Nanotechnology and Dental Implants

The long-term clinical success of dental implants is related to their early osseointegration. This paper reviews the different steps of the interactions between biological fluids, cells, tissues, and surfaces of implants. Immediately following implantation, implants are in contact with proteins and platelets from blood. The differentiation of mesenchymal stem cells will then condition the peri-implant tissue healing. Direct bone-to-implant contact is desired for a biomechanical anchoring of implants to bone rather than fibrous tissue encapsulation. Surfaces properties such as chemistry and roughness play a determinant role in these biological interactions. Physicochemical features in the nanometer range may ultimately control the adsorption of proteins as well as the adhesion and differentiation of cells. Nanotechnologies are increasingly used for surface modifications of dental implants. Another approach to enhance osseointegration is the application of thin calcium phosphate (CaP) coatings. Bioactive CaP nanocrystals deposited on titanium implants are resorbable and stimulate bone apposition and healing. Future nanometer-controlled surfaces may ultimately direct the nature of peri-implant tissues and improve their clinical success rate

Cold plasma-treated ringer’s saline: a weapon to target osteosarcoma

Osteosarcoma (OS) is the main primary bone cancer, presenting poor prognosis and difficult treatment. An innovative therapy may be found in cold plasmas, which show anti-cancer effects related to the generation of reactive oxygen and nitrogen species in liquids. In vitro models are based on the effects of plasma-treated culture media on cell cultures. However, effects of plasma-activated saline solutions with clinical application have not yet been explored in OS. The aim of this study is to obtain mechanistic insights on the action of plasma-activated Ringer’s saline (PAR) for OS therapy in cell and organotypic cultures. To that aim, cold atmospheric plasma jets were used to obtain PAR, which produced cytotoxic e ects in human OS cells (SaOS-2, MG-63, and U2-OS), related to the increasing concentration of reactive oxygen and nitrogen species generated. Proof of selectivity was found in the sustained viability of hBM-MSCs with the same treatments. Organotypic cultures of murine OS confirmed the time-dependent cytotoxicity observed in 2D. Histological analysis showed a decrease in proliferating cells (lower Ki-67 expression). It is shown that the selectivity of PAR is highly dependent on the concentrations of reactive species, being the differential intracellular reactive oxygen species increase and DNA damage between OS cells and hBM-MSCs key mediators for cell apoptosis.Peer ReviewedPostprint (published version

Vertical bone regeneration with synthetic biomimetic calcium phosphate onto the calvaria of rats

Bone regeneration is often required to provide adequate oral rehabilitation before dental implants. Vertical ridge augmentation is the most challenging of all situations, and often requires the use of autologous bone grafting. However, autologous bone grafting induces morbidity, and the harvestable bone is limited in quantity. Alternatives to autologous bone grafting include bovine-bone-derived biomaterials, which provide good clinical results and synthetic bone substitutes that still fail to provide a reliable clinical outcome. Synthetic biomimetic calcium phosphate (SBCP) biomaterials, consisting of precipitated apatite crystals that resemble in composition and crystallinity to the mineral phase of bone, arise as alternatives to both bovine bone and the current sintered bone substitutes. This study aims to compare the vertical bone regeneration capacity of the SBCP (MimetikOss, Mimetis Biomaterials) with that of a deproteinized bovine bone matrix (DBBM, Bio-Oss®; Geistlich Biomaterials) on the calvaria of rats. To model vertical bone augmentation, hemispherical cups were filled with the two types of biomaterial granules and implanted onto the skull of rats, while empty cups were used as controls. After 4 and 8 weeks of healing, bone growth was determined by microcomputed tomography and histomorphometry. After 4 weeks of implantation, a significantly higher bone growth was found in the case of SBCP compared with DBBM and left empty controls. At 8 weeks, no statistically significant differences were found between the two bone substitutes. These results are promising since vertical bone regeneration was faster in the case of SBCP than for DBBM.Peer ReviewedPostprint (author's final draft

Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive β-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion

Computational model combined with in vitro experiments to analyse mechanotransduction during mesenchymal stem cell adhesion.

The shape that stem cells reach at the end of adhesion process influences their differentiation. Rearrangement of cytoskeleton and modification of intracellular tension may activate mechanotransduction pathways controlling cell commitment. In the present study, the mechanical signals involved in cell adhesion were computed in in vitro stem cells of different shapes using a single cell model, the so-called Cytoskeleton Divided Medium (CDM) model. In the CDM model, the filamentous cytoskeleton and nucleoskeleton networks were represented as a mechanical system of multiple tensile and compressive interactions between the nodes of a divided medium. The results showed that intracellular tonus, focal adhesion forces as well as nuclear deformation increased with cell spreading. The cell model was also implemented to simulate the adhesion process of a cell that spreads on protein-coated substrate by emitting filopodia and creating new distant focal adhesion points. As a result, the cell model predicted cytoskeleton reorganisation and reinforcement during cell spreading. The present model quantitatively computed the evolution of certain elements of mechanotransduction and may be a powerful tool for understanding cell mechanobiology and designing biomaterials with specific surface properties to control cell adhesion and differentiation

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (ß-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic ß-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of ß-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to ß-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered ß-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to ß-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.Peer ReviewedPostprint (author's final draft

Physico-chemical and thermochemical studies of the hydrolytic conversion of amorphous tricalcium phosphate into apatite

The conversion of amorphous tricalcium phosphate with different hydration ratio into apatite in water at 25 °C has been studied by microcalorimetry and several physical–chemical methods. The hydrolytic transformation was dominated by two strong exothermic events. A fast, relatively weak, wetting process and a very slow but strong heat release assigned to a slow internal rehydration and the crystallization of the amorphous phase into an apatite. The exothermic phenomenon related to the rehydration exceeded the crystalline transformation enthalpy. Rehydration occurred before the conversion of the amorphous phase into apatite and determined the advancement of the hydrolytic reaction. The apatitic phases formed evolved slightly with time after their formation. The crystallinity increased whereas the amount of HPO4 2− ion decreased. These data allow a better understanding of the behavior of biomaterials involving amorphous phases such as hydroxyapatite plasma-sprayed coating

Split

The nucleation and growth of a calcium phosphate (Ca-P) coating deposited on titanium implants from simulated body fluid was investigated by using atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Forty titanium alloy plates were assigned into two groups. One group with a smooth surface having a maximum roughness Rmax<0.10 μm (s-Ti6Al4V) and a group with a rough surface with an Rmax<0.25 μm (r-Ti6Al4V) were used. Titanium samples were immersed in SBF concentrated by five (SBF×5) from 10 min to 5 h and examined by AFM and ESEM. Scattered Ca-P deposits of approximately 15 nm in diameter appeared after only 10 min of immersion in SBF×5. These Ca-P deposits grew up to 60–100 nm after 4 h on both s- and r-Ti6Al4V substrates. With increasing immersion time, the packing of Ca-P deposits with size of tens of nanometers in diameter formed larger globules and then a continuous Ca-P film on titanium substrates. A direct contact between the Ca-P coating and the Ti6Al4V surface was observed. The Ca-P coating was composed of nanosized deposits and of an interfacial glassy matrix. This interfacial glassy matrix might ensure the adhesion between the Ca-P coating and the Ti6Al4V substrate. In the case of s-Ti6Al4V substrate, failures within this interfacial glassy matrix were observed overtime. Part of the glassy matrix remained on s-Ti6Al4V while part detached with the Ca-P film. The Ca-P coating detached from the smooth substrate, whereas the Ca-P film extended onto the whole rough titanium surface over time. In the case of r-Ti6Al4V, the Ca-P coating covered evenly the substrate after immersion in SBF×5 for 5 h. The present study suggested that the heterogeneous nucleation of Ca-P on titanium was immediate and did not depend on the Ti6Al4V surface topography. The further growth and mechanical attachment of the final Ca-P coating strongly depended on the surface, for which a rough topography was beneficial