Effect of biofunctionalized implant surface on osseointegration: a histomorphometric study in dogs

Among the different properties that influence bone apposition around implants, the chemical or biochemical composition of implant surface may interfere on its acceptance by the surrounding bone. The aim of this study was to investigate if a biofunctionalization of implant surface influences the bone apposition in a dog model and to compare it with other surfaces, such as a microstructured created by the grit-blasting/acid-etching process. Eight young adult male mongrel dogs had the bilateral mandibular premolars extracted and each one received 6 implants after 12 weeks, totaling 48 implants in the experiment. Four groups of implants were formed with the same microrough topography with or without some kind of biofunctionalization treatment. After histomorphometric analysis, it was observed that the modified microstructured surface with a "low concentration of the bioactive peptide" provided a higher adjacent bone density (54.6%) when compared to the other groups (microstructured + HA coating = 46.0%, microstructured only = 45.3% and microstructured + "high concentration of the bioactive peptide" = 40.7%), but this difference was not statistically significant. In conclusion, biofunctionalization of the implant surface might interfere in the bone apposition around implants, especially in terms of bone density. Different concentrations of bioactive peptide lead to different results.Entre as diferentes propriedades de uma superfície capazes de influenciar a deposição óssea ao redor de implantes, a composição química e bioquímica pode atuar no reconhecimento do tecido ósseo circundante. O presente trabalho investigou a influência da biofuncionalização de superfícies de implante na deposição óssea ao redor dos mesmos em um modelo animal, comparando-as com outras superfícies, como a microtexturizada obtida pelo processo de jateamento e ataque ácido. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos e após 12 semanas foram instalados 6 implantes em cada cão, constituindo uma amostra de 48 implantes. Dos 4 grupos experimentais de diferentes superfícies, todos continham a mesma microtopografia rugosa, porém possuindo ou não alguma biofuncionalização. A análise histomorfométrica revelou que a superfície microtexturizada modificada pela adição de baixa concentração peptídica obteve uma maior densidade óssea adjacente (54,6%) quando comparada aos outros grupos (microtexturizada + HA = 46%, somente microtexturizada = 45,3% e microtexturizada com adição de alta concentração peptídica = 40,7%), no entanto estas diferenças numéricas não foram estatisticamente significantes. Dentro deste contexto, conclui-se que a biofuncionalização da superfície de implantes pode interferir na aposição óssea, em particular na densidade óssea, e que diferentes concentrações peptídicas podem conduzir a diferentes resultados.FAPES

Biofunctionalization of REDV elastin-like recombinamers improves endothelialization on CoCr alloy surfaces for cardiovascular applications

To improve cardiovascular implant success, metal-based stents are designated to modulate endothelial cells adhesion and migration in order to prevent restenosis and late thrombosis diseases. Biomimetic coatings with extra-cellular matrix adhesive biomolecules onto stents surfaces are a strategy to recover a healthy endothelium. However, the appropriate bioactive sequences to selective promote growth of endothelium and the biomolecules surface immobilization strategy remains to be elucidated. In this study, biofunctionalization of cobalt chromium, CoCr, alloy surfaces with elastin-like recombinamers, ELR, genetically modified with an REDV sequence, was performed to enhance metal surfaces endothelialization. Moreover, physical adsorption and covalent bonding were used as biomolecules binding strategies onto CoCr alloy. Surfaces were activated with plasma and etched with sodium hydroxide previous to silanization with 3-chloropropyltriethoxysilane and functionalized with the ELR. CoCr alloy surfaces were successfully biofunctionalized and the use of an ELR with an REDV sequence, allows conferring bioactivity to the biomaterials surface, demonstrating a higher cell adhesion and spreading of HUVEC cells on the different CoCr surfaces. This effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique, covalent bonding, and the increase of surface charge electronegativity. Our strategy of REDV elastin-like recombinamers immobilization onto CoCr alloy surfaces via covalent bonding through organosilanes provides a bioactive surface that promotes endothelial cell adhesion and spreading. (C) 2015 Elsevier B.V. All rights reserved.Peer ReviewedPostprint (author’s final draft

All-in-one trifunctional strategy: A cell adhesive, bacteriostatic and bactericidal coating for titanium implants

Strategies to inhibit initial bacterial adhesion are extremely important to prevent infection on biomaterial surfaces. However, the simultaneous attraction of desired eukaryotic cells remains a challenge for successful biomaterial-host tissue integration. Here we describe a method for the development of a trifunctional coating that repels contaminating bacteria, kills those that adhere, and promotes osteoblast adhesion. To this end, titanium surfaces were functionalized by electrodeposition of an antifouling polyethylene glycol (PEG) layer and subsequent binding of a peptidic platform with cell-adhesive and bactericidal properties. The physicochemical characterization of the samples via SEM, contact angle, FTIR and XPS analysis verified the successful binding of the PEG layer and the biomolecules, without altering the morphology and topography of the samples. PEG coatings inhibited protein adsorption and osteoblast-like (SaOS-2) attachment; however, the presence of cell adhesive domains rescued osteoblast adhesion, yielding higher values of cell attachment and spreading compared to controls (p < 0.05). Finally, the antibacterial potential of the coating was measured by live/dead assays and SEM using S. sanguinis as a model of early colonizer in oral biofilms. The presence of PEG layers significantly reduced bacterial attachment on the surfaces (p < 0.05). This antibacterial potential was further increased by the bactericidal peptide, yielding values of bacterial adhesion below 0.2% (p < 0.05). The balance between the risk of infection and the optimal osteointegration of a biomaterial is often described as “the race for the surface”, in which contaminating bacteria and host tissue cells compete to colonize the implant. In the present work, we have developed a multifunctional coating for a titanium surface that promotes the attachment and spreading of osteoblasts, while very efficiently inhibits bacterial colonization, thus holding promise for application in bone replacing applications.Peer ReviewedPostprint (author's final draft

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO3 or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO3-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.Peer Reviewe

Biomimetic implant surface functionalization with liquid L-PRF products: in vitro study

Abstract Objective: Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. Methods: Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). Results: Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. Conclusions: Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products

Surface guidance of stem cell behavior: Chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitro and bone formation in vivo

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor a5ß1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs.Peer ReviewedPostprint (author's final draft

Cell adhesive peptides functionalized on CoCr alloy stimulate endothelialization and prevent thrombogenesis and restenosis

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion.Postprint (author's final draft

Surface immobilization and bioactivity of TGF-ß1 inhibitor peptides for bone implant applications

TGF-ß1 is the most related cytokine with the production of fibrotic tissue. It plays an important role on the production of collagen by fibroblasts and other types of cells. The inhibition of this cytokine reduces fibrosis in various types of tissue. Biofunctionalization of dental and orthopedic implants with biomolecules enables modification of the physical, chemical and biochemical properties of their surfaces to improve its biological and clinical performance. Our objective was to develop a reliable method to immobilize oligopeptides on Ti surfaces to obtain a surface with TGF-ß1 inhibitory activity that will potentially minimize fibrotic encapsulation of implants during the process of osseointegration. We covalently immobilized TGF-ß1 inhibitor P17-peptides on Ti surfaces and assessed by characterizing each step of the process that we successfully biofunctionalized the implant surfaces. High amounts of peptides were anchored and homogeneously distributed on the surfaces with mechanical and thermochemical stability after in vitro simulated challenges. Notably, the immobilized peptides retained their TGF-ß1 inhibitory activity in vitro. Thus, these biofunctional coatings are potential candidates for inducing a fast and reliable osseointegration in vivo.Preprin

Unión de péptidos a superficies de titanio para la mejora de la osteointegración

El presente estudio pretende mejorar el proceso de osteointegración del implante de titanio mediante la biofuncionalización de su superficie. Para ello se ha realizado una previa silanización de la superficie y una posterior unión covalente entre el silano y un péptido el cual posee la secuencia RGD. Se han estudiado diferentes aspectos como es la estabilidad tanto de conjunto titanio-silano como silano-péptido. Finalmente se ha realizado un ensayo de adhesión celular utilizando células osteoblásticas MG63 cuyas muestras de titanio han sido previamente biofuncionalizadas con el proceso estudiado.This study aims to improve the osseointegration process of the titanium implant through surface biofunctionalization. Samples were silanised on the surface and afterwards a peptide with the RGD sequence was covalently attached to the silane. Specific characteristics of the stability of the union titanium-silane as well as silane-peptide were studied. Finally cell adhesion studies to samples biofunctionalized with the technique studied in this work were made with MG63 osteoblastic cells.Peer Reviewe

Universal scattering behavior of co-assembled nanoparticle-polymer clusters

Water-soluble clusters made from 7 nm inorganic nanoparticles have been investigated by small-angle neutron scattering. The internal structure factor of the clusters was derived and exhibited a universal behavior as evidenced by a correlation hole at intermediate wave-vectors. Reverse Monte-Carlo calculations were performed to adjust the data and provided an accurate description of the clusters in terms of interparticle distance and volume fraction. Additional parameters influencing the microstructure were also investigated, including the nature and thickness of the nanoparticle adlayer.Comment: 5 pages, 4 figures, paper published in Physical Review