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PubMed2001

Genetically engineered mice and their use in aging research.

Genetically engineered animal models have been and will continue to be invaluable for exploring the basic mechanisms involved in the aging process as well as in extending our understanding of diseases found to be more prevalent in the older human population. Continued development of such in vivo systems will allow scientists to further dissect the role genetic and environmental factors play in aging and in age-related disease states and to enhance our understanding of these processes. In this article we discuss techniques involved in the development of such models and review some examples of laboratory mouse strains that have been used to study either normal aging or select diseases associated with aging.

Aging↗

PubMed2005

Developing the concept of adoptive cellular gene therapy of rheumatoid arthritis.

Progressive destruction of articular cartilage and bone is the pivotal problem of rheumatoid arthritis (RA). Joint destruction is the cause of severe disability and determines the long-term outcome of disease. Conventional therapy does not control this destructive process sufficiently and the anti-rheumatic drugs available today can cause severe systemic adverse effects. Local application of chondroprotective and osteoprotective agents by means of gene therapy would be an attractive alternative to conventional therapy of RA and could provide long-term expression of the therapeutic agents and minimize systemic adverse effects. For this purpose, we have developed the concept of adoptive cellular gene therapy. This treatment strategy is based on using genetically engineered cells that home specifically to sites of autoimmune inflammation and thus allow local delivery of therapeutic gene products. Ex vivo transduction of these cells avoids systemic exposure of the host to the transgene-encoding vector and thus adds to the safety of this approach. In this article of the CIS Spring School in Autoimmune Diseases 2005 proceedings, we review our work on developing the strategy of adoptive cellular gene therapy and summarize recent advances in the evaluation of therapeutic effects and the identification of novel therapeutic targets.

Adoptive Transfer↗

PubMed2006

Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus.

Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.

Animals↗

PubMed2006

Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB.

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Becaplermin↗

PubMed2005

Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation.

The development of biocompatible photopolymerizing polymers for biomedical and tissue engineering applications has the potential to reduce the invasiveness and cost of biomaterial implants designed to repair or augment tissues. However, more information is needed about the cellular toxicity of the compounds and initiators used in these systems. The current study evaluates the cellular toxicity of three ultraviolet sensitive photoinitiators on six different cell populations that are used for engineering numerous tissues. The photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959) caused minimal toxicity (cell death) over a broad range of mammalian cell types and species. It was also demonstrated that different cell types have variable responses to identical concentrations of the same photoinitiator. While inherent differences in the cell lines may contribute to the variable cytotoxicity, a correlation between cellular proliferation rate (population doubling time) and increased cytotoxicity of the photoinitiator was observed. Cell lines that divided more quickly were more sensitive to photoinitiator-induced cell death. In summary, the photoinitiator Irgacure 2959 is well tolerated by many cell types over a range of mammalian species. Cell photoencapsulation strategies may be optimized to improve cell survival by manipulating proliferation rate.

Biocompatible Materials↗

PubMed2003

Pharmacologic, biologic, and genetic engineering approaches to potentiation of donor-derived dendritic cell tolerogenicity.

There are various approaches to the enhancement of dendritic cell (DC) tolerogenicity for the promotion of cell or organ allograft survival. Both pharmacologic and biologic agents, including several commonly used immunosuppressive drugs, and specific anti-inflammatory cytokines inhibit DC maturation, whereas co-stimulation-blocking agents can also promote the induction of antigen-specific T-cell unresponsiveness by DC. Delivery of genes encoding molecules that subvert T-cell responses by various mechanisms, and targeting of DC migration by selective manipulation of chemokine and chemokine receptor expression, represent additional promising strategies. In this short review, the authors consider those approaches that have been used to promote the tolerogenicity of donor-derived DC in experimental models. Whereas most work to date has focused on myeloid DC, manipulation of other DC subsets may also offer potential for improving the outcome of transplantation and enhancing tolerance induction.

Animals↗

PubMed2003

Tissue engineering: in vitro embryonal nidation in a murine endometrial construct.

The epithelial and mesothelial cellular components of organs can be obtained as dissociated cells using adequate procedures of enzymatic digestion followed by pycnotic separation on density gradients. Using a specially developed procedure for tissue dissociation, the epithelial and connective tissue components of endometria from pseudopregnant mice were grown in culture using a combination of three dimensional culture of connective tissue components in collagen gel, with the superimposition of epithelial components in liquid medium on the surface of the gells. After a few days of growth, when the cultures became dense, murine blastocysts obtained on postcoital day 4.5 by fallopian flushing of hormonally primed and mated mice, were transferred onto the imitated endometria. The blastocysts hatched and grew on the endometrial epithelium as spherical coherent conglomerates of cells quite different from hatched blastocysts grown on the surface of a petri dish, in which the presumtive trophoblasts spred around the central mass. Light and electronmicroscopy of resin embeded sections (2 days after nidation on the simulated endometria) revealed that at least two populations of cell types were recognisable as layers. This is interpreted as an early sign of morphogenesis and the first visible steps of differentiation. The presence of mitotic figures indicates viability and continuing growth. Electronmicroscopy of cell types grown under conditions simulating in vivo tissue architectonics showed overtly less cytopathology and better cell function. Simulated endometria may, therefore, serve as an attractive model for studying early mammalian embryogenesis and the effects of toxic agents.

Animals↗

PubMed2005

In vivo behavior of decellularized vein allograft.

BACKGROUND: We are investigating decellularized vein allograft as a scaffold to engineer a non-synthetic, small-diameter vascular graft. This study examines the in vivo behavior of this scaffolding after implantation into the arterial circulation. MATERIALS AND METHODS: Canine animals underwent bilateral carotid interposition grafting using jugular vein implanted as either: 1) fresh autograft, 2) fresh allograft, or 3) decellularized allograft. Decellularization was achieved using sodium dodecyl sulfate. Grafts were examined with duplex ultrasound biweekly to determine luminal diameter, thrombosis, stenosis, or anastomotic breakdown. After perfusion fixation at 2 or 8 weeks, grafts underwent histological, morphometric, and immunohistochemical examination. RESULTS: All animals survived without neurological or hemorrhagic complication. No deterioration of graft integrity (rupture, aneurysm) was observed in any group. Luminal narrowing was observed in both allograft groups, but secondary to different pathology. Fresh allografts had significant mononuclear cell infiltrate, intimal hyperplasia, and intramural hemorrhage consistent with rejection. Conversely, decellularized allografts had minimal evidence of rejection but instead had a compact fibrin layer formed along their lumen. This fibrin layer was absent in the peri-anastomotic regions where endothelium had migrated from the native artery. By 8 weeks, decellularized grafts had repopulated with cells staining positive for smooth muscle alpha-actin. CONCLUSIONS: After 8 weeks of arterial flow, decellularized vein allograft exhibits satisfactory strength, reduced antigenicity compared to fresh allograft, and supports cellular repopulation. These characteristics make it satisfactory for further tissue engineering; combined with luminal vascular cell seeding, it may prove useful as a small-diameter arterial bypass graft.

Animals↗

PubMed2001

[Bone grafts using tissue engineering].

An overview of bone grafts and, in particular, the allografts is presented. The availability of bone allografts, has promoted their use at the expense of the autograft. However, the loss of the cellular activity in an allograft, makes them less performant than an autograft. The use of an allograft in a small size defect can be advocated provided that the implantation technique is stringent. In case of a large segmental bone defect, an allograft can be considered whereas an autograft is not anymore possible. A massive bone allograft allows an anatomical reconstruction and the preservation of strong tendon insertions. In tumor surgery, a bone allograft has become one of the best options to reshape the skeleton. To offset the poor remodeling of the massive bone allografts, and to improve the take of small size bone allografts, researches are presently carried on, using tissue engineering in order to recover a cellular population. The aim is to combine an acellular bone graft with the cells of the recipient. Cells are procured from the bone marrow. Stromal cells are isolated, cultured, so that they will grow with an osteoblastic phenotype. They can be used alone or in association with a bone graft. It is believed that tomorrow such cellular therapy will become a routine procedure.

Bone Transplantation↗

PubMed2006

Reference models for mitral valve tissue engineering based on valve cell phenotype and extracellular matrix analysis.

The advance of mitral valve repair techniques through tissue engineering is impeded by the lack of information regarding the cellular and extracellular components of the mitral valve. The present study aims to expand our understanding of the mitral valve structure by analysing the synthesis of extracellular matrix (ECM) proteins and the expression of nitric oxide synthase (NOS). Valvular endothelial cells (VECs) and valvular interstitial cells (VICs) were isolated from porcine mitral valves. Immunochemical staining of ECM components, including type I, II, III, IV and V collagen, laminin, fibronectin, elastin and chondroitin sulphate (CS), was performed on both mitral valve tissue and cell cultures. Reverse transcription polymerase chain reaction and immunochemistry were used to analyse NOS expression in native valve and in culture. Both VECs and VICs synthesised the basement membrane components, laminin and type IV collagen both in vivo and in vitro, amongst other fibrous ECM proteins. Synthesis of type I collagen and CS was absent in VEC cultures. Each cell type had a characteristic profile of NOS expression. VECs synthesised endothelial NOS both in vivo and in vitro, with a minority of VICs expressing neuronal NOS in vitro. The present study reports newly recognised aspects of the mitral valve structure and the in vitro behaviour of mitral valve cell populations based on ECM synthesis and NOS expression. The presented profiles can be used as base tools for the generation of data necessary for the selection of ideal cell sources and for the design of appropriate scaffolds for the development of effective tissue-engineered mitral valves.

Actins↗

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↗

PubMed2000

[SV40 and cell immortalization].

OBJECTIVE: To explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.

Animals↗

PubMed2006

Mechanism of bone induction by KUSA/A1 cells using atelocollagen honeycomb scaffold.

In order to induce new bone formation, mesenchymal stem cells were seeded onto atelocollagen honeycomb scaffold. We evaluated the mechanism of bone induction by KUSA/A1 cells combined with honeycomb atelocollagen scaffold. Scaffold alone, KUSA/A1 cells alone and with scaffold were implanted in the subcutaneous pockets of 4-week-old male SCID mice. The transplants were subjected to radiographical, histological and immunohistochemical examinations after 2 and 4 weeks of implantation. Radiographically, both KUSA/A1 cells alone and KUSA/A1-Scaffold showed some radiopaque areas formation but the latter disclosed a larger amount. Scaffold alone did not show any radiopacity. Histologically, Scaffold alone demonstrated only fibrous connective tissues in the periphery of the scaffold. KUSA/A1 cells alone showed few small islands of new bone formation surrounded by a thin layer of cellular proliferation. On the other hand, KUSA/A1-Scaffold revealed abundant new bone formation as well as cellular proliferation. We also determined the immunolocalization of type I collagen, CD34, Osteocalcin and PCNA in this newly formed bone. Our results indicated that less amount of stem cells are capable to induce the more amount of new bone in tissue engineering. This study support that atelocollagen honeycomb scaffold plays an important role in cellular anchorage and in vessel invasion, giving the precise shape and size for the new bone formation.

Animals↗

PubMed2004

Fetal bone cells for tissue engineering.

We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1alpha,25-dihydroxyvitamin D3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering.

Adult↗

PubMed2005

The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.

Cartilage engineering consists of re-constructing functional cartilage by seeding chondrocytes in suitable biomaterials in vitro. The characteristics of neocartilage differ upon the type of biomaterial chosen. This study aims at determining the appropriate scaffold material for articular cartilage reconstruction using non articular chondrocytes harvested from rat sternum. For this purpose, the use of polysaccharide hydrogels such as alginate (AA) and hyaluronic acid (HA) was investigated. Several ratios of AA/HA were used as well as three derivatives obtained by chemical modification of HA (HA-C18, HA-C12(2.3), HA-C12(2.5)-TEG0.5). Sternal chondrocytes were successfully cultured in 3D alginate and alginate/HA scaffolds. HA retention in alginate beads was found to be higher in beads seeded with cells than in beads without cells. HA-C18 improved HA retention in beads but inhibited the chondrocyte synthesis process. Cell proliferation and metabolism were enhanced in all biomaterials when beads were mechanically agitated. Preliminary results have shown that the chondrocyte neo-synthesised matrix had acquired articular characteristics after 21 days culture.

Alginates↗

PubMedDate not supplied

Metabolic engineering: techniques for analysis of targets for genetic manipulations.

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated.

Animals↗

PubMed2006

Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three-dimensional collagen gels.

Tissue-engineered ligament substitutes have the potential to become an alternative graft source for ligament reconstruction. If this approach is to become viable, one must first understand and define the mechanisms responsible for creation, maintenance, and remodeling of the native anterior cruciate ligament. It is well accepted that mechanical load alters fibroblast phenotypic expression in a variety of cell sources; however, the mechanosensitive pathways responsible for alteration in matrix production, remodeling, and alignment are unknown. We hypothesize that cell surface integrins play a role in this mechanotransduction process, and as such respond to application of cyclic tensile load. Linear 3D collagen gels containing canine ACL fibroblasts were created in Flexercell Tissue-Train Culture Plates. Gels were untethered (control), tethered without external strain (tethered), or tethered and exposed to 2.5% cyclic strain for 2 h per day for 4 days (strain). Quantitation of alpha1, alpha5, and beta1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of alpha5 and beta1 integrin subunits was increased significantly in fibroblasts in tethered and strained 3D collagen gels compared with the control, unloaded constructs (p < 0.05). These integrins are known to function as mechanotransducers in other tissues, implicating a similar role in mechanotransduction in ACL fibroblasts. Histologic analysis of the tethered and strained gels demonstrated a linear arrangement of cells and parallel collagen fibril architecture. In contrast, cell distribution and collagen alignment were disorganized in the control, unloaded gels. The alignment of cells and collagen in the 3D gels parallel to applied strain is similar to the in vivo state. These data add to our understanding of the behavior of ACL fibroblasts in vitro. The ability to manipulate signal transduction pathways may enhance our ability to engineer implantable ACL grafts or to modify ACL healing response.

Animals↗

PubMed2006

Development of antibodies and chimeric molecules for cancer immunotherapy.

Monoclonal antibodies are among the most rapidly expanding class of therapeutics for cancer treatment. Monoclonal antibodies targeting non-Hodgkin's lymphoma (NHL), Her-2/neu highly expressing metastatic breast cancer, colorectal cancer, acute myelogenous leukemia, and B-cell chronic lymphocytic leukemia (CLL) have received FDA approval. Promising new targets for antibody therapy include cellular growth factor receptors, mediators of tumor-driven neo-angiogenesis, as well as host negative immunoregulatory checkpoints that impede an effective immune response to neoplasia. Antibody efficacy has been increased by genetic engineering to humanize the antibodies and to increase their effector functions including antibody dependent cellular cytotoxicity. Furthermore, antibodies have been armed with cytokines, chemotherapeutic agents, toxins, and radionuclides to augment their efficacy as tumor cytotoxic agents. As a consequence of these advances, 30 years after their first development, monoclonal antibodies have become an important standard approach for the therapy of neoplasia with 19 therapeutic monoclonal antibodies now approved by the FDA including 8 for the treatment of cancer.

Animals↗