with 10 FCSand 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin at37 C in a humidified atmosphere of 5 CO2 After 1 35 and 7 days the cell viability was determined by 3-(4 5-dimethyl-2-thiazolyl)-2 5-diphenyl-2H-tetrazolium bromide(MTT) assay 10 μl of MTT solution in PBS (05 mg mlminus1) wasadded at each well and incubated for 4 h at 37 C Scaffoldswithout cells were used as blanks After solubilisation withMTT solvent (01 N HCl in isopropanol) the optical densitywas read at 570 nm by a Microplate Reader (Model 680Biorad Lab Inc California) MTT data were reported as themean ( plusmn SD) of triplicate experiments

As a comparison an MTT assay was also performed onMG-63 and HPFs seeded without the scaffold at a density of20000 cellswell and cultured with complete MEM mediumsupplemented with 010 μM 020 μM 025 μM 030 μM ofDHA for 1 3 5 and 7 days

28 HRSEM of the cells cultured on the scaffold

To evaluate the cell adhesion on the scaffold surface and thecell morphology HRSEM analysis was carried out on the

Figure 1 HRSEM analysis of gelatinndashDHA scaffold (bar 100 μm)The average diameter pore size is 10941 μm Ten measures of poresizes were performed on ten HRSEM images of magnification100 times

scaffold samples containing cells seeded and cultured by thesame method reported in section 25 At each experimentalpoint the scaffold samples were fixed with a solution of 25glutaraldehyde in 01 M phosphate buffer (Sigma Aldrich StLouis USA) for 2 h at 4 C and subsequently post-fixed with1 OsO4 in 01 M phosphate buffer (Societa Italiana ChimiciRoma Italy) for 1 h at RT After several washes in a 015 Mphosphate buffer the samples were dehydrated in an ascendingalcohol series and critical point dried (CPD 030 Balzers LeicaMicrosystems GmbH Wetzlar Germany) The samples werethen metal coated with a thin layer of carbonplatinum (CPD030 Balzers Leica Microsystems GmbH Wetzlar Germany)and observed under HRSEM (JSM 890 Jeol Company TokioJapan) with 7 kV accelerate voltage and 1 times 10minus11 mA

29 Statistical analysis

All values in the figures and text are expressed as mean plusmn SDof N experiments (with N 3) Statistical data analysis wascarried out using Studentrsquos t-test Data sets were examinedby analysis of variance and P values less than 005 wereconsidered statistically significant

3 Results

31 Characterisation of the scaffold

The microstructure of the gelatinndashDHA scaffold is shown infigure 1 The presence of a very porous structure in whichthe pores are uniformly distributed in the solid matrix andare separated with thin walls can be seen The pore sizevaried between 90 and 200 μm The pore size distributionappeared almost unimodal The total porosity obtained by MPwas 56 plusmn 39

3

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 2 Water uptake of gelatinndashDHA scaffold The experimentswere carried out for 10 days Data were expressed as mean of tenexperiments plusmn SD (lowastP lt 005)

32 Water uptake

The water uptake was quite efficient providing a final weightof the hydrated scaffold about 6-fold its initial dry weightThe maximum water uptake corresponding to the maximumhydration ability of the scaffold was obtained only after 1 dayand lasted for the whole duration of the experiment (figure 2)

33 Degradation of the scaffold

The scaffold degradation provided a progressive weight losswith a degradation rate almost constant over time up to day 9Between day 9 and 10 a sharp increase in the degradationrate was observed (figure 3) which preceded the scaffolddissolution taking place at day 10

34 Release of DHA from the scaffold

The DHA released from the scaffold was characterized bya rate which progressively increased over time (figure 4)The presence of cells did not significantly influence the DHAreleased as no significant difference was observed between thedifferent systems (figure 4)

35 Cell viability

The viability of the tumor cells in the scaffold stronglydecreased over time to reach complete cell death at day 7In contrast to this the viability of the normal cells was highlyimproved in the scaffold A progressive increase in viabilitywas observed over time reaching 25-fold the viability of thecontrol at day 7 (figure 5)

HPFs incubated in MEM medium without any scaffoldand with different concentrations of DHA showed a high andconstant cell viability during the treatment (figure 6(a)) whileMG63 showed a decrease of cell viability after 5 days fromthe start of the treatment (figure 6(b))

36 Cell adhesion

HRSEM showed the presence of HPFs inside themacroporosity of the gelatinndashDHA scaffold after 24 h of cellculture (figure 7(a)) Cells showed a round shape morphology

Figure 3 GelatinndashDHA scaffold degradation The experiments werecarried out for 10 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005)

with the cell membrane covered by thin microvilli connectedwith their migratory behaviour (figure 7(a)) After 5 days ofcell culture HPFs showed a fibroblastic like morphology withseveral thin cytoplasmic extroflessions which allowed a stronginteraction with the scaffold surface (figure 7(b))

MG63 cells colonized the scaffold surface and itsmacroporosities after 24 h of cell culture (figure 7(c)) Theyshowed a round shape morphology as described for HPFsAfter 5 days of cell culture they still showed a round shapemorphology suggesting the lack of a strong adhesion to thescaffold surface (figure 7(d)) The cell and scaffold surfaceswere covered by several bubbles and cell debris which suggesta strong cell sufferance (figure 7(d)) in agreement with thereduction of cell viability

4 Discussion

Porous scaffolds for tissue regeneration have been the subjectof several studies especially in the presence of tumor diseaseto promote connective tissue regeneration in defective areascaused by radiotherapy chemotherapy chirurgical resectionor reabsorption by tumor activity Until now attention hasbeen focused mainly on the physicochemical characteristicsof the scaffolds such as mechanical strength degradation rateand porosity in order to find out suitable supports for cellproliferation and differentiation The possibility for a scaffoldto promote normal cell proliferation and differentiation whilepreventing tumor cell development has rarely been addresseduntil now in spite of its great importance in medicine asa part of the antitumor approach Indeed scaffolds fortissue regeneration are mainly employed in patients alreadytreated for the presence of tumor disease In this case thepossibility that residual tumor cells remain in the body afterantitumor treatment strongly exposes the patient to the threatof a disease relapse if these cells infiltrate the scaffoldand proliferate In this work we prepared a novel scaffoldbased on gelatin crosslinked with DHA and evaluated itsability to promote normal cell proliferation while preventing

4

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 4 Release of DHA from gelatinndashDHA scaffold containing cells compared to the gelatinndashDHA scaffold without cells Data wereexpressed as mean of three experiments plusmn SD (lowastP lt 005)

Figure 5 Cell viability of MG-63 and HPFs seeded ongelatinndashDHA scaffold up to 7 days Data were expressed as mean ofthree experiments plusmn SD (lowastP lt 005) Abs absorbance

tumor cell development Gelatin has been selected for itsproven ability to play the role of scaffold for supportingcell attachment and proliferation and due to its bioadhesiveproperties ascribable to the presence of the RGD sequenceinvolved in cell attachment and formation of intercellularmatrix [22] DHA has been used as the crosslinking agentfor gelatin due to the presence of two ketone groups on itsmolecule which may form covalent bonds with the aminegroups of gelatin Crosslinking is indeed necessary to obtainstable gelatin scaffolds at physiological temperatures To thispurpose bifunctional ketones or aldehydes such as butadioneor glutaraldehyde are generally used as crosslinking agentsbut their release following scaffold degradation usually raisesconcerns about scaffold toxicity [23] In contrast the DHA

(a)

(b)

Figure 6 Cell viability of HPFs (a) and MG-63 (b) seeded withoutscaffolds up to 7 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005) Abs absorbance

released by scaffold degradation is not expected to raisetoxicity concerns as the biocompatibility of ascorbic acid andits derivatives is well documented [24] In addition DHArelease may provide antitumor activity and favor collagen

5

Biomed Mater 8 (2013) 035011 M Falconi et al

(a) (b)

(c) (d)

Figure 7 HRSEM of MG-63 cells and HPFs seeded on gelatinndashDHA scaffold for 24 h and for 5 days (a) HPFs after 24 h of cell culturesPorosity of the scaffold is colonized by cells (bar 10 μm) (b) After 5 days of cell culture HPFs showed a fibroblast like morphology Thinfilaments raising from the cell membrane allowed the interaction with the scaffold surface (white arrows) (bar 10 μm) (c) MG-63 cellscultured for 24 h on gelatinndashDHA scaffold Cells showed a round shape morphology (bar 10 μm) (d) MG-63 cells after 5 days of cellculture Cell membrane and scaffold surface were covered by several cell debris (white arrows) suggesting cell death (bar 10 μm)

deposition for connective tissue formation The antitumoractivity of DHA is both intrinsic due to inhibition of thekinases IKKβ and IKKα [15] and it is also provided byits reduction to ascorbic acid inside the cells after DHAabsorption through the glucose transport mechanism [15 25]As it concerns the physicochemical characteristics of thisnovel gelatinndashDHA scaffold HRSEM indicated the presenceof pores uniformly distributed in the solid matrix whosedimensions correlated with the range 90ndash200 μm which hasbeen recognized as being suitable for use in tissue regeneration(figure 1) [26] Also its total porosity correlated with the bestcharacteristics of the scaffolds used for tissue regenerationbeing higher than 50 the total volume Moreover thehydrophilic nature of both gelatin and DHA made the scaffoldvery hydrophilic Indeed the water uptake was quite efficientproviding after only 1 day a 568-fold increase of the scaffoldweight (figure 2) The scaffold hydration ability together witha controlled porosity are considered key features for cellattachment and proliferation as they allow the scaffold tobecome a gelled tridimensional environment upon hydrationable to mimic the extracellular matrix High levels of scaffold

hydration make cell attachment and growth easier due toan increase in cell diffusion in the gelled structure whichfavours cell spreading and colonization The gelatinndashDHAscaffold was also stable towards matrix disaggregation in anaqueous environment in the absence of hydrolytic enzymesIndeed once it reached its complete hydration it maintaineda constant weight for the whole length of the experimentas indicated by the plateau in figure 2 In contrast to thisin the presence of hydrolytic enzymes such as lysozyme adecrease in the scaffold weight was obtained over time dueto degradation During degradation hydrolysis of the DHAcrosslinked gelatin takes place with diffusion of the hydrolizedresidues from the gelled matrix towards the external aqueousenvironment As long as degradation takes place the releaseof the hydrolysed chain residues to the aqueous environmentprovides a progressive weight decrease of the gelled matrixwhich is usually about constant over time as long as thematrix integrity is maintained When the matrix collapsesdue to the advancement of degradation a sharp weight lossis obtained followed by matrix dissolution The degradationof the DHA-gelatin scaffold in the presence of lysozyme

6

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

The values were expressed as the mean plusmn standard deviation(n = 3)

25 Release of DHA from the scaffold

The release of DHA from the scaffold was evaluated bothin the presence of cells and without cells Scaffold samples(5 times 1 times 5 mm 59 mg plusmn 004) were placed in 96 well plateseither without cells or in the presence of cells which wereseeded upon their surfaces (30000 cellswell) They wereincubated for 1 2 5 7 or 10 days in the presence of 100 μl ofMEM supplemented with 10 FCS 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin At each time point the medium wascollected 200 μl of acetonitrile was added and the medium wascentrifuged at 5000 rpm for 10 min to separate proteins Thesupernatant was collected and analysed by high performanceliquid chromatography-mass spectrometry (HPLC) for itsDHA content using the same conditions reported in section 21Scaffolds without cells and cells seeded on the plates withoutscaffolds were used as blanks at the experimental time points

26 Primary culture of human fibroblasts

Dental pulps were obtained from human third molarsextracted for orthodontic reasons Informed consent wasobtained from patients Pulp explants were dissected insmall pieces (3ndash4 mm3) and cultured in MEM mediumsupplemented with 10 FCS and 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin The human pulp fibroblasts (HPFs)obtained were cultured at 37 C in a humidified atmosphereof 5 CO2 Cells from passages 3 to 10 were utilized for thefollowing experiments

27 Cell viability in the presence of the scaffold

HPFs and MG-63 cells were seeded onto the surface of scaffoldsamples (5 times 1 times 5 mm 59 mg plusmn 004) contained in 96-wellplates The cells were seeded at a density of 30 000 cellswelland cultured in MEM medium supplemented with 10 FCSand 100 IU mlminus1 penicillinndash50 μg mlminus1 streptomycin at37 C in a humidified atmosphere of 5 CO2 After 1 35 and 7 days the cell viability was determined by 3-(4 5-dimethyl-2-thiazolyl)-2 5-diphenyl-2H-tetrazolium bromide(MTT) assay 10 μl of MTT solution in PBS (05 mg mlminus1) wasadded at each well and incubated for 4 h at 37 C Scaffoldswithout cells were used as blanks After solubilisation withMTT solvent (01 N HCl in isopropanol) the optical densitywas read at 570 nm by a Microplate Reader (Model 680Biorad Lab Inc California) MTT data were reported as themean ( plusmn SD) of triplicate experiments

As a comparison an MTT assay was also performed onMG-63 and HPFs seeded without the scaffold at a density of20000 cellswell and cultured with complete MEM mediumsupplemented with 010 μM 020 μM 025 μM 030 μM ofDHA for 1 3 5 and 7 days

28 HRSEM of the cells cultured on the scaffold

To evaluate the cell adhesion on the scaffold surface and thecell morphology HRSEM analysis was carried out on the

Figure 1 HRSEM analysis of gelatinndashDHA scaffold (bar 100 μm)The average diameter pore size is 10941 μm Ten measures of poresizes were performed on ten HRSEM images of magnification100 times

scaffold samples containing cells seeded and cultured by thesame method reported in section 25 At each experimentalpoint the scaffold samples were fixed with a solution of 25glutaraldehyde in 01 M phosphate buffer (Sigma Aldrich StLouis USA) for 2 h at 4 C and subsequently post-fixed with1 OsO4 in 01 M phosphate buffer (Societa Italiana ChimiciRoma Italy) for 1 h at RT After several washes in a 015 Mphosphate buffer the samples were dehydrated in an ascendingalcohol series and critical point dried (CPD 030 Balzers LeicaMicrosystems GmbH Wetzlar Germany) The samples werethen metal coated with a thin layer of carbonplatinum (CPD030 Balzers Leica Microsystems GmbH Wetzlar Germany)and observed under HRSEM (JSM 890 Jeol Company TokioJapan) with 7 kV accelerate voltage and 1 times 10minus11 mA

29 Statistical analysis

All values in the figures and text are expressed as mean plusmn SDof N experiments (with N 3) Statistical data analysis wascarried out using Studentrsquos t-test Data sets were examinedby analysis of variance and P values less than 005 wereconsidered statistically significant

3 Results

31 Characterisation of the scaffold

The microstructure of the gelatinndashDHA scaffold is shown infigure 1 The presence of a very porous structure in whichthe pores are uniformly distributed in the solid matrix andare separated with thin walls can be seen The pore sizevaried between 90 and 200 μm The pore size distributionappeared almost unimodal The total porosity obtained by MPwas 56 plusmn 39

3

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 2 Water uptake of gelatinndashDHA scaffold The experimentswere carried out for 10 days Data were expressed as mean of tenexperiments plusmn SD (lowastP lt 005)

32 Water uptake

The water uptake was quite efficient providing a final weightof the hydrated scaffold about 6-fold its initial dry weightThe maximum water uptake corresponding to the maximumhydration ability of the scaffold was obtained only after 1 dayand lasted for the whole duration of the experiment (figure 2)

33 Degradation of the scaffold

The scaffold degradation provided a progressive weight losswith a degradation rate almost constant over time up to day 9Between day 9 and 10 a sharp increase in the degradationrate was observed (figure 3) which preceded the scaffolddissolution taking place at day 10

34 Release of DHA from the scaffold

The DHA released from the scaffold was characterized bya rate which progressively increased over time (figure 4)The presence of cells did not significantly influence the DHAreleased as no significant difference was observed between thedifferent systems (figure 4)

35 Cell viability

The viability of the tumor cells in the scaffold stronglydecreased over time to reach complete cell death at day 7In contrast to this the viability of the normal cells was highlyimproved in the scaffold A progressive increase in viabilitywas observed over time reaching 25-fold the viability of thecontrol at day 7 (figure 5)

HPFs incubated in MEM medium without any scaffoldand with different concentrations of DHA showed a high andconstant cell viability during the treatment (figure 6(a)) whileMG63 showed a decrease of cell viability after 5 days fromthe start of the treatment (figure 6(b))

36 Cell adhesion

HRSEM showed the presence of HPFs inside themacroporosity of the gelatinndashDHA scaffold after 24 h of cellculture (figure 7(a)) Cells showed a round shape morphology

Figure 3 GelatinndashDHA scaffold degradation The experiments werecarried out for 10 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005)

with the cell membrane covered by thin microvilli connectedwith their migratory behaviour (figure 7(a)) After 5 days ofcell culture HPFs showed a fibroblastic like morphology withseveral thin cytoplasmic extroflessions which allowed a stronginteraction with the scaffold surface (figure 7(b))

MG63 cells colonized the scaffold surface and itsmacroporosities after 24 h of cell culture (figure 7(c)) Theyshowed a round shape morphology as described for HPFsAfter 5 days of cell culture they still showed a round shapemorphology suggesting the lack of a strong adhesion to thescaffold surface (figure 7(d)) The cell and scaffold surfaceswere covered by several bubbles and cell debris which suggesta strong cell sufferance (figure 7(d)) in agreement with thereduction of cell viability

4 Discussion

Porous scaffolds for tissue regeneration have been the subjectof several studies especially in the presence of tumor diseaseto promote connective tissue regeneration in defective areascaused by radiotherapy chemotherapy chirurgical resectionor reabsorption by tumor activity Until now attention hasbeen focused mainly on the physicochemical characteristicsof the scaffolds such as mechanical strength degradation rateand porosity in order to find out suitable supports for cellproliferation and differentiation The possibility for a scaffoldto promote normal cell proliferation and differentiation whilepreventing tumor cell development has rarely been addresseduntil now in spite of its great importance in medicine asa part of the antitumor approach Indeed scaffolds fortissue regeneration are mainly employed in patients alreadytreated for the presence of tumor disease In this case thepossibility that residual tumor cells remain in the body afterantitumor treatment strongly exposes the patient to the threatof a disease relapse if these cells infiltrate the scaffoldand proliferate In this work we prepared a novel scaffoldbased on gelatin crosslinked with DHA and evaluated itsability to promote normal cell proliferation while preventing

4

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 4 Release of DHA from gelatinndashDHA scaffold containing cells compared to the gelatinndashDHA scaffold without cells Data wereexpressed as mean of three experiments plusmn SD (lowastP lt 005)

Figure 5 Cell viability of MG-63 and HPFs seeded ongelatinndashDHA scaffold up to 7 days Data were expressed as mean ofthree experiments plusmn SD (lowastP lt 005) Abs absorbance

tumor cell development Gelatin has been selected for itsproven ability to play the role of scaffold for supportingcell attachment and proliferation and due to its bioadhesiveproperties ascribable to the presence of the RGD sequenceinvolved in cell attachment and formation of intercellularmatrix [22] DHA has been used as the crosslinking agentfor gelatin due to the presence of two ketone groups on itsmolecule which may form covalent bonds with the aminegroups of gelatin Crosslinking is indeed necessary to obtainstable gelatin scaffolds at physiological temperatures To thispurpose bifunctional ketones or aldehydes such as butadioneor glutaraldehyde are generally used as crosslinking agentsbut their release following scaffold degradation usually raisesconcerns about scaffold toxicity [23] In contrast the DHA

(a)

(b)

Figure 6 Cell viability of HPFs (a) and MG-63 (b) seeded withoutscaffolds up to 7 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005) Abs absorbance

released by scaffold degradation is not expected to raisetoxicity concerns as the biocompatibility of ascorbic acid andits derivatives is well documented [24] In addition DHArelease may provide antitumor activity and favor collagen

5

Biomed Mater 8 (2013) 035011 M Falconi et al

(a) (b)

(c) (d)

Figure 7 HRSEM of MG-63 cells and HPFs seeded on gelatinndashDHA scaffold for 24 h and for 5 days (a) HPFs after 24 h of cell culturesPorosity of the scaffold is colonized by cells (bar 10 μm) (b) After 5 days of cell culture HPFs showed a fibroblast like morphology Thinfilaments raising from the cell membrane allowed the interaction with the scaffold surface (white arrows) (bar 10 μm) (c) MG-63 cellscultured for 24 h on gelatinndashDHA scaffold Cells showed a round shape morphology (bar 10 μm) (d) MG-63 cells after 5 days of cellculture Cell membrane and scaffold surface were covered by several cell debris (white arrows) suggesting cell death (bar 10 μm)

deposition for connective tissue formation The antitumoractivity of DHA is both intrinsic due to inhibition of thekinases IKKβ and IKKα [15] and it is also provided byits reduction to ascorbic acid inside the cells after DHAabsorption through the glucose transport mechanism [15 25]As it concerns the physicochemical characteristics of thisnovel gelatinndashDHA scaffold HRSEM indicated the presenceof pores uniformly distributed in the solid matrix whosedimensions correlated with the range 90ndash200 μm which hasbeen recognized as being suitable for use in tissue regeneration(figure 1) [26] Also its total porosity correlated with the bestcharacteristics of the scaffolds used for tissue regenerationbeing higher than 50 the total volume Moreover thehydrophilic nature of both gelatin and DHA made the scaffoldvery hydrophilic Indeed the water uptake was quite efficientproviding after only 1 day a 568-fold increase of the scaffoldweight (figure 2) The scaffold hydration ability together witha controlled porosity are considered key features for cellattachment and proliferation as they allow the scaffold tobecome a gelled tridimensional environment upon hydrationable to mimic the extracellular matrix High levels of scaffold

hydration make cell attachment and growth easier due toan increase in cell diffusion in the gelled structure whichfavours cell spreading and colonization The gelatinndashDHAscaffold was also stable towards matrix disaggregation in anaqueous environment in the absence of hydrolytic enzymesIndeed once it reached its complete hydration it maintaineda constant weight for the whole length of the experimentas indicated by the plateau in figure 2 In contrast to thisin the presence of hydrolytic enzymes such as lysozyme adecrease in the scaffold weight was obtained over time dueto degradation During degradation hydrolysis of the DHAcrosslinked gelatin takes place with diffusion of the hydrolizedresidues from the gelled matrix towards the external aqueousenvironment As long as degradation takes place the releaseof the hydrolysed chain residues to the aqueous environmentprovides a progressive weight decrease of the gelled matrixwhich is usually about constant over time as long as thematrix integrity is maintained When the matrix collapsesdue to the advancement of degradation a sharp weight lossis obtained followed by matrix dissolution The degradationof the DHA-gelatin scaffold in the presence of lysozyme

6

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 2 Water uptake of gelatinndashDHA scaffold The experimentswere carried out for 10 days Data were expressed as mean of tenexperiments plusmn SD (lowastP lt 005)

32 Water uptake

The water uptake was quite efficient providing a final weightof the hydrated scaffold about 6-fold its initial dry weightThe maximum water uptake corresponding to the maximumhydration ability of the scaffold was obtained only after 1 dayand lasted for the whole duration of the experiment (figure 2)

33 Degradation of the scaffold

The scaffold degradation provided a progressive weight losswith a degradation rate almost constant over time up to day 9Between day 9 and 10 a sharp increase in the degradationrate was observed (figure 3) which preceded the scaffolddissolution taking place at day 10

34 Release of DHA from the scaffold

The DHA released from the scaffold was characterized bya rate which progressively increased over time (figure 4)The presence of cells did not significantly influence the DHAreleased as no significant difference was observed between thedifferent systems (figure 4)

35 Cell viability

The viability of the tumor cells in the scaffold stronglydecreased over time to reach complete cell death at day 7In contrast to this the viability of the normal cells was highlyimproved in the scaffold A progressive increase in viabilitywas observed over time reaching 25-fold the viability of thecontrol at day 7 (figure 5)

HPFs incubated in MEM medium without any scaffoldand with different concentrations of DHA showed a high andconstant cell viability during the treatment (figure 6(a)) whileMG63 showed a decrease of cell viability after 5 days fromthe start of the treatment (figure 6(b))

36 Cell adhesion

HRSEM showed the presence of HPFs inside themacroporosity of the gelatinndashDHA scaffold after 24 h of cellculture (figure 7(a)) Cells showed a round shape morphology

Figure 3 GelatinndashDHA scaffold degradation The experiments werecarried out for 10 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005)

with the cell membrane covered by thin microvilli connectedwith their migratory behaviour (figure 7(a)) After 5 days ofcell culture HPFs showed a fibroblastic like morphology withseveral thin cytoplasmic extroflessions which allowed a stronginteraction with the scaffold surface (figure 7(b))

MG63 cells colonized the scaffold surface and itsmacroporosities after 24 h of cell culture (figure 7(c)) Theyshowed a round shape morphology as described for HPFsAfter 5 days of cell culture they still showed a round shapemorphology suggesting the lack of a strong adhesion to thescaffold surface (figure 7(d)) The cell and scaffold surfaceswere covered by several bubbles and cell debris which suggesta strong cell sufferance (figure 7(d)) in agreement with thereduction of cell viability

4 Discussion

Porous scaffolds for tissue regeneration have been the subjectof several studies especially in the presence of tumor diseaseto promote connective tissue regeneration in defective areascaused by radiotherapy chemotherapy chirurgical resectionor reabsorption by tumor activity Until now attention hasbeen focused mainly on the physicochemical characteristicsof the scaffolds such as mechanical strength degradation rateand porosity in order to find out suitable supports for cellproliferation and differentiation The possibility for a scaffoldto promote normal cell proliferation and differentiation whilepreventing tumor cell development has rarely been addresseduntil now in spite of its great importance in medicine asa part of the antitumor approach Indeed scaffolds fortissue regeneration are mainly employed in patients alreadytreated for the presence of tumor disease In this case thepossibility that residual tumor cells remain in the body afterantitumor treatment strongly exposes the patient to the threatof a disease relapse if these cells infiltrate the scaffoldand proliferate In this work we prepared a novel scaffoldbased on gelatin crosslinked with DHA and evaluated itsability to promote normal cell proliferation while preventing

4

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 4 Release of DHA from gelatinndashDHA scaffold containing cells compared to the gelatinndashDHA scaffold without cells Data wereexpressed as mean of three experiments plusmn SD (lowastP lt 005)

Figure 5 Cell viability of MG-63 and HPFs seeded ongelatinndashDHA scaffold up to 7 days Data were expressed as mean ofthree experiments plusmn SD (lowastP lt 005) Abs absorbance

tumor cell development Gelatin has been selected for itsproven ability to play the role of scaffold for supportingcell attachment and proliferation and due to its bioadhesiveproperties ascribable to the presence of the RGD sequenceinvolved in cell attachment and formation of intercellularmatrix [22] DHA has been used as the crosslinking agentfor gelatin due to the presence of two ketone groups on itsmolecule which may form covalent bonds with the aminegroups of gelatin Crosslinking is indeed necessary to obtainstable gelatin scaffolds at physiological temperatures To thispurpose bifunctional ketones or aldehydes such as butadioneor glutaraldehyde are generally used as crosslinking agentsbut their release following scaffold degradation usually raisesconcerns about scaffold toxicity [23] In contrast the DHA

(a)

(b)

Figure 6 Cell viability of HPFs (a) and MG-63 (b) seeded withoutscaffolds up to 7 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005) Abs absorbance

released by scaffold degradation is not expected to raisetoxicity concerns as the biocompatibility of ascorbic acid andits derivatives is well documented [24] In addition DHArelease may provide antitumor activity and favor collagen

5

Biomed Mater 8 (2013) 035011 M Falconi et al

(a) (b)

(c) (d)

Figure 7 HRSEM of MG-63 cells and HPFs seeded on gelatinndashDHA scaffold for 24 h and for 5 days (a) HPFs after 24 h of cell culturesPorosity of the scaffold is colonized by cells (bar 10 μm) (b) After 5 days of cell culture HPFs showed a fibroblast like morphology Thinfilaments raising from the cell membrane allowed the interaction with the scaffold surface (white arrows) (bar 10 μm) (c) MG-63 cellscultured for 24 h on gelatinndashDHA scaffold Cells showed a round shape morphology (bar 10 μm) (d) MG-63 cells after 5 days of cellculture Cell membrane and scaffold surface were covered by several cell debris (white arrows) suggesting cell death (bar 10 μm)

deposition for connective tissue formation The antitumoractivity of DHA is both intrinsic due to inhibition of thekinases IKKβ and IKKα [15] and it is also provided byits reduction to ascorbic acid inside the cells after DHAabsorption through the glucose transport mechanism [15 25]As it concerns the physicochemical characteristics of thisnovel gelatinndashDHA scaffold HRSEM indicated the presenceof pores uniformly distributed in the solid matrix whosedimensions correlated with the range 90ndash200 μm which hasbeen recognized as being suitable for use in tissue regeneration(figure 1) [26] Also its total porosity correlated with the bestcharacteristics of the scaffolds used for tissue regenerationbeing higher than 50 the total volume Moreover thehydrophilic nature of both gelatin and DHA made the scaffoldvery hydrophilic Indeed the water uptake was quite efficientproviding after only 1 day a 568-fold increase of the scaffoldweight (figure 2) The scaffold hydration ability together witha controlled porosity are considered key features for cellattachment and proliferation as they allow the scaffold tobecome a gelled tridimensional environment upon hydrationable to mimic the extracellular matrix High levels of scaffold

hydration make cell attachment and growth easier due toan increase in cell diffusion in the gelled structure whichfavours cell spreading and colonization The gelatinndashDHAscaffold was also stable towards matrix disaggregation in anaqueous environment in the absence of hydrolytic enzymesIndeed once it reached its complete hydration it maintaineda constant weight for the whole length of the experimentas indicated by the plateau in figure 2 In contrast to thisin the presence of hydrolytic enzymes such as lysozyme adecrease in the scaffold weight was obtained over time dueto degradation During degradation hydrolysis of the DHAcrosslinked gelatin takes place with diffusion of the hydrolizedresidues from the gelled matrix towards the external aqueousenvironment As long as degradation takes place the releaseof the hydrolysed chain residues to the aqueous environmentprovides a progressive weight decrease of the gelled matrixwhich is usually about constant over time as long as thematrix integrity is maintained When the matrix collapsesdue to the advancement of degradation a sharp weight lossis obtained followed by matrix dissolution The degradationof the DHA-gelatin scaffold in the presence of lysozyme

6

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

Figure 4 Release of DHA from gelatinndashDHA scaffold containing cells compared to the gelatinndashDHA scaffold without cells Data wereexpressed as mean of three experiments plusmn SD (lowastP lt 005)

Figure 5 Cell viability of MG-63 and HPFs seeded ongelatinndashDHA scaffold up to 7 days Data were expressed as mean ofthree experiments plusmn SD (lowastP lt 005) Abs absorbance

tumor cell development Gelatin has been selected for itsproven ability to play the role of scaffold for supportingcell attachment and proliferation and due to its bioadhesiveproperties ascribable to the presence of the RGD sequenceinvolved in cell attachment and formation of intercellularmatrix [22] DHA has been used as the crosslinking agentfor gelatin due to the presence of two ketone groups on itsmolecule which may form covalent bonds with the aminegroups of gelatin Crosslinking is indeed necessary to obtainstable gelatin scaffolds at physiological temperatures To thispurpose bifunctional ketones or aldehydes such as butadioneor glutaraldehyde are generally used as crosslinking agentsbut their release following scaffold degradation usually raisesconcerns about scaffold toxicity [23] In contrast the DHA

(a)

(b)

Figure 6 Cell viability of HPFs (a) and MG-63 (b) seeded withoutscaffolds up to 7 days Data were expressed as mean of threeexperiments plusmn SD (lowastP lt 005) Abs absorbance

released by scaffold degradation is not expected to raisetoxicity concerns as the biocompatibility of ascorbic acid andits derivatives is well documented [24] In addition DHArelease may provide antitumor activity and favor collagen

5

Biomed Mater 8 (2013) 035011 M Falconi et al

(a) (b)

(c) (d)

Figure 7 HRSEM of MG-63 cells and HPFs seeded on gelatinndashDHA scaffold for 24 h and for 5 days (a) HPFs after 24 h of cell culturesPorosity of the scaffold is colonized by cells (bar 10 μm) (b) After 5 days of cell culture HPFs showed a fibroblast like morphology Thinfilaments raising from the cell membrane allowed the interaction with the scaffold surface (white arrows) (bar 10 μm) (c) MG-63 cellscultured for 24 h on gelatinndashDHA scaffold Cells showed a round shape morphology (bar 10 μm) (d) MG-63 cells after 5 days of cellculture Cell membrane and scaffold surface were covered by several cell debris (white arrows) suggesting cell death (bar 10 μm)

deposition for connective tissue formation The antitumoractivity of DHA is both intrinsic due to inhibition of thekinases IKKβ and IKKα [15] and it is also provided byits reduction to ascorbic acid inside the cells after DHAabsorption through the glucose transport mechanism [15 25]As it concerns the physicochemical characteristics of thisnovel gelatinndashDHA scaffold HRSEM indicated the presenceof pores uniformly distributed in the solid matrix whosedimensions correlated with the range 90ndash200 μm which hasbeen recognized as being suitable for use in tissue regeneration(figure 1) [26] Also its total porosity correlated with the bestcharacteristics of the scaffolds used for tissue regenerationbeing higher than 50 the total volume Moreover thehydrophilic nature of both gelatin and DHA made the scaffoldvery hydrophilic Indeed the water uptake was quite efficientproviding after only 1 day a 568-fold increase of the scaffoldweight (figure 2) The scaffold hydration ability together witha controlled porosity are considered key features for cellattachment and proliferation as they allow the scaffold tobecome a gelled tridimensional environment upon hydrationable to mimic the extracellular matrix High levels of scaffold

hydration make cell attachment and growth easier due toan increase in cell diffusion in the gelled structure whichfavours cell spreading and colonization The gelatinndashDHAscaffold was also stable towards matrix disaggregation in anaqueous environment in the absence of hydrolytic enzymesIndeed once it reached its complete hydration it maintaineda constant weight for the whole length of the experimentas indicated by the plateau in figure 2 In contrast to thisin the presence of hydrolytic enzymes such as lysozyme adecrease in the scaffold weight was obtained over time dueto degradation During degradation hydrolysis of the DHAcrosslinked gelatin takes place with diffusion of the hydrolizedresidues from the gelled matrix towards the external aqueousenvironment As long as degradation takes place the releaseof the hydrolysed chain residues to the aqueous environmentprovides a progressive weight decrease of the gelled matrixwhich is usually about constant over time as long as thematrix integrity is maintained When the matrix collapsesdue to the advancement of degradation a sharp weight lossis obtained followed by matrix dissolution The degradationof the DHA-gelatin scaffold in the presence of lysozyme

6

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

(a) (b)

(c) (d)

Figure 7 HRSEM of MG-63 cells and HPFs seeded on gelatinndashDHA scaffold for 24 h and for 5 days (a) HPFs after 24 h of cell culturesPorosity of the scaffold is colonized by cells (bar 10 μm) (b) After 5 days of cell culture HPFs showed a fibroblast like morphology Thinfilaments raising from the cell membrane allowed the interaction with the scaffold surface (white arrows) (bar 10 μm) (c) MG-63 cellscultured for 24 h on gelatinndashDHA scaffold Cells showed a round shape morphology (bar 10 μm) (d) MG-63 cells after 5 days of cellculture Cell membrane and scaffold surface were covered by several cell debris (white arrows) suggesting cell death (bar 10 μm)

deposition for connective tissue formation The antitumoractivity of DHA is both intrinsic due to inhibition of thekinases IKKβ and IKKα [15] and it is also provided byits reduction to ascorbic acid inside the cells after DHAabsorption through the glucose transport mechanism [15 25]As it concerns the physicochemical characteristics of thisnovel gelatinndashDHA scaffold HRSEM indicated the presenceof pores uniformly distributed in the solid matrix whosedimensions correlated with the range 90ndash200 μm which hasbeen recognized as being suitable for use in tissue regeneration(figure 1) [26] Also its total porosity correlated with the bestcharacteristics of the scaffolds used for tissue regenerationbeing higher than 50 the total volume Moreover thehydrophilic nature of both gelatin and DHA made the scaffoldvery hydrophilic Indeed the water uptake was quite efficientproviding after only 1 day a 568-fold increase of the scaffoldweight (figure 2) The scaffold hydration ability together witha controlled porosity are considered key features for cellattachment and proliferation as they allow the scaffold tobecome a gelled tridimensional environment upon hydrationable to mimic the extracellular matrix High levels of scaffold

hydration make cell attachment and growth easier due toan increase in cell diffusion in the gelled structure whichfavours cell spreading and colonization The gelatinndashDHAscaffold was also stable towards matrix disaggregation in anaqueous environment in the absence of hydrolytic enzymesIndeed once it reached its complete hydration it maintaineda constant weight for the whole length of the experimentas indicated by the plateau in figure 2 In contrast to thisin the presence of hydrolytic enzymes such as lysozyme adecrease in the scaffold weight was obtained over time dueto degradation During degradation hydrolysis of the DHAcrosslinked gelatin takes place with diffusion of the hydrolizedresidues from the gelled matrix towards the external aqueousenvironment As long as degradation takes place the releaseof the hydrolysed chain residues to the aqueous environmentprovides a progressive weight decrease of the gelled matrixwhich is usually about constant over time as long as thematrix integrity is maintained When the matrix collapsesdue to the advancement of degradation a sharp weight lossis obtained followed by matrix dissolution The degradationof the DHA-gelatin scaffold in the presence of lysozyme

6

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

took place at a constant rate until day 9 Between day 9and 10 a sharp weight decrease was observed indicative ofthe matrix collapse (figure 3) At day 105 complete scaffolddissolution was observed The DHA release is a consequenceof the scaffold degradation Indeed during degradation boththe peptide bonds of gelatin and the crosslinking bonds of DHAwith the amine groups of gelatin are hydrolyzed providingother than chain residues free DHA molecules The freeDHA molecules are released towards the external aqueousenvironment at a rate depending on both their concentrationand diffusibility inside the gelled matrix At the start ofdegradation the free DHArsquos concentration and diffusibilityare expected to be very low causing the low numbers ofhydrolyzed bonds but as the degradation proceeds morebonds are hydrolyzed This is providing that more free DHAmolecules are inside the gel and also provide a looser gellednetwork favouring DHA diffusion As a consequence the rateof DHA release is expected to increase over time according tothe model proposed for drug release from matrices undergoingbulk biodegradation [27 28] The DHA release from thegelatinndashDHA scaffold is indeed characterized by an initial slowrelease whose rate increases over time (figure 4) The presenceof cells on the scaffold did not significantly influence the DHArelease pattern both in terms of released concentration at eachtime point and kinetic behaviour (figure 4) Nevertheless theDHA release provided different effects on normal and tumorcell proliferation In the scaffold containing fibroblasts theDHA release strongly enhanced cell proliferation with the cellviability being about 2-fold the control at day 5 and furtherimproving at day 7 (figure 5) The improvement of fibroblastproliferation has been correlated to the DHA ability to promoteconnective tissue formation [16] Scaffold biodegradation after10 days is compatible with start of tissue regeneration [29ndash31]In contrast to this the DHA release from the scaffoldcontaining the tumor cells significantly inhibited cell growthand proliferation with the cell viability being about 80the control at day 1 about 6 at day 5 and 0 at day 7(figure 5) The inhibition of tumor cell growth has beencorrelated to both the intrinsic antitumor activity of DHAand its reduction to AA inside the cells [15] HRSEM imagesshowed a facilitated attachment of HPF cells their progressivemigration and adaptation inside the scaffold and an initial MG-63 cells colonization at day 1 with a subsequent decrease incell number in accordance with the viability studies

5 Conclusions

A novel scaffold based on gelatin crossliked withdehydroascorbic acid has been prepared to promote healthytissue regeneration while preventing tumor cell proliferationThe antitumor activity of dehydroascorbic acid releasedfrom the scaffold during degradation prevented proliferationof tumor cells while promoting normal fibroblasts growthand proliferation due to its ability to favour connectivetissue regeneration This peculiar combination of gelatin anddehydroascorbic acid represents a novel approach in the useof scaffolds for tissue regeneration especially in patients whounderwent antitumor therapy as the possible persistence of

residual tumor cells in the body may allow their attachmentand proliferation in the scaffold thus exposing the patient tothe risk of a relapse of the disease

Acknowledgment

This work was supported by the Italian Ministry ofResearch and Technology (MURST) with a FIRB grant(RBAP10MLK7_005) and a PRIN 2009 grant

References

[1] Tabata Y 2003 Tissue regeneration based on growth factorrelease Tissue Eng 9 5ndash15

[2] Yarlagadda P K Chandrasekharan M and Shyan J Y 2005Recent advances and current developments in tissuescaffolding Biomed Mater Eng 15 159ndash77

[3] Mastrogiacomo M Muraglia A Komlev V Peyrin FRustichelli F Crovace A and Cancedda R 2005 Tissueengineering of bone search for a better scaffold OrthodCraniofac Res 8 277ndash84

[4] Gunatillake P Mayadunne R and Adhikari R 2006 Recentdevelopments in biodegradable synthetic polymersBiotechnol Annu Rev 12 301ndash47

[5] Link D P van den Dolder J Wolke J G and Jansen J A 2007The cytocompatibility and early osteogenic characteristicsof an injectable calcium phosphate cement Tissue Eng13 493ndash500

[6] Byrne D P Lacroix D Planell J A Kelly D Jand Prendergast P J 2007 Simulation of tissuedifferentiation in a scaffold as a function of porosityyoungrsquos modulus and dissolution rate application ofmechanobiological models in tissue engineeringBiomaterials 28 5544ndash54

[7] OrsquoBrien F J Harley B A Waller M A Yannas I V Gibson L Jand Prendergast P J 2007 The effect of pore size onpermeability and cell attachment in collagen scaffolds fortissue engineering Technol Health Care 15 3ndash17

[8] Karageorgiou V and Kaplan D 2005 Porosity of 3Dbiomaterial scaffolds and osteogenesis Biomaterials26 5474ndash91

[9] Raucci M G DrsquoAnto V Guarino V Sardella E Zeppetelli SFavia P and Ambrosio L 2010 Biomineralized porouscomposite scaffolds prepared by chemical synthesis forbone tissue regeneration Acta Biomater 6 4090ndash9

[10] Kim H W Knowles J C and Kim H E 2004Hydroxyapatitepoly(ε-caprolactone) composite coatingson hydroxyapatite porous bone scaffold for drug deliveryBiomaterials 25 1279ndash87

[11] Hedberg E L Shih C K Lemoine J J Timmer M DLiebschner M A K and Jansen J A 2005 In vitro degradationof porous poly(propylene fumarate)poly(DL-lactic-co-glycolic acid) composite scaffolds Biomaterials16 3215ndash25

[12] Komlev V S Mastrogiacomo M Pereira R C Peyrin FRustichelli F and Cancedda R 2010 Biodegradation ofporous calcium phosphate scaffolds in an ectopic boneformation model studied by x-ray computedmicrotomograph Eur Cell Mater 19 136ndash46

[13] Gupta V et al 2011 Repair and reconstruction of a resectedtumor defect using a composite of tissue flapndashnanotherapeuticndashsilk fibroin and chitosan scaffold AnnBiomed Eng 39 2374ndash87

[14] Rohanizadeh R Swain M V and Mason R S 2008 Gelatinsponges (Gelfoam) as a scaffold for osteoblasts J MaterSci Mater Med 19 1173ndash82

7

Biomed Mater 8 (2013) 035011 M Falconi et al

[15] Carcamo J M Pedraza A Borquez-Ojeda O Zhang BSanchez R and Golde D W 2004 Vitamin C is a kinaseinhibitor dehydroascorbic acid inhibits IKB-alfa kinasebeta Mol Cell Biol 24 6645ndash52

[16] Kim A Y Lee E M Lee E J Min C W Kang K K Park J KHong I H Ishigami A Tremblay J P and Jeong K S 2012Effects of vitamin C on cytotherapy-mediated muscleregeneration Cell Transplant at press

[17] Dhariwal K R Hartzell W O and Levine M 1991 Ascorbicacid and dehydroascorbic acid measurements in humanplasma and serum Am J Clin Nutr 54 712ndash6

[18] Karlsen A Blomhoff R and Gundersen T E 2005High-throughput analysis of vitamin C in human plasmawith the use of HPLC with monolithic column andUV-detection J Chromatogr B Analyt Technol BiomedLife Sci 824 132ndash8

[19] Sanderson M J and Schorah C J 1987 Measurement ofascorbic acid and dehydroascorbic acid in gastric juice byHPLC Biomed Chromatogr 2 197ndash202

[20] Azevedo H S and Reis R L 2005 Biodegradable Systems inTissue Engineering and Regenerative Medicine vol 12 edR L Reis and J F San Roman (Boca Raton FL CRC Press)pp 214ndash45

[21] Ratanavaraporn J Damrongsakkul S Sanchavanakit NBanaprasert T and Kanokpanont S 2006 Comparison ofgelatin and collagen scaffolds for fibroblast cell cultureJ Met Mater Mineral 16 31ndash6

[22] Stanton J S Salih V Bentley G and Downes S 1995 Thegrowth of chondrocytes using gelfoam as a biodegradablescaffold J Mater Sci Mater Med 6 739ndash44

[23] Zeiger E Gollapudi B and Spencer P 2005 Genetic toxicityand carcinogenicity studies of glutaraldehydemdasha reviewMutat Res 589 136ndash51

[24] Wilson J X 2002 The physiological role of dehydroascorbicacid FEBS Lett 527 5ndash9

[25] Vera J C Rivas C I Fischbarg J and Golde D W 1993Mammalian facilitative hexose transporters mediate thetransport of dehydroascorbic acid Nature 364 79ndash82

[26] Yang S Leong K F Du Z and Chua C K 2002 The design ofscaffolds for use in tissue engineering part II Rapidprototyping techniques Tissue Eng 1 1ndash11

[27] Gopferich A 1996 Mechanisms of polymer degradation anderosion Biomaterials 17 103ndash14

[28] Tsuji H and Ishizaka T 2001 Blends of aliphatic polyesters VILipase-catalyzed hydrolysis and visualized phase structureof biodegradable blends from poly(ε-caprolactone) andpoly(L-lactide) Int J Biol Macromol 29 83ndash9

[29] Li R D et al 2012 Biphasic effects of TGFβ1 onBMP9-induced osteogenic differentiation of mesenchymalstem cells BMB Rep 45 509ndash14

[30] Phadke A Hwang Y Hee Kim S Hyun Kim S Yamaguchi TMasuda K and Varghese S 2013 Effect of scaffoldmicroarchitecture on osteogenic differentiationof human mesenchymal stem cells Eur Cell Mater25 114ndash29

[31] Thibault R A Baggett L S Mikos A G and Kasper F K 2010Osteogenic differentiation of mesenchymal stem cells onpregenerated extracellular matrix scaffolds in the absence ofosteogenic cell culture supplements Tissue Eng A16 431ndash40

8