Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy

Abstract

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α₂β₁ integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α₂β₁ signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α₂ integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces.Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)₂D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the α₂ integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α₂β₁ integrin signaling.

Introduction

Biomaterial surface properties play a significant role in determining host cellular responses to implant materials used in tissue engineering and regenerative medicine applications. Modifications to surface microarchitecture, chemistry, or energy can alter cell adhesion, proliferation, and gene expression [1], [2], [3]. By designing materials to present specific surface properties, there is the potential to control cell responses to achieve the desired application.

Titanium (Ti) is a widely used biomaterial in the orthopaedic and dental industries because of the biocompatibility and resistance to wear of the Ti oxide layer that forms on its surface. In vitro studies have shown that modifications to Ti surface microtopography affect the attachment and differentiation of osteoblasts, including MG63 and MC3T3-E1 cell lines, as well as fetal rat calvarial cells and normal human osteoblasts [4]. MG63 cells cultured on Ti surfaces with microrough topographies that resemble osteoclast resorption pits display a more differentiated phenotype than cells grown on smooth Ti substrates, characterized by decreased alkaline phosphatase specific activity and higher levels of osteocalcin [5]. The combination of microstructure and high surface energy further enhances osteoblast differentiation on Ti surfaces [6]. In vivo, microstructured implant surfaces support greater bone-to-implant contact than smooth surfaces do, resulting in greater removal torque strength [7], [8], [9].

Osteoblasts interact with their substrate via integrin binding to extracellular matrix proteins [10]. On tissue culture polystyrene (TCPS) surfaces, osteoblasts primarily express α₅β₁ integrins [11]. However, when grown on Ti substrates, expression of α₂ and β₁ integrin subunits is increased [12], suggesting that the surface roughness dependent differentiation of osteoblasts may be mediated specifically through α₂β₁ signaling. Knockdown of either the α₂ or β₁ integrin subunits in MG63 cells blocks surface roughness dependent differentiation of those cells [13], [14], supporting this hypothesis.

The overall success of biomaterial implants in orthopaedic and dental applications however, is not only dependent on achieving the desired cellular response at the host tissue/implant interface but also by the integration of the implant into the surrounding host tissue. Angiogenesis, the sprouting of new capillary blood vessels from the pre-existing vasculature, is a critical process during embryonic development and in several physiological conditions, including the formation of new bone and bone fracture healing [15], [16], as well as during bone regeneration and osseointegration of implanted materials [17]. This suggests that materials that support peri-implant bone formation may support angiogenesis as well as osteogenesis.

The formation of blood vessels in vivo is a complex process and involves the coordination of multiple growth factors and events. Among the many identified growth factors that serve to initiate and control angiogenesis are vascular endothelial growth factor-A (VEGF-A) [18], basic fibroblast growth factor (FGF-2) [19], epidermal growth factor (EGF) [20], and angiopoietin-1 (Ang-1) [21]. Both VEGF-A and FGF-2 are two of the growth factors necessary for initiating angiogenesis and both are chemotactic for endothelial cells [22]. VEGF-A is produced by multiple cell types, including osteoblasts [23] and hypertrophic chondrocytes [24], and affects vascular permeability in vivo [25]. The interaction of VEGF with its receptors Flt-1 and Flk-1/KDR is one of the first signal transduction pathways activated during angiogenesis in endothelial cells [26]. FGF-2 is a heparin-binding polypeptide that induces proliferation, migration, and protease production in cultured endothelial cells and promotes neovascularization in vivo [27]. EGF has also been implicated in angiogenesis by stimulating the proliferation of endothelial cells through its interaction with the tyrosine kinase EGF receptor [28]. EGF treatment of prostate cancer cells increases VEGF mRNA expression suggesting that EGF may also exert its effect by stimulating VEGF production [29]. Ang-1, a member of the angiopoietin family of signaling molecules, binds to its cognate receptor tyrosine kinase Tie1 present on the surface of endothelial cells, inducing signaling events that serve to control later stages of blood vessel formation, such as the stabilization of the endothelial sprout and its interaction with pericytes [30].

Recent studies suggest that osteoblasts may play a role in directly stimulating endothelial cells. Osteoblasts produce VEGF-A [31] and FGF-2 [32], and levels of these angiogenic factors are regulated by factors that stimulate osteogenesis in vivo, including 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] [33], 17β-estradiol [34], and bone morphogenetic protein-2 (BMP-2) [35]. Others have noted that neovascularization is increased in peri-implant bone when microstructured Ti implants are used [36].

Mesenchymal stem cells (MSCs) that have been induced to become osteoblasts produce greater levels of angiogenic factors than unstimulated MSCs [37]. This suggests that this is a function of mature secretory cells and those factors that enhance osteoblast differentiation may also enhance their ability to promote angiogenesis. While it has been established that Ti surface properties influence osteoblast maturation and differentiation and enhance osseointegration in vivo, the potential role that surface properties may have in enhancing angiogenesis surrounding the implant surface through the secretion of angiogenic stimulators by osteoblasts has not been investigated.

In this study, we examined the production of the pro-angiogenic growth factors VEGF-A, FGF-2, EGF and Ang-1 by MG63 human osteoblast-like cells and normal human osteoblasts cultured on Ti surfaces with varying microtopographies and surface energies. In addition, we investigated whether surface dependent production of those factors is sensitive to systemic regulation by treating the cells with 1α,25(OH)₂D₃. We verified that factors produced by the cells were angiogenic by assessing endothelial cell differentiation in response to the conditioned media from MG63 cell cultures from the different Ti substrates. The specific contribution of VEGF-A was determined by treating the endothelial cell cultures with conditioned media in the presence of a neutralization antibody to VEGF-A. Finally, we examined whether the production of angiogenic growth factors is modulated through specific integrin adhesion receptor signaling by silencing of the α₂ integrin subunit.