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PubMed2004

Genetic modification of stem cells to enhance bone repair.

Orthopaedic surgeons are often faced with difficult bone loss problems. Conventional bone grafting is usually accomplished with autogenous iliac crest bone graft that provides osteogenic cells, osteoinductive growth factors, and an osteoconductive matrix. Cadaveric bone allograft and bone graft substitutes are inferior to autogenous bone graft because they fail to supply osteogenic cells or a significant amount of osteoinductive growth factors. Recombinant growth factors such as bone morphogenetic protein-2 and osteogenic protein-1 are currently in clinical use but these proteins require supraphysiologic dosing and considerable expense while failing to provide a sustained osteoinductive signal at the implantation site. Mesenchymal stem cells capable of differentiating into mesodermal tissues have been isolated and expanded in culture from several different sources including bone marrow, adipose tissue, and muscle. In the presence of appropriate growth factors these cells can differentiate into osteoblast lineage cells that will form bone in vitro and in vivo. Recent attention has focused on genetic modification of mesenchymal stem cells to both produce and respond to osteogenic growth factors with the goal of developing a tissue engineering strategy for bone repair. This review examines the current potential and limitations of these cellular systems for bone repair.

Animals↗
PubMed1996

Role of material surfaces in regulating bone and cartilage cell response.

Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Studies comparing chondrocyte response to TiO2 of differing crystallinities show that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. Optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biologicals is another.

Adsorption↗
PubMedDate not supplied

Cell-based therapy for disc repair.

BACKGROUND CONTEXT: One of the most promising therapies for symptomatic disc degeneration involves the implantation of therapeutic cells into the degenerative disc. PURPOSE: In this article, the rationale and approaches for cell-based tissue engineering of the intervertebral disc are discussed. STUDY DESIGN: The scientific literature related to cell-based tissue engineering of the intervertebral disc is reviewed. METHODS: A variety of cell types have been used in various research models to affect matrix repair of the intervertebral disc. The use of cellular scaffolds and growth factors or genes also appears promising for achieving meaningful tissue repair of the intervertebral disc. RESULTS: Disc tissue engineering is a promising approach for achieving repair of the intervertebral disc. Using cell-based approaches, various research models suggest that improvements in the complex matrix of the disc may be achieved. CONCLUSION: A cell-based approach to repair of the intervertebral disc appears promising. More research is needed to define the optimal cell type, cellular scaffold and mixture of growth factors that may allow meaningful repair of the human symptomatic degenerative disc.

Animals↗
PubMed1989

Human cell models for genetic engineering.

Technological advances made in molecular biology and culture of human and other mammalian cells in vitro have increased the ability to introduce functional genes into a variety of cell types. Subgenomic components of both DNA and RNA viruses are used as the eukaryotic components of many recombinant DNA vector constructs which are used for transfection either by a variety of methods to facilitate DNA uptake or as virions. This paper reviews some of the factors involved in expression, persistence, and recombination of the introduced genes. Also considered are the use of DNA transfection models to study human cancer or other diseases, the current status of gene therapy, and the use of human cells to produce biologicals. The need to better develop well-characterized human cell culture models from multiple organ sites and various cell types is a recurrent theme for all of these applications, including the role that such cells will play in understanding the functions for genes which will be identified in the human genome cloning project. With continued study and a better understanding of pathophysiology at the cellular and molecular levels should come the logical next step of adding genetically engineered human cells to the repertoire of acceptable biomedical technologies.

Cells, Cultured↗
PubMed2000

Intracellular trehalose improves the survival of cryopreserved mammalian cells.

We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus alpha-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.

3T3 Cells↗
PubMedDate not supplied

Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation.

To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.

Adipose Tissue↗
PubMed1999

Effects of microgravity on growing cultured skin constructs.

Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue. The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated. LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix. Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B). All groups were cultured for an average of 3 additional days. LSEs were processed for histologic evaluation. Skin cells were stained for cytokeratin to evaluate function. Images were digitally captured and processed for analysis. Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured. Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001). No significant difference in cellular function as measured by protein expression [stained cytokeratin area (micro(2)) per cell] was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227). In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups. Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001). Elimination of the air/liquid interface enhanced proliferation of keratinocytes. This effect was further enhanced in the presence of microgravity. No significant effect on cell function was noted with the use of this microgravity environment. We hypothesize that the increased epidermal contact plays a role in this proliferation. Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.

Air↗
PubMed2006

Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium.

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)-derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6-10-fold because of induction of proliferation on purification. Long-term engraftment (4-5 months) was observed when co-transplanting selected ES cell-derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell-derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Animals↗
PubMed1991

Antibodies against vertebrate microfilament proteins in the analysis of cellular motility and adhesion.

Microinjection of specific antibodies can be an alternative and a supplement to genetic engineering in dissecting the function of individual cytoskeletal components. In this report, we describe some of the requirements for using this technique, its potential application in conjunction with morphological and biochemical analyses, and its limitations. Examples are given for the injection of antibodies to alpha-actinin, vinculin and myosin, and the effects of such treatment on adhesion, motility and cytokinesis of the recipient cells.

Animals↗
PubMed2006

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.

Actins↗
PubMed2001

Chemical engineering of cell penetrating antibodies.

Antibodies, being exquisitely specific tools in biology, are routinely used to detect and identify intra-cellular structures. However, current intra-cellular application of antibodies requires that the membrane be rendered leaky, resulting in the death of cells. Here, we present a novel method to allow antibodies to penetrate the cellular membrane of living cells without affecting cell viability. A peptide (MTS, membrane transport sequence) that facilitates transport across membranes has been site-specifically attached to antibodies. MTS-antibodies enter the living cells in culture and can be detected by immunofluorescence and ELISA after extraction. Cellular structures are visualized in living cells using a specific MTS-antibody. Antibodies with membrane penetrating properties can become an important tool for the study of intra-cellular processes in living cells. Furthermore, such membrane penetrating antibodies can be used to selectively stimulate or suppress functions of the cellular machinery.

3T3 Cells↗
PubMed2000

Genomes and form. The case for teleomorphic recursivity.

The genotype-phenotype (genome-form) distinction is considered by many to be fundamental to modern evolutionary thinking. Indeed, the premises that: DNA solely constitutes the genotype; that the phenotype is a transient product of the genotype, with the latter not only describing, but also implementing the construction of the former; and that the constructed materials and systems of the cell have no impact on the genotype, have become dogmas. Yet a vast body of data from molecular genetics reveals that cellular systems, directly and indirectly, alter the genome. Some of these data are reviewed. Proteins can influence mutations along the chromosomes, heritably modify the information content of DNA sequences, and, in some instances, reorganize the germline or somatic genome via DNA engineering pathways. These data suggest that the constructed (proteins, chromatin arrays, and metabolic pathways) has an important role in shaping the descriptor. Insofar as it is biochemically possible for states adopted by cellular structures to be stabilized and eventually memorized by engineering chromosomes, semantic closure can be transcended--meaning can be transferred from the domain of form to the genome, and this presumably ongoing process is termed teleomorphic recursivity. Throughout the paper, I implicitly argue that the genome-form partition is strictly a formal one, with no deeply material basis.

Biological Evolution↗
PubMed2000

Tissue-engineered valved conduits in the pulmonary circulation.

OBJECTIVE: Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue-engineering techniques. METHODS: Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (+/-3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. RESULTS: Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. CONCLUSION: This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue-engineered structure continues for at least 6 months.

Animals↗
PubMed2002

Microengineering neocartilage scaffolds.

Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.

Absorbable Implants↗
PubMed2002

Drug-releasing scaffolds fabricated from drug-loaded microspheres.

Biodegradable scaffolds serve a central role in many strategies for engineering tissue replacements or in guiding tissue regeneration. Typically, these scaffolds function to create and maintain a space and to provide a support for cell adhesion. However, these scaffolds also can serve as vehicles for the delivery of bioactive factors (e.g., protein or DNA) in order to manipulate cellular processes within the scaffold microenvironment. This study presents a novel approach to fabricate tissue-engineering scaffolds capable of sustained drug delivery whereby drug-loaded microspheres are fabricated into structures with controlled porosity. A double-emulsion process was used to fabricate microspheres with encapsulated DNA that retained its integrity and was released from the microspheres within 24 h. These DNA-loaded microspheres subsequently were formed into a nonporous disk or an interconnected open-pore scaffold (>94% porosity) via a gas-foaming process. The disks and scaffolds exhibited sustained plasmid release for at least 21 days and had minimal burst during the initial phase of release. This approach of assembling drug-loaded microspheres into porous and nonporous structures may find great utility in the fabrication of synthetic matrices that direct tissue formation.

Biocompatible Materials↗
PubMed2002

Advances in molecular labeling, high throughput imaging and machine intelligence portend powerful functional cellular biochemistry tools.

Cellular behavior is complex. Successfully understanding systems at ever-increasing complexity is fundamental to advances in modern science and unraveling the functional details of cellular behavior is no exception. We present a collection of prospectives to provide a glimpse of the techniques that will aid in collecting, managing and utilizing information on complex cellular processes via molecular imaging tools. These include: 1) visualizing intracellular protein activity with fluorescent markers, 2) high throughput (and automated) imaging of multilabeled cells in statistically significant numbers, and 3) machine intelligence to analyze subcellular image localization and pattern. Although not addressed here, the importance of combining cell-image-based information with detailed molecular structure and ligand-receptor binding models cannot be overlooked. Advanced molecular imaging techniques have the potential to impact cellular diagnostics for cancer screening, clinical correlations of tissue molecular patterns for cancer biology, and cellular molecular interactions for accelerating drug discovery. The goal of finally understanding all cellular components and behaviors will be achieved by advances in both instrumentation engineering (software and hardware) and molecular biochemistry.

Animals↗
PubMed2006

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs.

Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three-dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue-development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue-development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues.

Adult↗
PubMed1995

Plasmid mutagenesis by PCR for high-level expression of para-hydroxybenzoate hydroxylase.

We report a PCR deletion mutagenesis method for the exact positioning of a foreign gene (pobA) in the lac operon of an expression plasmid in place of the lacZ protein code. This method requires the synthesis of four oligonucleotides and three PCR reactions to delete unwanted bases and retain the nucleotide sequence naturally found between the lac promoter and the protein code. The engineered plasmid results in the production of at least 40% of the cellular protein as the foreign polypeptide. In the example presented the expression of the protein is high even with a substantial difference in codon usage between the host (Escherichia coli) and a foreign gene from Pseudomonas aeruginosa. Some of the polypeptide produced has the ame properties as native protein and is easily purified. The remainder is present as insoluble inclusion bodies. This method of plasmid refinement may be applicable to the expression of many proteins.

4-Hydroxybenzoate-3-Monooxygenase↗