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

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

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

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

Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells: study of their biodistribution in the early time points after injection

INTRODUCTION: Osteonecrosis of the femoral head (ONFH) is a degenerative disease progressing to a femoral head (FH) collapse. Injection of osteoprogenitor cells like bone marrow mesenchymal stromal cells (BMSCs) into the FH appears to be a good therapeutic treatment. However, safety and efficacy of BMSCs to treat bone defect are the main preclinical data required for clinical application. Efficacy and the lack of risk of cell transformation after amplification of BMSCs have been extensively described. The main objectives of this study were to develop a simple and usable procedure for clinicians and control its feasibility by evaluating the biodistribution of BMSCs after injection into the FH in a large animal model. The impact of this approach was evaluated on one natural pig ONFH. METHODS: BMSCs were directly injected in the pig FH, and then the biodistribution of grafted cells was detected by quantitative real-time polymerase chain reaction, cytometry, or a combination of classic histology analysis and in situ hybridization (ISH). BMSC efficacy on bone regeneration was evaluated by magnetic resonance imaging (MRI) and histology. RESULTS: After 30-minute and 24-hour follow-up, grafted cells were detected at the injection site and no BMSCs were detected in filter organs or body fluids. The combination of classic histology analysis and ISH showed a good homogeneity of cell distribution in FH. Local delivery of BMSCs onto a bone scaffold associated with bone formation in vivo confirmed the preferential tropism of BMSCs to the bone tissue as well as their efficacy to form bone. Treatment of a natural pig ONFH by autologous BMSCs indicated a beginning of bone healing as early as 2 weeks with a complete healing after 9 weeks. At this stage, MRI and histological analysis were similar to those of a normal FH. CONCLUSIONS: Intra-osseous injection of BMSCs in FH seems to be a good strategy for ONFH treatment as the safety concerning the biodistribution of BMSCs is ensured. Moreover, the efficacy of BMSCs in natural ONFH seems to indicate that this is a promising approach. Altogether, these results constitute the preclinical data necessary for the setup of a clinical application with expanded BMSCs in the context of advanced therapy medicinal products

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

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

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

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