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PubMed2006

Readily available porcine aortic valve matrices for use in tissue valve engineering. Is cryopreservation an option?

The clinical use of acellular biological valves as scaffolds in tissue valve engineering would require them to be readily available. This study examines the feasibility of cryopreserving porcine aortic valve matrices for use in tissue valve engineering. Matrices prepared using an enzymatic-detergent decellularization protocol were examined before and after cryopreservation. The biochemical status of tissues were evaluated by collagen and uronic acid (proteoglycan) determination and their mechanical properties were determined using a burst test. The histological and ultrastructural properties were evaluated by light and electron microscopy. Cryopreservation did not significantly affect the collagen and uronic acid content of aortic leaflet matrices. Histological and ultrastructural sections, however, confirmed extensive disruption of the extracellular collagen matrix and inter-fibrillar proteoglycan associations following cryopreservation. Although neither the breakage force nor the maximum force at failure was significantly different in matrices before and after cryopreservation, the strain observed in matrices was significantly higher after cryopreservation. To our knowledge this is the first study to investigate the effects of cryopreservation on aortic valve matrices. Cryopreservation did not significantly alter the biochemical properties of porcine aortic valve matrices. Nevertheless, cryopreservation had significant adverse effects on the structural and mechanical properties of matrices. Cryopreserved matrices showed significantly higher strain when stressed compared to non cryopreserved matrices. While, theoretically matrices are only expected to be functional for a limited time until regenerated in vivo, further mechanical testing is necessary to evaluate the effects of these changes on the durability of porcine aortic valve matrices for use in tissue valve engineering.

Animals↗
PubMed2006

Genetic engineering of a Ca(2+) dependent chemical switch into the linear biomotor kinesin.

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.

Amino Acid Sequence↗
PubMed2005

Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases.

Pure chiral molecules are needed in the pharmaceutical and chemical industry as intermediates for the production of drugs or fine chemicals. Microorganisms represent an attractive alternative to chemical synthesis since they have the potential to generate single stereoisomers in high enantiomeric excess (ee). The baker's yeast Saccharomyces cerevisiae can notably reduce dicarbonyl compounds (in particular alpha- and beta-diketones and keto esters) to chiral alcohols with high ee. However, products are formed at a low rate. Moreover, large amounts of co-substrate are required for the regeneration of NADPH that is the preferred co-factor in almost all the known dicarbonyl reductions. Traditionally, better ee, reduction rate and product titre have been achieved via process engineering. The advent of recombinant DNA technology provides an alternative strategy to improve productivity and yield by strain engineering. This review discusses two aspects of strain engineering: (i) the generation of strains with higher reductase activity towards dicarbonyl compounds and (ii) the optimisation of co-substrate utilisation for NADPH cofactor regeneration.

Alcohol Oxidoreductases↗
PubMed2004

Engineering large fragment insertions into the chromosome of Escherichia coli.

An effective DNA replacement system has been established for engineering large fragment insertions into the chromosome of Escherichia coli. The DNA replacement plasmid, pHybrid I, was first constructed based on the bacterial artificial chromosome (BAC) vector. Two fragments of the E. coli genome, 5.5 and 6.5 kb in length, were introduced into the vector for homologous recombination. In addition to the chloramphenicol gene, a second gene neo was introduced for double marker screening for recombinant clones. By shot-gun cloning and homologous recombination techniques, using our new recombinant vector (pHybrid I), a 20-kb fragment from Lactococcus lactis genomic DNA has been successfully integrated into the chromosome of the E. coli strain J93-140. Plating tests and PCR amplification indicated that the integration remained stable after many generations in cell culture. This system will be especially useful for the chromosome engineering of large heterologous fragment insertions, which is necessary for pathway engineering.

Chromosomes, Bacterial↗
PubMed2005

Structure function relationships of transgenic starches with engineered phosphate substitution and starch branching.

Potato tuber starch was genetically engineered in the plant by the simultaneous antisense suppression of the starch branching enzyme (SBE) I and II isoforms. Starch prepared from 12 independent lines and three control lines were characterised with respect to structural and physical properties. The lengths of the amylopectin unit chains, the concentrations of amylose and monoesterified phosphate were significantly increased in the transgenically engineered starches. Size exclusion chromatography with refractive index detection (SEC-RI) indicated a minor decrease in apparent molecular size of the amylose and the less branched amylopectin fractions. Differential scanning calorimetry (DSC) revealed significantly higher peak temperatures for gelatinisation and retrogradation of the genetically engineered starches whereas the enthalpies of gelatinisation were lower. Aqueous gels prepared from the transgenic starches showed increased gel elasticity and viscosity. Principle component analysis (PCA) of the data set discriminated the control lines from the transgenic lines and revealed a high correlation between phosphate concentration and amylopectin unit chain length. The PCA also indicated that the rheological characteristics were primarily influenced by the amylose concentration. The phosphate and the amylopectin unit chain lengths had influenced primarily the pasting and rheological properties of the starch gels.

Amylopectin↗
PubMed2004

Engineered delta ribozymes can simultaneously knock down the expression of the genes encoding uracil phosphoribosyltransferase and hypoxanthine-xanthine-guanine phosphoribosyltransferase in Toxoplasma gondii.

Engineered delta ribozymes or HDV ribozymes were used as gene expression modulators in Toxoplasma gondii. The substrate recognition sequence of the trans-acting delta ribozyme, which was derived from the self-cleaving motif located on the antigenomic strand of the hepatitis delta virus genome, was modified to target T. gondii transcripts. The mRNA encoding two well-documented genes, namely uracil phosphoribosyltransferase (UPRT) and hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) of T. gondii were chosen as the targets for the ribozymes. UPRT and HXGPRT are the operative enzymes of the pyrimidine and purine salvage pathway, respectively. The knockdown of UPRT or HXGPRT expression by the engineered ribozymes resulted in parasites with lower levels of the corresponding transcripts and diminished their abilities to assimilate radioactive pyrimidine or purine analogs. Five out of six engineered ribozymes could cleave their substrates and gave rise to the products, which were detected by primer extension assays. Upon electroporation of individual active ribozymes against the UPRT gene, the uracil incorporation was decreased. Similarly, the ribozymes against HXGPRT caused decreased incorporation of hypoxanthine. When the most active ribozyme against UPRT was used in a combination with the best HXGPRT specific ribozyme, the incorporation of both uracil and hypoxanthine were decreased. Northern blot analysis revealed that the target transcripts were lowered to an undetectable level when specific ribozymes were used, and that the transcripts of the housekeeping gene remained intact. The ribozyme system should thus prove to be effective for the study of gene expression in T. gondii.

Animals↗
PubMed2006

Lipidic implants for controlled release of bioactive insulin: effects on cartilage engineered in vitro.

Controlled release systems for growth factors and morphogens are potentially powerful tools for the engineering or the treatment of living tissues. However, due to possible instabilities of the protein during manufacture, storage, and release, in the development of new release systems it is paramount to investigate into the maintenance of bioactivity of the protein. Within this study, recently developed protein releasing lipid matrix cylinders of 2 mm diameter and 2 mm height made from glycerol tripalmitate were manufactured in a compression process without further additives. Insulin in different concentrations (0.2%, 1%, and 2%) served as model protein. The bioactivity of the protein released from the matrices was investigated in a long-term cartilage engineering culture for up to four weeks; additionally, the release profiles were determined using ELISA. Insulin released from the matrices increased the wet weights of the cartilaginous cell-polymer constructs (up to 3.2-fold), the amount of GAG and collagen in the constructs (up to 2.4-fold and 3.2-fold, respectively) and the GAG and collagen content per cell (1.8-fold and 2.5-fold, respectively), compared to the control. The dose-dependent effects on tissue development correlated well with release profiles from the matrices with different insulin loading. In conclusion, the lipid matrices, preserving the bioactivity of incorporated and released protein, are suggested as a suitable carrier system for use in tissue engineering or for the localized treatment of tissues with highly potent protein drugs such as used in the therapy of brain cancer or neurodegenerative CNS diseases.

Animals↗
PubMed2005

Guidance of engineered tissue collagen orientation by large-scale scaffold microstructures.

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale ( approximately 200 microm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 microm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds (n=6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124-127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.

Animals↗
PubMed2005

Yarn design for functional tissue engineering.

Tissue engineering requires the ability to design scaffolds with mechanical properties similar to those of the native tissue. Here, B. mori silk yarns are used as a model system to demonstrate the potential benefits and drawbacks of several textile methods used to fabricate tissue engineering scaffolds. Fibers are plied, twisted, cabled, braided, and/or textured to form several geometries with a wide range of mechanical outcomes. Predictable changes in ultimate tensile strength and stiffness are demonstrated following processing and as a function of test environment. The mechanical effects of increasing turns per inch and combining groups of fibers into higher-order yarn structures are demonstrated. Braids, one of the most commonly used textile structures, are shown to be limited by a change in stiffness following the locking-angle and therefore, potentially not the ideal structure for tissue engineering. Cabled yarns appear to allow the most flexibility in mechanical outcomes with a highly organized geometry. Twisted yarns, while more economical than cabled yarns, result in a higher stiffness and lower percent elongation at break than cabled yarns.

Animals↗
PubMed2006

Osteochondral tissue engineering.

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patient's own cells with three-dimensional (3D) porous biomaterials of pre-defined size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reflects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging field.

Bioartificial Organs↗
PubMed2004

Genetically engineered polymers: status and prospects for controlled release.

Genetic engineering methodology has enabled the synthesis of protein-based polymers with precisely controlled structures. Protein-based polymers have well-defined molecular weights, monomer compositions, sequences and stereochemistries. The incorporation of tailor-made motifs at specified locations by recombinant techniques allows the formation of hydrogels, sensitivity to environmental stimuli, complexation with drugs and nucleic acids, biorecognition and biodegradation. Accordingly, a special interest has emerged for the use of protein-based polymers for controlled drug and gene delivery, tissue engineering and other biomedical applications. This article is a review of genetically engineered polymers, their physicochemical characteristics, synthetic strategies used to produce them and their biomedical applications with emphasis on controlled release.

Animals↗
PubMed2006

Cell delivery in regenerative medicine: the cell sheet engineering approach.

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Acrylic Resins↗
PubMed2026

Optimizing eco-engineering pedogenesis of bauxite residues: Synergistic effects of humus and FeSO4/sulfur on microbial community and function.

Eco-engineered pedogenesis represents a promising approach for soil amelioration of bauxite residues (BRs) through exogenous organic matter. However, the role of humus in mediating this process remains poorly understood, significantly impeding the eco-engineering rehabilitation of BRs. In this study, we conducted pot experiments and subsequent microbial analysis to evaluate the individual improvement of humic acid (HA), fulvic acid (FA), and corn straw (SWZ) on the BRs' pedogenesis. High-throughput sequencing analysis revealed that both FA and SWZ were more effective than HA in steering microbial community assembly, as community diversity, dominant taxa enrichment, and species' interaction were all significantly higher (p < 0.05) in the FA/SWZ treatments than in HA treatments. Notably, the combination of FA with FeSO4 specifically enriched halophilic taxa, while FA coupled with sulfur (S) significantly improved the connectivity and complexity of the microbial network, as the average connection degree increasing from 1.008 to 1.113. Hydrolytic enzyme activity assays further indicated that FA, especially when combined with S, was the most effective treatment in restoring microbial function during BR pedogenesis. These findings highlight FA as a critical driver of microbial restructuring and functional recovery in BRs. Moreover, its efficacy can be enhanced by co-amendment with FeSO4 or S. This study provides important theoretical and practical insights for optimizing organic-inorganic amendment strategies to accelerate the eco-engineering pedogenesis of bauxite residues.

Humic Substances↗
PubMed2005

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.

Binding Sites↗
PubMed2005

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.

Amino Acid Sequence↗
PubMed2003

The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading.

OBJECTIVE: Functional tissue engineering (FTE) of articular cartilage involves the use of physiologically relevant mechanical signals to encourage the growth of engineered constructs. The goal of this study was to determine the utility of deformational loading in enhancing the mechanical properties of chondrocyte-seeded agarose hydrogels, and to investigate the role of initial cell seeding density and nutrient supply in this process. DESIGN: Chondrocyte-seeded agarose hydrogels were cultured in free-swelling conditions or with intermittent deformational loading (10% deformation, 1 Hz, 1 h on/ 1 h off, 3 h per day, five days per week) over a two-month culture period. Disks were seeded at lower (10 million cells/ml) and higher (60 million cells/ml) seeding densities in the context of a greater medium supply than previous studies (decreasing the number of cells/ml feed medium/day) and with an increasing concentration of fetal bovine serum (10 or 20% FBS). RESULTS: Under these more optimal nutrient conditions, at higher seeding densities and high serum concentration (20% FBS), dynamically loaded constructs show >2-fold increases in material properties relative to free-swelling controls. After two months of culture, dynamically loaded constructs achieved a Young's modulus of approximately 185 kPa and a dynamic modulus (at 1 Hz) of approximately 1.6 MPa, with a frequency dependent response similar to that of the native tissue. These values represent approximately 3/4 and approximately 1/4 the values measured for the native tissue, respectively. While significant differences were found in mechanical properties, staining and bulk measurements of both proteoglycan and collagen content of higher seeding density constructs revealed no significant differences between free-swelling and loading groups. This finding indicates that deformational loading may act to increase material properties via differences in the structural organization, the production of small linker ECM molecules, or by modulating the size of macromolecular proteoglycan aggregates. CONCLUSIONS: Taken together, these results point to the utility of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs and the necessity for increasing feed media volume and serum supplementation with increasing cell seeding densities.

Animals↗
PubMed2004

Reconstruction of mandibular defects with autologous tissue-engineered bone.

PURPOSE: Maxillofacial reconstructive procedures often require bone graft harvesting, which results in donor site morbidity; the use of tissue-engineered bone would eliminate this problem. In this study, a novel scaffold design and new fabrication protocol were used to produce autologous tissue-engineered constructs (scaffolds seeded with cells) to reconstruct segmental mandibular defects in a minipig model. MATERIALS AND METHODS: Porcine mesenchymal stem cells were isolated from the ilium. They were expanded in culture and seeded onto poly-dl-lactic-coglycolic acid scaffolds. The constructs were placed in a bioreactor and incubated for 10 days in medium and osteogenic supplements. Four full-thickness bony defects (2 x 2 cm) were created in the same pig's mandible. The constructs (n = 2) were placed into 2 of the defects as autologous grafts. One unseeded scaffold and 1 empty defect served as controls. At 6 weeks postimplantation, the pig was sacrificed, the mandible was harvested, and the grafted sites were evaluated by clinical, radiographic, and histologic methods. RESULTS: The construct-implanted defects appeared to be filled with hard tissue resembling bone, whereas controls were filled with fibrous tissue. Radiographically, the tissue-engineered constructs were uniformly radiodense with bone distributed throughout. The interface between native bone and constructs was indistinct. Complete bone ingrowth was not observed in control defects. Osteoblasts, osteocytes, bone trabeculae, and blood vessels were identified throughout the defects implanted with constructs. CONCLUSION: This "proof-of-principle" study indicates that porcine mandibular defects can be successfully reconstructed by in vitro cultured autologous porcine mesenchymal stem cells on a biodegradable polymer scaffold with penetration of bone and blood vessels.

Animals↗
PubMed2004

Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes.

PURPOSE: This study examined the effects of amniocyte-based engineered tendons on partial diaphragmatic replacement. METHODS: Ovine mesenchymal amniocytes were labeled with green fluorescent protein (GFP), expanded, and seeded into a collagen hydrogel. Composite grafts (20 to 25 cm2) based on acellular dermis (group I), or acellular small intestinal submucosa (group II) received either a cell-seeded or an acellular hydrogel within their layers. Newborn lambs (n = 20) underwent partial diaphragmatic replacement with either an acellular or a cellular autologous construct from either group. At 3 to 12 months' postoperatively, implants were subjected to multiple analyses. RESULTS: Diaphragmatic hernia recurrence was significantly higher in animals with acellular grafts (5 of 5) then in animals with cellular ones (1 of 4) in group I (P <.05) but not in group II (3 of 6 and 4 of 5, respectively). Cellular grafts had higher modular (5.27 +/- 1.98 v. 1.27 +/- 0.38 MPa) and ultimate (1.94 +/- 0.70 v. 0.29 +/- 0.05 MPa) tensile strength than acellular implants in group I (P <.05), but not in group II. Quantitative analyses showed no differences in extracellular matrix components between cellular and acellular implants in either group. All cellular implants showed GFP-positive cells. CONCLUSIONS: Diaphragmatic repair with an autologous tendon engineered from mesenchymal amniocytes leads to improved mechanical and functional outcomes when compared with an equivalent acellular bioprosthetic repair, depending on scaffold composition. The amniotic fluid may be a preferred cell source for engineered diaphragmatic reconstruction.

Amniotic Fluid↗