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PubMed2001

[Cryopreservation of vascular mixed cell for tissue engineering in cardiovascular surgery].

Tissue engineering (TE) is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffold. Various vascular and valvular grafts have been tried to create with this TE approach. In clinical use of this technique, harvested and cultured cells have to keep viability until implantation as tissue engineered tissue. But few research for cryopreservation of vascular mixed cells has been performed. So, we investigated the proper method for cryopreservation of vascular mixed cells harvested from femoral artery and vein of dogs. Cells were cultured and divide into three groups, A: cryopreserving in 5% dimethylsulfoxide (DMSO), hydroxyethyl starch (HES), and fetal bovine serum (FBS) with -80 degrees C freezer; B: cryopreserving in 10% DMSO and FBS with programmed freezer; C: control (continuous culture in media). After rapid thawing at 40 degrees C, group A showed higher viability than group B with flow cytometry. The results means that vascular mixed cells can be successfully cryopreserved in the DMSO/HES mixture simply and inexpensively, without rate controlled freezing.

Animals↗
PubMed2004

Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.

Absorbable Implants↗
PubMedDate not supplied

Tissue engineering research: the engineer's role.

Tissue engineering holds the promise of revolutionary advances for health care. Academic, clinical and industrial efforts are increasingly directed towards the development of autogenic substitutes to restore, maintain, or improve tissue and organ functions. This article summarizes the role of the engineer in the multidisciplinary environment of tissue engineering research.

Bioreactors↗
PubMed2000

Tissue engineering and skeletal diseases.

Tissue engineering, a cross between the science of the living organism and that of engineering, aims to replace, maintain or improve human tissue functions, by means of tissue substitutes containing living elements. Thus, it is about production of artificial tissue, using (alone or in combination) cells, matrix or bioactive factors. Their association gives rise to a hybrid biomaterial combining biological components (cells, growth factors or adhesion proteins) and materials (polymers, ceramics). The applications are wide-ranging, from the skin, to the liver, or to the cornea as well as to the locomotor system. Bone tissue engineering has advanced the most in this field, partly because of the progress made by research into bone substitutes, although cartilage and tendons are also concerned. This technology requires cell culture (committed cells or more often bone marrow stem cells), biomaterials (porous materials with controlled architecture and cements), growth factors (such as 'Bone Morphogenetic Proteins'), the proteins implicated in cell adhesion (such as fibronectin or the aminoacid sequences specifically recognised by integrin subunits) or gene therapy (notably using transfected stem cells). Tissue engineering and regenerative stimulation of tissue are now booming on experimental and industrial levels and clinical applications are increasingly numerous. Considering the potential of these technologies, they should continue to develop widely.

Biomedical Engineering↗
PubMed2003

The fundamentals of tissue engineering: scaffolds and bioreactors.

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate, and (iii) bioreactors that provide the conditions necessary for the cells to regenerate functional cartilaginous tissues. Here we explore the paradigm of tissue-engineered cartilage repair that is based on the generation of immature but functional constructs in vitro, and the remodelling and maturation of these constructs in vivo.

Adult↗
PubMed2002

Bioreactor studies of native and tissue engineered cartilage.

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.

Animals↗
PubMed2002

Bioreactors and bioprocessing for tissue engineering.

Bioreactor design in tissue engineering is complex, and at the early stages of its development. Design of biologically effective, yet scalable, devices requires intimate collaboration between engineers and biologists to ensure that all aspects are considered fully. Growth conditions, harvesting time, scale-up, storage, and sterility issues all need to be considered and incorporated into the design of bioreactors. Each tissue-engineered product will likely require individualized bioreactor design. However, without a comprehensive understanding of each of these components, bioreactor design and tissue growth to manufacture product will remain at a relatively rudimentary and limited level. Increased fundamental understanding of the issues can have a dramatic impact on the ability to generate tissue-engineered product safely, economically, and in the numbers that are required to fully address the patient populations in need.

Animals↗
PubMed2003

Effect of GLP-2 on mucosal morphology and SGLT1 expression in tissue-engineered neointestine.

Using tissue-engineering techniques, we have developed a neointestine that regenerates the structural and dynamic features of native small intestine. In this study, we tested neointestinal responsiveness to glucagon-like peptide 2 (GLP-2). Neointestinal cysts were engineered by seeding biodegradable polymers with neonatal rat intestinal organoid units. The cysts were matured and anastomosed to the native jejunum of syngeneic adult recipients. Animals were treated with GLP-2 [Gly2] (twice daily, 1 microg/g body wt) or vehicle alone (control) for 10 days. Rats were then killed, and tissues were harvested for analysis. Na+-glucose cotransporter (SGLT1) mRNA expression was assessed with Northern blotting and in situ hybridization. SGLT1 protein was localized by using immunofluorescence. GLP-2 administration resulted in 1.8- and 1.7-fold increases (P < 0.05) in neointestinal villus height and crypt depth, respectively. GLP-2 administration also resulted in a 2.4-fold increase (P < 0.01) in neomucosal SGLT1 mRNA expression. SGLT1 mRNA expression was localized to enterocytes throughout the villi, and SGLT1 protein was localized to the brush border of enterocytes along the entire length of villi from the neointestine of GLP-2-treated animals. The response of tissue-engineered neointestine to exogenous GLP-2 includes mucosal growth and enhanced SGLT1 expression. Therefore, tissue-engineering principles may help in dissecting the regulatory mechanisms mediating complex processes in the intestinal epithelium.

Animals↗
PubMed2004

Assessment of polyglycolic acid mesh and bioactive glass for soft-tissue engineering scaffolds.

Sufficient neovascularization of neotissue is currently a limiting factor for the engineering of large tissue constructs. 45S5 Bioglass has been investigated extensively in bone tissue engineering but there has been relatively little previous research on its application to soft-tissue engineering. The objectives of this study were to investigate the use of 45S5 Bioglass in soft-tissue engineering scaffolds using in vitro and in vivo models. A fibroblast cell line (208F) was used for in vitro evaluation of surfaces coated with 45S5 Bioglass. Increased proliferation of fibroblasts was observed after growth on polystyrene surfaces coated with low concentrations (0.01-0.2%wt/vol) of 45S5 Bioglass for 24 h in vitro, determined as a change in total cell number by measuring lactate dehydrogenase. At higher concentrations of 45S5 Bioglass and longer periods of incubation (48 and 72 h) on coated surfaces, cell proliferation was reduced. Light microscopy revealed that the morphology of fibroblasts grown on 45S5 Bioglass-coated surfaces was not altered at low concentrations, but at higher concentrations fibroblasts became vacuolated. Enzyme-linked immunosorbent assay of conditioned culture medium collected from fibroblasts grown for 24 h on surfaces coated with low concentrations of 45S5 Bioglass (0.01%wt/vol) was found to contain significantly higher concentrations of vascular endothelial growth factor. Histological examination of polyglycolic acid (PGA)/45S5 Bioglass composite scaffolds that had been implanted subcutaneously into rats revealed that 45S5 Bioglass-coated meshes were well tolerated. Light microscopy revealed that neovascularization into 45S5 Bioglass-coated meshes was significantly increased at 28 and 42 days. Electron microscopy revealed fibroblasts adhering closely to the PGA mesh but not to 45S5 Bioglass particles. The apparent ability of 45S5 Bioglass incorporated into scaffolds to increase neovascularization would be extremely beneficial during the engineering of larger soft-tissue constructs.

Animals↗
PubMed2001

Tissue engineering's impact on dentistry.

Tissue engineering is a novel and exciting field that aims to re-create functional, healthy tissues and organs in order to replace diseased, dying, or dead tissues. The field has developed due to the inadequate supply of organs and tissues for patients requiring organ and tissue replacement. The following review first describes three major tissue engineering strategies. Although similar in their objectives, these strategies each maintain a unique component. Next, several examples of preclinical and clinical progress engineering oral-maxillofacial tissues are presented. Each of these examples highlights specific tissue engineering applications to different tissues of the oral-maxillofacial apparatus. Finally, practical implications are addressed as well as challenges that must be met in order for tissue engineering to reach its full potential.

Biocompatible Materials↗
PubMed2001

Tissue engineering in plastic reconstructive surgery.

Tissue engineering (TE) is a new interdisciplinary field of applied research combining engineering and biosciences together with clinical application, mainly in surgical specialities, to develop living substitutes for tissues and organs. Tissue engineering approaches can be categorized into substitutive approaches, where the aim is the ex vivo construction of a living tissue or organ similar to a transplant, vs. histioconductive or histioinductive concepts in vivo. The main successful approaches in developing tissue substitutes to date have been progresses in the understanding of cell-cell interactions, the selection of appropriate matrices (cell-matrix interaction) and chemical signalling (cytokines, growth factors) for stimulation of cell proliferation and migration within a tissue-engineered construct. So far virtually all mammalian cells can be cultured under specific culture conditions and in tissue specific matrices. Future progress in cell biology may permit the use of pluripotent stem cells for TE. The blueprint for tissue differentiation is the genome: for this it is reasonable to combine tissue engineering with gene therapy. The key to the progress of tissue engineering is an understanding between basic scientists, biochemical engineers, clinicians, and industry.

Animals↗
PubMed2001

Growth factor delivery for bone tissue engineering.

Bone is a dynamic tissue that undergoes significant turnover during the life cycle of an individual. Despite having a significant regenerative capability, trauma and other pathological scenarios commonly require therapeutic intervention to facilitate the healing process. Bone tissue engineering, where cellular and biological processes at a site are deliberately manipulated for a therapeutic outcome, offers a viable option for the treatment of skeletal diseases. In this review paper, we aim to provide a brief synopsis of cellular and molecular basis of bone formation that are pertinent to current efforts of bone healing. Different approaches for engineering bone tissue were presented with special emphasis on the use of soluble (diffusible) therapeutic agents to accelerate bone healing. The latter agents have been used for both local bone repair (i.e. introduction of agents directly to a site of repair) as well as systemic bone regeneration (i.e. delivery for regeneration throughout the skeletal system). Critical drug delivery and targeting issues pertinent for each mode of bone regeneration are provided. In addition, future challenges and opportunities in bone tissue engineering are proposed from the authors' perspective.

Bone Morphogenetic Proteins↗
PubMed2006

Diffusion of bioactive molecules through the walls of the medial tissue-engineered hybrid ePTFE grafts for applications in designs of vascular tissue regeneration.

Strategies of better vascular tissue engineering may require delivery of soluble bioactive signals in cell culture medium to the cells in tissue-regenerating constructs. We measured the diffusivity and permeability of model tissue-engineering bioactive molecules such as water and heparin through the walls of both a hybrid ePTFE graft and a porcine carotid artery, a model vascular tissue. While diffusivities of H(3)-water and H(3)-heparin were measured as 3.9 x 10(-) (6) and 1.6 x 10(-) (6) cm(2)/s in the artery, respectively, under diffusional circulation of cell culture medium through the lumens of the carotid arteries, their corresponding permeabilities were 4.7 x 10(-) (5) and 2.0 x 10(-) (5) cm/s. On the other hand, diffusivities of H(3)-water and H(3)-heparin were also measured as 5.1 x 10(-) (6) and 4.7 x 10(-) (6) cm(2)/s, respectively, in the tissue-engineered hybrid ePTFE grafts; their corresponding permeabilities were 5.1 x 10(-) (5) and 3.7 x 10(-) (5) cm/s. The hybrid graft tissues were engineered by replacing the biodegradable, porous poly(lactide-co-glycolide) layers coated on the ePTFE surfaces with smooth muscle cell-derived tissues for 6 weeks. We analyzed the morphologies of the artery and the engineered hybrid ePTFE tissues with scanning electron microscopy and H&E stains. While the artery had its typical structure properties with layers of intima, media and adventitia, the tissue-engineered ePTFE hybrid graft had two layers of engineered tissues on the inner and outer surfaces of the ePTFE. There were no significant differences among the luminal tissue morphologies of the test samples from the effects of diffusion flow applications, with minor changes on their luminal surfaces. The results of water and heparin diffusion experiments indicated that these bioactive molecules were well transported from the cell culture medium to the tissue-engineering cells, enough to support tissue regeneration. We hope that these transport results may elucidate the transport behaviors of soluble nutrient molecules and biological signals through the vascular constructs under tissue engineering processes.

Absorbable Implants↗
PubMed2004

The role of bioreactors in tissue engineering.

Ex vivo engineering of living tissues is a rapidly developing area with the potential to impact significantly on a wide-range of biomedical applications. Major obstacles to the generation of functional tissues and their widespread clinical use are related to a limited understanding of the regulatory role of specific physicochemical culture parameters on tissue development, and the high manufacturing costs of the few commercially available engineered tissue products. By enabling reproducible and controlled changes of specific environmental factors, bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In addition, by automating and standardizing tissue manufacture in controlled closed systems, bioreactors could reduce production costs, thus facilitating a wider use of engineered tissues.

Bioreactors↗
PubMed2000

The impact of tissue engineering on dentistry.

BACKGROUND: Tissue engineering is a novel and highly exciting field of research that aims to repair damaged tissues as well as create replacement (bioartificial) organs. OVERVIEW: The authors provide a general review of the principles underlying key tissue engineering strategies, as well as the typical components used. Several examples of preclinical and clinical progress are presented. These include passive approaches, such as dental implants, and inductive approaches that activate cells with specific molecular signals. PRACTICE IMPLICATIONS: Tissue engineering will have a considerable effect on dental practice during the next 25 years. The greatest effects will likely be related to the repair and replacement of mineralized tissues, the promotion of oral wound healing and the use of gene transfer adjunctively.

Animals↗
PubMed2003

Materials as morphogenetic guides in tissue engineering.

Within native tissues cells are held within the extracellular matrix (ECM), which has a role in maintaining homeostasis, guiding development and directing regeneration. Efforts in tissue engineering have aimed to mimick the ECM to help guide morphogenesis and tissue repair. Studies have not only looked at ways to mimick the structure and characteristics of the ECM, but have also considered ways to reproduce its molecular properties including its bioadhesive character, proteolytic susceptibility and ability to bind growth factors.

Animals↗
PubMed2000

Early in vivo experience with tissue-engineered trileaflet heart valves.

BACKGROUND: Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. METHODS AND RESULTS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 microm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell-seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were <20 mm Hg on echocardiography. The maximum gradient was 10 mm Hg with direct pressures. Macroscopically, the tissue-engineered constructs were covered with tissue, and there was no thrombus formation on any of the specimens. Scanning electron microscopy showed smooth flow surfaces during the follow-up period. Histological examination demonstrated laminated fibrous tissue with predominant glycosaminoglycans as extracellular matrix. 4-Hydroxyproline assays demonstrated an increase in collagen content as a percentage of native pulmonary artery (1 week 45.8%, 17 weeks 116%). DNA assays showed a comparable number of cells in all explanted samples. There was no tissue formation in the acellular control. CONCLUSIONS: Tissue-engineered heart valve scaffolds fabricated from polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.

Absorbable Implants↗