Low-power ultrasounds as a tool to culture human osteoblasts inside cancellous hydroxyapatite

Abstract

Bone graft substitutes and cancellous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of cancellous hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering. In this study, we have followed a tissue-engineering strategy where ultrasonically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix inside a porous hydroxyapatite scaffold. The ultrasonic stimulus had the following parameters: average power equal to 149 mW and frequency of 1.5 MHz. In comparison with control conditions, the ultrasonic stimulus increased the cell proliferation and the surface coating with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The mechanical stimulus aimed at obtaining a better modification of the biomaterial internal surface in terms of cell colonization and coating with bone matrix. The modified biomaterial could be used, in clinical applications, as an implant for bone repair.

Figure 1

SEM image of unseeded hydroxyapatite, bar equal to 100 μm.

Figure 2

SEM image of the static culture, bar equal to 30 μm. The osteoblasts are in the “backscattered depressions” near the juxtaposed asterisks: at the end of the culture period, statically cultured cells were few and, essentially, not surrounded by extracellular matrix; therefore, wide biomaterial regions remained devoid of cell-matrix complexes.

Figure 3

SEM image of the ultrasonic culture, bar equal to 30 μm. During the culture period, the physical stimulus caused a wide-ranging coat of the internal surface of the biomaterial: several osteoblasts proliferated and the biomaterial was tending to be hidden by cell-matrix layers (asterisks).

Figure 4

Immunolocalization of type-I collagen (panels a and c, green) and cellular nuclei (panels b and d, blue) in the static culture (panels a and b) and in the ultrasonic culture (panels c and d), bars equal to 80 μm. During the culture period, in the control (panels a and b), the osteoblasts built a scanty amount of bone matrix, whereas, in the stimulated culture (panels c and d), the osteoblasts secreted a wide amount of matrix. The immunolocalization of osteocalcin, osteopontin, and type-III collagen revealed similar results.

Figure 5

Immunolocalization of decorin (panels a and c, green) and cellular nuclei (panels b and d, blue) in the static culture (panels a and b) and in the ultrasonic culture (panels c and d), bars equal to 80 μm. During the culture period, in the control (panels a and b), the osteoblasts produced a very little amount of decorin, a key regulator for matrix spatial organization, whereas, in the stimulated culture (panels c and d), the osteoblasts secreted a larger amount of 3D organized bone matrices.