Tissue-engineered small intestine: ontogeny of the immune system

BACKGROUND: Using tissue-engineering techniques, we have developed a that regenerates structural and transporter properties of native jejunum. The purpose of this study was to characterize the mucosal immune system of the engineered neointestine. We hypothesized that the neointestinal mucosa is capable of developing a mature immunocyte population and that exposure to luminal stimuli is critical to this development. METHODS: Neointestinal cysts were engineered by implanting polymer-organoid constructs into syngeneic adult recipients. Neointestine (cysts left nonanastomosed [NA] and cysts anastomosed to native bowel [AN]) and native jejunum were harvested serially (3-56 weeks postoperatively). Immune cell subsets were characterized by the immunohistochemical detection of cell-specific antigens (T cells [CD3], B cells [CD32], NK cells [CD56], and macrophages [CD68]) combined with computer-based morphometry. RESULTS: Intraepithelial and lamina propria immunocyte population densities and subset distributions were identical in AN cysts harvested 20 weeks postoperatively and in native jejunum. Mucosal immunocyte population densities were lower in AN cysts harvested 10 weeks postoperatively and only rudimentary in NA cysts, even those harvested 20 weeks postoperatively. CONCLUSIONS: These results suggest that tissue-engineered intestine has the capacity to develop a mucosal immune system with an immunocyte population similar to that of native small intestine. The development of this immune system is a function of both exposure to luminal stimuli and the duration of this exposure. Tissue-engineered intestine offers promise as a new therapeutic approach for patients who have intestinal insufficiency.     

A novel model for simultaneous study of neointestinal regeneration and intestinal adaptation

The use of autologous grafts, fabricated from tissue‐engineered neointestine, to enhance insufficient compensation of intestinal adaptation for severe short bowel syndrome is a compelling idea. Unfortunately, current approaches and knowledge for neointestinal regeneration, unlike intestinal adaptation, are still unsatisfactory. Thus, we have designed a novel model of intestinal adaptation with simultaneous neointestinal regeneration and evaluated its feasibility for future basic research and clinical application. Fifty male Sprague‐Dawley rats weighing 250–350 g underwent this procedure and sacrificed at 4, 8, and 12 weeks postoperatively. Spatiotemporal analyses were carried out by gross, histology, and DNA/protein quantification. Three rats died of operative complications. In early experiments, the use of hard silicone stent as tissue scaffold in 11 rats was unsatisfactory for neointestinal regeneration. In later experiments, when a soft silastic tube was used, the success rate increased up to 90.9%. Further analyses revealed that no neointestine developed without donor intestine; regenerated lengths of mucosa and muscle were positively related to time postsurgery but independent of donor length with 0.5 or 1 cm. Other parameters of neointestinal regeneration or intestinal adaptation showed no relationship to both time postsurgery and donor length. In conclusion, this is a potentially important model for investigators searching for solutions to short bowel syndrome.     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Endocrine cell and nerve regeneration in autologous in situ tissue-engineered small intestine

BACKGROUND: The purpose of this study was to regenerate a larger size of small intestinal tissue than that of our previous study and to evaluate the regeneration of the endocrine cells (ECC) and nerve system of autologous tissue-engineered small intestine. The effect of implantation of large numbers of smooth muscle cells (SMC) for the regeneration of small intestine was also investigated. METHODS: Two types of scaffolds with different cell densities were fabricated: low density (LD) of SMC in the scaffold and high density (HD) of SMC in the scaffold. Both scaffolds were implanted into defects of isolated ileum in a canine model. Animals were sacrificed at 8, 12, 18, and 24 weeks. RESULTS: The area of engineered small intestine in the HD group was four times larger than that in the LD group, although that was smaller in size than the original size of the defect. There were no significant changes in the thickness of regenerated smooth muscle layer (SML) in the LD and HD groups. The numbers of endocrine cells gradually increased after implantation. At 18 weeks of regeneration, the number of ECC reached levels comparable to that of normal mucosa. The nerve fibers extended to the center of the graft area and were observed in regenerated SML and regenerated villi at 24 weeks. CONCLUSIONS: The ECC and nerve fibers were regenerated in autologous in situ tissue-engineered small intestine. Seeding a large number of SMC was not sufficient for the regeneration of the small intestine in a tubular configuration.     

Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine

BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer tubes. This study investigated methods of stimulation for optimizing neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes and implanted into the omentum of adult Lewis rats in the following groups: (1) the control group (group C), implantation alone (n=9); (2) the small bowel resection (SBr) group, after 75% SBr (n=9); (3) the portacaval shunt (PCS) group, after PCS (n=8); and (4) the partial hepatectomy (PH) group, after 75% PH (n=8). Neointestinal cyst size was recorded using ultrasonography. Constructs were harvested at 10 weeks and were examined using histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program (NIH Image, version 1.59). RESULTS: Cyst development was noted in all animals. Cyst lengths and diameters were significantly larger in the SBr group at 7 and 10 weeks compared with the other three groups (P<0.05; analysis of variance [ANOVA], Fisher's protected least significant difference). Histology revealed a well-vascularized tissue with a neomucosa lining the lumen with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated a significantly greater villus number, height, area, and mucosal surface in the SBr group compared with the other three groups and a significantly greater crypt number and area in the PCS group compared with group C (P<0.05; ANOVA, Fisher's protected least significant difference). CONCLUSIONS: Intestinal epithelial organoid units transplanted on porous biodegradable polymer tubes can successfully vascularize, survive, and regenerate into complex tissue resembling small intestine. SBr and, to a lesser extent, PCS provide significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered small intestine.     

Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel

BACKGROUND: Our laboratory has investigated the fabrication of a tissue-engineered intestine using biodegradable polymer scaffolds. Previously we reported that isolated intestinal epithelial organoid units on biodegradable polymer scaffolds formed cysts and the neointestine was successfully anastomosed to the native small bowel. The purpose of this study was to observe the development of tissue-engineered intestine after anastomosis and to demonstrate the effect of the anastomosis over a 9-month period. METHODS: Microporous biodegradable polymer tubes were created from polyglycolic acid. Intestinal epithelial organoid units were harvested from neonatal Lewis rats and seeded onto the polymers, which were implanted into the abdominal cavity of adult male Lewis rats followed by 75% small bowel resection (n=24). Three weeks after implantation, the unit/polymer constructs were anastomosed to the native jejunum in a side-to-side fashion. The anastomosed tissue-engineered intestine was measured by laparotomy 10, 24, and 36 weeks after the implantation (n= 14). During the laparotomy, all rats with an obstruction in their anastomosis were killed and excluded from the statistical analysis. Another five rats were also killed at 10 and 36 weeks for histological and morphometric studies. RESULTS: All analyzed rats survived this study and significantly increased their body weight by 36 weeks. Obstruction of the anastomosis was observed in one rat at 24 weeks and in two rats at 36 weeks; however, the anastomosis was patent in the other 11 rats by 36 weeks. The tissue-engineered intestine of these 11 rats increased in length and diameter at 10, 24, and 36 weeks after anastomosis; there were statistically significant differences between each time point except between the length of 10 and 24 weeks (P<0.016 by Wilcoxon signed rank test). Histologically the inner surface of the tissue-engineered intestine was lined with well-developed neomucosa at 10 and 36 weeks; however, there were small bare areas lacking neomucosa in the tissue-engineered intestine at 36 weeks. Morphometric analysis demonstrated no significant differences in villus number, villus height, and surface length of the neomucosa at 10 and 36 weeks. CONCLUSIONS: Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after operation and maintained a high patency rate for up to 36 weeks. The tissue-engineered intestine increased in size and was lined with well-developed neomucosa for the duration of the study.     

Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture

OBJECTIVE: Novel production and in vitro characterization of tissue engineered colon. SUMMARY BACKGROUND DATA: The colon provides important functions of short chain fatty acid production, sodium and water absorption, and storage. We report the first instance of tissue-engineered colon (TEC) production from autologous cells and its in vitro characterization. METHODS: Organoid units, mesenchymal cell cores surrounded by a polarized epithelia derived from full thickness sigmoid colon dissection from neonatal Lewis rats, adult rats, and tissue engineered colon itself, were implanted on a polymer scaffold into the omentum of syngeneic hosts. TEC was either anastomosed at 4 weeks or excised for Ussing chamber studies or histology, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay. RESULTS: TEC was generated by 100% of all animals without regard to tissue source, the first instance of engineered intestine from adult cells or an engineered tissue. TEC architecture is identical to native with muscularis propria staining for actin, acetylcholinesterase detected in a linear distribution in the lamina propria, S100-positive cells, ganglion cells, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay similar to native colon. Ussing chamber data indicated in vitro function consistent with mature colonocytes, and a positive short circuit current response to theophylline indicating intact ion transfer. TEM showed normal microarchitecture. Colon architecture was maintained in anastomosis with gross visualization of fluid uptake. CONCLUSIONS: TEC can be successfully produced with fidelity to native architecture and in vitro function from neonatal syngeneic tissue, adult tissue, and TEC itself.     

Effects of copper and cross-linking on the extracellular matrix of tissue-engineered arteries

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix. but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.     

Effects of copper and cross-linking on the extracellular matrix of tissue-engineered arteries

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.     

Tissue-engineered neomucosa: morphology,enterocyte dynamics,and SGLT1 expression topography

BACKGROUND: The standard therapy for short bowel syndrome is total parenteral nutrition, which is expensive and associated with significant morbidity and mortality. New therapeutic approaches for this disorder are needed. We have applied the techniques of tissue engineering to develop a prototype neointestine. We hypothesized that anastomosis of this neointestine to the native bowel would result in regeneration of mucosal morphology and enterocyte dynamics. METHODS: Biodegradable polymers seeded with neonatal rat intestinal organoid units were implanted into the omenta of adult rats to form neointestinal cysts. Five weeks after implantation, side-to-side cyst-jejunal anastomoses were fashioned in one cohort of rats. Tissues were harvested from all rats at 5 months after implantation. Native jejunal (J) and non-anastomosed (N-N) and anastomosed (A-N) neointestinal tissues were assessed for morphology, epithelial cell proliferation (5-bromo-2-deoxyuridine immunohistochemistry), apoptotic rates (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay), and SGLT1 in situ hybridization. RESULTS: Mucosal morphology, rates and topography of enterocyte proliferation, and transporter expression in A-N neointestine recapitulated those of native jejunum. Each of these features was rudimentary in N-N neointestine. CONCLUSIONS: These results suggest that the tissue-engineered neomucosa can develop structural and dynamic features of the normal jejunum. Anastomosis to the native intestine is an essential step for neomucosal development. Tissue engineering offers promise as a novel approach to the treatment of patients suffering from short bowel syndrome.     

Tumor lymphangiogenesis in transitional cell carcinoma of the upper urinary tract: association with clinicopathological features and prognosis

PURPOSE: Lymph node metastasis is an important prognostic factor in many types of cancer. Recently several specific markers for lymphatic endothelium were developed that facilitate the quantification of lymphangiogenesis in human cancer tissues. We investigated the clinical and prognostic significance of lymphangiogenesis in patients with transitional cell carcinoma of the upper urinary tract. MATERIALS AND METHODS: We measured lymph vessel density and relative lymphatic vascular area in 125 specimens by quantitative immunohistochemical staining for D2-40 antibody (DakoCytomation, Glostrup, Denmark). These parameters were examined in the intratumor and peritumor areas, and measured using image analysis software. RESULTS: Peritumor lymph vessel density and peritumor lymphatic vascular area correlated with lymph node metastasis and tumor grade. In the intratumor area lymphatic vessels were detected in only 16.0% of specimens. However, the presence of intratumor lymphatic vessels was associated with lymph node metastasis (p = 0.002). Multivariate analysis identified high peritumor lymphatic vascular area and the presence of intratumor lymphatic vessels as significant and independent factors of metastasis-free survival after surgery (OR = 5.11, p = 0.020 and OR = 2.92, p = 0.025, respectively). Multivariate analysis also identified the presence of intratumor lymphatic vessels as the only independent predictive factor of cause specific survival (OR = 3.89, p = 0.049). CONCLUSIONS: Lymphangiogenesis may have important roles in tumor metastasis and survival in patients with transitional cell carcinoma of the upper urinary tract. Quantification of lymphatic vessels, especially peritumor lymphatic vascular area and intratumor lymphatic vessels, was useful for predicting metastasis-free survival. In addition, the presence of intratumor lymphatic vessels was an independent predictor of cause specific survival.     

Successful anastomosis between tissue-engineered intestine and native small bowel

BACKGROUND: Previous work from this laboratory has shown that isolated intestinal epithelial organoid units on porous biodegradable polymer scaffolds formed vascularized cysts lined by a neomucosa. The purpose of this study was to demonstrate anastomosis between tissue-engineered intestine and the native small bowel and to observe the effect of this anastomosis on cyst growth. METHODS: Intestinal epithelial organoid units from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes made of polyglycolic acid, and they were implanted into the omentum of adult male Lewis rats. Three weeks after implantation, the unit-polymer constructs were anastomosed in a side-to-side fashion to the native jejunum in 20 rats (group 1). The other 18 rats were closed without anastomosis (group 2). All 38 tissue-engineered constructs were harvested 10 weeks after implantation. Four rats underwent upper gastrointestinal (GI) study before they were killed. RESULTS: The rats in group 1 increased their body weights equal to those in group 2, and there was no statistically significant difference between the two groups. Upper GI examinations revealed no evidence of either bowel stenosis or obstruction at the anastomotic site. Grossly, the patency of the anastomosis was 90% and the lumen of the cyst was visualized by the upper GI study. At the second operation, there was no significant difference in the size of the cysts in either group: however, at the time the rats were killed, the length of the cysts in group 1 was significantly longer than that in group 2 (P<0.05 using Mann-Whitney U test). Histological examination showed that cysts after anastomosis were lined by a neomucosa in continuity to native small bowel across the anastomotic site and also demonstrated crypt-villus structures. Morphometric study demonstrated that cysts in group 1 had significantly greater villus number, height, and surface length than did those in group 2. CONCLUSIONS: Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after the operation, kept a high patency rate, and maintained mucosal continuity between the tissue-engineered intestine and native small bowel. Furthermore, anastomosis had a positive effect on cyst size and development of the mucosa in the tissue-engineered intestine.     

改良小鼠后肢淋巴水肿模型的构建

目的通过调整辐射剂量和手术时机,构建一种更为稳定和持久的小鼠后肢淋巴水肿模型。方法将36只8周龄雌性C57小鼠随机分为3组:术前放疗组(R+S)、术后放射组(S+R)、术前及术后放射组(r+S+r)。采用淋巴结摘除手术联合局部放射的方法进行模型构建。R+S组于术前3 d接受4.5 Gy的局部放射,S+R组于术后2周接受4.5 Gy的局部放射,r+S+r组于术前3 d和术后2周分别接受2.25 Gy的局部放射。术后采用排水法测量动物后肢体积变化。术后24周时在后肢注射FDNG以显示体内淋巴管的分布,并结合免疫组化计数微淋巴管数目来评价淋巴管新生的情况。结果在我们为期6个月的观察中,所有小鼠均成功形成淋巴水肿。尤以r+S+r组形成的淋巴水肿最为明显、稳定持久,且死亡率和并发症率较低。术后24周的淋巴管造影显示,R+S及S+R组后肢浅表淋巴管密度增加,r+S+r组少有淋巴管再生。免疫组化结果显示,r+S+r组的淋巴管数量要少于R+S及S+R组(P<0.05)。结论在术前3 d及术后2周分别接受2.25 Gy的局部放射,可以成功构建稳定持久的小鼠后肢淋巴水肿模型。     

冻存对组织工程骨膜生物活性的影响

目的 观察冻存复苏过程对组织工程骨膜生物学特性的影响.方法 体外培养新西兰大白兔骨髓间充质干细胞(BMSCs),与猪小肠黏膜下层(SIS)构建组织工程骨膜.冻存后复苏培养,扫描电镜(SEM)观查复苏后种子细胞生长状况、噻唑蓝(MTT)比色法绘生长曲线、钙-钴法检测碱性磷酸酶(ALP)、生物化学法定量分析ALP的表达.结果 冻存复苏后BMSCs仍能在SIS上保持良好的生长状况;组织工程骨膜复苏后成骨诱导培养5d时ALP含量达峰值,10～15d时稳定在较低的量,与对照组比较差异有统计学意义(P<0.05). 结论组织工程骨膜冻存复苏后仍保持较稳定的生物活性.     

Decellularized native and engineered arterial scaffolds for transplantation

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use. In contrast, decellularized tissue-engineered arteries could serve as grafts for immediate implantation, as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. The goal of this study was to quantify the effects of decellularization on vascular matrix and mechanical properties. Specifically, we compared cellular elimination, extracellular matrix retention, and mechanical characteristics of porcine carotid arteries before and after treatment with three decellularization methods. In addition, for the first time, tissue-engineered arteries were decellularized. Decellularized native arteries were also used as a scaffold onto which vascular cells were seeded. These studies identified a decellularization method for native and engineered arteries that maximized cellular elimination, without greatly compromising mechanical integrity. We showed that engineered tissues could be decellularized, and demonstrated the feasibility of reseeding decellularized vessels with vascular cells.     

A Model of Tissue-Engineered Ventral Hernia Repair

We have developed a tissue-engineered ventral hernia repair system using our novel aligned collagen tube and autologous skeletal muscle satellite cells. In this model system, skeletal muscle satellite cells were isolated from a biopsy, expanded in culture, and incorporated into our collagen tube scaffold, forming the tissue-engineered construct. We characterized the results of the repaired hernias on both the gross and microscopic scales and compared them to an unrepaired control, an autologous muscle repair control, and a collagen-tube-only repair. Untreated animals developed a classic hernia sac, devoid of abdominal muscle and covered only with a thin layer of mesothelial tissue. Significant muscle, small-diameter blood vessels, and connective tissue were apparent in both the autologous control and the engineered muscle repairs. The engineered muscle repairs became cellularized, vascularized, and integrated with the native tissue, hence becoming a “living” repair. A tissue-engineered construct repair of ventral hernias with subsequent incorporation and vascularization could provide the ultimate in anterior wall myofascial defect repair and would further the understanding of striated muscle engineering. The knowledge gained from our model system would have immediate application to mangled extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.     

Tissue-engineered trachea from sheep marrow stromal cells with transforming growth factor beta2 released from biodegradable microspheres in a nude rat recipient

OBJECTIVE: The purpose of this study was to evaluate the feasibility of using autologous sheep marrow stromal cells cultured onto polyglycolic acid mesh to develop helical engineered cartilage equivalents for a functional tracheal replacement. We also explored the potential benefit of local delivery of transforming growth factor beta 2 with biodegradable gelatin microspheres. METHODS: Bone marrow was obtained by iliac crest aspiration from 6-month-old sheep and cultured in monolayer for 2 weeks. At confluence, the cells were seeded onto nonwoven polyglycolic acid fiber mesh and cultured in vitro with transforming growth factor beta 2 and insulin-like growth factor 1 for 1 week. Cell-polymer constructs were wrapped around a silicone helical template. Constructs were then coated with microspheres incorporating 0.5 microg transforming growth factor beta 2. The cell-polymer-microsphere structures were then implanted into a nude rat. On removal, glycosaminoglycan content and hydroxyproline were analyzed in both native and tissue-engineered trachea. Histologic sections of both native and tissue-engineered trachea were stained with hematoxylin and eosin, safranin-O, and a monoclonal anti-type II collagen antibody. RESULTS: Cell-polymer constructs with transforming growth factor beta 2 microspheres formed stiff cartilage de novo in the shape of a helix after 6 weeks. Control constructs lacking transforming growth factor beta 2 microspheres appeared to be much stiffer than typical cartilage, with an apparently mineralized matrix. Tissue-engineered trachea was similar to normal trachea. Histologic data showed the presence of mature cartilage. Glycosaminoglycan and hydroxyproline contents were also similar to native cartilage levels. CONCLUSIONS: This study demonstrates the feasibility of engineering tracheas with sheep marrow stromal cells as a cell source. Engineering the tracheal equivalents with supplemental transforming growth factor beta 2 seemed to have a positive effect on retaining a cartilaginous phenotype in the newly forming tissue.     

A model of tissue-engineered ventral hernia repair.

We have developed a tissue-engineered ventral hernia repair system using our novel aligned collagen tube and autologous skeletal muscle satellite cells. In this model system, skeletal muscle satellite cells were isolated from a biopsy, expanded in culture, and incorporated into our collagen tube scaffold, forming the tissue-engineered construct. We characterized the results of the repaired hernias on both the gross and microscopic scales and compared them to an unrepaired control, an autologous muscle repair control, and a collagen-tube-only repair. Untreated animals developed a classic hernia sac, devoid of abdominal muscle and covered only with a thin layer of mesothelial tissue. Significant muscle, small-diameter blood vessels, and connective tissue were apparent in both the autologous control and the engineered muscle repairs. The engineered muscle repairs became cellularized, vascularized, and integrated with the native tissue, hence becoming a "living" repair. A tissue-engineered construct repair of ventral hernias with subsequent incorporation and vascularization could provide the ultimate in anterior wall myofascial defect repair and would further the understanding of striated muscle engineering. The knowledge gained from our model system would have immediate application to mangled extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.     

毛囊单位促进组织工程皮肤形成的体外实验研究

目的 通过插入分离的毛囊单位,构建带有皮肤附属器的组织工程皮肤,探讨毛囊对组织工程皮肤形成的作用.方法 将头皮分离成毛囊单位,插入由Ⅰ型鼠尾胶原、成纤维细胞和角质形成细胞构建的组织工程皮肤模型中,浸没培养1周,气-液界面培养3周.通过镜下观察、HE染色和免疫组化检测组织工程皮肤和毛囊的生长状态.结果 相对于悬浮培养的毛囊,实验组毛囊体外生长期延长,结构更完整.HE染色显示,实验组组织工程皮肤可见毛囊和皮脂腺存在.免疫组化染色显示,带有毛囊的组织工程皮肤中可见反映基底膜完整性的Laminin和Ⅳ型胶原呈线状连续分布于表皮、真皮连接处;而反应表皮成熟度的CK4和CK10/13则呈阳性分布于基底上层非角化细胞.结论 复合毛囊单位可以促进组织工程皮肤表皮组织的分化和成熟.本方法为组织工程皮肤的构建和毛囊的体外培养提供了新的思路和方法.