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At least 865 records · Page 48Linked to original sources
PubMed2003

System-engineered cartilage using poly(N-isopropylacrylamide)-grafted gelatin as in situ-formable scaffold: in vivo performance.

Our previous study showed that cartilaginous tissue can be engineered in vitro with articular chondrocytes and poly(N-isopropylacrylamide)-grafted gelatin. This short-term in vivo study for cartilage repair was performed to screen a candidate method for a long-term study. In our previous in vitro study, however, two potential problems with the tissue-engineered cartilage were identified: (1). leakage of the transplant due to temperature decline and (2). concave deformation of transplant due to compressive loading. To solve these problems, we investigated in this study the usefulness of suturing with two different covering materials (periosteum or collagen film) and preculturing an engineered tissue for 2 weeks. PNIPAAm-gelatin-based engineered cartilage samples were evaluated at 5 weeks after operation by gross and microscopic examination. Leakage occurred only in specimens without precultured tissue and with a collagen film. Minimal surface deformation occurred in all specimens with precultured tissue. The score on gross examination showed that transplants with precultured tissue acquired a higher score than did the others. Histological evaluation showed a minimal foreign-body response of PNIPAAm-gelatin in all specimens and higher maturity as a cartilaginous tissue in specimens with precultured tissue. These results indicate that transplantation with precultured tissue may be a suitable method for a long-term in vivo study.

Acrylic Resins↗
PubMed2003

Gene therapy in tissue-engineered blood vessels.

Cardiovascular disease is the leading cause of morbidity and mortality in Western society. More than 1 million arterial bypass procedures are performed annually in the United States, where either autologous veins or synthetic grafts are used to replace arteries in the coronary or peripheral circulation. Tissue engineering of blood vessels from autologous cells has the potential to produce biological grafts for use in bypass surgery. Ex vivo development of vascular grafts also provides an ideal target of site-specific gene therapy to optimize the physiology of the developing conduit, and for the possible delivery of other therapeutic genes to a vascular bed of interest. In this article, we demonstrate that by using a novel retroviral gene delivery system, a target gene of interest can be specifically delivered to the endothelial cells of a developing engineered vessel. Further, we demonstrate that this technique results in stable incorporation of the delivered gene into the target endothelial cells for more than 30 days. These data demonstrate the utility of the retroviral gene delivery approach for optimizing the biologic phenotype of engineered vessels. This also provides the framework for testing an array of genes that may improve the function of engineered blood vessels after surgical implantation.

3T3 Cells↗
PubMed2003

Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering.

Bone tissue engineering has the potential to provide us with an autologous bone substitute. Despite extensive research to optimize the technique, little is known about the survival and function of the cells after implantation. To monitor the cells, in vivo labeling is the method of choice. In this study we investigated the use of the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) to label cells used in bone tissue engineering. When applying label concentrations up to 50 microM, cells could be labeled efficiently without negative effects on cell vitality, proliferation, or bone-forming capacity. Porous hydroxyapatite scaffolds were seeded with labeled cells, and up to 6 weeks after implantation in nude mice cells could be traced inside tissue-engineered bone. However, contrary to other reports concerning intramembranous labels, transfer of the label from labeled to unlabeled cells was detected. Transfer occurred both in vitro and in vivo between vital cells and between dead and living cells. To determine when in vivo label transfer happened, devitalized, labeled constructs were implanted for various time periods in nude mice. The presence of vital labeled cells inside these constructs, when evaluated at different implantation periods, indicated transfer of the label. Transfer occurred at 7 days postimplantation when 40 microM label was applied, whereas 10 microM labeled constructs showed transfer 10 days after implantation. These findings indicate that CM-Dil label is useful for in vivo tracing of cells for follow-up periods up to 10 days. This makes the label particularly useful for cell survival studies in tissue-engineered implants.

Bone and Bones↗
PubMed2003

Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach.

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.

Animals↗
PubMed2003

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats.

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Animals↗
PubMed2003

Tissue engineering by cocultivating human elastic chondrocytes and keratinocytes.

To date, there is no optimal way to reconstruct an external ear in cases of microtia or after trauma or burns damaging the external ear. However, success in the area of tissue engineering has indicated that autologous elastic cartilage produced in vitro might be of great importance in the future treatment of these patients. In the present study we have engineered human, elastic cartilage in vitro by culturing chondrocytes in fibrin glue. Furthermore, the engineered elastic cartilage was seeded with human keratinocytes to investigate the possibility of combining these two tissues into one integrated structure. Histological analysis and immunohistochemistry were done every second week for 10 weeks. The elastic chondrocytes were shown to grow well in the matrix and proliferated in a dense pattern. After 10 weeks a matrix containing elastin was shown by staining with orcein, indicating that an elastic cartilage had been formed. The seeded keratinocytes adhered to the cartilage, proliferated, and formed a stratified epidermal layer, which was shown by routine histological staining and immunohistochemistry. This study shows that human elastic chondrocytes can be cultured in fibrin glue and that human keratinocytes can be cocultured with this engineered cartilage, which might be of great importance in future reconstruction of ears.

Biomarkers↗
PubMedDate not supplied

Fetal liver as a source of autologous progenitor cells for perinatal tissue engineering.

Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.

Adipocytes↗
PubMedDate not supplied

Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.

Animals↗
PubMed2005

Interleukin 7-engineered stromal cells: a new approach for hastening naive T cell recruitment.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Animals↗
PubMed1998

Voluntary guidance for the development of tissue-engineered products.

Tissue Engineering is an emerging field of medical research in which there is tremendous activity. Many of these products rely on the use of a cellular component co-formulated with a natural or synthetic biomaterial. At this time, though, there are no consensus safety or efficacy standards for tissue-engineered products. We describe general approaches for assessment of the safety and efficacy of cell-based tissue-engineered products which will lead to reliable medical products for human use. This article provides a general summary of the factors that should be considered in the design and development of cell- and tissue-based products. Seven areas are considered: cell and tissue sourcing; cell and tissue characterization; biomaterials testing; quality assurance; quality control; and nonclinical testing and clinical evaluation. Factors relevant to these areas have been discussed to provide a set of recommendations on which development of products can be standardized. Where relevant, the discussion has been separated in each area to issues that are independent or dependent on cell source. Also, examples are provided of how these guidelines would be applied to two product types that represent somewhat extreme ends of the spectrum for tissue engineering applications. The first example is a product whose mechanism of action is to provide locally-acting structural repair or enhancement in vivo. The second example is a product whose mechanism of action involves systemically distributed physiologically or pharmacologically active products. In general, we have limited the discussion of product types to those that are implanted into the patient for relatively long periods of time. We believe that adoption of these voluntary guidelines would lead to products that are more consistent in quality and performance as well as more rapidly developed.

Animals↗
PubMedDate not supplied

Potential of nanofiber matrix as tissue-engineering scaffolds.

Tissue-engineering scaffolds should be analogous to native extracellular matrix (ECM) in terms of both chemical composition and physical structure. Polymeric nanofiber matrix is similar, with its nanoscaled nonwoven fibrous ECM proteins, and thus is a candidate ECM-mimetic material. Techniques such as electrospinning to produce polymeric nanofibers have stimulated researchers to explore the application of nanofiber matrix as a tissue-engineering scaffold. This review covers the preparation and modification of polymeric nanofiber matrix in the development of future tissue-engineering scaffolds. Major emphasis is also given to the development and applications of aligned, core shell-structured, or surface-functionalized polymer nanofibers. The potential application of polymer nanofibers extends far beyond tissue engineering. Owing to their high surface area, functionalized polymer nanofibers will find broad applications as drug delivery carriers, biosensors, and molecular filtration membranes in future.

Animals↗
PubMedDate not supplied

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

Algorithms↗
PubMedDate not supplied

Myocardial engineering in vivo: formation and characterization of contractile, vascularized three-dimensional cardiac tissue.

Engineering cardiac tissue in three dimensions is limited by the ability to supply nourishment to the cells in the center of the construct. This limits the radius of an in vitro engineered cardiac construct to approximately 40 microm. This study describes a method of engineering contractile three-dimensional cardiac tissue with the incorporation of an intrinsic vascular supply. Neonatal cardiac myocytes were cultured in vivo in silicone chambers, in close proximity to an intact vascular pedicle. Silicone tubes were filled with a suspension of cardiac myocytes in fibrin gel and surgically placed around the femoral artery and vein of adult rats. At 3 weeks, the tissues in the chambers were harvested for in vitro contractility evaluation and processed for histologic analysis. By 3 weeks, the chambers had become filled with living tissue. Hematoxylin and eosin staining showed large amounts of muscle tissue situated around the femoral vessels. Electron micrographs revealed well-organized intracellular contractile machinery and a high degree of intercellular connectivity. Immunostaining for von Willebrand factor demonstrated neovascularization throughout the constructs. With electrical stimulation, the constructs were able to generate an average active force of 263 microN with a maximum of 843 microN. Electrical pacing was successful at frequencies of 1 to 20 Hz. In addition, the constructs exhibited positive inotropy in response to ionic calcium and positive chronotropy in response to epinephrine. As engineering of cardiac replacement tissue proceeds, vascularization is an increasingly important component in the development of three-dimensional structures. This study demonstrates the in vivo survival, vascularization, organization, and functionality of transplanted myocardial cells.

Action Potentials↗
PubMed2006

Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.

Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ.

Animals↗
PubMed2006

Review. Hyaluronan: a powerful tissue engineering tool.

Hyaluronan (HA) is a versatile molecular tool with considerable potential for tissue engineering applications. The inclusion of HA has created biocompatible biomaterials and engineered tissues that can be crosslinked or degraded controllably and can facilitate angiogenesis, osteointegration, and cell phenotype preservation. The utility of HA in tissue engineering has been broadened further by the recently identified HA synthases, which can be manipulated to stimulate the endogenous production of HA by cells seeded within biomaterial scaffolds. Overall, HA shows great promise in the development of engineered tissues and biomaterials for a variety of biomedical needs including orthopedic, cardiovascular, pharmacologic, and oncologic applications.

Animals↗
PubMed2006

Novel geometries for tissue-engineered tendonous collagen constructs.

A promising approach to addressing the performance limitations of currently available mechanical and bioprosthetic heart valves lies in tissue engineering. Tissue-engineered valves should incorporate the complex microstructure of the native valves to mimic their unique mechanics. This would include a layered topology, mesh networks, and branched collagen fiber bundles. Our approach to heart valve tissue engineering is to develop the functional components of the aortic valve cusps separately in vitro and, once they are mature, integrate them into a composite valve structure. Here we report on our efforts to create more complex collagenous structures, suitable for heart valve tissue engineering. Collagen fiber bundles were fabricated using the principle of directed collagen gel contraction, using neonatal rat aortic smooth muscle cells and acid-soluble type I rat-tail tendon collagen. The collagen gels were cast into rectangular or branched wells with porous end holders that constrained the gels longitudinally but allowed contraction to occur transverse to the long axis. Pairs of such constructs were placed in direct contact with each other and cultured further to determine whether they integrated to form continuous tissue. After 6-8 weeks of culture, highly compacted and aligned collagen fiber bundles formed. Mechanical testing revealed that linear constructs (2 free ends) with an 8:1 aspect ratio were significantly stronger than similar constructs with an aspect ratio of 2:1 (mean +/- SD, 298 +/- 90 kPa vs. 152 +/- 49 kPa; p < .001). Branching reduced mechanical strength considerably. Constructs fabricated with 4 free ends were significantly weaker than constructs with 3 ends (31 +/- 32 kPa vs. 116 +/- 66 kPa; p < .003). Histologic images demonstrated the integration of the crossed collagen bundles, with a bonding strength of 2.1 +/- 1.1 g (0.02 N). We found that the geometry of the molds into which the collagen constructs are cast can greatly affect their mechanical strength: multibranched constructs were the weakest, and long, linear constructs were the strongest. We also found that integration of collagen constructs occurs in vitro and that the fabrication of a composite structure in vitro is probably feasible.

Absorbable Implants↗
PubMed2006

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Animals↗
PubMed2006

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.

Aggrecans↗