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Keywords:

  • Angiogenesis;
  • brun;
  • capillary;
  • tissue engineering;
  • transplantation;
  • skin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The major limitation for the application of an autologous in vitro tissue-engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary-like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14-day period was necessary to achieve a similar result with the non-endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue-engineered organs requiring a rapid vascularization.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Wound coverage with an autologous in vitro tissue engineered reconstructed skin (RS) made of both dermis and epidermis may be the best alternative to split-thickness grafts for patients with full-thickness cutaneous wounds (1,2). Unfortunately, various pitfalls have delayed the clinical application of RS (3). Lack of vascularization after grafting has been suggested as one of the major reasons for poor graft take and survival of RS (4). This lack of adequate blood supply is also an obstacle to the development of many tissue-engineered organs. For many decades, an intriguing phenomenon of cadaver skin grafts take was well described in burn patients. It was shown by clinical evidence of early skin blood flow in these transplants (5–8). Young et al. have described the two mechanisms responsible for the phenomenon of revascularization of human full thickness skin graft on nude mice: inosculation and neovascularization (9). Thus, when human skin was grafted on athymic mice, initial vascularization was limited to anastamosis of the human vessels in the graft to mouse vessels from the recipient's bed and occurred between 2 and 5 days after grafting (9). Also, the combination of mice and human endothelial cells was observed and described as chimeric microvessels (10). Neovascularization, the growth of completely new capillaries from the recipient's bed into the graft, is a latter process. In fact, Krejci et al. have shown that when autologous keratinocytes were seeded on an acellular dermis and then engrafted into athymic mice, the acellular dermis was repopulated within 14 days by fibroblasts and blood vessels of host origin (1,2). Since that RS did not possess a capillary network, it survived by diffusion of nutrient through the graft (imbibition) until neovascularization occurs (Figure 1).

image

Figure 1. Schematization of the three different processes of graft nutrition after transplantation. Imbibition, the passive diffusion of nutrients from the wound bed to the graft, is a rapid process, but has a limited range of diffusion. Neovascularization, the migration of new blood vessels originating from the wound bed by angiogenesis, is a much slower process, which depends on the graft thickness. Inosculation, the connection of the blood vessel-like network of the graft with the host's vasculature, is a rapid process (less than 4 days in our experiments) and should be independent of the graft thickness.

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We have developed an endothelialized reconstructed skin (ERS) in which a network of human capillary-like structures (CLS) was spontaneously formed within the dermis (11,12). The ERS showed the organization of CLS into a complex network with a branching morphology. Since this RS contained its own capillary-like network, we hypothesized it could be vascularized in the same manner as full thickness human skin grafts with the establishment of blood flow through the pre-existing capillary-like network by the inosculation phenomenon (9) (Figure 1).

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human keratinocyte and dermal fibroblast isolation

Human keratinocytes and fibroblasts were isolated from human skin biopsies after breast reductive surgeries as previously described (13,14). Keratinocytes were grown in Dulbecco-Vogt modification of Eagle's medium (DMEM) with Ham's F12 in a ratio 3:1 (Invitrogen, Burlington, Canada), supplemented with 24.3 μg/mL adenine (Sigma, Oakville, Canada), 10 ng/mL human epidermal growth factor (Austral, San Ramon, CA), 5 μg/mL bovine insulin (Sigma), 0.4 μg/mL hydrocortisone (Calbiochem, LaJolla, CA), 10−10 M cholera toxin (ICN, St-Laurent, Canada), 100 U/mL penicillin (Sigma) and 25 μg/mL gentamicin (Schering, Pointe Claire, Canada), and 5% newborn calf serum (NCS, FetalClone II, HyClone, Logan, Utah), and used at the second passage as previously described (13). Fibroblasts at the fifth passage were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS) (HyClone), antibiotics (100 U/mL of penicillin G, and 25 μg/mL of gentamicin) in 8% CO2 at 37°C.

Human umbilical vein endothelial cell isolation

Human umbilical vein endothelial cells (HUVEC) were obtained from healthy newborns by enzymatic digestion with 0.250 μg/mL thermolysin (Sigma). Veins were cannulated at both ends and washed with calcium-free HEPES solution (ICN). A thermolysin solution was then injected to rinse and fill the vein and the cord was placed in calcium-free HEPES at 37°C. After 30 min of incubation, the veins were perfused with M199 medium (Sigma) containing 10% FetalClone II (HyClone) and antibiotics (100 U/mL of penicillin G, and 25 μg/mL of gentamicin).

Endothelial cells (EC) were centrifuged and resuspended in M199 medium supplemented with 20% NCS, 2.28 mM glutamine (Invitrogen), 40 IU/mL heparin (Leo Laboratories, Pickering, Canada), 20 μg/mL EC growth supplement (Sigma) and antibiotics (100 U/mL of penicillin G, and 25 μg/mL of gentamicin) (15). HUVEC were plated on gelatin-coated tissue culture flasks, characterized as previously described, (16) and cultured at the fourth passage in EGM-2 medium (Clonetics, San Diego, CA).

Collagen/ glycosaminoglycan/ chitosan biopolymer preparation

The collagen/ glycosaminoglycan/ chitosan biomaterial was prepared as previously described (17,18). These sponges were prepared by mixing type I, III bovine collagen (Laboratoire Perouse Implant, Chaponost, France), chitosan (SADUC, Lyon, France) and chondroitin 4–6 sulfates (SADUC) dissolved in 0,1% acetic acid. Then, 2.5 mL/well (9 cm2) of the final solution was poured into six well plates (Becton Dickinson, Toronto, Canada) and frozen overnight at −80°C. The frozen plates were then lyophilized in a Genesis 12EL vacuum lyophilizer (Virtis, Gardiner, NY).

RS and ERS preparation

Two different tissues were produced: (i) The standard reconstructed dermis was prepared by adding a suspension of 2.1 × 105 fibroblasts/cm2 on top of the biopolymer; (ii) The endothelialized reconstructed dermis was produced by seeding a suspension 1:1 ratio of fibroblasts and HUVEC (11). Both were cultured for 10 days in a medium containing 50 μg/mL ascorbic acid (Sigma) and EGM-2 medium. The medium was changed thrice a week. Human keratinocytes were plated on the reconstructed dermis and the endothelialized reconstructed dermis at a concentration of 2.1 × 105 cells/cm2. All RSs were cultured in complete DME-Ham's F12 medium with 10% NCS (as described above) and 100 μg/mL ascorbic acid under submerged conditions for 7 days. The tissues 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 bovine insulin, 100 μg/mL ascorbic acid and antibiotics.

Animals and surgical manipulations

Adult male athymic nu/nu mice (42 days old) (Charles River Laboratories, Lasalle, Canada) were injected with ceftazidime (3 mg/mouse, Glaxo, Toronto, Canada) 24 and 48 h before surgery to prevent infections (18,19). Ampicillin and gentamicine sulfate (100 U/mL and 26 mg/L, respectively; Novopharm Limited, Toronto, Canada) were also added to the mice's sterile drinking water. Animals were anesthetized by inhalation of 3% Isoflurane USP (Schein Pharmaceutical, Etobicoke, Canada). A 2-cm incision was made through the dorsal skin. The loose connective tissue under the panniculus carnosus was excised. A silicone Fusenig chamber (20) was implanted and stitched to the mouse skin. Human skins from breast reductive surgery were used as positive controls. The skin was washed in DMEM to remove the remaining blood. A 9-cm2 human skin was cut to be the same size as the RS and the ERS. All three different samples were deposited directly on the dorsal muscle of the mouse. A cap was used to close the chamber for all post-grafting time. Mice were divided in 3 groups, 12 mice transplanted with human skin, 12 with RS and 12 with endothelialized RS. Four mice of each group were sacrificed at 4, 7 and 14 days post-grafting for analysis. All manipulations of animals were done according to the rules established by the Canadian Council on Animal Care.

Immunohistochemical analysis

On each grafted mouse, four biopsies were taken and embedded in O.C.T. compound (Somengen, Edmonton, Canada). Frozen sections (6 μm) were blocked in phosphate-buffered saline containing 1% (w/v) bovine serum albumin. Immunofluorescence studies were performed on consecutive sections. On the first slice, a mouse monoclonal anti-human leucocyte antigen antibody (HLA), (produced in our laboratory by Dr. Rochon) (1/100 dilution) coupled with Alexa 488 antibody (Molecular Probes, Eugene, OR) was used, in addition to a goat monoclonal anti-mouse and human von Willebrand factor antibody (1/200 dilution) (Cedarlane, Hornby, Canada), immunostained with a TRITC-conjugated rabbit anti-goat IgG (1/200 dilution) (Bio/Can Scientific, Mississauga, Canada). On the second consecutive slice, a rabbit polyclonal anti-mouse red blood cell antibody (1/800 dilution) (Cedarlane) and a rat monoclonal anti-mouse PECAM-1 antibody (1/100 dilution) (BD PharMingen, Mississauga, Canada) were used. These antibodies did not react with human blood and human ECs, respectively (data not shown). The second antibodies were Alexa 594-conjugated chicken anti-rabbit IgG (1/400 dilution) (Molecular Probes) and Fuorescein-coupled goat anti-rat IgG (1/400 dilution) (Chemicon, Temecula, CA), respectively. The second antibodies were mixed with Hoescht (1/100 dilution) (Sigma) to observe the cell nuclei and merge images exactly. Sections were examined using a Nikon Eclipse E600 fluorescence microscope.

The confocal image was obtained using a Nikon C1 confocal microscope on a 25-μm thick tissue section. Human ECs were stained with Ulex Europae-1 lectin conjugated with Rhodamine (Sigma), and mouse ECs, with rat monoclonal anti-mouse PECAM-1 revealed with Alexa 488-conjugated anti-rat antibody.

Histological analysis

Samples were fixed with Histochoice's solution (Armesco, Solon, OH) and embedded in paraffin. Six micrometer sections were cut and stained using Masson's trichrome to determine the presence of CLS with blood cells in the graft.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The RS was considered as a negative control while human skin was used as a positive control of the inosculation process. Before grafting, the two types of RS showed a well-differentiated epidermis (Figure 2A, B). Some CLS were observed under the epidermis in the ERS before graft (Figure 2B) and in the human skin (Figure 2C), while none could be observed in the conventional RS (Figure 2A). Moreover, red blood cells within capillaries could be detected on the fourth day after grafting the human skin (Figure 2F). These erythrocytes came from the mouse because no red blood cells had been observed in the capillaries of the human skin before transplantation (Figure 2C).

image

Figure 2. RS, ERS and human skin histology before and after transplantation. The sections were stained with Masson's trichrome. The RS (A) or the ERS (B) presented before graft and 4 days after graft (D and E, respectively) a well-stratified epidermis. We can observe the presence of CLS in the ERS (B and E; arrows) like the human skin (C and F; white arrows) but these structures were absent from the RS (A and D). We have also detected the presence of red blood cell 4 days after graft in capillaries under the epidermis of human skin (F; black arrow). These red blood cells were coming from the mouse because no red blood cell could be identified in the capillaries before the graft (C; black arrow). Bar = 100 μm.

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To identify human ECs, a double staining was performed on consecutive tissue sections. The human cells were characterized with an antibody against the human leucocyte antigen (in green), and ECs were recognized with an antibody against von Willebrand factor (in red) (Figure 3A, D, G, J) that stained both human and mouse cells. However, the mouse ECs were detected by a specific antibody against the mouse PECAM-1 antigen (in green), and the re-establishment of blood flow within the vessels was demonstrated with an antibody specific for the mouse red blood cells (in red) (Figure 3B, E, H, K). Four days after transplantation of the human skin, the microvessels near the epithelial layers were solely detected by the double staining of HLA and von Willebrand factor (Figure 3A; arrows), but no mouse EC could be detected with the specific anti-PECAM-1 antibody (Figure 3B). Vessels filled with mouse erythrocytes were homogeneously distributed in the human skin grafts (Figure 3B; arrows). For the RS, capillaries containing mouse red blood cells were only observed in the wound bed, but not in the graft (Figure 3D, E, F). Four days after graft, no blood vessels could be detected in the RS under the epidermis (Figure 3E, F). However, 14 days after graft, PECAM-1-immunoreactive mouse capillaries filled with blood could be observed near the epidermis (Figure 3H, I; arrows). These vessels also expressed von Willebrand factor (Figure 3G; arrows), while the graft was still made of a large number of HLA-positive human endothelial cells (Figure 3G).

image

Figure 3. Immunohistochemical characterization of revascularization of the RS, the human skin and ERS. Immunofluorescence studies were performed on consecutive frozen sections, In (A), (D), (G) and (J), sections were immunostained with a marker of the cell nucleus (Hoechst, in blue), an antibody against the human leukocyte antigen (HLA) (in green) and an antibody against the mouse and human von Willebrand factor (in red). In (B), (E), (H) and (K), frozen sections were immunostained with Hoechst (in blue), with an antibody specific to the mouse PECAM-1, a marker of endothelial cells (in green), and with an antibody specific to mouse red blood cells (in red). (C), (F), (I) and (L) were mergers of (A–B), (D–E), (G–H) and (J–K) respectively; white dotted lines indicate dermal-epidermal junction. In the human skin (A–C) that already contained capillaries (A), red blood cells were observed 4 days after graft (B) near the epidermis in capillaries that were not made of mouse endothelial cells (PECAM-1 negative in B). In the RS (D–I), 4 days after graft, no endothelial cell tube containing red blood cells was observed (E) under the epidermis, while some capillaries connected to the blood flow were detected in the bottom-half part of the graft thickness. Nevertheless, 14 days after graft, a homogeneous endothelial cells network (G) filled with red blood cells (H) was detected under the epidermis of the RS. Four days after graft of the ERS (J–L), human capillaries, characterized by a double staining of HLA and von Willebrand Factor (arrows, J), which contained red blood cells (K), were observed close to the epidermis. Mouse capillaries, stained by mouse specific anti-PECAM-1 antibody (K) were only observed in the bottom-half part of the graft. Bar = 200 μm in (A–C), in (D–F) and in (G–I). Bar = 50 μm in (J–L).

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Four days after graft, blood-containing vessels were observed in the ERS underneath the epidermis (Figure 3J, K, L). These capillaries were detected with a double staining of both antibodies against HLA and von Willebrand's factor in the upper part of the dermal compartment (Figure 3J), demonstrating that the vessels under the epidermal layer were populated by human ECs. Furthermore, mouse ECs could not be detected under the epidermis, and were located lower in the graft, as observed with RS (Figure 3E, K).

Fourteen days after graft, a co-localization between human and mouse ECs was sometimes observed in the graft using confocal microscopy ischimeric microvessels. Some mouse capillaries stained in green with anti-PECAM-1 specific to mouse ECs were colocalized with human capillaries stained in red with UEA-1 lectin in the ERS 14 days after transplantation (Figure 4A). Some of these co-localizations could correspond to branching patterns between the two types of vessels (Figure 4A, white arrow-heads), while more complex associations were also observed (Figure 4A, white arrows). Vessels only made of human ECs were also detected (Figure 4B, C, D, same stainings than Figure 3). The presence in their lumen of red blood cells was a clear demonstration of their inosculation with mouse recipient capillaries (Figure 4C, D).

image

Figure 4. Immunohistochemical characterization of the tubular structures formed in the ERS 14 days after graft. In A, human endothelial cells were immunostained in red with UEA-1 lectin, and mouse endothelial cells, were immunostained in green with an antibody specific to the mouse PECAM-1, on a 25-μm thick section observed using a Nikon C1 confocal microscope. In B, immunofluorescence studies were performed on 5-μm thick consecutive sections. Section was immunostained with a marker of the cell nucleus (Hoechst, in blue), an antibody against the human leukocyte antigen (HLA) (in green) and an antibody against the von Willebrand factor (in red). In (C), section was immunostained with Hoechst (in blue), with an antibody specific to the mouse PECAM-1, a marker of endothelial cells (in green), and with a mouse red blood cell antibody (in red). (D) was a merger of (B) and (C). The co-localization of human and mouse endothelial cells in a part of the same capillary was also sometimes observed in a branching-like pattern (A, arrow-heads) or in a closer association (A, arrows). More often, human capillaries containing blood (B–D) but devoid of mouse endothelial cells (C) were detected. Bar = 10 μm in (A) and 20 μm in (B–D).

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Finally, the total number of human and mouse capillaries per tissue section was evaluated for the three different types of graft (n = 4 for each time point). A two times significant decrease was observed in the number of human capillaries present in the human skin graft between 4 and 14 days after transplantation (Figure 5; p < 0.02). This could be due to a degeneration of supernumerary vessels that were not connected to mouse blood flow. Such a decrease was not observed in the ERS. Nevertheless, a significant increase in the number of mouse capillaries was detected in the ERS (p < 0.01), and in the RS 14 days after graft, in contrast with the human skin, in which only few mouse vessels were observed. This could be explained by the lower density of the reconstructed tissue, which might facilitate mouse blood vessel migration through the graft, as observed in the RS, in contrast with human skin.

image

Figure 5. Variation in the number of human and mouse vessels between 4 and 14 days after graft in human skin, ERS and RS. Human endothelial cells were immunostained with UEA-1 and mouse endothelial cells with PECAM-1. The number of CLS was counted on the biopsy section of samples from four different mice for each group, and divided by the biopsy surface. Results were considered significantly different when p < 0.05 using the Student-t test with n = 4. A significant decrease in the number of vessels was observed (*) between 4 and 14 days after graft of the human skin, but not in the ERS. A significant increase in the number of mouse vessels was detected in the ERS 14 days after graft, but not in the human skin.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

One of the main restrictions to the use of RS for wound coverage is its delayed vascularization, due to the thickness of the dermis, compared with an epithelial sheet. This thicker dermis is an advantage because it protects efficiently the reconstructed epidermis against mechanical, chemical and bacterial aggressions and promotes a far better healing of the wound (18). But as a drawback, it lengthens the time necessary for the host's blood vessels to vascularize the whole graft thickness. This delay could ultimately diminish the graft take and provoke the necrosis of the epidermis because of a lack of nutrition (4). We hypothesized that the transplantation on wounds of a RS containing a capillary-like network should promote a faster vascularization by inosculation between the capillaries of the graft with the wound bed vasculature, as observed with full-thickness human skin transplanted on wounds (Figure 1) (9,21). We had previously developed an ERS that promotes the spontaneous formation of CLS with human EC in basic culture conditions (11,12). These CLS featured a well-formed lumen in the entire length of the tubules, with basement membrane proteins on the basal side of the EC (11). In the present study, we described that the inclusion of EC in a RS accelerated the process of vascularization compared to conventional RS.

The human skin grafts experiments allowed the validation of our methods. Four days after graft, we observed human capillaries filled with red blood cells near the epidermis. These blood-containing vessels were homogeneously distributed in the graft. Thus, we confirmed the results previously obtained by Young et al. (9).

Similar results were obtained with the ERS. Human capillaries filled with mouse red blood cells were detected underneath the epidermis only 4 days after transplantation, although no mouse blood vessel was present in this area at that time. The fact that a human capillary was filled with mouse blood was itself a demonstration that this vessel was connected to the mouse vasculature. Furthermore, we observed that the establishment of blood circulation occurred faster within the ERS than the conventional RS underneath the epidermis, and as fast as observed with human skin. Indeed, capillaries filled with red blood cells were detected only in the lower and middle part of the dermal portion of the RS 4 days after graft. In addition, vascularized blood vessels were seen close to the epidermis of the RS only 14 days after grafting. Such a 14 days delay is thought to be related to the RS thickness (1 mm) and thus should increase proportionately with an augmentation of the graft thickness. In contradistinction, the inosculation process should remain independent of tissue thickness.

Krejci et al. obtained similar results with a comparable RS thickness. They used an acellular reticular dermis (AlloDerm™) (22). Autologous keratinocytes were seeded on the acellular dermis in order to form a RS that was grafted on athymic mice. The acellular dermis was re-populated by fibroblasts and blood vessels after 2 weeks (2,18,23–25).

Our results demonstrated that the initial process of vascularization observed during the first 4 days in the ERS was the result of the anastomosis of the human CLS with the host's blood vessels (inosculation). Indeed, mouse capillaries, extended by the neovascularization process, could not be detected under the epidermal layer of the ERS until 14 days after the graft. The inosculation of vessels from the graft to the wound vasculature was completed within 2–5 days, as described in the literature (9,26). To our knowledge, this is the very first experiment that demonstrates an inosculation process in less than 4 days between normal human capillaries reconstructed in vitro by tissue engineering, with the wound bed vasculature.

In the literature, the usual procedure to enhance the vascularization of engineered tissues by incorporation of ECs is to inject cells in a scaffold, without promoting a capillary-like formation during an in vitro maturation period before transplantation (10,27–30). Koike et al implanted in cranial windows of SCID mice a collagen/fibronectin gel mixed with HUVEC and 10T1/2 mesenchymal precursor cells after 1 day of in vitro maturation. These cells promoted the formation of a well-organized capillary network, which was perfused by the host blood in the first 2 weeks after graft (31).

Two other groups have shown that the subcutaneous implantation of matrices injected with Bcl-2-transfected human ECs led to the organization of cells into functional microvessels that were anastomosed with mouse vasculature in less than 7 days (10,30) or 14 days (28,29). Meanwhile, such an anastomosis was demonstrated in less than 4 days in our ERS by the presence of human capillaries containing mouse blood under the epidermis. This can be explained by the fact that the ECs in the ERS formed CLS by initiation of a vasculogenesis process in vitro before transplantation, while ECs added in the sponge model used by Nör and co-workers were not organized in CLS, prior to grafting, and thus necessitated the organization of EC into vessels before inosculation could occur (30). In addition, it should be noticed that ECs in our ERS did not necessitate the transfection of the Bcl-2 anti-apoptotic factor to be enhanced and form CLS, in contrast with other groups (28–30). For a clinical application, this Bcl-2 transfection is a too complex procedure to be readily applied in such a setting.

Supp et al. also transplanted skin substitutes containing ECs on athymic mice, but after only 16 days of in vitro maturation, in contrast with the 31-day culture of our ERS. They observed the formation of CLS only in the upper part of the dermis before grafting but not in the middle part, in contrast to our ERS. After 2 weeks, they observed a colocalization of human and mouse ECs but did not prove if these CLS were functional (i.e., contained red blood cells) (25). We have previously demonstrated by confocal microscopy that the capillary-like network reconstructed in our ERS extended throughout the thickness of the tissue (12). This could explain that the inosculation in our ERS was achieved in only 4 days, compared with the much longer delay of 2 weeks in the Supp et al. model.

The clinical application of our ERS requires the simultaneous extraction of keratinocytes, fibroblasts and ECs from a single biopsy harvested on the patient. Different techniques have been described to isolate microvascular EC from dermis and adipose tissue (32–37). The work of Supp et al. demonstrated the efficacy of HDMEC to form CLS and the feasibility of preparing autologous ERS (25). In addition, methods that allow the isolation of EC and/or their precursors from peripheral blood and their differentiation in culture are promising alternatives (38,39). The angioblasts isolated from blood circulation can participate in different postnatal physiological and pathological neovascularization phenomenon (40). These results are very interesting for the clinical application of the ERS. As another alternative, the use of specifically treated heterologous ECs could also be considered (41).

Furthermore, it would be interesting to perform long-term grafts of ERS in order to analyze the remodeling of the human capillaries. Nör et al. have demonstrated that 21 days after graft, non-functional CLS disappeared. They suggested that blood flow mediates signals, which stabilize branched microvessels while the absence of blood connection led to vessels regression. They also observed that 28 days after graft, mouse ECs took the place of the human ECs (10). Thus, it would be worthwhile to determine the survival of human EC within the graft in long-term studies.

The results obtained in this study showed that the presence of a CLS network in an ERS prior to the graft markedly increased the speed of vascularization by inosculation of its capillary network with the host's vasculature. This inosculation-mediated vascularization might be even more significant with thicker organs (over 1 mm thick), by making the difference between survival and necrosis of organs during the first week after transplantation. Thus, the reconstruction of a network of CLS in tissue-engineered organs could solve the major obstacle of vascularization deficiency that hinders the development of most tissue-engineered organs. The ERS appears to be a very efficient alternative to drastically improve vascularization of tissue-engineered organs after their transplantation in the human body.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We wish to thank Mrs. Anne-Marie Moisan, Sarah Poulin, Nathalie Tremblay and Danielle Larouche for their excellent technical assistance.

This study was supported by the Canadian Institutes of Health Research (CIHR) grant MOP-14364, “Fondation de l'Hôpital du Saint-Sacrement”, and “Fondation des Pompiers du Québec pour les Grands Brûlés”. P.-L. Tremblay was recipient of a scholarship from Fonds de Recherche en Santé du Québec (FRSQ); F. Berthod was recipient of a Fellowship from FRSQ; L. Germain was recipient of a Canada Research Chair on stem cells and tissue engineering.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Hansbrough JF. Current status of skin replacements for coverage of extensive burn wounds. J Trauma 1990; 30: S155S160.
  • 2
    Krejci NC, Cuono CB, Langdon RC, McGuire J. In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. J Invest Dermatol 1991; 97: 843848.DOI: 10.1111/1523-1747.ep12491522
  • 3
    Berthod F, Damour O. In vitro reconstructed skin models for wound coverage in deep burns. Br J Dermatol 1997; 136: 809816.
  • 4
    Boyce S. Cultured skin substitutes: a review. Tissue Eng 1996; 2: 255266.
  • 5
    Converse JM, Ballantyne DLJ. Distribution of diphosphopyridine nucleotide diaphrose in rat skin autografts and homografts. Plast Reconstr Surg 1962; 30: 415425.
  • 6
    Haller JA, Billingham RE. Studies of the origin of the vasculature in free skin grafts. Ann Surg 1967; 166: 896901.
  • 7
    Lambert PB. Vascularization of skin grafts. Nature 1971; 232: 279280.
  • 8
    Demarchez M, Hartmann D, Prunieras M. An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse. Transplantation 1987; 43: 896903.
  • 9
    Young DM, Greulich KM, Weier HG. Species-specific in situ hybridization with fluorochrome-labeled DNA probes to study vascularization of human skin grafts on athymic mice. J Burn Care Rehabil 1996; 17: 305310.
  • 10
    Nör JE, Peters MC, Christensen JB et al. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab Invest 2001; 81: 453463.
  • 11
    Black A, Berthod F, L'Heureux N, Germain L, Auger FA. In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent. FASEB J 1998; 12: 13311340.
  • 12
    Hudon V, Berthod F, Black AF, Damour O, Germain L, Auger FA. A tissue-engineered endothlialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro. Br J Dermatol 2003; 148: 10941104.DOI: 10.1046/j.1365-2133.2003.05298.x
  • 13
    Germain L, Rouabhia M, Guignard R, Carrier L, Bouvard V, Auger FA. Improvement of human keratinocyte isolation and culturing using thermolysin. Burns 1993; 19: 99104.
  • 14
    Auger FA, López Valle CA, Guignard R et al. Skin equivalent produced with human collagen. In Vitro Cell Dev Biol Anim 1995; 31: 432439.
  • 15
    Gordon PB, Sussman II, Hatcher VB. Long-term culture of human endothelial cells. In Vitro Cell Dev Biol 1983; 9: 661671.
  • 16
    L'Heureux N, Germain L, Labbe R, Auger FA. In vitro construction of a human blood vessel from cultured vascular cells: a morphologic study. J Vasc Surg 1993; 17: 499509.DOI: 10.1067/mva.1993.38251
  • 17
    Berthod F, Saintigny G, Chretien F, Hayek D, Collombel C, Damour O. Optimization of thickness, pore size and mechanical properties of a biomaterial designed for deep burn coverage. Clin Mater 1994; 15: 259265.
  • 18
    Berthod F, Germain L, Li H, Xu W, Damour O, Auger FA. Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice. Matrix Biol 2001; 20: 463473.
  • 19
    Li H, Berthod F, Xu W, Damour O, Germain L, Auger FA. Use of in vitro reconstructed skin to cover skin flap donor site. J Surg Res 1997; 73: 143148.DOI: 10.1006/jsre.1997.5229
  • 20
    Worst PK, Valentine EA, Fusenig NE. Formation of epidermis after reimplantation of pure primary epidermal cell cultures from perinatal mouse skin. J Natl Cancer Inst 1974; 53: 10611064.
  • 21
    Converse JM, Smahel J, Ballantyne DLJ, Harper AD. Inosculation of vessels of skin graft and host bed: a fortuitous encounter. Br J Plast Surg 1975; 28: 274282.
  • 22
    Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995; 21: 243248.
  • 23
    Gentzkow GD. Dermagraft, a cultured human dermis, to treat diabetic foot ulcers. Diabetes Care 1996; 19: 350354.
  • 24
    Ishihara T, Ono T. Analysis of the vascularity of an atelocollagen sponge substitute dermis in the human. J Dermatol 2001; 28: 360368.
  • 25
    Supp DM, Wilson-Landy K, Boyce ST. Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. FASEB J 2002; 16: 797804.DOI: 10.1096/fj.01-0868com
  • 26
    Boyce ST, Warden GD. Principles and practices for treatment of cutaneous wounds with cultured skin substitutes. Am J Surg 2002; 183: 445456.
  • 27
    Skovseth DK, Yamanaka T, Brandtzaeg P, Butcher EC, Haraldsen G. Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice. Am J Pathol 2002; 160: 16291637.
  • 28
    Schechner JS, Nath AK, Zheng L et al. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Proc Natl Acad Sci USA 2000; 97: 91919196.DOI: 10.1073/pnas.150242297
  • 29
    Schechner JS, Crane SK, Wang F et al. Engraftment of a vascularized human skin equivalent. FASEB J 2003; 17: 22502256.DOI: 10.1096/fj.03-0257com
  • 30
    Nör JE, Christensen J, Liu J et al. Up-Regulation of Bcl-2 in microvascular endothelial cells enhances intratumoral angiogenesis and accelerates tumor growth. Cancer Res 2001; 61: 21832188.
  • 31
    Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK. Tissue engineering: creation of long-lasting blood vessels. Nature 2004; 428: 138139.DOI: 10.1038/428138a
  • 32
    Marks RM, Czerniecki M, Penny R. Human microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture. In Vitro Cell Dev Biol Anim 1985; 21: 627635.
  • 33
    Karasek MA. Microvascular endothelial cell culture. J Invest Dermatol 1989; 93: 33S38S.DOI: 10.1111/1523-1747.ep12580906
  • 34
    Hewett PW, Murray JC, Price EA, Watts ME, Woodcock M. Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. In Vitro Cell Dev Biol 1993; 29A: 523531.
  • 35
    Conrad-Lapostolle V, Bordenave L, Baquey C. Optimization of use of UEA-1 magnetic beads for endothelial cell isolation. Cell Biol Toxicol 1996; 12: 189197.
  • 36
    Gupta K, Ramakrishnan S, Browne PV, Solvey A, Hebel RP. A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented with vascular endothelial growth-factor. Exp Cell Res 1997; 230: 244251.DOI: 10.1006/excr.1996.3421
  • 37
    Richard L, Velasco P, Detmar M. A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells. Exp Cell Res 1998; 240: 16.DOI: 10.1006/excr.1998.3936
  • 38
    Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells of angiogenesis. Science 1997; 275: 964967.DOI: 10.1126/science.275.5302.964
  • 39
    Boyer M, Townsend LE, Vogel LM et al. Isolation of endothelial cells and their progenitor cells from human peripheral blood. J Vasc Surg 2000; 31: 181189.
  • 40
    Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221228.
  • 41
    Laumonier T, Mohacsi PJ, Matozan KM et al. Endothelial cell protection by dextran sulfate: a novel strategy to prevent acute vascular rejection in xenotransplantation. Am J Transplant 2004; 4: 181187.DOI: 10.1046/j.1600-6143.2003.00306.x