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Chapitre III: MANUSCRITDE:«Human epidermal sheets cultured on plastic and fibrin gels: a comparative study»

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Les brûlures sévères détruisent la fonction barrière de la peau, rendent l’organisme sujet aux infections et aux pertes fluidiques et protéiques. Il est donc crucial de restaurer cette barrière. La greffe de peau autologue sur les sites affectés est préconisée. Celle-ci est toutefois limitée par l’état et le nombre de sites donneurs. La régénération des dits sites par des feuillets épidermiques autologues, accélère leur guérison. L’utilisation de la dispase, requise pour détacher les feuillets, restreint son utilisation. Des feuillets cultivés sur un substrat greffable, comme le gel de fibrine ne requiert pas de dispase. Cette étude vise à comparer les feuillets épidermiques cultivés sur fibrine à ceux cultivés sur plastique. Les analyses ont révélé que les deux types des feuillets épidermiques possédaient des caractéristiques histologiques et immunohistochimiques similaires. Il apparaît alors que la culture des cellules épithéliales sur un gel de fibrine peut produire des feuillets épidermiques confluent normaux.

Human epidermal sheets cultured on plastic and fibrin gels: a comparative study

Eric Boucher, Claudia Fugère, François Auger et Lucie Germain.

Laboratoire d’Organogénèse Expérimentale, Hôpital du Saint-Sacrement du Centre Hospitalier Affilié à l’Université Laval, Sainte-Foy, Québec, Canada and Département de Chirurgie, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada

Running title: Effect of fibrin glue

Key words: fibrin glue, stem cells, keratinocytes, cultured epithelial sheets

Address reprint requests to:

Dr Lucie Germain
Laboratoire d’Organogénèse Expérimentale
Hôpital du Saint-Sacrement
1050, chemin Sainte-Foy, Québec
Québec, Canada, G1S 4L8
Tel: 418-682-7696 Fax: 418-682-8000
E-mail: lucie.germain@chg.ulaval.ca
www.fmed.ulaval.ca/LOEX

Extensive burns destroy the protective barrier of the skin, rendering the organism susceptible to bacterial infection. Important fluidic and loss of proteins can also ensue, leading to high metabolic stress. Thus, rapid restoration of this barrier is crucial to patient recovery. The preferred approach involves grafting autologous split-thickness skin over the wound bed. This approach is however greatly limited by the surface area of the body that was not affected by burns. One method used to circumvent this problem involves accelerating the regeneration of the donor sites by grafting them with cultured autologous epithelial sheets. Traditional epithelial sheets culture requires a dispase treatment that could potentially be detrimental to cell viability and requires costly logistics. Culturing epithelial cells on a readily graftable substrate, such as fibrin gel can side step this costly procedure. Epithelial sheets cultured on fibrin gels have already been successfully grafted onto patients in Europe. Moreover, in vitro testing revealed that sub-confluent epithelial cells cultured on a fibrin matrix share similar proliferative potential and differentiation markers with those cultured on plastic. This study compares fully confluent epithelial sheets cultured on plastic and on fibrin. We found significant histological differences between fibrin and plastic cultured epithelial sheets, likely due to contraction of the plastic cultured epithelial sheet once it was detached. Immunohistological analyses did not reveal any significant differences between the epithelial sheets produced by the two culture systems. Thus, our results show that epithelial cells cultured on a fibrin matrix can produce graftable, fully confluent epithelial sheets.

The aim of the treatments for patients suffering from extensive full-thickness burns is to recover their wounds with functional skin to regenerate the skin barrier to infection and water loss. The preferred approach involves grafting autologous split-thickness skin over the wound bed. A way to accelerate the regeneration of the barrier is to regenerate donor site epidermis by grafting them with autologous cultured epithelial sheets. Traditional epithelial sheets culture requires a dispase treatment that could potentially be detrimental to cell viability and requires costly logistics. Alternative methods of culture that could eliminate the need of dispase treatment, facilitate manipulation of the epithelial sheets or produce a more complete skin equivalent (i.e. with an epidermis and a dermis) have been investigated: keratinocyte cultured on collagen gels [9-11], in artificial or biological membranes [12, 13] and keratinocyte cultured on fibrin gels [14-17]. Fibrin glue is a commercially available (as Tisseel VH®) mixture of human plasma derived fibrinogen, plasmafibronectin, plasminogen, factor XIII, thrombin and bovine aprotinin. Formation of the fibrin gel prepared in vitro mimics blood clotting in vivo . Briefly, the fibrinogen zymogen is activated by thrombin and thus gives rise to fibrin monomers. Factor XIII and calcium then help in the formation and the stabilization of the fibrin polymer. It is in use in many European countries as well as Canada and has been shown to be safe and effective for sealing, gluing and reestablishing hemostasis in many types of surgery [18]. Its biodegradability properties make it an ideal substrate for epidermal sheets culture and grafting. Earlier results have shown that spraying fibrin glue on the wound bed of an athymic mouse modelenhanced mechanical stability, leads to better graft take of cultured human epithelial sheets and reformation of the basement membrane formation, although delayed, was not inhibited [19]. Use of fibrin glue on the wound bed of severe burn patients also led to a to better graft take of cultured human epithelial sheets and faster revascularisation . Other teams adapted traditional epithelial cell culture methods (plastic substrate) to use fibrin as an alternate culture substrate. Cells were either cultured directly onto [14, 15, 17] or embedded in fibrin gels [16, 20, 21]. When cultured onto a fibrin matrix, epithelial sheets are able to successfully reach confluence and even form epidermal sheets with an efficiency comparable to cells cultured on plastic [15, 17]. Consequently, fibrin-cultured epithelial sheets have been successfully transplanted onto corneal [16, 22] and burns related defects [15, 17].

Moreover, epithelial cells culture on a fibrin substrate did not alter their clonogenic potential negatively and the epithelial stem cells were conserved as effectively as on plastic [15, 16, 22]. Even though, previous studies examined differentiation markers in epithelial cells cultured on fibrin gels [16], a comparative study between the properties of epithelial sheets cultured on plastic and those cultured on a fibrin gel have yet to be made. This study compares the epithelial sheets produced by the two culture systems on the basis of their mechanical properties, histology, keratin expression and proliferation markers.

Here we show that human epithelial sheets cultured on fibrin gels display normal keratin patterning similar to their counterparts cultured on plastic. Furthermore, absence of contraction of the fibrin cultured epithelial sheets (FCES) made it possible for the cells to retain a normal cuboïdal shape whereas cells from the control sheets seemed more isometric and loosely connected. Thus, it appears that the use of fibrin for the culture of epithelial sheets could be beneficial for the treatment of extensive burn patients. It does not seem to negatively influence the quality of the produced epithelial sheets, but diminishes the number of sheets necessary to cover a given area thus decreasing the cost of treatment.

Biopsies were taken directly from epithelial sheets cultured on fibrin or plastic two days after confluence. Sheets cultured on fibrin were detached by removing the HDPE ring from the petri dish. As previously described, sheets cultured on plastic required enzymatic treatment with dispase to detach them from the petri dish. Biopsies were taken from sheets at different levels of contractions. Biopsies were either embedded in OCT and cryopreserved for immunohistological staining or fixed in Bouin or Histochoice and embedded in paraffin. Five-micrometer-thick paraffin embedded tissue sections were processed for standard Masson’s trichrome staining. Acetone-fixed 5μm-thick cryosections were labeled by indirect immunofluorescence using the antibodies listed below. Briefly, cryosections were fixed with acetone (10 minutes at -20°C) as previously described. Sections were incubated with the primary antibody for 45 minutes, followed by incubation with the conjugated secondary antibody for 30 minutes. Cell nuclei were counterstained with Hoechst reagent 33258 (Sigma, Oakvill, Ontario, Canada). The antibodies used included mouse monoclonal AE3, an antibody specific to epithelial keratins (ICN), anti-human keratin 10 (clone K8,60; Sigma), anti-Ki67 (Plasminogen), anti-filaggrin (BTI), human transglutaminase (BTI), rabbit anti-human keratin 14 directed against peptide (kind gift from Dr. Normand Marceau) and rabbit polyclonal anti-human involucrin (Biomedical technologies). DTAF-conjugated goat anti-mouse secondary antibody (Chemicon) and DTAF-conjugated goat anti-rabbit secondary antibody (Chemicon) were used. Direct immunofluorescence was also performed with a mouse monoclonal anti-human keratin 19 (clone A53-B/A6; kind gift from Dr. Uwe Karsten) conjugated to fluorescein-isothiocyanate (FITC).Negative controls consisted of omission of the primary antibody during the labeling reaction. Cells were observed under a Nikon Optiphot (Tokyo, Japan) microscope equipped with epifluorescence and photographed with a CCD camera (Sensys).

Figure I

The histological apparence of the cultured epidermal sheets on plastic (A) and on a fibrin gel (B), produced from newborn cells. The histological cross-section are phase-contrast micrographs stained by Masson’s trichrome. Note that keratinocytes cultured on plastic or on the fibrin gel are well organized. Biopsies were taken two days after confluence. Time to reach confluence: 7 days. Scale bar: 50μM .

Total keratin (K) expression in epidermal sheets was detected using AE3 which stains all layers of the epidermis in normal skin [26, 27]. Normal patterns of specific keratins expression were expressed in both fibrin and plastic cultured epithelium ( Fig II ) as their AE3 labeling shows ( Fig II G-P) . In normal human skin, as well as in our epithelial sheets cultured on fibrin and on plastic cells in the basal layer (referred as basal cells) are undifferentiated cells and express K14 ( Fig II I-R) . The more differentiated cells (suprabasal cells) stop expressing K14 and start expressing K10. Since we did not let our cultured epithelia fully differentiate, both plastic and fibrin cultured epithelial

sheets did not expressed K10 ( Fig II H-Q) . To further evaluate the differentiation of our cultured epithelia, the presence of proteins specific to the differentiation process were also evaluated. As expected, only moderate labeling can be observed in either epithelia for markers such as involucrin ( Fig IV H;Q) and transglutaminase ( Fig IV I;R) in the suprabasal region of the epithelia. Fillagrin on the other hand did not produce significant signal ( Fig IV G-P) .

Figure III

Histologies and immunohistologies of plastic (A-F) and fibrin (G-L) cultured epidermal sheets. Cryopreserved sections were stained with Ab directed K19 (E;K), and proliferation marker Ki67 (F;L) Tissues were also counter-stained with Hoechst 33258 (C-D; I-J).

Normal epithelium is regenerated every 26 days. This feat is accomplished by a small proliferating subpopulation of the basal cells: the transitory amplification cells (TA cells). The TA cells are thought to come from an even smaller sub-population of epithelial cells, the epithelial stem cells. Although definite markers for this subpopulation do not exist, epithelial cells are routinely identified on the basis of their proliferative potential and of the expression of K19, a keratin restricted to a subpopulation of cells in the basal layer and to Merkels cells in human skin epithelium. Epidermal sheets cultured on fibrin ( Fig III K) and on plastic ( Fig III E ) both shows keratin 19 expression.The differences in labelling intensity could be explained by the fact that the plastic cultured sheets contracted and their fibrin cultured counterparts did not. The actively dividing population was detected using the Ki67 antibody. Again, epithelial sheets cultured on fibrin ( Fig III L ) and plastic ( Fig III F ) contained positive cells. Again, differences in labeling activity are probably connected to the contraction of the plastic cultured sheet. It then follows that the human epithelial sheets cultured on plastic displays markers patterning similar to their plastic-cultured counterparts.

Figure IV

Histologies and immunohistologies of plastic (A-I) and fibrin (J-R) cultured epidermal sheets. Cryopreserved sections were stained with Ab directed against differentiation markers filagrin (G;P), involucrin (H;Q) and transglutaminase(I;R). Tissues were also counter-stained with Hoechst 33258 (D-F; M-O).

Extensive third degree burns can be permanently covered by the autologous transplantation of cultured keratinocytes sheets [17]. Although very effective, this technique could be improved if the enzymatic treatment could be avoided. The fact that released epithelial sheets can contract to as much as 50% of their original surface area increases the number of epithelial sheets required for treatment, and thus increases the cost of treatment. Another limitation is the infrastructure and personnel necessary to detach the epithelial sheets on the site of operation since it had been shown that viability of the graft depends on the time between detachment and grafting. One of the most attractive means to improve this technique is to culture epithelial sheets on a graftable support that is easily harvested from plastic without enzymatic treatment, like fibrin. Epithelial sheets cultured on fibrin gels have in fact already been used to treat corneal defects [16, 22] and severe burn defects [14, 15, 17, 22] in European countries. Use of the fibrin matrix facilitated manipulation and grafting of the epithelial sheets.

Ronfard and colleagues demonstrated that the clonogenic properties of human keratinocytes grown on a fibrin matrix did not significally differ from those grown on plastic [17]. It was also shown that the culture on a fibrin substrate is effective at conserving epidermal stem cells in sub-confluent cultures [15]. Because these studies were made in sub-confluent cultures, the characteristics of a confluent epithelial sheets cultured on fibrin was not available. Thus, our study compared confluent epithelial sheets grown 48hrs post-confluence, which is the standard procedure for epithelial sheets used to treat patient in our laboratory. Three different donor sites were studied: newborn foreskin, scalp and facelift skin (data not shown). These sites were chosen because past experiments have shown that they yield varying numbers of stem cells.

In epithelial sheets cultured on fibrin gels, absence of contraction made it possible for the cells to retain normal cuboïdal shape, whereas cells from the control sheets seemed more isometric and loosely connected. Impact of this major histological difference on the take and/or long-term viability of the epithelial sheets as yet to be established. First, epithelial sheets cultured on fibrin gels were easily manipulable, a sharp contrast with the traditional fragile epithelial sheets. Also, we found that human epithelial sheets cultured on plastic display normal keratin patterning similar to their counterparts cultured on plastic. Furthermore, adequate keratin patterning and absence of staining for late differentiation markers such as transglutaminase, involucrin and fillagrin demonstrate that keratinocytes cultured on fibrin differentiates normally and forms similar epithelial sheets to those that can be cultured in a traditional culture flaks. Finally, immunostaining of the epithelial sheets cultured on fibrin gels with an antibody directed against K19 as also shown that culturing epithelial sheets on a fibrin matrix has no adverse effects on the conservation of stem cells which are vital for the long term survival of the grafted tissue.

One of the main drawbacks that could be envision is the time required for the fibrin matrix to be degraded by the host. Results from previous and current grafts show that this is nothing to be concerned about, because the fibrin matrix is usually degraded in a few days. Another deterrent to the use of fibrin is the risk of HIV or other blood transmitted diseases. The rigorous testing of donated plasma used to produce commercially available fibrin glue backed by the fact that there are no documented transmissions makes the risk of such infection to be reasonable. Alternatively, this problem could probably be sidestepped by isolating fibrin from either the patient himself (if advisable) or from a healthy friend or relative. Bovine aprotinin generally used could also be replaced by recombinant human aprotinin. Thus, it appears that the use of fibrin for the culture of epithelial sheets could be a beneficial addition to epidermal sheets culture. It does not seem to negatively influence the quality of the produced epithelial sheet, but diminishes the number of sheets necessary to cover, facilitates manipulation, transport and an additional flexibility in the grafting time frame.

The authors gratefully thanks Drs Roy and Hébert for skin biopsies and Dr. Normand Marceau and Dr. Uwe Karsten for the antibodies directed against K14 and K19 respectively.This work was supported by the Fondation des Pompiers du Québec pour les Grands Brûlés and theFonds de la Recherche en Santé du Québec. L.G. was recipient of a studentship from the Fonds de la Recherche en Santé du Québec and Canadian Institutes for Health Research and is the holder of the Canadian Research Chair on stem cells and tissue engineering.

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© Éric Boucher, 2005