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PubMed2003

Long-term cyclic distention enhances the mechanical properties of collagen-based media-equivalents.

In this study, we sought to identify the key parameters involved in long-term cyclic distension (CD) as they pertain to the development of collagen-based media-equivalents (MEs). By using only highly compacted, cross-linked constructs, we avoided the complicating issues of irrecoverable creep and transient alignment, and isolated the effects of cyclic mechanical loading on ME development. Our system allowed us to study this development over a wide range of parameters including strain amplitude, pulse frequency, pulse shape, and culture time. We found that in most cases involving cyclic distension, MEs were both stronger and stiffer than constructs that were grown under static conditions. The mechanical properties were not significantly different from static controls after two weeks of CD, however, five weeks of CD was sufficient to note significant increases in both stiffness and strength. The strain, stretch time, and relaxation time were all important variables in determining ME mechanical properties. While we were unable to detect a significant net change in the amount of total collagen, we observed significant deposition of insoluble elastin in our CDMEs, something that has never been previously reported using adult smooth muscle cells. Finally, these changes in ME development did not depend on the age of the MEs prior to the initiation of CD.

Adaptation, Physiological↗
PubMed2006

Mouse chromosome engineering for modeling human disease.

Chromosomal rearrangements are frequently in humans and can be disease-associated or phenotypically neutral. Recent technological advances have led to the discovery of copy-number changes previously undetected by cytogenetic techniques. To understand the genetic consequences of such genomic changes, these mutations need to be modeled in experimentally tractable systems. The mouse is an excellent organism for this analysis because of its biological and genetic similarity to humans, and the ease with which its genome can be manipulated. Through chromosome engineering, defined rearrangements can be introduced into the mouse genome. The resulting mouse models are leading to a better understanding of the molecular and cellular basis of dosage alterations in human disease phenotypes, in turn opening new diagnostic and therapeutic opportunities.

Animals↗
PubMed2000

Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes. Comparison with rat.

Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a beta-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [(3)H]phenylalanine-labeled protein content. T3 and NE also increased alpha-myosin heavy chain (MyHC) mRNA and reduced beta-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including alpha(1)-adrenergic agonists, endothelin-1, prostaglandin F(2alpha), interleukin 1beta, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or beta-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal-regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and beta-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C-coupled agonists do not.

Adrenergic alpha-Agonists↗
PubMed1997

Wnt-11 is expressed in early avian mesoderm and required for the differentiation of the quail mesoderm cell line QCE-6.

The beginning of mesodermal development involves the aggregation of newly gastrulated cells into epithelial fields, as a prelude to organ formation. To analyze the molecular regulation of this initial patterning, we have focused on the Wnt family of secreted signaling proteins, molecules which have been shown to promote embryonic patterning by regulating cell-cell associations. In this study, we show that the Wnt-11 gene is expressed by newly gastrulated mesoderm cells within avian embryos. The expression pattern of Wnt-11 also suggests that it may be involved in formation of the cardiogenic fields and somites. Subsequently, we utilized the quail mesoderm cell line QCE-6 as a culture model for examining the influence of Wnt-11 on early mesoderm cell differentiation. This cell line has been shown to be representative of early nondifferentiated mesoderm cells and has the potential to differentiate into cardiomyocytes, endothelial or red blood cells. Similar to early mesoderm cells, QCE-6 cells express Wnt-11. We have engineered stable transfectants of these cells that produce either diminished or enhanced levels of Wnt-11 protein. Our studies show that Wnt-11 regulates cellular interactions of QCE-6 cells, as demonstrated by alterations in contact-inhibited growth, tight and gap junction formation and plakoglobin expression. Both the morphology and growth factor-induced differentiation of QCE-6 cells are regulated in a cooperative fashion by Wnt-11 and fibronectin. These results, described in detail below, demonstrate the uniqueness of QCE-6 cells as a culture system for analyzing Wnt activity. In particular, QCE-6 cells are the first cell line that has demonstrated: (1) Wnt-dependent differentiation; (2) concentration-variable responses to Wnt protein; and (3) altered cell phenotypes as a direct response to Wnt-5a class proteins (e.g. Wnt-4 and Wnt-11).

Amino Acid Sequence↗
PubMed2001

The place of pegvisomant in the management of acromegaly.

Conventional treatments for acromegaly include surgery, radiotherapy, dopamine agonists and somatostatin (SMS) analogues, which effect disease control by lowering circulating growth hormone (GH). Due to variability in tumour characteristics, combinations of these treatment modalities leave a significant number of patients with sub-optimal serum GH and insulin-like growth factor-I (IGF-I) levels, which have been linked to increased morbidity and mortality. The GH receptor antagonist pegvisomant is a genetically engineered analogue of GH that prevents functional dimerisation of the growth hormone receptor (GHR); a process that is critical to GH action at the cellular level. A crucial amino acid substitution at Gly(120) to Arg(120) within the third alpha helix of the antagonist prevents functional GHR dimerisation. Pegvisomant represents a novel treatment for acromegaly as, unlike existing treatment modalities, the effectiveness of pegvisomant is independent of pituitary tumour characteristics. Initial clinical studies in patients with active acromegaly have demonstrated serum IGF-I normalisation in over 90% of patients receiving 20 mg per day, such that, in terms of serum IGF-I normalisation, pegvisomant now represents the most effective medical treatment for acromegaly. Although there are limited long-term data on the use of pegvisomant and questions regarding pituitary tumour growth and altered liver function remain, this therapy offers the prospect of serum IGF-I normalisation in the vast majority of patients with active acromegaly.

Acromegaly↗
PubMed2005

Current and emerging therapies for the lysosomal storage disorders.

Targeted treatments for the lysosomal storage disorders (LSDs), in the form of enzyme replacement and/or substrate depletion, have been shown to be relatively safe and effective in reversing core disease features in selected clinical subtypes (including Gaucher disease types I and III, Fabry disease and the Hurler-Scheie syndrome). These approaches have expanded the therapeutic options available to patients with rare genetic disorders, beyond palliative measures (such as liver or kidney transplantation for end-organ failure) and cellular replacement through bone marrow transplantation. Present efforts are focused on the development of novel strategies, including chaperone-mediated enzyme enhancement and genetically engineered stem cell therapy. In the coming decades, a broadening therapeutic horizon for patients with inborn errors of metabolism is anticipated, and the growing experience in the management of patients with LSDs will serve as an instructive model. Among the many challenges will be determination of the extent to which these therapies have modified the course of disease beyond merely extending the age of survival, but also enabling a meaningful patient quality of life, and the minimisation of current resource use. The projected lifetime acquisition costs of newly introduced therapeutic options also raises several issues, related to equitable access and the large opportunity costs for other therapeutic areas, that will need to be addressed by healthcare policy makers and third-party payers.

Animals↗
PubMed2005

[An immunological study on adenovirus mediated human bone morphogenetic protein 2 gene therapy].

OBJECTIVE: To evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repair of tibial defects. METHODS: Twelve goats were made 2.1 cm segmental defects in the tibial diaphysis and divided into 2 groups. Adv-hBMP-2 transfected marrow mesenchymal stem cells (MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n = 7) and untransfected group (n = 5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. RESULTS: More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213 +/- 1.278) and the untransfected group(- 0.310 +/- 0.147, P < 0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544 +/- 0.957) and the untransfected group (3.104 +/- 0.644, P > 0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359 +/- 0.226, 2.297 +/- 0.200, 2.214 +/- 0.215 and 2.297 +/- 0.210 in transfected group, and - 0.175 +/- 0.335, - 0.419 +/- 0.171, 0 +/- 0.171 and 0.874 +/- 0.524 in untransfected group, being significant differences between two groups (P < 0.05). CONCLUSION: Adenovirus mediated BMP-2 gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.

Adenoviridae↗
PubMed2006

Physiologic pulsatile flow bioreactor conditioning of poly(ethylene glycol)-based tissue engineered vascular grafts.

Mechanical conditioning represents a potential means to enhance the biochemical and biomechanical properties of tissue engineered vascular grafts (TEVGs). A pulsatile flow bioreactor was developed to allow shear and pulsatile stimulation of TEVGs. Physiological 120 mmHg/80 mmHg peak-to-trough pressure waveforms can be produced at both fetal and adult heart rates. Flow rates of 2 mL/sec, representative of flow through small diameter blood vessels, can be generated, resulting in a mean wall shear stress of approximately 6 dynes/cm(2) within the 3 mm ID constructs. When combined with non-thrombogenic poly(ethylene glycol) (PEG)-based hydrogels, which have tunable mechanical properties and tailorable biofunctionality, the bioreactor represents a flexible platform for exploring the impact of controlled biochemical and biomechanical stimuli on vascular graft cells. In the present study, the utility of this combined approach for improving TEVG outcome was investigated by encapsulating 10T-1/2 mouse smooth muscle progenitor cells within PEG-based hydrogels containing an adhesive ligand (RGDS) and a collagenase degradable sequence (LGPA). Constructs subjected to 7 weeks of biomechanical conditioning had significantly higher collagen levels and improved moduli relative to those grown under static conditions.

Animals↗
PubMed2000

Endogenous carriers and ligands in non-immunogenic site-specific drug delivery.

Targeted drug delivery has gained recognition in modern therapeutics and attempts are being made to explore the potentials and possibilities of cell biology related bioevents in the development of specific, programmed and target oriented systems. The components which have been recognized to be tools include receptors and ligands, where the receptors act as molecular targets or portals, and ligands, with receptor specificity and selectivity, are trafficked en route to the target site. Although ligands of exogenous or synthetic origin contribute to the selectivity component of carrier constructs, they may impose immunological manifestations of different magnitudes. The latter may entail a continual quest for bio-compatible, non-immunogenic and target orientated delivery. Endogenous serum, cellular and extracellular bio-ligands interact with the colloidal carrier constructs and influence their bio-fate. However, these endogenous bio-ligands can themselves serve as targeting modules either in their native form or engineered as carrier cargo. Bio-regulatory, nutrient and immune ligands are sensitive, specific and effective site directing handles which add to targeted drug delivery. The present review provides an exhaustive account of the identified bio-ligands, which are not only non-immunogenic in nature but also site-specific. The cell-related bioevents which are instrumental in negotiating the uptake of bio-ligands are discussed. Further, a brief account of ligand-receptor interactions and the set of biological events which ensures ligand-driven trafficking of the ligand-receptor complex to the cellular interior is also presented. Since ligand-receptor interaction is a critical pre-requisite for negotiating cellular uptake of endogenous ligands and anchored carrier cargo, an attempt has been made to identify differential expression of receptors and bio-ligands under normal and etiological conditions. Studies which judiciously utilized bio-ligands or their analogs in negotiating site-specific drug delivery have been reviewed and presented. Targeted delivery of bioactives using endogenous bio-ligands offers enormous options and opportunities through carrier construct engineering and could become a future reality in clinical practice.

Animals↗
PubMed1997

Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry.

The increasing accessibility of genetically engineered receptor systems for the study of drug-receptor interaction has led to a corresponding increase in the testing of new drug entities in recombinant receptor systems. In this article Terry Kenakin illustrates some possible conditions within these recombinant systems where the relative stoichiometry of the receptors to other cellular components may differ from that found in natural systems and where this difference may lead to anomalies in drug testing.

Animals↗
PubMed2002

Tissue engineering: TGF-beta superfamily members and delivery systems in bone regeneration.

The induction of bone formation requires three parameters that interact in a highly regulated process: soluble osteoinductive signals, capable responding cells, and a supporting matrix substratum or insoluble signal. The use of recombinant and naturally derived bone morphogenetic proteins and transforming growth factor beta(s) (TGF-beta(s)) has increased our understanding of the functions of these morphogens during the induction of endochondral bone formation. In addition, growing understanding of the cellular interactions of living tissues with synthetic biomaterials has led to the in vivo induction of bone formation using porous biomimetic matrices as an alternative to the use of autografts for bone regeneration. This review outlines the basis of bone tissue engineering by members of the TGF-beta superfamily, focusing on their delivery systems and the intrinsic induction of bone formation by specific biomimetic matrices with a defined geometry.

Animals↗
PubMed1999

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary molecular determinants.

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component. A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria. Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform. Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles. Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria. The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.

Animals↗
PubMed2006

Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution.

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.

Amino Acid Sequence↗
PubMed2004

Skeletal volume enhancement: implants and osteotomies.

PURPOSE OF REVIEW: Facial plastic surgeons are concerned with improving or restoring function and form. Most surgeons perform primarily soft tissue procedures, which alone are often sufficient. However, deficiencies in the underlying craniomaxillofacial skeleton must also be addressed. Facial skeletal augmentation remains an essential aspect of cosmetic and reconstructive surgery. This article reviews the basic alloplastic biomaterials available for facial volume enhancement, discusses the zygomatic sandwich osteotomy for malar augmentation, and describes recent applications of distraction osteogenesis in the craniomaxillofacial region. An update in tissue engineering and computer modeling is also provided. RECENT FINDINGS: High-porosity expanded polytetrafluoroethylene has been developed to provide a softer feel with less shrinkage and migration because of better biointegration and cellular ingrowth. Long-term results with porous polyethylene have demonstrated superior biocompatibility and minimal complications. Hydroxyapatite cement has been associated with an immunoguided delayed inflammatory reaction that leads to thinning of the overlying skin and exposure of the implant.Applications of distraction osteogenesis are rapidly expanding and include deformities of the mandible, midface, and cranium. There has been a trend toward the use of internal hardware, and internal devices are being developed to deliver a greater degree of vector control. Biodegradable devices have been developed to eliminate the second surgical procedure necessary for hardware removal. In the future, successful tissue engineering could eliminate many of the drawbacks associated with implants and osteotomies. The ability to stimulate stem cells to generate autogenous bone has been demonstrated in the laboratory. A novel application of computer technology that integrates laser surface scanning and digitizing with computer-aided design and manufacturing to produce facial prostheses has been described. SUMMARY: An abundance of alternatives exist for skeletal volume enhancement including alloplastic implants, standard osteotomies, and distraction osteogenesis. The surgeon must evaluate the pros and cons of each technique in the context of each individual patient to determine the most appropriate option. Technologic advances in biomaterials, distraction hardware, computer modeling, and tissue engineering will continue to supply the surgeon's repertoire with improved methods to augment and restore the craniomaxillofacial skeleton.

Absorbable Implants↗
PubMed2006

Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration.

Synthetic biodegradable polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) (50:50) modified by porcine type II collagen and an Arg-Gly-Asp (RGD)-containing protein were evaluated as scaffolds for cartilage regeneration in this study. Cytocompatibility of the polymer films was tested using immortalized chondrocytes. Neocartilage formation in vitro on cell-seeded scaffolds was further examined using primary porcine chondrocytes. The inflammatory response of the scaffolds was evaluated subcutaneously in rats. A pilot animal study was conducted, in which rabbit allogeneic chondrocyte-seeded scaffolds were implanted to repair the defected rabbit knee cartilage. The results demonstrated that PLGA as well as its blends with PLLA had better cell growth than pure PLLA, and that type II collagen enhanced, but RGD inhibited cell proliferation. Scaffolds made of blended PLLA/PLGA had larger dynamic compressive modulus compared to scaffolds made of PLLA or PLGA single polymer. Chondrocyte-seeded scaffolds modified by type II collagen without RGD had the greater number of cells as well as higher glycosaminoglycan (GAG) and collagen contents compared to scaffolds without type II collagen modification or scaffolds further modified with RDG. Type II collagen modification prevented infiltration by host tissue and capsule formation. Unmodified PLLA and PLLA/PLGA constructs demonstrated persisting inflammatory response after 6 months, while all type II collagen-modified PLLA/PLGA constructs showed complete repair and no inflammation. Partial or full repair was observed after 2 months of postimplantation in type II collagen-modified PLLA/PLGA constructs, with equal cellularity and 75-80% matrix contents of a normal rabbit articular cartilage. It was concluded that PLLA/PLGA blended scaffolds modified by type II collagen were a potential tissue engineering scaffold for cartilage regeneration.

Analysis of Variance↗
PubMed1992

Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.

Amino Acid Sequence↗
PubMed2006

Degradation behavior of hydrophilized PLGA scaffolds prepared by melt-molding particulate-leaching method: comparison with control hydrophobic one.

Porous PLGA/PVA scaffolds as hydrophilized PLGA scaffolds for tissue engineering applications were fabricated by a novel melt-molding particulate leaching method (non-solvent method). The prepared scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200-300 microm; porosity, about 90%). The in vitro degradation behavior of the PLGA and PLGA/PVA scaffolds was compared at 37 degrees C in PBS (pH 7.4) with and without the solution change everyday to see the effect of solution pH as well as scaffold hydrophilicity on the degradation behavior. The changes in dimension, molecular weight, mechanical properties (maximum load and modulus), and morphology of the scaffolds were examined with degradation time. The degradation behavior of the PLGA and PLGA/PVA scaffolds was further investigated in vivousing a rat model (subcutaneously implantation). It was observed that both PLGA and PLGA/PVA scaffolds in decreasing pH condition (PBS no change) showed faster degradation than those in constant pH condition (PBS change everyday), owing to the enhanced intramolecular depolymerization by the increment of chain hydrophilicity caused by carboxylate groups as well as the autocatalysis of carboxylic acids accumulated in the solution by the cleavage of PLGA backbone ester bonds. The scaffolds in vivo condition also showed faster degradation than those in vitro, probably due to the aid of foreign body giant cells or enzymes. The PLGA/PVA scaffold showed slightly faster degradation than the PLGA scaffold for both in vitro and in vivo conditions.

Animals↗