RESUMO
Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.
Assuntos
Adipócitos/citologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/isolamento & purificação , Osteogênese , Células Estromais/citologia , Engenharia Tecidual/métodos , Cicatrização , Adipócitos/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Células Estromais/metabolismoRESUMO
The binary ligand-receptor complex of human growth and differentiation factor 5 (GDF5) bound to its type I receptor BMP receptor IA (BRIA) was prepared and crystallized. By utilizing the GDF5 variant R57A, which exhibits a high affinity in the subnanomolar range for BRIA, the binary complex of GDF5R57A bound to the extracellular domain of BRIA could be produced and purified. Crystals of this complex belonged to a monoclinic space group: either I2, with unit-cell parameters a = 63.81, b = 62.85, c = 124.99 Å, ß = 95.9°, or C2, with unit-cell parameters a = 132.17, b = 62.78, c = 63.53 Å, ß = 112.8°.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/química , Fator 5 de Diferenciação de Crescimento/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/isolamento & purificação , Cristalização , Cristalografia por Raios X , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/isolamento & purificação , Humanos , Ligantes , Mutação , Ligação ProteicaRESUMO
The ligand-receptor complex of GDF5 bound to its type I and type II receptors BRIB and ActRIIB was produced and crystallized. Crystals of the GDF5-BRIB complex could only be obtained if a ternary complex comprising GDF5, BRIB and the extracellular domain of the type II receptor ActRIIB was used in crystallization; however, the type II receptor ActRIIB was lost during crystallization. Crystals of this complex belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 76.46, c = 82.78 A. Small changes in the crystallization condition resulted in crystals with a different morphology. These crystals consisted of the full ternary complex GDF5-BRIB-ActRIIB, but only diffracted to low resolution.
