Ph.D., Biomaterials and Cell Biology
Postdoctoral Fellow, Biomedical Engineering
"Understanding if there is one or hundreds of molecules in e-cigarette vapor that negatively affect craniofacial development is a difficult task because the number of commercially available e-liquids is in the thousands." René Olivares-Navarrete, D.D.S., Ph.D., an assistant professor in the Department of Biomedical Engineering, said. "But finding these answers would give us a better understanding of the possible adverse effects of e-cigarettes."view more
For co-author René Olivares-Navarrete, the study has revealed just a small glimpse at the potential dangers and outcomes of e-cigarettes. “Understanding if there is one or hundreds of molecules in e-cigarette vapor that negatively affect craniofacial development is a difficult task because the number of commercially available e-liquids is in the thousands,” comments Olivares-Navarrete. “But finding these answers would give us a better understanding of the possible adverse effects of e-cigarettes.”view more
The Atlantic online
Many of these chemicals have never been tested on whether they’re safe to breathe in. And that makes vaping’s already unclear effects on health even murkier, because different flavors could be more or less dangerous. A recent study investigated the effects of six different e-cig vapors on tadpoles, as a proxy test for how vaping while pregnant might affect human embryos. Some of the exposed tadpoles developed “clefts” in the bone behind the upper lip, somewhat similar to cleft palate in humans. These clefts only appeared in tadpoles exposed to two particular flavors out of six tested. When the researchers exposed tadpoles to nicotine-free versions of the same flavors, those tadpoles still developed clefts in the same ratios. These initial studies are “just small steps,” says René Olivares-Navarrete, a bioengineering assistant professor at VCU and another coauthor on the tadpole study. As with Tarran’s cell lines, results in tadpoles and mice may or may not translate to humans. Olivares-Navarette says he hopes that e-cigarettes are as safe as vaping advocates claim, but first, “people need to have the information about what is possible.”view more
Hindustan Times online
Smoking e-cigarettes during pregnancy could cause facial defects in the babies, a study warns. The findings suggested that e-cigarettes pose health risks despite being widely considered a safer alternative to tobacco cigarettes. Researchers exposed frog embryos and samples of mammalian neural crest cells to saline infused with e-cigarette vapour. “Neural crest cells are extremely important in the development of craniofacial structures because they can form many different tissues like bones, cartilage, skin, teeth and glands,” Olivares-Navarrete said. E-cigarettes are connected to the pump, which “inhales” the vapour.view more
Smoking e-cigarettes during pregnancy could cause birth defects of the face and oral cavity, new research suggests. Researchers at the Virginia Commonwealth University exposed frog embryos and mammalian neural crest cells to chemicals in to test whether or not they cause defects, to find out what happens to fetuses when e-cigarettes are used during pregnancy. Frogs, like other vertebrates, are similar to humans embryonically. Publishing their findings in the PLOS One journal, they wrote that the same processes and genes govern major developmental events, such as craniofacial development, or the formation of the skull and face. “Neural crest cells are extremely important in the development of craniofacial structures because they can form many different tissues like bones, cartilage, skin, teeth and glands,” Mr Olivares-Navarrete said.view more
Medical Xpress online
Using e-cigarettes during pregnancy could cause birth defects of the oral cavity and face, according to a recent Virginia Commonwealth University study. The findings, published in the journal PLOS ONE in late September, add to growing scientific evidence suggesting that e-cigarettes pose health risks despite being widely considered a safer alternative to tobacco cigarettes. Damage to cells in utero can cause craniofacial birth defects, said co-investigator René Olivares-Navarrete, D.D.S., Ph.D., an assistant professor in the Department of Biomedical Engineering. "Neural crest cells are extremely important in the development of craniofacial structures because they can form many different tissues like bones, cartilage, skin, teeth and glands," Olivares-Navarrete said.view more
A recent study from Virginia Commonwealth University found that using e-cigarettes during pregnancy could cause birth defects. The defects would affect the baby’s oral cavity and face, according to the study. The research findings add to scientific evidence that e-cigs do pose health risks, despite them being considered a safer alternative to traditional tobacco cigarettes.view more
Health Canal online
VCU researchers aim to educate the public about the dangers of e-cigarettes and produce results that would compel tighter government regulation, said primary investigator Rene Olivares-Navarrete, D.D.S., Ph.D., an assistant professor in the Department of Biomedical Engineering. “We’re not going to be able to stop everyone from using e-cigarettes,” Olivares-Navarrete said. “But at least we can tell them this is your choice and give strong evidence about what can happen.”view more
(e) Science News online
In one project, Coulter Department research scientist Rene Olivares-Navarrete and Williams are examining individuals with craniosynostosis to identify genes that influence suture fusion. Determining the genes that control suture closure may help the researchers identify potential therapeutic targets to prevent premature suture fusion.view more
Human mesenchymal stem cells (MSCs) showed larger differentiation into osteoblasts on nanoscale amorphous titanium oxide (TiO2 ) coatings in comparison to polycrystalline TiO2 coatings or native oxide layers. In this article, we showed that the subtle alterations in the surface properties due to a different atomic ordering of titanium oxide layers could substantially modify the osteoblastic differentiation of MSCs. Amorphous (a) and polycrystalline (c) TiO2 coatings were deposited on smooth (PT) and microstructured sandblasted/acid-etched (SLA) Ti substrates using a magnetron sputtering system. The surface roughness, water contact angle, structure, and composition were measured using confocal microscopy, drop sessile drop, X-ray diffraction, and X-ray photoelectron spectroscopy, respectively. The ∼70-nm-thick coatings presented a well-passivated and uniform TiO2 (Ti4+ ) surface composition, while the substrates (native oxide layer) showed the presence of Ti atoms in lower valence states. The polycrystalline TiO2 -coated surfaces (cPT and cSLA) showed the same cell attachment as the uncoated metallic surfaces (PT and SLA), and in both cases, it was lower on the rough than on the smooth surfaces. However, attachment and differentiation were significantly increased on the amorphous TiO2 -coated surfaces (aPT and aSLA). The amorphous coated Ti surfaces presented the highest expression of integrins and production of osteogenic proteins in comparison to the uncoated and crystalline-coated Ti surfaces.view more
Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffnessview more
Microstructured implant surfaces created by grit blasting and acid etching titanium (Ti) support osseointegration. This effect is further enhanced by storing in aqueous solution to retain hydrophilicity, but this also leads to surface nanostructure formation. The purpose of this study was to assess the contributions of nanostructures on the improved osteogenic response of osteoblast lineage cells to hydrophilic microstructured Ti. Human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) were cultured separately on non-nanostructured/hydrophobic (SLA), nanostructured/hydrophilic (modSLA), or nanostructured/hydrophobic (SLAnano) Ti surfaces. XPS showed elevated carbon levels on SLA and SLAnano compared to modSLA. Contact angle measurements indicated only modSLA was hydrophilic. Confocal laser microscopy revealed minor differences in mean surface roughness. SEM showed the presence of nanostructures on modSLA and SLAnano. MSCs and NHOst cells exhibited similar morphology on the substrates and osteoblastic differentiation and maturation were greatest on modSLA. These results suggest that when the appropriate microstructure is present, hydrophilicity may play a greater role in stimulating MSC and NHOst osteoblastic differentiation and maturation than the presence of nanostructures generated during storage in an aqueous environment.view more
The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible for this induction. Sharks are an especially interesting model for cartilage regeneration because their entire skeleton is composed of cartilage and they do not undergo endochondral ossification. Culturing human MSCs on porcine and shark cartilage ECM gels directly, with ECM gel conditioned media, or degradation products increased mRNA levels of chondrogenic factors while decreasing angiogenic factors. These studies indicate that xenogeneic cartilage ECMs have potential as biodegradable scaffolds capable of stimulating chondrogenesis while preventing angiogenesis for regenerative medicine applications and that ECM species selection can yield differential effects.view more
The aim of this study was to examine messenger RNA (mRNA) levels of bone morphogenetic protein (BMP) ligands, receptors, and soluble inhibitors in cells isolated from single-suture synostoses from fused coronal, metopic, sagittal, and lambdoid sutures. Methods: Cells were isolated from bone collected from patients undergoing craniotomies at Children's Healthcare of Atlanta. Real-time polymerase chain reaction was used to examine mRNA levels in cells isolated from fused sutures or patent sutures in comparison with levels in normal bone from the same patient. Results: Cells isolated from fused sutures in cases of sagittal and coronal synostosis highly expressed BMP2, while cells isolated from fused metopic or lambdoid synostosis expressed high BMP4. Noggin, a BMP inhibitor, was lower in fused sutures and had high expression in patent sutures. Conclusions: These results suggest that BMPs and inhibitors play a significant role in the regulation of suture fusion as well in the maintenance of patency in the normal suture.view more
OBJECTIVES: To determine the effects of dental implant surface chemistry and energy on macrophage activation in vitro.
MATERIALS AND METHODS: Disks made from two clinically used implant materials (titanium [Ti], titanium zirconium alloy [TiZr]) were produced with two different surface treatments (sandblast/acid-etch [SLA], hydrophilic-SLA [modSLA]). Surface roughness, energy, and chemistry were characterized. Primary murine macrophages were isolated from 6- to 8-week-old male C57Bl/6 mice and cultured on test surfaces (Ti SLA, TiZr SLA, Ti modSLA, TiZr modSLA) or control tissue culture polystyrene. mRNA was quantified by quantitative polymerase chain reaction after 24 h of culture. Pro- (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-4, IL-10) protein levels were measured by ELISA after 1 or 3 days of culture.
RESULTS: Quantitatively, microroughness was similar on all surfaces. Qualitatively, nanostructures were present on modSLA surfaces that were denser on Ti than on TiZr. modSLA surfaces were determined hydrophilic (high-energy surface) while SLA surfaces were hydrophobic (low-energy surface). Cells on high-energy surfaces had higher levels of mRNA from anti-inflammatory markers characteristic of M2 activation compared to cells on low-energy surfaces. This effect was enhanced on the TiZr surfaces when compared to cells on Ti SLA and Ti modSLA. Macrophages cultured on TiZr SLA and modSLA surfaces released more anti-inflammatory cytokines.
CONCLUSIONS: The combination of high-energy and altered surface chemistry present on TiZr modSLA was able to influence macrophages to produce the greatest anti-inflammatory microenvironment and reduce extended pro-inflammatory factor release.
Metals like titanium (Ti) are common in orthopaedics and dentistry due to their ability to integrate with surrounding tissue and good biocompatibility. Roughness- and wettability-increasing surface modifications promote osteogenic differentiation of stem cells on Ti. While these modifications increase production of osteoblastic factors and bone formation, little is known about their effect on immune cells. The initial host response to a biomaterial is controlled primarily by macrophages and the factors they secrete in response to the injury caused by surgery and the material cues. Here we demonstrate the effect of surface roughness and wettability on the activation and production of inflammatory factors by macrophages. Control of inflammation will inform the design of surface modification procedures to direct the immune response and enhance the success of implanted materials.view more
Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra < 0.4 μm], rough [Ra ≥ 3.4 μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition.view more
Osteoblasts are sensitive to surface microtopography and chemistry. Osteoblast differentiation and maturation are higher in vitro and bone formation and osseointegration enhanced in vivo on microstructured titanium (Ti) compared to smooth surfaces. Cells increased BMP2 expression on microtextured Ti alloy, suggesting a paracrine role in regulating osteoblast maturation. However, recent studies show that exogenous BMP2 inhibits osteoblast production of anti-inflammatory cytokines and osteocalcin, indicating that control of BMP-signaling may be involved. This study examined whether cells modulate BMP ligands, receptors, and inhibitors during osteoblast maturation on Ti, specifically focusing on the roles of BMP2 and Noggin (NOG). mRNA and protein for BMP2, BMP4, and BMP7 and receptors BMPR1A, BMPR1B, and BMPR2, and BMP inhibitors were upregulated on microtextured surfaces in comparison to smooth surfaces. Maturation on microstructured Ti was slightly enhanced with exogenous BMP2 while NOG addition inhibited osteoblast maturation. Cells with NOG knocked down significantly increased osteoblast maturation. These results demonstrate that BMP-related molecules are controlled during osteoblast maturation on microstructured Ti surfaces and that endogenous NOG is an important regulator of the process. Modifying paracrine BMP signaling may yield more robust bone formation than application of exogenous BMPs.view more
Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Raview more
The aim of this study was to examine whether the inflammatory microenvironment generated by cells on titanium-aluminum-vanadium (Ti-alloy, TiAlV) surfaces is affected by surface microtexture and whether it differs from that generated on poly-ether-ether-ketone (PEEK).
SUMMARY OF BACKGROUND DATA: Histologically, implants fabricated from PEEK have a fibrous connective tissue surface interface whereas Ti-alloy implants demonstrate close approximation with surrounding bone. Ti-alloy surfaces with complex micron/submicron scale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation whereas PEEK favors fibrous tissue formation.
METHODS: Human mesenchymal stem cells were cultured on tissue culture polystyrene, PEEK, smooth TiAlV, or macro-/micro-/nano-textured rough TiAlV (mmnTiAlV) disks. Osteoblastic differentiation and secreted inflammatory interleukins were assessed after 7 days. Fold changes in mRNAs for inflammation, necrosis, DNA damage, or apoptosis with respect to tissue culture polystyrene were measured by low-density polymerase chain reaction array. Data were analyzed by analysis of variance, followed by Bonferroni's correction of Student's t-test.
RESULTS: Cells on PEEK upregulated mRNAs for chemokine ligand-2, interleukin (IL) 1β, IL6, IL8, and tumor necrosis factor. Cells grown on the mmnTiAlV had an 8-fold reduction in mRNAs for toll-like receptor-4. Cells grown on mmnTiAlV had reduced levels of proinflammatory interleukins. Cells on PEEK had higher mRNAs for factors strongly associated with cell death/apoptosis, whereas cells on mmnTiAlV exhibited reduced cytokine factor levels. All results were significant (P < 0.05).
CONCLUSION: These results suggest that fibrous tissue around PEEK implants may be due to several factors: reduced osteoblastic differentiation of progenitor cells and production of an inflammatory environment that favors cell death via apoptosis and necrosis. Ti alloy surfaces with complex macro/micro/nanoscale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation.
Muscle regeneration in severe muscle injuries is complex, requiring a sequence of events to promote healing and not fibrosis. Aligned biomaterials that recapitulate muscle environments hold potential to facilitate regeneration, but it is important to understand cell-substrate signaling to form functional muscle. A critical component of muscle signaling is integrin α7β1, where mice lacking α7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of α7β1 signaling in myoblast differentiation on aligned biomaterials. α7-silenced myoblasts were found to regulate myogenic differentiation and demonstrate defective fusion. Our data shows reduced levels of myogenin and myosin heavy chain protein, while MyoD remains unchanged. These results support the hypothesis that α7β1 signaling plays a role in substrate-dependent tissue engineering strategies.view more
Craniosynostosis occurs in approximately 1 in 2,000 children and results from the premature fusion of ≥1 cranial sutures. If left untreated, craniosynostosis can cause numerous complications as related to an increase in intracranial pressure or as a direct result from cranial deformities, or both. More than 100 known mutations may cause syndromic craniosynostosis, but the majority of cases are nonsyndromic, occurring as isolated defects. Most cases of craniosynostosis require complex cranial vault reconstruction that is associated with a high risk of morbidity. While the first operation typically has few complications, bone rapidly regrows in up to 40% of children who undergo it. This resynostosis typically requires additional surgical intervention, which can be associated with a high incidence of life-threatening complications. This article reviews work related to the dental and maxillofacial implications of craniosynostosis and discusses clinically relevant animal models related to craniosynostosis and resynostosis. In addition, information is provided on the imaging modalities used to study cranial defects in animals and humans.view more
Although titanium (Ti) is commonly used for dental implants, Ti alloy materials are being developed to improve their physical material properties. Studies indicate that osteoblast differentiation and maturation of human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) respond to microstructured Ti and titanium-aluminum-vanadium (Ti6Al4V) surfaces in a similar manner. The goal of this study was to determine whether this is the case for osteoblast lineage cells grown on microstructured TiZr surfaces and whether their response is affected by surface nanotexture and hydrophilicity.
MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) disks were modified by large grit sand-blasting and acid-etching with storage in saline solution, resulting in a complex microstructured and hydrophilic surface corresponding to the commercially available implants SLActive® and Roxolid® SLActive® (Institut Straumann AG, Basel, Switzerland). The subsequent Ti modSLA and TiZr modSLA surfaces were characterized and osteogenic markers were measured.
RESULTS: Evaluation of physical parameters revealed that the fabrication method was capable of inducing a microstructured and hydrophilic surface on both the Ti and TiZr disks. Overall, the surfaces were similar, but differences in nanostructure morphology/density and surface chemistry were detected. On Ti modSLA and TiZr modSLA, osteoblastic differentiation and maturation markers were enhanced in both MSCs and NHOsts, while inflammatory markers decreased compared with TCPS.
CONCLUSIONS: These results indicate a similar positive cell response of MSCs and NHOsts when cultured on Ti modSLA and TiZr modSLA. Both surfaces were hydrophilic, indicating the importance of this property to osteoblast lineage cells.
The prevalence of osteoarthritis is higher in women than in men in every age group, and overall prevalence increases with advancing age. Sex-specific differences in the properties of osteoarthritic joint tissues may permit the development of sex-specific therapies. Sex hormones regulate cartilage and bone development and homeostasis in a sex-dependent manner. The purpose of this study was to determine if sex-specific differences exist in synovial fluid and knee tissues isolated from male and female patients with severe knee osteoarthritis. We determined the presence of vitamin D3 metabolites, inflammatory cytokines, growth factors, and matrix metalloproteinases (MMPs) in synovial fluid and assessed responses of articular chondrocytes and subchondral osteoblasts to 17β-estradiol, dihydrotestosterone, and 1α,25(OH)2D3.
METHODS: Samples from knee joints of 10 Caucasian male and 10 Caucasian female patients with advanced osteoarthritis aged 65 to 75 years were obtained from total knee arthroplasty. Vitamin D metabolites, cytokines, MMPs, and growth factors in the synovial fluid were measured. Primary cultures of chondrocytes were isolated from fibrillated articular cartilage adjacent to osteoarthritis lesions and minimally affected cartilage distal to the lesion. Osteoblasts were isolated from the subchondral bone. Expression of receptors for 17β-estradiol and 1α,25(OH)2D3 was assessed by real-time PCR. Chondrocytes and osteoblasts were treated with 10(-8) M 17β-estradiol, dihydrotestosterone, or 1α,25(OH)2D3 and effects on gene expression and protein synthesis determined.
RESULTS: Histology of the articular cartilage confirmed advanced osteoarthritis. Sex differences were found in synovial fluid levels of vitamin D metabolites, cytokines, and metalloproteinases as well as in the cellular expression of receptors for 17β-estradiol and 1α,25(OH)2D3. Male cells were more responsive to 1α,25(OH)2D3 and dihydrotestosterone, whereas 17β-estradiol-affected female cells.
CONCLUSIONS: These results demonstrate that there are underlying sex differences in knee tissues affected by osteoarthritis. Our findings do not address osteoarthritis etiology but have implications for different prevention methods and treatments for men and women. Further research is needed to better understand these sex-based differences.
Dental implants will be exposed to a complex ecosystem once they are placed in the oral cavity. The bacterial colonization and biofilm formation on these devices will depend not only on the physicochemical surface implant properties but also on the periodontal health conditions of the patients, as these devices are exposed.
PURPOSE: The aim of this study was to correlate the subgingival microbial profile with the composition of initial biofilm formed on different microstructured titanium (Ti) surfaces.
MATERIALS AND METHODS: Ten periodontitis and 10 periodontally healthy subjects were included in this study. The subjects wore a removable acrylic device with four different fixed Ti surfaces for 48 hours. Microbial samples of subgingival plaque and the biofilm formed on each Ti surface were individually analyzed by the checkerboard DNA-DNA hybridization technique.
RESULTS: Despite the roughness or hydrophilicity of the Ti surfaces, a characteristic pattern of bacterial adhesion was observed on each of the study groups. However, significant differences in the proportion of the species that colonized the Ti surfaces were found between the periodontitis and periodontally healthy groups. Treponema denticola, Neisseria mucosa, Eikenella corrodens, and Tannerella forsythia were detected in higher proportions on the Ti disks placed in the periodontitis subjects, while significant higher proportions of Capnocytophaga sputigena, Fusobacterium periodonticum, Prevotella melaninogenica, and Streptococcus mitis were detected on the Ti disks placed in the periodontally healthy group.
CONCLUSIONS: The results obtained in this study shows that the composition and the proportion of the species that initially colonize Ti surfaces are highly influenced by the periodontal status more than the surface characteristics of the Ti implant.
Wnt5a and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] regulate endochondral ossification. 1α,25(OH)2D3 initiates its calcium-dependent effects via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3). 1α,25(OH)2D3 binding to Pdia3 triggers the interaction between Pdia3 and phospholipase A2 (PLA2)-activating protein (PLAA), resulting in downstream activation of calcium/calmodulin-dependent protein kinase II (CaMKII), PLA2, and protein kinase C (PKC). Wnt5a initiates its calcium-dependent effects via binding its receptors Frizzled2 (FZD2) and Frizzled5 (FZD5) and receptor tyrosine kinase-like orphan receptor 2 (ROR2), activating intracellular calcium release and stimulating PKC and CaMKII. Recent efforts to determine the inter-relation between Wnt5a and 1α,25(OH)2D3 signaling pathways have demonstrated that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade in chondrocytes and osteoblasts in which the components of the Pdia3 receptor complex were required. Furthermore, ROR2, but not FZD2 or FZD5, was required to mediate the calcium-dependent actions of 1α,25(OH)2D3. This review provides evidence that 1α,25(OH)2D3 and Wnt5a mediate their calcium-dependent pathways via similar receptor components and proposes that these pathways may interact since they are competing for the same receptor complex components.
Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1.view more
Surface microroughness plays an important role in determining osteoblast behavior on titanium. Previous studies have shown that osteoblast differentiation on microtextured titanium substrates is dependent on alpha-2 beta-1 (α2β1) integrin signaling. This study used focused ion beam milling and scanning electron microscopy, combined with three-dimensional image reconstruction, to investigate early interactions of individual cells with their substrate and the role of integrin α2β1 in determining cell shape. MG63 osteoblast-like cells on sand blasted/acid etched (SLA) Ti surfaces after 3 days of culturing indicated decreased cell number, increased cell differentiation, and increased expression of mRNA levels for α1, α2, αV, and β1 integrin subunits compared to cells on smooth Ti (PT) surfaces. α2 or β1 silenced cells exhibited increased cell number and decreased differentiation on SLA compared to wild-type cells. Wild-type cells on SLA possessed an elongated morphology with reduced cell area, increased cell thickness, and more apparent contact points. Cells on PT exhibited greater spreading and were relatively flat. Silenced cells possessed a morphology and phenotype similar to wild-type cells grown on PT. These observations indicate that surface microroughness affects cell response via α2β1 integrin signaling, resulting in a cell shape that promotes osteoblastic differentiation.view more
This study assessed contributions of micron-scale topography on clinically relevant titanium (Ti) to differentiation of osteoprogenitor cells and osteoblasts; the interaction of this effect with 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3); and if the effects are sex-dependent. Male and female rat bone marrow cells (BMCs) were cultured on acid-etched (A, R a = 0.87 μm), grit-blasted (GB, R a = 3.90 μm), or grit-blasted/acid-etched (SLA, R a = 3.22 μm) Ti. BMCs were sensitive to surface topography and underwent osteoblast differentiation. This was greatest on SLA; acid etching and grit blasting contributed additively. Primary osteoblasts were also sensitive to SLA, with less effect from individual structural components, demonstrated by enhanced local factor production. Sex-dependent responses of BMCs to topography varied with parameter whereas male and female osteoblasts responded similarly to surface treatment. 1α,25(OH)2D3 enhanced cell responses on all surfaces similarly. Effects were sex-dependent and male cells grown on a complex microstructured surface were much more sensitive than female cells. These results indicate that effects of the complex SLA topography are greater than acid etching or grit blasting alone on multipotent BMCs and committed osteoblasts and that individual parameters are sex-specific. The effect of 1α,25(OH)2D3 was sex dependent. The results also suggest that levels of 1α,25(OH)2D3 in the patient may be important in osseointegration.view more
Titanium (Ti) and Ti alloys are used in orthopaedic/spine applications where biological implant fixation, or osseointegration, is required for long-term stability. These implants employ macro-scale features to provide mechanical stability until arthrodesis, features that are too large to influence healing at the cellular level. Micron-scale rough Ti alloy (Ti-6Al-4V) increases osteoblastic differentiation and osteogenic factor production in vitro and increases in vivo bone formation; however, effects of overall topography, including sub-micron scale and nanoscale features, on osteoblast lineage cells are less well appreciated. To address this, Ti6Al4V surfaces with macro/micro/nano-textures were generated using sand blasting and acid etching that had comparable average roughness values but differed in other roughness parameters (total roughness, profile roughness, maximum peak height, maximum valley depth, root-mean-squared roughness, kurtosis, skewness) (#5, #9, and #12). Human mesenchymal stem cells (HMSCs) and normal human osteoblasts (NHOst) were cultured for 7 days on the substrates and then analyzed for alkaline phosphatase activity and osteocalcin content, production of osteogenic local factors, and integrin subunit expression. All three surfaces supported osteoblastic differentiation of HMSCs and further maturation of NHOst cells, but the greatest response was seen on the #9 substrate, which had the lowest skewness and kurtosis. The #9 surface also induced highest expression of α2 and β1 integrin mRNA. HMSCs produced highest levels of ITGAV on #9, suggesting this integrin may play a role for early lineage cells. These results indicate that osteoblast lineage cells are sensitive to specific micro/nanostructures, even when overall macro roughness is comparable and suggest that skewness and kurtosis are important variables.view more
Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter.view more
1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/- heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/- mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/- mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/- mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/- heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3's actions in regulating skeletal development.view more
Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)2D3 initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A2 (PLA2)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA2, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA2 inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)2D3 reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)2D3-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)2D3-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)2D3-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)2D3 on PKC and CaMKII. 1α,25(OH)2D3 membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)2D3 or Wnt5a treatment. This study demonstrates that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.view more
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.view more
Recent studies of new surface modifications that superimpose well-defined nanostructures on microrough implants, thereby mimicking the hierarchical complexity of native bone, report synergistically enhanced osteoblast maturation and local factor production at the protein level compared to growth on surfaces that are smooth, nanorough, or microrough. Whether the complex micro/nanorough surfaces enhance the osteogenic response by triggering similar patterns of integrin receptors and their associated signaling pathways as with well-established microrough surfaces, is not well understood. Human osteoblasts (hOBs) were cultured until confluent for gene expression studies on tissue culture polystyrene (TCPS) or on titanium alloy (Ti6Al4V) disks with different surface topographies: smooth, nanorough, microrough, and micro/nanorough surfaces. mRNA expression of osteogenesis-related markers such as osteocalcin (BGLAP) and bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2), BMP4, noggin (NOG) and gremlin 1 (GREM1) were all higher on microrough and micro/nanorough surfaces, with few differences between them, compared to smooth and nanorough groups. Interestingly, expression of integrins α1 and α2, which interact primarily with collagens and laminin and have been commonly associated with osteoblast differentiation on microrough Ti and Ti6Al4V, were expressed at lower levels on micro/nanorough surfaces compared to microrough ones. Conversely, the αv subunit, which binds ligands such as vitronectin, osteopontin, and bone sialoprotein among others, had higher expression on micro/nanorough surfaces concomitantly with regulation of the β3 mRNA levels on nanomodified surfaces. These results suggest that the maturation of osteoblasts on micro/nanorough surfaces may be occurring through different integrin engagement than those established for microrough-only surfaces.view more
Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention.view more
Porcine enamel matrix derivative (pEMD), a complex mixture of proteins and peptides including full-length amelogenin protein, splice variants, and proteolytic peptides, is used clinically with a carrier to regenerate supportive tissue around teeth. During application, pEMD self-assembles as nanospheres and precipitates as a three-dimensional matrix to facilitate cell migration and differentiation. Amelogenin, the primary constituent of pEMD, stimulates osteoblast differentiation, but it is unclear what specific roles other components of pEMD play in determining biological response. This study examined the potential of one constituent of pEMD, the N-terminal amelogenin peptide (NTAP), to promote osteoblastic differentiation of human mesenchymal stem cells (MSCs) and to elucidate possible signaling pathways involved. Effects of porcine NTAP on MSC cultures were compared to those of recombinant human amelogenin. While amelogenin induced MSC osteoblastic differentiation, a more robust osteoblastic response was seen after NTAP treatment. A phospho-kinase proteasome array measuring phosphorylation of 35 proteins indicated that protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and β-catenin were highly phosphorylated by NTAP. This was confirmed by measuring PKC activity and levels of phospho-ERK1/2 and β-catenin. Both amelogenin and NTAP increased PKC, but NTAP induced higher phosho-ERK1/2 and phospho-β-catenin than amelogenin. ERK1/2 inhibition blocked both amelogenin- and NTAP-induced increases in RUNX2, ALP, OCN, COL1, and BMP2. The results demonstrate that NTAP induces osteogenic differentiation of MSCs via PKC and ERK1/2 activation and β-catenin degradation. NTAP may be an active bone regeneration component of amelogenin, and may play this role in pEMD-stimulated periodontal regeneration.view more
Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young's modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.view more
Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner.view more
Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin α2β1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK.
PURPOSE: The purpose of this study was to determine if osteoblasts generate an environment that supports angiogenesis and reduces osteoclastic activity when grown on smooth titanium alloy, rough titanium alloy, or PEEK.
STUDY DESIGN: This in vitro study compared angiogenic factor production and integrin gene expression of human osteoblast-like MG63 cells cultured on PEEK or titanium-aluminum-vanadium (titanium alloy).
METHODS: MG63 cells were grown on PEEK, smooth titanium alloy, or rough titanium alloy. Osteogenic microenvironment was characterized by secretion of osteoprotegerin and transforming growth factor beta-1 (TGF-β1), which inhibit osteoclast activity and angiogenic factors including vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and angiopoietin-1 (ANG-1). Expression of integrins, transmembrane extracellular matrix recognition proteins, was measured by real-time polymerase chain reaction.
RESULTS: Culture on titanium alloy stimulated osteoprotegerin, TGF-β1, VEGF-A, FGF-2, and angiopoietin-1 production, and levels were greater on rough titanium alloy than on smooth titanium alloy. All factors measured were significantly lower on PEEK than on smooth or rough titanium alloy. Culture on titanium alloy stimulated expression of messenger RNA for integrins that recognize Type I collagen in comparison with PEEK.
CONCLUSIONS: Rough titanium alloy stimulated cells to create an osteogenic-angiogenic microenvironment. The osteogenic-angiogenic responses to titanium alloy were greater than PEEK and greater on rough titanium alloy than on smooth titanium alloy. Surface features regulated expression of integrins important in collagen recognition. These factors may increase bone formation, enhance integration, and improve implant stability in interbody spinal fusions.
Protein disulfide isomerase family A, member 3 (Pdia3) mediates many of the plasma membrane (PM)-associated rapid responses to 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3). It is not well understood how Pdia3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1α,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for Pdia3's ER chaperone function, and examined their role in responses to 1α,25(OH)2D3. Pdia3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for Pdia3. Finally, we determined whether palmitoylation and/or myristoylation were required for Pdia3-mediated responses to 1α,25(OH)2D3. Overexpressing the Pdia3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1α,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overexpressing wild-type Pdia3 (Pdia3Ovr cells). Cells overexpressing Pdia3 with K214A and C406S mutations had PKC activity comparable to untreated controls, indicating that the native response to 1α,25(OH)2D3 also was blocked. Overexpressing Pdia3[-KDEL] increased PM localization and augmented baseline PKC, but the stimulatory effect of 1α,25(OH)2D3 was comparable to that seen in wild-type cultures. In contrast, 1α,25(OH)2D3 increased prostaglandin E2 in Pdia3[±KDEL] cells. Although neither palmitoylation nor myristoylation was required for PM association of Pdia3, myristoylation was needed for PKC activation. These data indicate that both the chaperone functional domains and the subcellular location of Pdia3 control rapid membrane responses to 1α,25(OH)2D3.view more
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.view more
Craniosynostosis is the premature fusion of cranial sutures early in development. Mice are commonly used to study the mechanisms driving both normal and pathologic cranial suture development. Despite their frequency of use as a model, the time course of bone formation and mineralization during fusion of mouse posterior frontal suture is not well defined.
METHODS: To address this, C57Bl/6J mice were euthanized at ages ranging from 6 to 107 days, and the posterior frontal sutures were imaged using micro-computed tomography. Scans were analyzed with an image-processing algorithm that was previously validated with serial histology to quantify both suture fusion and mineral content. The expression profile of genes associated with key developmental time points was examined using real-time polymerase chain reaction in both the bone and the dura.
RESULTS: Results demonstrate that the bones of the posterior frontal suture come together during days 10 to 20 and then increase in mineral content and volume between days 21 and 45. The onset of posterior frontal suture fusion was associated with an increase in cartilage-associated genes on day 12. Later mineralization of the suture was associated with an increase in mRNAs for osteoblast differentiation markers, bone morphogenetic proteins, and bone morphogenetic protein inhibitors.
CONCLUSIONS: Complete analysis fusion posterior frontal suture shows that it occurs in a discontinuous biphasic manner. The first phase is from days 10 to 20 and involves production of cartilage. A second mineralization phase from days 21 to 45 was seen with both the imaging algorithm and changes in gene expression.
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 the 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 blockSmad (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.view more
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 (1.5mm by 2.5mm) 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 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.view more
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.view more
The goal of this study was to develop a method for increasing the yield of multipotent adipose-derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271(+), CD73(+) and CD45(-)) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11(+) and OCN(+). Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µM) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two-dimensional (2D) cultures as a function of alkaline phosphatase-specific activity and osteocalcin production. In addition, cells were seeded onto three-dimensional (3D) poly-ε-caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro-CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol-treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy.view more
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.view more
The interrelationships among suture fusion, basicranial development, and subsequent resynostosis in syndromic craniosynostosis have yet to be examined. The objectives of this study were to determine the potential relationship between suture fusion and cranial base development in a model of syndromic craniosynostosis and to assess the effects of the syndrome on resynostosis following suturectomy. To do this, posterior frontal and coronal suture fusion, postnatal development of sphenooccipital synchondrosis, and resynostosis in Twist1(+/+) (WT) and Twist1(+/-) litter-matched mice (a model for Saethre-Chotzen syndrome) were quantified by evaluating μCT images with advanced image-processing algorithms. The coronal suture in Twist(+/-) mice developed, fused, and mineralized at a faster rate than that in normal littermates at postnatal days 6-30. Moreover, premature fusion of the coronal suture in Twist1(+/-) mice preceded alterations in cranial base development. Analysis of synchondrosis showed faster mineralization in Twist(+/-) mice at postnatal days 25-30. In a rapid resynostosis model, there was an inability to fuse both the midline posterior frontal suture and craniotomy defects in 21-day-old Twist(+/-) mice, despite having accelerated mineralization in the posterior frontal suture and defects. This study showed that dissimilarities between Twist1(+/+) and Twist1(+/-) mice are not limited to a fused coronal suture but include differences in fusion of other sutures, the regenerative capacity of the cranial vault, and the development of the cranial base.view more
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.view more
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. Whereas 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, vascular endothelial growth factor (VEGF)-A, and active transforming growth factor (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)(2)D(3) 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)(2)D(3) 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-old) mice yielded lower bone-to-implant contact and neovascularization in response to Ti surface roughness and energy compared to younger (2-month-old) 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.view more
One of the most difficult challenges in medical imaging is the accurate segmentation of mineralized tissues. This process is complicated when studying developmental or regenerative processes due to the changes in mineral density that these tissues undergo over time. To address these limitations an algorithm was developed to enable the use of computed tomography (CT) to study tissues of varying and heterogeneous mineralization. To examine and validate this algorithm a study was performed on the development of murine cranial sutures. C57Bl/6J mice ranging in age from 6 to 25 days were imaged using micro-CT (μCT). The algorithm was developed to segment the bones of both the posterior frontal (PF) and coronal (COR) sutures. For the curved COR suture, an addition to the algorithm was developed to reconstruct images that were perpendicular to the suture about all three axes. The algorithm showed excellent linear correlation (R (2) > 0.96) with serial histomorphometry and nearly a 1:1 relationship between all measures. The algorithm was validated with serial histology. The algorithm showed that the PF suture fused between days 12 and 20 but then showed a significant increase in bone volume after day 20. The algorithm developed provides an accurate method to segment the irregular sutures of the mouse calvaria.view more
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.view more
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.view more
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.view more
Protein kinase C (PKC) signaling can be activated rapidly by 17β-estradiol (E(2)) via nontraditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERβ. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [(3)H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.view more
The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation.
MATERIALS AND METHODS: Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [R(a)
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.
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 (S(a)=0.09±0.01), PEEK (S(a)=0.43±0.07), and rTiAlV (S(a)=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.
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 three-dimensional 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 implants, or more appropriately the 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 nanofiber meshes with different surface microroughness and nanofiber 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 microroughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nanofiber 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.view more
The success rate of titanium implants for dental and orthopedic applications depends on the ability of surrounding bone tissue to integrate with the surface of the device, and it remains far from ideal in patients with bone compromised by physiological factors. The electrical properties and electrical stimulation of bone have been shown to control its growth and healing and can enhance osseointegration. Bone cells are also sensitive to the chemical products generated during corrosion events, but less is known about how the electrical signals associated with corrosion might affect osseointegration. The metallic nature of the materials used for implant applications and the corrosive environments found in the human body, in combination with the continuous and cyclic loads to which these implants are exposed, may lead to corrosion and its corresponding electrochemical products. The abnormal electrical currents produced during corrosion can convert any metallic implant into an electrode, and the negative impact on the surrounding tissue due to these extreme signals could be an additional cause of poor performance and rejection of implants. Here, we review basic aspects of the electrical properties and electrical stimulation of bone, as well as fundamental concepts of aqueous corrosion and its electrical and clinical implications.view more
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.view more
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra=0.2 μm), sand-blasted and acid-etched SLA (Ra=3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.view more
Micrometer- and submicrometer-scale surface roughness enhances osteoblast differentiation on titanium (Ti) substrates and increases bone-to-implant contact in vivo. However, the low surface wettability induced by surface roughness can retard initial interactions with the physiological environment. We examined chemical modifications of Ti surfaces [pretreated (PT), R(a) ≤ 0.3 μm; sand blasted/acid etched (SLA), R(a) ≥ 3.0 μm] in order to modify surface hydrophilicity. We designed coating layers of polyelectrolytes that did not alter the surface microstructure but increased surface ionic character, including chitosan (CHI), poly(L-glutamic acid) (PGA), and poly(L-lysine) (PLL). Ti disks were cleaned and sterilized. Surface chemical composition, roughness, wettability, and morphology of surfaces before and after polyelectrolyte coating were examined by X-ray photoelectron spectroscopy (XPS), contact mode profilometry, contact angle measurement, and scanning electron microscopy (SEM). High-resolution XPS spectra data validated the formation of polyelectrolyte layers on top of the Ti surface. The surface coverage of the polyelectrolyte adsorbed on Ti surfaces was evaluated with the pertinent SEM images and XPS peak intensity as a function of polyelectrolyte adsorption time on the Ti surface. PLL was coated in a uniform thin layer on the PT surface. CHI and PGA were coated evenly on PT, albeit in an incomplete monolayer. CHI, PGA, and PLL were coated on the SLA surface with complete coverage. The selected polyelectrolytes enhanced surface wettability without modifying surface roughness. These chemically modified surfaces on implant devices can contribute to the enhancement of osteoblast differentiation.view more
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.view more
Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)(2)D(3). In intestinal epithelium and chondrocytes, 1,25(OH)(2)D(3) stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)(2)D(3) stimulates phospholipase A(2) (PLA(2))-dependent rapid release of prostaglandin E(2) (PGE(2)), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)(2)D(3) failed to stimulate PKC and PGE(2) responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)(2)D(3) were augmented. Downstream mediators of Pdia3, PLA(2)-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)(2)D(3). Treatment of MC3T3-E1 cells with 1,25(OH)(2)D(3) for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)(2)D(3) treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)(2)D(3)-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.view more
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.
his 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 beta1 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 beta1 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 beta1 mRNA noted previously in confluent cultures were evident in pre-confluent cells as well, supporting the hypothesis that beta1 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.view more
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.view more
Biomaterial surface properties such as microtopography and energy can change cellular responses at the cell-implant interface. Phospholipase D (PLD) is required for the differentiation of osteoblast-like MG63 cells on machined and grit-blasted titanium surfaces. Here, we determined if PLD is also required on microstructured/high-energy substrates and the mechanism involved. shRNAs for human PLD1 and PLD2 were used to silence MG63 cells. Wild-type and PLD1 or PLD1/2 silenced cells were cultured on smooth-pretreatment surfaces (PT); grit-blasted, acid-etched surfaces (SLA); and SLA surfaces modified to have higher surface energy (modSLA). PLD was inhibited with ethanol or activated with 24,25-dihydroxyvitamin-D(3) [24R,25(OH)(2)D(3)]. As surface roughness/energy increased, PLD mRNA and activity increased, cell number decreased, osteocalcin and osteoprotegerin increased, and protein kinase C (PKC) and alkaline phosphatase specific activities increased. Ethanol inhibited PLD and reduced surface effects on these parameters. There was no effect on these parameters after knockdown of PLD1, but PLD1/2 double knockdown had effects comparableto PLD inhibition. 24R,25(OH)(2)D(3) increased PLD activity and the production of osteocalcin and osteoprotegerin, but decreased cell number on the rough/high-energy surfaces. These results confirm that surface roughness/energy-induced PLD activity is required for osteoblast differentiation and that PLD2 is the main isoform involved in this pathway. PLD is activated by 24R,25(OH)(2)D(3) in a surface-dependent manner and inhibition of PLD reduces the effects of surface microstructure/energy on PKC, suggesting that PLD mediates the stimulatory effect of microstructured/high-energy surfaces via PKC-dependent signaling.view more
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.view more
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 [Raview more
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, Raview more
Efforts to improve bone response to biomaterials have focused on ligands that bind alpha5beta1 integrins. However, antibodies to alpha5beta1 reduce osteoblast proliferation but do not affect differentiation when cells are grown on titanium (Ti). beta1-silencing blocks the differentiation stimulus of Ti microtopography, suggesting that other beta1 partners are important. Stably alpha2-silenced MG63 human osteoblast-like cells were used to test whether alpha2beta1 specifically mediates osteoblast response to Ti surface micron-scale structure and energy. WT and alpha2-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 (R(a)) < 0.02 microm], PT surfaces that were grit-blasted and acid-etched (SLA; R(a) = 4 microm), and SLA with high surface energy (modSLA). Alkaline phosphatase (ALP), alpha2 and beta1 mRNA, but not alpha5, alpha v, beta3, type-I collagen, or osteocalcin, increased on SLA and modSLA at 6 days. Alpha2 increased at 8 days on TCPS and PT, but remained unchanged on SLA and modSLA. Alpha2-protein was reduced 70% in alpha2-siRNA cells, whereas alpha5-mRNA and protein were unaffected. Alpha2-knockdown blocked surface-dependent increases in beta1 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 E(2), osteoprotegerin, latent and active TGF-beta1, and stimulatory effects of 1alpha,25(OH)(2)D(3) on these parameters]. This finding indicates that alpha2beta1 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.view more
Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. beta(1) integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1alpha,25(OH)(2)D(3) in a surface-dependent manner. To determine if beta(1) has a role in mediating osteoblast response, we silenced beta(1) expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human beta(1) to block ligand binding. beta(1)-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta(1), prostaglandin E(2), and osteoprotegerin in comparison with control cells. Moreover, beta(1)-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)(2)D(3). Anti beta(1) antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1alpha,25(OH)(2)D(3) on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta(1) plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)(2)D(3). The results also show that beta(1) mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)(2)D(3).view more
Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. The observations that beta1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1alpha,25(OH)2D3 in a surface-dependent manner, suggest that beta1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced beta1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the beta1-silenced osteoblasts to surface microtopography and 1alpha,25(OH)2D3. To better understand the role of beta1, MG63 cells were also treated with two different monoclonal antibodies to human beta1 to block ligand binding. beta1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, beta1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)2D3. Anti beta1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1alpha,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)2D3. The results also show that beta1 mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)2D3.view more
Amorphous carbon (a-C), carbon nitride (a-CN) and titanium films were deposited on stainless steel substrates (SS) using a dc magnetron sputtering system attached to a high vacuum chamber. Films were deposited using a base pressure of 1.3x10(-4) Pa. For the carbon films a pure graphite target was eroded in an Argon plasma. For the case of the a-CN films, the Ar flux was substituted by 100% N2 gas. Titanium films were deposited in a different chamber, using a pure Ti target and an argon plasma. In vitro studies were carried out on the coated samples using human osteoblasts cells. Cytotoxicity of carbon films was assessed by cellular adhesion and proliferation, as determined by direct cellular counting using a spectroscopic technique and a well-defined standard curve. Osteoblasts cells were also grown on uncoated steel and prepared Petri dishes for comparison. The percentage of osteoblasts adhesion measured at 24 hrs attained maximum values for the a-C films. Similarly, cellular proliferation evaluated at three, five and seven days showed an outstanding increase of osteoblasts cells for the a-C and Ti coatings in contrast to the uncoated steel. The cell functionality was evaluated by the MTT test after incubation periods of 3, 5 and 7 days. The absorbance values obtained for a-C, a-CN and Ti surfaces resulted significantly higher with respect to the positive control, indicating that the surface did not induce any toxic effect. Preliminary bio-mineralization was evaluated by measuring the elemental composition of the mineral grown on the substrates after periods up to 14 days.
Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration.view more