Daniela Baccelli Silveira Mendonça

Advancing dental implant surface technology--from micron- to nanotopography.

Current trends in clinical dental implant therapy include use of endosseous dental implant surfaces embellished with nanoscale topographies. The goal of this review is to consider the role of nanoscale topographic modification of titanium substrates for the purpose of improving osseointegration. Nanotechnology offers engineers and biologists new ways of interacting with relevant biological processes. Moreover, nanotechnology has provided means of understanding and achieving cell specific functions. The various techniques that can impart nanoscale topographic features to titanium endosseous implants are described. Existing data supporting the role of nanotopography suggest that critical steps in osseointegration can be modulated by nanoscale modification of the implant surface. Important distinctions between nanoscale and micron-scale modification of the implant surface are presently considered. The advantages and disadvantages of nanoscale modification of the dental implant surface are discussed. Finally, available data concerning the current dental implant surfaces that utilize nanotopography in clinical dentistry are described. Nanoscale modification of titanium endosseous implant surfaces can alter cellular and tissue responses that may benefit osseointegration and dental implant therapy.

The effects of implant surface nanoscale features on osteoblast- specific gene expression

This study investigated the influence of nanoscale implant surface features on osteoblast differentiation. Titanium disks (20.0 x 1.0 mm) with different nanoscale materials were prepared using sol-gel-derived coatings and characterized by scanning electron microscopy, atomic force microscopy and analyzed by X-ray Photoelectron Spectrometer. Human Mesenchymal Stem Cells (hMSCs) were cultured on the disks for 3-28 days. The levels of ALP, BSP, Runx2, OCN, OPG, and OSX mRNA and a panel of 76 genes related to osteogenesis were evaluated. Topographical and chemical evaluation confirmed nanoscale features present on the coated surfaces only. Bone-specific mRNAs were increased on surfaces with superimposed nanoscale features compared to Machined (M) and Acid etched (Ac). At day 14, OSX mRNA levels were increased by 2-, 3.5-, 4- and 3-fold for Anatase (An), Rutile (Ru), Alumina (Al), and Zirconia (Zr), respectively. OSX expression levels for M and Ac approximated baseline levels. At days 14 and 28 the BSP relative mRNA expression was significantly up-regulated for all surfaces with nanoscale coated features (up to 45-fold increase for Al). The PCR array showed an up-regulation on Al coated implants when compared to M. An improved response of cells adhered to nanostructured-coated implant surfaces was represented by increased OSX and BSP expressions. Furthermore, nanostructured surfaces produced using aluminum oxide significantly enhanced the hMSC gene expression representative of osteoblast differentiation. Nanoscale features on Ti implant substrates may improve the osseointegration response by altering adherent cell response.

The combination of micron and nanotopography by H2SO4/H2O2 treatment and its effects on osteoblast-specific gene expression of hMSCs

H(2)SO(4)/H(2)O(2) treatment of titanium implants imparts nanofeatures to the surface and alters the osteoblast response. The aim of this study was to evaluate the effect of H(2)SO(4)/H(2)O(2) treatment of commercially pure Titanium (cpTi) surfaces on gene expression of human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. Commercially pure grade IV titanium disks (20.0 mm x 1.0 mm) were polished or polished and subsequently treated by grit blasting or grit-blasting/acid etching with an H(2)SO(4)/H(2)O(2) solution. The surfaces were divided into three groups: smooth (S), grit-blasted (Gb), and nanostructured: grit-blasted/acid etched (Nano). Surfaces were examined by scanning electron microscopy and atomic force microscopy. HMSCs were grown on the disks. The data points analyzed were at 3, 7, 14, and 28 days. Real-time PCR was used to measure the mRNA levels of ALP, BSP, Runx2, OCN, OPN, and OSX. The housekeeping gene GAPDH was used as a control. Descriptive statistics were calculated using Microsoft Excel. T-test was performed for comparison of mRNA levels when compared with S surfaces (p < 0.05). All osteoblast-specific genes were regulated in surface-dependent patterns and most of them were upregulated on the Nano surfaces. Runx2 and OSX mRNAs were more than threefold upregulated at days 14 and 28 on Nano. Higher levels for ALP (38-fold), BSP (76-fold), and OCN (3-fold) were also observed on the Nano surfaces. A grit-blasted surface imparted with nanofeatures by H(2)SO(4)/H(2)O(2) treatment affected adherent cell bone-specific gene expression. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Titanium surface topography affects collagen biosynthesis of adherent cells

Collagen-dependent microstructure and physicochemical properties of newly formed bone around implant surfaces represent key determinants of implant biomechanics. This study investigated the effects of implant surface topography on collagen biosynthesis of adherent human mesenchymal stem cells (hMSCs). hMSCs were grown for 0 to 42 days on titanium disks (20.0 × 1.0 mm) with smooth or rough surfaces. Cell attachment and spreading were evaluated by incubating cells with Texas-Red-conjugated phalloidin antibody. Quantitative real-time PCR was used to measure the mRNA levels of Col1α1 and collagen modifying genes including prolyl hydroxylases (PHs), lysyl oxidases (LOXs) and lysyl hydroxylases (LHs). Osteogenesis was assessed at the level of osteoblast specific gene expression and alizarin red staining for mineralization. Cell layer-associated matrix and collagen content were determined by amino acid analysis. At 4h, 100% cells were flattened on both surfaces, however the cells on smooth surface had a fibroblast-like shape, while cells on rough surface lacked any defined long axis. PH, LH, and most LOX mRNA levels were greater in hMSCs grown on rough surfaces for 3 days. The mineralized area was greater for rough surface at 28 and 42 days. The collagen content (percent total protein) was also greater at rough surface compared to smooth surface at 28 (36% versus 26%) and 42 days (46% versus 29%), respectively (p<.05). In a cell culture model, rough surface topography positively modulates collagen biosynthesis and accumulation and the expression of genes associated with collagen cross-linking in adherent hMSC. The altered biosynthesis of the collagen-rich ECM adjacent to endosseous implants may influence the biomechanical properties of osseointegrated endosseous implants.

Management of fractured dental implants: a case report.

An implant fracture may be one of the major causes of late implant failures. Complications, such as loosening or fracture of the prosthesis restorative components, or even fracture of the implant, may occur and dental professionals should be aware of the causes of these complications. This study reports a clinical situation involving a patient restored with a mandibular overdenture that presented a fractured implant 2 years after placement. The probable cause of the implant fracture was dueto biomechanical overload caused by parafunctional habits. The implant head was flattened to make it smooth, retapping the internal screw, installing a new abutment (longer), and fabricating part of the overdenture bar. This treatment was timesaving for the patient in that the prosthesis was repaired in the shortest time possible.

NF-κB suppresses HIF-1α response by competing for P300 binding

Hypoxia has emerged as a key determinant of osteogenesis. HIF-1α is the transcription factor mediating hypoxia responses that include induction of VEGF and related bone induction. Inflammatory signals antagonize bone repair via the NF-κB pathway. The present investigation explored the functional relationship of hypoxia (HIF-1α function) and inflammatory signaling (NF-κB) in stem like and osteoprogenitor cell lines. The potential interaction between HIF-1α and NF-κB signaling was explored by co-transfection studies in hFOB with p65, HIF-1α and 9x-HRE-luc or HIF-1α target genes reporter plasmids. Nuclear cross-talk was directly tested using the mammalian Gal4/VP16 two-hybrid, and confirmed by co-immunoprecipitation/western blotting assays. The results show that inflammatory stimulation (TNF-α treatment) causes a marked inhibition of HIF-1α function at the HRE in all cell lines studied. Also, co-transfection with p65 expression vector leads to reduced hVEGFp transcription after DFO-induced hypoxia. However, TNF-α treatment had little effect on HIF-1α mRNA levels. The functional interaction of Gal4-HIF-1α and VP16-p300 fusion proteins is effectively blocked by expression of p65 in a dose dependent manner. It was concluded that NF-κB-mediated inflammatory signaling is able to block HIF-1α transactivation at HRE-encoding genes by direct competition for p300 binding at the promoter. Inflammation may influence the stem cell niche and tissue regeneration by influencing cellular responses to hypoxia.

RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography‐Dependent

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and ‐mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano‐scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. J. Cell. Physiol. 231: 568–575, 2016.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow–derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Mammalian two-hybrid assays for studies of interaction of p300 with transcription factors.

The two-hybrid system is a powerful genetic assay that allows the interaction between two proteins to be detected in vivo. It was originally described in 1989 and since then it has been one of the main techniques used to identify interactions between proteins from different cellular organisms. Here we describe the methods to study the interaction of p300 with other transcription factors, specifically between p300 and two transcription factors related to hypoxia and inflammation, HIF-1α and NF-κB-p65, respectively.

Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration

Mammalian EAK-7 forms an alternative mTOR complex. Nematode EAK-7 (enhancer-of-akt-1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mTOR, whereas overexpression of mEAK-7 results in enhanced lysosomal localization of mTOR. Deletion of the carboxyl terminus of mEAK-7 significantly decreases mTOR interaction. mEAK-7 knockdown decreases cell proliferation and migration, whereas overexpression of mEAK-7 enhances these cellular effects. Constitutively activated S6K rescues mTOR signaling in mEAK-7–knocked down cells. Thus, mEAK-7 activates an alternative mTOR signaling pathway through S6K2 and 4E-BP1 to regulate cell proliferation and migration.