Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

Abstract

Pluripotent adipose tissue-derived stem cells (hASCs) can differentiate into various mesodermal cell types such as osteoblasts, chondroblasts, and myoblasts. We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA), polylactide-co-glycolide/hydroxyapatite (PLGA/HA), and trabecular titanium scaffolds (Ti6Al4V). Pore size can influence certain criteria such as cell attachment, infiltration, and vascularization. The aim of this study was to investigate the performance of PLGA and PLGA/HA scaffolds with a higher porosity, ranging between 75% and 84%, with respect to Ti scaffolds but with smaller pore size, seeded with hASCs to develop a model that could be used in the treatment of bone defects and fractures. Osteogenesis was assessed by ELISA quantitation of extracellular matrix protein expression, von Kossa staining, X-ray microanalysis, and scanning electron microscopy. The higher amount of protein matrix on the Ti scaffold with respect to PLGA and PLGA/HA leads to the conclusion that not only the type of material but the structure significantly affects cell proliferation.

Figure 1

Images of PLGA (a) and PLGA/HA (b) scaffolds obtained using the porogen particle leaching method as reported in “Materials and methods”; (c) image of trabecular titanium scaffold (Ti6Al4V).

Figure 2

Phenotypic characterization of hASCs: CD105, 73, and 90 mesenchymal stem cells markers are positive; CD34 and 45 haematopoietic stem cells markers are negative.

Figure 3

(a) hASCs after the 3rd passage in control medium show a fibroblast-like shape; (b) hASCs in osteogenic medium after 28 days show a more spherical shape if compared to the undifferentiated cells (Toluidine blue). Mag. 10x.

Figure 4

Scanning electron microscopic images of unseeded titanium scaffolds (a) and seeded titanium scaffolds (Ti6Al4V) with hASCs in osteogenic medium (b). Panel A shows the innovative multi-planar hexagonal structure of the scaffold imitating the structure of the trabecular bone, bar = 2 mm. In Panel B, cells appear to cover the surface of the trabecular scaffold uniformly and completely, bar = 100 μm; (c) Extracellular matrix between pores, bar = 20 μm.

Figure 5

Scanning electron microscopic images of unseeded PLGA scaffold (a), bar = 500 μm and seeded PLGA scaffold (b and c) with hASCs in osteogenic medium for 28 days. Panel b shows cells embedded in their extracellular matrix over the scaffold surface bar = 100 μm. Panel c shows round cells bar = 100 μm.

Figure 6

Scanning electron microscopic images of unseeded PLGA/HA scaffold (a), bar = 200 μm and seeded PLGA/HA scaffolds (b and c) with hASCs in osteogenic medium for 28 days. In panel B, cells appear have a round morphology, bar = 100 μm. Panel C, at greater magnification, clusters of cells embedded in their matrix and inside the pores of the scaffold, bar = 20 μm.

Figure 7

von Kossa staining of hASCs grown in osteogenic medium for 28 days in a culture monolayer. (a) Negative control, (b) positive sample; the secreted calcified extracellular matrix are shown as black nodules Mag. 20x. (c) X-ray microanalysis performed on trabecular titanium, (d) PLGA scaffolds, (e) PLGA/HA scaffolds seeded with hASCs in osteogenic medium for 28 days. Calcium and Phosphatum peaks were detected, inferring that hydroxyapatite was formed.