In vitro and in vivo proposal of an artificial esophagus Maurizio Marzaro,1 Simonetta Vigolo,2 Barbara Oselladore,2 Maria Teresa Conconi,2 Domenico Ribatti,3 Stefano Giuliani,1 Beatrice Nico,3 Giampiero Perrino,1 Gastone Giovanni Nussdorfer,4 Pier Paolo Parnigotto2 1 Department of Pediatric Surgery, Treviso Regional Hospital, Treviso, Italy 2 Department of Pharmaceutical Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy 3 Department of Human Anatomy and Histology, University Of Bari Medical School, Bari, Italy 4 Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Italy Received 29 July 2005; revised 8 September 2005; accepted 21 October 2005 Published online 30 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30666 Abstract: Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. This study evaluates the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of homologous esophageal acellular matrix and autologous smooth muscle cells (SMCs). Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker and expressed bFGF as protein, showing angiogenic activity in vivo on the chick embryo chorioallantoic membrane (CAM). Moreover, they supported cell adhesion, and inasmuch as just after 24 h from seeding, the scaffold appeared completely covered by SMCs. To verify the biocompatibility of our constructs, defects created in the porcine esophageal wall were covered using homologous acellular matrices with and without cultures of autologous
SMCs. At 3 week from surgery, the patches composed of only acellular matrices showed a more severe inflammatory response and were negative for ␣-smooth muscle actin immunostaining. In contrast, the cell-matrix implants presented ingrowth of SMCs, showing an early organization into small fascicules. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous SMCs may represent a promising tissueengineering approach for the repair of esophageal injuries. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res 77A: 795– 801, 2006
INTRODUCTION
any complication, like those ones typical of the use of the colon or other intestinal segments. To produce esophageal substitutes, a variety of biomaterials have been tested. Autologous tissues or homografts, such as gastric fundus, ileal pedicle, pleura, pericardium, skin, fascia and intercostal muscle, have been already used in humans.4 – 8 Nevertheless, all these materials lead to complications like stenosis and leakage at long-term follow-up. Artificial materials, such as teflon and polypropylene,9 often lead to development of fibrosis. Over the last 5 years, promising results have been obtained by various studies performed on animal models, using polyvinilidene fluoride (PVDF) mesh,10 collagen sponge,11 and decellularized matrices, such as Alloderm12 and acellular porcine aorta.13 All patches were colonized by host cells with neovascularization and no signs of clinical esophageal dysfunction were observed. To improve the biocompatibility of the implant, other investigators used a tissue-engineered neoesophagus composed of human autologous esophageal epithelial
All major esophageal accidents requiring reconstruction procedures, like long-gap esophageal atresia, esophageal burns, or cancer, represent until today an important challenge for the surgeons. Several surgical techniques for different situations, such as stretching, circular myotomy, and interposition of stomach or colon, have been used according to the lesion, but leakage, stricture, anomalous elongation of the interposed tissue, altered transit time and, definitely, a bad quality of life can occur.1–3 Moreover, in the pediatric patients, the prognosis has to be the best that the surgeon can achieve, and the technique must be free of Contract grant sponsor: Regione Veneto Correspondence to: M.T. Conconi; Department of Pharmaceutical Sciences, via Marzolo 5, 35131 Padova, Italy; e-mail:
[email protected] © 2006 Wiley Periodicals, Inc.
Key words: esophagus; acellular matrix; smooth muscle cells; chick embryo chorioallantoic membrane; tissue engineering
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cells on collagen gel.14 The cell-matrix construct implanted in the latissimus dorsi muscle of athymic mice showed neovascularization and an epithelial cell layer similar to normal esophageal epithelium. We have already reported that acellular matrices can be used as tissue substitutes in animal models, because they are in vivo repopulated by the host cells and remodelled in a living tissue.15,16 Moreover, we have observed that previously seeding the matrices with autologous cells improves in vivo biocompatibility of the implants.17,18 On the ground of this basis, the aim of this study was to investigate whether (i) esophageal acellular matrix could represent a valuable scaffold for the in vitro culture of esophageal smooth muscle cells (SMCs) and (ii) the biocompatibility of homologous acellular matrix—autologous cell constructs in an animal model of esophageal defect.
MATERIALS AND METHODS Animals and reagents Pigs (3– 4 days old) were purchased from Azienda Agricola Zanato Michele (Ponso, Padova). The experiments were performed according to DLgs 116/92 which warrants care of experimental animals in Italy. The research project was approved by the Italian Health Department according to the art. 7 of the aforementioned DL. Antibiotic and antimicotic solution (AF), formalin, b-FGF, sodium deoxycholate, DNase-I, NaCl, gelatine, Dulbecco’s modified Eagle’s medium (DMEM), and monoclonal anti-␣smooth muscle actin antibody were purchased from Sigma Chemical Company (St Louis, MO). Fetal calf serum (FCS) was obtained from Biochrom-Seromed (Berlin, Germany). Monoclonal anti-MHC class I OX27, anti-MHC class II OX4, and anti-bFGF antibodies were provided by Abcam (Cambridge, UK). The Large Volume Dako LSAB Peroxidase Kit was purchased from Dako (Glostrup, Denmark). Gelatin sponges were purchased from Gelfoam Upjohn (Kalamazoo, MI). Glutaraldehyde was obtained from Merck (Darmstadt, Germany) and sodium cacodylate was provided by Prolabo (Paris, France).
Acellular matrix of pig esophagus Esophagus, obtained from newborn pig, was rinsed four times in phosphate buffered saline (PBS) containing 1% AF and then treated, according to Meezan et al.,19 to obtain an acellular matrix. Samples were processed five times as follow: distilled water for 72 h at 4°C, 4% sodium deoxycholate for 4 h, and 2000 kU DNase-I in 1M NaCl (Sigma) for 3 h. The absence of cells was confirmed histologically (hematoxylin-eosin staining), and acellular matrices were stored in PBS at 4°C.
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In vitro cultures of SMCs SMCs were isolated from a cervical esophagus biopsy in newborn pigs via a surgical cervical approach. Briefly, muscolaris externa was separated and minced. The fragments were placed in 1% gelatin-coated Petri dishes. Cells were cultured in DMEM containing 20% FCS for 3 days and then supplemented with 10% FCS.
Immunochemistry After each detergent-enzymatic treatment, aliquots of esophagus were fixed with 10% neutral buffered formalin for 24 h and embedded in paraffin. Five micrometer transversal sections were incubated for 30 min at room temperature with PBS containing 10% FCS. Samples were then incubated at 37°C for 1 h with monoclonal anti-MHC class I OX27, anti-MHC class II OX4, and anti-bFGF antibodies diluted in 1% FCS-PBS (1:400), and then labeled with avidin– biotin amplified immunoperoxidase method, using the Large Volume Dako LSAB Peroxidase Kit. Cell purity of explant preparations (passage 2– 4) was determined by ␣-actin immunostaining. Cell cultures were fixed for 10 min at 4°C in cold methanol. After further washing, fixed cells were incubated for 30 min at room temperature with PBS containing 10% FCS. Samples were then incubated at 37°C for 1 h with a monoclonal anti-␣smooth muscle actin antibody diluted in 1% FCS-PBS (1:400) and then labeled as described earlier. Negative controls were carried out by treating matrices and cultures similarly and omitting the primary antibody.
CAM assay Fertilized White Leghorn chicken eggs (20 for each experimental group) were incubated at 37°C at constant humidity. On day 3 of incubation a square window was opened in the egg shell and 2–3 mL of albumen was removed of so as to detach the developing CAM from the shell. The window was sealed with a glass and the eggs were returned to the incubator. On day 8, 1-mm thick cross section of an acellular matrix of pig esophagus cut with a scissor was implanted on top of the growing CAM under sterile conditions. Sterilized gelatin sponges (1 mm3) adsorbed with 500 ng/embryo of FGF-2 dissolved in 1 L PBS or with PBS alone, used as positive and negative control, respectively, were implanted on day 8 on the top of some CAM, as previously reported.20 CAM were examined daily until day 12 and photographed in ovo with a stereomicroscope equipped with a Camera System MC 63 (Zeiss, Oberkochen, Germany). Blood vessels entering the implants or the sponges within the focal plane of the CAM were counted by two observers in a doubleblind fashion at 50⫻ magnification. Means ⫾ standard deviation (SD) were determined for all variables. The statistical significance of the differences between mean values was determined by the Student’s t-test for unpaired data.
IN VITRO AND IN VIVO PROPOSAL OF AN ARTIFICIAL ESOPHAGUS
Figure 1. Smooth muscle cell cultures. A: Phase-contrast microscopy of cultures 4 days after isolation. B: Scanning electron microscopy of primary cultures. C: Secondary cultures immunostained with monoclonal anti-␣-smooth muscle actin antibody. Magnification: (A) ⫻40, (C) ⫻100. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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Figure 2. Transversal sections (5 m) of esophageal acellular matrices treated (A, C) and not treated (B) with 5 cycles of detergent-enzymatic treatment. A,B: Immunostaining with monoclonal anti-MHC class I antibody. C: Immunostaining with monoclonal anti-bFGF antibody. Magnification: ⫻40. [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]
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Figure 3. Macroscopic pictures of an esophageal acellular matrix (A) and a gelatin sponge soaked with FGF-2 (B) implanted onto the chick embryo chorioallantoic membrane on day 12 of incubation. Note numerous allantoic vessels developing radially toward the implants. C: A chorioallantoic membrane on day 12 of incubation, incubated for 4 days with a sponge adsorbed with PBS, and no vascular response is detectable around the sponge. Magnification: (A) and (C) ⫻50. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
In vitro cultures of SMCs and homologous acellular matrix Secondary cultures of SMCs were detached, seeded (5 ⫻ 105 cm⫺2) on the external side of homologous acellular matrix, and then cultured with DMEM supplemented with 10% FCS. After 24 h from seeding, cultures were fixed with 3% gluteraldehyde in 0.1M cacodylate buffer (pH 7.2). After critical point drying and gold sputtering, cultures were examined by a scanning electron microscopy (SEM; Stereoscan-205 S, Cambridge, UK).
Implantation Pigs (3– 4-week-old) underwent general anesthesia via tracheal intubation and using pentotal fluotane, curare, nitrous oxide, atropine and fentanyl. Through an extrapleural approach, the thoracic esophagus was exposed and a 2-cm diameter defect in the tonaca muscolaris of its wall was created. This defect was covered with the scaffold, which was sutured with 6.0 absorbable (poliglycolid acid) material and the wall closed. Antibiotics (ceftizoxima) were administrated during the operation and the days immediately after while no immunosuppressive therapy was used. Soon after the surgery, the animals resumed oral feeding. A group of animals (n ⫽ 3) were implanted with homologous acellular matrix, and another group (n ⫽ 3) with homologous acellular matrix seeded with autologous SMCs. All animals survived and no complications occurred. Pigs were killed on the third postoperative week. The implants were recovered, fixed in 10% neutral buffered formalin, and embedded in paraffin. Five-micrometer thick transversal sections of the implants were immunostained with monoclonal anti-␣smooth muscle actin antibody, as described earlier.
RESULTS SMC cultures Cells emerged from the isolated tissue as early as 4 days after isolation [Fig. 1(A)]. The migrating cells Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
spread around the fragments and proliferated, forming a confluent monolayer in 1 week. These cells displayed the elongated shape of smooth muscle cells [Fig. 1(B)] and were positively stained by the anti-␣smooth muscle actin antibody [Fig. 1(C)].
Acellular matrices Histological examination of the matrices obtained by 5 cycles of detergent-enzymatic treatment of pig esophagus revealed the absence of cells [Fig. 2(A)]. Moreover, MHC class I and MHC class II antigens [Fig. 2(A)] disappeared in comparison with not treated tissue [Fig. 2(B)] and a wide positivity to bFGF was visible [Fig. 2(C)].
CAM assay Macroscopic observation of CAM treated with acellular matrices showed that the implants on day 12 of incubation were surrounded by allantoic vessels (mean number of vessels around the implants ⫽ 28 ⫾ 4; p ⬍ 0.001 vs vehicle alone) that developed radially towards the implant in a “spoked-wheel” pattern [Fig. 3(A)] as compared to the same implants on day 8 of incubation (mean number of vessels around the implants ⫽ 7 ⫾ 2). The angiogenic response was comparable to that exerted by a well-known angiogenic cytokine, namely FGF-2 (mean number of vessels around the implants ⫽ 32 ⫾ 4; p ⬍ 0.001 vs vehicle alone) [Fig. 3(B)], while on day 12 of incubation, no vascular reaction was detectable around the sponge treated with vehicle alone (mean number of vessels around the implants ⫽ 8 ⫾ 2) [Fig. 3(C)].
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In vitro cultures of SMCs on homologous acellular matrix When examined by SEM, the matrix surface appeared irregular [Fig. 4(A)], and after 24 h from seeding, it was completely covered by SMCs [Fig. 4(B)]. Although the large part of cells possessed a round shape, some of them started to flatten [Fig. 4(C)].
Implantation Three weeks after surgery, an inflammatory response, with granulocyte and macrophage infiltration, was noted in pigs implanted with homologous acellular matrices [Fig. 5(A,B)], but not signs of rejection were seen. Capillary ingrowth was visible inside the patches. Immunohistochemistry revealed that these implants did not present SMCs, whereas the grafts composed of acellular matrix and cells were positively stained by the anti-␣-smooth muscle actin antibody [Fig. 5(C)]. Moreover, SMCs started to organize in small fascicles [Fig. 5(D)].
DISCUSSION In the last years, evidences has been accumulated that acellular matrices could be successfully employed to repair skin,21 intestinal,15 urethral,16 and skeletal muscle17,18 defects in experimental animals. These biocompatible scaffolds function as templates that provide a structural support during tissue development. Moreover, decellularized matrices obtained from human skin12 or porcine aorta13 have been already used for esophageal replacement. We proposed the use of homologous esophageal acellular matrix, because it presents thickness and structure close to the native tissue. The detergent-enzymatic method employed to obtain the acellular matrices preserves matrix integrity,22 which represents an important factor to avoid their in vivo destruction ensuing from the obvious inflammatory response.23 Moreover, the decellularization process abolishes the risk of rejection,24 since it completely removes the major histocompatibility complex markers (the MHC class I and MHC class II antigens). In our previous works aimed to repair skeletal muscle defects in rat,17,18 we observed that homologous muscle acellular matrix alone was quickly remodeled into fibrous tissue, whereas the presence of autologous satellite cells on the scaffold preserved the structural integrity and improved in vivo biocompatibility. Hence, we obtained an in vitro esophageal substitute composed of autologous SMCs seeded on homolo-
Figure 4. Scanning electron microscopy of esophageal acellular matrix (A) and SMCs cultured on acellular matrices after 24 h from seeding (B and C).
gous esophageal acellular matrix. Our findings demonstrate that esophageal acellular matrix was able to support cell adhesion, and inasmuch as just after 24 h Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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Figure 5. Transversal sections of esophageal acellular matrix implants without (A and B) and with autologous cells (C and D), 3 weeks after surgery, immunostained with monoclonal anti-␣-smooth muscle actin antibody. Magnification: (A) and (B), ⫻40; (C), ⫻250; (D) ⫻400. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
from seeding, the scaffold appeared completely covered by muscle smooth cells. In the tissue engineering, the improvement of vascularization of the regenerating tissue is essential to change a non-permissive injury environment to a regeneration-permissive one, and thus to allow the survival of the implanted cells.25 The use of angiogenic factors, such as VEGF and bFGF,26 –28 is a popular approach to induce neovascularization. Angiogenic growth factors are required for endothelial cell (EC) proliferation and blood vessel formation. To enhance new capillary growth from the host vascular network, slow releasing source of an angiogenic factor into the damaged or bioengineered tissue can be incorporate prior to implantation.29 Alternatively, cells can be genetically altered to secrete angiogenic factors.30 Our data indicate that acellular esophageal matrices maintained the expression of bFGF as protein and showed angiogenic activity on CAM. To verify the biocompatibility of our constructs, defects created in the porcine esophageal wall were Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
bridged using either homologous acellular matrices alone or cultures of autologous SMCs on homologous acellular matrix. At 3 weeks from surgery, histological examination revealed that both patches were infiltrated by mononuclear cells and fibroblasts without signs of rejection. Moreover, neovascularization was well visible. Nevertheless, the patches composed of only acellular matrices showed a severe inflammatory response and were negative for ␣-smooth muscle actin immunostaining. In contrast, the cell-matrix implants presented ingrowth of SMCs, showing an early organization into small fascicules. Although preliminary, our data confirm that the presence of autologous cells improves the integration of the graft into the recipient tissues. Actually, we have not established how long the implanted cells remain viable. However, we can hypothesize that autologous cells may represent a signal, which lowers inflammatory response and induces the migration of host smooth muscle cells from the edges of the implant. The ingrowth of tissue represents a key step in the regeneration process, avoiding
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the formation of an amotile and not functional scar tissue. Regeneration of esophageal wall layers was successfully achieved using collagen sponge,31 PVDF mesh,10 and esophagus organoid units32 in rat models. In conclusion, our study suggests that patches composed of homologous esophageal acellular matrix and autologous SMCs may represent a promising tissue engineering approach for the repair of esophageal injuries. Further long-term studies must be performed to evaluate whether this construct, when used to replace a full-thickness defect, could allow a full regeneration of the host tissue without strictures or leakage. Moreover, it must be established whether the autologous cells could secrete growth factors favouring the ingrowth of the host cells and their contribution to the regeneration process.
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