ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

Abbreviations�CorrespondE-mail addr

1Contributed

Biomaterials 28 (2007) 650–660

www.elsevier.com/locate/biomaterials

A Nd:YAG laser-microperforated poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-basal membrane matrix composite film as substrate

for keratinocytes

Fernando Serranoa,�, Laura Lopez-Ga, Maria Jadraqueb, Marielle Kopera, Gary Ellisc,Pilar Canoc, Margarita Martınb,1, Leoncio Garridoc,1

aFundacion Hospital de Alcorcon, Avda Villaviciosa 1, Alcorcon E-28922, SpainbInstituto de Quımica-Fısica Rocasolano, CSIC, Serrano 119, E-28006 Madrid, Spain

cInstituto de Ciencia y Tecnologıa de Polımeros, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain

Received 7 June 2006; accepted 14 September 2006

Available online 6 October 2006

Abstract

Epithelia cultured for the treatment of ulcers, burns and for gene therapy applications require a flexible biomaterial for growth and

transplantation that is adaptable to body contours. We tested several materials and found that a poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) (PHBHV) polyester provided support for keratinocytes, although adhesion to this material proved to be suboptimal.

Since epithelia adhere to the mesoderm through basal membranes, we engineered a basal membrane surrogate by preparing composites

of PHBHV with basal membrane matrix (BMM). To allow cell migration into injuried areas the polyester film was micromachined to

insert high-density micropores through a Nd:YAG laser ablation process. These flexible composites provided firm attachment for

keratinocytes from the outer root sheath of human hair allowing keratinocyte migration through micropores. Films of microperforated

PHBHV-BMM may be of use for the replacement of diseased or injured skin epithelia.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: PHBHV; Basal membrane matrix; Epithelium; Microperforated biomaterial

1. Introduction

There is an increased demand for epithelium substitutesfor the treatment of severe burns, congenital and acquiredepidermal disorders (epidermolysis bullosa, pemphigoid),and as vehicles for gene therapy applications [1,2]. Theintegrity of an artificial epithelium relies on the existence ofstem cells to maintain its ability to generate differentiatedprogeny [3,4]. Stem cells reside in a microenvironmenttermed the stem cell niche, where they receive intrinsic andextrinsic signals from neighboring cells, the extracellularmatrix, basal membrane (BM) and growth factors.Whereas epidermal cells can be cultured and expanded to

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.09.018

: H-E, Hematoxilin-eosin; Ph/C, Phase contrast.

ing author. Tel.: +3491 5619845; fax: +34 91 5619845.

ess: fserrano@fhalcorcon.es (F. Serrano).

equally to this work.

some degree [5], transplant of pure epidermal sheets issometimes unsatisfactory [1,6]. Engraftment is poor whendermal integrity has been compromised (second-degreeburns). This led to engineering of epidermal/dermalequivalents on which the cultured epidermis is supportedby an underlying fibroblast layer that simulates the dermis,which is then able to graft [1,2,7–9].In vivo, epithelial stem cells do not interact directly with

the underlying connective tissue, but rather through a BM[10]. The principal BM components are collagen IV andlaminins arranged to form a matrix (BMM) [11,12].Laminins appear to maintain the undifferentiated statusof the basal epithelial layer through interaction withintegrins a3b1 and a6b4 [4]. The major epidermal lamininis laminin 5, which is also associated with other epithelialtissue types such as cornea, mammary gland, oral mucosaand pancreatic b cells [10,13–15]. When cultured keratino-cytes are placed in suspension, they withdraw from the cell

ARTICLE IN PRESSF. Serrano et al. / Biomaterials 28 (2007) 650–660 651

cycle and undergo differentiation, which is partiallyinhibited by anti-b1 integrin antibodies [4]. Thus, laminin5/integrin signaling is the main event in keratinocyte stemcell maintenance [16,17].

To simulate an epithelial niche to which basal cells canattach, expand, and provide a sheet for transplant, westudied the use of biocompatible substrates onto which abasal lamina could be assembled. Initial experimentssuggested that keratinocytes could be cultured on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) polye-ster membranes; this material is compatible with mamma-lian blood and other tissues, and can be degraded bynon-specific esterases [18,19]. Whereas attempts have beenmade to modify PHBHV hydrophobicity and improveepithelial adhesion [20,21], our approach consisted ofengineering composite films of PHBHV and BMM. Weshow that the use of this composite promotes rapid adhesionof keratinocytes and morphological changes indicative oftight adhesion. The biomaterial can be micromachined viaUV laser ablation at 266 nm. These composite films allowthe growth and transport of a keratinocyte sheet, and maybe of use in the treatment of burns and ulcers, as well asvehicles for epidermal gene therapy.

2. Materials

PHBHV containing 11% (mol:mol) hydroxyvalerate was purchased

from Aldrich (Sigma-Aldrich Co., Ltd., Gillingham, Dorset, UK).

2.1. Methods

2.1.1. Polymer films

To produce a flexible membrane, PHBHV films were prepared by

solvent casting from polymer solutions in chloroform at different

concentrations into a Petri dish. After casting, chloroform was slowly

evaporated, the membrane cut into 30mm diameter disks (1–50mmthickness), then sterilized with ethanol and desiccated. Polycarbonate and

nylon membranes were purchased from Millipore (Billerica, MA). Fibrin

films were made by coagulating a 3mg/ml fibrinogen solution with

thrombin (1U/ml) (Sigma-Aldrich Co., Ltd., Gillingham, Dorset, UK).

Cell adhesion was measured after a phosphate-buffered saline (PBS) wash

to remove non-adherent cells and attached cells counted in cytometer.

2.1.2. Basal membrane matrix

Insoluble 804G matrix rich in laminin 5 was obtained from 804G

bladder carcinoma cells (American Type Culture Collection, Manassas,

VA) and was made as described [22]. Briefly, 50Gy lethally irradiated

804G cells were cultured for 48 h in the surface where the matrix was to be

deposited. Treatment of the culture area with 20mM NH4OH detached all

cells from plastic, leaving a protein-rich acellular material (matrix) that

was washed several times with water to remove ammonia. This biomaterial

is stable for weeks if wet. The PHBHV-BMM composite was prepared by

assembling laminin 5-rich 804G matrix onto one surface of the PHBHV

film disks. For other experiments, a soluble fraction enriched in a fragment

of laminin 5 secreted into 804G-conditioned medium was obtained by

ammonium sulfate precipitation, as described [23].

Presence of laminin 5 in the composite was monitored by western blot

employing the J-18 rabbit antiserum, kindly donated by J.C. Jones

(Northwestern University, Chicago, IL). This antibody raised against

804G matrix recognizes three major protein bands in the matrix that

correspond to the three chains of the laminin 5 heterotrimer. Western blot

was made as described [23].

2.1.3. HaCat keratinocytes

HaCat cells are non-transformed, immortal human keratinocytes used

as surrogates of interfollicular keratinocytes, and were a kind gift of N.E.

Fusening (German Cancer Research Center, Heidelberg, FRG). Attach-

ment experiments were done with 105 cells/well in 6 well plates in DMEM

(Gibco, Gaithersburg, MD) supplemented with 10% fetal calf serum.

2.1.4. Primary keratinocyte derivation and culture

Individual occipital hairs were plucked from informed donors and

immediately microdissected to expose the bulge region of the outer root

sheath (ORS). Hairs were then cultured in 60% DMEM, 30% F-12

(Gibco, Gaithersburg, MD) with 10% fetal calf serum with adenine, OH-

cortisone, cholera toxin, T3, and bovine insulin (all from Sigma).

Recessive Dystrophic Epidermolysis Bullosa keratinocytes where obtained

from the hair of a informed patient and a normal control and were

immortalized using a Moloney Leukemia Retroviral Vector containing

oncogenic c-myc and ras (plx–myc–ras) pseudotyped with the ecotropic

envelope and a similar retroviral vector containing the ecotropic receptor

(both kindly provided by M. Serrano, CNIO, Madrid, Spain). Retroviral

supernatant production and keratinocyte transduction was done as

described [24]. When necessary primary cultures where detached with a

20min treatment with trypsin and a single-cell suspension was prepared by

pipetting and filtering through a 30mm nylon mesh. The number of

clonogenic cells giving rise to a colony was calculated by plating the cell

suspension into a 100mm diameter dish and scoring the number of

colonies present in plate after a 10-day culture period [25].

2.1.5. Electron microscopy

Cultures were fixed with glutaraldehyde (1:100), then dehydrated and

processed for electron microscopy. Scanning electron microscopy of gold-

coated samples was performed on a microscope Philips XL30 (Philips,

Eindhoven, NL) at ambient temperature by using the parameters

indicated in each photograph.

2.1.6. Laser ablation

The fourth harmonic (266 nm) of a Nd:YAG laser (Ultra CFR, Big Sky

Laser Quantel, Les Ulis Cedex, FR) was employed to ablate the PHBHV

films. Maximum energy per pulse was 4mJ and pulse duration 6 ns. The

laser beam was focused with a quartz lens of 4 cm focal distance.

The ablation threshold at 266 nm, was measured by acoustic detection

methods. The acoustic signal generated in the ablation was detected by a

microphone placed at a distance of 1 cm from the target surface. The

signal was amplified (LF356 amplification circuit) and recorded by a TDS

3020 oscilloscope (Textronix, Richardson, TX). Energy measurements

were made with a pyroelectric detector Joulemeter ED-200. A custom-

build platform was constructed to micromachine the biomaterial.

A computer controlled with Labview software commands three stepper

motors (103H546-0440 Sanyo, Tokyo JP) that allow movement in the

three axis for controlled micromachining.

2.1.7. Infrared spectroscopy

Attenuated total reflectance (ATR) microspectroscopy was performed

on an i-Series IMAGE microscope coupled to a Spectrum 2000 FTIR

spectrometer (Perkin-Elmer, Beaconsfield, UK). Optical micrographs were

obtained on the microscope in the visible image mode, and served to define

the sampling positions. Infrared spectra were recorded using a Germa-

nium ATR crystal with contact face of 100mm diameter, employing a

spectral resolution of 4 cm�1.

Synchrotron infrared microspectroscopy was performed on a con-

tinuum mm infrared microscope coupled to a Magna 860 FTIR spectro-

meter (Thermo, Madison, USA) installed on beamline U10B at the

National Synchrotron Light Source of the Brookhaven National

Laboratory, Upton, NY, USA. Infrared spectra were recorded at a

spectral resolution of 4 cm�1 through an 8 mm confocal aperture.

ARTICLE IN PRESSF. Serrano et al. / Biomaterials 28 (2007) 650–660652

2.1.8. Migration assay

Human primary keratinocytes were transduced with lentiviral super-

natants of prrl.sin 18 CMV EGFP wpre vector (kindly provided by

L. Naldini, TIGEM, Milano, IT) in 60mm diameter dishes as described

elsewhere. EGFP labelled keratinocytes from a confluent dish were

deposited in the upper surface of three microporous PHBHV-BMM

composite where an area of 1 cm2 had been microdrilled. A steel

cylindrical chamber of 12mm diameter kept the keratinocytes in contact

with the upper side of the biomaterial only. The following day the

biomaterial with the keratinocytes attached in one side was lifted, gently

washed with PBS to detach non-adherent cells and placed of top of a 5mm

thick fibrin gel doped with 105 human fetal dermal fibroblast/cc in a 6 well

transwell plate (Corning, Acton, MN) with the keratinocyte side up

exposed to air and subsequently fed from the bottom. Cell migration

through pores was monitored using and inverted microscope equipped

with EGFP epiluminiscence filters. After 20 days of culture the film was

fixed and embedded in paraffin for histology.

3. Results

3.1. Keratinocyte attachment to polymeric films

To identify a flexible membrane that would supportkeratinocyte growth, we tested various biomaterials forpromotion of keratinocyte adhesion after 24h culture (Fig. 1).These materials included bovine fibrinogen, PHBHV,polycarbonate, polycarbonate coated with hydrophilicpolyvinylpyrrolidone, plus collagen, fibronectin or vitro-nectin, non-adhesive nylon, and collagen-coated nylon.The number of HaCat keratinocytes adhered to rigid tissueculture plastic (Falcon) served as a 100% adhesionstandard. Compared to tissue culture-treated plastic

100%25% 50% 75%

Fibrinogen

PHBHV

Cb

Cb-PVP

Nylon

Nylon-Col

Keratinocyte Adhesion 24h

Cb-PVP-Col

Cb-PVP-Fn

Cb-PVP-Vn

T.C. Plastic

Fig. 1. Keratinocytes adhere to PHBHV surfaces. HaCat keratinocytes

were plated onto polymerized bovine fibrinogen, polyhydroxybutyrate-co-

hydroxyvalerate with 12% hydroxyvalerate (PHBHV), polycarbonate

(Cb), hydrophilic polycarbonate coated with polyvinylpyrrolidone (Cb-

PVP), collagen (Cb-PVP-Col), fibronectin (Cb-PVP-Fn), vitronectin (Cb-

PVP-Vn), non-adhesive nylon and collagen-coated nylon (Nylon-Col).

Rigid plasma-treated tissue culture polystyrene (TCP) served as positive

control of adhesion.

(100%) and nylon (0%), these keratinocytes adhered alsoto fibrinogen (30%) and PHBHV (75%).

3.2. Keratinocyte attachment to PHBHV

We monitored the growth dynamics of HaCat keratino-cytes on PHBHV surfaces compared to tissue cultureplastic. After a 24 h culture the tissue culture plastic controlshowed well-defined polygonal cell arrangements withflattened morphology (Fig. 2A). In PHBHV cell morphol-ogy was rounded and they did not spread (Fig. 2B). After48 h, spreading and growth in tissue culture plastic(Fig. 2C) was superior to that observed on PHBHVsurfaces (Fig. 2D). These differences persisted, with cells ontissue culture plastic showing rapid XY-axis growth(Fig. 2E), whereas culture on PHBHV gave rise to slow-growing keratinocyte colonies with rounded edges thattended to grow in the Z-axis (Fig. 2F).

3.3. Keratinocyte attachment to PHBHV-BMM composite

To improve epithelial cell adhesion to PHBHV, weinvestigated whether laminin 5 could be incorporated intoPHBHV, to generate a composite (protein/polyester) film,that may permit a growth pattern similar to that on tissueculture plastic. Laminin 5 is a BM component thatpromotes strong adhesion between the dermis/mesodermand basal epithelial stem cells. An insoluble deposit of BMproteins (matrix, BMM) was obtained by culturing 804Gepithelial cells, and then removing the cells with atreatment that preserves BM integrity. This proteinaceousmatrix is composed mainly of a three-dimensional arrange-ment of laminin 5. Fig. 3 shows the presence of laminin 5 inthe composite PHBHV-BMM material by western blottingusing a polyclonal antibody. Bands detected overlappedwith major protein bands in silver-stained gels.HaCat cells in plastic tissue culture plasma-treated

polystyrene (TCP) on which a BMM was assembled showrapid spreading after 30min and 4 h (Fig. 4B, D) comparedto the same cells on untreated plates (Fig. 4A, C). Westudied whether BMM also promoted rapid keratinocyteattachment to PHBHV surfaces. Cell attachment toPHBHV alone was suboptimal (Fig. 4E), whereas attach-ment and spreading improved markedly on PHBHV-BMMcomposite (Fig. 4F). Soluble laminin 5 was concentratedfrom 804-conditioned medium by ammonium sulfateprecipitation, used to coat PHBHV, and tested in the 4 hadhesion assay. Compared to PHBHV alone (Fig. 4G),keratinocyte attachment was more rapid to PHBHVmembranes preincubated with soluble laminin 5 (Fig. 4H).

3.4. Keratinocyte colony growth in PHBHV-BMM

composite vs. PHBHV alone

Since PHBHV-BMM composite promoted rapid kerati-nocyte attachment and spreading, we analyzed whether thecolony growth pattern in these composite was also

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Fig. 2. Adhesion of basal keratinocytes to PHBHV surfaces is suboptimal. HaCat keratinocytes were plated in TPC and on PHBHV; phase contrast

microscopy (Ph/C). TCP control at (A) 24 h, (C) 48 h and (E) 5 days. PHBHV at (B) 24 h, (D) 48 h and (F) 5 days.

F. Serrano et al. / Biomaterials 28 (2007) 650–660 653

indicative of firm attachment. On PHBHV surfaces, the cellleading edge adheres poorly, while cells at the colony centershow better attachment (Fig. 5A), giving rise to a three-dimensional colony that grows in the Z-axis. Poor cell edgeattachment is indicated by the absence of focal contactpoints and loss of cell contact to the PHBHV surface as aresult of fixation (Fig. 5B). Keratinocyte colonies growingon PHBHV-BMM composite show extensive morphologi-cal changes. Colonies are virtually flat, as they grow in theXY-axes (Fig. 5C, D), and display flattened edges withclose contact to the material that is not disrupted byfixation. Cell leading edges show focal contacts and highlyflattened morphology (Fig. 5E), indicative of tight adhe-sion between the cell sheet and the PHBHV-BMMcomposite (Fig. 5F).

3.5. Outer root sheath keratinocyte culture in PHBHV-

BMM composite

Epidermal stem cells are believed to reside in a G0 phasein the bulge region of the ORS, in an area adjacent to the

attachment of the arrector pili muscle (Fig. 6A). Tomonitor the growth of epidermal cells from single hairs, weconstructed 30mm diameter PHBHV films cast in flexible,5–50 mm-thick membranes. Each disk was coated withBMM and received a single human hair with an intact ORS(Fig. 6B). Twelve of 20 hairs grew in the wells with thePHBHV-BMM composite, compared to 8 in control TCPwells and 18 in TCP wells with BMM; no hairs formedcolonies on PHBHV alone.A scanning electron micrograph of a keratinocyte colony

formed from the ORS of a seeded human hair on PHBHV-BMM composite is shown in Fig. 6C–E, with an overallview of the colony at 16� magnification (Fig. 6C).Fixation artifacts allow definition of cell leading edgegrowth at 100� magnification showing a cobblestonekeratinocyte pattern, as a result of outward growth of theORS stem cells (Fig. 6D). At high magnification (4000� ),the leading edge shows a flattened morphology and closecontact between the cell and the composite (Fig. 6E).Transmission electron microscopy of HaCat keratinocytesgrowing on a disk showed close contact between the cell

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3T3J2 MATRIX 804G MATRIX

TCP PHB TCP PHB kDa

160

105

Fig. 3. Presence of laminin 5 in PHBHV-BMM composite biomaterial. A structured laminin 5-rich matrix (BMM) was deposited on TCP and PHBHV.

Insoluble matrix deposited was analyzed by western blotting.

F. Serrano et al. / Biomaterials 28 (2007) 650–660654

and the composite, with a dense basal membrane-likematerial between the cell and the PHBHV-BMMcomposite that may correspond to the laminin 5 matrix(Fig. 6F).

3.6. Clonogenic assays in PHBHV-BMM and

complementation of keratinocyte attachment deficits

A biomaterial able to support stem cell integrity shouldmaintain the ability of those cells to self renew. Thus, wemeasured if the composite biomaterial maintained clono-genicity. PHBHV-BMM and PHBHV control disks werebound to 35mm dishes and its colony output compared toa TCP standard. Fig. 6G shows that after an input of akeratinocyte cell dose from a biopsy of known clonogeniccontent, the PHBHV-BMM composite disk and TCP wereentirely covered with a keratinocyte sheet, that after a 10day culture period resulted in a 6-fold expansion in thenumber of clonogenic precursors (5 to 30 and 33clonogenic cells/cm2). However, in the PHBHV disk, thegrowth of cells in the Z-axis resulted in squamousdifferentiation and lost of clonogenicity. This effect wasmost pronounced when these cells where replated intosimilar disks and the number of secondary clonogenicprecursors measured.

Recessive Dystrophic Epidermolysis Bullosa (RDEB)keratinocytes are unable to synthesize a proper extracel-lular matrix. RDEB keratinocytes have a deep deficit ofinitial adhesion to culture biomaterials [26]. After initialobservations that RDBE keratinocytes did not adhere toPHBHV in the 4 h adhesion assay, we measured if BMMcould complement this deficit. Fig. 6H shows results of a

4 h adhesion assay normalized for the attachment ofkeratinocytes of a control donor. A collagen matrixprovided by 3T3-J2 fibroblast improved adhesion of donorkeratinocytes to PHBHV but failed to restore adhesion ofRDEB keratinocytes to basal donor levels. Addition of theBMM greatly (3 fold) improved adhesion of donorkeratinocytes but most notably complemented the earlyadhesion deficit of RDEB cells.

3.7. Laser ablation and pore migration assay

Epithelial cells are polarized. In order to maintain rightgraft polarity in a putative transplantation setting, westudied the insertion of pores in films. The 4th harmonic ofa Nd:YAG laser with a wavelength of l ¼ 266 nm, close tothe absorption band of the carbonyl group in PHBHV, wasused to perforate the polymer at a fluence of 6.7 J cm�2.Five laser shots were delivered on the same spot of thetarget at a repetition rate of 4Hz, yielding micropores eachone with a well defined oval hole of 150� 100 mm(�11.7� 10�3mm2) apparently free of alterations.A micromachining platform was built to microfabricate alattice of dense microholes by minimizing InterNodalDistance (IND) between holes to allow maximal cellmigration. It was possible to decrease IND up to 200 mm.Fig. 7A shows a PHBHV film of 200 mm IND. Thethreshold fluence at this laser wavelength was of 1.2 J cm�2,as shown in Fig. 7B. Energy transmission at edges of themicropore resulting in some chemical modification of thepolymer films compared to the untreated areas was studiedby infrared ATR microspectroscopy. The variationsare illustrated in Fig. 7C, where several differences were

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Fig. 4. PHBHV-BMM composite improve keratinocyte attachment compared to PHBHV (Ph/C). (A) HaCat keratinocytes on tissue culture plastic 30min

post-plating. (B) Cells as in (A) on tissue culture plastic coated with BMM. (C) As in (A), at 4 h post-plating. (D) As in B, at 4 h post-plating. (E) HaCat

cells in PHBHV 4h post-plating. (F) As in E, in PHBHV-BMM composite. (G, H) Soluble laminin 5 partially mimics the effect of BMM. (G) HaCat cells

on PHBHV, 4 h post-plating. (H) As in G, PHBHV coated with soluble laminin 5.

F. Serrano et al. / Biomaterials 28 (2007) 650–660 655

observed, including a reduction in the amplitude of theabsorption band associated to the carbonyl group.Transmission synchrotron infrared microspectroscopywas employed to confirm this observation with 1 mm thickfree-standing films (Fig. 7D).

Porous films of 200 mm IND were employed in amigration assay. HaCat cells were labeled with a retroviralvector containing the EGFP fluorescent protein. Fig. 7E, F

shows adhesion at irradiated edges with microporeinvasion by HaCat cells. Fig. 7G shows epifluorescenceand white light view of 200 mm IND microperforatedcomposite biomaterial after seeding of primary EGFPkeratinocytes at the start of culture. At the end of the20-day culture period, a thick dense multilayered epidermiswas present on both sides of microperforated biomaterial(Fig. 7H).

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Fig. 5. Surface scanning electron microscopy (SEM) of HaCat keratinocyte colonies. (A) PHBHV, 470� ; (B) PHBHV, 2000� ; (C, D) PHBHV-BMM

composite, 350� ; (E) PHBHV-BMM composite, 5000� ; (F) HaCat in PHBHV-BMM composite (H–E).

F. Serrano et al. / Biomaterials 28 (2007) 650–660656

4. Discussion

There is considerable interest in the development ofepidermal equivalents for biomedical uses such as treat-ment of second-degree burns, chronic ulcers and genetherapy applications [24,27–32].

We tested several materials for their ability to supportkeratinocyte attachment and proliferation. Attachmentand growth pattern appearance were studied using HaCatcells, a non-transformed keratinocyte cell line [23]. Initialexperiments showed suboptimal attachment to PHBHVsurfaces, a material previously reported to supportepidermal growth [18–21,33]. PHBHV is a non-toxic, fullybiodegradable polyester that is broken down by esterasesto D-3 hydroxybutyrate, a normal human serum compo-nent, and polymers of the polyhydroxyalcanoate familydo not cause foreign body reaction when implanted. Toimprove cell adhesion to PHBHV surfaces we engineered asurrogate BM to which keratinocytes could attach[34–39]. A structured laminin 5-rich matrix (BMM) wasassembled by culturing the well-studied laminin 5-deposit-

ing 804G cell line upon PHBHV films [22,40–45]. Aftercell removal, a composite material was generated compris-ing a polyester layer that provides integrity and weakattachment, plus a laminin 5-rich arrangement thatsignals the cell to attach firmly as in a BM (Fig. 7I). Thismaterial has optimal attachment and growth propertiesfor keratinocytes, resulting in morphological changesindicative of strong adhesion between the cell and thecomposite.We seeded composite disks with individual adult hairs, a

source of keratinocyte stem cells. ORS keratinocytesproliferated and covered the entire surface of these30mm disks in 4 weeks. PHBHV-BMM compositemaintained keratinocyte clonogenicity and complementedthe adhesion deficit of cells that cannot synthesize a propermatrix. These assays point that the composite biomaterialmay maintain the functionality of epidermal stem cells.Future work will include competitive long-term reconstitu-tion of the live animal to ascertain this point.Finally, micropores were inserted through laser ablation

[46]. It was found that Nd:YAG pulsed irradiation at the

ARTICLE IN PRESS

40

Input

Co

lon

ies/

cm2

10

20

30

Primary Secondary

350%

Basal

Per

cen

tag

e o

f ad

hes

ion

50%

100%

125%

3T· J2 Matrix 804G BM Matrix

(G) (H)

(A) (B)

(D)

(F)(E)

(C)

Fig. 6. Growth of primary keratinocyte colonies from outer root sheath (ORS) hair cells on PHBHV-BMM composite disks. (A) Plucked human hair;

arrow indicates ORS. (B) Keratinocyte sheets obtained from the ORS after a 3-week culture period (8� ) (Ph/C). (C–E) SEM. (C) Colony, 16� . (D)

Leading edge and ORS, 100� . (E) Leading edge, 4000� . (F) Transmission electron micrograph. (G) Maintenance of clonogenic precursors in PHBHV-

BMM composite. Columns: black TCP, open PHBHV-BMM, shaded PHBHV. (H) PHBHV-BMM composite complement the defect of RDEB

keratinocytes. Columns: black normal donor keratinocytes, open RDEB keratinocytes.

F. Serrano et al. / Biomaterials 28 (2007) 650–660 657

wavelength of 266 nm, resulted in micropores of desiredsize while energy transfer modified the chemical composi-tion at the irradiated zones but did not altered cell adhesionat irradiated pore edges. A micropore dense lattice wasconstructed by micromachining resulting in a microperfo-

rated film of 30 mm thickness with a nominal IND of200 mm (2500micropores/cm2) that maintains physical filmstability and provides a large pore area (29.4% of total)for the migration of subconfluent keratinocytes into awounded area.

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8

Fluence / Jcm-2

Mic

roph

one

Vol

tage

/ V

1

6

5

1

2

3

4

2 3 4 5 76

3600

(B)

(C) (D)2800 2000 1600 1200 800

Wavenumber,cm-1

0

0.2

0.4

0.6

A

3600 2800 2000 1600 1200

Wavenumber,cm1

A

100 µm 100 µm

100 µm 100µm

1 mm

νC=OA

00 10050 150 200

50

100

150

µm

µm

00 10050 150 200

50

100

150

µm

µm

00 10050 150 200

50

100

150

µm

µm

00 10050 150 200

50

100

150

µm

µm

νC=O

νOH

(I)

(A)

(E) (F)

(H)(G)

F. Serrano et al. / Biomaterials 28 (2007) 650–660658

ARTICLE IN PRESSF. Serrano et al. / Biomaterials 28 (2007) 650–660 659

5. Conclusions

Laser-microperforated PHBHV-BMM (200 IND) com-posite films are an alternative surface for the culture ofepidermal equivalents. This biomaterial may be of interestfor the treatment of burns, ulcers and for use in cutaneousgene therapy protocols.

Acknowledgments

The authors thank C. Mark for editorial assistance.A. Monforte electronic technician of M.M and later ofF. S. constructed the computer-controlled platform underguidance of M.M. Some experiments were done inA. Bernad laboratory at Centro Nacional de Biotecnolo-gıa, CSIC. This work was financed in part by the FundacioPrivada de Hemofilia Catalana and the Bayer YoungInvestigator Hemophilia Award 2003 Grant Program toF.S. L.G. acknowledge the financial support provided bythe MSyC, FIS01/1070; F.S. and L.G. of FIS PI05/1847and M.M. and L.G. of CSIC-PIF 2004. The infraredmeasurements undertaken at the National SynchrotronLight Source, Brookhaven National Laboratory, which issupported by the US Department of Energy, Division ofMaterials Sciences and Division of Chemical Sciences,under Contract No. DE-AC02-98CH10886.

References

[1] Boyce ST, Warden GD. Principles and practices for treatment

of cutaneous wounds with cultured skin substitutes. Am J Surg

2002;183(4):445–56.

[2] Kirsner RS, Falanga V, Eaglstein WH. The development of

bioengineered skin. Trends Biotechnol 1998;16(6):246–9.

[3] Li A, Simmons PJ, Kaur P. Identification and isolation of candidate

human keratinocyte stem cells based on cell surface phenotype. Proc

Natl Acad Sci USA 1998;95(7):3902–7.

[4] Watt FM. Role of integrins in regulating epidermal adhesion, growth

and differentiation. EMBO J 2002;21(15):3919–26.

[5] Gallico III GG, O’Connor NE, Compton CC, Kehinde O, Green

H. Permanent coverage of large burn wounds with autologous

cultured human epithelium. N Engl J Med 1984;311(7):448–51.

[6] Muhart M, McFalls S, Kirsner R, Kerdel F, Eaglstein WH.

Bioengineered skin. Lancet 1997;350(9085):1142.

[7] Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Living tissue formed in

vitro and accepted as skin-equivalent tissue of full thickness. Science

1981;211(4486):1052–4.

[8] Daniel JT, Kearney JN, Ingham E. Human keratinocyte isolation

and cell culture: a survey of current practices in the UK. Burns

1996;22(1):35–9.

[9] Hansbrough JF, Mozingo DW, Kealey GP, Davis M, Gidner A,

Gentzkow GD. Clinical trials of a biosynthetic temporary skin

replacement, dermagraft-transitional covering, compared with

Fig. 7. Micropore machining and cell migration through PHBHV-BMM. (

PHBHV in air. (C) Infrared attenuated total reflectance spectra of pore edge

microspectra of polymer and pore edge. Inset shows approximate sampled posi

(Ph/C epiluminiscence). (E) EGFP-labelled HaCat cells after attachment. (F)

initial attachment. (H) G after culture (20 days) (H–E). (I) Schematic diagra

monomer. (I-right) PHBHV-BMM composite.

cryopreserved human cadaver skin for temporary coverage of excised

burn wounds. J Burn Care Rehabil 1997;18(1 Part 1):43–51.

[10] Colognato H, Yurchenco PD. Form and function: the laminin family

of heterotrimers. Dev Dynamics 2000;218(2):213–34.

[11] Tunggal P, Smyth N, Paulsson M, Ott MC. Laminins: structure and

genetic regulation. Microsc Res Tech 2000;51(3):214–27.

[12] Petitclerc E, Boutaud A, Prestayko A, Xu J, Sado Y, Ninomiya Y,

et al. New functions for non-collagenous domains of human collagen

type IV: novel integrin ligands inhibiting angiogenesis and tumor

growth in vivo. J Biol Chem 2000;275(11):8051–61.

[13] Patarroyo M, Tryggvason K, Virtanen I. Laminin isoforms in tumor

invasion, angiogenesis and metastasis. Semin Cancer Biol 2002;12(3):

197–207.

[14] Fleischmajer R, Utani A, MacDonald ED, Perlish JS, Pan TC, Chu

ML, et al. Initiation of skin basement membrane formation at the

epidermo-dermal interface involves assembly of laminins through

binding to cell membrane receptors. J Cell Sci 1998;111(Part 14):

1929–40.

[15] Michelson PH, Tigue M, Jones JC. Human bronchial epithelial cells

secrete laminin 5, express hemidesmosomal proteins, and assemble

hemidesmosomes. J Histochem Cytochem 2000;48(4):535–44.

[16] Baker SE, DiPasquale AP, Stock EL, Quaranta V, Fitchmun M,

Jones JC. Morphogenetic effects of soluble laminin-5 on cultured

epithelial cells and tissue explants. Exp Cell Res 1996;228(2):262–70.

[17] Patthy L. A family of laminin-related proteins controlling ectodermal

differentiation in Drosophila. FEBS Lett 1992;298(2–3):182–4.

[18] Holland SJ, Yasin M, Tighe BJ. Polymers for biodegradable medical

devices. VII. Hydroxybutyrate–hydroxyvalerate copolymers: degra-

dation of copolymers and their blends with polysaccharides under in

vitro physiological conditions. Biomaterials 1990;11:206–15.

[19] Holland SJ, Jolly AM, Yasin M, Tighe BJ. Polymers for biodegrad-

able medical devices. II. Hydroxybutyrate–hydroxyvalerate copoly-

mers: hydrolytic degradation studies. Biomaterials 1987;8(4):289–95.

[20] Tezcaner A, Bugra K, Hasirci V. Retinal pigment epithelium cell

culture on surface modified poly(hydroxybutyrate-co-hydroxyvale-

rate) thin films. Biomaterials 2003;24(25):4573–83.

[21] Yasin M, Holland SJ, Jolly AM, Tighe BJ. Polymers for biodegrad-

able medical devices. VI. Hydroxybutyrate–hydroxyvalerate copoly-

mers: accelerated degradation of blends with polysaccharides.

Biomaterials 1989;10(6):400–12.

[22] Langhofer M, Hopkinson SB, Jones JC. The matrix secreted by 804G

cells contains laminin-related components that participate in hemi-

desmosome assembly in vitro. J Cell Sci 1993;105(Part 3):753–64.

[23] Hormia M, Falk-Marzillier J, Plopper G, Tamura RN, Jones JC,

Quaranta V. Rapid spreading and mature hemidesmosome formation

in HaCat keratinocytes induced by incubation with soluble laminin-

5r. J Invest Dermatol 1995;105(4):557–61.

[24] Del Rio M, Larcher F, Serrano F, Meana A, Munoz M, Garcia M,

et al. A preclinical model for the analysis of genetically modified

human skin in vivo. Hum Gene Ther 2002;13(8):959–68.

[25] Papini S, Cecchetti D, Campani D, Fitzgerald W, Grivel JC, Chen S,

et al. Isolation and clonal analysis of human epidermal keratinocyte

stem cells in long-term culture. Stem Cells 2003;21(4):481–94.

[26] Chen M, Kasahara N, Keene DR, Chan L, Woodley DT.

Restoration of type VII collagen expression and function in

dystrophic epidermolysis bullosa. Nat Genet 2002;32:670–5.

[27] Saffle JR, Davis B, Williams P. Recent outcomes in the treatment of

burn injury in the United States: a report from the American Burn

Association Patient Registry. J Burn Care Rehabil 1995;16(3 Part 1):

288–9.

A) Microporous 200mm IND lattice, (20� ). (B) Ablation threshold of

compared to non-irradiated area. (D) Transmission synchrotron infrared

tions. (E, F) Cell migration through PHBHV-BMM composite micropores

E after culture (5 days). (G) Primary EGFP-labelled keratinocytes after

m of the PHBHV-BMM composite film. (I-left) Heterotrimeric (a3b3g2)

ARTICLE IN PRESSF. Serrano et al. / Biomaterials 28 (2007) 650–660660

[28] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med

1999;341(10):738–46.

[29] Larcher F, Del Rio M, Serrano F, Segovia JC, Ramirez A, Meana A,

et al. A cutaneous gene therapy approach to human leptin

deficiencies: correction of the murine ob/ob phenotype using leptin-

targeted keratinocyte grafts. FASEB J 2001;15(9):1529–38.

[30] Serrano F, Del Rio M, Larcher F, Garcia M, Munoz E, Escamez MJ,

et al. A comparison of targeting performance of oncoretroviral versus

lentiviral vectors on human keratinocytes. Hum Gene Ther 2003;

14(16):1579–85.

[31] Higham MC, Dawson R, Szabo M, Short R, Haddow DB, MacNeil

S. Development of a stable chemically defined surface for the culture

of human keratinocytes under serum-free conditions for clinical use.

Tissue Eng 2003;9(5):919–30.

[32] Lam PK, Chan ES, To EW, Lau CH, Yen SC, King WW.

Development and evaluation of a new composite laserskin graft.

J Trauma 1999;47(5):918–22.

[33] Yasin M, Holland SJ, Tighe BJ. Polymers for biodegradable medical

devices. V. Hydroxybutyrate–hydroxyvalerate copolymers: effects of

polymer processing on hydrolytic degradation. Biomaterials 1990;

11(7):451–4.

[34] DeHart GW, Healy KE, Jones JC. The role of alpha 3 beta 1 integrin in

determining the supramolecular organization of laminin-5 in the

extracellular matrix of keratinocytes. Exp Cell Res 2003;283(1):67–79.

[35] Baker SE, Hopkinson SB, Fitchmun M, Andreason GL, Frasier F,

Plopper G, et al. Laminin-5 and hemidesmosomes: role of the alpha 3

chain subunit in hemidesmosome stability and assembly. J Cell Sci

1996;109(Part 10):2509–20.

[36] Eble JA, Wucherpfennig KW, Gauthier L, Dersch P, Krukonis E,

Isberg RR, et al. Recombinant soluble human alpha 3 beta 1 integrin:

purification, processing, regulation, and specific binding to laminin-5

and invasin in a mutually exclusive manner. Biochemistry

1998;37(31):10945–55.

[37] Grose R, Hutter C, Bloch W, Thorey I, Watt FM, Fassler R, et al. A

crucial role of beta 1 integrins for keratinocyte migration in vitro

and during cutaneous wound repair. Development 2002;129(9):

2303–15.

[38] Haase I, Hobbs RM, Romero MR, Broad S, Watt FM. A role for

mitogen-activated protein kinase activation by integrins in the

pathogenesis of psoriasis. J Clin Invest 2001;108(4):527–36.

[39] Evans RD, Perkins VC, Henry A, Stephens PE, Robinson MK, Watt

FM. A tumor-associated beta 1 integrin mutation that abrogates

epithelial differentiation control. J Cell Biol 2003;160(4):589–96.

[40] Falk-Marzillier J, Domanico SZ, Pelletier A, Mullen L, Quaranta V.

Characterization of a tight molecular complex between integrin alpha

6 beta 4 and laminin-5 extracellular matrix. Biochem Biophys Res

Commun 1998;251(1):49–55.

[41] Lissitzky JC, Cantau P, Martin PM. Heterotrimeric configuration is

essential to the adhesive function of laminin. J Cell Biochem

1992;48(2):141–9.

[42] Kariya Y, Ishida K, Tsubota Y, Nakashima Y, Hirosaki T, Ogawa T,

et al. Efficient expression system of human recombinant laminin-5.

J Biochem (Tokyo) 2002;132(4):607–12.

[43] Kariya Y, Tsubota Y, Hirosaki T, Mizushima H, Puzon-McLaughlin

W, Takada Y, et al. Differential regulation of cellular adhesion and

migration by recombinant laminin-5 forms with partial deletion or

mutation within the G3 domain of alpha3 chain. J Cell Biochem

2003;88(3):506–20.

[44] Lefebvre VH, Otonkoski T, Ustinov J, Huotari MA, Pipeleers DG,

Bouwens L. Culture of adult human islet preparations with

hepatocyte growth factor and 804G matrix is mitogenic for duct

cells but not for beta-cells. Diabetes 1998;47(1):134–7.

[45] Riddelle KS, Hopkinson SB, Jones JC. Hemidesmosomes in the

epithelial cell line 804G: their fate during wound closure, mitosis and

drug induced reorganization of the cytoskeleton. J Cell Sci

1992;103(Part 2):475–90.

[46] Kruger J, Niino H, Yabe A. Investigation of excimer laser ablation

threshold of polymers using a microphone. Appl Surf Sci 2002;197:

800.