JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2008
Cristina Olivo
Abstract Mesenchymal stem cells (MSCs) represent a powerful tool for applications in regenerative medicine. In this study, we used in vivo bioluminescence imaging to noninvasively investigate the fate and the contribution to bone formation of adult MSCs in tissue engineered constructs. Goat MSCs expressing GFP-luciferase were seeded on ceramic scaffolds and implanted subcutaneously in immune-deficient mice. The constructs were monitored weekly with bioluminescence imaging and were retrieved after 7 weeks to quantify bone formation by histomorphometry. With increasing amounts of seeded MSCs (from 0 to 1,×,106 MSC/scaffold), a cell-dose related increase in bioluminescence was observed at all time points, correlating with increased bone formation at 7 weeks. To investigate the relevance of MSC proliferation to bone deposition, cell-seeded scaffolds were irradiated. The irradiated cells were functional with respect to oxygen consumption but no increase in bioluminescence was observed in vivo, and only minimal bone was produced. Proliferating MSCs are likely required for initiation of bone formation in tissue engineered constructs in vivo. Bioluminescence is a useful tool to monitor cellular responses and predict bone formation in vivo. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:901,909, 2008 [source]

Biomorphic Silicon Carbide Coated with an Electrodeposition of Nanostructured Hydroxyapatite/Collagen as Biomimetic Bone Filler and Scaffold,

ADVANCED ENGINEERING MATERIALS, Issue 8 2010
M. Lelli
Abstract The paper describes the method of preparation and chemical/physical characterization of a new biomaterial to be used as a bone substitute and bone-tissue engineering scaffold, which synergistically joins a porous bio-inspired morphology and the mechanical properties of biomorphic silicon carbide (BioSiC) with the surface bioactivity of a nanostructured hydroxyapatite/collagen biomimetic coating. FT-IR spectroscopy and XRD techniques are utilized to determine the chemical coating's composition. The morphology and size of the inorganic and protein components are investigated by TEM. The characteristic morphology of BioSiC channels and pores, which differ as a function of the transversal or longitudinal cross-section and with etching time, are investigated by SEM. Natural wood transformed into BioSiC acts as a cathode in an electrochemically assisted process that produces on its surface a biomimetic coating of hydroxyapatite nanocrystals and reconstituted type I collagen fibrils, producing an innovative apatite/collagen biomimetic porous bone filler and scaffold for tissue engineering. [source]

Monodisperse Mesoporous Silica Spheres Inside a Bioactive Macroporous Glass,Ceramic Scaffold,

ADVANCED ENGINEERING MATERIALS, Issue 7 2010
Renato Mortera
In the field of bone tissue engineering, monosized MCM-41 spheres have been incorporated inside a bioactive glass,ceramic macroporous scaffold belonging to the SiO2CaOK2O (SCK) system so obtaining a multiscale hierarchical composite. The MCM-41-SCK system was prepared by dipping the glass,ceramic scaffold into the MCM-41 synthesis solution and was characterized by means of XRD, micro-XRD, N2 sorption and scanning electron microscopy. The MCM-41 spheres inside the scaffold are highly uniform in diameter, as those synthesized in powder form. The adsorption capacity of the composite toward ibuprofen is three times higher than that of the MCM-41-free scaffold, because of the presence of the ordered mesoporous silica. Also the release behavior in SBF at 37,°C is strongly affected by the presence of MCM-41 inside the scaffold macropores. [source]

Antheraea assama Silk Fibroin-Based Functional Scaffold with Enhanced Blood Compatibility for Tissue Engineering Applications,

ADVANCED ENGINEERING MATERIALS, Issue 5 2010
Naresh Kasoju
Abstract The architecture and surface chemistry of a scaffold determine its utility in tissue engineering (TE). Conventional techniques have limitations in fabricating a scaffold with control over both architecture and surface chemistry. To ameliorate this, in this report, we demonstrate the fabrication of an Antheraea assama silk fibroin (AASF)-based functional scaffold. AASF is a non-mulberry variety having superior qualities to mulberry SF and is largely unexplored in the context of TE. First, a 3D scaffold with biomimetic architecture is fabricated. The scaffold is subsequently made blood compatible by modifying the surface chemistry through a simple sulfation reaction. EDX and FTIR analysis demonstrate the successful sulfation of the scaffold. SEM observations reveal that sulfation has no any effect on the scaffold architecture. TGA reveals that it has increased thermal stability. The sulfation reaction significantly improves the overall hydrophilicity of the scaffold, as is evident from the increase in water holding capacity; this possibly enhances the blood compatibility. The enhancement in blood compatibility of the sulfated scaffold is determined from in vitro haemolysis, protein adsorption and platelet adhesion studies. The sulfated scaffold is non-toxic and supports cell adhesion and growth, as revealed by indirect and direct contact-based in vitro cytotoxicity assays. This study reveals that the AASF-based functional scaffold, which has biomimetic architecture and blood-compatible surface chemistry, could be suitable for TE applications. [source]

Optimization of the Azobenzene Scaffold for Reductive Cleavage by Dithionite; Development of an Azobenzene Cleavable Linker for Proteomic Applications

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2010
Geoffray Leriche
Abstract In this paper we conducted an extensive reactivity study to determine the key structural features that favour the dithionite-triggered reductive cleavage of the azo,arene group. Our stepwise investigation allowed identification of a highly reactive azo,arene structure 25 bearing a carboxylic acid at the ortho position of the electron-poor arene and an ortho - O -alkyl-resorcinol as the electron-rich arene. Based on this 2-(2,-alkoxy-4,-hydroxyphenylazo)benzoic acid (HAZA) scaffold, the orthogonally protected difunctional azo,arene cleavable linker 26 was designed and synthesized. Selective linker deprotection and derivatization was performed by introducing an alkyne reactive group and a biotin affinity tag. This optimized azo,arene cleavable linker led to a total cleavage in less than 10 s with only 1 mM dithionite. Similar results were obtained in biological media. [source]

Short Synthesis of Orthogonally Protected 3,,12,-Diamino-5,-cholan-24-oic Acid, a Dipodal Steroid Scaffold for Combinatorial Chemistry

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 11 2007
Dieter Verzele
Abstract A short, practical, multigram-scale synthesis of C3,-NHAlloc, C12,-NHBoc-diamino-5,-cholan-24-oic acid 2 was developed, applying a new, straightforward synthetic strategy. Key features are the conservation of the carboxyl moiety at C24 during oxime reduction, the late differentiation between the C3 and C12 amino groups and the gradual separation of diastereomers during the synthesis. This orthogonally protected diamino steroid derivative can be used as starting point for the generation of steroid based dipodal peptide and non-peptide combinatorial libraries. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source]

An Efficient Asymmetric Synthesis of 2-Substituted 1,4-Benzodiazepin-3-one as a Potential Molecular Scaffold

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 8 2005
Nuria Cabedo
Abstract 2-Substituted 1,4-benzodiazepine-2-one compounds (9,12) were obtained by a highly diastereoselective alkylation of a seven-membered ring benzolactam (8) in the presence of (R)-phenylglycinol as a chiral inductor. The corresponding acid derivative (16) afforded a conformationally constrained structure suitable for preparing peptidomimetic analogues useful as a novel molecular scaffold. After cleavage of the chiral appendage this approach might also lead efficiently to enantiomerically pure 2-substituted benzodiazepines (15). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source]

Synthesis of an Optically Active Decahydro-6-isoquinolone Scaffold with a Quaternary Stereocenter

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 12 2004
Jens Christoffers
Abstract A straightforward synthesis of optically active decahydro-6-isoquinolone derivative 3, containing a quaternary stereocenter, is reported. The starting (R)-configured enantiopure enone 2, which is readily accessible through a copper-catalyzed, L -valine amide mediated Michael reaction and a subsequent Robinson annulation, was hydrogenated with Pd/C in iPrOH to give the decahydroisoquinolone 4. Treatment of 4 with ethyleneglycol in the presence of PPTS yielded the dioxolane-protected derivative 7. A sequence of ester reduction with LiAlH4 and subsequent Ley oxidation of the resulting primary alcohol 10 accomplished the synthesis. Enantiomerically pure aldehyde 3, with three groups for further functionalization, was thus obtained in 63% overall yield. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source]

D -Glucose as a Pentavalent Chiral Scaffold

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 8 2003
Till Opatz
Abstract A novel carbohydrate-based scaffold for combinatorial chemistry has been developed. This scaffold allows the selective attachment of five different side chains, giving rise to products of enormous structural diversity. As a demonstration of its usefulness, a series of model compounds has been prepared in high purity and yield by multistep parallel synthesis on a solid phase. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source]

Engineering LacI for Self-Assembly of Inorganic Nanoparticles on DNA Scaffold through the Understanding of LacI Binding to Solid Surfaces

ADVANCED FUNCTIONAL MATERIALS, Issue 8 2009
Haibin Chen
Abstract The potential of utilizing the DNA binding protein lac repressor (LacI) to organize inorganic nanoparticles (NPs) is explored in this study. A peptide cognitive of both SiO2 and TiO2 simultaneously (STB1, -CHKKPSKSC-) is genetically engineered into the C-terminus of LacI to give LacI-STB1, and the inserted STB1 peptides in the context of LacI-STB1 molecules are shown to actively interact with both SiO2 and TiO2. Wild-type LacI is found to interact with the two surfaces at its flexible N-terminal DNA binding domain, and LacI-STB1 exhibits much stronger binding affinity to both surfaces by harnessing a second binding region (STB1 peptide) fused at its C-terminus. The quantitative analysis of binding kinetics reveals that, compared to wild-type LacI with one binding region (N-terminus), two remote binding regions (N-terminus and C-terminus) in LacI-STB1 do not lead to faster adsorption rates to the two surfaces, but remarkably slow down the desorption rates. Finally, using LacI-STB1 as a linker, the successful assembly of a sandwich nanostructure of DNA/LacI-STB1/TiO2 NPs is demonstrated using surface plasmon resonance (SPR) measurements and TEM. The demonstrated LacI-STB1-mediated assembly of TiO2 NPs on DNA scaffold may provide a generic platform for controlled spatial arrangement of various nanoparticles of engineering interest. [source]

Temperature-Induced Hydrogels Through Self-Assembly of Cholesterol-Substituted Star PEG- b -PLLA Copolymers: An Injectable Scaffold for Tissue Engineering,

ADVANCED FUNCTIONAL MATERIALS, Issue 8 2008
Koji Nagahama
Abstract Partially cholesterol-substituted 8-arm poly(ethylene glycol)- block -poly(L -lactide) (8-arm PEG- b -PLLA-cholesterol) has been prepared as a novel star-shaped, biodegradable copolymer derivative. The amphiphilic 8-arm PEG- b -PLLA-cholesterol aqueous solution (polymer concentration, above 3,wt%) exhibits instantaneous temperature-induced gelation at 34,°C, but the virgin 8-arm PEG- b -PLLA does not, irrespective of concentration. Moreover, an extracellular matrix (ECM)-like micrometer-scale network structure has been created with favorable porosity for three-dimensional proliferation of cells inside the hydrogel. This network structure is mainly attributed to specific self-assembly between cholesterol groups. The 10 and 20,wt% hydrogels are eroded gradually in phosphate buffered saline at 37,°C over the course of a month, and after that the gel becomes completely dissociated. Moreover, L929 cells encapsulated into the hydrogel are viable and proliferate three-dimensionally inside the hydrogels. Thus, in-vitro cell culture studies demonstrate that 8-arm PEG- b -PLLA-cholesterol is a promising candidate as a novel injectable cellular scaffold. [source]

A New Approach to Pyrrolocoumarin Derivatives by Palladium-Catalyzed Reactions: Expedient Construction of Polycyclic Lamellarin Scaffold

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009
Lei Chen
Abstract A new and efficient protocol for straightforward synthesis of chromeno[3,4- b]pyrrol-4(3H)-one derivatives by palladium-catalyzed sequential coupling/cyclization reactions has been developed. The key strategy relies on creation of pyrrole ring through palladium-catalyzed intramolecular hydroamination of related acetylenic aminocoumarins. The synthetic utility of the obtained chromeno[3,4- b]pyrrol-4(3H)-one product has been demonstrated by the expedient synthesis of polycyclic lamellarin scaffold in four steps. It provides a new entry to synthesis of potentially valuable lamellarin analogues. [source]

Partially Hydrogenated 1,1,-Binaphthyl as Ligand Scaffold in Metal-Catalyzed Asymmetric Synthesis

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 5 2003
T.-L., Terry
Abstract Although chiral binaphthyl-type ligands are already known to be effective over a broad spectrum of reactions, they sometimes fail in providing high enantioselectivities in some catalytic asymmetric reactions. This article summarizes recent attempts to elevate their performance by partly hydrogenating the naphthyl components of the binaphthyl. The synthetic routes to some of these ligands are briefly outlined. Positive results are observed in asymmetric hydrogenation, alkylation, borane reduction, epoxidation and hetero-Diels,Alder reactions. The function of the partially reduced binaphthyl skeleton, however, can sometimes be disadvantageous or ambiguous as illustrated in reactions such as asymmetric ring-closing metathesis, 1,4-conjugate addition, epoxidation, allylic alkylation, trimethylsilylcyanation, epoxide ring-opening and hydroformylation. [source]

A Novel Biphasic Bone Scaffold: ,-Calcium Phosphate and Amorphous Calcium Polyphosphate

JOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 4 2009
Gaoxiang Chen
Calcium polyphosphate (CPP) was added to hydroxyapatite (HA) to develop a novel biphasic calcium phosphate (BCP). The effects of varying CPP dosage on the sintering property, the mechanical strength, and the phase compositions of HA were investigated. Results showed that CPP reacted with HA and produced ,-calcium phosphate (,-TCP) and H2O and that an excessive dosage of CPP (>10 wt%) obtained a novel BCP of ,-TCP/amorphous-CPP, while a lesser dosage of CPP (<10 wt%) obtained a traditional BCP (HA/,-TCP). The porous ,-TCP/amorphous-CPP scaffolds (porosity of 66.7%, pore diameter of 150,450 ,m, and compressive strength of 6.70±1.5 MPa) were fabricated and their in vitro degradation results showed a significant improvement of degradation with the addition of CPP. [source]

State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2007
Dietmar Werner Hutmacher
Abstract Scaffold-based bone tissue engineering aims to repair/regenerate bone defects. Such a treatment concept involves seeding autologous osteogenic cells throughout a biodegradable scaffold to create a scaffold,cell hybrid that may be called a tissue-engineered construct (TEC). A variety of materials and scaffolding fabrication techniques for bone tissue engineering have been investigated over the past two decades. This review aims to discuss the advances in bone engineering from a scaffold material point of view. In the first part the reader is introduced to the basic principles of bone engineering. The important properties of the biomaterials and the scaffold design in the making of tissue engineered bone constructs are discussed in detail, with special emphasis placed on the new material developments, namely composites made of synthetic polymers and calcium phosphates. Advantages and limitations of these materials are analysed along with various architectural parameters of scaffolds important for bone tissue engineering, e.g. porosity, pore size, interconnectivity and pore-wall microstructures. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Porous Biodegradable Scaffold: Predetermined Porosity by Dissolution of Poly(ester-anhydride) Fibers from Polyester Matrix

MACROMOLECULAR BIOSCIENCE, Issue 7 2009
Jaana Rich
Abstract A novel selective leaching method for the porogenization of the biodegradable scaffolds was developed. Continuous, predetermined pore structure was prepared by dissolving fast eroding poly(, -caprolactone)-based poly(ester-anhydride) fibers from the photo-crosslinked poly(, -caprolactone) matrix. The porogen fibers dissolved in the phosphate buffer (pH 7.4, 37,°C) within a week, resulting in the porosity that replicated exactly the single fiber dimensions and the overall arrangement of the fibers. The amount of the porosity, estimated with micro-CT, corresponded with the initial amount of the fibers. The potential to include bioactive agents in the porogen fibers was demonstrated with the bioactive glass. [source]

Self-Organization of a Chiral D -EAK16 Designer Peptide into a 3D Nanofiber Scaffold

MACROMOLECULAR BIOSCIENCE, Issue 8 2008
Zhongli Luo
Abstract Self-assembling peptide nanofiber scaffolds are an excellent material for applications such as tissue repair, tissue regeneration, instant stopping of bleeding, and slow drug release. We report a new self-assembling peptide D -EAK16 consisting purely of D -amino acids. D -EAK16 and L -EAK16 display mirror-image CD spectra at 20,°C. Like L -EAK16, D -EAK16 self-assembles into nanofibers, thus demonstrating that chiral self-assembling peptide nanofiber scaffolds can be made from both L - and D -amino acids. We also show that D -peptide nanofibers are resistant to natural proteases and may thus be useful in biotechnology, nanobiotechnology, tissue repair and tissue regeneration as well as other medical applications. [source]

Solid-Phase Preparation of a Library Based on a Phenylalanine Scaffold

MOLECULAR INFORMATICS, Issue 8 2005
Aina Colombo
Abstract A convenient strategy (preliminary study, preproduction and production) for the solid-phase preparation of a library using 4-iodophenylalanine as a scaffold is described. The aromatic ring was first modified via the Suzuki reaction and the amino position was subsequently derivatized into amides, sulfonamides, amines, carbamates and ureas. The scope and limitations of all of the reactions carried out in parallel are discussed. The solid-phase synthesis of a library of 315 individual compounds was attempted by using seven boronic acids and nine representative compounds from each of the following classes: carboxylic acids, sulfonyl chlorides, aldehydes, alcohols and isocyanates. Owing to the failure of the amine derivatization, 297 compounds were ultimately obtained. [source]

Interaction Between N-terminal Loop and , -Scaffold of Photoactive Yellow Protein,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Miki Harigai
During the photoreaction cycle of photoactive yellow protein (PYP), a physiologically active intermediate (PYPM) is formed as a consequence of global protein conformational change. Previous studies have demonstrated that the photocycle of PYP is regulated by the N-terminal loop region, which is located across the central , -sheet from the p -coumaric acid chromophore. In this paper, the hydrophobic interaction between N-terminal loop and , -sheet was studied by characterizing PYP mutants of the hydrophobic residues. The rate constants and structural changes of the photocycle of L15A and L23A possibly participating in such an interaction were more similar to wild-type than F6A, showing that the CH/, interaction between Phe6 and Lys123 is the most essential as reported previously. To better understand the interactions between N-terminal tail and , -sheet of PYP, Phe6 and Phe121 were replaced by Cys and linked by a disulfide bond. Since the photocycle kinetics, structural change and thermal stability of F6C/F121C were similar to F6A, the CH/, interaction between Phe6 and Lys123 is not substitutable. It is likely that the detachment of position 6 from position 123 substantially alters the nature of PYP. [source]

Penile Enhancement Using Autologous Tissue Engineering with Biodegradable Scaffold: A Clinical and Histomorphometric Study

THE JOURNAL OF SEXUAL MEDICINE, Issue 9 2010
Sava V. Perovic PhD
ABSTRACT Introduction., Autologous tissue engineering with biodegradable scaffolds is a new treatment option for real penile girth enhancement. Aim., The aim of this article is to evaluate tissue remodeling after penile girth enhancement using this technique. Methods., Between June 2005 and May 2007, a group of 12 patients underwent repeated penile widening using biodegradable scaffolds enriched with expanded autologous scrotal dartos cells. Clinical monitoring was parallel to histological investigation of tissue remodeling. During second surgical procedure, biopsies were obtained 10,14 months after first surgery (mean 12 months, N = 6) and compared with those obtained after 22,24 months (mean 23 months, N = 6), and control biopsies from patients who underwent circumcision (N = 5). Blind evaluation of histomorphometrical and immunohistochemical finding was performed in paraffin sections. Main Outcome Measurements., Penile girth gain in a flaccid state ranged between 1.5 and 3.8 cm (mean 2.1 ± 0.28 cm) and in full erection between 1.2 and 4 cm (mean 1.9 ± 0.28 cm). Patients' satisfaction, defined by a questionnaire, was good (25%) and very good (75%). Results., In biopsies obtained 10,14 months after first surgery, highly vascularized loose tissue with collagen deposition associated with small foci of mild chronic and granulomatous inflammation surrounding residual amorphous material was observed. Fibroblast-like hyperplasia and small vessel neoangiogenesis occurred intimately associated with the progressive growth of vascular-like structures from accumulation of CD34 and alpha-smooth muscle actin-positive cells surrounding residual scaffold-like amorphous material. Capillary neoangiogenesis occurred inside residual amorphous material. In biopsies obtained after 22,24 months, inflammation almost disappeared and tissue closely resembled that of the dartos fascia of control group. Conclusions., Autologous tissue engineering using expanded scrotal dartos cells with biodegradable scaffolds is a new and promising method for penile widening that generates progressive accumulation of stable collagen-rich, highly vascularized tissue matrix that closely resemble deep dartos fascia. Perovic SV, Sansalone S, Djinovic R, Ferlosio A, Vespasiani G, and Orlandi A. Penile enhancement using autologous tissue engineering with biodegradable scaffold: A clinical and histomorphometric study. J Sex Med 2010;7:3206,3215. [source]

Long-Term Survival of Nonhuman Primate Islets Implanted in an Omental Pouch on a Biodegradable Scaffold

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2009
D. M. Berman
The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and five allogeneic diabetic monkey transplants under the cover of steroid-free immune suppression (SFIS) were undertaken. Fasting blood glucose (FBG) and C-peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/mL following transplant, a 66,92% posttransplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well-granulated and well-vascularized, insulin-positive islets, surrounded by T-cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n = 20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation. [source]

Directed Evolution of Orthogonal Ligand Specificity in a Single Scaffold,

ANGEWANDTE CHEMIE, Issue 42 2009
Michael J. McLachlan
Zwei hochempfindliche Ligand-Rezeptor-Paare, die auf einem einzigen Proteingerüst beruhen, sind zum nativen Ligand-Rezeptor-Paar und zueinander vollständig orthogonal. Alle drei Rezeptoren werden durch ihre jeweiligen Liganden (rote Elemente im Bild) spezifisch aktiviert, wenn mit ihnen die Expression von grün fluoreszierendem (obere Reihe), mCherry- (mittlere Reihe) und gelb fluoreszierendem Protein (untere Reihe) in Hefe nachgewiesen werden soll. [source]

Cryopreservation of Fibroblasts Immobilized Within a Porous Scaffold: Effects of Preculture and Collagen Coating of Scaffold on Performance of Three-Dimensional Cryopreservation

ARTIFICIAL ORGANS, Issue 7 2010
Hirotoshi Miyoshi
Abstract As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs. [source]

Novel Strategy to Engineer Trachea Cartilage Graft With Marrow Mesenchymal Stem Cell Macroaggregate and Hydrolyzable Scaffold

ARTIFICIAL ORGANS, Issue 5 2010
Liangqi Liu
Abstract Limited donor sites of cartilage and dedifferentiation of chondrocytes during expansion, low tissue reconstruction efficiency, and uncontrollable immune reactions to foreign materials are the main obstacles to overcome before cartilage tissue engineering can be widely used in the clinic. In the current study, we developed a novel strategy to fabricate tissue-engineered trachea cartilage grafts using marrow mesenchymal stem cell (MSC) macroaggregates and hydrolyzable scaffold of polylactic acid,polyglycolic acid copolymer (PLGA). Rabbit MSCs were continuously cultured to prepare macroaggregates in sheet form. The macroaggregates were studied for their potential for chondrogenesis. The macroaggregates were wrapped against the PLGA scaffold to make a tubular composite. The composites were incubated in spinner flasks for 4 weeks to fabricate trachea cartilage grafts. Histological observation and polymerase chain reaction array showed that MSC macroaggregates could obtain the optimal chondrogenic capacity under the induction of transforming growth factor-,. Engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans. PLGA scaffold degraded totally during in vitro incubation and the engineered cartilage graft was composed of autologous tissue. Based on this novel, MSC macroaggregate and hydrolyzable scaffold composite strategy, ready-to-implant autologous trachea cartilage grafts could be successfully fabricated. The strategy also had the advantages of high efficiency in cell seeding and tissue regeneration, and could possibly be used in future in vivo experiments. [source]

Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro Study

ARTIFICIAL ORGANS, Issue 2 2010
Zheng-Hui Wang
Abstract To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte,fibrin suspension, which was polymerized by submerging the constructs into thrombin,calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering. [source]

Designing a Three-dimensional Expanded Polytetrafluoroethylene,Poly(lactic-co-glycolic acid) Scaffold for Tissue Engineering

ARTIFICIAL ORGANS, Issue 4 2009
Hung-Jen Shao
Abstract:, The purpose of this study was to design a three-dimensional expanded polytetrafluoroethylene (ePTFE),poly(lactic-co-glycolic acid) (PLGA) scaffold for tissue engineering. To test the feasibility of this composite scaffold, a series of two-dimensional culture experiments were performed to investigate the behavior of anterior cruciate ligament (ACL) cells on the ePTFE and PLGA membranes. It was found PLGA provided a cell-favorable substrate for cell adhesion, migration, and growth, indicating PLGA is an ACL cell-conductive material. Conversely, poor adhesion and proliferation of ACL cells were observed on the ePTFE, even on the collagen-coated ePTFE. Therefore, the scaffold was not fabricated by coating PLGA on the ePTFE surface because it is difficult to coat anything on the extremely hydrophobic ePTFE surface. Instead, the ePTFE embedded in the PLGA matrix was prepared by immersing ePTFE scrim yarns into the PLGA solution, and then precipitating PLGA to form a three-dimensional construction with porous morphology. The role of ePTFE is regarded as a reinforcing constituent to improve the mechanical strength of porous PLGA matrix to provide early repair strength for tissue healing. However, porous PLGA matrix acts as a supportive environment for allowing cell adhesion, migration, and growth to guide the repair and regeneration of ligament tissue. To test this assumption, a preliminary animal experiment of rabbit ACL wound healing with this three-dimensional ePTFE,PLGA scaffold was performed. These results are very encouraging because such a new scaffold made of ePTFE scrim yarns embedded in PLGA may serve as ACL prostheses in the ligament tissue engineering. [source]

Development and Validation of Small-diameter Vascular Tissue From a Decellularized Scaffold Coated With Heparin and Vascular Endothelial Growth Factor

ARTIFICIAL ORGANS, Issue 3 2009
Min Zhou
Abstract To overcome shortcomings of current small-diameter vascular prostheses, we developed a novel allogenic vascular graft from a decellularized scaffold modified through heparin immobilization and vascular endothelial growth factor (VEGF) coating. The VEGF coating and release profiles were assayed by enzyme-linked immunosorbent assay, the biological activity of modified surface was validated by human umbilical vein endothelial cells seeding and proliferation for 10 days in vitro. In vivo, we implanted either a modified or a nonmodified scaffold as bilateral carotid allogenic graft in canines (n = 15). The morphological examination of decellularized scaffolds showed complete removal of cellular components while the extracellular matrix structure remained intact. After modification, the scaffolds possessed local sustained release of VEGF up to 20 days, on which the cells cultured showed significantly higher proliferation rate throughout the time after incubation compared with the cells cultured on nonmodified scaffolds (P < 0.0001). After 6 months of implantation, the luminal surfaces of modified scaffolds exhibited complete endothelium regeneration, however, only a few disorderly cells and thrombosis overlay the luminal surfaces of nonmodified scaffolds. Specifically, the modified scaffolds exhibited significantly smaller hyperplastic neointima area compared with the nonmodified, not only at midportion (0.56 ± 0.07 vs. 2.04 ± 0.12 mm2, P < 0.0001), but also at anastomotic sites (1.76 ± 0.12 vs. 3.67 ± 0.20 mm2, P < 0.0001). Moreover, modified scaffolds had a significantly higher patency rate than the nonmodified after 6 months of implantation (14/15 vs. 7/15, P = 0.005). Overall, this modified decellularized scaffold provides a promising direction for fabrication of small-diameter vascular grafts. [source]

Tissue Engineered Heart Valves: Autologous Cell Seeding on Biodegradable Polymer Scaffold

ARTIFICIAL ORGANS, Issue 5 2002
Toshiharu Shinoka
Abstract: We previosly reported on the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts then subsequently coated with endothelial cells. Using these methods, autologous cell/polymer constructs were implanted in 6 animals. In 2 additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, using cardiopulmonary bypass, the right-posterior leaflet of the pulmonary valve was resected completely and replaced with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. After 6 h and 1, 6, 7, 9, and 11 weeks, the animals were sacrificed and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. Animals receiving leaflets made from polymer without cell seeding were sacrificed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were degraded completely leaving no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group; 4-hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. In the tissue-engineered heart valve leaflet, transplanted autologous cells generated proper matrix on the polymer scaffold in a physiologic environment at a period of 8 weeks after implantation. [source]

Use of the chicken lysozyme 5, matrix attachment region to generate high producer CHO cell lines

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2005
Pierre-Alain Girod
Abstract Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures. © 2005 Wiley Periodicals, Inc. [source]

Hydrogel-Perfluorocarbon Composite Scaffold Promotes Oxygen Transport to Immobilized Cells

BIOTECHNOLOGY PROGRESS, Issue 2 2008
Kyuongsik Chin
Cell encapsulation provides cells a three-dimensional structure to mimic physiological conditions and improve cell signaling, proliferation, and tissue organization as compared to monolayer culture. Encapsulation devices often encounter poor mass transport, especially for oxygen, where critical dissolved levels must be met to ensure both cell survival and functionality. To enhance oxygen transport, we utilized perfluorocarbon (PFC) oxygen vectors, specifically perfluorooctyl bromide (PFOB) immobilized in an alginate matrix. Metabolic activity of HepG2 liver cells encapsulated in 1% alginate/10% PFOB composite system was 47,104% higher than alginate systems lacking PFOB. A cubic model was developed to understand the oxygen transport mechanism in the alginate/PFOB composite system. The theoretical flux enhancement in alginate systems containing 10% PFOB was 18% higher than in alginate-only systems. Oxygen uptake rates (OURs) of HepG2 cells were enhanced with 10% PFOB addition under both 20% and 5% O2 boundary conditions, by 8% and 15%, respectively. Model predictions were qualitatively and quantitatively verified with direct experimental OUR measurements using both a perfusion reactor and oxygen sensing plate, demonstrating a greater OUR enhancement under physiological O2 boundary conditions (i.e., 5% O2). Inclusion of PFCs in an encapsulation matrix is a useful strategy for overcoming oxygen limitations and ensuring cell viability and functionality both for large devices (>1 mm) and over extended time periods. Although our results specifically indicate positive enhancements in metabolic activity using the model HepG2 liver system encapsulated in alginate, PFCs could be useful for improving/stabilizing oxygen supply in a wide range of cell types and hydrogels. [source]