Requirement for both micron- and submicron scale structure for synergistic responses of osteoblasts to substrate surface energy and topography

Abstract

Objective: Surface roughness and surface free energy are two important factors that regulate cell responses to biomaterials. Previous studies established that titanium (Ti) substrates with micron-scale and submicron scale topographies promote osteoblast differentiation and osteogenic local factor production and that there is a synergistic response to micro-rough Ti surfaces that have retained their high surface energy via processing that limits hydrocarbon contamination. This study tested the hypothesis that the synergistic response of osteoblasts to these modified surfaces depends on both surface micro-structure and surface energy.

Methods: Ti disks were manufactured to present three different surface structures: smooth pretreatment (PT) surfaces with R(a) of 0.2 microm; acid-etched surfaces (A) with a submicron roughness R(a) of 0.83 microm; and sandblasted/acid-etched surfaces (SLA) with R(a) of 3-4 microm. Modified acid-etched (modA) and modified sandblasted/acid-etched (modSLA) Ti substrates, which have low contamination and present a hydroxylated/hydrated surface layer to retain high surface energy, were compared with regular low surface energy A and SLA surfaces. Human osteoblast-like MG63 cells were cultured on these substrates and their responses, including cell shape, growth, differentiation (alkaline phosphatase, osteocalcin), and local factor production (TGF-beta1, PGE(2), osteoprotegerin (OPG)) were analyzed (N=6 per variable). Data were normalized to cell number.

Results: There were no significant differences between smooth PT and A surfaces except for a small increase in OPG. Compared to A surfaces, MG63 cells produced 30% more osteocalcin on modA, and 70% more on SLA. However, growth on modSLA increased osteocalcin by more than 250%, which exceeded the sum of independent effects of surface energy and topography. Similar effects were noted when levels of latent TGF-beta1, PGE(2) and OPG were measured in the conditioned media.

Conclusions: The results demonstrate a synergistic effect between high surface energy and topography of Ti substrates and show that both micron-scale and submicron scale structural features are necessary.

Figure 1

Morphology of MG63 osteoblast-like cells cultured on Ti surfaces. MG63 cells were grown on PT, A, modA, SLA and modSLA surfaces for 2 h, 4 h, 8 h, 1 d, 3 d and 6 d and their morphology imaged by scanning electron microscopy. Areas of lower cell density were selected to facilitate observation of individual cell shapes. The images of the cells shown in the selected micrographs are typical of cells throughout the culture.

Figure 2

Immunofluorescent staining of MG63 cells cultured on tissue culture polystyrene (TCPS) and Ti surfaces. MG63 cells were seeded on TCPS, PT, A, modA, SLA and modSLA surfaces for 2, 4, 8, and 24 hours. Cells were stained for vinculin (red), actin (green) and nucleus (blue). The yellow color represents the co-localization of vinculin and actin. Scale bar = 20µm.

Figure 3

Effect of surface microstructure and surface energy on cell number. MG63 cells were cultured on tissue culture polystyrene (plastic), PT, A, mod A, SLA and modSLA surfaces. Cell number was determined 24-hours after cells reached confluence on plastic surfaces. Values are means + SEM of six independent cultures. Data are from one of two separate experiments, both with comparable results. Data were analyzed by ANOVA and significant differences between groups determined using the Bonferroni modification of Student’s t-test. *p<0.05, Ti surfaces v. plastic; #p<0.05, v. PT; ●p<0.05, v. SLA surface.

Figure 4

Effect of surface microstructure and surface energy on cell differentiation. MG63 cells were cultured on tissue culture polystyrene (plastic), PT, A, mod A, SLA and modSLA surfaces. (A) Alkaline phosphatase specific activity was measured in isolated cells. (B) Osteocalcin levels were measured in conditioned media of confluent cultures. Values are means ± SEM of six independent cultures. Data are from one of two separate experiments, both with comparable results. Data were analyzed by ANOVA and significant differences between groups determined using the Bonferroni modification of Student’s t-test. *p<0.05, Ti surfaces v. plastic; #p<0.05, v. A; ●p<0.05, v. SLA surface.

Figure 5

Effect of surface microstructure and surface energy on local factor levels. MG63 cells were cultured on tissue culture polystyrene (plastic), PT, A, mod A, SLA and modSLA surfaces. (A) Osteoprotegerin levels of conditioned media were determined by ELISA kit. (B) PGE₂ contents of the conditioned media were determined by RIA. Active TGF-β1 and latent TGF-β1 (C) in the conditioned media were measured using an ELISA kit. Values are means ± SEM of six independent cultures. Data are from one of two separate experiments, both with comparable results. Data were analyzed by ANOVA and significant differences between groups determined using the Bonferroni modification of Student’s t-test. *p<0.05, Ti surfaces v. plastic; #p<0.05, v. A; ●p<0.05, v. SLA surface.