摘要
This study evaluated chondrogenesis within a nanofiber polymeric scaffold seeded with isolated untreated chondrocytes, isolated chondrocytes genetically engineered with adenoviral (Ad) bone morphogenetic protein (BMP)-2, or isolated chondrocytes genetically engineered with green fluorescent protein (Ad-GFP). Electrospun polycaprolactone scaffolds (150-200 m thickness, 700 m fiber diameter, 30 m pore size) were optimally seeded with 1 x 107 isolated chondrocytes by using a 20% serum gradient culture system. Chondrocyte-scaffold constructs (untreated, Ad-B- MP-2 and Ad-GFP) were generated from 5 adult horses, cultured in triplicate for 7 or 14 days, and quantitatively analyzed for cell proliferation (DNA content;Hoechst assay), viability, morphology (confocal microscopy), matrix production (proteoglycan content;DMMB assay), and mRNA expression of collagen I, collagen II, and aggrecan. Chondrocytes transduced with Ad-BMP-2 demonstrated greater cell numbers and significantly greater expression of chondrogenic markers including aggrecan, collagen II, and proteoglycan through 14 days of culture as compared to untransduced or Ad-GFP controls. This study demons- trated that chondrocytes can be driven to seed a polycaprolactone nanofiber scaffold by serum gradient and a polycaprolactone nanofiber scaffold containing Ad-BMP2 transduced chondrocytes resulted in grea- ter and accelerated chondrogenesis than controls. This cell engineered construct has potential use in one-step cartilage repair in vivo.
This study evaluated chondrogenesis within a nanofiber polymeric scaffold seeded with isolated untreated chondrocytes, isolated chondrocytes genetically engineered with adenoviral (Ad) bone morphogenetic protein (BMP)-2, or isolated chondrocytes genetically engineered with green fluorescent protein (Ad-GFP). Electrospun polycaprolactone scaffolds (150-200 m thickness, 700 m fiber diameter, 30 m pore size) were optimally seeded with 1 x 107 isolated chondrocytes by using a 20% serum gradient culture system. Chondrocyte-scaffold constructs (untreated, Ad-B- MP-2 and Ad-GFP) were generated from 5 adult horses, cultured in triplicate for 7 or 14 days, and quantitatively analyzed for cell proliferation (DNA content;Hoechst assay), viability, morphology (confocal microscopy), matrix production (proteoglycan content;DMMB assay), and mRNA expression of collagen I, collagen II, and aggrecan. Chondrocytes transduced with Ad-BMP-2 demonstrated greater cell numbers and significantly greater expression of chondrogenic markers including aggrecan, collagen II, and proteoglycan through 14 days of culture as compared to untransduced or Ad-GFP controls. This study demons- trated that chondrocytes can be driven to seed a polycaprolactone nanofiber scaffold by serum gradient and a polycaprolactone nanofiber scaffold containing Ad-BMP2 transduced chondrocytes resulted in grea- ter and accelerated chondrogenesis than controls. This cell engineered construct has potential use in one-step cartilage repair in vivo.
