Terminal urothelium differentiation of engineered neoureter after in vivo maturation in the "omental bioreactor"

OBJECTIVE: Long ureteric defects may theoretically be repaired with the use of tissue-engineered neoureter. However, attempts to construct such a neoureter in animal models have failed because of major inflammatory response. Avoidance of such inflammation requires a well-differentiated urothelium. We investigated whether omental maturation of a seeded construct in a pig model could achieve terminal differentiation of the urothelium to allow construction of a stricture-free neoureter. MATERIAL AND METHOD: Bladder biopsies were taken to allow urothelial and smooth muscle cell cultures. These cultured cells were used to seed small intestinal submucosa (SIS) matrix. After 2 wk of cell growth, the in vitro SIS-seeded construct was shaped around a silicone drain and wrapped by the omentum to obtain neoureters. These neoureters were left in the omentum without any contact with urine, and then harvested 3 wk later for histologic and immunohistochemical studies. RESULTS: Before implantation, the in vitro constructs were composed of a mono- or bilayer of undifferentiated urothelium overlying a monolayer of smooth muscle cells. After 3 wk of omental maturation, these constructs were vascularized and comprised a terminally differentiated multilayered urothelium with umbrella cells over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. CONCLUSION: We obtained, for the first time, with this model of in vivo maturation in the omentum, a mature neoureter composed of a well-differentiated multilayered urothelium.     

Development of a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for bladder tissue engineering

OBJECTIVES: Tissue engineering is very promising in bladder reconstruction. However, one of the main problems is to limit the development of ischaemic fibrosis during tissue maturation. We describe a model using the omentum as an in vivo bioreactor for a previously seeded scaffold. METHODS: Bladder biopsies were taken from five female pigs, from which both urothelial and smooth muscle cells cultures were made. These cultured cells were used to seed a sphere-shaped small intestinal submucosa (SIS) matrix, which was transferred into the omentum after 3 wk of cell growth. The grafts were harvested 3 wk later and histologic, immunohistochemical, and functional studies were performed. RESULTS: We obtained a highly vascularized tissue-engineered construct that contracted in response to acetylcholine stimulation. The wall thickness was 4mm, on average. Histologic and immunostaining analysis of the construct confirmed the presence of a multilayer urothelium on the luminal aspect and deeper fascicles organised tissue composed of differentiated smooth muscle cells and mature fibroblasts without evidence of inflammation or necrosis. Large- and small-diameter vessels were clearly identified histologically in the tissue obtained. CONCLUSION: The omentum permitted in vivo maturation of seeded scaffolds with the development of a dense vascularisation that is anticipated to prevent fibrosis and loss of contractility. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.     

Bioartifizielles autologes Urothel etabliert aus Spülungen der Harnblase

Reconstructive surgery of lower urinary tract disorders can be limited by a shortage of adequate autologous tissue. Tissue engineering is an option for surgical reconstruction with evolved biological substitutes. Urethral repair with bioartificial urothelial implants can be an innovative method for sustained urothelial regeneration in situ. The needed urothelial cells are commonly isolated from native urothelium requiring surgery.The aim of this study was to establish primary human urothelial cell cultures from bladder washings in serum-free media and to generate urothelial tissue without seeding of matrices in a feeder cell-free system. It could be demonstrated that under these conditions bioartificial urothelium can be developed successfully from bladder washings. Its multilayered cellular structure and the initial differentiation in vitro, similar to native-grown urothelial tissue, are promising with regard to intended clinical application. Current work focuses on establishing cell culture techniques according to legal regulations, terminal differentiation of the urothelial constructs in vitro, and techniques to surgically implant lab-grown bioartificial urothelium.     

Porcine small intestine submucosa as a flexor tendon graft

An attractive strategy for tendon tissue engineering is the use of natural extracellular matrices as scaffold materials. One matrix that has been shown to promote healing and regeneration of neotissue in various applications is porcine-derived small intestinal submucosa. It was the objective of this study to investigate small intestinal submucosa for intrasynovial flexor tendon grafting in a canine model. We hypothesized that at 6 weeks small intestinal submucosa grafts would undergo host cell infiltration, neovascularization, and replacement by host neotendon. We also hypothesized that small intestinal submucosa grafts would be incorporated by the host without extensive adhesions to surrounding tissues and therefore maintain normal digit function. An intrasynovial tendon autograft was used as a gold standard. At 6 weeks the intrasynovial tendon autografts remained viable, contained normal numbers of cells along their length, and had minimal peritendinous adhesions. Four of six autografts had normal function as determined by rotation of the distal interphalangeal joint. Also at 6 weeks, the small intestinal submucosa grafts had host cell infiltration, neovascularization, and wavy, oriented tissue. However, ubiquitous adhesions together with impaired function in all cases suggest that small intestinal submucosa grafts in the configuration used are not suitable as full-length intrasynovial grafts in this tendon and animal model.     

In vitro three-dimensional reconstruction of human bladder mucosa

ObjetiveThe purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for graftingMaterial and MethodsBiopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was madeResultsA three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membraneConclusionsIn vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature     

Temporal differentiation and maturation of regenerated rat urothelium

OBJECTIVE

To determine if porcine small intestinal submucosa (SIS)‐regenerated urothelium expresses markers of urothelial differentiation, uroplakin and zona occludens‐1 (ZO‐1), and whether their expression correlates with the histological appearance of the urothelium.

MATERIALS AND METHODS

In all, 15 rats underwent partial cystectomy and bladder replacement with SIS. Regenerated bladders were harvested at either 2, 7, 14, 28, or 56 days after SIS grafting. Histological examination with haematoxylin and eosin staining was conducted to assess tissue regeneration. Immunohistochemistry was performed with uroplakin and ZO‐1 antibodies.

RESULTS

By 14 days after SIS grafting, the urothelial layer was completely confluent over the SIS. Expression of uroplakin and ZO‐1, evident at 2 days after SIS grafting, progressed from a cytoplasmic pattern of expression to a mature pattern of cytoplasmic and membrane expression by 56 days after SIS grafting.

CONCLUSION

In vivo tissue regeneration produces histologically and phenotypically mature urothelium within 2 weeks of SIS implantation. Regeneration of functional urothelium is probably essential for the subsequent development of the remaining bladder.     

The long-term culture of porcine urothelial cells and induction of urothelial stratification.

OBJECTIVE: To assess porcine urothelial cell cultures and the in vitro induction of urothelial stratification in long-term cultures, to study their morphological, functional and genetic behaviour, and thus provide potential autologous urothelium for tissue-engineered substitutes for demucosalized gastric or colonic tissue. MATERIALS AND METHODS: Primary cultures of porcine urothelium were established and the cells passaged thereafter. Cell specificity was confirmed by cytokeratin analysis, cell membrane stability assessed using lactate dehydrogenase leakage, cell de-differentiation by gamma-glutamyl transferase activity and genomic stability by karyotype investigations. Histology and scanning electron microscopy were performed to study the cultured cells and the stratified constructs. Furthermore, collagen matrices were tested as cell scaffolds. RESULTS: The cells were cultured for 180 days; 10 subcultures were established during this period. Stratification was induced in a culture flask and on a collagen matrix. Cytokeratins 7, 8, 17 and 18 were expressed in all cultures, and cell membranes were stable, with no evident de-differentiation. The cultures were stable in their genotype and no chromosomal aberrations were found. The histology and immunohistochemistry of the stratified porcine constructs, and cell membrane stability and cell de-differentiation, were compared with those in the human system. CONCLUSION: Pig and human urothelial cells can be cultured over a long period with no signs of senescence. Urothelial stratification can be induced in vitro. The collagen matrix seems to be an excellent scaffold, allowing cell adherence and growth.     

Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps

OBJECTIVE: To evaluate urothelium regeneration by grafting viable cultured urothelial cell sheets, harvested from temperature-responsive culture surfaces, on demucosalized gastric flaps in a dog model. MATERIALS AND METHODS: Viable urothelium was obtained from eight beagle dogs by partial cystectomy. Harvested urothelial cells were seeded on temperature-responsive culture dishes modified with the thermally sensitive polymer, poly(N-isopropylacrylamide). Urothelial cells cultured for 3 weeks generated contiguous urothelial cell sheets that were noninvasively harvested with no enzymatic treatment from these dishes, by reducing culture temperature. Urothelial cell sheets were autografted onto surgically demucosalized gastric flaps. Three weeks after autografting the dogs were killed and the gastric flaps with the urothelial cell sheets were examined. Cell and tissue characteristics were compared between these urothelial cell sheet-grafted gastric flaps and native urothelium. Ultrafine structures were also examined by electron microscopy. RESULTS: Five of the eight urothelial cell sheet-grafted flaps showed viable urothelial regeneration. Urothelial cell sheets attached spontaneously to demucosalized tissue surfaces completely, with no suture or fixing, and developed into a stratified viable epithelium very similar to native urothelium. Regenerated urothelium remained unstained by antiproton pump antibody, which typically stains epithelial cells positively in gastric mucosal layers. On three of the eight flaps where there were severe haematomas, grafted cell sheets were not adherent and there was no urothelial regeneration. CONCLUSIONS: Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suture or fixation, generating a multilayered urothelium in vivo. The novel intact cell-sheet grafting method rapidly produces native-like epithelium in vivo. This versatile technology should prove useful in urinary tract tissue engineering and surgical reconstruction.     

FIBRIN GLUE AS A DELIVERY VEHICLE FOR AUTOLOGOUS UROTHELIAL CELL TRANSPLANTATION ONTO A PREFABRICATED POUCH

PurposeThe purpose of the study was to investigate how in vivo expanded urothelium can be transplanted onto a prefabricated capsule-pouch for urinary reconstruction.Materials and MethodsUrothelial cells from 40 rats were harvested for culture. A tissue expander was used to induce a capsule-pouch on which the cell culture were reimplanted ten days later. As delivery vehicle we compared standard culture media and fibrin glue.ResultsThe histology demonstrated viable, multilayered clusters of urothelium cells only in the group using the fibrin glue delivery vehicle.ConclusionWe conclude that cultured urothelial cells can be successfully reimplanted onto a prefabricated capsule-pouch via fibrin glue, showing potential for urinary reconstructions.     

猪角朊细胞复合小肠黏膜下层体外培养实验

目的 探讨脱细胞小肠黏膜下层（SIS）作为组织工程皮肤支架的可行性.方法 将猪第二代角朊细胞接种于SIS上,接种后第11、13、15、17天通过直接染色、石蜡组织切片、HE染色、抗层粘连蛋白免疫组化染色、透射电镜等方法,观察角朊细胞在SIS上的生长情况.结果 猪角朊细胞接种在SIS上11d形态良好,部分区域形成2～3层细胞组成的复层结构,免疫组织化学染色显示在角朊细胞与SIS之间的细胞外基质中抗层粘连蛋白呈连续阳性表达.13、15、17 d细胞复层结构增多,细胞之间连接紧密,透射电镜下可见细胞内有张力微丝,细胞间有桥粒连接,角朊细胞与SIS之间可见基底膜形成.结论 在猪角朊细胞体外培养中,SIS是良好的细胞黏附支架.     

小肠粘膜下层的制备及细胞相容性的实验研究

目的了解猪小肠粘膜下层(SIS)的细胞相容性,探讨用SIS为生长载体复合骨髓基质干细胞(BMSCs)构筑组织工程骨的可能性。方法用物理和化学方法处理猪小肠粘膜下层,将兔骨髓基质干细胞与SIS进行体外复合培养,分别进行组织学、相差显微镜和扫描电镜观察。结果经物理和化学处理的SIS纯度高,孔隙多,胶原纤维未受损;BMSCs在SIS材料上生长、粘附、增殖,并能长入材料的孔隙内,分泌大量的细胞外基质成分。结论SIS的细胞相容性良好,不影响BMSCs的形态,对细胞生长和功能表达无抑制作用,可以用作骨组织工程的支架材料。     

Immunoreactivity of p63 in monolayered and in vitro stratified human urothelial cell cultures compared with native urothelial tissue

OBJECTIVE: To investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs. METHODS: p63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated. RESULTS: In native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen. CONCLUSIONS: p63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.     

Tissue engineering of small caliber vascular grafts.

OBJECTIVE: Previous tissue engineering approaches to create small caliber vascular grafts have been limited by the structural and mechanical immaturity of the constructs. This study uses a novel in vitro pulse duplicator system providing a 'biomimetic' environment during tissue formation to yield more mature, implantable vascular grafts. METHODS: Vascular grafts (I.D. 0.5 cm) were fabricated from novel bioabsorbable polymers (polyglycolic-acid/poly-4-hydroxybutyrate) and sequentially seeded with ovine vascular myofibroblasts and endothelial cells. After 4 days static culture, the grafts (n=24) were grown in vitro in a pulse duplicator system (bioreactor) for 4, 7, 14, 21, and 28 days. Controls (n=24) were grown in static culture conditions. Analysis of the neo-tissue included histology, scanning electron microscopy (SEM), and biochemical assays (DNA for cell content, 5-hydroxyproline for collagen). Mechanical testing was performed measuring the burst pressure and the suture retention strength. RESULTS: Histology showed viable, dense tissue in all samples. SEM demonstrated confluent smooth inner surfaces of the grafts exposed to pulsatile flow after 14 days. Biochemical analysis revealed a continuous increase of cell mass and collagen to 21 days compared to significantly lower values in the static controls. The mechanical properties of the pulsed vascular grafts comprised supra-physiological burst strength and suture retention strength appropriate for surgical implantation. CONCLUSIONS: This study demonstrates the feasibility of tissue engineering of viable, surgically implantable small caliber vascular grafts and the important effect of a 'biomimetic' in vitro environment on tissue maturation and extracellular matrix formation.     

Investigations of urothelial cells seeded on commercially available small intestine submucosa

OBJECTIVES: To examine adherence and viability of human urothelial cells seeded on commercially available small intestine submucosa (SIS) specimens under serum-free conditions. MATERIALS AND METHODS: Before seeding, SIS was either washed with incubation medium or coated with collagen A, fibronectin, or pronectin. A possible influence of SIS itself on the viability of urothelial cells was analysed with conditioned cell culture medium obtained by incubation of SIS for 24hours. In addition, untreated SIS and a setting without SIS were used as controls. Viability of urothelial cells was analysed with the WST-1 assay until day 9. Histology of seeded and unseeded SIS specimens was investigated after Papanicolaou staining. To demonstrate urothelial cell adherence on SIS, immunohistology was performed with a mixture of monoclonal AE1 and AE3 anticytokeratin antibodies. RESULTS: Urothelial cells seeded on SIS revealed no measurable cell viability. SIS-conditioned cell culture medium was cytotoxic for urothelial cells after 24 hours. Histology only demonstrated cell nuclei and no cytoplasm both in seeded and unseeded SIS specimens, thus indicating porcine DNA. Expression of the cell type-specific marker proteins AE1/AE3 could not be demonstrated. CONCLUSION: Since the commercially available SIS specimens used contained porcine DNA residues and demonstrated cytotoxic effects on urothelial cells, SIS is not suitable for in vitro construction of urothelial cell-matrix implants.     

Laparoscopy in ureteral engineering: a feasibility study

Objective

We recently bioengineered a ureter substitute from a seeded scaffold implanted by open surgery in the omentum. In view of the development of laparoscopy in the treatment of benign conditions of the ureter, obtaining a ureter substitute by minimally invasive techniques would be a desirable objective. However, conflicting results about the biological impact of carbon dioxide insufflation on the microcirculation of intra-abdominal organs prompted us to investigate first whether the results obtained by open surgery, in terms of vascular supply and maturation, could be reproduced laparoscopically.

Materials and methods

Bladder full-thickness tissue was harvested laparoscopically from three pigs for urothelial and smooth muscle cell primary cultures subsequently used to seed a small intestinal submucosa (SIS) matrix. After 2 wk, the in vitro seeded constructs were shaped around silicone drains and transferred laparoscopically into the abdomen for omental maturation. Three weeks later, the constructs were harvested for histological, immunohistochemical, and electron microscopic analysis.

Results

The laparoscopic procedures were performed successfully in all animals. After omental maturation, the constructs were vascularized and comprised of a well-differentiated multilayered urothelium with umbrella cells, over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. Electron microscopic analysis showed characteristics of a terminally differentiated urothelium.

Conclusion

As shown by conventional microscopy, immunochemistry, and electron microscopy, carbon dioxide insufflation does not impact cell growth and differentiation. These findings validate the laparoscopic approach for omental maturation of ureter substitutes.     

Histomorphometry and cell kinetics of normal human bladder mucosa in vitro

Summary In this study, we have quantified the morphologic and kinetic parameters of explant cultured normal adult human urinary bladder mucosa. Quantitative parameters studied were urothelial height, cell density, labelling index and mitotic index. For these studies, urinary bladders from seven adults with no previous history of urologic disease were obtained at autopsy. Mucosal explants were maintained in rocking culture on Gelfoam rafts for up to 33 days using supplemented CMRL 1066 medium. Prior to sampling, cultures were treated with tritiated thymidine and colchicine to investigate tissue kinetics. Data was based on histologic autoradiograms. During culture, urothelial cells retained normal polarity. During the first week of culture, urothelial height increased and cell density decreased. DNA synthesis and mitotic activity occurred primarily among basal cells. DNA synthesis was first noted on day 2 of culture; mitotic activity began after 3 days of culture. Morphologically, human urothelium was well maintained; DNA synthesis and mitotic activity was variable but continued throughout culture.This work was supported by NIH Grant CA-28013     

Tissue Engineering der Harnblase

In tissue engineering of the urinary bladder with autologous cell transplantation, high differentiation of the cells cultivated in vitro on biocompatible membranes is essential for the functionality of the tissue constructs after implantation. The terminal differentiation of superficial urothelial cells has a key role because of the barrier function of these cells against urine. The aim of this study was to determine optimized conditions for the creation of terminally differentiated urothelium to cover large membrane surfaces. This can bring us closer to the goal of using functioning tissue constructs in clinical trials.     

Fibroblast growth factor-7 regulates stratification of the bladder urothelium.

PURPOSE: The cellular and molecular mechanisms that regulate the organization of bladder urothelium into basal, intermediate and superficial cell layers remain poorly understood. We tested the hypothesis that fibroblast growth factor (FGF)-7 is essential for generating a multilayered stratified bladder epithelium. MATERIALS AND METHODS: The morphological and molecular characteristics of bladder urothelium in age and sex matched FGF-7 +/+ wild-type and -/- null mice were evaluated. In addition, the effect of exogenous FGF-7 on the growth and differentiation of primary murine urothelial cells was assessed. RESULTS: Morphometric analyses demonstrate that FGF-7 null urothelium is markedly thinned compared with wild-type urothelium. Electron microscopy revealed that null urothelium lacks the intermediate cell layers and molecular marker analyses confirmed this observation. In vitro cell culture experiments indicated that FGF-7 regulates urothelial cell growth, differentiation and stratification. Primary urothelial cultures maintained without FGF-7 ceased to divide and expressed proteins characteristic of terminally differentiated umbrella cells. In contrast, cultures maintained with exogenous FGF-7 contained proliferating epithelial cells with protein expression patterns consistent with those of intermediate cells in addition to terminally differentiated, post-mitotic umbrella cells. Importantly, isolated urothelial cells maintained with exogenous FGF-7 formed a multilayered epithelium in vitro. CONCLUSIONS: Collectively these data indicate that FGF-7 is essential for normal bladder urothelial stratification, specifically the formation of the intermediate cell layers. Fibroblast growth factor-7 stimulates urothelial proliferation and delays the differentiation of these cells into post-mitotic umbrella cells.     

Growth, differentiation and senescence of normal human urothelium in an organ-like culture

OBJECTIVE: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model. MATERIALS AND METHODS: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous beta-galactosidase activity and p16(INK4a) mRNA expression. RESULTS: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor alpha (TGF alpha) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16(INK4a) mRNA expression and beta-galactosidase activity increased on day 25, giving signs of senescence. CONCLUSIONS: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.     

骨髓基质干细胞复合小肠黏膜下层体外构建组织工程骨膜的实验研究

目的 探讨用猪小肠粘膜下层（SIS）作支架,复合骨髓问充质干细胞（BMSC）体外构建组织工程骨膜的可行性。方法用常规方法培养BMSC,再与SIS进行体外复合培养,分别进行相差显微镜、透射电镜、扫描电镜和组织学检查。观察BMSC在SIS上的生长、分化、增殖及细胞分泌细胞外基质情况。结果BMSC在SIS材料上粘附、增殖,分泌大量的细胞外基质成份,细胞功能活跃,在SIS上成多层生长,厚度随复合培养时间的延长而增加,类似生物骨膜。结论SIS与BMSC在体外复合培养可以构建出类似生物骨膜的组织工程骨膜,为进一步研究体内成骨奠定基础。