A Novel and Faster Method to Obtain a Differentiated 3-Dimensional Tissue Engineered Bladder.

PURPOSE: We report what is to our knowledge a novel approach that led to the rapid development of a 3-dimensional bladder model, including a differentiated urothelium reconstructed without a period of exposure to the air-liquid interface. MATERIALS AND METHODS: Bilayered bladder constructs were produced using anchored mesenchymal cell seeded collagen gels to create the mesenchymal layer. Gels were coated with urine for 20 minutes before urothelial cell seeding. The 3-dimensional bladder models were cultured under submerged conditions for 15 days. RESULTS: Pure urine coating of the collagen matrix surface combined with its intermittent presence during urothelial development was found to be best to maintain urothelial cell properties. Immunohistological and ultrastructural analyses showed the formation of a pseudostratified urothelium devoid of abnormal K14 expression, allowing for uroplakin trafficking and forming an asymmetrical unit membrane at the apical surface. CONCLUSIONS: Such tissues could be adapted for clinical applications, including bladder repair. In the context of basic science this model could serve as a good alternative to animal use for fundamental and pharmacological studies of normal or pathological bladder tissues.

New ligament healing model based on tissue-engineered collagen scaffolds.

The anterior cruciate ligament (ACL) is often the target of knee trauma. This ligament does not heal very well, leading to joint instability. Long-term instability of the knee can lead to early arthritis and loss of function. To develop efficient strategies to stimulate posttraumatic ACL regeneration in vivo, a good healing model is needed in vitro. Such a model must remain as simple as possible, but should include key features to provide relevant answers to precise questions about the clinical problem addressed. Here, we report tissue-engineered type I collagen scaffolds developed to establish an ACL healing model in vitro and a potential ACL substitute in vivo. Such scaffolds were used to evaluate ACL cell growth, migration, and the capacity to synthesize and assemble collagen fibers for up to 40 days in vitro and up to 180 days in vivo. They were anchored with two bone plugs to allow their static stretching in culture and to facilitate their surgical implantation in knee joints. Our results have shown that living ACL fibroblasts can attach, migrate, and colonize this type of scaffold. In vitro, the cells populated the scaffolds and expressed mRNAs coding for the prolyl-4-hydroxylase, involved in collagen fibers' assembly. In vivo, acellular implants were vascularized and populated with caprine cells that migrated from the osseous insertions toward the center of the grafts. This model is a very good tool to study ACL repair and identify the factors that could accelerate its healing postsurgery.

Second Generation of Tissue-Engineered Ligament Substitutes for Torn ACL Replacement: Adaptations for Clinical Applications.

The anterior cruciate ligament (ACL) of the knee joint is one of the strongest ligaments of the body and is often the target of traumatic injuries. Unfortunately, its healing potential is limited, and the surgical options for its replacement are frequently associated with clinical issues. A bioengineered ACL (bACL) was developed using a collagen matrix, seeded with autologous cells and successfully grafted and integrated into goat knee joints. We hypothesize that, in order to reduce the cost and simplify the model, an acellular bACL can be used as a substitute for a torn ACL, and bone plugs can be replaced by endobuttons to fix the bACL in situ. First, acellular bACLs were successfully grafted in the goat model with 18% recovery of ultimate tensile strength 6 months after implantation (94 N/mm2 vs. 520). Second, a bACL with endobuttons was produced and tested in an exvivo bovine knee model. The natural collagen scaffold of the bACL contributes to supporting host cell migration, growth and differentiation in situ post-implantation. Bone plugs were replaced by endobuttons to design a second generation of bACLs that offer more versatility as biocompatible grafts for torn ACL replacement in humans. A robust collagen bACL will allow solving therapeutic issues currently encountered by orthopedic surgeons such as donor-site morbidity, graft failure and post-traumatic osteoarthritis.

Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses.

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Modulated expression of a nuclear-associated glycoprotein during normal rat liver development and in various hepatoma cells.

Liver plays a major role in systemic detoxification and drug metabolism. NF-164, a protein of 164 kDa predominantly localized in hepatocyte nuclei, was found to be present in increasing amounts during liver maturation. In addition, fetal rat hepatocytes had ten times, and neonatal five times less of this protein than adult hepatocytes. It was also detected in an albumin producing hepatoma cell line, but not in three other lines that have lost several differentiated functions. These data suggest that NF-164 expression is development-dependent and that it may be a marker for both normal and malignant hepatocyte differentiation. NF-164 seems to be liver-specific, since it was not detected in rat brain, spleen, kidney, lung and bovine thymus. It was purified from adult rat hepatocyte nuclei. Its estimated pI is 6.8. Its total amino acid composition and partial amino acid sequence is also being reported. Despite major differences between their respective contents in amino acids, partial sequences showed homologies with carbamyl phosphate synthetase I (CPSI). These observations may suggest that NF-164 also shares some functional features with this enzyme.

Tissue-engineered human asthmatic bronchial equivalents.

The isolation of human bronchial epithelial (HBEC) and fibroblastic cells (HBFC) from biopsies of asthmatic and non-asthmatic volunteers provided unique cellular materials to be used for the production of bioengineered bronchial equivalents (BE) in vitro. The HBEC are grown on a mesenchymal layer seeded with HBFC and the BE can be maintained for at least 15 days in culture. Under the BE culture conditions established previously, HBEC undergo differentiation into ciliated and goblet cells, within a pseudostratified organization comparable to human bronchi. We published previously the results from histologic and functional analyses of such BE produced exclusively using non-asthmatic HBEC and HBFC. We report here the comparative analyses of BE produced with non-asthmatic and asthmatic living HBEC and HBFC (naBE and aBE, respectively). Our data indicated that all asthmatic HBEC populations grown on a mesenchymal layer, containing non-asthmatic HBFC, slowly reached a confluent state but then detached from the matrix upon culture time. These BE appear to be very good models to study the mechanisms involved in asthma in vitro.

[Tissue engineering: a tool to understand the physiological mechanisms]. / Le génie tissulaire au service de la compréhension du vivant.

Tissue engineering is a new domain, which allows some very unique studies of many human physiological mechanisms. This technology, based on cell capacity to reproduce a three-dimensional tissue with or without the help of biomaterials, is an interesting approach to study cells in an environment quite similar to the in vivo context. This article summarizes the LOEX's (laboratory of experimental organogenesis) scientific endeavor in tissue engineering in order to better understand some physiological or pathological mechanisms. Thus wound healing, stem cells, graft vascularization and cell interactions are domains where tissue engineering has already made a significant impact.

Potential of skin fibroblasts for application to anterior cruciate ligament tissue engineering.

Fibroblasts isolated from skin and from anterior cruciate ligament (ACL) secrete type I and type III collagens in vivo and in vitro. However, it is much easier and practical to obtain a small skin biopsy than an ACL sample to isolate fibroblasts for tissue engineering applications. Various tissue engineering strategies have been proposed for torn ACL replacement. We report here the results of the implantation of bioengineered ACLs (bACLs), reconstructed in vitro using a type I collagen scaffold, anchored with two porous bone plugs to allow bone-ligament-bone surgical engraftment. The bACLs were seeded with autologous living dermal fibroblasts, and grafted for 6 months in goat knee joints. Histological and ultrastructural observations ex vivo demonstrated a highly organized ligamentous structure, rich in type I collagen fibers and cells. Grafts' vascularization and innervation were observed in all bACLs that were entirely reconstructed in vitro. Organized Sharpey's fibers and fibrocartilage, including chondrocytes, were present at the osseous insertion sites of the grafts. They showed remodeling and matrix synthesis postimplantation. Our tissue engineering approach may eventually provide a new solution to replace torn ACL in humans.

Identification of functional markers in a self-assembled skin substitute in vitro.

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collagenases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.

Cross-sectional profiles and volume reconstructions of soft tissues using laser beam measurements.

Precise geometric reconstruction is a valuable tool in the study of soft tissues biomechanics. Optical methods have been developed to determine the tissue cross section without mechanical contact with the specimen. An adaptation of the laser micrometer developed by Lee and Woo [ASME J. Biomech. Eng., 110 (2), pp. 110-114]. is proposed in which the laser-collimated beam rotates around and moves along a fixed specimen to reconstruct its cross sections and volume. Beam motion is computer controlled to accelerate data acquisition and improve beam positioning accuracy. It minimizes time-dependent shape modifications and increases global reconstruction precision. The technique is also competent for the measurement of immersed collagen matrices.