Direct and indirect effects of microstructured titanium substrates on the induction of mesenchymal stem cell differentiation towards the osteoblast lineage

Abstract

Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved. MSCs exhibited osteoblast characteristics (alkaline phosphatase, RUNX2, and osteocalcin) when grown on microstructured Ti; this effect was more robust with increased hydrophilicity. Factors produced by osteoblasts grown on microstructured Ti were sufficient to induce co-cultured MSC differentiation to osteoblasts. Silencing studies showed that this was due to signaling via alpha2beta1 integrins in osteoblasts on the substrate surface and paracrine action of secreted Dkk2. Thus, human MSCs are sensitive to substrate properties that induce osteoblastic differentiation; osteoblasts interact with these surface properties via alpha2beta1 and secrete Dkk2, which acts on distal MSCs.

2010 Elsevier Ltd. All rights reserved.

Figures

Figure 1

Effect of titanium surface microstructure and hydrophilicity on human mesenchymal stem cell differentiation. MSCs were plated on TCPS, PT, SLA and modSLA surfaces and grown to confluence. At confluence, cell number (A), alkaline phosphatase specific activity (B), osteocalcin (C), osteoprotegerin (D), VEGF-A (E), and TGF-β1 (F) were measured. Data represented are mean ± SEM of six independent samples. *p

Figure 2

Effect of titanium surface microstructure and hydrophilicity on human mesenchymal stem cell gene expression. MSCs were plated on TCPS, PT, SLA and modSLA surfaces and grown to confluence. At confluence, mRNA expression of (A) RUNX2, (B) Osteocalcin, (C) ITGα2, (D) ITGβ1, (E) DKK1, and (F) DKK2 was measured by Real-time PCR. Data represented are mean ± SEM of four independent samples. *p

Figure 3

Indirect effect of titanium surface microstructure and hydrophilicity on differentiation of human mesenchymal stem cells. MSCs were cultured together with MG63 cells grown on TCPS, PT, SLA, and modSLA surfaces. After 12 days of co-culture, MSCs were harvested and cell count (A), alkaline phosphatase specific activity (B), osteocalcin (C), osteoprotegerin (D), VEGF-A (E), and TGF-β1 (F) were measured. Data represented are mean ± SEM of six independent samples. *p

Figure 4

Indirect effect of ITGα2 silencing, titanium surface microstructure and hydrophilicity on differentiation of human mesenchymal stem cells. MSCs were cultured together with wild type MG63 and ITGα2-silenced cells grown on TCPS, PT, SLA, and modSLA surfaces. After 12 days of co-culture, MSCs were harvested and cell count (A), alkaline phosphatase specific activity (B), osteocalcin (C), osteoprotegerin (D), VEGF-A (E), and TGF-β1 (F) were measured. Data represented are mean ± SEM of six independent samples. *p

Figure 5

Indirect effect of Dkk2 silencing, titanium surface microstructure and hydrophilicity on differentiation of human mesenchymal stem cells. MSCs were cultured together with wild type MG63 and Dkk2-silenced cells grown on TCPS, PT, SLA, and modSLA surfaces. After 12 days of co-culture, MSCs were harvested and cell count (A), alkaline phosphatase specific activity (B), osteocalcin (C), osteoprotegerin (D), VEGF-A (E), and TGF-β1 (F) were measured. Data represented are mean ± SEM of six independent samples. *p