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(The FASEB Journal. 1998;12:1331-1340.)
© 1998 FASEB

RESEARCH COMMUNICATION

In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent

Annie F. Black1,a, François Berthoda, Nicolas L'heureux2,a, Lucie Germaina, and François A. Augera,1

a Laboratoire d'Organogénèse Expérimentale/LOEX, Centre Hospitalier Affilié, Pavillon Saint-Sacrement and Department of Surgery, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada G1S 4L8


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types—keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells—were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.—Black, A. F., Berthod, F., L'Heureux, N., Germain, L., Auger, F. A. In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent. FASEB J. 12, 1331–1340 (1998)


Key Words: angiogenesis • endothelial cell • extracellular matrix • inosculation • skin substitute


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
THE TREATMENT OF severely burned patients is a complex endeavor. However various studies have shown that early surgery and wound coverage lead to better survival rates (1). Nevertheless, this approach is at odds with the diminishing possibility of treating these patients when the burned body surface increases. Thus, with every increment in the extent of the burn wound, the available spared skin for harvesting and grafting dwindles. The classic therapeutic options, such as cadaver skin or xenografts, are temporary and have various drawbacks (1). In recent years, the reconstruction of human skin by tissue engineering has offered a therapeutic alternative for this clinical challenge. This method already permits the production of human epidermis in large quantities from a small initial skin biopsy (2). Further amelioration of these skin substitutes is directed toward a complete cutaneous replacement comprising dermal and epidermal components (3–7). Even though this type of complete tissue reconstruction offers the ultimate possibility of a one-step burn wound closure, there seem to be a few drawbacks (8). The exact nature of these disadvantages is not evident since these transplantation experiments have been few and on a small scale clinically. However, an overall picture of limited engraftment with unpredictable viability of the epidermal component is apparent. These unfavorable events may be attributed to the delayed vascularization of the dermal element of such tissue reconstruction with an ensuing deleterious effect on epidermal survival, since this skin layer is nourished by diffusion from the dermal capillaries (9, 10). In contradistinction, there is a favorable chain of events in the `take' of cryopreserved skin allograft as a result of microanastomosis in these transplants (11). The phenomenon has been explained by inosculation, which is the connection of the capillary plexus present in the grafts with the vascular components from the patient's wound (10). The precedent observations have led our group to create a tissue-engineered skin equivalent that would allow the rapid reappearance of functional vascular components. We also took advantage of our experience in tissue-engineered human blood vessel production, which gave us key tools in endothelial cell production and coculture with various cell types (12).

A number of in vitro models have been developed to generate capillary-like structures and reproduce angiogenesis. In vitro angiogenesis models usually combine endothelial cells (13) to various purified extracellular matrix (ECM)4 components such as collagen (14–19), fibrin (20), fibrinogen (21), or multimolecular matrices such as Matrigel (22). Most of these models were made of animal cells of various origin. Indeed, bovine endothelial cells are known to differentiate spontaneously into capillary-like structures in basic culture conditions, although human endothelial cells degenerate. This observation can be explained by a lower growth factor requirement of animal cells. Work accomplished with various in vitro models suggests that angiogenesis can be observed in human endothelial cell cultures without the cooperation of any other cell types, but necessitates the presence of various angiogenesis promoting agents such as phorbol 12-myristate 13-acetate (PMA), specific growth factors [basic fibroblasts growth factor (bFGF), vascular endothelial growth factor (VEGF)], and high amounts of fetal calf serum (FCS, 20% or more) (17, 18, 23). Since PMA is a tumor promoting agent, its use for clinical application is not desirable.

Angiogenesis results from a sequential set of events that can be locally modulated in vivo by both cell–cell contacts (24–26) and cell–matrix interactions (21, 27). Thus, in the present work we hypothesize that the higher growth factor requirement of human endothelial cells compared to bovine or rat cells cultured in vitro could be overcome by culturing HUVEC in presence of 1) other cell types such as the main cell types of the skin (i.e., fibroblasts and keratinocytes), and 2) a highly differentiated extracellular matrix.

Thus, the aim of this study was to reconstruct an endothelialized skin equivalent (ESE) in which capillary-like structures could be reproduced in vitro. We chose an ESE that was made of a chitosan-linked collagen-glycosaminoglycan sponge, since its structure allowed fibroblasts and endothelial cells to migrate (28) and we had previous experience with this material (29).

We demonstrated that the ESE enables the formation of capillary-like structures without addition of external angiogenesis promoting agents such as PMA. Immunohistochemical analysis and transmission electron microscopy of the ESE showed the formation of small tubes. The nature of the cells involved in the formation of these capillary-like structures was confirmed by the expression of specific cytological and structural markers of endothelial cells [von Willebrand factor (vWF) and Weibel-Palade bodies]. Furthermore, morphological characteristics associated with in vivo microvasculature were observed in the ESE (basement membrane deposition and intercellular junction formation). Moreover, we have demonstrated that the neosynthesis of extracellular matrix by fibroblasts is a key event in this in vitro angiogenesis process. To our knowledge, this is the first attempt to produce such a network of human capillary-like structures within a skin equivalent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Human keratinocyte and dermal fibroblast isolation
Keratinocytes and dermal fibroblasts were isolated from human skin biopsies after breast reductive surgeries as previously described (30, 31). Keratinocytes were grown in Dulbecco-Vogt modification of Eagle's medium (DMEM) and Ham's F12 in a ratio of 3:1 (Flow Lab., Mississauga, Canada) supplemented with 24.3 µg/ml adenine, 10 µg/ml human epidermal growth factor (Chiron Corp., Emeryville, Calif.), 0.4 µg/ml hydrocortisone (Calbiochem, La Jolla, Calif.), 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 2 x 10-9 M 3, 3', 5', triiodo-L-thyronin, 10-10 M cholera toxin (Schwarz/Mann, Cleveland, Ohio), 100 U/ml penicillin, 25 µg/ml Gentamicin (Schering, Pointe-Claire, Canada), and 10% newborn calf serum (NCS, fetal clone II, Hyclone, Logan, Utah), and used at the second passage as previously described (30). Fibroblasts were grown in DMEM (Flow Lab., Mississauga, Canada) supplemented with 10% FCS (Immunocorp, Montreal, Canada), antibiotics in 8% CO2 at 37°C.

Endothelial cell isolation
Human umbilical vein endothelial cells (HUVEC) were obtained from healthy newborns by enzymatic digestion with 0.153 IU/mg collagenase H (Boehringer Mannheim, Laval, Canada), as previously described (12, 32). Briefly, veins were cannulated at both ends and washed with calcium-free HEPES solution (Flow Laboratories, Mississauga, Canada). A collagenase solution was then injected to rinse and fill the vein and the cord was placed in HEPES with 5 mM CaCl2 at 37°C. After a 15 min incubation, the veins were perfused with M199 medium (Sigma, Mississauga, Ontario) containing 10% NCS fetal clone II (Hyclone, Logan, Utah) and antibiotics.

The endothelial cells isolated were centrifuged and suspended in M199 medium supplemented with 20% NCS, 2.28 mM glutamine, 25,000 IU/ml heparin (Leo Laboratories, Pickering, Canada), 19 µg/ml endothelial cell growth supplement (Sigma, Mississauga, Canada), and antibiotics (33). HUVEC were plated on gelatin-coated tissue culture flasks. The culture medium was changed three times a week. Endothelial cell characterization was assessed as previously described (32). The cells were used at passages three or four.

Chitosan/collagen biopolymer preparation
Chitosan/collagen biopolymers were prepared as previously described (28, 34). Briefly, type I, III bovine collagen, chitosan (95% deacetylated) (SADUC, Lyon, France), and chondroitin 4–6 sulfates (SADUC, Lyon, France) were dissolved in 0.1% acetic acid and mixed. After mixing, 1 ml/well (3.8 cm2) of the final solution was poured into 12-well plates (Becton Dickinson, Toronto, Canada) and frozen overnight at -70°C. The frozen plates were then lyophilized in a Genesis 12EL vacuum lyophilizer (Virtis, Gardiner, N.Y.), submerged in 70% ethanol for 24 h, rinsed and equilibrated in 3 ml of supplemented DMEM, and incubated at 37°C with 8% CO2 for a minimum 24 h.

Dermal, endothelial, and dermal–endothelial equivalent preparation
Three different dermal equivalents were produced. 1) The standard dermal equivalent (DE) was prepared by adding a suspension of 2.1 x 105 fibroblasts/cm2 in each well on top of the biopolymer; 2) the endothelial equivalent was produced by seeding 2.1 x 105 HUVEC/cm2 on top of the biopolymer; 3) the endothelial-fibroblast dermal equivalent (EDE) was prepared by seeding a suspension of 1:1 ratio of fibroblasts and of HUVEC. All equivalents were cultured for 10 days in a medium containing 50 µg/ml ascorbic acid (Sigma) and DME supplemented with 10% FCS and M199 supplemented with 20% NCS in a 1:1 ratio. The DE were fed daily with 3 ml of medium. A total of 85 EDE were produced in 12 different experiments.

Skin equivalent preparation
Human keratinocytes were plated on all three types of DE at a concentration of 2.1 x 105 cells/cm2. All skin equivalents (SE) were cultured in complete medium with 10% NCS (as described above) and 50 µg/ml ascorbic acid under submerged conditions for 7 days. The SE were then elevated at the air–liquid interface for the remaining 14 days in DME-Ham's F12, supplemented with 10% NCS, 0.4 µg/ml hydrocortisone, 5 µg/ml insulin, 50 µg/ml ascorbic acid, and antibiotics. A total of 94 ESE were produced in 12 different experiments.

Histological analysis
Equivalents were fixed with Bouin's solution and embedded in paraffin. Sections 6 µm in thickness were cut and stained using Masson's trichrome.

Immunofluorescence analysis
Frozen sections (4 µm) were blocked in phosphate-buffered saline containing 1% (w/v) bovine serum albumin. Immunofluorescence studies were performed using mouse monoclonal anti-human vWF antibody (1/100 dilution) and rabbit polyclonal anti-human type IV collagen antibody (1/100 dilution) (both purchased from Chemicon, Montreal, Canada) and mouse monoclonal anti-laminin antibody (1/100 dilution) (BIO/CAN Scientific Inc., Mississauga, Canada). The second antibody was FITC-conjugated sheep anti-rabbit IgG (1/100 dilution) or goat anti-rat IgG (1/100 dilution) (Cederlane, Hornby, Canada). The second antibody was mixed with 0.1% Evans blue in order to reduce the nonspecific staining of the biopolymer network (35). The primary antibody was omitted in controls.

Transmission electron microscopy
Equivalents were fixed with 0.1 M sodium cacodylate buffer containing 2% glutaraldehyde, pH 7.5, at 4°C overnight and postfixed with 1% osmium tetroxide (Sigma, Mississauga, Canada). Tissues were stained with 0.5% uranyl acetate for 1 h at 4°C. After dehydration, the samples were embedded in Epon 812. Contrasted sections (uranyl acetate and lead citrate) were observed on a Philips EM300 electron microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Importance of cell–matrix interactions in the formation of capillary-like tubular structures
The HUVEC cultured alone in the biopolymer spread on the scaffold, but most showed features of dying cells. Very limited ECM production by HUVEC was observed in the equivalents ( Fig. 1A). In contrast, fibroblasts cultured alone migrated, attached to the scaffold of the biopolymer, synthesized ECM, and proliferated within its structure ( Fig. 1B). When both cell types were seeded together on the biopolymer, the fibroblasts still proliferated and synthesized new ECM ( Fig. 1C). Moreover, HUVEC migrated and formed capillary-like tubular structures in the newly synthesized ECM by the fibroblasts ( Fig. 1C, E, F). Histological analyses of all dermal equivalents were done after 31 days of culture.



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  		Figure 1. Histological staining of in vitro tissue-engineered skin equivalents. Biopolymers were seeded with HUVEC (A), fibroblasts alone (B), HUVEC and fibroblasts (C, E, F), and HUVEC, fibroblasts, and keratinocytes (ESE) (D) and cultured for 31 days as described in Materials and Methods. Sections were stained with Masson's trichrome. Note that fibroblasts were numerous and filled the pores with newly synthesized ECM either when cultured alone (B) or with HUVEC (C, E, F). In contrast, when HUVEC were alone in the biopolymer, endothelial cells were scarce (A). In coculture of fibroblasts and HUVEC, the formation of capillary-like tubular structures in the newly synthesized ECM was observed (E, F). In the ESE, keratinocytes produced a differentiated epidermis. Moreover, tubular structures (arrowhead) were present in the dermal portion of the ESE (D). Bars indicate 60 µm (A), 40 µm (E), and 10 µm (F).

Histological analysis of ESE stained using Masson's trichrome showed well-differentiated basal layer, stratum spinosum, stratum granulosum, and stratum corneum ( Fig. 1D). The underlying dermis was populated with dermal fibroblasts embedded in newly synthesized ECM on top and within the biopolymer porous structure. Seven days after seeding, keratinocytes covered the surface of the equivalent, which is coated with ECM secreted by mesenchymal cells. After 17 days of culture under submerged conditions, the equivalent was then raised at the air–liquid interface to further enhance keratinocyte differentiation and promote fibroblast migration within the structure of the biopolymer. After 14 days of culture at the air–liquid interface, a multistratified differentiated epidermis was observed ( Fig. 1D). All DE, EDE, SE, and ESE were cultured for a total period of 31 days.

In the EDE and ESE, a network of capillary-like tubular structures was formed ( Fig. 1C–F), whereas none could be observed in the absence of HUVEC ( Fig. 1B). Histological studies revealed segments of ramified structures ( Fig. 1E). Such structures were mostly observed in the thickest areas of the dermis.

Immunofluorescent colabeling of vWF, laminin, and type IV collagen shows basement membrane deposition around capillary-like structures
The specific morphological and functional features of HUVEC were previously defined by their positive immunofluorescent staining with an anti-human vWF monoclonal antibody and Ac-LDL-Dil uptake (32). The presence of vWF was assessed throughout the entire culture period. A distinct granular signal was observed from day 1 to 31 ( Fig. 2A).



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  		Figure 2. Immunohistochemical characterization of tubular structures. ESE were produced by coculturing HUVEC, fibroblasts, and keratinocytes for 15 (C) or 31 (A, B, D) days of culture as described in Materials and Methods. Frozen sections were immunostained for human vWF (A), type IV collagen (B), and laminin (C, D). Note that cells surrounding tubular structures contained vWF (A), indicating that they are endothelial cells. Moreover, the basement membrane constituents, type IV collagen (B) and laminin (C, D), were observed surrounding tubular structures (arrows) at 15 and 31 days of culture. Laminin was also detected at the dermal–epidermal junction (open arrow) (D). Bar indicates 30 µm (A, B, D) and 60 µm (C).

A positive laminin staining was detected in ESE sections 5 days after initial seeding of HUVEC and fibroblasts (not shown). At day 15 of culture, immunostaining of ESE sections with antibodies specific to laminin demonstrated the deposition of a basement membrane around the tubular structures ( Fig. 2C). On the 31st day of culture, a stronger laminin signal was detected both under the basal layer of the epidermis and around the capillary-like structures ( Fig. 2D). Type IV collagen was also detected around the tubular structures ( Fig. 2B).

To further confirm the integrity of the newly formed capillary-like structures, immunofluorescent colabeling of vWF, laminin, and type IV collagen was performed on ESE serial frozen sections. As expected, vWF, laminin, and type IV collagen fluorescent signals were detected and colocalized in the same region of all ESE sections ( Fig. 3B, C, D). The biopolymer matrix ( Fig. 3A) remained unstained by the specific antibodies used, confirming that the capillary-like structure formation resulted from intrinsic HUVEC organization.



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  		Figure 3. Hg agents inactivate 4F2hc-induced amino acid uptake by modification of extracellular cysteine residue(s). Three days after injection with saturating amounts of human 4F2hc cRNA, groups of seven or eight 4F2hc-injected and noninjected oocytes were incubated with 1 mM of the indicated organic mercury agents (pCMBS or pCMB) for 5 min. These agents were dissolved in 100 mM choline Cl medium (see ref 15 and Materials and Methods) containing 0.01% DMSO and 10 µM EDTA (to chelate free Hg2+) for pCMB and 10 µM EDTA for pCMBS. Control oocytes were incubated in the same conditions and medium, but without the organic mercury agents and other chemicals. Previous experiments showed that 0.01% DMSO and 10 µM EDTA did not modify 4F2hc-induced amino acid uptake (data not shown). Then, oocytes treated with organic mercury agents and control oocytes were rinsed three times in 3 ml of 100 mM choline Cl medium and incubated for 5 min with 100 mM choline Cl medium with or without 5 mM ;hb-mercaptoethanol, as indicated. Prior to 50 µM L-[3H]arginine uptake measurement in 100 mM choline Cl medium, oocytes were again rinsed three times. Uptake values in the noninjected oocytes were (pmol/15 min per oocyte; mean ± SEM): 11 ± 1, control; 1.2± 0.3, pCMBS; 1.2 ± 0.2, pCMB; 11 ± 2, pCMBS + ß-mercapto[chethanol; 8.5 ± 1.2, pCMB + ß-mercaptoethanol. 4F2hc-induced uptake (see Materials and Methods) is expressed in pmol/15 min per oocyte (mean ± SEM).

Ultrastructural characterization of capillary-like structures shows features of native microvasculature
Ultrastructural observations of the capillary-like structures newly formed in the ESE showed the organization of HUVEC defining an internal lumen with intercellular junctions ( Fig. 4A, Fig. 5A). The presence of intracellular Weibel-Palade bodies was also observed ( Fig. 4A, B, Fig. 5A). Longitudinal sections of capillary-like structures were also found, as seen in Fig. 5A, where one is formed by at least 13 HUVEC linked together by intercellular junctions and delimiting an internal lumen. The internal lumen contained cellular debris but no ECM components. Moreover, a basement membrane was partially deposited between the basal side of HUVEC and the ECM within the ESE ( Fig. 5B, C). Such complex structures could not be seen in the equivalents seeded with HUVEC or fibroblasts alone (data not shown).



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  		Figure 4. Transmission electron microscopy of a capillary-like structure observed in a 31-day-old ESE. A closed tube formed by seven cells showed an internal lumen and intercellular junctions (arrow). Weibel-Palade bodies were also seen (arrowheads) (A, B). Note that the lumen was filled with cellular debris, but did not contain dense ECM components (A). B) An enlargement of panel A. Bar indicates 1 µm (A) and 0.1 µm (B).



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  		Figure 5. Transmission electron microscopy of a capillary-like structure observed in a 31-day-old ESE. An endothelial capillary-like tube formed by at least 13 HUVEC was observed along its longitudinal axis, delimiting an internal lumen (A). Weibel-Palade bodies were also seen (arrowheads) (A). Note that lumens were filled with cellular debris, but did not contain dense ECM components (A, B). The intercellular junctions between individual HUVEC can be seen (arrows) (A). Discontinuous deposits of basement membrane material were observed (open arrows) (B, C). C) An enlargement of panel B. Bar indicates 1 µm (A, B) and 0.1 µm (C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As human cadaver skin grafts were introduced as temporary biological dressing for burn wound coverage, the clinicians marveled at the usefulness of these grafts. Furthermore, they observed a much delayed rejection process compared with the well-described phenomenon in nontraumatized and immunologically competent patients (1). Indeed, not only was the level of "take" high, but these allografts seemed to revitalize (1). The immunological process involved was not readily unraveled (36). It also took many decades before the neovascularization phenomenon was clearly explained and appreciated as a fundamental step in keeping the graft viable within these critical first few days.

Thus, Young and colleagues (10) have suggested that split-thickness skin grafts survive first by diffusion of nutrients through the graft (imbibition), then initial vascularization by inosculation, and finally by neovascularization, which is thought to be a late process (10). Since composite skin substitutes do not contain a capillary network, they cannot vascularize as well as a real full-skin graft. Nutrient supply of the epidermis of these avascularized reconstructed skin substitutes is then provided only by imbibition and neovascularization. Imbibition does not seem to be sufficient to support the permanent implantation of skin substitutes until neovascularization is established (10). This sequence of events explains in part the poor engraftment rates of composite skin grafts. In the present study, we strove to demonstrate that the formation of a capillary-like network in vitro within our skin equivalent may lead to new fundamental and better clinical applications for these tissue-engineered constructs.

Angiogenesis has been studied by many research groups with a variety of in vivo and in vitro models. Unfortunately, none of these in vitro models were designed for clinical use. In an attempt to reproduce angiogenesis in vitro, some groups have included rat (26) or bovine endothelial cells (25) or freshly isolated rat microvessel fragments (17) in their models. Their results showed that endothelial cells of bovine origin spontaneously formed capillary-like tubes by a process called `sprouting'. Thus, a subpopulation of cells formed a reticular network of vessels on top of a cell monolayer (37). However, when HUVEC were used in a similar manner, most of the cells detached and only the remaining cells spontaneously formed tubes. Most of these cells underwent cell death as nutrients in the medium became depleted (38). Thus, the formation of such capillary-like tubes from human cells appears to be a rare and fugitive event. Bovine endothelial cells may have a lower growth factor requirement than HUVEC and may tolerate longer growth factor depletion periods in culture (39). To induce the same capillary-like structures in human endothelial cells in monolayer, the addition of PMA, collagen, or Matrigel is needed to produce the sprouting patterns observed spontaneously in bovine tube formation (15, 22, 40). PMA is a tumor promoter in mice skin carcinogenesis that acts on cell proliferation by protein kinase C activation (15, 38).

Our purpose was to produce in vitro an endothelialized reconstructed skin designed for grafting on deep wounds, in which vascular-like tube formation was promoted. This ESE was made of human cells in the absence of specific endothelial growth factors or tumor promoting agents such as PMA. Grafting of this ESE in humans may enhance revascularization through inosculation of its preexisting capillary-like network with the patient's own blood vessels.

In a clinical setting, the capillary-like network formed in the ESE would have to be of autologous origin to prevent immunologic rejection posttransplantation. Several methods were proposed to isolate endothelial cells from human skin or fat biopsies (41–43). Such approaches could be applied to harvest autologous endothelial cells from the patient simultaneously with dermal fibroblasts and keratinocytes. Our own experience with vascular equivalent reconstruction through tissue engineering (12) has shown the possibility of such an approach. Thus, the collection, culture, and expansion of adult human endothelial cells is a demanding but quite feasible step in such a skin tissue reconstruction. In addition, new techniques to improve and facilitate endothelial cell extraction and purification from skin microvasculature are under development. Indeed, the use of magnetic beads or a panning technique in combination with either lectin, Ulex europaeus agglutinin-1, or a monoclonal antibody specific to endothelial cells allows a pure microvascular endothelial cell population to be obtained from a skin biopsy (42, 43).

The results reported here strongly suggest that HUVEC can reorganize themselves into an extensive network of capillary-like structures when cocultured with human dermal fibroblasts. These structures were absent when each cell type was cultured separately in the biopolymer. The capillary-like tubes were clearly visible from days 15 to 31 in culture. Phase contrast microscopy observations revealed the presence of round and elongated tubes formed by HUVEC, as demonstrated by their positive labeling with vWF. Basement membrane components such as laminin and type IV collagen were detected in the vicinity of these tubules and at the dermal–epidermal junction of ESE. Ultrastructural analysis of ESE containing HUVEC cocultured with dermal fibroblasts confirmed the presence of capillary-like structures with open lumen. These structures were composed of polarized HUVEC connected through intercellular junctions. A basement membrane was observed beneath the fine HUVEC network. The discontinuous deposition of basement membrane may be explained by the slow formation of the capillary-like network. Moreover, Weibel-Palade bodies were present in the HUVEC that formed the capillary-like structures. Finally, such observations strongly suggest a differentiated endothelium, sharing some features with native microvasculature. We explain the spontaneous capillary-like formation in the ESE on one hand by the cell–cell contacts of HUVEC with fibroblasts and, on the other hand, by cell–matrix interactions. Indeed, fibroblasts have been shown to have angiogenic properties and increase both the number and life span of microvessels in vitro (26). They also produce high amounts of a well-organized human ECM when cultured in a 3-dimensional porous structure (29, 34, 44). Likewise, keratinocytes have been shown to express VEGF in culture (45), which is known to be a potent angiogenic agent. Therefore, we suggest that the absence of specific angiogenesis promoting agents in our model is compensated mainly by the direct contact of endothelial cells with dermal fibroblasts, the secretion of growth factors, and newly synthesized ECM by both dermal fibroblasts and keratinocytes. Furthermore, in vitro angiogenesis-like behavior is expressed more rapidly and among a greater variety of endothelial cells in culture systems where the cells are placed in contact with ECM (46). Indeed, cells must adhere to matrix proteins to survive and proliferate. It has been shown that HUVEC cultured under conditions that prevent adhesion and spreading stop growing and loose viability (47–49) whereas ECM components stabilize growing microvessels (26, 50). We believe that coculture of fibroblasts with endothelial cells in a 3-dimensional culture system that promotes the deposition of a human collagen matrix favored the differentiation of endothelial cells demonstrated by the formation of capillary-like structures.

Angiogenesis is a complex process that involves the synthesis by HUVEC of proteases (23, 51), expression of ß1 integrins (52), of CD44 (53), and changes in membrane localization pattern of platelet endothelial cell adhesion molecule-1 (PECAM-1) (54) to promote cell migration. Thus, in addition to deposition by human fibroblasts of a highly differentiated extracellular matrix in our 3-dimensional culture system, capillary-like formation might be also promoted by fibroblast and keratinocyte paracrine secretions. Indeed, the complexity of the human ECM remodeled in the ESE may explain why the addition of angiogenic agents was not required to form capillary-like structures in contrast to other 3-dimensional culture systems, such as in a preformed bovine collagen gel (14) or fibrin matrices (20, 55) that demand the addition of bFGF or PMA.

We have developed an endothelialized reconstructed skin in which capillary-like tubes were formed. This ESE should accelerate the revascularization process of the dermis after transplantation on humans by inosculation of the preexisting capillary-like tubes with growing blood vessels from the wound. Additional studies of grafts of human ESE on athymic mice and analysis of the resulting vascular network connection between human and murine endothelial cells will be required to test this hypothesis.

In addition to having advantageous clinical possibilities, the ESE seems to be a promising in vitro angiogenesis model. Neovasculature is an essential feature of solid tumor cancers. Insight into the regulatory role of cell-matrix interactions in early neoplastic progression has been hindered by the lack of tissue models. This novel ESE holds the potential of a novel approach to the understanding of cancer biology by providing a new tool for the study of the proteolytic and extracellular matrix systems implicated in the formation of new blood vessels.


   ACKNOWLEDGMENTS
 
The authors acknowledge Dr. Odile Damour for allowing the use of their patented biopolymer and for careful review of this manuscript; Aristide Pusterla, Nathalie Tremblay, Rina Guignard, and Julie Bergeron for excellent technical assistance; and Claude Marin for photographic assistance. This study was supported by the Medical Research Council of Canada, `Fondation de l'Hôpital du Saint-Sacrament', `Réseau des Grands Brûlés du Fonds de la Recherche en Santé du Québec (FRSQ)', `Fondation des Pompiers du Québec pour les Grands Brûlés', `Club Richelieu', and `France-Québec' exchange program. L.G. and F.A.A. were recipients of Scholarships from FRSQ and N.L'H. was the recipient of a Studentship from the Fonds FCAR du Québec.


   FOOTNOTES
 
1 Current address: Laboratoire des Substituts Cutanés, Université Claude Bernard Lyon 1, Fédération de Biochimie, Bâtiment 5, Hôpital Edouard Herriot, 5 Place d'Arsonval, 69437 Lyon cedex 03.

2 Current address: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412, USA.

1 Correspondence: Laboratoire d'Organogénèse Expérimentale, Hôpital Saint-Sacrement, 1050 chemin Sainte-Foy, Quebec City, Quebec, Canada G1S 4L8. E-mail: HYPERLINK mailto:Francois.auger{at}chg.ulaval.ca

4 Abbreviations: bFGF, basic fibroblast growth factor; DE, dermal equivalent(s); ECM, extracellular matrix; EDE, endothelialized dermal equivalent; ESE, endothelialized skin equivalent; FCS, fetal calf serum; HUVEC, human umbilical vein endothelial cells; NCS, newborn calf serum; PECAM-1, platelet endothelial cell adhesion molecule 1; PMA, phorbol 12-myristate 13-acetate; SE, skin equivalent(s); VEGF, vascular endothelial growth factor; vWF, von Willebrand factor; DMEM, Dulbecco's modified Eagle's medium.

Received for publication February 24, 1998. Accepted for publication May 15, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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