Capsule induction technique in a rat model for bladder wall replacement: an overview

The search for a reliable technique for functional genitourinary tissue replacement remains a challenging task. The most recent advances in cell biology and tissue engineering have utilized various avascular and acellular collagen scaffolds with or without seeded cells. These techniques, however, are frequently complicated by tissue necrosis, contracture and resorption due to limited vascularization. We employed a new three-stage, evolving animal model with stage I optimizing the culture delivery vehicle, stage II employing a seeded vascularized capsule flap, and stage III adding a contractile matrix in the form of pedicled gracilis muscle prelaminated with autologous, in vitro-expanded urothelial cells to reconstruct an entire supratrigonal bladder-wall defect in rats.Specimens stained with hematoxylin and eosin (H&E), alpha(1)-actin staining, and a specific immunohistochemical staining (AE(1)&AE(3)-anticytoceratin monoclonal antibody stain) showed a continuous, multilayered, functioning urothelial lining along the transposed prelaminated gracilis flap in the animals of the final-stage experiment.Successful urinary reconstruction requires a contractile neoreservoir resistant to resorption over time and a stable, protective urothelial lining. We demonstrated that a gracilis muscle flap can be seeded with autologous cultured urothelial cells suspended in fibrin glue. This prelaminated flap can be safely transposed onto its pedicle and become successfully integrated into the remaining bladder wall, demonstrating urothelial lining and the potential to contract. Further studies in larger animals with urodynamic assessment is warranted to determine if this type of bladder-wall replacement technique is suitable for urinary reconstruction in humans.     

胶原海绵支架体外构建组织工程膀胱黏膜***◆

背景：组织工程膀胱黏膜层的构建在组织工程膀胱修复中占有重要的地位,但目前并没有最合理的构建方法。 目的：探讨胶原海绵支架复合猪膀胱尿路上皮细胞体外构建膀胱黏膜结构的可行性。 方法：刮取猪膀胱黏膜层后用酶消化方式进行猪膀胱尿路上皮细胞原代培养,并进行尿路上皮细胞标志物免疫荧光和RT-PCR鉴定。制备疏松多孔的胶原海绵支架材料,将第3代尿路上皮细胞接种在胶原海绵支架上,体外培养4~8 d后观察尿路上皮细胞和胶原海绵材料的复合情况。 结果与结论：原代培养的猪膀胱尿路上皮细胞呈“多角形”、“铺路石”样,以克隆团形式生长。免疫荧光鉴定AE1/AE3阳性,RT-RCR检测uroplakin-ⅠA、uroplakin-Ⅱ阳性。胶原海绵复合尿路上皮细胞体外培养4~8 d后,细胞在胶原海绵支架上生长良好,覆盖胶原材料的表面并长入材料内部,保持了尿路上皮细胞的特性。体外培养6 d时,尿路上皮细胞与胶原支架复合效果最好,同时细胞数量也最多。结果初步表明了胶原海绵复合膀胱尿路上皮细胞可以构建组织工程膀胱黏膜,且体外培养6 d为最佳时间点。     

A Dynamically Cultured Collagen/Cells-Incorporated Elastic Scaffold for Small-Diameter Vascular Grafts

Abstract

There is an essential demand for tissue-engineered autologous small-diameter vascular grafts, which offer temporary supports and guides for vascular tissue organization, repair and remodeling. This study reports on the effect of collagen/smooth muscle cells (SMCs) mixtures under dynamic cultures and SMC-endothelial cell (ECs) co-culture on cell proliferation, uniform cell distribution, extracellular matrix deposition, and endothelial cells monolayer formation in tissue-engineered tubular arterial constructs of 4 mm inner diameter. Rabbit aortic SMCs were infiltrated with collagen solution in poly(L-lactide-co-?-caprolactone) (PLCL) scaffolds under vacuum to form collagenous gel and subjected to dynamic strain by culturing them in a dynamic perfusion bioreactor. The construct lumen was subsequently seeded with ECs and experiments were completed to create ECs–SMCs co-culture constructs. The collagen/SMCs incorporated elastic scaffold cultured under dynamic culture conditions promoted matrix deposition, leading to the development of tissue-engineered vascular constructs, and induced SMC to have more uniform cell distribution. Scanning electron microscopic examination and von Willebrand Factor staining demonstrated the presence of ECs spread over the lumen. Quantitative analysis of elastin contents demonstrated that the engineered vessels acquired similar elastin contents as native arteries. The collagen/SMCs/ECs incorporated PLCL scaffolds under dynamic culture conditions can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.     

膀胱组织工程研究进展

膀胱作为储尿排尿器官具有重要的生理功能,许多病理原因导致的膀胱损伤往往需要进行膀胱重建。临床上膀胱重建的金标准是胃肠道代膀胱术,但是胃肠道的结构和功能与膀胱有着本质的区别,导致肠段置换术易产生血尿、排尿困难、结石、肿瘤等并发症。近年来,随着组织工程及再生医学的诞生及发展,为膀胱缺损修复提供了新的思路和技术手段。本文就膀胱组织工程的三个方面--种子细胞、支架材料及生长因子的最新进展进行了综述。     

Direct magnetic tubular cell seeding: a novel approach for vascular tissue engineering

Optimizing seeding efficiency, reducing delayed culture periods and mimicking native tissue architecture are crucial requirements for the development of seeding procedures in tissue engineering. In vascular applications, the tubular geometry of the grafts further hampers the efficient delivery of cells onto the scaffold. To overcome these limitations, a novel technology based upon the use of magnetic fields is presented in this study: a radial magnetic force drives the cells immediately onto the luminal surface of a tubular scaffold and immobilizes the cells on the substrate's surface promoting cell attachment. Human smooth muscle cells (SMCs) labeled with CD44 magnetic Dynabeads were successively seeded onto the luminal surface of a tubular shaped collagen membrane. After 5 h, one additional layer of human umbilical vein endothelial cells (HUVECs) labeled with CD31 magnetic Dynabeads was seeded onto the luminal SMCs. The co-culture was incubated during 5 days prior to analysis. Cell viability and expression profiles were preserved during the entire seeding process. Histological examination of the constructs highlighted densely packed multilayers of SMCs covered by a monolayer of endothelial cells. SEM inspection confirmed a heterotypic multilayer assembly formed by multiple layers of elongated SMCs covered by a single layer of endothelial cells. Seeding kinetics of HUVECs and SMCs showed over 90% seeding efficiency after 20 and 40 min magnetic exposure respectively. Magnetically induced cell seeding provides a valuable tool for rapid seeding procedures of tubular scaffolds while complying with the histological architecture of tissue.     

Co-expression of elastin and collagen leads to highly compliant engineered blood vessels

Elastin synthesis and physiologic compliance are significant challenges in blood vessel tissue engineering. Here, we report that a biocompatible elastomeric scaffold can support the co-expression of elastin and collagen, which likely yielded the physiologic compliance in the constructs. A biodegradable elastomer, poly(glycerol sebacate), was fabricated into highly porous tubular scaffolds. Primary baboon arterial smooth muscle cells (SMCs) were seeded in the lumen of the scaffolds followed by a 1-week culture under gentle perfusion. Circulating endothelial progenitor cells (EPCs) isolated from baboon peripheral blood was seeded directly on the smooth muscle layer in the lumen on day 8. The constructs were perfused using a pulsatile flow system for another 2 weeks before characterization. In another set of experiments, the SMCs were cultured for 7 weeks and were co-cultured for 1 week with the EPCs. Constructs obtained using either set of culture conditions contained elastin and collagen: Masson's trichrome stain showed a circumferential collagen band in the constructs, and elastin was evident from its characteristic autofluorescence, Verhoff's stain, and amino acid analysis of insoluble remnants after hot alkali digestion. All constructs had a confluent cellular lumen with cells well-dispersed throughout the scaffolds. At physiologic pressures, the compliance of the 8-week construct was comparable to human arteries as observed in pressure-diameter testing. Combination of elastomeric scaffolds, co-culture of EPC and SMC, and mechanical conditioning appears to encourage the expression of a more natural extracellular matrix and lead to physiologically-relevant compliance; both are major challenges in blood vessel tissue engineering.     

What's in the pipeline about bladder reconstructive surgery? Some remarks on the state of the art

The fusion of engineering with cell biology and advances in biomaterials may lead to de novo construction of implantable organs. Engineering of neobladder from autologous urothelial and smooth muscle cells cultured on biocompatible, either synthetic or naturally-derived substrates, is now feasible in preclinical studies and may have clinical applicability in the near future. The development of a bioartificial bladder would warrant the prevention of both the metabolic and neoplastic shortcomings of the intestinal neobladder. Two tissue-engineering techniques for bladder reconstruction have been tested on animals: 1) the in vivo technique involves the use of naturally-derived biomaterials for functional native bladder regeneration 2) the in vitro technique involves the establishment of autologous urothelial and smooth muscle cell culture from the host's urinary tract, after which the cells are seeded on the biodegradable matrix-scaffold to create a composite graft that is implanted into the same host for complete histotectonic regeneration. Waiting for the creation of a complete tissue-engineered bladder with a trigone-shaped base, we suggest, in surgical oncology after radical cystectomy, the realization of conduit or continent pouch using tissue-engineered material.     

A Collagen/Smooth Muscle Cell-Incorporated Elastic Scaffold for Tissue-Engineered Vascular Grafts

Biodegradable tubular scaffolds have been developed for vascular graft application. This study was focused to improve the adhesion and proliferation of vascular smooth muscle cells (SMCs) in a tubular scaffold. Tubular scaffolds (ID 4 mm, OD 6 mm) were fabricated from a biodegradable elastic polymer, poly(L-lactide-co-ε-caprolactone) (PLCL) (50:50, M _n 1.58 × 10⁵), by an extrusion/particulate leaching method. SMCs suspended in a collagen solution were infiltrated in tubular PLCL scaffolds under vacuum and incubated for 1 h at 37°C to form a collagenous gel. Results from SEM image analysis showed that collagen was infiltrated into the inside of the scaffolds. Cell adhesion and proliferation rate increased in collagen/SMC-incorporated tubular PLCL scaffolds as compared with the scaffolds in which only SMCs were seeded. From SEM image and histological analysis, we further found that SMCs grew on the inside as well as on the surface of collagen/SMCs-incorporated scaffolds and the cells continued to grow as a monolayer on collagen fibers. In particular, cell proliferation and elastin contents were the highest in a PLCL scaffold with 50–100 μm pore size than any other scaffolds used in this experiment. A collagen/SMC-incorporated PLCL scaffold may support SMC growth and functions and can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.     

In vitro construction of urinary bladder wall using porcine primary cells reseeded on acellularized bladder matrix and small intestinal submucosa

BACKGROUND: Partial or radical cystectomy requires replacement of the urinary reservoir normally achieved by using small or large bowel segments. Our aim was to establish tissue engineering of an bioartificial bladder wall using primary cultures of porcine urothelial (pUC) and bladder smooth muscle cells (pSMC) to be reseeded on different acellular biological matrices. METHODS: Primary porcine cultures of pUC and pSMC were established from open bladder biopsy material 25 mm2 in size. Acellular matrix was generated either from a) porcine bladder wall segments or b) tubular small intestinal submucosa with the still attached decellularized muscularis layer. Reseeding of these matrices with primary cells was done in a two-dimensional static model and in a three-dimensional rotating bioreactor perfused with cell culture medium for a period of 6 weeks. RESULTS: Prior to reseeding the cultured cells were characterized as pUC and pSMC by immunohistochemical staining with either anti-keratin 7 or anti-alpha actin. For both matrices a reseeded double layer cell system of pUC and pSMC could be identified after incubation in the described systems for 6 weeks. CONCLUSIONS: Our results document successful generation of tissue engineered urinary bladder wall, which can be used in further large animal transplantation experiments.     

'UroMaix' scaffolds: novel collagen matrices for application in tissue engineering of the urinary tract

Reconstruction of bladder and ureter tissue is indicated in cases of injury, stenosis, infection or tumor. Substitution by ileum, colon or pure synthetic polymers generates a variety of complications. Biohybrid tissue mimicking structural and functional attributes of the multilayered wall architecture of the urinary conduit may be the solution to current problems. This study reports on porcine urinary tract cells isolated and placed on UroMaix matrices with different degrees of cross-linking produced from highly purified type I collagen from medically approved porcine tissue. A patented procedure revealed membrane structures composed of a dense fibrous side and an open fibrous side. These scaffolds with the porcine urinary tract cells were incubated in a batch culture system for up to 14 days. Cell growth and topographical orientation were examined. Urothelial cells showed maximum attachment and a significant increase of living cells on the dense fiber layer of UroMaix-1. No attachment of urothelial cells occurred on the other prototypes. Smooth muscle cells showed similar behavior within the open fiber layer of all UroMaix matrices. Both urothelial and smooth muscle cells retained their phenotypes as demonstrated by the immunostaining of epithelial cytokeratin 18 and the smooth muscle myosin heavy chain respectively. Thus we could show that UroMaix scaffolds support the attachment and proliferation of urinary tract cells. The elastomeric properties of the collagenous matrices promise attractive applications in the tissue engineering of the urinary tract with its high mechanical demands.     

Biocompatibility and remodeling potential of pure arterial elastin and collagen scaffolds

Surgical therapy of cardiovascular disorders frequently requires replacement of diseased tissues with prosthetic devices or grafts. In typical tissue engineering approaches, scaffolds are utilized to serve as templates to support cell growth and remodeling. Decellularized vascular matrices have been previously investigated as scaffolds for tissue engineering. However, cell migration into these scaffolds was inadequate due to the very tight matrix organization specific to the aortic structure. To address this problem, we prepared two types of decellularized scaffolds from porcine vascular tissues. Pure elastin scaffolds and pure collagen scaffolds were prepared by selectively removing the collagen component or elastin, respectively. In the current study, we use a subdermal implantation model to demonstrate that arterial elastin and collagen scaffolds exhibit enhanced potential for repopulation by host cells in vivo. Notably, numerous new collagen fibers and bundles were found within the remodeled elastin scaffolds and new elastin fibers within collagen scaffolds, respectively, clearly indicating their ability to support de novo extracellular matrix synthesis. We also show that biological cues such as growth factors are required for efficient repopulation of elastin and collagen scaffolds. Finally, we bring evidence that these scaffolds can be endothelialized in vitro for thrombosis resistance and thus can serve as promising candidates for cardiovascular tissue engineering.     

Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells

This study demonstrates the engineering of biologically active heart valve leaflets using prenatally available human umbilical cord-derived progenitor cells as the only cell source. Wharton's Jelly-derived cells and umbilical cord blood-derived endothelial progenitor cells were subsequently seeded on biodegradable scaffolds and cultured in a biomimetic system under biochemical or mechanical stimulation or both. Depending on the stimulation, leaflets showed mature layered tissue formation with functional endothelia and extracellular matrix production comparable with that of native tissues. This demonstrates the feasibility of heart valve leaflet fabrication from prenatal umbilical cord-derived progenitor cells as a further step in overcoming the lack of living autologous replacements with growth and regeneration potential for the repair of congenital malformation.     

Tissue engineering of urinary organs

Tissue engineering can serve as an alternative treatment for a malfunctioning or lost organ. Isolated and expanded cells adhere to a temporary scaffold, proliferate, and secrete their own extracellular matrices (ECM) replacing the biodegrading scaffold. The genitourinary system, composed of the kidney, ureter, bladder, urethra, and genital organs, is exposed to a variety of possible injury sites from the time of fetal development. All the urinary organs are mainly composed of smooth muscle and uroepithelial cells and which may be approached by tissue engineering techniques. A large number of materials, including naturally-derived and synthetic polymers have been utilized to fabricate prostheses for the genitourinary system. Usually, whenever there is a lack of native urologic tissue, reconstruction is considered with native non-urologic tissue, such as, gastrointestinal segments, or skin or mucosa from multiple body sites. Engineering tissues using selective cell transplantation may provide a means to create functional new genitourinary tissues. This review concerns urinary tissues reconstructed with bladder uroepithelial cells and smooth muscle cells (SMCs) implanted on biodegradable polymer matrices.     

Evaluation of smooth muscle cell response using two types of porous polylactide scaffolds with differing pore topography

The goal of tissue engineering is to create bioartificial tissues for the replacement of failed or nonfunctional tissue. Porous tissue-engineered scaffolds may be created through a solvent-casting/porogen-leaching technique. Almost exclusively, sodium chloride (NaCl) is the porogen of choice. Previous studies have demonstrated the importance of porosity and pore size in cell adhesion and tissue development, yet the impact of porogen morphology and the chemical effect of porogen residual has not been fully explored. Poly-L-lactide (PLLA) scaffolds were manufactured by a solvent-casting, particulate-leaching method with either glucose or NaCl porogen in an effort to vary pore characteristics and, subsequently, cell adhesion and tissue development. Porogen influence on scaffold morphology and topography was compared via histological techniques and qualitative surface characteristics. Using an in vitro model, scaffolds were seeded with rat aortic smooth muscle cells (SMCs) and evaluated over a 28-day period. Cell attachment and proliferation were subsequently evaluated. Results indicate that initial SMC attachment is higher for scaffolds manufactured with NaCl rather than glucose. The proliferation of SMCs was higher for scaffolds manufactured with glucose and, by day 28, scaffolds manufactured with glucose supported a higher cell population than those processed using NaCl porogen.     

Allogeneic stem cells seeded tissue engineered bladder: a possible alternative for bladder reconstruction and treatment to bladder cancer

Bladder cancer remains a difficult management problem, because of the high recurrence rate or the functional disorder of bladder post radical cystectomy. The autologous tissue engineering techniques have been advanced enough to reconstruct functional bladders for patients with myelomeningocele, but not capable for bladder cancer patients. On the other hand, allogeneic stem cells share the same multipotential, self-renewing and multi differentiated abilities with autologous ones, and would present anti-tumor effects when transplanted to recipients. Since the stem cells seeded tissue engineering techniques for bladder regeneration have already been feasible, a hypothesis was then proposed that the allogeneic stem cells seeded tissue engineered bladder would be a possible alternative for functional bladder reconstructions and treatments for bladder cancer recurrences and latent metastases postoperatively.     

In vivo biocompatibilty and degradation behavior of elastic poly(L-lactide-co-epsilon-caprolactone) scaffolds

Tubular scaffolds were fabricated from very elastic poly(L-lactide-co-epsilon-caprolactone) (PLCL, 50:50). The scaffolds were seeded with smooth muscle cells (SMCs) and implanted in nude mice to investigate the tissue compatibility and in vivo degradation behavior. Histological examination of all the implants with haematoxylin and eosin staining, masson trichrome staining, SM alpha-actin antibody, and CM-DiI labeling confirmed that the regular morphology and biofunction of the SMCs seeded and the expression of the vascular smooth muscle matrices in PLCL scaffolds. The implanted PLCL scaffolds displayed a slow degradation on time, where caprolactone units were faster degraded than lactide did. This could be explained by the fact that amorphous regions composed of mainly CL moieties degraded earlier than hard domains where most of the LA units were located. From these results, the scaffolds applied in this study were found to exhibit excellent tissue compatibility to SMCs and might be very useful for vascular tissue engineering.     

Tissue engineering of small intestinal tissue using collagen sponge scaffolds seeded with smooth muscle cells

In a previously reported attempt to regenerate small intestine with autologous tissues, collagen scaffolds were used without cell seeding or with autologous mesenchymal stem cell seeding. However the regenerated intestine lacked a smooth muscle layer. To accomplish regeneration of a smooth muscle layer, this present study used collagen scaffolds seeded with the smooth muscle cells (SMC) in a canine model. Autologous SMC were isolated from stomach wall and cultured. Two types of scaffolds were fabricated: in SMC (+), cultured SMCs were mixed with collagen solution and poured into a collagen sponge; and in SMC (-), SMCs were omitted. Both scaffolds were implanted into defects of isolated ileum as a patch graft. Animals were euthanized at 4, 8, and 12 weeks; for the last time point, the ileal loop had been reanastomosed at 8 weeks. At 12 weeks, the SMC (-) group showed a luminal surface covered by a regenerated epithelial cell layer with very short villi; however only a thin smooth muscle layer was observed, representing the muscularis mucosae. In the SMC (+) group, the luminal surface was covered completely by a relatively well-developed epithelial layer with numerous villi. Implanted SMCs were seen in the lamina propria and formed a smooth muscle layer. Thus, we concluded that collagen sponge scaffolds seeded with autologous SMCs have a potential for small intestine regeneration.     

Preparation,characterization and in vitro analysis of novel structured nanofibrous scaffolds for bone tissue engineering

In a previous study, a three-dimensional nanofibrous spiral scaffold for bone tissue engineering was developed, which showed enhanced human osteoblast cell attachment, proliferation and differentiation compared with traditional cylinder scaffolds, owing to the incorporation of spiral structures and nanofiber. However, the application of these scaffolds to bone tissue engineering was limited by their weak mechanical strength. This limitation triggered the design for novel structured scaffolds with reinforced physical characteristics. In this study, spiral polycaprolactone (PCL) nanofibrous scaffolds were inserted into poly(lactide-co-glycolide) (PLGA) microsphere sintered tubular scaffolds to form integrated scaffolds to provide mechanical properties and bioactivity appropriate for bone tissue engineering. Four experiment groups were designed: PLGA cylinder scaffold; PLGA tubular scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular scaffold with PCL nanofiber containing spiral structured inner core. The morphology, porosity and mechanical properties of the scaffolds were characterized. Furthermore, human osteoblastic cells were seeded on these scaffolds, and the cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds were evaluated. The integrated scaffolds had Young’s modulus 250–300 MPa, and compressive strength 8–11 MPa under uniaxial compression. With the addition of an inner highly porous insert to the tubular shell, human osteoblast cells seeded on the integrated scaffolds showed slightly higher cell proliferation, 20–25% more alkaline phosphatase expression and twofold higher calcium deposition than those on the cylinder and tubular scaffolds. Furthermore, compared with sintered PLGA cylinder scaffolds, the integrated scaffolds allowed better cellular infiltration Therefore, this design demonstrates great potential for integrated scaffolds in bone tissue engineering applications.     

全生物化组织工程血管构建的初步研究

为探索在体外初步构建全生物化组织工程血管,采用以酶消化为主的方法制备猪颈总动脉脱细胞支架,再种植犬胸主动脉的平滑肌细胞,培养四周。经组织学染色和电镜观察显示:在猪颈总动脉脱细胞支架上,犬血管平滑肌细胞大量生长,组织学和电镜结构与正常血管壁结构类似。结果表明,我们初步成功地构建了全生物化组织工程血管,为临床血管替代物的基础研究提供了有意义的实验资料。     

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype

The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of alpha(5) integrin and THY-1, and increased expression of alpha-smooth muscle actin (alphaSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.