Rene Olivares-Navarrete

The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation

Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.

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

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.

Integrin α2β1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates

Efforts to improve bone response to biomaterials have focused on ligands that bind α5β1 integrins. However, antibodies to α5β1 reduce osteoblast proliferation but do not affect differentiation when cells are grown on titanium (Ti). β1-silencing blocks the differentiation stimulus of Ti microtopography, suggesting that other β1 partners are important. Stably α2-silenced MG63 human osteoblast-like cells were used to test whether α2β1 specifically mediates osteoblast response to Ti surface micron-scale structure and energy. WT and α2-silenced MG63 cells were cultured on tissue culture polystyrene (TCPS) and Ti disks with different surface microtopographies: machined pretreatment (PT) surfaces [mean peak to valley roughness (Ra) < 0.02 μm], PT surfaces that were grit-blasted and acid-etched (SLA; Ra = 4 μm), and SLA with high surface energy (modSLA). Alkaline phosphatase (ALP), α2 and β1 mRNA, but not α5, αv, β3, type-I collagen, or osteocalcin, increased on SLA and modSLA at 6 days. α2 increased at 8 days on TCPS and PT, but remained unchanged on SLA and modSLA. α2-protein was reduced 70% in α2-siRNA cells, whereas α5-mRNA and protein were unaffected. α2-knockdown blocked surface-dependent increases in β1 and osteocalcin and decreases in cell number and increases in ALP and local factors typical of MG63 cells grown on SLA and modSLA [e.g., prostaglandin E2, osteoprotegerin, latent and active TGF-β1, and stimulatory effects of 1α,25(OH)2D3 on these parameters]. This finding indicates that α2β1 signaling is required for osteoblastic differentiation caused by Ti microstructure and surface energy, suggesting that conclusions based on cell behavior on TCPS are not predictive of behavior on other substrates or the mechanisms involved.

The Roles of Titanium Surface Micro/Nanotopography and Wettability on the Differential Response of Human Osteoblast Lineage Cells

Surface micro- and nanostructural modifications of dental and orthopedic implants have shown promising in vitro, in vivo and clinical results. Surface wettability has also been suggested to play an important role in osteoblast differentiation and osseointegration. However, the available techniques to measure surface wettability are not reliable on clinically relevant, rough surfaces. Furthermore, how the differentiation state of osteoblast lineage cells impacts their response to micro/nanostructured surfaces, and the role of wettability on this response, remain unclear. In the current study, surface wettability analyses (optical sessile drop analysis, environmental scanning electron microscopic analysis and the Wilhelmy technique) indicated hydrophobic static responses for deposited water droplets on microrough and micro/nanostructured specimens, while hydrophilic responses were observed with dynamic analyses of micro/nanostructured specimens. The maturation and local factor production of human immature osteoblast-like MG63 cells was synergistically influenced by nanostructures superimposed onto microrough titanium (Ti) surfaces. In contrast, human mesenchymal stem cells cultured on micro/nanostructured surfaces in the absence of exogenous soluble factors exhibited less robust osteoblastic differentiation and local factor production compared to cultures on unmodified microroughened Ti. Our results support previous observations using Ti6Al4V surfaces showing that recognition of surface nanostructures and subsequent cell response is dependent on the differentiation state of osteoblast lineage cells. The results also indicate that this effect may be partly modulated by surface wettability. These findings support the conclusion that the successful osseointegration of an implant depends on contributions from osteoblast lineage cells at different stages of osteoblast commitment.

Implant osseointegration and the role of microroughness and nanostructures: lessons for spine implants.

The use of spinal implants for spine fusion has been steadily increasing to avoid the risks of complications and donor site morbidity involved when using autologous bone. A variety of fusion cages are clinically available, with different shapes and chemical compositions. However, detailed information about their surface properties and the effects of such properties on osteogenesis is lacking in the literature. Here we evaluate the role of surface properties for spinal implant applications, covering some of the key biological processes that occur around an implant and focusing on the role of surface properties, specifically the surface structure, on osseointegration, drawing examples from other implantology fields when required. Our findings revealed that surface properties such as microroughness and nanostructures can directly affect early cell behavior and long-term osseointegration. Microroughness has been well established in the literature to have a beneficial effect on osseointegration of implants. In the case of the role of nanostructures, the number of reports is increasing and most studies reveal a positive effect from the nanostructures alone and a synergistic effect when combined with microrough surfaces. Long-term clinical results are nevertheless necessary to establish the full implications of surface nanomodifications.

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy.

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the alpha(2)beta(1) 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 alpha(2)beta(1) signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for alpha(2) 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 1 alpha,25(OH)(2)D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the alpha(2) 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 alpha(2)beta(1) integrin signaling.

Effect of cleaning and sterilization on titanium implant surface properties and cellular response

Titanium (Ti) has been widely used as an implant material due to the excellent biocompatibility and corrosion resistance of its oxide surface. Biomaterials must be sterile before implantation, but the effects of sterilization on their surface properties have been less well studied. The effects of cleaning and sterilization on surface characteristics were bio-determined using contaminated and pure Ti substrata first manufactured to present two different surface structures: pretreated titanium (PT, Ra=0.4 μm) (i.e. surfaces that were not modified by sandblasting and/or acid etching); (SLA, Ra=3.4 μm). Previously cultured cells and associated extracellular matrix were removed from all bio-contaminated specimens by cleaning in a sonicator bath with a sequential acetone-isopropanol-ethanol-distilled water protocol. Cleaned specimens were sterilized with autoclave, gamma irradiation, oxygen plasma, or ultraviolet light. X-ray photoelectron spectroscopy (XPS), contact angle measurements, profilometry, and scanning electron microscopy were used to examine surface chemical components, hydrophilicity, roughness, and morphology, respectively. Small organic molecules present on contaminated Ti surfaces were removed with cleaning. XPS analysis confirmed that surface chemistry was altered by both cleaning and sterilization. Cleaning and sterilization affected hydrophobicity and roughness. These modified surface properties affected osteogenic differentiation of human MG63 osteoblast-like cells. Specifically, autoclaved SLA surfaces lost the characteristic increase in osteoblast differentiation seen on starting SLA surfaces, which was correlated with altered surface wettability and roughness. These data indicated that recleaned and resterilized Ti implant surfaces cannot be considered the same as the first surfaces in terms of surface properties and cell responses. Therefore, the reuse of Ti implants after resterilization may not result in the same tissue responses as found with never-before-implanted specimens.

Mechanisms Regulating Increased Production of Osteoprotegerin by Osteoblasts Cultured on Microstructured Titanium Surfaces

Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2microm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4microm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4microm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-beta1, PKC, and alpha2beta1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-beta1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-beta1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-beta type II receptor or by reducing TGF-beta1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of alpha2 or a double knockdown of alpha2beta1 also reduced OPG, as well as production of TGF-beta1. These results indicate that substrate-dependent OPG production is regulated by TGF-beta1, PKC, and alpha2beta1 and suggest a mechanism by which alpha2beta1 signaling increases PKC, resulting in TGF-beta1 production and TGF-beta1 then acts on its receptor to increase transcription of OPG.

Differential Responses of Osteoblast Lineage Cells to Nanotopographically-Modified, Microroughened Titanium-Aluminum-Vanadium Alloy Surfaces

Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications.

Mediation of osteogenic differentiation of human mesenchymal stem cells on titanium surfaces by a Wnt-integrin feedback loop.

Peri-implant bone formation depends on the ability of mesenchymal cells to colonize the implant surface and differentiate into osteoblasts. Human mesenchymal stem cells (HMSCs) undergo osteoblastic differentiation on microstructured titanium (Ti) surfaces in the absence of exogenous factors, but the mechanisms are unknown. Wnt proteins are associated with an osteoblast phenotype, but how Wnt signaling regulates HMSC differentiation on microstructured Ti surfaces is not known. HMSCs were cultured on tissue culture polystyrene or Ti (PT [Sa = 0.33 μm, θ = 96°], SLA [Sa = 2.5 μm, θ = 132°], modSLA [hydrophilic-SLA]). Expression of calcium-dependent Wnt ligand WNT5A increased and canonical Wnt pathway ligands decreased on microstructured Ti in a time-dependent manner. Treatment of HMSCs with canonical ligand Wnt3a preserved the mesenchymal phenotype on smooth surfaces. Treatment with Wnt5a increased osteoblastic differentiation. Expression of integrins ITGA1, ITGA2, and ITGAV increased over time and correlated with increased WNT5A expression. Treatment of HMSCs with Wnt5a, but not Wnt3a, increased integrin expression. Regulation of integrin expression due to surface roughness and energy was ablated in WNT5A-knockdown HMSCs. This indicates that surface properties regulate stem cell fate and induce osteoblast differentiation via the Wnt calcium-dependent pathway. Wnt5a enhances osteogenesis through a positive feedback with integrins and local factor regulation, particularly though BMP signaling.