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Biomaterials xxx (2009) 1–10

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Concentrated collagen hydrogels as dermal substitutes

Christophe Helary a,*, Isabelle Bataille b,c, Aicha Abed b,c, Corinne Illoul a, Annie Anglo a, Liliane Louedec b,Didier Letourneur b,c, Anne Meddahi-Pelle b,c, Marie Madeleine Giraud-Guille a

a CMCP - CNRS UMR7574, EPHE, University Pierre and Marie Curie, 4 place Jussieu, 75005 Paris, Franceb INSERM, U698, CHU Xavier Bichat, Bat. Inserm, 46 rue Henri-Huchard,75018 Paris, Francec BPC, Institut Galilee, Universite Paris 13, 93430 Villetaneuse, France

a r t i c l e i n f o

Article history:Received 14 June 2009Accepted 18 September 2009Available online xxx

Keywords:Dermal substitutesConcentrated collagen hydrogelFibroblast growthContractionGelatinase AIn vivo integration

* Corresponding author. Tel./fax: þ33 1 44 27 65 64E-mail address: chelary@snv.jussieu.fr (C. Helary).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.09.073

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a b s t r a c t

Collagen hydrogels first appeared promising for skin repair. Unfortunately, their extensive contraction andtheir poor mechanical properties constituted major disadvantages toward their utilization as permanentgraft. The present study has investigated a way to correct these drawbacks by increasing the collagenconcentration in controlled conditions. Concentrated collagen hydrogels (CCH) at 1.5, 3 and 5 mg/ml wereobtained. The effect of raised collagen concentration on contraction, cell growth and remodeling activitieswas evaluated for 21 days in culture. Subsequently, in vivo integration of CCH and normal collagenhydrogels (NCH) was assessed. Compared to NCH, CCH contraction was delayed and smaller. At day 21,surface area of CCH at 3 mg/ml was 18 times more important than that of NCH. Whatever the initialfibroblast density, CCH favored cell growth that reached about 10 times the initial cell number at day 21;cell proliferation was inhibited in NCH. Gelatinase A activities appeared lower in CCH than within NCH. Invivo studies in rats revealed a complete hydrolysis of NCH 15 days after implantation. In contrast, CCH at3 mg/ml was still present after 30 days. Moreover, CCH showed cell colonization, neovascularization and nosevere inflammatory response. Our results demonstrate that concentrated collagen hydrogels can beconsidered as new candidates for dermal substitution because they are is easy to handle, do not contractdrastically, favor cell growth, and can be quickly integrated in vivo.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Skin substitutes are among the first examples of tissue engi-neered devices. They are used as permanent grafts for treatment ofburns or as temporary dressing for chronic wounds and foot ulcers[1–3]. The challenge to perform a good skin substitute is to find theright balance between restoring basic function of the skin andcreating an environment to regenerate the tissue. These bioma-terials are often composed of two layers: an epidermal layer anda dermal layer. The epidermal layer, composed of sheets of autol-ogous keratinocytes, is commercialized under the brand nameEpicel� [1]. The dermal scaffold is produced with artificial ornatural polymers and can include cells [2–5]. The disadvantages ofartificial scaffolds are the lack of cell recognition and sometimesthe low biocompatibility [6]. Therefore, these biomaterials have tobe functionalized and sometimes removed from host organismafter healing [4]. Biopolymers such as collagen or fibronectin havenumerous advantages in tissue engineering such as low toxicity,

.

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, et al., Concentrated collage

low immunogenicity and biodegradability [6–8]. The first scaffoldused as dermal substitute was an acellular sponge of collagen andproteoglycans crosslinked with glutaraldehyde and commercial-ized under the brand name Integra� [3,9]. To colonize this scaffold,cells have to migrate into large pores. The contacts between Inte-gra structure and cells are in two dimensions and can be consid-ered as non physiological [10]. More recently, another acellularscaffold, a dense collagen matrix concentrated at 40 mg/ml hasbeen developed with interesting mechanical properties [11–13]. Invitro, human dermal fibroblasts migrate into the dense collagennetwork to reach a distance of about 350 mm after 28 days.

Several studies have shown the advantages to include fibro-blasts during dermal substitute fabrication [14,15]. Dermal fibro-blasts secrete extra-cellular matrix macromolecules, soluble factorsthat diffuse to the overlying epidermis and cytokines which havea role in neovascularization [16]. Fibroblasts have a key role inwound healing and the environment properties determine theirbehavior [14]. A system in which physiological 3D contacts existbetween fibroblasts and the scaffold is the normal collagenhydrogel developed by Bell and co-workers [17]. Collagen hydrogelcombined with keratinocyte sheets are used as skin substitutes fortreatment of chronic or acute wound under the name Apligraf�

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

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[18]. Nevertheless, this biomaterial has drawbacks explaining whyit is not used as permanent graft. Classical hydrogels has a lowinitial collagen concentration (0.66 mg/ml) and exhibit poormechanical properties [10,19]. In addition, they are sensitive tometalloproteinases, while cell proliferation and collagen synthesisare quickly inhibited [20–24]. Lastly, collagen hydrogels contractwith cells to represent only less than 3% of their initial surface after14 days in culture. This contraction increases as cell densityincreases [17,19]. As a consequence, they are not the appropriatesubstrates for growth and cell delivery on wound location. Someimprovements have been made, such as enhancing the mechanicalproperties of collagen hydrogels using plastic compression [25].The materials with improved mechanical resistance had a highercollagen concentration, were very thin (40 mm) and dried easily.

In this study, we have developed concentrated collagen hydro-gels (CCH) obtained in controlled conditions. The effect of a highercollagen concentration on hydrogel contraction, fibroblast prolif-eration and metalloproteinase activities were evaluated. Moreover,the biocompatibility and the ability of concentrated collagenhydrogels to integrate a host organism were assessed in rat.

2. Materials and methods

2.1. Fibroblast cell culture

Normal human dermal fibroblasts were obtained from Promocell�. Fibroblastswere grown in Dubelco’s Modified Eagle Culture Medium (DMEM, Gibco BRL)supplemented with 10% Fetal Calf Serum (FCS, Gibco BRL), 100 units/ml penicillinand 100 mg/ml streptomycin (Gibco BRL) 0,25 mg/ml Fungizone (Gibco BRL), 50 mg/ml ascorbate (VWR France). 75 cm2 culture flasks were kept at 37 �C in 95% humidityand 5% CO2 atmosphere. At confluence, fibroblasts were removed from culturedflasks by treatment with 0.1% trypsin and 0.02% EDTA. Cells were rinsed andhomogenized in the above culture medium at a density of one million/flask.Fibroblasts were used at passage 6 for the experiments.

2.2. Preparation of collagen hydrogels

A stock solution of type I collagen at 5 mg/ml in 0.1% acetic acid was eitherdiluted to make a 2 mg/ml solution or concentrated to make 10 or 15 mg/ml solu-tions. The concentration process was carried out by controlled evaporation of thesolvent in sterile conditions [11]. The collagen concentration was then estimated byhydroxyproline titration [26]. Each collagen solution (at 2, 5, 10 or 15 mg/ml) and theculture medium were placed at 25 �C for 1 h before processing the hydrogels.Collagen hydrogels with fibroblasts were prepared by mixing in ice 0.6 ml of eachcollagen solution, 1.1 ml of DMEM with 10% FCS, 0.25 ml of 0.1 M NaOH, and 0.25 mlof cell suspension (7.5�104 cells per hydrogel). The mixtures were poured in35 mm-diameter Petri dishes and set into an incubator at 37 �C. Normal collagenhydrogels at 0.66 mg/ml (NCH) and concentrated collagen hydrogel at 1.5 mg/ml(CCH1.5), 3 mg/ml (CCH3) and 5 mg/ml (CCH5) were obtained from collagen solu-tions at 2, 5, 10, and 15 mg/ml respectively. NCH were carried out according to theBell’s method [17] and were the control hydrogels in our experiments. NCH andCCH3 hydrogels were also prepared with a high cell number (2.5�105 cells perhydrogels) to assess the effect of cell density on hydrogel contraction. After 30 min at37 �C, the hydrogels with fibroblasts were gently shaken free from dishes in order tostudy hydrogel contraction over time.

Collagen hydrogels used for in vivo implantations were performed withoutdermal fibroblasts. NCH and CCH3 were carried out by mixing 3.25 ml of phosphatebuffer saline (PBS), 0.25 ml of 0.1 M NaOH and 0.6 ml of type I collagen solution.These mixtures were poured in a 1 cm2 template and set into an incubator at 37 �C totrigger gelling. After 24 h at 37 �C, hydrogels without cells were stock in a sterile PBSsolution at 4 �C.

2.3. Rheological measurements

Shear oscillatory measurements on hydrogel disks were performed on a BohlinGemini rheometer (Malvern) equipped with a plane acrylic 40 mm diametergeometry. Both base and geometry surfaces were rough in order to avoid sampleslipping during measurement. All tests were performed at 37 �C. Mechanicalspectra, namely storage, G0 and loss, G00 moduli versus frequency, were recorded atan imposed 2% strain, which corresponded to non-destructive conditions, aspreviously checked (data not shown). In order to test all hydrogels in same condi-tions, before each run the gap between base and geometry was chosen so thata slight positive normal force was applied on gels during measurement. Foursamples of each hydrogel type were tested at day 0.

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2.4. In vitro hydrogel analyses

2.4.1. 3D cell culture and samplingCollagen hydrogels were kept at 37 �C in 95% humidity and 5% CO2 atmosphere.

Culture media were replaced every week with fresh media. Collagen hydrogelsseeded with dermal fibroblasts were analyzed after 1, 4, 7, 14 and 21 days of culture.Contractile capability was evaluated by measuring the diameter of collagen hydro-gels for the 21 days of culture (n¼ 10). At each culture time point, hydrogels weresampled for cell counting (n¼ 6) or fixed in 4% paraformaldehyde in PBS for 24 h(n¼ 4). Then, these samples were dehydrated in increasing alcohol solutions andembedded in paraffin. At each time culture point, the corresponding culture mediawere collected and frozen at �20 �C for gelatin zymography analyses.

2.4.2. Cell counting and collagen titrationCollagen hydrogels were hydrolyzed at 37 �C by 2 ml of 2 mg/ml type II collage-

nase solution (VWR France). After centrifugation at 400 G for 5 min, supernatantswere removed and stocked at�20 �C. Cellular pellets were then homogenized in 1 mlof Trypan blue solution 0.1% and counted with a Malassez hematocymeter (n¼ 6).Dead cells stained in blue were not counted. Collagen concentration in supernatantswas assessed by a hydroxyproline titration (n¼ 6) [26]. Collagenase solution andhydrogel volume were assessed by weighting to calculate the dilution factor. There-after, real collagen concentrations were calculated for each hydrogel. Experimentswere carried out twice and the results were expressed as the mean value� standardderivation (SD).

2.4.3. In vitro histological analysis and collagen III immunodetectionTen micro meter thick sections, transverse to the hydrogel surface were per-

formed with a manual microtome (Stiassnie France). Paraffin sections were thenrehydrated and stained with hemalun [11]. The sections were dehydrated, mountedbetween slide and coverslip and observed with an optical microscope (Nikon E600POL). Photos of hydrogel slides were taken with a CCD camera (Nikon).

For indirect immunodetection, after rehydration and an extended wash inphosphate-buffered saline solution (PBS), the rehydrated sections were incubated5 min at room temperature with 0.2% pepsin in acetic acid 10% (v/v). The sectionswere rinsed in PBS and incubated for 30 min in PBS containing 1% glycin. Afteranother wash in PBS, the sections were incubated 60 min at room temperature witha blocking solution (0.05% tween PBS, 1% bovine serum albumin, 10% calf serum).Primary antibody against Collagen III (Sigma, France) diluted 1/50 was added ina moist chamber overnight. The following day, section were incubated in moist anddark chamber for 90 min with a secondary antibody anti mouse coupled withRhodamine (Molecular Probe). After three rinses in PBS, section were then incubatedfor 10 min in a DAPI bath (dilution: 1/50,000 v/v). Finally, slides were rinsed threetimes in PBS and observed with a fluorescence microscope AXIO 100 (Zeiss).

The percentage of cells expressing collagen III was estimated on each section bythe ratio (number of Collagen III þ/total cell number). The results were expressed asthe mean value� SD (n¼ 10).

2.4.4. Gelatin zymographyElectrophoresis was carried out using miniprotean III system (Bio-Rad France).

Polyacrylamide migration gel was performed at 10% (ratio Acrylamide/Bis acryl-amide 37:1) in 0.375 M Tris-HCl pH 8.8 contained 1 mg/ml gelatin and 0.1% sodiumdodecyl sulfate (SDS). Stacking gel contained 4% polyacrylamide in 0.125 M Tris, pH6.8 and 0.1% SDS. Gels were polymerized by adding 50 ml of 10% ammonium per-sulfate and 10 ml of 0.1% TEMED. At each culture time point, media from hydrogelsNCH or CCH3 were half-diluted in 0.125 M Tris, pH 6.8, 50% glycerol, 0.4% bromo-phenol blue and deposited within gel wells (n¼ 6). Then, gels were run underLaemmli conditions (24 mM Tris, 192 mM glycine, 3.47 mM SDS; 40 mA, 1 h).Following electrophoresis, gels were washed twice (30 min each) in 200 ml of 2.5%Triton X-100 under constant mechanical stirring and then incubated in 100 mM Tris-HCl pH 7.4, 30 mM CaCl2 for 19 h at 37 �C. Gels were stained with Coomassie blueR-250 for 2 h (50% methanol, 10% acetic acid) and destained appropriately(40% ethanol, 10% acetic acid). Proteinase activity was evidenced as cleared area.Finally, the gels were rinsed for 1 h in 5% ethanol, 7.5% acetic acid and kept in sealedbags containing distilled water. The stained polyacrylamide gels were observed witha CFR 126 Video camera and black and white images were converted into differentgray levels. Complete digestion of gelatin substrate corresponded to 255 gray levelsand absence of digestion corresponded to the 0 gray level. The average surface ofeach lysis band from gelatin zymograms was determined semi-automaticallyfollowing its contour with a calibrated electronic slide on a BIOCOM station.Gelatinase activity was expressed as arbitrary units U¼ surface (in pixels) X graylevels of the lysis band. A control was performed with culture medium collected ona collagen hydrogel without fibroblasts. Raw data were divided by the cell numberpreviously assessed (n¼ 6). The results were then expressed as the mean value� SDfor each culture time point and compared to a calibration point: value¼ 150 is themean value of the results obtained with hydrogel NCH at day 7.

2.4.5. Scanning electron microscopy (SEM) analysisSamples were fixed using 3.63% glutaraldehyde in cacodylate/saccharose

buffer (0.05 M/0.3 M, pH 7.4) for 1 h at 4 �C. Following fixation, samples were

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

Table 1Storage, G0 and loss, G00 moduli of collagen hydrogels

NCH CCH1.5 CCH3 CCH5

G0 (Pa) 13.1� 3.9 49.9� 6.9* 290� 54 * 189� 60*G00(Pa) 2.5� 1.3 6.2� 2.0* 44.1� 9.3 * 29.3� 10.3*

*P< 0.05.

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washed three times in cacaodylate/saccharose buffer (0.05 M/0.3 M, pH 7.4) anddehydrated through successive raising concentration ethanol baths from 70% to100% alcohol. Thereafter, samples were dried in a critical point dryer and goldsputtered for analysis. Samples were observed with Jeol JSM 5510LV SEM oper-ating at 10 kV.

2.4.6. Transmission electron microscopy (TEM) analysisSamples were fixed as described above. Following fixation and washing, samples

were post fixed using 2% osmium tetra-oxide in cacodylate/saccharose buffer(0.05 M/0.3 M, pH 7.4) for 1 h at 4 �C. Samples were then washed three times incacodylate/saccharose buffer, dehydrated with ethanol and embedded in araldite.Thin araldite transverse sections (100–200 nm) were performed by an Ultracutultramicrotome (Reichert, France) and contrasted by phosphotungstic acid. Slideswere then analyzed with a Philips CM 12 electron microscope operating at 120 kV.

2.5. In vivo collagen hydrogel implantation

2.5.1. Surgical procedureBoth the procedure and the animal treatment complied with the Principles of

Laboratory Animal Care formulated by the National Society for Medical Research.The studies were carried out under authorization no. 006235 of the Ministere del’Agriculture, France. Twelve adult Wistar male rats weighing 250 g (Wi/Wi, Charles-Rivers France) were anaesthetized by intraperitoneal injection of sodium pento-barbital solution (30 mg/kg, Centravet France). The abdomen was shaved and dis-infected. A vertical incision was made on the abdominal midline, and the 1 cm2

hydrogels NCH or CCH3 were implanted in subcutaneous pocket (n¼ 6). The skinand the muscle layer were then sutured (Vicryl� 4/0). After 15 or 30 days postsurgery, the rats were euthanized by intraperitoneal injection of sodium pento-barbital (60 mg/Kg). The hydrogels were then sampled and fixed in 4% para-formaldehyde (Merck France) in PBS for 24 h, dehydrated and embedded in paraffin(n¼ 3).

2.5.2. Histological analysis and indirect immunodetectionThick sections of 7 mm, transverse to the hydrogel surface were dewaxed,

rehydrated, stained with eosin-hemalun or Von Kossa, dehydrated and mountedbetween slide and coverslip. Observation was performed as described above. Theexperiments of indirect immunodetection were carried out as described above(chapter 2.4.3) until the step of incubation with blocking solution. Then, macro-phages and endothelial cells were visualized using appropriate primary antibodies(Euromedex, France) in moist chamber overnight. RECA-1 antibody was diluted inblocking solution 1/10 v/v and anti CD68 1/1000 v/v. In the case of RECA-1 detection,the sections were incubated in moist chamber for 90 min with appropriatedsecondary antibody from DAKO (anti-mouse IgG biotin conjugated). After three PBSwashes, endogenous peroxidases were inhibited by incubation at 37 �C with 3%H2O2. After washing, the samples were incubated 45 min with streptavidin/perox-ydase complex from DAKO diluted 1/300 in PBS 3% NaCl. After three rinses in PBS 3%NaCl, Peroxydase labeling was revealed for 15 min in a dark chamber using 3-30

diaminobenzindine tetrahydrochloride (Sigma) in Tris-HCl, pH 7.6 and observedwith Nikon E600 POL microscope. To exclude nonspecific binding, controls wereperformed by omitting primary antibodies or by using irrelevant secondary anti-bodies. In the case of CD 68 detection, section were incubated in moist and darkchamber for 90 min with a secondary antibody anti mouse coupled with Rhodamine(Molecular Probe). After three rinses in PBS, section were then incubated for 10 minin a DAPI bath (dilution: 1/50,000 v/v). Finally, slides were rinsed three times in PBSand observed with a fluorescence microscope AXIO 100 (Zeiss).

Fig. 1. Macroscopic view of hydrogels. Handling of collagen hydrogels including dermal fibro0.66 mg/ml (NCH) exhibited poor mechanical properties. Deposited on a spatula, they sliphydrogels at 3 mg/ml (CCH3) kept their original shape and were easily handled (B). Bar: 5

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2.6. Statistical analysis

All experiments were carried out at least twice and the results were expressed asthe mean value� standard derivation (SD). Statistical significance was determinedwith the Student Test by Microsoft Exel Software 2003. A P value< 0.05 wasconsidered significant. Statistical analysis was carried out between CCHs and NCHfor rheological analysis, cell contraction, cell growth and zymography analyses.

3. Results

3.1. Handling, mechanical properties and cell contraction ofcollagen hydrogels

The handling of the collagen hydrogels was assessed 24 h aftergelling when bubbles inside the hydrogels had disappeared.Deposited on a spatula, normal collagen hydrogel at 0.66 mg/ml(NCH) and concentrated collagen hydrogel at 1.5 mg/ml (CCH1.5) didnot keep their original shape. These hydrogels slipped from thespatula and resembled a water drop (Fig. 1A). These hydrogels werethus difficult to handle. In contrast concentrated collagen hydrogel at3 mg/ml (CCH3) and at 5 mg/ml (CCH5) kept their original shape andwere easy to handle (Fig. 1B). Mechanical properties of collagenhydrogels were then investigated by rheological measurements atday 0. Storage, G0 and loss, G00 moduli were measured versusfrequency. In each case, G0 was around one decade superior to G00

which was to be related to a mainly elastic behavior and both G0 andG00 were almost independent of frequency (data not shown). Bothfeatures were typical of three-dimensional gels. As a summary G0 andG00 values were averaged at a unique frequency (1 Hz) for each geland are given in Table 1. Both moduli G0 and G00 increased withcollagen concentration from NCH to CCH3 which shows that collagenconcentration plays a role in material rigidity. The elastic modulus(G0) measured in CCH3 was about twenty two times higher thanNCH. As this modulus is related to hydrogel stiffness, CCH3 wasdrastically stiffer than NCH. From CCH3 to CCH5, increasing collagenconcentration did not seem to be useful since CCH5 hydrogels tendedto creep and to be thus less elastic. The CCH5 mechanical propertiescan be explained by the non-homogeneity of these hydrogels (bothdense and loose regions scattered within the collagen hydrogel)(Fig. 2A). For these reasons, CCH5 was not used further.

blasts (7.5�104 cells/hydrogel) at day one. Normal collagen hydrogels concentrated atped, became deformed, and were handled with difficulty (A). Concentrated collagenmm.

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

CCH3

CCH5 Non

homogeneous

hydrogels

Day 1 Day 7 Day 21

CCH1.5

NCH

A

0

20

40

60

80

100

0 7 14 21

Time (days)

Pe

rc

en

ta

ge

o

f in

itia

l a

era

NCH

CCH1.5

CCH3

B

* * *

*

*

*

*

*

Fig. 2. Hydrogel contraction by dermal fibroblasts (7.5�104 cells/hydrogel). (A):Macroscopic view of collagen hydrogels during cell culture. Contraction depended oninitial collagen concentration within hydrogel. As collagen concentration rose from0.66 to 3 mg/ml, contraction was delayed. When reaching 5 mg/ml, collagen hydrogelsappeared non homogenous. (B): Surface shrinkage of collagen hydrogels during cellculture. NCH contracted quickly to reach after two weeks 3% of their initial surface.CCH1.5 contraction started at day 4 to reach about 20% of the initial area at day 21.Contraction of CCH3 started at day 7 and was linear to reach about 56% of their initialsurface at day 21. Whatever the culture time point, contraction was statistically (*:P< 0.05) more important in NCH than in CCH1.5 or CCH3 (n¼ 10).

0

2

4

6

8

0 7 14 21

Time (days)

To

ta

l c

ell n

um

be

r (X

10

5)

NCH

CCH1.5

CCH3

*

*

*

**

Fig. 3. Cell growth within collagen hydrogels. Kinetic analysis of the total cell numberwithin collagen hydrogels. Seeded at 75,000 cells per hydrogel, the number of fibro-blasts increased gradually from day one. The cell growth depended on the initialcollagen concentration of hydrogels and reached a maximum in the case of CCH3. Fromday 7, cell number was statistically (*: P< 0.05) higher in CCH1.5 and CCH3 than inNCH (n¼ 6).

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Capacity of dermal fibroblasts to contract collagen hydrogels isrelated to collagen concentration (Fig. 2A). The contraction of NCHstarted the first day in culture whereas that of CCH1.5 and CCH3 wasdelayed to day 4 and day 7 respectively (Fig. 2B). NCH contractionwascharacterized bya rapid phase up to day 7 and reached a maximum atday 14. At this culture time point, NCH surface was only 3% of theinitial area (Fig. 2B). Contraction of concentrated hydrogels wasslower and did not reach a plateau phase at day 21. CCH1.5 contrac-tion was quasi linear from day 4 to day 14, was slower from day 14 today 21 and CCH1.5 surface reached about 20% of its initial area at day21. CCH3 exhibited a latent phase up to day 7 and then a linearcontraction up to day 21. The hydrogel surface was about 56% of theinitial area at day 21, e.g. 18 times higher than NCH area (Fig. 2B).

Table 2Hydrogel collagen concentrations (mg/ml)

Day 0 Day 1 Day 7 Day 14 Day 21

NCH 0.66 2.62� 0.32 5.20� 0.67 10.49� 1.1 37.39� 5.89CCH1.5 1.5 3.31� 0.45 3.17� 0.86 5.32� 1.89 18.61� 2.67CCH3 3 3.80� 0.56 3.54� 0.45 8.00� 0.99 11.03� 1.98

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Contraction by dermal fibroblasts led to an increase of thecollagen concentration within collagen hydrogels (Table 2).Collagen concentration increased quickly within NCH to reach37.39� 5.89 mg/ml at day 21. In contrast, collagen concentrationincreased slowly within CCH1.5 and CCH3 to reach respectively18.61�2.67 and 11.03�1.98 mg/ml at day 21.

3.2. Cell growth

The number of dermal fibroblasts within all hydrogels increasedgradually from day 1 to day 14 (Fig. 3). From day 14 to 21, cell growthreached a plateau within NCH and CCH1.5 but was still increasingwithin CCH3. The most important growth rate was evaluatedbetween day 7 and day 14. From day 7, the number of fibroblastswithin CCH1.5 and CCH3 hydrogels was significantly (P< 0.05)higher than in NCH. At day 21, the number of dermal fibroblastsrepresented 2.3, 5.2, and 9.2 times the initial number for NCH,CCH1.5 and CCH3 respectively.

The volume of collagen hydrogels varied during the course ofthe experiment and was related to the initial collagen concentra-tion. Therefore, cell density was assessed within collagen hydrogels(Table 3). Cell density increased rapidly for NCH that contracted themost to reach 5728� 780 cells/mm3 at day 21. On the opposite, celldensity for CCH1.5 and CCH3 increased slowly to reach after threeweeks 1979�150; 1728� 190 cells/ml respectively.

Analyses of cell contraction, cell growth and handling evidencedthat CCH3 is the most promising material. Analyses of histologicalsections after cell culture with human fibroblasts revealed a homog-enous cell distribution within both NCH and CCH3 (Fig. 4A and B). Athigher magnification, fibroblasts within NCH exhibited roundedshape (Fig. 4C) whereas cells within CCH3 were more spread witha spindle shape (Fig. 4D).

Table 3Fibroblast density within collagen hydrogels (cells/mm3)

Day 0 Day 1 Day 4 Day 7 Day 14 Day 21

NCH 30 86� 17 142� 43 599� 9 1085� 175 5728� 780CCH1.5 30 31� 6 72� 6 90� 6 528� 75 1979� 150CCH3 30 38� 12 57� 5 79� 9 551� 108 1728� 190

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

Fig. 4. Morphology of dermal fibroblasts within collagen hydrogels. At day 21, fibroblasts within NCH were well distributed (A) and had a rounded shape (C). In CCH3, fibroblastswere also well distributed within the collagen network (B) and exhibited a spindle shape (D). Bar: 100 mm.

C. Helary et al. / Biomaterials xxx (2009) 1–10 5

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3.3. In vitro remodeling within hydrogels

Gelatinase A and B activity of dermal fibroblasts seeded in vitrowithin collagen hydrogels was observed by gelatin zymography ofthe culture media. Bands at 72 kDa and 66 kDa were detected,corresponding respectively to pro-gelatinase A (pro-MMP2) andactivated gelatinase A (MMP2) (Fig. 5A). No bands were observed at92 and 88 kDa, corresponding to gelatinase B (MMP9) forms. At Day1, no active MMP2 was detected in the culture media of NCH andCCH3. At day 7, bands at 66 kDa (active MMP2) appeared and theirintensities normalized for 105 fibroblasts remained unchangeduntil the end of the culture (Fig. 5B). The MMP2 activity quantifiedwith CCH3 media was significantly lower (3–5 times) than this withNCH media whatever the culture time point studied (Fig. 5B). Theactivation ratio of MMP2 (activated form/latent form) was also lowerwith CCH3 than with NCH, with values at day 21: 1.41�0.11 and0.76� 0.21 in NCH and CCH3 respectively.

After one day in culture, immunolabeling of Collagen III wasobserved within the cellular bodies of the entire dermal fibroblastpopulation in NCH and CCH3. At day 21, the percentage of cellscollagen III positive was 65� 4.1% and 91�2.5% within NCH andCCH3 respectively (Supporting data 1).

3.4. Ultrastructural observation of hydrogels

Observation in transmission electron microscopy revealeda homogenous size of collagen fibrils within NCH and CCH3(diameter: about 50 nm). Furthermore from day 7, all fibrils hada cross-striated pattern (Supporting data 2).

Observed in scanning electron microscopy (SEM), few minutesafter their fabrication (Day 0), NCH appeared as a homogenousnetwork of thin and regular fibrils (Fig. 6). The SEM observation at

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day 7 and 21 revealed that NCH contraction gave rise to bundles ofcollagen fibrils. From day 7, the collagen network became hetero-geneous and 5 mm large fibril bundles appeared. In addition, theporosity within normal hydrogels decreased drastically. In contrast,CCH3 exhibited a homogeneous network of thin fibrils during thecell culture (Fig. 6). The hydrogel contraction led to the shrinkage ofthe network mesh but without a fibril bundling. Moreover, CCH3porosity appeared more important than in NCH and pores were stillvisible at day 21.

3.5. Influence of cell density on hydrogel contraction and cellgrowth

Collagen hydrogels were then seeded with fibroblasts at a highcell density (2.5�105 cells/hydrogel). The NCH-HD (HD: highdensity) contraction was characterized by a rapid phase during thefirst day of culture to reach 20% of the initial hydrogel area, followedby a slow contraction phase observed until the end of the culture(Fig. 7A). At day 21, the NCH-HD surface was about 0.08% of theinitial area. In the case of CCH3-HD, the contraction only started atday 4, and then decreased up to 21 days. At day 4, CCH3-HD wasstill 100% of the initial area, versus 3�1% for NCH-HD. At the end ofthe experiment, CCH3-HD surface was about 13% of the initial areaand was 16 times more than the NCH-HD surface. In comparisonwith collagen hydrogels seeded with a low cell density (7.5�104

cells/hydrogel), the contraction observed was faster and moreimportant with for instance a latent phase of 4 days for CCH3-HDinstead of 7 days. The contraction assessed at day 21 was 4.3 timesmore important than the contraction observed within CCH3 seededwith a low cell density.

The number of dermal fibroblasts within H3-HD increasedgradually for the culture period to reach 2,6� 0.19�106 at day 21

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

0

50

100

150

200

1 7 14 21

Time (days)

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Fig. 5. Remodeling of collagen hydrogels. (A): Detection of gelatinase A (MMP2) byzymography in the culture media of normal collagen hydrogels (NCH) and concentratedcollagen hydrogels at 3 mg/ml (CCH3) at day 21. In each condition, two bands at 72 and66 kDa were detected, corresponding to the latent and active form of MMP2 respectively.Control sample was obtained with culture medium of NCH without fibroblasts andcultured over 21 days. (B): Diagram showing gelatinase A (MMP2) activity detected in theculture media at day 1, 7, 14, and 21 for both types of collagen hydrogels. From day 7,MMP2 activity was significantly (*: P< 0.05) lower within CCH3 than within NCH (n¼ 6).

Fig. 6. Hydrogel ultrastructure analyzed by scanning electronic microscopy. A parallel packinday 21 of the cell culture. The aggregation and contraction led to a decrease of porosity. Cnetwork whatever the culture time point. Inset is the macroscopic view of the hydrogel. B

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(Fig. 7B). The most important growth rate (0.27�106 cells/day)was observed between days 1 and 4. Within NCH-HD, the numberof fibroblasts increased slightly from day 1 to 4 to reach0.36� 0.09�106 cells, was stable from day 4 to 14 and decreasedslightly to reach 0.30� 0.01�106 cells at day 21. At the end of theexperiment, number of fibroblasts within CCH3-HD and NCH-HDwas respectively 10.3 and 1.2 times the number of seeded cells. Thenumber of fibroblasts within CCH3-HD was always significantlyhigher than in NCH-HD (P< 0.05) during the cell culture (4.05, 5.15and 8.59 times at day 7, 14 and 21 respectively).

3.6. In vivo integration of collagen hydrogels

Hydrogels CCH3 and NCH, without dermal fibroblastsembedded within the hydrogel, were implanted in subcutaneouspockets (performed within rat abdomen). At the end of the gran-ulation tissue formation (15 days after implantation), NCH haddisappeared (Fig. 8A). In contrast, CCH3 had kept its initial surfaceand was surrounded by a thin fibrous cap (Fig. 8C). CCH3 was stillvisible 30 days after implantation, during the matrix remodelingprocess with a 50% surface reduction (Fig.8D).

Histological analysis of CCH3 revealed a complete colonizationat day 15 and 30 by the host cells with presence of tracks observedwithin implants (Fig. 9A and B). MMP2 immunodetection evi-denced that migration into the hydrogel by host cells relied onmatrix breakdown (Supporting data 3). Some fibrous tissue waspresent above and below the implant indicating remodeling CCH3.Von Kossa staining was negative on CCH3 slides at day 30 thatevidenced absence of mineralization (data not shown). The CD68immunolabeling of CCH3 evidenced a little inflammatory reactionat day 15 post surgery (data not shown). At day 30, no macrophageswere observed within CCH3 (Fig. 9C). Endothelial cells weredetected by RECA-1 immunodetection at day 15 within CCH3hydrogels and endothelial cells organized in tubules-like capillariesand blood vessel were observed at day 30 (Fig. 9D).

g of collagen fibrils was observed within normal collagen hydrogel (NCH) from day 7 tooncentrated collagen hydrogel at 3 mg/ml (CCH3) exhibited a homogeneous collagen

ar: 5 mm.

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

0

20

40

60

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0 7 14 21

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rc

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tal cell n

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1

0 5)

A

B

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*

*

*

*

*

*

**

Fig. 7. Influence of fibroblast density on hydrogel contraction and cell growth. (A):Hydrogel contraction by a high cell density (2.5�105 cells/hydrogel). A rapid contractionwas observed in NCH-HD at day 1 to reach a maximum at day 4. For CCH3-HD, a moredelayed contraction (*: P< 0.05), starting at day 4, was observed reaching 13% of the initialhydrogel surface at day 21 (n¼ 10). (B): Cell growth within collagen hydrogels seeded athigh density (2.5�105 cells/hydrogel). From day 1, the number of cells grew graduallywithin CCH3-HD whereas it was stable within NCH-HD (n¼ 6) (*: P< 0.05 for CCH3-HDversus NCH-HD).

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4. Discussion

The aim of the present work was to improve classical hydrogels(NCH) by raising the initial collagen concentration as much aspossible. Thereafter, concentrated collagen hydrogels populated bydermal fibroblasts were characterized, and their potential utilizationas dermal substitutes evaluated. In this purpose, the effect of collagenconcentration on hydrogel contraction, cell growth and remodelingactivities was evaluated for 21 days of culture. Taking into account theinhibition of contraction, homogeneity and the stimulation of prolif-eration, hydrogels concentrated at 3 mg/ml appeared to be the best toimprove normal hydrogel drawbacks. In vivo integration of collagenhydrogels concentrated at 3 mg/ml was compared to that of 0.66 mg/ml collagen hydrogels (Bell and co-workers method). Colonization byhost cells, host inflammatory response and neovascularization wereassessed in both types of hydrogels.

4.1. Benefit of increased collagen concentration on hydrogelstructure and stability

Normal collagen hydrogels obtained by Bell and co-workers(NHC) are characterized by a poor stability. Dermal fibroblasts

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contract the collagen network few hours after the beginning of cellculture, and shrink the hydrogel area up to 3% of the initial valueafter two weeks [17,19,27]. Extensive contraction constitutes themajor disadvantage toward the utilization of normal collagenhydrogels in tissue engineering [19]. Another drawback is thedifficulty for surgeons to handle and use hydrogels immediatelyafter their fabrication. For instance, it is impossible to suture thehydrogel on a wound bed which explains the necessity to work invivo with contracted hydrogels [15]. The dermal layer of Apligraf� isa normal collagen hydrogel contracted for at least 6 days ona porous membrane [18]. When contracted, the hydrogel porosityis drastically reduced. As a consequence, they do not succeed tointegrate the host organism because of a weak neovascularizationwithin hydrogels [10]. This fact can explain why Apligraf is not usedas a permanent graft but only as a biological dressing. Actually, themajor advantage of Apligraf seems to be the cytokine delivery bydermal fibroblasts that promotes wound healing [14].

Our results show that homogenous concentrated collagenhydrogels can be obtained with a collagen concentration up to 3 mg/ml (CCH3). The hydrogels concentrated at 5 mg/ml (CCH5) appearedgranulous and represent the limit of the technique. An increasedcollagen concentration enhances hydrogel stability and structure.Indeed, CCH3 did not contract after one week in culture. The SEManalyses confirmed the stability of the collagen network and revealeda good porosity which is a critical point for gas and fluid exchanges[10]. At 3 mg/ml, concentrated collagen hydrogels (CCH3) exhibitedgood mechanical properties compared to NCH. As consequence, thesehydrogels can be easily handled and sutured in a wound bed. Evenafter a contraction of about 50% of the initial area at day 21, thenetwork was still homogenous (observation in SEM). The inhibition ofthe contraction within concentrated hydrogels can be explained bytheir stiffness [28–30]. When the collagen concentration is increasedwithin a hydrogel, its stiffness is also increased and its handlingproperties improved. Hence, concentrated collagen hydrogels can beused just after their fabrication without waiting for the end of thecontraction.

4.2. Fibroblast behavior within concentrated collagen hydrogels

To be validated as a suitable device for tissue engineering,a scaffold has to provide the right signals that regulate cell growth,new tissue deposition, and favor the remodeling of the 3D structure[10]. The concentrated collagen hydrogels CCH3 can be considered asgood cell carriers because they stimulate cell growth. Whatever theinitial cell density seeded within 3 mg/ml hydrogels (CCH3), thefibroblast population increased during the cell culture and wasmultiplied by ten at day 21. In contrast, cell proliferation appearedinhibited within normal collagen hydrogels (NCH) after 4 days inculture as previously reported [31]. Several authors suggest thatproliferation is inhibited by increased collagen fibrils/cells contacts[20,22]. Contrary to NCH, hydrogels CCH3 did not exhibit compactfibril bundles and showed a better porosity. So matrix/cells contactswere less important in CCH3 than within NCH and could explain theimproved growth in CCH3. According to Hadjipanayi and co-workers, the regulation of cell proliferation is mainly controlled bymatrix stiffness [32,33]. By compression technique, they obtainedcollagen hydrogels at different concentrations. Mechanical studiesrevealed positive correlation between collagen concentration, stiff-ness and proliferation. They show that fibroblast proliferation isstimulated by an increased stiffness due to an increased collagenconcentration. As CCH3 have an initial concentration five timeshigher than NCH and exhibit better mechanical properties, the sus-tained proliferation can be explained by the higher stiffness. Hence,using CCH3 as dermal substitute allows delivering an importantnumber of cells in the wound bed. Interestingly, at day 14, cell

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

Fig. 8. Macroscopic view of collagen hydrogels 15 and 30 days after implantation in rats. (A, B): NCH had completely disappeared from the implantation site 15 days afterimplantation. (C, D): CCH3 was still visible at day 15 and 30 with a thin fibrous cap surrounding them. At day 30, about 50% of hydrogel surface had disappeared. Bar: 10 mm.

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density within concentrated collagen hydrogels at 3 mg/ml reachedthe one evaluated within dermis in vivo [34].

The cytokine secretion by cells is a key factor for wound healing[14]. For instance, growth factors secreted by dermal fibroblasts favorthe epidermal development but the effects depend on fibroblastdensity. A high cell density increases proliferation of keratinocyteswhereas a low cell density favors keratinocyte differentiation[35,36]. With the aim of evaluating CCH3 as biological dressing forthe treatment of chronic wound or leg ulcer, we could use CCH3 athigh cell density to assess the stimulation of keratinocyte prolifera-tion and CCH3 at low cell density to form a differentiated epidermis.

It was shown that environment properties determine the fate offibroblasts. For instance, apoptosis of fibroblasts embedded withincollagen hydrogels has been observed by different authors [37–39].We also observed this phenomenon within our normal collagenhydrogel (NHC) from day 14. On the opposite, the number of cellsdid not decrease in concentrated collagen hydrogels. One possibilityis that apoptosis is cell density dependant which could explain theabsence of this phenomenon at the beginning of cell culture in NCH[34]. Another hypothesis is that the main stimulus might be the lossof mechanical tension which occurs at the end of contraction [37].The absence of apoptosis in concentrated hydrogels could beexplained by a persisting tension existing within hydrogel duringcell culture. The absence of apoptotic cells within CCH3 is a majoradvantage for an application in tissue engineering. The temporaryrestoration of skin function by implanted cells would be improved,and would let the time for host cells to colonize and remodel thehydrogel.

In all cases, a homogeneous cell distribution was observed withinCCH3 and NCH which is an advantage in comparison with collagensponges. Indeed, within collagen sponges, a cell density gradientexists from the superior to lower levels [40,41]. The fibroblasts within

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concentrated collagen gels had a spindle shape and were morespread than cells within NCH. The fibroblasts within concentratedhydrogels were more stressed by the matrix stiffness and mayassume a synthetic phenotype [22,42]. The anabolic activities wereinvestigated by collagen III detection within hydrogels. Collagen IIIrepresents about 10% of the collagen content within neosynthesizedcollagen fibrils and is a good marker of remodeling. Our results showthat CCH3 favors collagen III synthesis by fibroblasts. In contrast theexpression decreased within NCH from day 1 to day 21 showing aninhibition of the synthesis. These results are consistent witha previous study which evidenced the inhibition of collagen I secre-tion within NCH [21,23]. Therefore, the collagen concentration withinconcentrated hydrogels gives the right signals that favor remodelingby the secretion of matrix macromolecules. Nevertheless thesesynthetic activities must be quantified further.

Gelatinase A (MMP2) is a good marker of matrix breakdownprocess. On one hand, this enzyme performs the complete collagendegradation and on the other hand MMP2 is activated by a collage-nase: MT1-MMP [43]. The activation process depends on biologicalsignals and is stimulated by the link integrin a2b1/collagen fibrils[44]. Moreover, remodeling by metalloproteinases depends onphysical state of the hydrogel. When fibroblasts are stressed by thematrix stiffness, metalloproteinases activities decrease [22]. Thehydrolysis activities detected within concentrated collagen hydro-gels were less important than within normal hydrogels. MMP2activity and the ratio active MMP2/latent MMP2 was higher in NCHthan in CCH3. The hydrolysis activities detected within CCH3resemble those quantified within crosslinked collagen sponges[40,41]. These results can be explained by the stiffness of concen-trated hydrogels. When the collagen concentration is increasedwithin hydrogels, the hydrogel stiffness also increases and cells aremore stressed. As a consequence, hydrolysis activities are inhibited

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

Fig. 9. Histological analysis of CCH3 integration in wistar rats. (A, B): colonization of CCH3 by host cells 15 days (A) and 30 days (B) after implantation (inset is a high magnification of thearea marked with a star). (C): Absence of CD68 immunodetection at day 30. No macrophage was detected within implants. Nucleus of cells was observed by DAPI staining (blue). (D):Detection of endothelial cells by RECA-1 immunodetection. Endothelial cells were detected and were organized in tubular-likes 30 days after implantation (black arrow). Bar 50 mm.

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and concentrated collagen hydrogels are more persistent to let timefor new tissue synthesis.

4.3. In vivo collagen hydrogel integration

Integration of a biomaterial in a host organism depends on itsbiocompatibility, host cell colonization, and initial implant remod-eling by resident cells [6,10]. Acellular classical hydrogels, concen-trated at 0.66 mg/ml (NCH), exhibited a good porosity which favorscell colonization. Unfortunately, besides their poor mechanicalproperties and the difficulty of handle them, hydrogels NCH werealso poorly resistant to hydrolysis by metalloproteinases [10]. Twoweeks after their implantation, NCH were completely hydrolyzed. Asconsequence, the scaffold is not resistant enough to allow cells toremodel and create a neodermis; therefore they cannot be validatedas good candidates for permanent grafting.

In contrast, 3 mg/ml collagen hydrogels (CCH3) have good inte-gration abilities. Two weeks after implantation, CCH3 was completelycolonized by host cells, a neovascularization was observed, and theimplant did not trigger a severe inflammatory response. In addition,some tracks were observed which evidenced CCH3 remodeling byhost cells. The absence of host response comes from the biocompat-ibility of collagen and confirms the advantage of using this naturalpolymer. The ability of a biomaterial to be vascularizated is a criticalpoint for a good integration because blood feeds the resident cell andcarries oxygen. So, using newly prepared CCH3 is clearly an advantage

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in comparison with contracted normal collagen hydrogels (i.e. Apli-graf�) in which a poor neovascularization is observed [5]. Thehydrogel porosity observed in scanning electron microscopy of CCH3favors a rapid colonization by host cells [10]. This colonization ishowever based on hydrolysis because cells which migrated into CCH3secreted MMP2. Finally, no mineralization was observed in opposi-tion with non crosslinked collagen sponges [41]. CCH3 can beimplanted within an organism host since it shows a good persistencewithout any chemical crosslinking. To enhance the colonizationspeed, some growth factors in microspheres could be incorporatedwithin concentrated hydrogels [45].

5. Conclusions

We have demonstrated that concentrated collagen hydrogels at3 mg/ml populated by dermal fibroblasts (CCH3) can be consideredas good candidates for dermal substitution. Contrary to normalcollagen hydrogels (NCH), CCH3 are easy to handle, favor cell growthand are stable over one week with the absence of any contraction. So,increasing collagen concentration improves the hydrogel propertiesand corrects the drawbacks of normal hydrogels. In vivo studies haverevealed that acellular 3 mg/ml collagen hydrogels integrate rapidlya host organism. Hence, CCH3 including fibroblasts could beimplanted in rats. Cells within CCH3 could grow and restore thefunction of dermis. Meanwhile, new blood vessels and other hostcells could colonize the implant, and remodel the system.

n hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/

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Acknowledgments

This work was supported by CNRS, INSERM, Universities Paris 6,7, 13 and EPHE. We thank Gervaise Mosser for her experience intransmission electronic microscopy and Sylvie Igondjo-Tchen forher help in zymography. We would like to thank Thibaud Coradinfor his advices in the preparation of this manuscript.

Appendix

Figures with essential colour discrimination. Most of the figuresin this article have parts that are difficult to interpret in black andwhite. The full colour images can be found in the online version, atdoi: 10.1016/j.biomaterials.2009.09.073

Appendix. Supplementary data

Supplementary data associated with this article can be found inonline version at doi: 10.1016/j.biomaterials.2009.09.073.

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