Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract iatrogenic defects

Introduction
Many experimental studies focus on obtaining a substitute of the digestive tract for the treatment of short small bowel syndrome. In this respect all the efforts have been directed to obtain a tissue engineered digestive segment with normal absorption and peristaltic movements. These functions can be possible only by reproducing all the layers of digestive tract. Although promising, the experimental results could not yield a segment of neointestine long enough to be capable of solving the clinical problems of the short small bowel syndrome. However, the positive achievements reported with tissue-engineering grafts on experimental animal studies allow us viewing the future application of these bioconstructs as grafts for the treatment of a devastating postoperative complication: the digestive fistula (1). The future clinical usage of these bioengineered grafts to treat digestive fistula is much more feasible than in the case of short bowel syndrome. The restoration of the digestive defect does not specifically need the reconstruction of a digestive wall with normal absorptive and peristaltic function. The aims of using the bioengineered grafts to patch a digestive hole are to stop the external leakage of the digestive secretions and facilitate the complete regeneration of the digestive wall.
We hypothesized that the digestive wall defect encountered in digestive fistula can be covered by a patch with enough durability to ensure the regeneration of the digestive layers. We aimed not only to obtain the digestive wall repair, but also a structure with a high similarity with the native digestive wall consisting in all four layers. MSCs are similar to embryonic stem cells in that they have extensive proliferation, clonal multilineage and differentiation potential in vitro and in vivo to form cells of three germ layers (2,3), therefore we assumed that the use of these cells may be beneficial for the repair of digestive wall defects.
For the development of an innovative method of digestive wall replacement using MSCs, a design of an adequate support for cell survival and differentiation was developed, tested and analyzed in an experimental animal model.

Materials and methods
Obtaining the collagen-agarose sponge
The collagen-agarose matrix was obtained using a mixture of collagen type I and agarose (Sigma Chemical Company, St. Louis, MO, USA) in a proportion of 1:0.5. Collagen was extracted and purified in our laboratory from cow Achillean tendons using an enzymatic extraction protocol with pepsin and acetic acid. The sponge-type matrix was obtained by lyophilization of the mixture at a temperature of -40ºC. The freeze-dried materials were exposed to UV radiation for 8 h, in a sterilization cabinet with Vilber-Lourmat UV-tubes.
The collagen-agarose sponge was processed and analyzed in low vacuum model under scanning electron microscopy (SEM) using an ESEM, Quanta 400, FEI, Philips (Holland) for the characterization of both the 3D aspect and porosity.
Procurement and preservation of aortic vessels
Aortic vessels were procured either from cadaver human donors or sacrificed pigs.
Thoracic aortas of cadaver human donors were harvested during the procurement of other organs for transplantation. The aorta segments were rapidly immersed in cryopreservation solution Custodiol® HTK (Methapharm Inc., Brantford, Ontario, Canada), transported and stored at 4°C, prior to further processing. Under sterile condition the aorta segments were cleared of adipose tissue and introduced in a solution of glycerol for cryopreservation at -196°C.
Thoracic and abdominal aortas of adult large pigs were obtained from a local slaughterhouse by sterile harvesting. They were immediately immersed in Hanks' balanced salt solution (HBSS, Gibco BRL, Grand Island, NY, USA). The transportation and storage were similar to those used for human aortas.
For the construction of the patch, the aorta segments were thawed and transported at 4°C. After arriving at the laboratory, the aortas were placed in a solution of phosphate-buffered saline (PBS) with penicillin (200 U/ml) and streptomycin (200 mg/ml) (Biochrom, Berlin, Germany) and stored at 4°C prior to further processing. For endothelial decellularization, vessels were placed in 0.25% trypsin with 0.2% EDTA (Biochrom), for 20 minutes at 37°C, under continuous shaking. Vessels were then washed with PBS several times to remove residual substances, and used for graft preparation.
Harvesting, isolation and culture of MSCs
MSCs used for the patches were of human origin in 9 experiments. There were 3 human volunteers for 30 ml bone marrow harvesting. For the other 29 cases MSCs were harvested from rat (Table 1). Fresh rat bone marrow was extracted from their femurs under sterile conditions after sacrificing them. The animals were kept and treated according to the animal welfare regulations.
Mononuclear cells and MCSs were isolated from bone marrow aspirate by centrifugation (30 minutes, 400 g, density 1.077 g/ml) over a Biocoll layer (Biochrom). After centrifugation, the cells were cultivated in Alpha MEM (Gibco BRL, Grand Island, NY, USA), supplemented with 20% fetal calf serum (Sigma-Aldrich Corp., St. Louis, MO, USA), bFGF 10 ng/ml (Sigma-Aldrich Corp.), penicillin (200 U/ml), and streptomycin (200 mg/ml). The medium was changed after the first 24 hours and then twice a week. When cells reached 80% confluence, they were detached using 0.25% trypsin with 0.2% EDTA. Cells were cultivated for 3 passages, and MSCs were selected by adherence.
MSCs characterisation by flow cytometry
The surface marker identification of the cultured MSCs was performed on an Epics XL FACS analyzer (Beckman Coulter Inc, CA, USA), using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies staining to CD105+, CD90+, CD34-, CD3- and phycoerythrin (PE)-labeled monoclonal antibodies staining for CD45- and CD14- (all from BD Pharmingen, San Diego, CA, USA). To assess the unspecific binding, appropriate isotype controls were included.
Viability, cell proliferation, and microscopy assays
Scaffold's biocompatibility was assessed by monitoring cell viability with trypan blue test, and the proliferative capacity with Cell Titer 96 Test (Promega Corporation, Madison, WI, USA), after seeding similar number of cells (5x104) in 2D, and 3D culture systems, respectively, in the medium described above. For counting, 3D collagen scaffolds were completely degraded with type I collagenase 0.05% for 30 minutes.
Cell dispersion into the scaffold was observed by micros-copy techniques: collagen seeded scaffolds were stained with propidium iodide 0.01 mg/ml at 37°C after ethanol treatment.
Graft preparation
5x104 cells were seeded into 5-7 mm 3D collagen-agarose scaffolds and placed onto appropriate decellularized arterial fragment in 24 well dishes in culture medium. The resulted 3D patches were used for the experimental transplantation. Following preparation, patches were maintained in culture media allowing MSCs proliferation and dispersion to the whole structure.
To test the biodegradability, the 3D patches were subjected to digestion in vitro by type I collagenase (0.05%), showing a complete degradation in 30 minutes.
Animals
Adult Wistar rats, weighting 230-350 g, were used after minimum 7 days of acclimatization. Animals were housed in cages in a circadian rhythm light (dark and temperature controlled environment) and fed with standard rat chow and water ad libitum. The animals were only allowed to drink water ad libitum, 24-36 hours before the experiment. All procedures were carried out in accordance with "Guide for the Care and Use of Laboratory Animals" National Research Council; Washington: National Academy Press, 1996.
Animal experiments
Anesthesia was induced using a gas mixture of halotane 4% and 4 l/min O2, in a sealed chamber. Prior to administration of the i.m. anesthetic cocktail, each subject received s.c. injections with 0.05 mg atropine and 2.5 ml of 5% glucose in two different regions. Anesthesia was maintained injecting i.m. the mixture of ketamine 80 mg/kg and xylazine 7 mg/kg.
The rats were prepared for a sterile operation. An upper midline laparotomy was performed through a 2.5 - 3 cm -long incision. The stomach or the jejunum was identified and isolated with wet gauzes. A small 5-7 mm diameter portion of the anterior stomach wall or small intestine was removed with microscissors. The resulted defect was repaired using a MSCs enriched 3D patch. The patch was sutured at the edges of the digestive wall defect, by microsurgical techniques, using interrupted 8-0 Prolene or Vicryl sutures (Ethicon, Cincinnati, OH, USA). The 3D patches with MSCs were mounted on iatrogenic defects of stomach (21 subjects) or jejunum (17 subjects). Prior to the abdominal closure the peritoneal cavity was irrigated with 10-30 ml isotonic saline. The abdomen was closed using a continuous suture of 4-0 Vicryl. The skin was closed using either separate 4-0 Silk sutures or staplers. Postoperatively, rats were housed in individual cages and allowed water ad libitum immediately. Standard solid rat chow was introduced after 48 hours. Animals were checked on a daily basis for signs of distress, and i.m. acetaminophen was administered as needed.
Macroscopic assessment of intestinal suture healing
At predetermined time-points, all surviving animals were killed by administration of an overdose of intravenous pentobarbital followed by potassium chloride. If animals appeared ill or septic, they were euthanized. The abdomen of the rat was opened, and suture was identified, inspected, and resected 2 cm proximal and distal to the suture line. Specimen was gently flushed with isotonic saline and skeletonised free from adherent tissues, and longitudinally cut to inspect the luminal aspect.
Histology
Excised pieces of stomach or jejunum were placed in 10% neutral buffered formalin or 0.9 % saline for further histological and immunofluorescence studies. The specimens were evaluated by light microscopy on 4 µm hematoxylin and eosin stained tissue sections. The examiner was unaware of the identity of the experimental groups. The time-points provided the opportunity to examine the acute and chronic cellular response to the xenogenic implants, neoangiogenesis, neomatrix deposition, cellular colonization, proliferation and differentiation, and scaffold degradation.
Immunofluorescence
Human MSCs were identified in the transplanted intestine by staining with anti-human HLA-FITC (Sigma, St. Louis, USA). The specimens obtained from animals in which we used patches with human MSCs were analyzed by immunofluorescence. For detection of the human cells at the level of patch-induced digestive wall regeneration, the mouse monoclonal antibody anti-HLA class I antigen FITC conjugate (Sigma) was used at a dilution of 1:5, employing a direct fluorescence staining method. Briefly, the frozen sections (6 µ) were layered on slides (Fisher Superfrost) and fixed with cold acetone, and then rinsed and equilibrated 5 minutes in 0.01 phosphate buffered saline (PBS), pH 7.2 (Sigma). The sections were then incubated with 1.5% bovine serum albumin (BSA) (Sigma), 0.05 % TritonX-100 (Boehringer Mannheim GmbH, Mannheim, Germany) in PBS for permeabilization and non-specific sites blocking (30 minutes, at room temperature, in a humidified chamber). After decanting the blocking solution and rinse with PBS-BSA 1.5%, the sections were incubated with the FITC conjugated antibody diluted in PBS-BSA 1.5%, 2 hours at room temperature in a humidified chamber. After washing 3x5 minutes in PBS, the sections were incubated with Hoechst stain (Invitrogen Corporation, Carlsbad, California), 15 minutes, at room temperature in a humidified chamber for nuclei staining. Finally, the sections were washed 3x5 minutes with PBS, counterstained with hematoxiline and mounted in fluorescent mounting medium (Dako-Cytomation Inc., Carpinteria, CA, USA), then examined under a fluorescence microscope.
Transmission electron microscopy (TEM)
For electron microscopy, tissue fragments were fixed with 2% glutaraldehyde in 0.1% cacodylate buffer, cut into small pieces of about 2 mm3, and postfixed with 1% OsO4 in the buffer, for 2 hours. They were dehydrated in a graded series of ethanol and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined at a TEM (Philips EM 208S).

Statistical Analysis
The results of the cell proliferation assay were analyzed using paired t tests. Fisher's exact test was applied to analyze clinical data. Significance was set at p<0.05.

Results
Clinical outcome of the transplanted rats
The clinical results did not depend upon the type of digestive defect and the provenience of the aortic fragments and MSCs, as demonstrated by the Fisher's exact test. The overall mortality rate was 15.78% (6 of 38 cases). The cause of death was diffuse peritonitis (5 cases) and intraabdominal abscess (1 case). These complications occurred in 4 cases due to the failure of the anastomosis between digestive wall and the patch. In the other 2 cases no disruptions of the anastomosis or the patch were found. In the other 32 cases the transplanted rats had no complications. The provenance of the 3D patches with MSCs and the clinical outcome are presented in Table 1.
Biocompatible, biodegradable, and MSCs enriched transplantable 3D patches
The bone marrow cells were maintained in vitro through 3 serial passages in order to obtain a uniform pattern of MSCs (Fig. 1A) with high proliferative potential to be seeded on the biomatrix. The collagen-agarose sponge appeared in SEM images as a honeycomb structure with pores of 200 mm size, and a homogenous mixture of the two substances (Fig. 1B). Our scaffold's pore size permitted cell multiplication and diffusion from seeding points (Fig. 1C). In order to ensure the proper structural integrity of patches, similar to that of the digestive tract wall, we used our scaffolds together with aortic fragments (Fig. 1D). Swine vessels were used as support in 22 cases. In other 16 cases aortic segments were of human provenience (Table 1). Due to the highly immunogenic potential, native cells of the aorta were removed. The remaining matricial fragments were used as support for the 3D collagen-agarose scaffolds seeded with MSCs (Fig. 1D).
The cultured cells obtained from bone marrow had a representative phenotype for MSCs, expressing peptide SH2 (CD105+) and receptors for adhesion molecules (CD90+), but not the hematopoietic lineage antigens (CD34-, CD45-, CD3-, CD14-) (Fig. 2A).
The viability and proliferation assays confirmed that the collagen scaffold was not toxic for the cells, and provided an optimal environment for MSCs growth, as shown by cell proliferation curves. Cell growth assay performed with a similar number of cells plated in both 2D and 3D culture systems, showed that in 3D scaffold system the number of cells was significantly higher than in 2D classical culture system at 14 days (p</=0.01) (Fig. 2B).

Regeneration of the intestinal wall
In all transplanted animals the extraluminal surface of the patch was partially or totally covered by adjacent organs. In the case of the stomach patches, adhesions developed to liver or great omentum. In the case of the small intestine, adhesions between patch and other intestinal segments were encountered. The adhesions in the surrounding area made the identification of the patched area easier. After adhesiolysis the outer surface of the regenerated digestive wall was almost indistinguishable from the normal digestive surface after 6 weeks. In two cases there was a circular ridge of hypertrophied mucosa and submucosa. After this period of time the differentiation between the regenerated and the normal digestive wall could be made only by inspection of the inner face of the regenerated digestive wall. The diameter of the regenerated stomach or intestine was not altered proximal and distal to the patched zone.
By microscopic examination, three days postoperatively, stem cells were observed at the level of the engrafted patch (Fig. 4 A, B). The regeneration of the digestive wall was facilitated by the colonization of the 3D patch by cells from the digestive wall (Fig. 4C), and the occurrence of the granulation tissue, which in time receded, in parallel with digestive wall reconstruction. Full regeneration of all four layers (mucosa, submucosa, muscularis and serosa) of the stomach and small intestine was observed, with the complete resorption of the aortic vascular structure at 48 days (Fig. 4D). The particularities of the regenerated digestive wall were the presence of squamous pluristratified epithelium and inflammatory infiltrate, and the lack of mucous glands (Fig. 4D). The inflammatory infiltrate was more significant after the use of patches with human MSCs. The aspect of the regenerated digestive tissue was not influenced by the origin of aortic vessels used for the patch preparations and was in accordance with the digestive segment involved.
Human cells were identified in the regenerated digestive wall of the rats in the presence of an intact immune system. Identification of human origin cells was performed at 48 days by immunofluorescence examinations using antibodies anti HLA-FITC (Fig. 5).
Three days postoperatively cells were identified by TEM at the luminal aspect of the patch (Fig. 6A). The aspect of these cells is highly representative for stem cells: cells with large nuclei and scarce cytoplasm. Ten days postoperatively, a lower density of round-oval cells is found near the aortic fragment (Fig. 6B), and a higher density of cells with a pavimentous-like distribution at the luminal aspect of the patch (Fig. 6C). Numerous intercellular connections are also noticed. At 180 days the structure is highly organized with numerous neovessels and reorganization of collagen fibers (Fig. 6D).

Discussion
In the present study we tested for the first time a combination of MSCs and biological matrix with the purpose of digestive wall regeneration in vivo. During the present research we used decellularized pig vessels as support, covered by a collagen-agarose sponge seeded with MSCs.
Tissue engineering has emerged as a need for restoration, maintenance, and improvement of tissue functions. There are three general strategies to create new tissues: (1) the use of isolated cells or cell substitutes, (2) the use of tissue-inducing substances, and (3) the use of cells placed on or within matrices (4).
The ideal support for the reconstitution of the digestive wall has not been found yet. Generally, the ideal graft should be biocompatible to avoid unwanted host tissue responses, promote cell attachment, infiltration and proliferation, favor neovascularization, and hence tissue development, possess adequate mechanical and physical properties to maintain the structure and function during tissue repair, and be bio-degradable in a controlled manner (5).
When using simple scaffolds without viable cells, the reconstruction of the digestive wall is the result of the self-tissue regeneration. Using scaffolds embedded with cells, the reconstruction of the digestive continuity is accomplished by both the donor tissue and host tissue.
The first report on the successful use of prosthetic nonabsorbable materials as patch for repairing digestive wall defects was made by Harmon et al. (6). As it has been demonstrated by other studies, nonabsorbable materials appear to be a poor choice for tissue ingrowth because the regenerated tissue has architectural disarray (7).
Thompson investigated for the first time the use of an absorbable synthetic patch: polyglycolic acid mesh (8). Polymeric scaffolds may not interact with cells in a desired manner since their surface chemistry does not promote adequate cell adhesion and may also induce toxic and inflammatory reactions (9). In addition there is concern of potential infection regarding the use of synthetic grafts (10).
Nowadays the studies focus on the use of biomaterials which have some advantages over the synthetic materials: are biodegradable in time, promote cellular interaction, growth, tissue development, and are resistant to infection. The ultimate success of tissue engineering may rest on the selection of the appropriate type of biomaterials. Collagen is the most frequently used biomaterial for cell culture in various fields. Collagen is advantageous in that it contains information that may facilitate cell attachment, migration, proliferation, and maintenance of differentiated function (11). Furthermore, exogenous collagen can be incorporated into the granulation tissue being a fuel for tissue reconstruction (12). 3D cell cultures have been obtained with collagen sponges, gels, films, and sandwiches of them (11, 13, 14). Collagen-based biomaterials such as Contigen and extracellular matrix-based materials such as AlloDerm, small intestinal mucosa (SIS), urinary bladder submucosa (UBS) are examples of commercially available products. AlloDerm was used for patching the defects in the cervical esophagus of dogs (15). SIS was used as a patch for esophageal (16) and small bowel (17-19) regeneration in an in situ xenograft model. UBS was used as a resorbable xenograft scaffold for repair of esophageal defects in a dog model (16). SIS and UBS have been shown to contain angiogenic growth factors (e.g. basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF), and also components that support angiogenesis (fibronectin, heparin, collagen I, III, IV, and V) which could explain the important role of these scaffolds in neovascularization (16, 17, 20).
Porcine aorta is commonly used in experiments which aim the development of a vascular substitute (21). Kajitani et al. repaired major duodenal injury with an elastin patch created from porcine aorta (22). The use of pig aortic vessels is justified not only by their resistance to the digestive luminal content but also by their high content of collagen and elastin which have a high impact on tissue healing.
The limitation of the xenograft is its antigenicity which can be precluded by decellularization. The decellularization techniques include chemical, enzymatic and mechanical means of removing cellular components, leaving a material composed essentially of extracellular matrix components (23). There is a patented method of decellularization: SynerGraft processing by CryoLife Inc, Kennesaw, GA, USA (24, 25). The enzymatic treatment used by us was optimized through serial trials in order to avoid that arterial fragments become too friable. The treatment preserves extracellular matrix architecture consisting in structural proteins, proteoglycans, collagen and elastin fibers, as was previously proved by Teebken (26).
Synthetic and biologic scaffolds have been in vitro populated with different viable cells depending on the tissue needed to be repaired. Vacanti et al. have been the pioneers in tissue engineered neointestine using organoids seeded onto a scaffold made of polyglycolic acid and polylactic acid (27). Their studies were based on the work of Evans and Tait (28, 29). Intestinal organoids appear to be superior to intestinal cells for seeding the biomaterial because the organoids contain stromal elements in addition to crypt cells that appear to facilitate the generation of neointestine (7). Moreover these organoids may also contain intestinal stem cells.
The role of MSCs in tissue regeneration is well-established (30). They have the capacity to differentiate, under appropriate in vitro conditions, to form osteoblasts, chondrocytes, tenocytes, myoblasts, neurons, and cells of visceral mesoderm (endothelial cells) (31). MSCs have the ability to engraft into healthy as well as injured tissue and can differentiate into several cell types in vivo (31). In vivo MSCs are able to repair damaged tissue from kidney, heart, liver, pancreas and gastrointestinal tract (32). Efficiency of bone marrow-derived cells in regeneration of the stomach after induction of ethanol-induced ulcers was showed in experimental studies in rats (33). Sustaining the MSCs implication in the repair of gastrointestinal tract, successful results of their clinical usage in the treatment of post radiation enteritis (34) and inflammatory bowel disease (35) were reported.
The potential benefit of MSCs in the repair of full-thickness digestive wall defects is a new concept. Due to their ex vivo and in vivo multiplication and differentiation properties, we presumed MSCs to be of great importance in the repair of digestive wall defects.
We hypothesized that the most proper delivery of MSCs for the repair of digestive wall defects is the use of a local patch with a high number of MSCs. The use of a high concentration of MSCs implies their availability. Due to the possibility of in vitro cultivation and multiplication of these cells, the single limitative factor in their availability could be their harvesting. Bone marrow stroma is the most recurrent tissue source utilized in obtaining mesenchymal stem cells. In our study the harvesting of sufficient quantity of bone marrow necessitated the sacrifice of the rats but for larger animals these cells can be easily harvested from the same animal by bone marrow aspiration.
Bone marrow MSCs-seeded SIS scaffold was successfully used for bladder regeneration in a canine model (36, 37). In contrast, cell migration into the decellularized aortic scaffolds is inadequate both in vitro (26) and in vivo (38), since the extracellular matrix of aorta has a very tight organization. To address this problem we embedded the MSCs into a collagen-agarose sponge which was then applied on the aortic scaffold. In collagen-agarose 3D matrices, embedded MSCs remain healthy (39). By our innovative method we aimed to obtain a 3D patch with specific properties: biocompatibility, biodegradability, provision of structural integrity, and adequate porosity. The honeycomb structure was chosen for the cell seeding due to its advantage of providing a high-density number of cells since the total area of the sponge is very great (11). By using the collagen-rich scaffolds enriched with MSCs, we intended not only to obtain but also to accelerate the proper regeneration of the digestive wall. With the future intention to develop a new patch for the treatment of digestive fistula in human patients, we aimed to obtain a 3D patch containing in vitro expanded autologous MSCs. The human MSCs can be easily harvested by 15-20 ml bone marrow aspiration from the iliac spina. After delivery into the organism, MSCs need a friendly medium for survival. The lumen of the digestive tube is the most unfamiliar medium for these cells, due to the presence of a high concentration of enzymes, acids, bases, and germs. A possibility to protect the cells of being killed by this unfamiliar medium is to deliver them into a matrix resistant to enzymes, acids, and bases. For this purpose the matrix of collagen-agarose was chosen because collagen is the most resistant biomaterial to the action of digestive secretions (40), and agarose is a polysaccharide inert to the action of enzymes, acids, and bases. Collagen-agarose sponge used for our study had a high porosity and an adequate pore size, which are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. However the use of the matrix of collagen-agarose enriched with MSCs by itself is not of sufficient resistance to cover the digestive wall defect. The use of a more resistant support is necessary till the regeneration of the digestive wall but the biodegradability is also essential since scaffolds need to be absorbed by the surrounding tissues without the necessity of a surgical removal. The rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation. This means that the scaffold is able to provide structural integrity, while cells are fabricating their own natural matrix, and eventually the scaffold will break down, leaving the newly formed tissue to take over the mechanical load. The complete regeneration of the digestive wall and graft degradation might take up to 90 days as it was depicted by different authors regarding the use of different biomaterials (16, 18). In our study the complete regeneration of the digestive wall was observed at 48 days. The relationship of scaffold thickness and size to the rate of its degradation and remodeling remains to be determined.
The patches were used as xenografts due to the origin of aorta wall (swine, human) and/or MSCs (human). Owing to pretreatment decellularization, the aortic segments were immunologically inert. MSCs are considered to be hypo-immunogenic, displaying low expression levels of human leukocyte antigen (HLA) major histocompatibility complex (MHC) class I but no expression of human leukocyte antigen (HLA) class II (31). Although MSCs are known to have a lower expression of HLA class I, the positive immunofluorescence at 48 days for human cells can be explained by the fact that these cells begin to express these antigens along with differentiation into adult cells. In the present study there was no evidence of rejection of the MSCs-enriched patch in rats without immunosuppresion, although previous xenotransplantation studies suggested that human MSCs are not intrinsically immunoprivileged, and may determine rejection in hosts with an intact immune system (41).
In this study we presented our results regarding repair of stomach and intestinal iatrogenic defects using 3D patches with MSCs, by demonstrating the macroscopic and microscopic reconstruction of the digestive wall at the patch site.
The mechanism of action of MSCs in the repair of digestive wall defect has not been elucidated yet. Recent work showed the contribution of MSCs to collagen deposition (42) and epithelization (43). Seeded on an optimized scaffold these cells may guide angiogenesis and tissue healing. The implication of MSCs in the digestive wall repair has been proven in the present study by identifying the xenografted cells in the regenerated digestive wall of the immunocompetent rats.
The application of 3D patch with autologous MSCs might represent a promising solution in solving the refractory digestive fistula.

Conclusions
Our study described isolation, culture and characterization of bone marrow MSCs, and preparation by an innovative procedure of a bioconstruct made of MSCs-enriched collagen-agarose scaffold, associated with a decellularized aortic wall fragment, with the aim of transplantation for the repair of digestive wall defects. Our results showed that MSCs-enriched 3D patches promoted repair of the digestive wall defects with the reconstruction of all four layers. The colagen-agarose scaffold was proven to be biocompatible and biodegradable, and to facilitate MSCs engraftment. The aortic component of the patch was a good mechanical support for the other components and was completely resorbed in the regenerating tissue. Transplanted MSCs clearly improved engraftment of the patch and complete tissue regeneration without the need of immunosuppression.

Acknowledgments
The study was supported by the research grant CEEX 139/2006 from the Romanian Ministry of Education and Research.

References
1. Sitges-Serra, A., Jaurrieta, E., Sitges-Creus, A. - Management of postoperative enterocutaneous fistulas: the roles of parenteral nutrition and surgery. Br. J. Surg., 1982, 69:147.
2. Abdallah, B.M., Kassem, M. - Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther., 2008, 15:109.
3. Shieh SJ, Vacanti JP. - State-of-the-art tissue engineering: From tissue engineering to organ building. Surgery, 2005, 137:1.
4. Langer, R. - Tissue engineering: a new field and its challenges. Pharm. Res., 1997, 14:840.
5. Lu Q, Ganesan K, Simionescu DT, Vyavahare NR. - Novel porous aortic elastin and collagen scaffolds for tissue engineering. Biomaterials, 2004, 25:5227.
6. Harmon, J.W., Wright, J.A., Noel, J., Cogan, M. - Fate of Dacron prostheses in the small bowel of rabbits. Surg. Forum, 1979, 30:365.
7. Chen, M.K., Beierle, E.A. - Animal models for intestinal tissue engineering. Biomaterials, 2004, 25:1675.
8. Thompson, J.S., Kampfe, P.W., Newland, J.R., Vanderhoof, J.A. - Growth of intestinal neomucosa on prosthetic materials. J. Surg. Res., 1986, 41:484.
9. Walles, T., Herden, T., Haverich, A., Mertsching, H. - Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24:1233.
10. Wang, Z.Q., Watanabe, Y., Kagawa, A.T. - Experimental assessment of small intestinal submucosa as a small bowel graft in a rat model. J. Pediatr. Surg., 2003, 38:1596.
11. Itoh, H., Aso, Y., Furuse, M., Noishiki, Y., Miyata, T. - A honeycomb collagen carrier for cell culture as a tissue engineering scaffold. Artif. Organs, 2001, 25:213.
12. Mutter, D., Aprahamian, M., Damge, C., Sonzini, P., Marescaux, J. - Biomaterial supports for colonic wall defect healing. An experimental study in the rat. Biomaterials, 1996, 17:1411.
13. Feng, Z., Yamato, M., Akutsu, T., Nakamura, T., Okano, T., Umezu, M. - Investigation on the mechanical properties of contracted collagen gels as a scaffold for tissue engineering. Artif. Organs, 2003, 27:84.
14. Chen SS, Revoltella RP, Papini S, Michelibi M, Fitzgerald W, Zimmerberg J, Margolis L. - Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems. Stem Cells, 2003, 21:281.
15. Isch JA, Engum SA, Ruble CA, Dacis MM, Grosfeld JL. - Patch esophagoplasty using AlloDerm as a tissue scaffold. J. Pediatr. Surg., 2001, 36:266.
16. Badylak, S., Meurling, S., Chen, M., Spievack, A., Simmons-Byrd, A. - Resorbable bioscaffold for esophageal repair in a dog model. J. Pediatr. Surg., 2000, 35:1097.
17. Demirbilek, S., Kanmaz, T., Özardah, I., Edali, M.N., Yücesan, S. - Using porcine small intestinal submucoasa in intestinal regeneration. Pediatr. Surg. Int., 2003, 19:588.
18. Chen, M.K., Badylak, S.F. - Small bowel tissue engineering using small intestinal submucoasa as a scaffold. J. Surg. Res., 2001, 99:352.
19. Wang, Z.Q., Watanabe, Y., Noda, T., Yoshida, A., Oyama, T., Toki, A. - Morphologic evaluation of regenerated small bowel by small intestinal submucosa. J. Pediatr. Surg., 2005, 40:1898.
20. de la Fuente, S.G., Gottfried, M.R., Lawson, D.C., Harris, M.B., Mantyh, C.R., Pappas, T.N. - Evaluation of porcine-derived small intestine submucosa as a biodegradable graft for gastrointestinal healing. J. Gastrointest. Surg., 2003, 7:96.
21. Nerem, R.M., Seliktar, D. - Vascular tissue engineering. Annu Rev. Biomed. Eng., 2001, 3:225.
22. Kajitani, M., Wadia, Y., Xie, H., Hinds, M.T., Shalaby, S.W., Swartz, K.R., Gregory, K.W. - Use of a new elastin patch and glue for repair of a major duodenal injury. ASAIO J., 2000, 46:409.
23. Schmidt, C.E., Baier, J.M. - Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials 2000; 21:2215-2231.
24. Goldstein, S. - Method of preparing transplant tissue to reduce immunogenicity upon implantation. Cryolife, Inc. (Marietta, GA) (US patent 5613982) 1997.
25. Goldstein, S. - Treated tissue for implantation and methods of preparation. Cryolife, Inc. (Marietta, GA) (US patent 5899936) 1999.
26. Teebken, O.E., Bader, A., Steinhoff, G., Haverich, A. - Tissue engineering of vascular grafts: human cell seeding of decellularized porcine matrix. Eur. J. Vasc. Endovasc. Surg., 2000, 19:381.
27. Grikscheit, T.C., Siddique, A., Ochoa, E.R., Srinivasan, A., Alsberg, E., Hodin, R.A., Vacanti, J.P. - Tissue-engineered small intestine improves recovery after massive small bowel resection. Ann. Surg., 2004, 240:748.
28. Evans, G.S., Flint, N., Somers, A.S., Eyden, B., Potten, C.S. - The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J. Cell Sci., 1992, 101:219.
29. Tait, I.S., Flint, N., Campbell, F.C., Evans, G.C. - Generation of neomucosa in vivo by transplantation of dissociated postnatal small intestinal epithelium. Differentiation, 1994, 56:91.
30. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R. - Multilineage potential of adult human mesenchymal stem cells. Science, 1996, 5411:143.
31. Chamberlain, G., Fox, J., Ashton, B., Middleton, J. - Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells, 2007, 25:2739.
32. Bianco, P., Robey, P.G., Simmons, P.J. - Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell, 2008, 10:313.
33. Komori, M., Tsuji, S., Tsujii, M., Murata, H., Iijima, H., Yasumaru, M., Nishida, T., Irie, T., Kawano, S., Hori, M. - Efficiency of bone marrow-derived cells in regeneration of the stomach after induction of ethanol-induced ulcers in rats. J. Gastroenterol., 2005, 40:591.
34. Sémont, A., François, S., Moiuseddine, M., François, A., Saché, A., Frick, J., Thierry, D., Chapel, A. - Mesenchymal stem cells increase self-renewal of small intestinal epithelium and accelerate structural recovery after radiation injury. Adv. Exp. Med. Biol., 2006, 585:19.
35. García-Olmo, D., García-Arranz, M., Herreros, D., Pascual, I., Peiro, C., Rodríguez-Montes, J.A. - A phase I clinical trial of the treatment of Crohn's fistula by adipose mesenchymal stem cell transplantation. Dis. Colon Rectum, 2005, 48:1416.
36. Zhang, Y., Lin, H.K., Frimberger, D., Epstein, R.B., Kropp, B.P. - Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int., 2005, 96:1120.
37. Chung, S.Y., Krivorov, N.P., Rausei, V., Thomas, L., Frantzen, M., Landsittel, D., Kang, Y.M., Chon, C.H., Ng, C.S., Fuchs, G.J. - Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition. J. Urol., 2005, 174:353.
38. Walles, T., Herden, T., Haverich, A., Mertsching, H. - Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24:1233.
39. Batorsky, A., Liao, J., Lund, A.W., Plopper, G.E., Stegemann, J.P. - Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenviroments. Biotechnol. Bioeng., 2005, 92:492.
40. Aprahamian, M., Damgé, C., Kerr-Conte, J., Mutter, D., Evrard, S., Marescaux, J. - In vitro resistance of artificial connective tissues to human bile and pancreatic juice. Biomaterials, 1992, 13:697.
41. Grinnemo, K.H., MAnsson, A., Dellgren, G., Klingberg, D., Wardell, E., Drvota, V., Tammik, C., Holgersson, J., Ringdén, O., Sylvén, C., Le Blanc, K. - Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J Thorac Cardiovasc. Surg., 2004, 127:1293.
42. Han, S.K., Yoon, T.H., Lee, D.G., Lee, M.A., Kim, W.K. - Potential of human bone marrow stromal cells to accelerate wound healing in vitro. Ann. Plast. Surg., 2005, 55:414.
43. Fu, X., Fang, L., Li, X., Sheng, Z. - Enhanced wound-healing quality with bone marrow mesenchymal stem cells autografting after skin injury. Wound Rep. Reg., 2006, 14:325.

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