Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Abstract

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both alpha5 and alphav-containing integrin heterodimers, whereas only alphav integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of alpha2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.

Fig. 1

Osteosarcoma cells and MSCs bind optimally to different matrix molecules. Cells were pre-loaded with a fluorescent dye and then seeded onto HA disks precoated with FBS, FN, Col I or denatured BSA (a negative control). Cells were allowed to adhere for 1 hour, and were then evaluated for cell adhesion using a fluorometric-based assay. As shown, MG-63 and Saos-2 cells demonstrated greater attachment to FN coated disks than to FBS or col-I-coated disks. This contrasts with the behavior of MSCs, which adhere better to disks coated with FBS or col-I as compared with FN. Results are from 3 independent experiments, each performed in triplicate. * denotes significant difference from FBS-coated samples (p < 0.05). Bars = means plus S.E.M.

Fig. 2

Osteosarcoma cells and MSCs demonstrate differential cell spreading on matrix molecules. Cells were allowed to adhere for 1 hour on HA disks pre-coated with pro-adhesive proteins. The cells were then fixed with formaldehyde, and the actin cytoskeleton was labeled using phalloidin conjugated to a green fluorescent dye. Maximal spreading of Saos-2 and MG-63 cells was observed on FN-coated HA disks, whereas only limited cell spreading was observed on disks coated with either col-I or FBS. The morphology of MSCs was very different from that of Saos-2 and MG-63 cells, in that MSCs appeared to spread better on FBS than on either collagen or FN.

Fig. 3

Osteosarcoma cells and MSCs synthesize FN and VN Cells were grown overnight on tissue culture plastic and then lysed in buffer containing 1% TX-100. Lysates were resolved by SDS-PAGE, transferred to PVDF membrane and immunoblotted for either FN (A) or VN (B). All blots were stripped and re-probed with an antibody against βactin to control for protein loading. Western blots were performed using 3 independent harvests of cell lysates, and band intensities were quantified by densitometry. Values for integrin levels were normalized to the β-actin control bands. Graphs represent the means and SEMs; * denotes a statistically significant difference (p < 0.05).

Fig. 4

Osteosarcoma cells use different integrin receptors than MSCs to adhere to fibronectin. MSCs, MG-63, and Saos-2 cells (labeled with cell tracker green) were preincubated with function blocking antibodies against α5 or αv integrins, or with a nonspecific IgG (IgG) as a control. Trials were also included in which no antibody was added (no Ab). The cells were then seeded onto FN-coated HA disks, and allowed to adhere for 1 hour at 37°C. The disks were then washed to remove non-adherent cells, and the number of adherent cells was quantified as previously described. Results represent the means and SEMs for 3 experiments performed in triplicate. * denotes significant difference from the no antibody samples (p < 0.05)

Fig. 5

Osteosarcoma cells and MSCs exhibit differential expression of integrin receptors. MSC, MG-63, and Saos-2 cell lysates were immunoblotted for α2 integrins (A), αv integrins (B) or α5 integrins (C). All blots were stripped and re-probed with an antibody against β-actin to control for protein loading. Western blots were performed using 3 independent harvests of cell lysates, and band intensities were quantified by densitometry. Values for integrin levels were normalized to the β-actin control bands. Graphs represent the means and SEMs; * denotes a statistically significant difference (p < 0.05).