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PubMed2004

Cells, scaffolds, and molecules for myocardial tissue engineering.

Unlike heart valves or blood vessels, heart muscle has no replacement alternatives. The most challenging goal in the field of cardiovascular tissue engineering is the creation/ regeneration of an engineered heart muscle. Recent advances in methods of stem cell isolation, culture in bioreactors, and the synthesis of bioactive materials promise to create engineered cardiac tissue ex vivo. At the same time, new approaches are conceived that explore ways to induce tissue regeneration after injury. The purpose of our review is to describe the principles, status, and challenges of myocardial tissue engineering with emphasize on the concept of in situ cardiac tissue engineering and regeneration.

Biocompatible Materials↗
PubMed2006

Localizing the reovirus packaging signals using an engineered m1 and s2 ssRNA.

Using in vitro engineered and transcribed reovirus m1 and s2 ssRNAs, we demonstrate that the nucleotides used to identify these ssRNAs are localized to the 5' and not the 3' termini. To demonstrate this, we used our previously reported S2-CAT reovirus and we report the creation of an engineered M1-CAT reovirus. The M1 gene of this virus retains 124 nucleotides from the wild type M1 gene preceding the CAT gene and 172 nucleotides from the wild type gene following the CAT gene. The engineered M1-CAT ssRNA is 1048 nucleotides in length, much shorter than the wild type M1 at 2304 nucleotides. We have used a set of chimeric s2.m1 ssRNAs to localize the packaging signals within these RNAs. By packaging signals we mean that the presence of these signals in engineered ssRNAs results in these ssRNAs being replicated to dsRNA and packaged into progeny virus. An engineered ssRNA with a 5' sequence identical with the wild type s2 ssRNA, supported by a 3' sequence from either the m1 or s2 ssRNA, is incorporated into a virus as an S2 dsRNA. Likewise, an ssRNA with an m1 5' end is incorporated as an M1 dsRNA.

Animals↗
PubMed2004

Impact of 'ome' analyses on inverse metabolic engineering.

Genome-wide or large-scale methodologies employed in functional genomics such as DNA sequencing, transcription profiling, proteomics, and metabolite profiling have become important tools in many metabolic engineering strategies. These techniques allow the identification of genetic differences and insight into their cellular effects. In the field of inverse metabolic engineering mapping of differences between strains with different degree of a certain desired phenotype and subsequent identification of factors conferring that phenotype are an essential part. Therefore, the tools of functional genomics in particular have the potential to promote and expand inverse metabolic engineering. Here, we review the use of functional genomics methods in inverse metabolic engineering, examples are presented, and we discuss the identification of targets for metabolic engineering with low fold changes using these techniques.

Animals↗
PubMed2003

Fetal tissue engineering: chest wall reconstruction.

BACKGROUND/PURPOSE: This study was aimed at applying fetal tissue engineering to chest wall reconstruction. METHODS: Fetal lambs underwent harvest of elastic and hyaline cartilage specimens. Once expanded in vitro, fetal chondrocytes were seeded onto synthetic scaffolds, which then were placed in a bioreactor. After birth, fetal cartilage constructs (n = 10) were implanted in autologous fashion into the ribs of all lambs (n = 6) along with identical, but acellular scaffolds, as controls (n = 6). Engineered and acellular specimens were harvested for analysis at 4 to 12 weeks postimplantation. Standard histology and matrix-specific staining were performed both before implantation and after harvest on all constructs. RESULTS: Regardless of the source of chondrocytes, all fetal constructs resembled hyaline cartilage, both grossly and histologically, in vitro. In vivo, engineered implants retained hyaline characteristics for up to 10 weeks after implantation but remodeled into fibrocartilage by 12 weeks postoperatively. Mononuclear inflammatory infiltrates surrounding residual PGA/PLLA polymer fibers were noted in all specimens but most prominently in the acellular controls. CONCLUSIONS: Engineered fetal cartilage can provide structural replacement for at least up to 10 weeks after autologous, postnatal implantation in the chest wall. Fetal tissue engineering may prove useful for the treatment of severe congenital chest wall defects at birth.

Animals↗
PubMed2003

Extracardiac total cavopulmonary connection using a tissue-engineered graft.

OBJECTIVE: Extracardiac and lateral tunnel total cavopulmonary connection are currently 2 major options for patients with a single ventricle physiology. However, each procedure has some disadvantages over the other. We developed a new technique of extracardiac total cavopulmonary connection using a tissue-engineered graft to overcome some of the disadvantages previously associated with both the extracardiac and lateral tunnel procedures. METHODS: Between February 2001 and October 2002, 8 patients underwent an extracardiac total cavopulmonary connection using a tissue-engineered graft in our institution. Collected bone marrow cells (1 x 10(8) mononucleocytes) from a patient (approximately 1-4 mL/kg body weight) were seeded onto a biodegradable scaffold composed of polycaprolactone-polylactic acid copolymer reinforced with woven polylactic acid. After a 2- to 4-hour cultivation, the seeded scaffold was implanted as an extracardiac conduit during the total cavopulmonary connection operation. RESULTS: There were no hospital or late deaths. At a mean follow-up of 13.4 months (range 4-25 months), all patients are alive and asymptomatic with no need for repeat surgery. A postoperative catheter examination or computed tomography showed all tissue-engineered grafts to be patent and revealed no stenosis, obstruction, or aneurysmal change in the 8 patients. CONCLUSION: We believe that extracardiac total cavopulmonary connection using a tissue-engineered graft has the potential to overcome some of the disadvantages previously associated with extracardiac or lateral tunnel total cavopulmonary connection. However, an extended follow-up period is required to clarify the long-term clinical outcome for the tissue-engineered graft.

Adolescent↗
PubMed2003

Bioaccumulation of nickel from aqueous solutions by genetically engineered Escherichia coli.

This study constructed a genetically engineered Escherichia coli JM109 which simultaneously expressed nickel transport system and metallothionein to remove and recover Ni(2+) from aqueous solution. Bioaccumulation process was rapid and followed linearized Langmuir isotherm. A more than six-fold increase of Ni(2+) binding capacity was obtained by genetically engineered E. coli cells compared with original host E. coli cells. A pH assay showed genetically engineered E. coli cells accumulated Ni(2+) effectively over a broad range of pH (4-10). The presence of 1000 mg/L Na(+) and Ca(2+), or 50mg/L Cd(2+) or Pb(2+) did not have a significant effect on Ni(2+) bioaccumulation, while Mg(2+), Hg(2+) and Cu(2+) posed a severe adverse influence on Ni(2+) uptake by genetically engineered E. coli. Furthermore, genetically engineered E. coli cells did not require extra nutrients for Ni(2+) bioaccumulation.

Biodegradation, Environmental↗
PubMed2005

Evolving concepts in bone tissue engineering.

The field of tissue engineering integrates the latest advances in molecular biology, biochemistry, engineering, material science, and medical transplantation. Researchers in the developing field of regenerative medicine have identified bone tissue engineering as an attractive translational target. Clinical problems requiring bone regeneration are diverse, and no single regeneration approach will likely resolve all defects. Recent advances in the field of tissue engineering have included the use of sophisticated biocompatible scaffolds, new postnatal multipotent cell populations, and the appropriate cellular stimulation. In particular, synthetic polymer scaffolds allow for fast and reproducible construction, while still retaining biocompatible characteristics. These criteria relate to the immediate goal of determining the ideal implant. The search is becoming a reality with widespread availability of biocompatible scaffolds; however, the desired parameters have not been clearly defined. Currently, most research focuses on the use of bone morphogenetic proteins (BMPs), specifically BMP-2 and BMP-7. These proteins induce osteogenic differentiation in vitro, as well as bone defect healing in vivo. Protein-scaffold interactions that enhance BMP binding are of the utmost importance, since prolonged BMP release creates the most osteogenic microenvironment. Transition into clinical studies has had only mild success and relies on large doses of BMPs for bone formation. Advances within the field of bone tissue engineering will likely overcome these challenges and lead to more clinically relevant therapies.

Animals↗
PubMed2006

Tissue engineering using adult stem cells.

Patients with a variety of diseases may be treated with transplanted tissues and organs. However, there is a shortage of donor tissues and organs, which is worsening yearly because of the aging population. Scientists in the field of tissue engineering are applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The stem cell field is also advancing rapidly, opening new options for cellular therapy and tissue engineering. The use of adult stem cells for tissue engineering applications is promising. This chapter discusses applications of these new technologies for the engineering of tissues and organs. The first part provides an overview of regenerative medicine and tissue engineering techniques; the second highlights different adult stem cell populations used for tissue regeneration.

Adult Stem Cells↗
PubMed2003

Tissue engineering, stem cells, and cloning for the regeneration of urologic organs.

Tissue engineering efforts are currently being undertaken for every type of tissue and organ within the urinary system. Most of the effort expended to engineer genitourinary tissues has occurred within the last decade. Tissue engineering techniques require a cell culture facility designed for human application. Personnel who have mastered the techniques of cell harvest, culture, and expansion as well as polymer design are essential for the successful application of this technology. Various engineered genitourinary tissues are at different stages of development, with some already being used clinically, a few in preclinical trials, and some in the discovery stage. Recent progress suggests that engineered urologic tissues may have an expanded clinical applicability in the future.

Biocompatible Materials↗
PubMedDate not supplied

Blood vessels engineered from human cells.

Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.

Aged↗
PubMed2006

Tissue-engineered autologous bladders for patients needing cystoplasty.

BACKGROUND: Patients with end-stage bladder disease can be treated with cystoplasty using gastrointestinal segments. The presence of such segments in the urinary tract has been associated with many complications. We explored an alternative approach using autologous engineered bladder tissues for reconstruction. METHODS: Seven patients with myelomeningocele, aged 4-19 years, with high-pressure or poorly compliant bladders, were identified as candidates for cystoplasty. A bladder biopsy was obtained from each patient. Urothelial and muscle cells were grown in culture, and seeded on a biodegradable bladder-shaped scaffold made of collagen, or a composite of collagen and polyglycolic acid. About 7 weeks after the biopsy, the autologous engineered bladder constructs were used for reconstruction and implanted either with or without an omental wrap. Serial urodynamics, cystograms, ultrasounds, bladder biopsies, and serum analyses were done. RESULTS: Follow-up range was 22-61 months (mean 46 months). Post-operatively, the mean bladder leak point pressure decrease at capacity, and the volume and compliance increase was greatest in the composite engineered bladders with an omental wrap (56%, 1.58-fold, and 2.79-fold, respectively). Bowel function returned promptly after surgery. No metabolic consequences were noted, urinary calculi did not form, mucus production was normal, and renal function was preserved. The engineered bladder biopsies showed an adequate structural architecture and phenotype. CONCLUSIONS: Engineered bladder tissues, created with autologous cells seeded on collagen-polyglycolic acid scaffolds, and wrapped in omentum after implantation, can be used in patients who need cystoplasty.

Adolescent↗
PubMed2003

Successful application of tissue engineered vascular autografts: clinical experience.

Foreign materials often used in cardiovascular surgery may cause unwanted side effects and reduced growth potential. To resolve these problems, we have designed a tissue-engineering technique that utilizes bone marrow cells (BMCs) in clinical treatments. To obtain tissue-engineered material, we harvested saphenous vein samples from patients, which were then minced, cultured and seeded onto a biodegradable scaffold. The first operation was performed in May 1999 as previously described (N. Engl. J. Med. 344 (7) (2001) 532) and this method was repeated on two other patients. From November 2001, we used aspirated BMCs as the cell source, which were seeded onto the scaffold on the day of surgery. This method was applied in 22 patients. There was no morbidity such as thrombogenic complications, stenosis or obstruction of tissue-engineered autografts, and no mortality due to these techniques. These results indicate that BMCs seeded onto a biodegradable scaffold to establish tissue-engineered vascular autografts (TEVAs) is an ideal strategy, and present strong evidence for the justification and validity of our protocol in clinical trials of tissue engineering.

Adolescent↗
PubMed2004

Tissue engineering of the meniscus.

Meniscus lesions are among the most frequent injuries in orthopaedic practice and they will inevitably lead to degeneration of the knee articular cartilage. The fibro-cartilage-like tissue of the meniscus is notorious for its limited regenerative capacity. Tissue engineering could offer new treatment modalities for repair of meniscus tears and eventually will enable the replacement of a whole meniscus by a tissue-engineered construct. Many questions remain to be answered before the final goal, a tissue-engineered meniscus is available for clinical implementation. These questions are related to the selection of an optimal cell type, the source of the cells, the need to use growth factor(s) and the type of scaffold that can be used for stimulation of differentiation of cells into tissues with optimal phenotypes. Particularly in a loaded, highly complex environment of the knee, optimal mechanical properties of such a scaffold seem to be of utmost importance. With respect to cells, autologous meniscus cells seems the optimal cell source for tissue engineering of meniscus tissue, but their availability is limited. Therefore research should be stimulated to investigate the suitability of other cell sources for the creation of meniscus tissue. Bone marrow stroma cells could be useful since it is well known that they can differentiate into bone and cartilage cells. With respect to growth factors, TGF-beta could be a suitable growth factor to stimulate cells into a fibroblastic phenotype but the problems of TGF-beta introduced into a joint environment should then be solved. Polyurethane scaffolds with optimal mechanical properties and with optimal interconnective macro-porosity have been shown to facilitate ingrowth and differentiation of tissue into fibro-cartilage. However, even these materials cannot prevent cartilage degeneration in animal models. Surface modification and/or seeding of cells into the scaffolds before implantation may offer a solution for this problem in the future.This review focuses on a number of specific questions; what is the status of the development of procedures for lesion healing and how far are we from replacing the entire meniscus by a (tissue-engineered) prosthesis. Subquestions related to the type of scaffold used are: is the degree of tissue ingrowth and differentiation related to the initial mechanical properties and if so, what is the influence of those properties on the subsequent remodelling of the tissue into fibro-cartilage; what is the ideal pore geometry and what is the optimal degradation period to allow biological remodelling of the tissue in the scaffold. Finally, we will finish with our latest results of the effect of tear reconstruction and the insertion of prostheses on articular cartilage degradation.

Animals↗
PubMed2004

The use of animal models in developing the discipline of cardiovascular tissue engineering: a review.

Cardiovascular disease remains one of the major causes of death and disability in the Western world. Tissue engineering offers the prospect of being able to meet the demand for replacement of heart valves, vessels for coronary and lower limb bypass surgery and the generation of cardiac tissue for addition to the diseased heart. In order to test prospective tissue-engineered devices, these constructs must first be proven in animal models before receiving CE marking or FDA approval for a clinical trial. The choice of animal depends on the nature of the tissue-engineered construct being tested. Factors that need to be considered include technical requirements of implanting the construct, availability of the animal, cost and ethical considerations. In this paper, we review the history of animal studies in cardiovascular tissue engineering and the uses of animal tissue as sources for tissue engineering.

Animals↗
PubMed2000

Protein engineering of subtilisin.

The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

Calcium↗
PubMed2003

Cryopreservation of tissue engineered constructs for bone.

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

Bone Substitutes↗
PubMed1998

A protein engineering analysis of the transition state for protein folding: simulation in the lattice model.

BACKGROUND: Protein engineering has been used extensively to evaluate the properties of transition states in protein folding. Although the method has proved useful, its limitations and the details of interpretation of the obtained results remain largely unexplored. RESULTS: Lattice model simulations are used to test and verify the protein engineering analysis of the transition state in protein folding. It is shown that in some cases - but not always - this method is able to determine the transition state with reasonable accuracy. Limitations of protein engineering are revealed and analyzed. In particular, the change in non-native interactions as a result of mutations is shown to influence the results of the protein engineering analysis. Furthermore, the temperature dependencies of phi values (which are a measure of the participation of a residue in the transition state) and the character of the transition state ensemble are studied. It is shown that as a general trend phi values decrease when the temperature decreases, a finding consistent with recent experimental results. Our analysis suggests that this trend results primarily from the formation of some contacts (native and non-native) in the unfolded state at a lower temperature, when the barrier for folding is energetic. CONCLUSIONS: Our analysis helps to interpret the results of protein engineering and allows observed φ values to be directly related to structural features of the unfolded state, the transition state and the native state.

Amino Acid Sequence↗
PubMed1999

Genetic engineering of crops as potential source of genetic hazard in the human diet.

The benefits of genetic engineering of crop plants to improve the reliability and quality of the world food supply have been contrasted with public concerns raised about the food safety of the resulting products. Debates have concentrated on the possible unforeseen risks associated with the accumulation of new metabolites in crop plants that may contribute to toxins, allergens and genetic hazards in the human diet. This review examines the various molecular and biochemical mechanisms by which new hazards may appear in foods as a direct consequence of genetic engineering in crop plants. Such hazards may arise from the expression products of the inserted genes, secondary or pleiotropic effects of transgene expression, and random insertional mutagenic effects resulting from transgene integration into plant genomes. However, when traditional plant breeding is evaluated in the same context, these mechanisms are no different from those that have been widely accepted from the past use of new cultivars in agriculture. The risks associated with the introduction of new genes via genetic engineering must be considered alongside the common breeding practice of introgressing large fragments of chromatin from related wild species into crop cultivars. The large proportion of such introgressed DNA involves genes of unknown function linked to the trait of interest such as pest or disease resistance. In this context, the potential risks of introducing new food hazards from the applications of genetic engineering are no different from the risks that might be anticipated from genetic manipulation of crops via traditional breeding. In many respects, the precise manner in which genetic engineering can control the nature and expression of the transferred DNA offers greater confidence for producing the desired outcome compared with traditional breeding.

Crops, Agricultural↗