RESUMEN
MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
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MicroARNs/genética , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I. METHODS: In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor ß (TGFß)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured. RESULTS: In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5. CONCLUSION: Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.
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Cartílago/crecimiento & desarrollo , Factor 5 de Diferenciación de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Tabique Nasal/crecimiento & desarrollo , Ingeniería de Tejidos , Humanos , InmunohistoquímicaRESUMEN
OBJECTIVE: To determine the effects of bone morphogenetic proteins (BMP)-2 and -7, and serum, on extracellular matrix production by human septal chondrocytes in alginate. STUDY DESIGN: Human nasal septal chondrocytes were expanded, suspended in alginate, and cultured in BMP-2 or 7, with and without serum. The optimal concentration of each growth factor was determined based on matrix production. Next, the synergistic effects of BMP-2 and -7 at optimal concentrations were determined on separate beads, based on matrix quantity and histology. RESULTS: Matrix content was highest with concentrations of BMP-2 and -7 of 100 ng/ml and 20 ng/ml, respectively, with serum. Adding both BMP-2 and -7, with serum, increased matrix content by factors of 5.1 versus serum-only cultures, 2.7 versus only BMP-2 with serum, and 2.4 versus only BMP-7 with serum. All comparisons were statistically significant. CONCLUSION: BMP-2 and -7 significantly increase production of extracellular matrix by septal chondrocytes suspended in alginate. The presence of serum improves matrix production. SIGNIFICANCE: BMP-2 and -7 have great potential for use in cartilage tissue engineering.
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Proteínas Morfogenéticas Óseas/farmacología , Condrocitos/efectos de los fármacos , Tabique Nasal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Alginatos , Sangre , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/administración & dosificación , Recuento de Células , Células Cultivadas , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Ácido Glucurónico , Glicosaminoglicanos/análisis , Ácidos Hexurónicos , Humanos , Tabique Nasal/citología , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
OBJECTIVES: Tissue engineering of septal cartilage provides ex vivo growth of cartilage from a patient's own septal chondrocytes for use in craniofacial reconstruction. To become clinically applicable, it is necessary to rapidly expand a limited population of donor chondrocytes and then stimulate the production of extracellular matrix on a biocompatible scaffold. The objective of this study was to determine favorable serum-free culture conditions for proliferation of human septal chondrocytes using various concentrations and combinations of four growth factors. STUDY DESIGN: Prospective, randomized, controlled study. METHODS: Nasal septal chondrocytes from six patient donors were isolated by enzymatic digestion and expanded in monolayer culture in both serum-free media (SFM) and 2% fetal bovine serum (FBS). Both of these groups were exposed to varying concentrations and combinations of transforming growth factor (TGF)-beta1, basic fibroblast growth factor (FGF)-2 both at 1, 5, and 25 ng/mL, and bone morphogenetic protein (BMP)-2 and insulin-like growth factor (IGF)-1, both at 5, 25, and 125 ng/mL in the medium during the expansion phase. Cell morphology was assessed throughout the culture duration. After 7 days of monolayer growth, cultures were assessed for cellularity and glycosaminoglycan (GAG) content. RESULTS: The addition of low-dose FBS in culture media consistently led to significantly greater cell proliferation and matrix deposition than the SFM cell cultures. FGF-2 and TGF-beta1 both alone and in combination led to the greatest proliferative effect compared with the other growth factors. In contrast, BMP-2 and IGF-1 led to the least cell proliferation although was most effective in retaining chondrocyte cell morphology. CONCLUSIONS: With the addition of TGF-beta1 and FGF-2 to culture media, the concentration of serum can be greatly decreased and possibly eliminated altogether without jeopardizing cell proliferation.