Improvement in endothelial cell adhesion and retention under physiological shear stress using a laminin-apatite composite layer on titanium

Abstract

Apatite (Ap), laminin-apatite composite (L5Ap, L10Ap, L20Ap and L40Ap) and albumin-apatite (AlbAp) composite layers were prepared on titanium (Ti) using a supersaturated calcium phosphate solution supplemented with laminin (0, 5, 10, 20 and 40 μg ml(-1)) or albumin (800 μg ml(-1)). With an increase in the concentrations of laminin in the supersaturated calcium phosphate solutions, the amounts of laminin immobilized on the Ti increased. The number of human umbilical vein endothelial cells (HUVECs) adhered to the laminin-apatite composite layers were remarkably higher than those to the untreated Ti, Ap layer and AlbAp composite layer. The number of cells adhered to the L40Ap was 4.3 times the untreated Ti. Moreover, cells adhered to the laminin-apatite composite layers showed significantly higher cell retention under the physiological shear stress for 1 h and 2 h than those to the untreated Ti, Ap layer and AlbAp composite layer. The number of cells remaining on the L40Ap under the physiological shear stress for 2 h was 9.5 times that of the untreated Ti. The laminin-apatite composite layer is a promising interfacial layer for endothelialization of blood-contacting materials.

Figure 1.

Scheme of in vitro HUVECs retention evaluation system (HUVECs were subjected to physiological shear stress then observed using a fluorescence microscope).

Figure 2.

XRD patterns of Ap, L5Ap, L10Ap, L20Ap, L40Ap and AlbAp on Ti. An asterisk denotes apatite and a T denotes titanium.

Figure 3.

SEM images of Ti, Ap, L5Ap, L10Ap, L20Ap, L40Ap and AlbAp.

Figure 4.

Amounts of calcium and phosphorus precipitated on Ti.

Figure 5.

Amounts of laminin or albumin precipitated on Ti.

Figure 6.

(a) Fluorescent images of HUVECs adhered to Ti, Ap, L5Ap, L10Ap, L20Ap, L40Ap and AlbAp after being cultured for 1 h (Ti-H, Ap-H, L40Ap-H and AlbAp-H are images of HUVECs adhered to the surfaces of Ti, Ap, L40Ap and AlbAp under higher magnification, respectively). (b) Numbers of HUVECs adhered to the surfaces of Ti, Ap, L40Ap and AlbAp after being cultured for 1 h.

Figure 7.

(a) Fluorescent images of HUVECs adhered to Ti, Ap, L40Ap and AlbAp after being subjected to physiological shear stress for 0, 1 and 2 h. (b) Number of and (c) percentage of cells remaining on the Ti, Ap, L40Ap and AlbAp after being subjected to physiological shear stress for 0, 1 and 2 h.