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1.  The Roles of Titanium Surface Micro/Nanotopography and Wettability on the Differential Response of Human Osteoblast Lineage Cells 
Acta biomaterialia  2012;9(4):6268-6277.
Surface micro and nanostructural modifications of dental and orthopaedic 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, remains unclear. In the current study, surface wettability analyses (optical sessile drop analysis, ESEM 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 (MSCs) 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.
doi:10.1016/j.actbio.2012.12.002
PMCID: PMC3618468  PMID: 23232211
commercially pure grade 2 titanium implants; osseointegration; bone; nanostructures; mesenchymal stem cell differentiation; dynamic contact angle
2.  Microstructured Titanium Regulates Interleukin Production by Osteoblasts, an Effect Modulated by Exogenous BMP-2 
Acta biomaterialia  2012;9(3):5821-5829.
Microtextured implant surfaces increase osteoblast differentiation in vitro and enhance bone-to-implant contact in vivo and clinically. These implants may be used in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2) to enhance peri-implant bone formation. However, the effect of surface modifications alone or in combination with rhBMP-2 on osteoblast-produced inflammatory microenvironment is unknown. MG63 cells were cultured on tissue culture polystyrene or titanium substrates: smooth pretreated (PT, Ra=0.2μm), sandblasted/acid-etched (SLA, Ra=3.2μm), or hydrophilic-SLA (modSLA). Expression and protein production of pro-inflammatory interleukins (IL1b, IL6, IL8, IL17) and anti-inflammatory interleukins (IL10) were measured in cells with or without rhBMP-2. To determine which BMP signaling pathways were involved, cultures were incubated with BMP pathway inhibitors to blocking Smad (dorsomorphin), TAB/TAK1 ((5Z)-7-oxozeaenol), or PKA (H-8) signaling. Culture on rough SLA and modSLA surfaces decreased pro-inflammatory interleukins and increased anti-inflammatory IL10. This effect was negated in cells treated with rhBMP-2, which caused an increase in pro-inflammatory interleukins and a decrease in anti-inflammatory interleukins through TAB/TAK signaling. The results suggest that surface microtexture modulates the inflammatory process during osseointegration, an effect that may enhance healing. However, rhBMP-2 in combination with microtextured titanium implants can influence the effect of cells on these surfaces, and may adversely affect cells involved in osseointegration.
doi:10.1016/j.actbio.2012.10.030
PMCID: PMC3618455  PMID: 23123301
Microstructure; Inflammation; BMP (bone morphogenetic protein); Titanium
3.  Rapid Re-synostosis Following Suturectomy in Pediatric Mice is Age and Location Dependent 
Bone  2012;53(1):284-293.
Craniosynostosis is the premature fusion of the cranial sutures early in development. If left untreated, craniosynostosis can lead to complications resulting from cranial deformities or increased intracranial pressure. The standard treatment involves calvarial reconstruction, which in many cases undergoes rapid re-synostosis. This requires additional surgical intervention that is associated with a high incidence of life threatening complications. To better understand this rapid healing, a pediatric mouse model of re-synostosis was developed and characterized. Defects (1.5 mm by 2.5 mm) over the posterior frontal suture were created surgically in weanling (21 days post-natal) and adolescent (50 days post-natal) C57Bl/6J mice. In addition, defects were created in the frontal bone lateral to the posterior frontal suture. The regeneration of bone in the defect was assessed using advanced image processing algorithms on micro-computed tomography scans. The genes associated with defect healing were assessed by real-time PCR of mRNA isolated from the tissue present in the defect. The results showed that the weanling mouse healed in a biphasic process with bone bridging the defect by post-operative (post-op) day 3 followed by an increase in the bone volume on day 14. In adolescent mice, there was a delay in bone bridging across the defect, and no subsequent increase in bone volume. No bridging of the defect by 14 days post-op was seen in identically sized defects placed lateral to the suture in both a weanling and adolescent animals. This study demonstrates that bone regeneration in the cranium is both age and location dependent. Rapid and robust bone regeneration only occurred when the defect was created over the posterior frontal suture in immature weanling mice.
doi:10.1016/j.bone.2012.11.019
PMCID: PMC3781584  PMID: 23201269
Craniosynostosis; Cranial defect; Micro-CT; Endochondral ossification; Re-synostosis
4.  BMP2 Induces Osteoblast Apoptosis in a Maturation State and Noggin-dependent Manner 
Journal of cellular biochemistry  2012;113(10):10.1002/jcb.24201.
Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast-like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG-MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z-7-oxozeaenol, dorsomorphin, H-8). Apoptosis was characterized by caspase-3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell-type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG-MG63 than MG63 cells. 5Z-7-oxozeaenol and dorsomorphin eliminated the BMP2-induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications.
doi:10.1002/jcb.24201
PMCID: PMC3852427  PMID: 22628200
Human osteoblasts; BMP (bone morphogenetic protein); Apoptosis; Noggin silencing; Human mesenchymal stem cells
5.  Differential Responses of Osteoblast Lineage Cells to Nanotopographically-Modified, Microroughened Titanium-Aluminum-Vanadium Alloy Surfaces 
Biomaterials  2012;33(35):8986-8994.
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.
doi:10.1016/j.biomaterials.2012.08.059
PMCID: PMC3618458  PMID: 22989383
(4 to 6) metallic implants; osteointegration; titanium aluminum vanadium alloy; bone; nanostructures; osteoblast differentiation
6.  Osteoblast Maturation and New Bone Formation in Response to Titanium Implant Surface Features are Reduced with Age 
The surface properties of materials contribute to host cellular response and play a significant role in determining the overall success or failure of an implanted biomaterial. Rough titanium (Ti) surface microtopography and high surface free energy have been shown to enhance osteoblast maturation in vitro and increase bone formation in vivo. While the surface properties of Ti are known to affect osteoblast response, host bone quality also plays a significant role in determining successful osseointegration. One factor affecting host bone quality is patient age. We examined both in vitro and in vivo whether response to Ti surface features was affected by animal age. Calvarial osteoblasts isolated from 1-, 3-, and 11-month-old rats all displayed a reduction in cell number and increases in alkaline phosphatase specific activity and osteocalcin in response to increasing Ti surface microtopography and surface energy. Further, osteoblasts from the three ages examined displayed increased production of osteocalcin and local factors osteoprotegerin, VEGF-A, and active TGF-β1 in response to increasing Ti surface roughness and surface energy. Latent TGF-β1 only increased in cultures of osteoblasts from 1- and 3-month-old rats. Treatment with the systemic osteotropic hormone 1α,25(OH)2D3 further enhanced the response of osteoblasts to Ti surface features for all three age groups. However, osteoblasts derived from 11-month-old animals had a reduced response to 1α,25(OH)2D3 as compared to osteoblasts derived from 1-or 3-month-old animals. These results were confirmed in vivo. Ti implants placed in the femoral intramedullary canal of old (9-month) mice yielded lower bone-to-implant contract and neovascularization in response to Ti surface roughness and energy compared to younger (2-month) mice. These results show that rodent osteoblast maturation in vitro as well as new bone formation in vivo is reduced with age. Whether comparable age differences exist in humans needs to be determined.
doi:10.1002/jbmr.1628
PMCID: PMC3835587  PMID: 22492532
7.  The responses to surface wettability gradients induced by chitosan nanofilms on microtextured titanium mediated by specific integrin receptors 
Biomaterials  2012;33(30):7386-7393.
Microtexture and chemistry of implant surfaces are important variables for modulating cellular responses. Surface chemistry and wettability are connected directly. While each of these surface properties can influence cell response, it is difficult to decouple their specific contributions. To address this problem, the aims of this study were to develop a surface wettability gradient with a specific chemistry without altering micron scale roughness and to investigate the role of surface wettability on osteoblast response. Microtextured sandblasted/acid-etched (SLA, Sa = 3.1 μm) titanium disks were treated with oxygen plasma to increase reactive oxygen density on the surface. At 0, 2, 6, 10, and 24 h after removing them from the plasma, the surfaces were coated with chitosan for 30 min, rinsed and dried. Modified SLA surfaces are denoted as SLA/h in air prior to coating. Surface characterization demonstrated that this process yielded differing wettability (SLA0 < SLA2 < SLA10 < SLA24) without modifying the micron scale features of the surface. Cell number was reduced in a wettability-dependent manner, except for the most water-wettable surface, SLA24. There was no difference in alkaline phosphatase activity with differing wettability. Increased wettability yielded increased osteocalcin and osteoprotegerin production, except on the SLA24 surfaces. mRNA for integrins α1, α2, α5, β1, and β3 was sensitive to surface wettability. However, surface wettability did not affect mRNA levels for integrin α3. Silencing β1 increased cell number with reduced osteocalcin and osteoprotegerin in a wettability-dependent manner. Surface wettability as a primary regulator enhanced osteoblast differentiation, but integrin expression and silencing β1 results indicate that surface wettability regulates osteoblast through differential integrin expression profiles than microtexture does. The results may indicate that both microtexture and wettability with a specific chemistry have important regulatory effects on osseointegration. Each property had different effects, which were mediated by different integrin receptors.
doi:10.1016/j.biomaterials.2012.06.066
PMCID: PMC3781581  PMID: 22835642
Wettability; Oxygen plasma; Chitosan; Titanium; Osteoblast; Integrin
8.  Osteogenic Differentiation of Stem Cells Alters Vitamin D Receptor Expression 
Stem Cells and Development  2011;21(10):1726-1735.
Pluripotent and multipotent stem cells adopt an osteoblastic phenotype when cultured in environments that enhance their osteogenic potential. Embryonic stem cells differentiated as embryoid bodies (EBs) in osteogenic medium containing β-glycerophosphate exhibit increased expression of bone markers, indicating that cells are osteoblastic. Interestingly, 1α,25-dihydroxyvitaminD3 (1,25D) enhances the osteogenic phenotype not just in EBs but also in multipotent adult mesenchymal stem cells (MSCs). 1,25D acts on osteoblasts via classical vitamin D receptors (VDR) and via a membrane 1,25D-binding protein [protein disulfide isomerase family A, member 3 (PDIA3)], which activates protein kinase C -signaling. The aims of this study were to determine whether these receptors are regulated during osteogenic differentiation of stem cells and if stem cells and differentiated progeny are responsive to 1,25D. mRNA and protein levels for VDR, PDIA3, and osteoblast-associated proteins were measured in undifferentiated cells and in cells treated with osteogenic medium. Mouse EBs expressed both VDR and PDIA3, but VDR increased as cells underwent osteogenic differentiation. Human MSCs expressed Pdia3 at constant levels throughout differentiation, but VDR increased in cells treated with osteogenic medium. These results suggest that both 1,25D signaling mechanisms are important, with PDIA3 playing a greater role during early events and VDR playing a greater role in later stages of differentiation. Understanding these coordinated events provide a powerful tool to control pluripotent and multipotent stem cell differentiation through induction medium.
doi:10.1089/scd.2011.0411
PMCID: PMC3376455  PMID: 22034957
9.  Use of polyelectrolyte thin films to modulate Osteoblast response to microstructured titanium surfaces 
Biomaterials  2012;33(21):5267-5277.
The microstructure and wettability of titanium (Ti) surfaces directly impact osteoblast differentiation in vitro and in vivo. These surface properties are important variables that control initial interactions of an implant with the physiological environment, potentially affecting osseointegration. The objective of this study was to use polyelectrolyte thin films to investigate how surface chemistry modulates response of human MG63 osteoblast-like cells to surface microstructure. Three polyelectrolytes, chitosan, poly(l-glutamic acid), and poly(l-lysine), were used to coat Ti substrates with two different microtopographies (PT, Sa = 0.37 µm and SLA, Sa = 2.54 µm). The polyelectrolyte coatings significantly increased wettability of PT and SLA without altering micron-scale roughness or morphology of the surface. Enhanced wettability of all coated PT surfaces was correlated with increased cell numbers whereas cell number was reduced on coated SLA surfaces. Alkaline phosphatase specific activity was increased on coated SLA surfaces than on uncoated SLA whereas no differences in enzyme activity were seen on coated PT compared to uncoated PT. Culture on chitosan-coated SLA enhanced osteocalcin and osteoprotegerin production. Integrin expression on smooth surfaces was sensitive to surface chemistry, but microtexture was the dominant variable in modulating integrin expression on SLA. These results suggest that surface wettability achieved using different thin films has a major role in regulating osteoblast response to Ti, but this is dependent on the microtexture of the substrate.
doi:10.1016/j.biomaterials.2012.03.074
PMCID: PMC3618464  PMID: 22541354
Wettability; Titanium; Surface roughness; Osteoblast
10.  Effect of cleaning and sterilization on titanium implant surface properties and cellular response 
Acta biomaterialia  2011;8(5):1966-1975.
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.
doi:10.1016/j.actbio.2011.11.026
PMCID: PMC3618465  PMID: 22154860
Titanium; Sterilization; Roughness; Hydrophilicity; MG63 cells
11.  Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production on titanium alloy substrates than on poly-ether-ether-ketone 
Background Context
Multiple biomaterials are clinically available to spine surgeons for performing interbody fusion. Poly-ether-ether-ketone (PEEK) is used frequently for lumbar spine interbody fusion, but alternative materials are also used, including titanium (Ti) alloys. Previously, we showed that osteoblasts exhibit a more differentiated phenotype when grown on machined or grit-blasted titanium aluminum vanadium (Ti6Al4V) alloys with micron-scale roughened surfaces than when grown on smoother Ti6Al4V surfaces or on tissue culture polystyrene (TCPS). We hypothesized that osteoblasts cultured on rough Ti alloy substrates would present a more mature osteoblast phenotype than cells cultured on PEEK, suggesting that textured Ti6Al4V implants may provide a more osteogenic surface for interbody fusion devices.
Purpose
The aim of the present study was to compare osteoblast response to smooth Ti6Al4V (sTiAlV) and roughened Ti6Al4V (rTiAlV) with their response to PEEK with respect to differentiation and production of factors associated with osteogenesis.
Study Design
This in vitro study compared the phenotype of human MG63 osteoblast-like cells cultured on PEEK, sTiAlV, or rTiAlV surfaces and their production of bone morphogenetic proteins (BMPs).
Methods
Surface properties of PEEK, sTiAlV, and rTiAlV discs were determined. Human MG63 cells were grown on TCPS and the discs. Confluent cultures were harvested, and cell number, alkaline phosphatase–specific activity, and osteocalcin were measured as indicators of osteoblast maturation. Expression of messenger RNA (mRNA) for BMP2 and BMP4 was measured by real-time polymerase chain reaction. Levels of BMP2, BMP4, and BMP7 proteins were also measured in the conditioned media of the cell cultures.
Results
Although roughness measurements for sTiAlV (Sa=0.09±0.01), PEEK (Sa=0.43±0.07), and rTiAlV (Sa= 1.81±0.51) varied, substrates had similar contact angles, indicating comparable wettability. Cell morphology differed depending on the surface. Cells cultured on Ti6Al4V had lower cell number and increased alkaline phosphatase specific activity, osteocalcin, BMP2, BMP4, and BMP7 levels in comparison to PEEK. In particular, roughness significantly increased the mRNA levels of BMP2 and BMP4 and secreted levels of BMP4.
Conclusions
These data demonstrate that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment without addition of exogenous factors, which may induce better and faster bone during interbody fusion.
doi:10.1016/j.spinee.2012.02.002
PMCID: PMC3618467  PMID: 22424980
Ti6Al4V; PEEK; Osteoblast; BMP; Roughness
12.  The roles of Wnt signaling modulators Dickkopf-1 (Dkk1) and Dickkopf-2 (Dkk2) and cell maturation state in osteogenesis on microstructured titanium surfaces 
Biomaterials  2009;31(8):2015-2024.
Osteoblast differentiation on tissue culture polystyrene (TCPS) requires Wnt/beta-catenin signaling, regulating modulators of the Wnt pathway like Dickkopf-1 (Dkk1) and Dkk2. Osteoblast differentiation is increased on microstructured titanium (Ti) surfaces compared to TCPS; therefore, we hypothesized that surface topography and hydrophilicity affect Dkk1 and Dkk2 expression and that their roles in osteoblast differentiation on Ti differs depending on cell maturation state. Human osteoblast-like MG63 cells, normal human osteoblasts (HOBs), and human mesenchymal stem cells (MSCs), as well as MG63 cells stably silenced for Dkk1 or Dkk2 were grown for 6 days on TCPS and Ti surfaces (PT [Ra<0.2 μm], SLA [Ra = 4 μm], modSLA [hydrophilic-SLA]). Dkk1 and Dkk2 mRNA and protein increased on SLA and modSLA for all cell types, but exogenous rhDkk1 and rhDkk2 affected MSCs differently than MG63 cells and HOBs. Silencing Dkk1 reduced MG63 cell number on TCPS and PT, but increased differentiation on these substrates. Silencing Dkk2 reduced stimulatory effects of SLA and modSLA on osteoblast differentiation; Dkk2 but not Dkk1 restored these effects. Antibodies to Dkk1 or Dkk2 specifically blocked substrate-dependent changes caused by the proteins, demonstrating their autocrine action. This indicates major roles for Dkk1 and the canonical Wnt pathway in early-stage differentiation, and for Dkk2 and Wnt/Ca2+-dependent signaling in late-stage differentiation on microstructured and hydrophilic surfaces, during osseointegration.
doi:10.1016/j.biomaterials.2009.11.071
PMCID: PMC3618462  PMID: 20004015
Osseointegration; Titanium; Osteoblast; Mesenchymal stem cell; Surface roughness; Cell signaling
13.  Effects of Structural Properties of Electrospun TiO2 Nano-fiber Meshes on their Osteogenic Potential 
Acta Biomaterialia  2011;8(2):878-885.
Ideal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the 3-D structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium (Ti) implants, or more appropriately the titania (TiO2) passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nano-fiber meshes with different surface micro-roughness and nano-fiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface micro-roughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nano-fiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factors.
doi:10.1016/j.actbio.2011.10.023
PMCID: PMC3309709  PMID: 22075122
nano structures; electrospinning; scaffold; titanium implant; tissue engineering; bone
14.  Mediation of Osteogenic Differentiation of Human Mesenchymal Stem Cells on Titanium Surfaces by a Wnt-Integrin Feedback Loop 
Biomaterials  2011;32(27):6399-6411.
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=0.33μm, θ=96°], SLA [Sa=2.5μm, 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.
doi:10.1016/j.biomaterials.2011.05.036
PMCID: PMC3350791  PMID: 21636130
Cell signaling; Surface roughness; Titanium; Stem cell; Growth factors
15.  Use of Molecular Beacons to Image Effects of Titanium Surface Microstructure on β1 Integrin Expression in Live Osteoblast-like Cells 
Biomaterials  2010;31(30):7640-7647.
This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation. A novel molecular beacon that is specific to the β1 integrin subunit mRNA was developed and used to image osteoblast-like MG63 cells in vitro on both glass and titanium surfaces of varying roughness. Specificity was verified by comparing the molecular beacon signal intensities to real-time PCR results in both wild-type cells and cells with shRNA knockdown of β1 integrin mRNA. The molecular beacon was able to detect changes due to both surface microtopography and silencing of the mRNA target. The results showed that effects of the substrate on β1 mRNA noted previously in confluent cultures were evident in pre-confluent cells as well, supporting the hypothesis that β1 integrin pairs are important in proliferation as well as differentiation of osteoblasts. This technique overcomes the limitations of traditional gene assays (PCR, immunofluorescence) by allowing for the real-time measurement and tracking of specific mRNAs in individual live cells prior to confluence.
doi:10.1016/j.biomaterials.2010.07.009
PMCID: PMC3350792  PMID: 20674005
Gene expression; Molecular imaging; Osteoblast; Titanium; Integrin
16.  Regulation of Angiogenesis during Osseointegration by Titanium Surface Microstructure and Energy 
Biomaterials  2010;31(18):4909-4917.
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.
doi:10.1016/j.biomaterials.2010.02.071
PMCID: PMC2896824  PMID: 20356623
Titanium; microstructure; surface energy; osteoblast; angiogenesis; VEGF
17.  Direct and Indirect Effects of Microstructured Titanium Substrates on the Induction of Mesenchymal Stem Cell Differentiation towards the Osteoblast Lineage 
Biomaterials  2010;31(10):2728.
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 α2β1 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 α2β1 and secrete Dkk2, which acts on distal MSCs.
doi:10.1016/j.biomaterials.2009.12.029
PMCID: PMC2821717  PMID: 20053436
18.  Sex dependent regulation of osteoblast response to implant surface properties by systemic hormones 
Background
Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)2D3 and 17β-oestradiol (E2)] and microstructured surfaces on osteoblasts are sex dependent.
Methods
Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)2D3, E2, or E2 conjugated to bovine serum albumin (E2-BSA).
Results
Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-β1, osteoprotegerin and prostaglandin E2 in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E2 and E2-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)2D3 increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells.
Conclusions
Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)2D3 had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones.
doi:10.1186/2042-6410-1-4
PMCID: PMC3010104  PMID: 21208469
19.  Beta-1 Integrins Mediate Substrate Dependent Effects of 1α,25(OH)2D3 on Osteoblasts 
Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. β1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)2D3 in a surface-dependent manner, suggesting that it has a role in mediating osteoblast response. Here, we silenced β1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the β1-silenced osteoblasts to surface microtopography and 1α,25(OH)2D3. MG63 cells were also treated with two different monoclonal antibodies to human β1 to block ligand binding. β1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, β1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)2D3. Anti β1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z decreased cell number and alkaline phosphatase and increased osteocalcin in a dose-dependent manner. Effects of 1α,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)2D3. The results also show that β1 mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)2D3.
doi:10.1016/j.jsbmb.2006.12.083
PMCID: PMC2689367  PMID: 17317155
1α; 25(OH)2D3; β1 integrin subunit; Ti surface roughness; osteoblasts

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