Cigarette smoke increases pro-inflammatory markers and inhibits osteogenic differentiation in experimental exposure model.

Tobacco smokers have slowed bone growth and regeneration and more frequent implant failures than non-smokers, but the effect of cigarette smoking on the host response to bone-dwelling biomaterials is poorly understood. Macrophages and mesenchymal stem cells (MSCs) are essential in the healing response after implant placement. This study examined the effects of an experimental model of cigarette smoke exposure using cigarette smoke extract (CSE) on bone architecture in vivo and differentiation and inflammatory cytokine production on clinically relevant microstructured surfaces in vitro. CSE was prepared by bubbling mainstream smoke from one research cigarette (3R4F) in 1 mL phosphate-buffered saline. For in vivo studies, bone morphometry was examined in femurs isolated from mice injected with diluted CSE for 25 days. For in vitro studies, osteogenic markers and interleukins were measured in human MSCs and murine macrophages cultured on rough or rough-hydrophilic titanium (Ti) surfaces in culture media ±â€¯CSE for seven days. In vivo, CSE exposure decreased in bone area, volume, and interconnectivity in a dose-dependent manner. In vitro, macrophages exposed to CSE increased production of pro-inflammatory cytokines, abolishing the increase in anti-inflammatory cytokines typically seen on rough-hydrophilic surfaces. MSCs exposed to CSE had lower mRNA expression of osteoblast differentiation markers, increased levels of pro-inflammatory mRNA, and reduced production of osteogenic proteins. Our results demonstrate that CSE decreases osteogenic differentiation and anti-inflammatory interleukin production while increasing pro-inflammatory interleukin production in macrophages and MSCs, suggesting that compounds in CSE strongly affect stem cell differentiation and may compromise bone formation following biomaterial placement. STATEMENT OF SIGNIFICANCE: The study of implantable materials' interaction with biological systems occurs nearly exclusively in healthy cell and animal models. However, 15% of the US population smokes cigarettes, which is known to modulate immune response and tissue regeneration. To explore this interaction, we created a method of capturing smoke compounds as CSE for in vivo and in vitro use. We found chronic injection into mice produced an osteoporotic, pro-inflammatory phenotype similar to direct smoke models. Furthermore, CSE attenuated osteogenic differentiation and promoted a pro-inflammatory profile in MSCs and macrophages, respectively, when cultured on titanium surfaces. These results demonstrate that this CSE model may be useful for predicting how chronic tobacco exposure may adversely affect the outcome of biomedical implants in pre-clinical models.

Craniosynostosis and Resynostosis: Models, Imaging, and Dental Implications.

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.

Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation.

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.

Role of the N-terminal peptide of amelogenin on osteoblastic differentiation of human mesenchymal stem cells.

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 ß-catenin were highly phosphorylated by NTAP. This was confirmed by measuring PKC activity and levels of phospho-ERK1/2 and ß-catenin. Both amelogenin and NTAP increased PKC, but NTAP induced higher phosho-ERK1/2 and phospho-ß-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 ß-catenin degradation. NTAP may be an active bone regeneration component of amelogenin, and may play this role in pEMD-stimulated periodontal regeneration.

Microstructured titanium regulates interleukin production by osteoblasts, an effect modulated by exogenous BMP-2.

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.

Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro.

OBJECTIVES: 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) <0.2 µm]), acid-etched (A [R(a) <0.8 µm]), A modified to be hydrophilic (modA), sand-blasted/acid-etched (SLA [R(a) =4 µm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA-DNA hybridization. RESULTS: Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva. CONCLUSIONS: The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used.

Electrical implications of corrosion for osseointegration of titanium implants.

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.

Factor de crecimiento epidermal y proteínas totales en saliva de fumadores y no fumadores / Epidermal growth factor and total protein in saliva of smokers and non smokers

La función del Factor de Crecimiento Epidermal (EGF) es mantener la integridad de los tejidos, su receptor se localiza en superficies celulares y se expresa principalmente en epitelios. Nuestro objetivo fue determinar la asociación entre el tabaquismo crónico, el pH, las proteínas totales (PT) y el EGF en saliva de fumadores. Se seleccionaron pacientes masculinos, entre 20 y 60 años, de la clínica de Registro del Postgrado de Odontología de la Universidad. Se tomaron muestras de saliva total estimulada (STHe) y no estimulada (STH) a un grupo de fumadores y no fumadores. Se midió el pH, se cuantificaron las PT por el método de Bradford y para el EGF la técnica de ELISA (ELISA Kit KHG0061 biosource). Los resultados fueron: el pH se encontró ligeramente alcalino en STHe en ambos grupos, la cantidad de PT fue de 1,11 ± 0,71 en no fumadores y 0,87 ± 0,31mg/mL en STH de fumadores. En STHe también existió un decremento en la cantidad de PT de 0,94 ± 0,470,66 ± 0,21 mg/mL. El EGF fue en STH de no fumadores 14,8 ± 5,62 comparado con los fumadores con un promedio de 11,4 ± 5,45. En STHe, el promedio del mismo factor fue en los no fumadores de 7,8 ± 5,60 yen los fumadores a 9,1 ± 6,53 ng/mL. Con una con una confiabilidad del 90% (AU)

The role of Epidermal Growth Factor (EGF) is to maintain tissue’s integrity. It’s receptor is located on cell surfaces and mainly expressed in epithelia. The aim of this study was to determine the association between chronicle tabaquism, pH, total proteins (TP) and EGF in smoker’s saliva. There were selected male volunteers between 20and 60 years old from the Registration Clinic of the Postgrade Division of UNAM’s Dentistry School. There were taken stimulated (sWHS) and not stimulated whole saliva (WHS) samples from groups of smokers and non-smokers. Total proteins (Bradford method), pH and EGF (ELISA Kit KHG0061 biosource) were measured. Results: Slightly alkaline pH in sWHS in both groups. TP were 1.11 ± 0.71 for non-smokers WHS and 0.87 ± 0.31 mg/mL for smokers WHS. In non-smokers WHS EGF were 14.8 ± 5.62 compared to smokers WHS which showed 11.4± 5.45. For sWHS the values were 7.8 ± 5.60 in non-smokers group and 9.10 ± 6.53 ng/mL in smokers group (AU)

Integrin alpha2beta1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates.

Efforts to improve bone response to biomaterials have focused on ligands that bind 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.