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PubMed2006

Tissue engineering of endothelial cells and the immune response.

BACKGROUND: While tissue engineering offers promise for organ and tissue transplantation, it can also be used to examine transplant and immune biology. Endothelial cells engrafted within 3-dimensional matrices create stable units that produce all of the factors of a functional quiescent endothelium. Perivascular implantation of tissue engineered endothelial cell constructs provides long-term control of vascular repair after injury. This control is established without restoration of the natural luminal:mural endothelium, and most intriguingly, without engendering host allo- and xenogeneic immune responses. We examined how endothelial immunogenicity is controlled by interaction with 3-dimensional matrices. MATERIALS AND METHODS: Human aortic endothelial cells (HAE) were either grown to confluence on polystyrene tissue culture plates or within 3-dimensional collagen-based matrices. Major histocompatibility complex (MHC) class II, integrin, interferon (IFN)-gamma receptor expression, and signaling were analyzed via confocal microscopy, flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), and microarray. Splenocyte proliferation was assayed by thymidine incorporation. RESULTS: Despite similar expression levels of IFN-gamma receptors, matrix-embedded HAE elicited far less STAT-1 phosphorylation upon IFN-gamma stimulation, and expressed 2-fold less MHC II than HAE grown to confluence on culture plates (P < .001). This effect correlated with reduced expression of integrin alpha(v) and beta(3) (P < .002), and muted proliferation of porcine splenocytes (P < .001). CONCLUSIONS: Matrix architecture is critical for modulation of endothelial immunogenicity. Embedding HAE within a physiologic 3-dimensional environment affects activity of intracellular signaling pathways, MHC II expression, and subsequent activation of immune cells. These findings might offer novel insights into our understanding of endothelial-mediated diseases and might enhance our ability to leverage the potential for cell-based therapies.

Animals↗

PubMed2006

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes.

Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and process-ability has demonstrated strong potential for skeletal tissue engineering. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and re-differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-beta1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.

Animals↗

PubMed2005

Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support.

OBJECTIVE: Treatment and management of congenital as well as post-traumatic trachea stenosis remains a challenge in pediatric surgery. The aim of this study was to reconstruct a trachea with human nasal septum chondrocytes by using the combination of biodegradable hydrogel and non-biodegradable high-density polyethylene (HDP) as the internal predetermined shape scaffold. METHODS: Human nasal septum cartilage was harvested as excessive tissue after elective septoplasty and digested in 0.6% collagenase II. Chondrocytes were cultured in an equal volume mix of Ham's F12 medium and Dulbecco's modified eagle medium added with 10% fetal bovine serum and basic fibroblast growth factor. After two passages, the cultured chondrocytes were trypsinized and mixed with biodegradable hydrogel Pluronic F127. The chondrocytes-hydrogel admixture was then painted over the HDP as the internal support in a predetermined trachea shape. The composite was then implanted subcutaneously in athymic mice. RESULTS: After 8 weeks of in vivo implantation, the tissue engineered trachea constructs were harvested. Macroscopic appearance of the tissue engineered trachea constructs demonstrated that the HDP were 80-90% covered with yellowish glistering cartilage like tissue without any sign of inflammation. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in basophilic matrix and stained red with Safranin-O staining denoting abundant proteoglycans production. Type II collagen gene which was expressed in native cartilage was highly expressed in this tissue engineered trachea cartilage. CONCLUSION: We have successfully reconstructed a trachea in vivo with human nasal septum chondrocytes using HDP as the internal support. This construct has the advantage of bio-inert and strength in which both are important properties in tracheal reconstruction.

Animals↗

PubMed2005

Tissue engineering of functional salivary gland tissue.

OBJECTIVES: Treatment of conditions that lead to salivary hypofunction, such as radiation induced xerostomia, is currently limited to the administration of saliva substitutes and sialogogues. The transient effect of these medications necessitates frequent administration, and systemic side effects may be intolerable. The creation of implantable, functional salivary gland tissue from autologous glandular cells would provide a physiologic solution to this problem. We investigated the feasibility of engineering such tissue in vivo. METHODS: Primary human salivary gland cells were grown, expanded, and seeded on biodegradable polymer scaffolds. A total of 48 scaffolds with cells and 16 control polymers without cells were implanted subcutaneously in athymic mice. The implants were retrieved 2, 4, and 8 weeks after the implantation for phenotypic and functional analyses. RESULTS: Human salivary gland epithelial cells retained their phenotypic and functional characteristics at all culture stages. Histologically, formation of acinar gland-like structures was observed within the engineered tissue by 4 weeks after implantation. Immunocytochemical and Western blot analyses of the implanted tissues demonstrated the expression of human a-amylase, cytokeratins AE1/AE3, and aquaporin-5 using cell-specific antibodies. Reverse-transcription polymerase chain reaction analyses confirmed the expression of human salivary type of alpha-amylase (sigma-amylase) mRNA. The retrieved tissues demonstrated the production of human alpha-amylase over time using a biochemical amylase detection system.

Animals↗

PubMed1996

Modification of translational control elements as a new approach to design of attenuated picornavirus strains.

The translation machineries of different host cells may exhibit varying requirements for a specific structure of cis-acting control elements in the viral RNA templates. Thus, the appropriately spaced oligopyrimidine/AUG tandem (OAT), a conserved control element in the 5' noncoding region of the picornavirus genomes, is dispensable for the growth of Theiler's murine encephalomyelitis virus (TMEV) in BHK-21 cells, but is essential for the neurovirulence of this virus. Also, the replacement of the cryptic (non-initiator) AUG moiety of the wild-type poliovirus OAT by the initiator AUG affects the viral reproduction in cultured cells only slightly, whereas neurovirulence of the relevant mutants is dramatically suppressed. These observations allow us to propose a rational way to construct novel attenuated viral strains by elimination or severe modification of host-specific regulatory regions in their genomes. The relevant genetic rearrangements may be so extensive that the probability of reversion to the virulent phenotype should be negligible. The feasibility of engineering of highly attenuated and genetically stable TMEV and poliovirus variants is illustrated.

Animals↗

PubMed2003

Identification and quantification of bisphenol A by gas chromatography and mass spectrometry in a lab-scale dual membrane system.

Endocrine disruptor contamination is an emerging issue of concern in the field of water quality engineering. In this study, a lab-scale microfiltration (MF) and reverse osmosis (RO) based water reclamation system was set up to monitor and evaluate the removal of bisphenol A (BPA), which is a known oestrogenic compound. The identification and quantification of BPA were performed by using gas chromatography coupled with mass spectrometry. It was noted that the detection method used in this study was able to achieve an average recovery ranging from 88.2 to 94.1% of BPA with standard deviations of less than 10% in different spiked samples. The detection limit of the analytical protocol was determined at 20 ng L(-1). Based on the analytical protocol, it was noted that a low level of BPA (1.18-3.04 microg L(-1)) could be detected in feed water (effluent of an activated sludge treatment system) to the dual membrane water reclamation system. The results obtained suggested that BPA could be easily chlorinated by sodium hypochlorite with a dosage of 4 to 5 mg L(-1) and a contact time of 1 to 2 min. In this lab-scale study, a satisfactory removal of BPA was readily obtained by RO and BPA was abated to an undetectable level in the product water. It was noted that the RO rejection characteristic of BPA was not sensitive to the variations in raw feed water characteristics experienced in this study. In addition, it was noted that BPA concentration present in raw feed water did not exert any significant impact on RO performance in terms of BPA rejection. The results of this study demonstrated that membrane technology could be effectively used for BPA removal.

Benzhydryl Compounds↗

PubMed2002

Telomerase recognizes its template by using an adjacent RNA motif.

Telomerase adds telomeric repeats to chromosome 3' ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication. The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA. Here, using circularly permuted variants of Tetrahymena thermophila telomerase RNA, we identify the features that allow recognition of the template region within the RNA. We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately. Only a short "template-recognition" element sequence tag is required to direct efficient use of adjacent 5' residues as a template for telomeric repeat synthesis. Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis.

Animals↗

PubMed1993

Improved method for expression of Kunitz-type serine proteinase inhibitor domain of beta-amyloid protein precursor in Escherichia coli and characterization of disulfide bonds of the product.

Kunitz-type serine proteinase inhibitor (KPI) domain of Alzheimer's disease-related beta-amyloid protein precursor (APP) was expressed in Escherichia coli as a fusion protein with a truncated form of Staphylococcus protein A. The fusion protein was purified from the cell culture medium using an IgG Sepharose column. The KPI domain was separated from the protein A portion by cleavage with human alpha-thrombin at the engineered recognition sequence, followed by purification on IgG Sepharose and reversed-phase HPLC columns. The recombinant KPI domain strongly inhibited trypsin; the inhibition constant (Ki) for bovine trypsin was 2.5 x 10(-11) M, comparable to those of the secreted forms of APP with the KPI domain. The recombinant protein contained three intramolecular disulfide bonds, which were determined to be located between Cys-6 (C1) and Cys-56 (C6), Cys-15 (C2) and Cys-39 (C4), and Cys-31 (C3) and Cys-52 (C5) of the recombinant KPI domain, respectively. These positions are highly homologous to those of disulfide bonds in bovine pancreatic trypsin inhibitor. The trypsin-inhibitory activity of the recombinant protein was abolished by preincubation with 0.4 mM dithiothreitol under non-denaturing conditions. By this mild reduction, all the disulfide bonds were completely cleaved. These results clearly indicate that the disulfide bonds play an important role in the function of the KPI domain of APP.

Alzheimer Disease↗

PubMed2006

Plasticization-enhanced hydrogen purification using polymeric membranes.

Polymer membranes are attractive for molecular-scale separations such as hydrogen purification because of inherently low energy requirements. However, membrane materials with outstanding hydrogen separation performance in feed streams containing high-pressure carbon dioxide and impurities such as hydrogen sulfide and water are not available. We report highly permeable, reverse-selective membrane materials for hydrogen purification, as exemplified by molecularly engineered, highly branched, cross-linked poly(ethylene oxide). In contrast to the performance of conventional materials, we demonstrate that plasticization can be harnessed to improve separation performance.

Journal Article↗

PubMed2004

Replication and transcription of the rotavirus genome.

Rotaviruses are an important cause of human morbidity and mortality, representing the primary pathogens responsible for acute dehydrating diarrhea in children under the age of 3. The infectious rotavirus particle is made up of three concentric layers of protein, and contains a genome consisting of eleven segments of double-stranded (ds)RNA. Upon infection, RNA polymerases associated with double-layered virus particles are activated, resulting in genome transcription and extrusion of the eleven viral mRNAs from such particles. The mRNAs not only direct protein synthesis, but also serve as templates for minus-strand synthesis to yield dsRNAs. Synthesis of the dsRNAs is an event that occurs following the gene-specific packaging of viral mRNAs into core-like assembly intermediates. Electron-dense cytoplasmic inclusions, termed viroplasms, function as sites of genome packaging and replication in the infected cell. Our understanding of key events in the viral life cycle has been advanced considerably by the development of cell-free systems that support mRNA synthesis from virion-derived double-layered particles and dsRNA synthesis from virion-derived core particles. The recent expression and purification of rotavirus recombinant proteins have also allowed progress to be made in defining the roles of viral proteins in genome replication and viroplasm formation. However, our efforts towards a full description of the viral life cycle, most notably an understanding of the events occurring during gene-specific packaging, remain hampered by the lack of a cell-free packaging system and a reverse genetics systems. The lack of a reverse genetics systems also confounds efforts towards the generation of molecular engineered second-generation vaccines.

Cell-Free System↗

PubMed2002

Gene expression: a review of clinical applications in otorhinolaryngology-head and neck surgery.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body or stimulating cells to grow into implanted matrix. In this short review, we aim to examine current techniques in gene expression analysis and their relevant clinical applications to the field of otorhinolaryngology-head and neck surgery.

Gene Expression↗

PubMed2004

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering.

BACKGROUND: In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering. METHODS: DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope (SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated. RESULTS: DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs. CONCLUSION: dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.

Bone Marrow Cells↗

PubMed2005

Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line.

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.

Animals↗

PubMed2004

Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

Animals↗

PubMed2004

Phenotypic expression of collagen type II and collagen type I gene in monolayer culture of human auricular chondrocytes.

Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.

Cells, Cultured↗

PubMed2002

Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.

Biocompatible Materials↗

PubMed2000

Stabilization of the U5-leader stem in the HIV-1 RNA genome affects initiation and elongation of reverse transcription.

Reverse transcription of the Human Immunodeficiency Virus type I (HIV-1) RNA genome is primed by a cellular tRNA-lys3 molecule that binds to the primer binding site (PBS). The PBS is predicted to be part of an extended RNA structure, consisting of a small U5-PBS hairpin and a large U5-leader stem. In this study we stabilized the U5-leader stem of HIV-1 to study its role in reverse transcription. We tested in vitro synthesized wild-type and mutant templates in primer annealing, initiation and elongation assays. Stabilization of the stem inhibits the initiation of reverse transcription, but not the annealing of the tRNA primer onto the PBS. These results suggest that stabilization of the stem results in occlusion of a sequence motif that is involved in an additional interaction with the tRNA-lys3 primer and that is needed to trigger the initiation of reverse transcription. The stable structure was also found to affect the elongation of reverse transcription, causing the RT enzyme to pause upon copying 7-8 bases into the extended base paired stem. The stabilizing mutations were also introduced into proviral constructs for replication studies, demonstrating that the mutant viruses have a reduced replication capacity. Analysis of a revertant virus demonstrated that opening of the stabilized U5-leader stem can restore both virus replication and reverse transcription.

Base Pairing↗

PubMed2002

Silk matrix for tissue engineered anterior cruciate ligaments.

A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy, DNA quantitation and the expression of collagen types I and III and tenascin-C markers. The results support the conclusion that properly prepared silkworm fiber matrices, aside from providing unique benefits in terms of mechanical properties as well as biocompatibility and slow degradability, can provide suitable biomaterial matrices for the support of adult stem cell differentiation toward ligament lineages. These results point toward this matrix as a new option for ACL repair to overcome current limitations with synthetic and other degradable materials.

Animals↗