Online measurement of collagen synthesis in smooth muscle cells. Toward non‐destructive analysis of matrix production in vascular tissue engineered grafts

Abstract

To provide vascular tissue engineerd grafts (VTEG) with sufficient suturing strength and burst pressure, appropriate cellular sources that produce sufficient collagen content are of vital importance. To define collagen production in VTEG, destructive methods like 4-hydroxyproline are usually applied. In this study we investigated the possibility to measure collagen synthesis in a non-destructive fashion by using a collagen 1α2 promoter driven secretable luciferase construct (pCol1a2). Primary porcine smooth muscle cells (pSMCs) were cultured and transfected with the pCol1a2 construct or empty vector and stimulated with TGFβ (0–20 ng/mL) up to 72 hours. Next, cells were analyzed for secreted luciferase activity, collagen -RNA and -protein production. High confluent SMC cultures stimulated with TGFβ gave a clear dose-response for both the luciferase and collagen protein production with good correlation. Overall, collagen mRNA levels lacked a relation with this new luciferase assay and protein levels in the medium. We conclude that this promoter construct can be used for medium throughput evaluation of collagen production during an early stage of culturing. An additional advantage is the non-destructive collagen assay, allowing adaption of the culture protocol to modulate collagen production.