Improving the cell distribution in collagen-coated poly-caprolactone knittings

Abstract

Adequate cellular in-growth into biomaterials is one of the fundamental requirements of scaffolds used in regenerative medicine. Type I collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support can be produced. However, in general, adequate cell in-growth and cell seeding has been suboptimal. In this study we prepared collagen scaffolds of different collagen densities and investigated the cellular distribution. We also prepared a hybrid polymer-collagen scaffold to achieve an optimal cellular distribution as well as sufficient mechanical strength. Collagen scaffolds [ranging from 0.3% to 0.8% (w/v)] with and without a mechanically stable polymer knitting [poly-caprolactone (PCL)] were prepared. The porous structure of collagen scaffolds was characterized using scanning electron microscopy and hematoxylin-eosin staining. The mechanical strength of hybrid scaffolds (collagen with or without PCL) was determined using tensile strength analysis. Cellular in-growth and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and human urothelium seeding. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a hybrid scaffold prepared from 0.4% collagen strengthened with knitting achieved the best cellular distribution.

FIG. 1.

Microscopic characterization of the cross-sectioned collagen scaffolds. Scanning electron microscopy (A–F) and hematoxylin-eosin of different collagen scaffolds (E–K) indicate that the interconnection is related to the collagen content. Scaffolds with lower collagen content show more interconnectivity between the pores (more open lamellae).

FIG. 2.

Schematic overview of preparation hybrid scaffold. Type I collagen (A) plus poly-caprolactone (PCL) knitting (B) is a hybrid scaffold (C). Collagen scaffold and PCL knitting were imaged by light microscopy (4×and 10×). Collagen is integrated into PCL knitting. Arrows indicate the collagen (D). Type I collagen and PCL were imaged by scanning electron microscope (E). Collagen is adequately connected to PCL. Taking a close look of PCL, collagen is well distributed through it (50×and 250×).

FIG. 3.

Tensile test of collagen scaffold with and without PCL (a). The mechanical strength increased slightly with the increase of collagen density. The strength of scaffolds with PCL is higher than that of collagen-alone scaffolds. (b, c) Individual tensile strength measurements of 0.8% collagen scaffold without (b) and with PCL (c). Please note plateau value at 7.8 N in (c), suggesting that beyond 7.8 N PCL distortion occurs.

FIG. 4.

Bead distribution in collagen scaffolds (0.8%–0.3%, A–F). Type I collagen was developed by Alexa 594 and the fluorescent beads were tagged with Alexa 488. Beads and collagen scaffolds were imaged by fluorescent microscopy (4×). Beads penetrate deeply into 0.3% and 0.4% collagen scaffolds. In 0.7% and 0.8% collagen scaffolds, beads mainly stay on the surface of scaffolds.

FIG. 5.

Immunofluorescence (IF) analysis of cell distribution in different densities (from 0.8% to 0.3%, A–F) of collagen scaffold. Type I collagen was developed by Alexa 594 (red), human bladder smooth muscle cells (HBSMCs) was developed by Alexa 488 (green), and all nuclei were counterstained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). HBSMCs and collagen scaffolds were imaged by fluorescence microscopy (10×). Smooth muscle cells (HBSMCs) are located in the scaffold. More HBSMCs are distributed through the 0.3% and 0.4% scaffolds and less cells penetrate 0.7% and 0.8% scaffolds. Cells on top of the surface that are not smooth muscle cell positive are urothelial cells.

FIG. 6.

IF double staining of HBSMCs and ScaBER in different densities (0.8%, 0.6%, and 0.4%) of collagen scaffold. HBSMCs were developed by Alexa 488 (green), ScaBER was developed by Alexa 594 (red), and all nuclei were counterstained by DAPI (blue). HBSMCs and ScaBER in scaffolds were imaged by fluorescence microscopy (10×and 40×). HBSMCs are located under the ScaBER cells as natural stratum for urothelial cells. ScaBER cells are always on top of the scaffolds regardless of the collagen density. More HBSMCs are distributed in the low-density scaffolds.