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PubMed2005

Dermal fibroblasts cultured on small intestinal submucosa: Conditions for the formation of a neotissue.

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial that has been used extensively as a soft tissue replacement, as a scaffold for tissue engineering, and as a substrate for the study of cells in 3D culture. The aim of this study is to define culture parameters that promote neotissue formation with the use of dermal fibroblasts and SIS. SIS sheets were seeded with dermal fibroblasts and cultured for 4 weeks. The resultant cell-scaffold composites (CSCs) were cultured with media alone, media supplemented with ascorbic acid, or fibronectin-pretreated SIS and ascorbic acid. CSCs were analyzed for cellular invasion into the scaffold, the rate of type I collagen production, MMP gelatinolytic activity, thickness, and ultrastructural morphology. CSCs treated with fibronectin and ascorbate showed an increase in Type I collagen production, no change in the MMP gelatinolytic activity, an increase in CSC thickness, and an organized neotissue on the surface of the SIS. Minimal cellular invasion was noted, suggesting that fibroblasts use the SIS as a template for neotissue growth rather than as a scaffold. These results indicate that fibronectin-treated SIS cultured with dermal fibroblasts in the presence of ascorbic acid will promote true neotissue formation for future cardiovascular tissue engineering efforts.

Ascorbic Acid↗

PubMed1999

In vivo tissue engineering: Part I. Concept genesis and guidelines for its realization.

A loss of function of an organ often represents a life-threatening situation. Transplantations are successful, but "replacement" availability, its compatibility with the host, and subsequent healing often pose serious questions. Tissue engineering, where a carefully prepared scaffold is populated, in vitro, by cells to form an artificial organ, addresses some of the problems mentioned above. Trauma associated with the implant introduction to the host often complicates the process. The novel concept of in vivo tissue engineering which is designed to mediate the healing and tissue regeneration process by providing an in vitro formed porous, microcellular scaffold is proposed. The scaffold (part or entire organ) is then populated by cells either spontaneously (the surrounding cells will spread and populate to inhabit the scaffold) or by cellular augmentation (encapsulated cells are delivered to this in statu nascendi scaffold). Minimally traumatic arthroscopic surgery combined with a unique polymer delivery system is envisioned for the introduction of this implant to a site to be repaired. Such an approach will require the formation of polymer in-situ, in a reasonable time. The scaffold-forming polymers will be, in principle, biodegradable. We propose to utilize biodegradable polyurethane systems for in vivo tissue engineering. Diversity of their structure/property relationships, relative "ease" of their preparation, and excellent biocompatibility predetermine polyurethanes to be the materials of choice. This paper describes the genesis of this concept and potentials for its realization. It is intended to initiate and stimulate discussion among the related scientific disciplines to form a basis for this field. The synthesis, application, and biodegradation of selected polyurethanes and variety of its medical utilization will be discussed in upcoming papers.

Artificial Organs↗

PubMedDate not supplied

Use of omentum as an in vivo cell culture system in tissue engineering.

Many modifications of in vitro culture techniques have been applied to promote tissue formation, resulting in limitations. Because the omentum is composed of lobes of adipose tissue with abundant blood vessels and has been used for organ reconstruction, we used the omentum as an in vivo culture system to promote cellular proliferation upon the scaffold. Two kinds of autogenous cells, oral epithelial cells and rib chondrocytes, obtained from canine were isolated and then seeded on porous poly-lactic-glycolic acid scaffolds of a pre-determined shape and size. Comparison was performed in two groups. In Group 1, cell-polymer constructs were cultured in vitro for 2 weeks, and in group 2, cell-polymer constructs were cultured in vitro for 1 week following the same protocol as group 1 but were then implanted into the omentum of same canines for the next week. We performed histologic analysis of tissue formation between the two groups. In group 1, seeded cells were presented spatially along the porous polymer surface only. However, in group 2, the cell-polymer constructs maintained their original dimensions and showed formation of a multicell layered structure with abundant blood vessels. We concluded that the use of the omentum as an in vivo culture medium offers possibilities as an efficient and effective method for tissue engineering with greater vascularization and more consistent cell spacing throughout the construct.

Animals↗

PubMedDate not supplied

Computational study of culture conditions and nutrient supply in cartilage tissue engineering.

Different culture conditions for cartilage tissue engineering were evaluated with respect to the supply of oxygen and glucose and the accumulation of lactate. A computational approach was adopted in which the culture configurations were modeled as a batch process and transport was considered within constructs seeded at high cell concentrations and of clinically relevant dimensions. To assess the extent to which mass transfer can be influenced theoretically, extreme cases were distinguished in which the culture medium surrounding the construct was assumed either completely static or well mixed and fully oxygenated. It can be concluded that severe oxygen depletion and lactate accumulation can occur within constructs for cartilage tissue engineering. However, the results also indicate that transport restrictions are not insurmountable, providing that the medium is well homogenized and oxygenated and the construct's surfaces are sufficiently exposed to the medium. The large variation in uptake rates of chondrocytes indicates that for any specific application the quantification of cellular utilization rates, depending on the cell source and culture conditions, is an essential starting point for optimizing culture protocols.

Cartilage↗

PubMed2006

Determination of structurally conservative amino acids of the HIV-1 protein gp120 V3 loop as promising targets for drug design by protein engineering approaches.

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.

Amino Acid Sequence↗

PubMed1999

Bone induction by implants coated with cultured osteogenic bone marrow cells.

The availability of osteoinductive coatings on dental and orthopedic implants will result in an improved fixation of these devices. Those cases where implants are placed in poor-quality bone or where high failure rates are obtained are especially expected to gain from such coatings. This paper presents a novel, biological approach to obtain bioactive and osteoinductive coatings on bone-replacement implant materials. This so-called tissue engineering approach utilizes osteogenic bone marrow cells that are cultured on an implant material to form a bone-like tissue. The implant materials used herein included porous calcium phosphate scaffolds and metallic plates, the latter of which were coated with a biomimetic calcium phosphate coating to facilitate cellular attachment. Bone marrow cells were obtained from a variety of species, including humans, and were grown to facilitate cellular proliferation. The cells were subsequently seeded onto the implants and cultured for an additional week to facilitate osteogenic differentiation and extracellular matrix production. The resulting hybrid implants, encompassing the biomaterial carrier and cultured bone-like tissue, were subsequently implanted subcutaneously in nude mice for 4 weeks, followed by histological examination for de novo bone formation. The results revealed that newly formed bone was seen both in porous implants and on flat metallic surfaces. This bone tissue engineering approach, therefore, offers great potential to enhance bony healing around implants in a compromised bone bed.

Aged↗

PubMed2004

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations.

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

Biomedical Engineering↗

PubMed2006

Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells.

Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.

Animals↗

PubMed1998

Genetically engineered antibodies in gene transfer and gene therapy.

Our ability to produce and engineer human monoclonal antibodies provides a basis for the development of novel therapeutical strategies against a variety of diseases. These strategies not only include improved passive immunotherapy but also more sophisticated antibody-based gene therapies involving gene transfer approaches. Four of the major applications of antibody gene engineering in the field of gene therapy are reviewed here. These are (1) the redefinition of viral vector tropism of infection for better transduction of cells of therapeutical interest, (2) the grafting of new cell recognition activities to effector cells of the immune system to kill cancer and pathogen-infected cells, (3) the inhibition of cellular and viral functions through intracellular expression of antibody-derived molecules, and (4) the systemic delivery of therapeutic monoclonal antibodies by non-B cells in living organisms.

Antibodies, Monoclonal↗

PubMed2001

Prospects for organ and tissue replacement.

Damage or loss of a tissue or organ is common, costly, and tragic. Advances in mechanical artificial organs and organ transplantation have improved the treatment of organ failure, and advances in molecular immunology, tissue engineering, and stem cell biology offer the promise of even better therapeutic modalities for treating organ failure in the future. Enhancement of immune tolerance of transplanted tissues, improved understanding of cellular differentiation and tissue development, and advances in biomaterials may enable the de novo creation of implantable tissue and organs for transplantation. Innovative techniques for prevention and treatment of tissue loss and organ failure should improve the quality and length of life.

Animals↗

PubMed2006

Corrosion rates of stainless steel under shear stress measured by a novel parallel-plate flow chamber.

A unique parallel-plate flow chamber has been engineered to assess the corrosion properties of implant materials in biological environments under shear flow. This parallel-plate flow chamber provides a novel approach to investigate hypotheses regarding cellular-material-mechanical-force interactions that influence the success or failure of implant devices. The results of the current study demonstrated that physiological stresses (0.5-50 dynes/cm2) from laminar flow from cell culture media did not significantly alter corrosion rates of stainless steel, providing baseline information for an extensive study of the cellular-material-mechanical-force interactions. Furthermore, this study demonstrated that this device is electrochemically stable and provides reproducible results within test parameters. In addition, the results were not significantly different from corrosion tests on bulk samples. Therefore, this system will be useful for investigating cell-material interactions under shear stress for implant alloys or other opaque materials. This information is currently lacking. The results of the present study also support further development of this test system to assess cellular responses to these materials under shear stresses.

Biocompatible Materials↗

PubMed2000

Suppression of IkappaBalpha expression is necessary for c-Jun N-terminal kinase-mediated enhancement of Fas cytotoxicity.

The role of c-Jun N-terminal kinase (JNK) in the regulation of Fas-mediated cell death was investigated. Murine L929 fibroblasts were pretreated with anisomycin for 1 h to activate JNK, followed by exposure to anti-Fas antibodies/actinomycin D (ActD) for 16-24 h. Compared to untreated controls, the induction of JNK activation failed to raise cellular sensitivity to anti-Fas/ActD killing. Notably, a significant increase in anti-Fas/ActD killing as induced by JNK preactivation was observed in L929 cells which were engineered to suppress IkappaBalpha protein expression by antisense mRNA. Restoration of the IkappaBalpha protein level in these cells by ectopic expression of a cDNA construct abolished the JNK-increased anti-Fas/ActD killing. Despite the suppression of IkappaBalpha, no constitutive p65 (RelA) NF-kappaB nuclear translocation was observed in the IkappaBalpha-antisense cells. Also, inhibition of NF-kappaB by curcumin failed to inhibit the JNK-increased Fas cytotoxicity, suggesting that NF-kappaB is not involved in the observed effect. Most interestingly, culturing of L929 cells on extracellular protein matrices resulted in partial suppression of IkappaBalpha expression and constitutive JNK and p42/44 MAPK activation. Upon stimulation with anisomycin, these matrix protein-stimulated cells further exhibited reduced IkappaBalpha expression and p42/44 MAPK activation, as well as became sensitized to JNK-increased anti-Fas/ActD killing. Again, ectopic expression of IkappaBalpha in these cells abolished the enhanced anti-Fas/ActD killing effect. Together, these results indicate that suppression of IkappaBalpha expression is essential for JNK-mediated enhancement of Fas cytotoxicity.

Animals↗

PubMed2005

Animal models in scleroderma.

Scleroderma or systemic sclerosis is an insidious connective tissue disease with no known cure. A hallmark feature of scleroderma is the excess synthesis and deposition of collagen resulting in a fibrotic state. In scleroderma, fibrosis is not confined only to the skin but impacts internal organs as well. In an effort to better understand the pathophysiology of this disease, researchers have developed a variety of animal models that display features of the human condition. This paper focuses on mouse models of scleroderma and summarizes work conducted with these experimental paradigms that is focused on understanding the cellular and molecular events associated with the onset and maintenance of fibrosis.

Animals↗

PubMed2006

Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches.

Environmental constraints that include abiotic stress factors such as salt, drought, cold and extreme temperatures severely limit crop productivity. Improvement of crop plants with traits that confer tolerance to these stresses was practiced using traditional and modern breeding methods. Molecular breeding and genetic engineering contributed substantially to our understanding of the complexity of stress response. Mechanisms that operate signal perception, transduction and downstream regulatory factors are now being examined and an understanding of cellular pathways involved in abiotic stress responses provide valuable information on such responses. This review presents genomic-assisted methods which have helped to reveal complex regulatory networks controlling abiotic stress tolerance mechanisms by high-throughput expression profiling and gene inactivation techniques. Further, an account of stress-inducible regulatory genes which have been transferred into crop plants to enhance stress tolerance is discussed as possible modes of integrating information gained from functional genomics into knowledge-based breeding programs. In addition, we envision an integrative genomic and breeding approach to reveal developmental programs that enhance yield stability and improve grain quality under unfavorable environmental conditions of abiotic stresses.

Adaptation, Physiological↗

PubMed2006

Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability.

Tetramerization of the human p53 tumor suppressor protein is required for its biological functions. However, cellular levels of p53 indicate that it exists predominantly in a monomeric state. Since the oligomerization of p53 involves the rate-limiting formation of a primary dimer intermediate, we engineered a covalently linked pair of human p53 tetramerization (p53tet) domains to generate a tandem dimer (p53tetTD) that minimizes the energetic requirements for forming the primary dimer. We demonstrate that p53tetTD self-assembles into an oligomeric structure equivalent to the wild-type p53tet tetramer and exhibits dramatically enhanced oligomeric stability. Specifically, the p53tetTD dimer exhibits an unfolding/dissociation equilibrium constant of 26 fM at 37 degrees C, or a million-fold increase in stability relative to the wild-type p53tet tetramer, and resists subunit exchange with monomeric p53tet. In addition, whereas the wild-type p53tet tetramer undergoes coupled (i.e. two-state) dissociation/unfolding to unfolded monomers, the p53tetTD dimer denatures via an intermediate that is detectable by differential scanning calorimetry but not CD spectroscopy, consistent with a folded p53tetTD monomer that is equivalent to the p53tet primary dimer. Given its oligomeric stability and resistance against hetero-oligomerization, dimerization of p53 constructs incorporating the tetramerization domain may yield functional constructs that may resist exchange with wild-type or mutant forms of p53.

Calorimetry, Differential Scanning↗

PubMed2004

A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.

Alkaline Phosphatase↗

PubMed2005

Growth and phenotype of low-density nasal septal chondrocyte monolayers.

OBJECTIVE: To analyze the growth patterns and differentiation of human septal chondrocyte monolayers of different seeding densities. STUDY DESIGN: Chondrocytes from 8 donors were plated at densities ranging from 20,000 cells/cm(2) (high density) to 300 cells/cm(2) (very low density). Confluency, cellularity, and glycosaminoglycan content were determined from days 1 to 15. RESULTS: Confluency was attained at 5.8, 8.3, 11.0, and 14.8 days for high-, intermediate-, low-, and very low-density monolayers, respectively (P < 0.001). Regression growth curves showed typical lag, logarithmic, and stationary phases. Confluent monolayers attained similar cellularity (power = 0.94) and differentiation (power = 0.88), regardless of initial density. CONCLUSIONS: Human septal chondrocyte monolayers reach confluency from very low initial densities. Growth patterns, cellularity, and differentiation are similar to other starting densities. SIGNIFICANCE: Very low-density monolayers expanded cell number 838-fold in 1 passage and therefore are sufficient for tissue-engineering purposes. This is important because of the requirement of maintaining differentiation and the limitation of small tissue harvest specimens.

Adult↗

PubMed2005

Cell-specific targeting of nanoparticles by multivalent attachment of small molecules.

Nanomaterials with precise biological functions have considerable potential for use in biomedical applications. Here we investigate whether multivalent attachment of small molecules can increase specific binding affinity and reveal new biological properties of such nanomaterials. We describe the parallel synthesis of a library comprising 146 nanoparticles decorated with different synthetic small molecules. Using fluorescent magnetic nanoparticles, we rapidly screened the library against different cell lines and discovered a series of nanoparticles with high specificity for endothelial cells, activated human macrophages or pancreatic cancer cells. Hits from the last-mentioned screen were shown to target pancreatic cancer in vivo. The method and described materials could facilitate development of functional nanomaterials for applications such as differentiating cell lines, detecting distinct cellular states and targeting specific cell types.

Animals↗