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PubMed2005

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation.

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.

Adjuvants, Immunologic↗
PubMed2006

Tissue engineering approach to the treatment of bone tumors: three cases of cultured bone grafts derived from patients' mesenchymal stem cells.

A novel approach to the treatment of bone tumors using tissue-engineered implants is reported in this study. The number of mesenchymal stem cells (MSCs) obtained from each patient's bone marrow cells was first increased, and the MSCs were forced to differentiate into osteoblasts followed by bone matrix formation on hydroxyapatite (HA) ceramics. The strong osteogenic ability of the implants, as evidenced by high osteoblastic activity, was confirmed. Consequently, the HA surface was covered with the patient's derived cultured osteoblast/bone matrix. The tissue-engineered HA was used to fill the patient's bone cavity after tumor curettage. Immediate healing potential was found by serial plain radiographs and computed tomograhy images, and no adverse reactions were noted in these patients. The results indicate that tissue-engineered osteogenic ceramics might be an alternative to autologous bone grafts.

Adolescent↗
PubMed2006

Engineering of vascular grafts with genetically modified bone marrow mesenchymal stem cells on poly (propylene carbonate) graft.

Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO(2) at 37 degrees C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency.

Animals↗
PubMed2005

Engineering craniofacial scaffolds.

OBJECTIVE: To develop an integrated approach for engineering craniofacial scaffolds and to demonstrate that these engineered scaffolds would have mechanical properties in the range of craniofacial tissue and support bone regeneration for craniofacial reconstruction. EXPERIMENTAL VARIABLE: Scaffold architecture designed to achieve desired elasticity and permeability. Scaffold external shape designed to match craniofacial anatomy. OUTCOME MEASURE: Final fabricated biomaterial scaffolds. Compressive mechanical modulus and strength. Bone regeneration as measured by micro-CT scanning, mechanical testing and histology. SETTING: Departments of Biomedical Engineering, Oral/Maxillofacial Surgery, and Oral Medicine, Pathology and Oncology at the University of Michigan. RESULTS: Results showed that the design/fabrication approach could create scaffolds with designed porous architecture to match craniofacial anatomy. These scaffolds could be fabricated from a wide range of biomaterials, including titanium, degradable polymers, and degradable calcium phosphate ceramics. Mechanical tests showed that fabricated scaffolds had compressive modulus ranging 50 to 2900 MPa and compressive strength ranging from 2 to over 56 MPa, within the range of human craniofacial trabecular bone. In vivo testing of designed scaffolds showed that they could support bone regeneration via delivery of BMP-7 transduced human gingival fibroblasts in a mouse model. Designed hydroxyapatite scaffolds with pore diameters ranging from 400 to 1200 microns were implanted in minipig mandibular defects for 6 and 18 weeks. Results showed substantial bone ingrowth (between 40 and 50% at 6 weeks, between 70 and 80% at 18 weeks) for all scaffolds, with no significant difference based on pore diameter. CONCLUSION: Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

Animals↗
PubMed1993

Genetically engineered vaccines. Comparison of active versus passive immunotherapy against solid tumors.

Late tumor bearers (LTB), that is, mice that had tumors 3 weeks or longer, can have a selective immune dysfunction and fail to respond to target antigens expressed by the cancer cells. Such mice, however, do respond to nonmalignant cells engineered to express the rejection antigen, and they can be vaccinated with such cells to reject growing tumors. In this study, we compared the efficacy of passive immunization with that of active immunization using the engineered vaccine. An allogeneic MHC class I molecule was used as model tumor antigen. We found that active immunotherapy was only effective for small tumors in early stages of growth. In a later stage of tumor growth, active immunotherapy did not cure any mice, whereas passive immunotherapy was successful in all animals. Reasons for the failure of these LTB to respond to active vaccination with the engineered vaccine may be related to the decreased primary or secondary response we observed in these mice after active immunization. It is suggested that normal antigen-presenting cells expressing the tumor rejection antigen can elicit, in the presence of IL-2, antigen-specific T-cell responses by LTB, and that such T cells may be curative when used in adoptive therapy. We also suggest that the stage of tumor growth and the immune status of LTB more closely simulate the conditions observed in cancer patients.

Animals↗
PubMed2026

Plant cis-regulatory grammar: Decoding the multidimensional code of transcriptional regulation for programmable crop engineering.

Cis-regulatory elements (CREs) orchestrate the spatiotemporal precision of gene expression that underlies plant development, adaptation, and domestication. Decoding the cis-regulatory grammar of plant genomes remains a central challenge in modern biology, with profound implications for programmable crop engineering. Here, recent conceptual and technological advances are synthesized to reshape our understanding of plant CREs. This review first argues that CRE function is not only an intrinsic property of DNA sequence alone but also emerges from a multidimensional context, including chromatin accessibility, histone modifications, three-dimensional genome topology, and cell type-specific regulatory landscapes. Furthermore, the convergence of single-cell epigenomics, high-throughput functional assays, and CRISPR-based dissection has begun to unravel this contextual grammar, revealing the computational principles governing transcriptional regulation. Critically, we propose that artificial intelligence (AI) platforms are catalyzing an ongoing transition from descriptive discovery to predictive engineering, wherein these platforms outperform natural evolution in designing synthetic CREs. Finally, a roadmap is outlined toward a plant regulatory grammar foundation model, which will enable truly predictive engineering of gene expression when fine-tuned for specific tasks. Collectively, the integration of single-cell resolution maps, precise genome editing, AI-driven design, and regulatory-compliant delivery systems promises to transform our ability to reprogram plant gene regulation for next-generation agriculture, bridging the gap between foundational regulatory biology and tangible crop improvement.

artificial intelligence↗
PubMed2005

New dimensions in tissue engineering: possible models for human physiology.

Tissue engineering is a discipline of great promise. In some areas, such as the cornea, tissues engineered in the laboratory are already in clinical use. In other areas, where the tissue architecture is more complex, there are a number of obstacles to manoeuvre before clinically relevant tissues can be produced. However, even in areas where clinically relevant tissues are decades away, the tissues being produced at the moment provide powerful new models to aid the understanding of complex physiological processes. This article provides a personal view of the role of tissue engineering in advancing our understanding of physiology, with specific attention being paid to musculoskeletal tissues.

Animals↗
PubMed2003

Principles of cell mechanics for cartilage tissue engineering.

The critical importance of mechanical signals to the health and maintenance of articular cartilage has been well demonstrated. Tissue engineers have taken a cue from normal cartilage physiology and incorporated the use of mechanical stimulation into their attempts to engineer functional cartilage. However, the specific types of mechanical stimulation that are most beneficial, and the mechanisms that allow a chondrocyte to perceive and respond to those forces, have yet to be elucidated. To develop a better understanding of these processes, it is necessary to examine the mechanical behavior of the single chondrocyte. This paper reviews salient topics related to chondrocyte biomechanics and mechanotransduction, and attempts to put this information into a context both appropriate and useful to cartilage tissue engineering. It also describes the directions this exciting field is taking, and lays out a vision for future studies that could have a significant impact on our understanding of cartilage health and disease.

Animals↗
PubMed2003

Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation.

Biochemical stimulation was applied in combination with cyclic mechanical strain to engineered vascular constructs made of isolated smooth muscle cells in a three-dimensional (3D) collagen type 1 matrix. Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) were added exogenously to the medium used to culture the constructs. Mechanical stimulation was applied using a bioreactor system that imparted a 10% circumferential strain at a frequency of 1 Hz. The parameters studied were gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA). Mechanical stimulation caused a characteristic increase in gel compaction and cell proliferation, relative to statically cultured controls. Stimulation with PDGF increased cell proliferation and decreased SMA expression in 3D gels, but inhibited the effects of mechanical stimulation and produced a more open matrix structure. TGF-beta strongly inhibited cell proliferation and increased SMA expression, especially in the presence of mechanical strain, and resulted in a dense matrix. These results show that cell phenotype can be modulated in engineered blood vessels by applying selected combinations of biochemical and mechanical stimuli, and suggest that such control over cell function can be used to tailor the properties of engineered tissues.

Actins↗
PubMed2003

A device for long term, in vitro loading of three-dimensional natural and engineered tissues.

In vitro studies of mechanical loads applied to three-dimensional tissue constructs are important to the design and production of functional, engineered bone tissue. This study reports the development and characterization of a mechanical device capable of subjecting a three-dimensional section of natural or engineered tissue to precise, reproducible four-point bending deformations over a range of programmable magnitudes and frequencies. To test the biological and mechanical capabilities of the system, a low-cycle (360 cycles/day), medium-range strain (2500 microstrain), long-term (16 day) loading regime was applied to rat bone marrow stromal cells cultured in porous DL-polylactic acid scaffolds. Cells proliferated in culture throughout the experiment, and with time showed an increase in alkaline phosphatase expression per cell. Calcium and phosphorus mineral deposition by the unloaded group was significantly greater (p<0.05) than that deposited by the loaded group. The molar ratio of calcium to phosphorus in the unloaded group (0.94:1) was significantly greater (p<0.05) than that of the loaded group (0.41:1). The loading device presented here is a tool which can be used to help elucidate contributions of mechanical loading/fatigue on biodegradable materials, as well as study the effects of mechanical loading on natural or engineered tissues in vitro.

Animals↗
PubMed2002

Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering.

Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).

Biomimetics↗
PubMed1998

Engineering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life.

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.

Bacillus subtilis↗
PubMed2000

Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains.

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2001

Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains.

Ni(2+)-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni(2+)-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni(2+)-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

Amino Acid Sequence↗
PubMed2005

Comparing antigenicity and immunogenicity of engineered gp120.

We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein. Importantly, serum mapping showed that both mutants did not elicit antibodies against a number of epitopes that had been targeted for dampening. The sera from rabbits immunized with the GDMR gp120 mutant neutralized some primary viruses at levels somewhat better than the wild-type gp120 immune sera as a result of an increased elicitation of anti-V3 antibodies. Unlike wild-type gp120 immune sera, GDMR gp120 immune sera failed to neutralize HXBc2, a T-cell line adapted (TCLA) virus. This was associated with loss of CD4bs/CD4-induced antibodies that neutralize TCLA but not primary viruses. The mCHO gp120 immune sera did not neutralize primary viruses to any significant degree, reflecting the masking of epitopes of even weakly neutralizing antibodies without eliciting b12-like antibodies. These results show that antibody responses to multiple epitopes on gp120 can be dampened. More precise focusing to a neutralizing epitope will likely require several iterations comparing antigenicity and immunogenicity of engineered proteins.

Amino Acid Substitution↗
PubMed2000

Metabolic engineering of Saccharomyces cerevisiae.

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.

Biotechnology↗
PubMed2006

Tissue engineering for tendon repair.

Tissue engineering aims to induce tissue self-regeneration in vivo or to produce a functional tissue replacement in vitro to be then implanted in the body. To produce a viable and functional tendon, a uniaxially orientated collagen type I matrix has to be generated. Biochemical and physical factors can potentially alter both the production and the organisation of this matrix, and their combination in a dose- and time-dependent manner is probably the key to in vitro engineered tendons. This review discusses the role of these different factors affecting tenocyte growth in a three-dimensional environment in vivo and in vitro, and underlines the future challenge of tendon tissue engineering.

Biocompatible Materials↗
PubMed1999

Historical cohort study of a New Zealand foundry and heavy engineering plant.

OBJECTIVES: To investigate the mortality of workers who had been exposed to asbestos, machining fluids and foundry work in a foundry and heavy engineering plant in the railway rolling stock manufacturing industry in New Zealand. METHODS: Historical cohort study design. RESULTS: For the total workforce of 3522 men employed between 1945 and 1991, follow up was 90% of person-years to 31 December 1991. Significantly increased standardised mortality ratios (SMRs) were found for all causes of death combined (SMR 1.07; 95% confidence interval (95% CI) 1.01 to 1.14), all malignancies (SMR 1.15; 95% CI 1.01 to 1.31), circulatory (SMR 1.16; 95% CI 1.07 to 1.27) and musculoskeletal diseases (SMR 3.06; 95% CI 1.39 to 5.84), all digestive cancers (SMR 1.29; 95% CI 1.04 to 1.59), all respiratory cancers (SMR 1.34; 95% CI 1.08 to 1.65), cancer of the oesophagus (SMR 1.97; 95% CI 1.01 to 3.45), and mesothelioma of the pleura (SMR 6.58; 95% CI 1.24 to 19.49). Three deaths from pleural mesothelioma were recorded, with latency times of 51, 53, and 57 years. There were no dose-response relations between exposure to asbestos, machining fluids or foundry work, or by duration of employment in the plant, and any cause of death. CONCLUSIONS: This study found small increases in risk for several causes of death among foundry and heavy engineering workers; however, these increases were small and the possible effects of smoking and other lifestyle factors could not be excluded. There was evidence of asbestos related disease in those involved in engineering work in the past.

Aged↗