RESUMEN
Craniofacial microsomia is a congenital deformity caused by asymmetric development of the skull (cranium) and face before birth. Current treatments include corrective surgery and replacement of the deformed structure using autograft tissue, which results in donor site morbidity. An alternative therapy can be achieved by developing a resorbable scaffold for skeletal muscle regeneration which will help restore the symmetry and function of the facial muscles and reduce donor site morbidity. Two resorbable weft knitted scaffolds were fabricated using poly(ε-caprolactone) multifilament yarns with unique auxetic design structures possessing negative Poisson's ratio (NPR). These scaffolds exhibit their NPR elasticity through an increase in total volume as well as no lateral narrowing when stretched longitudinally, which can provide orientated mechanical supports to the cell growth of skeletal muscle regeneration. These scaffolds were evaluated for the required physical properties, mechanical performance and biocompatibility by culturing them with neonatal human dermal fibroblasts so as to determine their cell metabolic activity, cell attachment and proliferation. This study can facilitate the understanding and engineering of textile-based scaffolds for tissues/organs. The work also paves a pathway to emerge the NPR textiles into tissue engineering, which has an extensive potential for biomedical end-uses.
RESUMEN
Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs) in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation of RUNX2 gene expression and downregulation of PPARG and IGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC for in tissue engineering applications.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Mecanotransducción Celular/genética , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Cilios/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Células Madre Mesenquimatosas/citología , Resistencia a la Tracción , Ingeniería de TejidosRESUMEN
At the present time, there is no successful synthetic, off-the-shelf small-caliber vascular graft (<6 mm) for the repair or bypass of the coronary or carotid arteries. This stimulates on-going investigations to fabricate an artificial vascular graft that has both sufficient mechanical properties as well as superior biological performance. Collagen has long been considered as a viable material to encourage cell recruitment, tissue regeneration, and revascularization, but its use has been limited by its inferior mechanical properties. In this study, novel electrochemically aligned collagen filaments were used to engineer a bilayer small-caliber vascular graft, by circular knitting the collagen filaments and electrospinning collagen nanofibers. The collagen prototype grafts showed significantly greater bursting strength under dry and hydrated conditions to that of autografts such as the human internal mammary artery and the saphenous vein (SV). The suture retention strength was sufficient under dry condition, but that under hydrated condition needs to be further improved. The radial dynamic compliance of the collagen grafts was similar to that of the human SV. During in vitro cell culture assays with human umbilical vein endothelial cells, the prototype collagen grafts also encouraged cell adhesion and promoted cell proliferation compared to the synthetic poly(lactic acid) grafts. In conclusion, this study demonstrated the feasibility of the use of novel collagen filaments for fabricating small caliber tissue-engineered vascular grafts that provide both sufficient mechanical properties and superior biological performance.
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Prótesis Vascular , Vasos Sanguíneos/fisiología , Colágeno/farmacología , Nanofibras/química , Ingeniería de Tejidos/métodos , Animales , Fenómenos Biomecánicos , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Poliésteres/química , Ratas , Suturas , Andamios del Tejido/químicaRESUMEN
Transdermal osseointegrated prostheses (TOPs) are emerging as an alternative to socket prostheses. Electron beam melting (EBM) is a promising additive manufacturing technology for manufacture of custom, freeform titanium alloy (Ti6Al4V) implants. Skin ongrowth for infection resistance and mechanical stability are critically important to the success of TOP, which can be influenced by material composition and surface characteristics. We assessed viability and proliferation of normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF) on several Ti6Al4V surfaces: solid polished commercial, solid polished EBM, solid unpolished EBM and porous unpolished EBM. Cell proliferation was evaluated at days 2 and 7 using alamarBlue(®) and cell viability was analyzed with a fluorescence-based live-dead assay after 1 week. NHDF and NHEK were viable and proliferated on all Ti6Al4V surfaces. NHDF proliferation was highest on commercial and EBM polished surfaces. NHEK was highest on commercial polished surfaces. All EBM Ti6Al4V discs exhibited an acceptable biocompatibility profile compared to solid Ti6Al4V discs from a commercial source for dermal and epidermal cells. EBM may be considered as an option for fabrication of custom transdermal implants.
Asunto(s)
Dermis/citología , Electrones , Células Epidérmicas , Ensayo de Materiales , Prótesis e Implantes , Titanio/química , Titanio/farmacología , Aleaciones , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Oseointegración/efectos de los fármacos , Transición de Fase , Propiedades de SuperficieRESUMEN
Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed "superlots," containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.
Asunto(s)
Tejido Adiposo/citología , Envejecimiento/fisiología , Sistema Musculoesquelético/metabolismo , Células Madre/citología , Ingeniería de Tejidos/métodos , Adipogénesis , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular , Proliferación Celular , Separación Celular , ADN/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Osteogénesis , Posmenopausia , Premenopausia , Proteínas/metabolismo , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.
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Tejido Adiposo/citología , Diferenciación Celular/fisiología , Cilios/fisiología , Células Madre Multipotentes/fisiología , Osteogénesis/fisiología , Análisis de Varianza , Cilios/ultraestructura , Técnicas de Silenciamiento del Gen , Ingeniería Genética/métodos , Humanos , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Incorporation of mammalian cells into nanofibers (cell electrospinning) and multilayered cell-nanofiber structures (cell layering) via electrospinning are promising techniques for tissue engineering applications. We investigate the viability of 3T3-L1 mouse fibroblasts after incorporation into poly(vinyl alcohol) nanofibers and multilayering with poly(caprolactone) nanofibers and analyze the possible factors that affect cell viability. We observe that cells do not survive cell electrospinning but survive cell layering. Assessing the factors involved in cell electrospinning, we find that dehydration and fiber stretching are the main causes of cell death. In cell layering, the choice of solvent is critical, as residual solvent in the electrospun fibers could be detrimental to the cells.
Asunto(s)
Fibroblastos/citología , Mamíferos/metabolismo , Nanocompuestos/química , Nanofibras/química , Ingeniería de Tejidos/métodos , Células 3T3-L1 , Animales , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibroblastos/ultraestructura , Ratones , Nanocompuestos/ultraestructura , Nanofibras/ultraestructura , Poliésteres/farmacología , Alcohol Polivinílico/farmacología , Soluciones , Solventes , Factores de TiempoRESUMEN
To study the potential effects of low-intensity pulsed ultrasound (LIPUS) on cell response in vitro, the ability to alter LIPUS parameters is required. However, commercial LIPUS systems have very little control over parameter selection. In this study, a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively). These findings promote the study of using LIPUS to induce osteogenic differentiation and further encourage the exploration of LIPUS parameter optimization. The custom LIPUS system was successfully designed to allow extreme parameter variation, specifically PRF, and encourages further studies.
Asunto(s)
Células Madre Adultas/diagnóstico por imagen , Ultrasonido/métodos , Tejido Adiposo/citología , Adulto , Células Madre Adultas/citología , Células de la Médula Ósea/citología , Calcio/análisis , Calcio/metabolismo , Procesos de Crecimiento Celular/fisiología , Células Cultivadas , ADN/química , Humanos , Reproducibilidad de los Resultados , Ultrasonido/instrumentación , UltrasonografíaRESUMEN
Human adipose-derived stem cells (hASCs) are a relatively abundant and accessible stem cell source with multilineage differentiation capability and have great potential for bone tissue engineering applications. The success of bone tissue engineering is intimately linked with the production of a mineralized matrix that mimics the natural mineral present within native bone. In this study, we examined the effects of ionic calcium levels of 1.8 (normal concentration in cell culture medium), 8, and 16 mM on hASCs seeded in both two-dimensional monolayer and three-dimensional electrospun scaffolds and cultured in either complete growth medium (CGM) or osteogenic differentiation medium (ODM). The impact of calcium supplementation on hASC viability, proliferation, and mineral deposition was determined. hASCs remained viable for all experimental treatments. hASC proliferation increased with the addition of 8 mM Ca(2+) CGM, but decreased for the 16 mM Ca(2+) CGM treatment. Materials deposited by hASCs were analyzed using four techniques: (1) histological staining with Alizarin Red S, (2) calcium quantification, (3) Fourier transform infrared spectroscopy, and (4) wide-angle X-ray diffraction. Mineral deposition was significantly enhanced under both growth and osteogenic medium conditions by increasing extracellular Ca(2+). The greatest mineral deposition occurred in the ODM 8 mM Ca(2+) treatment group. Fourier transform infrared spectroscopy analysis indicated that elevated calcium concentrations of 8 mM Ca(2+) significantly increased both PO(4) amount and PO(4) to protein ratio for ODM. X-ray diffraction indicated that mineral produced with elevated Ca(2+) in both CGM and ODM had a crystalline structure characteristic of hydroxyapatite. Ionic calcium should be considered a potent regulator in hASC mineralization and could serve as a potential treatment for inducing prompt ossification of hASC-seeded scaffolds for bone tissue engineering prior to implantation.
Asunto(s)
Adipocitos/citología , Calcio/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Ingeniería de Tejidos/métodos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Osteogénesis/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier , Células Madre/citología , Difracción de Rayos XRESUMEN
We investigated the effects of two types of cyclic tensile strain, continuous and rest inserted, on osteogenic differentiation of human adipose-derived adult stem cells (hASCs). The influence of these mechanical strains was tested on two hASC lines having different mineral deposition potential, with one cell line depositing approximately nine times as much calcium as the other hASC line after 14 days of culture in osteogenic medium on tissue culture plastic. Results showed that both continuous (10% strain, 1 Hz) and rest inserted cyclic tensile strain (10% strain, 1 Hz, 10 s rest after each cycle) regimens increased the amount and rate of calcium deposition for both high and low calcium depositing hASC lines as compared to unstrained controls. The response was similar for both types of tensile strain for a given cell line, however, cyclic tensile strain had a much stronger osteogenic effect on the high calcium depositing hASC line, suggesting that mechanical loading has a greater effect on cell lines that already have an innate ability to produce bone as compared to cell lines that do not. This is the first study to investigate the osteodifferentiation effects of cyclic tensile strain on hASCs and the first to show that both continuous (10%, 1 Hz) and rest inserted (10%, 1 Hz, 10 s rest) cyclic tensile strain accelerate hASC osteodifferentiation and increase calcium accretion.
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Modelos Biológicos , Osteogénesis/fisiología , Tejido Adiposo/citología , Células Madre Adultas , Calcio/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas , Estrés Mecánico , Resistencia a la Tracción , Ingeniería de Tejidos/métodosAsunto(s)
Tejido Adiposo/citología , Huesos/citología , Técnicas de Cultivo de Célula , Separación Celular/métodos , Células Madre Mesenquimatosas/citología , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Cultivadas , Criopreservación/métodos , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
Electrospun nanocomposite scaffolds were fabricated by encapsulating multi-walled carbon nanotubes (MWNT) in poly (lactic acid) (PLA) nanofibers. Scanning electron microscopy (SEM) confirmed the fabrication of nanofibers, and transmission electron microscopy identified the alignment and dispersion of MWNT along the axis of the fibers. Tensile testing showed an increase in the tensile modulus for a MWNT loading of 0.25 wt% compared with electrospun nanofibrous mats without MWNT reinforcement. Conductivity measurements indicated that the confined geometry of the fibrous system requires only minute doping to obtain significant enhancements at 0.32 wt%. Adipose-derived human mesenchymal stem cells (hMSCs) were seeded on electrospun scaffolds containing 1 wt% MWNT and 0 wt% MWNT, to determine the efficacy of the scaffolds for cell growth, and the effect of MWNT on hMSC viability and proliferation over two weeks in culture. Staining for live and dead cells and DNA quantification indicated that the hMSCs were alive and proliferating through day 14. SEM images of hMSCs at 14 days showed morphological differences, with hMSCs on PLA well spread and hMSCs on PLA with 1% MWNT closely packed and longitudinally aligned.
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Adipocitos/citología , Adipocitos/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Nanotubos de Carbono/química , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/química , Técnicas de Cultivo de Célula/métodos , Polaridad Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Electroquímica/métodos , Humanos , Ensayo de Materiales , Osteoblastos/citología , Osteoblastos/fisiología , RotaciónRESUMEN
Adipose-derived human mesenchymal stem cells (hMSCs) will be more valuable for tissue engineering applications if they can be extensively subcultured without loss of phenotype and multilineage differentiation ability. This study examined the effects of serial passaging on growth rate, gene expression, and differentiation potential of adipose-derived hMSCs. Differentiation was assessed by analyzing changes in messenger RNA (mRNA) expression of osteogenic and adipogenic marker genes and by determining production of calcium deposits and lipid vacuoles. Cells cultured in osteogenic medium for 2 weeks upregulated expression of alkaline phosphatase mRNA relative to cells in growth medium, and deposited calcium. Calcium deposition decreased in cells from passages 4 to 6 but returned to levels near or above those of primary cells by passage 10. Cells cultured in adipogenic medium upregulated expression of lipoprotein lipase and peroxisome proliferator activated receptor-gamma mRNA relative to cells in growth medium, and formed lipid vacuoles at all passages. By passage 8, however, cells in adipogenic medium also deposited calcium. Growth rate was stable through passage 5, then decreased. The results of this study indicate that adipose-derived hMSCs are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings) but that osteogenic differentiation may start to dominate at later passages.
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Adipocitos/citología , Adipogénesis/fisiología , Células Madre Mesenquimatosas/citología , Osteocitos/citología , Osteogénesis/fisiología , Ingeniería de Tejidos/métodos , Adipocitos/fisiología , Adulto , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Osteocitos/fisiologíaRESUMEN
This study investigated the differential effects of ramped and steady applications of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in 3-dimensional culture in the absence of transforming growth factor-beta (TGF-beta). A custom hydrostatic pressure system was designed and manufactured. hMSCs were seeded in agarose and exposed to steady (7.5 MPa) or ramped (1 MPa to 7.5 MPa over a 14-day period) CHP for 4 h/d at f = 1 Hz for 14 days. Real-time reverse transcriptase polymerase chain reaction analysis was performed on days 0, 4, 9, and 14 to determine changes in messenger ribonucleic acid (mRNA) expression levels of Sox9, aggrecan, collagen I, and collagen II. Collagen II and aggrecan mRNA expression remained unchanged. Collagen I increased at day 4 in CHP specimens before decreasing to levels at or below same-day unloaded controls at days 9 and 14. On average, ramped and steady regimens of CHP increased Sox9, with the largest upregulation occurring at day 4 in response to steady pressure. These findings indicate that hydrostatic pressure may induce chondrogenesis in hMSC-seeded agarose constructs without TGF-beta, and that hMSCs are capable of withstanding high initial pressures that may initiate chondrogenesis faster than lower pressures.
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Condrocitos/citología , Condrocitos/metabolismo , Condrogénesis/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/metabolismo , Ingeniería de Tejidos/métodos , Adolescente , Adulto , Materiales Biocompatibles/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Masculino , Mecanotransducción Celular/efectos de los fármacos , Mecanotransducción Celular/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Presión , Sefarosa/química , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
Human mesenchymal stem cells (hMSCs) differentiate down an osteogenic pathway with appropriate mechanical and/or chemical stimuli. This study describes the successful culture of hMSCs in 3D collagen matrices under mechanical strain. Bone marrow-derived hMSCs were seeded in linear 3D type I collagen matrices and subjected to 0%, 10%, or 12% uniaxial cyclic tensile strain at 1 Hz for 4 h/day for 7 or 14 days. Cell viability studies indicated that hMSCs remained viable throughout the culture period irrespective of the applied strain level. Real-time RT-PCR studies indicated a significant increase in BMP-2 mRNA expression levels in hMSCs strained at 10% compared to the same day unstrained controls after both 7 and 14 days. An increase in BMP-2 was also observed in hMSCs subjected to 12% strain, but the increase was significant only in the 14-day sample. This is the first report of the culture of bone marrow-derived hMSCs in 3D collagen matrices under cyclic strain, and the first demonstration that strain alone can induce osteogenic differentiation without the addition of osteogenic supplements. Induction of bone differentiation in 3D culture is a critical step in the creation of bioengineered bone constructs.
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Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Huesos/citología , Diferenciación Celular/fisiología , Colágeno Tipo I , Células Madre Mesenquimatosas/citología , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Adulto , Proteína Morfogenética Ósea 2 , Huesos/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Resistencia a la TracciónRESUMEN
PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.
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Pruebas Genéticas/normas , Técnicas de Diagnóstico Molecular/normas , Control de Calidad , Centers for Disease Control and Prevention, U.S. , Regulación Gubernamental , Humanos , Garantía de la Calidad de Atención de Salud/normas , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
BACKGROUND: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.
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Recolección de Muestras de Sangre , Línea Celular Transformada , Pruebas Genéticas/métodos , Herpesvirus Humano 4 , Linfocitos/citología , Enfermedades Genéticas Congénitas/diagnóstico , Humanos , Laboratorios , Biología Molecular , Mutación , Mutación Puntual , Eliminación de SecuenciaRESUMEN
CONTEXT: Bioelectronic sensors, which combine microchip and biological components, are an emerging technology in clinical diagnostic testing. An electronic detection platform using DNA biochip technology (eSensor) is under development for molecular diagnostic applications. Owing to the novelty of these devices, demonstrations of their successful use in practical diagnostic applications are limited. OBJECTIVE: To assess the performance of the eSensor bioelectronic method in the validation of 6 Epstein-Barr virus-transformed blood lymphocyte cell lines with clinically important mutations for use as sources of genetic material for positive controls in clinical molecular genetic testing. Two cell lines carry mutations in the CFTR gene (cystic fibrosis), and 4 carry mutations in the HFE gene (hereditary hemochromatosis). DESIGN: Samples from each cell line were sent for genotype determination to 6 different molecular genetic testing facilities, including the laboratory developing the DNA biochips. In addition to the bioelectronic method, at least 3 different molecular diagnostic methods were used in the analysis of each cell line. Detailed data were collected from the DNA biochip output, and the genetic results were compared with those obtained using the more established methods. RESULTS: We report the successful use of 2 applications of the bioelectronic platform, one for detection of CFTR mutations and the other for detection of HFE mutations. In all cases, the results obtained with the DNA biochip were in concordance with those reported for the other methods. Electronic signal output from the DNA biochips clearly differentiated between mutated and wild-type alleles. This is the first report of the use of the cystic fibrosis detection platform. CONCLUSIONS: Bioelectronic sensors for the detection of disease-causing mutations performed well when used in a "real-life" situation, in this case, a validation study of positive control blood lymphocyte cell lines with mutations of public health importance. This study illustrates the practical potential of emerging bioelectronic DNA detection technologies for use in current molecular diagnostic applications.
Asunto(s)
Técnicas Biosensibles/métodos , Fibrosis Quística/diagnóstico , Análisis Mutacional de ADN/métodos , Hemocromatosis/diagnóstico , Secuencia de Bases , Biotecnología/métodos , Línea Celular Transformada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Electrónica , Genotipo , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Proteínas de la Membrana/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.