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PubMed2002

Production and characterization of an in vitro engineered human oral mucosa.

The role of epithelial cells in oral pathologies is poorly understood. Until now, most studies have used normal or transformed epithelial cell monolayers, a system that largely bypasses oral mucosal complexity. To overcome these limitations, an engineered human oral mucosa (EHOM) model has been produced and characterized. Following histological and immunohistochemical analyses, EHOM showed well-organized and stratified tissues in which epithelial cells expressed proliferating keratins such as Ki-67, K14, and K19 and also differentiating keratin (K10). In this model, epithelial cells interacted with fibroblasts in the lamina propria by secreting basement membrane proteins (laminins) and by expressing integrins (beta1 and alpha2beta1). Cytokine analyses using cultured supernatants showed that cells in EHOM were able to secrete interleukins (IL) including IL-1beta and IL-8 and tumor necrosis factor alpha (TNF-alpha). Finally, cells in this engineered model were able to secrete different metalloproteinases such as gelatinase-A and gelatinase-B. In conclusion, using tissue engineering technology, we produced well-organized EHOM tissues. It is anticipated that this model will be useful for examining mechanisms involved in oral diseases under controlled conditions by modeling the interactions between mucosa and microorganisms in the oral cavity.

Animals↗
PubMed2004

Tissue engineering applications of therapeutic cloning.

Few treatment options are available for patients suffering from diseased and injured organs because of a severe shortage of donor organs available for transplantation. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for replacement therapy. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Animals↗
PubMed2006

Cell-based tissue engineering for lung regeneration.

Emphysema is a chronic lung disease characterized by alveolar enlargement and tissue loss. Tissue engineering represents an attractive potential for regeneration of several organ systems. The complex three-dimensional architectural structure of lung parenchyma requiring connections of alveolar units to airways and the pulmonary circulation makes this strategy less optimistic. In the present study, we used Gelfoam sponge as a scaffold material, supplemented with fetal rat lung cells as progenitors, to explore the potential application of cell-based tissue engineering for lung regeneration in adult rats. After injection into lung parenchyma, the sponge showed porous structures similar to alveolar units. It did not induce severe local inflammatory response. Fetal lung cells in the sponge were able to survive in the adult lung for at least 35 days, determined by CMTMR [5-(and-6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine] labeling. Proliferation of cells within the sponge was demonstrated in vivo by bromodeoxyuridine (BrdU) labeling. Cells formed "alveolar-like structures" at the border between the sponge and the surrounding lung tissue with positive immunohistochemical staining for epithelial and endothelial cells. Neovascularization of the sponge was demonstrated with India ink perfusion. The sponge degraded after several months. This study suggests that cell-based tissue engineering possesses the potential to regenerate alveolar-like structures, an important step towards our ultimate goal of lung regeneration.

Absorbable Implants↗
PubMed2001

Engineered cartilage, bone, joints, and menisci. Potential for temporomandibular joint reconstruction.

Reconstruction of many musculoskeletal structures can be accomplished by bone grafting and implantation of prostheses. Alternate approaches are needed, however, for repair of complex structures such as articular cartilage surfaces and the temporomandibular meniscus and joint. Tissue engineering, either cell-free or cell-based, offers promise because of recent advances in materials research and in our knowledge of the cellular and molecular mechanisms of tissue repair. There are three considerations in designing a construct for engineered tissue: the source of cells, if any; the nature of the carrier or scaffold; and use, if any, of genes, factors, or adjuvants. Autogenous cells, often expanded in vitro, have been useful for cartilage tissue engineering. Precursor/progenitor cells are advantageous for bone tissue. There are many natural and synthetic resorbable materials with good biocompatibility and tissue compatibility that can be modified to have the porosity and mechanical properties needed for specific applications. The scaffolds can also be modified to provide biological signals to augment repair and integration.

Animals↗
PubMed2005

Recent developments in durum wheat chromosome engineering.

Transfer of alien chromosome segments from various Triticeae species into cultivated wheats, commonly referred to as "chromosome engineering", is currently benefiting from the recent, impressive advancements in molecular genetics, cytogenetics and genomics, which are providing new insights into the genetic and physical organization of even complex plant genomes, such as those of the Triticeae. The powerful analytical tools presently available are making the assessment of desired genotypes in the course of chromosome engineering far more precise and effective than in the past, thus giving this transfer strategy renewed and increased potential for meaningful practical achievements. Examples are given here of the application of such tools to the engineering of the durum wheat genome with small alien segments containing genes with beneficial impact on disease resistance and quality traits.

Chromosomes, Plant↗
PubMed2005

Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering.

Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications.

Apoptosis↗
PubMed2006

Mesenchymal stem cells in tissue engineering.

The repair of diseased or damaged cartilage remains one of the most challenging problems of musculoskeletal medicine. Tissue engineering advances in cartilage repair have utilized autologous and allogenic chondrocyte and cartilage grafts, biomaterial scaffolds, growth factors, stem cells, and genetic engineering. The mesenchymal stem cell has specifically attracted much attention because of its accessibility, potential for differentiation, and manipulability to modern molecular, tissue and genetic engineering techniques. Mesenchymal stem cells provide invaluable tools for the study of tissue repair when combined with a carrier vehicle/matrix scaffold, and/or bioactive growth factors. However, an underappreciated source of knowledge lies in the relationship between fetal development and adult tissue repair. The multitude of events that take place during fetal development which lead from stem cell to functional tissue are poorly understood. A more thorough understanding of the events of development as they pertain to cartilage organogenesis may help elucidate some of the unknowns of adult tissue repair.

Animals↗
PubMed2006

Adipose tissue engineering by human adipose-derived stromal cells.

Tissue engineering has emerged as a promising alternative approach to current clinical treatments for restoration of soft tissue defects. The purpose of this study was to investigate adipose tissue formation in vitro and in vivo by using human adipose-derived stromal cells (ADSCs) utilizing a gelatin sponge (Gelform) as a scaffold. Adipogenic potentials of human ADSCs were demonstrated by Oil-O-red staining and cellular morphology. After seeding human ADSCs in a density of 3 x 10(6) cells/ml on three-dimensional gelatin sponges, tissue-engineered constructs were exposed to adipogenic differentiation medium for in vitro studies and implanted in the backs of severe combined immunodeficient (SCID) mice for in vivo adipose regeneration. Adipogenesis of ADSC-seeded gelatin sponges was confirmed by Oil-O-red staining after 4 weeks of in vitro incubation. The optical density of the elution from Oil-O-red staining of adipogenic constructs is significantly higher than that of the control group (p < 0.05, n = 4). With short-term in vitro differentiation, adipogenic constructs turned into fat tissue 4 weeks after in vivo implantation, confirmed by biochemical and immunohistochemical examination. No adipogenic-morphological change or fat formation was observed in in vitro or in vivo studies when ADSCs were exposed to a control medium without adipogenic stimulation. These results indicate that engineered adipose tissue can be achieved using human ADSCs and biocompatible and degradable gelatin sponges.

Adipose Tissue↗
PubMed2007

Different types of scaffolds for reconstruction of the urinary tract by tissue engineering.

INTRODUCTION: Tissue engineering is an important and expanding field in reconstructive surgery. The ideal biomaterial for urologic tissue engineering should be biodegradable and support autologous cell growth. We examined different scaffolds to select the ideal material for the reconstruction of the bladder wall by tissue engineering. MATERIALS AND METHODS: We seeded mouse fibroblasts and human keratinocytes in a co-culture model on 13 different scaffolds. The cell-seeded scaffolds were fixed and processed for electron microscopy, hematoxylin and eosin stain, and immunohistochemistry. Cell density and epithelial cell layers were evaluated utilizing a computer-assisted optical measurement system. RESULTS: Depending on the growth pattern, scaffolds were classified into the following three distinct scaffold types: carrier-type scaffolds with very small pore sizes and no ingrowth of the cells. This scaffold type induces a well-differentiated epithelium. Fleece-type scaffolds with fibers and huge pores. We found cellular growth inside the scaffold but no epithelium on top of it. Sponge-type scaffolds with pores between 20 and 40 microm. Cellular growth was observed inside the scaffold and well-differentiated epithelium on top of it. CONCLUSION: To our knowledge, this is the first time three distinct scaffold types have been reported. All types supported the cell growth. The structure of the scaffolds affects the pattern of cell growth.

Absorbable Implants↗
PubMed2006

Pulsatile myocardial tubes fabricated with cell sheet engineering.

BACKGROUND: Tissue engineering approaches involving the direct transplantation of cardiac patches have received significant attention as alternative methods for the treatment of damaged hearts. In contrast, we used cardiomyocyte sheets harvested from temperature-responsive culture dishes to create pulsatile myocardial tubes and examined their in vivo function and survival. METHODS AND RESULTS: Neonatal rat cardiomyocyte sheets were sequentially wrapped around a resected adult rat thoracic aorta and transplanted in place of the abdominal aorta of athymic rats (n=17). Four weeks after transplantation, the myocardial tubes demonstrated spontaneous and synchronous pulsations independent of the host heartbeat. Independent graft pressures with a magnitude of 5.9+/-1.7 mm Hg due to their independent pulsations were also observed (n=4). Additionally, histological examination and transmission electron microscopy indicated that the beating tubes were composed of cardiac tissues that resemble the native heart. Finally, when myocardial tubes used for aortic replacement were compared with grafts implanted in the abdominal cavity (n=7), we observed significantly increased tissue thickness, as well as expression of brain natriuretic peptide, myosin heavy chain-alpha, and myosin heavy chain-beta. CONCLUSIONS: Functional myocardial tubes that have the potential for circulatory support can be created with cell sheet engineering. These results also suggest that pulsation due to host blood flow within the lumen of the myocardial tubes has a profound effect on stimulating cardiomyocyte hypertrophy and growth. These results demonstrate a novel approach for the future development of engineered cardiac tissues with the ability for independent cardiac assistance.

Animals↗
PubMed2006

Functional growth in tissue-engineered living, vascular grafts: follow-up at 100 weeks in a large animal model.

BACKGROUND: Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model. METHODS AND RESULTS: Vascular grafts fabricated from biodegradable scaffolds (ID 18+/-l mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21 days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for < or = 100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography-angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries. CONCLUSIONS: This study provides evidence of growth in living, functional pulmonary arteries engineered from vascular cells in a full growth animal model.

Absorbable Implants↗
PubMed2006

Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds.

BACKGROUND: Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-beta1. Method and Results- Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2 x 10(6) cells/cm2, scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-beta1. After seeding at 15 x 10(6) cells/cm2, scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-beta1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31+, von Willebrand factor+, and alpha-smooth muscle actin (SMA)-) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31+ and alpha-SMA+) in response to TGF-beta1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-beta1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-beta1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-beta1-mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-beta1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-beta1-stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls. CONCLUSIONS: Stimulation of EPC-seeded tissue engineering scaffolds with TGF-beta1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-beta1 could offer new strategies to guide tissue formation in engineered cardiac valves.

Actins↗
PubMed1996

Engineering multiversion neural-net systems.

In this paper we address the problem of constructing reliable neural-net implementations, given the assumption that any particular implementation will not be totally correct. The approach taken in this paper is to organize the inevitable errors so as to minimize their impact in the context of a multiversion system, i.e., the system functionality is reproduced in multiple versions, which together will constitute the neural-net system. The unique characteristics of neural computing are exploited in order to engineer reliable systems in the form of diverse, multiversion systems that are used together with a "decision strategy" (such as majority vote). Theoretical notions of "methodological diversity" contributing to the improvement of system performance are implemented and tested. An important aspect of the engineering of an optimal system is to overproduce the components and then choose an optimal subset. Three general techniques for choosing final system components are implemented and evaluated. Several different approaches to the effective engineering of complex multiversion systems designs are realized and evaluated to determine overall reliability as well as reliability of the overall system in comparison to the lesser reliability of component substructures.

Algorithms↗
PubMed2002

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds.

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.

Coated Materials, Biocompatible↗
PubMed2004

Manufacture of elastic biodegradable PLCL scaffolds for mechano-active vascular tissue engineering.

A soft and very elastic poly(lactide-co-epsilon-caprolactone) (PLCL)(50:50, Mn 185 x 10(3)) was synthesized. Tubular scaffolds were prepared by an extrusion-particulate leaching method for mechano-active vascular tissue engineering. The copolymer was very flexible but completely rubber-like elastic. Even the high porous PLCL scaffolds (90% salt wt) exhibited 200% elongation, but recovery over 85% in a tensile test. Moreover, the PLCL scaffolds maintained their high elasticity also in culture media under cyclic mechanical strain conditions. The highly porous scaffold (90% salt wt) withstood for an initial 1 week without any deformation and sustained for 2 weeks in culture media under cyclic stress of 10% amplitude and at 1 Hz frequency which are similar to the natural vascular conditions. Vascular smooth muscle cells (VSMCs) were seeded on to the PLCL scaffolds. The cell adhesion and proliferation on the scaffolds of various pore-size were increased with increasing pore size. For the pore sizes of 50-100 microm, 100-150 microm, 150-200 microm and 200-250 microm, the ratios of cell numbers were about 1:1.2:1.9:2.2, respectively, at both 12 h and 5 days. Similarly, the higher porous scaffolds exhibited more cell adhesion and proliferation compared to lower porous one, where the effect was more pronounced in the longer proliferation period. SMC-seeded scaffolds were implanted subcutaneously in athymic nude mice to confirm the biocompatibility. Such a high elastic property and proper biocompatibility to SMCs of PLCL scaffolds prepared in this study will be very useful to engineer SM-containing tissues such as blood vessels under mechanically dynamic environments (mechano-active tissue engineering).

Animals↗
PubMed2005

Nanophononics: phonon engineering in nanostructures and nanodevices.

Phonons, i.e., quanta of lattice vibrations, manifest themselves practically in all electrical, thermal and optical phenomena in semiconductors and other material systems. Reduction of the size of electronic devices below the acoustic phonon mean free path creates a new situation for phonon propagation and interaction. From one side, it complicates heat removal from the downscaled devices. From the other side, it opens up an exciting opportunity for engineering phonon spectrum in nanostructured materials and achieving enhanced operation of nanodevices. This paper reviews the development of the phonon engineering concept and discusses its device applications. The review focuses on methods of tuning the phonon spectrum in acoustically mismatched nano- and heterostructures in order to change the phonon thermal conductivity and electron mobility. New approaches for the electron-phonon scattering rates suppression, formation of the phonon stopbands and phonon focusing are also discussed. The last section addresses the phonon engineering issues in biological and hybrid bio-inorganic nanostructures.

Acoustics↗
PubMed2006

Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.

PURPOSE: To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS: Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS: Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS: The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.

Actins↗
PubMed2005

Application of tissue engineering techniques for rotator cuff regeneration using a chitosan-based hyaluronan hybrid fiber scaffold.

BACKGROUND: The current surgical procedures for irreparable rotator cuff tears have considerable limitations. Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing these conditions. HYPOTHESIS: A chitosan-based hyaluronan hybrid scaffold could enhance type I collagen products with seeded fibroblasts and thereby increase the mechanical strength of regenerated tendon in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: The scaffolds were created from chitosan-based hyaluronan hybrid polymer fibers. Forty-eight rabbit infraspinatus tendons and their humeral insertions were removed to create defects. Each defect was covered with a fibroblast-seeded scaffold (n = 16) or a non-fibroblast-seeded scaffold (n = 16). In the other 16 shoulders, the rotator cuff defect was left free as the control. At 4 and 12 weeks after surgery, the engineered tendons were assessed by histological, immunohistochemical (n = 2), and biomechanical (n = 6) analyses. RESULTS: Type I collagen was only seen in the fibroblast-seeded scaffold and increased in the regenerated tissue. The tensile strength and tangent modulus in the fibroblast-seeded scaffold were significantly improved from 4 to 12 weeks postoperatively. The fibroblast-seeded scaffold had a significantly greater tangent modulus than did the non-fibroblast-seeded scaffold and the control at 12 weeks. CONCLUSION: This scaffold material enhanced the production of type I collagen and led to improved mechanical strength in the regenerated tissues of the rotator cuff in vivo. CLINICAL RELEVANCE: Rotator cuff regeneration is feasible using this tissue engineering technique.

Animals↗