The purpose of tissue engineering is the reconstruction of injured tissue through in vitro culture of autologous cells onto a porous scaffold.1 However, the supply of growth factors and nutrients is insufficient in in vitro culture methods; therefore, blood vessels cannot be formed. Scaffold size and culture time are also limitations in the in vitro cell culture method, because the extended cell culture time required to fill the pores of a scaffold can induce changes in the cell characteristics.2 For these reasons, in vivo culture methods such as omentum culture have been studied as alternatives.3–7
The omentum is an apron-like structure comprised of two mesothelial sheets; it also contains many small blood vessels and adipose tissue and is an important producer of vascular endothelial growth factor (VEGF).8–10 The blood vessels aid tissue formation by supplying various nutrients and oxygen difficult to obtain in vitro, but important for protection against necrosis and infection. In addition, because the omentum is located in the abdominal cavity, it has sufficient space to accommodate transplantation and can wrap uncovered transplant material with skin or muscle flap.11 The inflammatory reaction caused by omentum-transplanted artificial material stimulates angiogenesis and is a very important factor for cell survival and proliferation and tissue layer formation.12,13 The resulting tissue layer has density and hardness that allow it to maintain stability and prevent air or liquid leakage. This property is also very useful for the reconstruction of other internal organs that contain thick and/or ridged tissue. Therefore, tissue- engineered reconstruction studies of internal organs such as esophagus, spleen, intestine, bladder, and trachea have been performed with the omentum culture method.14–21 In our previous tissue engineering study using omentum, we identified that cells cultured on porous scaffold can be transplanted and survive on omentum.22 However, no analyses have been conducted to date concerning the variables associated with the use of omentum and the distribution of remaining transplanted cells. We and other previous studies concerned omentum as a bioreactor and its ability to form blood vessels and prevent infection.23–28 They not only include the omentum transplantation method, the transplanted cell status, and influences of omentum on scaffold, all of which are essential for artificial tissue formation by the tissue-engineered method on omentum but also can affect organ reconstruction.
In this study, we investigated these factors using the artificial trachea model. The trachea contains many blood vessels and hard tissue, is air tight, and has flexible conduit structure and large size. Therefore, because the status of newly formed tissue on omentum influences surgical procedures and reconstruction results, the trachea is very appropriate for the study of tissue-engineered reconstruction and transplantation using omentum. We analyzed the effectiveness of omentum usage from the perspectives of both cell culture and transplantation method and have made suggestions regarding how the omentum can be used effectively in future studies.
Materials and Methods
This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Samsung Biomedical Research Institute (SBRI). Samsung Biomedical Research Institute is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility and abide by the Institute of Laboratory Animal Resources (ILAR) guide.
In this study, we used 15 mongrel dogs and obtained autologous keratinocyte from them. They were individually housed and maintained at an environmental temperature of 20°C–24°C and on a 50/50 light/dark cycle. They were fed canine diet (Purina Korea Co., Korea) with automatic watering. All animals received humane care and were killed after experiments.
Preparation of PLGA Scaffold Prosthesis
Ten percent poly(lactic-co-glycolic acid) (PLGA)-gelatin solution was made by mixing 1 g PLGA particle (85:15 d,l-lactide-co-glycolide polymer, Medisorb, MW 110,000 Alkermes, OH) and 10 ml chloroform (Spectrometric grade, Sigma) and 14 g gelatin powder (Type B gelatin from bovine skin, MW 150,000–250,000, Sigma; St. Louis, MO). The gelatin power was sieved with 100–200 μm pore sized sieve before mixing. Seven polypropylene lines (9-cm length, 1-mm high, and 1-mm thick) were attached to a 6 × 10 cm piece of prolene mesh (polypropylene, nonabsorbable synthetic surgical mesh; Ethicon Co., Somerville, NJ) by heating at 7-mm intervals. The PLGA-gelatin solution was poured onto the mesh and dried for a day at room temperature, and then gelatin was dissolved in water for 2–3 days at 37°C. The resulting porous PLGA layer had a thickness of <0.5 mm.
Pieces of canine skin tissue, 4 × 5 cm in area, were obtained and washed three times with PBS (pH 7.4, without Ca2+ or Mg2+; BioWhitaker, Walkersville, MD). Subcutaneous tissue was removed with scissors, and the tissue samples were then trimmed to 1 × 1 cm2. One hundred milligrams of dispase II (1.20 U/mg; Gibco, Carlsbad, CA) was dissolved in 50 ml PBS and filtered through a 0.22-μm syringe filter (Millipore, Billerica, MA). The skin slices were immersed in this dispase II solution at 37°C for 5 hours to separate the epidermal layer. To obtain single cells, the epidermal layers were peeled from the skin slices and minced, incubated in trypsin for 10 minutes at 37°C, and then strained through a 100-μm cell strainer (BD Falcon, Franklin Lakes, NJ). Cells were centrifuged at 1,300g for 3 minutes and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and antibiotics. The medium was changed every 4 days.
Cell Labeling and Culture on PS Prosthesis
Skin epithelial cells were trypsinized from cell culture dishes and resuspended in 1 ml of diluent C solution. A 10 μl aliquot of PKH26 dye (PKH26GL-1KT; Sigma) was added to the cells and incubated for 5 minutes; thereafter, the cells were washed twice with serum-containing DMEM medium and centrifuged at 1,300g for 3 minutes. Approximately, 3 × 107 skin epithelial cells were resuspended in 0.5-ml culture medium and seeded onto a PS prosthesis by dropping technique. To increase cell adhesion, the PS prosthesis was treated with culture medium for 1 day before cell seeding. After 3 hours of cell attachment, culture medium was added, and the cells were cultured for 3 additional days in static condition.
Transplantation to Omentum and Trachea
An incision was made into the canine abdomen, and a PS prosthesis was placed onto the omentum. The prosthesis and omentum were sutured together and wrapped with polyurethane film to prevent adhesion between the upper layer of the PS prosthesis and surrounding tissue. After 1 week, the canine abdomen was opened again and status of PS prosthesis was identified. It could be transplanted on trachea; the diaphragm was penetrated and cervical skin near the trachea was incised. Poly(lactic-co-glycolic acid) scaffold (PS) prosthesis was moved to trachea from abdomen to diaphragm; 5-cm long part of the trachea was resected, and the PS prosthesis was transplanted onto the trachea by end to end anastomosis method. The surgical procedure is illustrated in Figures 1 and 2. After 1 month, the canines were killed. In the event of a short omentum, the PS prosthesis-omentum tissue was cut from the omentum and then sutured onto the trachea by end to end anastomosis method (Figure 3).
Observation of Material and Trachea
The PS prosthesis and labeled cells were observed by using fluorescence microscopy and a VersaDoc image analyzer (MP4000 Imaging System; Bio-Rad Laboratories, Hercules, CA). After 1 week omentum transplantation, transplantation status was identified through observation for external appearance and tissue section of artificial tissue. Cell distribution was observed on surface and section of artificial tissue with fluorescence microscopy. Animals were killed after 1 month, the transplanted tissue was removed from the trachea, and trachea lumen and section of specimens were observed. Also, relationships between omentum transplantation results and trachea reconstruction status were compared and estimated through appearance.
Poly(lactic-co-glycolic acid) scaffold prostheses were <1.5-mm thick and contained a thin porous PLGA layer (Figure 4A). PKH26-labeled cells (Figure 4, B and C) could be distinguished from nonlabeled cells by the presence of a strong red fluorescence on the PS prosthesis, in contrast to the PLGA scaffold, which emitted a weak red fluorescence (Figure 4D). One week after the transplantation of PS prostheses into omentum, transplantation was impossible in 5 of the 15 cases, and possible in the remaining 10 cases (Figures 5 and 6). The causes of failure were serious omentum-peritoneum adhesion (n = 1), short omentum length (n = 2), tissue formation failure (n = 1), and omentum-scaffold folding (n = 1; Figure 5). In the successful cases, the scaffold-omentum structure had fresh tissue and proper thickness, but the margin of the PS prosthesis was generally much thicker than the middle of the structure (Figure 6). The transplanted cells were scattered above and below the polypropylene line and mesh of the PS prosthesis, and they did not form an even cell layer (Figures 7 and 8). Many small blood vessels were formed around the polypropylene mesh (Figure 9).
One month after transplantation to the trachea, three different results were observed. In the case of transplantation without an omentum flap (short omentum length), PS prosthesis-omentum tissue contained a thick fibroblast layer and many small blood vessels, but trachea reconstruction was failed (Figure 10A). In 2 of the 10 successive cases, the formation of some thick tissue led to a narrow luminal diameter (Figure 10B). In the case of transplantation with an omentum flap, all tracheas (n = 10) had a normal inner diameter and stable surface (Figure 10C). The success rate of omentum transplantation was from 53% to 66%, which was low, but once proper tissue was made on omentum, success rate of trachea transplantation was 100%. The gross status of omentum and trachea transplantation and success rate are shown in Table 1.
In this study, we considered the omentum as a tool for both cell culture and transplantation, with the ultimate aim of engineering artificial organs. Therefore, different from other studies, we focused on influences of omentum usage for tissue-engineered cell transplantation using scaffold and transplantation method. PKH26-labeled cells were seeded evenly over the entire scaffold, but most of them remained localized to seeding points. One week after transplantation to the omentum, we observed transplanted cells scattered on and under the PS prosthesis; these cells did not form a stable and even layer. Therefore, it is assumed that many transplanted cells moved out from scaffold by leachate and intrusion of other kinds of cells. This result is inevitable.29 Because the formation of an even and stable cell layer with a specific function is crucial for successful tissue engineering, the porous scaffold used in this study is not appropriate for transplantation with omentum. Another type of material can be used for the effective use of omentum for cell culture and cell layer formation. Cell sheets have been shown to be helpful in increasing transplant efficiency.30,31 Natural materials such as small intestinal submucosa (SIS) are also very useful for making even and stable cell layers. In bladder reconstruction studies, even and stable keratinocyte layers were formed on SIS membrane.32,33 These studies achieved a stable tissue formation composed with transplanted cells on omentum.
We observed four different types of failed omentum transplantation. The first was an adhesion between peritoneum and omentum, which occurred even though the omentum and PS prosthesis were wrapped with polyurethane film. This problem can be easily solved, but severe adhesion can substantially increase the difficulty of the surgical procedure. The second was a shortening of the omentum length after transplantation. This occurrence was difficult to predict and regarded as failure, but the newly formed tissue had proper shape and then could be used for trachea reconstruction. However, in this study, such reconstructions were ultimately failed. This failure might be caused by the failure of blood vessel connection between the newly formed tissue and trachea or the decay of transplanted tissue on exposure to air. The third was a failure of tissue formation, despite omentum attachment and same procedure. We do not know the cause of this. The final type of omentum transplantation failure observed was omentum scaffold folding that occurred despite wrapping omentum scaffold structure and suturing at several points. Therefore, a more rigid cast should be applied in future studies, although this is associated with concerns regarding increased pain. In two cases of successful omentum transplantation, the narrowing of the luminal diameter was observed after trachea transplantation but not fatal. This was caused by the formation of some thick tissue layer on the omentum. Tissue layer formation is effective and important for air and liquid impermeability, tissue strength, and blood vessel formation.14 However, very thick tissue can limit tissue motion and cause structural and functional damage, for example, stenosis in trachea reconstruction. These results meant that successful omentum transplantation could guarantee successful trachea transplantation.
The omentum has the ability to form blood vessels and fibroblast layer, and it supplies oxygen and various nutrients. Although omentum is useful for tissue engineering using cells, some problems and a considerable associated risk obstruct tissue-engineered use of omentum. Therefore, we suggest three guidelines for the effective application of omentum to tissue engineering. First, formation of an even and stable cell layer composed exclusively of transplanted cells without the intrusion of other cell types has to be guaranteed. Second, transplantation method that maintains the shape of the transplant and confirms blood vessel connection has to be applied. Finally, the newly formed tissue layer must have a consistent thickness, adequate flexibility, and a sufficient dense structure.
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund; KRF-2006-311-D01014) and a Ministry of Health and Welfare (0405-MNO1-0604-0007) of Korea.
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