Nanoscale hybrid implant surfaces and Osterix-mediated osseointegration.

Endosseous implant surface topography directly affects adherent cell responses following implantation. The aim of this study was to examine the impact of nanoscale topographic modification of titanium implants on Osterix gene expression since this gene has been reported as key factor for bone formation. Titanium implants with smooth and nanoscale topographies were implanted in the femurs of Osterix-Cherry mice for 1-21 days. Implant integration was evaluated using scanning electron microscopy (SEM) to evaluate cell adhesion on implant surfaces, histology, and nanotomography (NanoCT) to observe and quantify the formed bone-to-implant interface, flow cytometry to quantify of Osterix expressing cells in adjacent tissues, and real-time PCR (qPCR) to quantify the osteoinductive and osteogenic gene expression of the implant-adherent cells. SEM revealed topography-dependent adhesion of cells at early timepoints. NanoCT demonstrated greater bone formation at nanoscale implants and interfacial osteogenesis was confirmed histologically at 7 and 14 days for both smooth and nanosurface implants. Flow cytometry revealed greater numbers of Osterix positive cells in femurs implanted with nanoscale versus smooth implants. Compared to smooth surface implants, nanoscale surface adherent cells expressed higher levels of Osterix (Osx), Alkaline phosphatase (Alp), Paired related homeobox (Prx1), Dentin matrix protein 1 (Dmp1), Bone sialoprotein (Bsp), and Osteocalcin (Ocn). In conclusion, nanoscale surface implants demonstrated greater bone formation associated with higher levels of Osterix expression over the 21-day healing period with direct evidence of surface-associated gene regulation involving a nanoscale-mediated osteoinductive pathway that utilizes Osterix to direct adherent cell osteoinduction.

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2.

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the ß-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

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.

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.

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.

Efeito de superfícies de implantes nano-estruturadas na expressão de genes de osteoblastos e no contato osso-implante in vivo / Effect of nanostructured implant surfaces on osteoblast related gene expression and bone-implant contact in vivo

As tendências atuais na terapia com implantes odontológicostêm incluído o uso de implantes com superfícies modificadasutilizando nanotecnologia. Ciência que permite a construçãode novos materiais e dispositivos pela manipulação de átomosindividuais e moléculas (escala menor do que 100nm). O objetivodeste trabalho foi avaliar o papel das modificações em escalananométrica de superfícies de implantes osseointegradospara melhorar o processo de osseointegração. Nanotecnologiaoferece a engenheiros e profissionais da área de biologia e saúdenovos meios para entender e otimizar funções e respostasespecíficas de células. As várias técnicas utilizadas para adicionarcaracterísticas nanométricas às superfícies de implantesosseointegrados são descritas neste trabalho. Vários trabalhostem apresentado os efeitos da nanotecnologia na modulaçãode etapas fundamentais do processo de osseointegração. Asvantagens e desvantagens da utilização da nanotecnologia nasuperfície de implantes também são discutidas nesse trabalho.Posteriormente, em uma série de experimentos in vitro e in vivo,foi possível avaliar o efeito específico destas modificações emdois diferentes modelos. Como efeitos observados da aplicaçãode nanoestruturas à superfície dos implantes osseointegradosfoi possível verificar-se uma melhor e mais rápida resposta deosseointegração destes materiais, atuando efetivamente na cascatade diferenciação de osteoblastos.

Current trends in clinical dental implant therapy include useof endosseous dental implant surfaces embellished with nanoscaletopographies. Nanotechnology deals with materials withat least one significant dimension less than 100nm. The goal ofthis study was to consider the role of nanoscale topographic modificationof titanium substrates for the purpose of improvingosseointegration. Nanotechnology offers engineers and biologistsnew ways of interacting with relevant biological processes.Moreover, nanotechnology has provided means of understandingand achieving cell specific functions. The various techniquesthat can impart nanoscale topographic features to titaniumendosseous implants are described. Existing data supportingthe role of nanotopography suggests that critical steps in osseointegrationcan be modulated by nanoscale modification ofthe implant surface. Important distinctions between nanoscaleand micron-scale modification of the implant surface are presentlyconsidered. The advantages and disadvantages of nanoscalemodification of the dental implant surface are discussed.Finally, available data concerning the current dental implantsurfaces that utilize nanotopography in clinical dentistry aredescribed. Nanoscale modification of titanium endosseous implantsurfaces can alter cellular and tissue responses that maybenefit osseointegration and dental implant therapy. In a seriesof in vitro and in vivo experiments it was possible to evaluatethe effect of this modifications in different study designs. Theadvantages of the use of nanocues added to the surface of theosseointegrated dental implants allowed to a better and fasterosseointegration response of these materials, by acting on thedifferentiation of the osteoblasts.

The combination of micron and nanotopography by H(2)SO(4)/H(2)O(2) 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.

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.

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.