In vitro calcification of chemically functionalized carbon nanotubes

Bone is composed of two phases. The organic phase is made of collagen fibrils assembled in broad fibers acting as a template for mineralization. The mineral phase comprises hydroxyapatite (HAP) crystals grown between and inside the collagen fibers. We have developed a biomimetic material using functionalized carbon nanotubes as scaffold to initiate in vitro mineralization. Biomimetic formation of HAP was performed on single-walled carbon nanotubes (SWCNTs) which have been grafted with carboxylic groups. Two types of nanotubes, HiPco(R) and Carbon Solutions(R), were oxidized via various acidic processes, leading to five different groups of carboxylated nanotubes, fully characterized by physical methods (thermogravimetric analysis, attenuated total reflectance infrared spectroscopy and X-ray photoelectron spectroscopy). All samples were dispersed in ultra-pure water and incubated for 2weeks in a synthetic body fluid, in order to induce the calcification of the SWCNTs. Scanning electron microscopy (SEM) and energy-dispersive X-ray analysis studies showed that Ca(2+) and PO(4)(3-) ions were deposited as round-shaped nodules (calcospherites) on the carboxylated SWCNTs. Fourier transform infrared and Raman spectroscopic studies confirmed the HAP formation, and image analysis made on SEM pictures showed that calcospherites and carboxylated SWCNTs were packed together. The size of calcospherites thus obtained in vitro from the HiPco(R) series was close to that issued from calcospherites observed in vivo. Functionalization of SWCNTs with carboxylic groups confers the capacity to induce calcification similar to woven bone

Carboxylated and esterified SWCNTs : characterization and contribution as components of biomaterials and solar cells

Date du colloque&nbsp;: 04/2010</p

In vitro assessment of osteoblast and macrophage mobility in presence of β-TCP particles by videomicroscopy

β-TCP is widely used to repair bone defects due to its good biocompatibility, macroporosity (favoring bone ingrowth) and bioresorbability. However, cell interactions with the biomaterial at the first times of implantation remain largely unknown. We have observed cell behaviors in direct contact with β-TCP particles using long-term culture under videomicroscopy. Osteoblastlike cells (SaOs-2) and macrophages (J774.2 and mouse peritoneal macrophages) were cultured in the presence of β-TCP particles. For each experiment, images from 20 independent fields were acquired and stored every 15 min during 8 days. At the end of the culture, they were combined to generate time lapse videos; coverslips were fixed and observed by scanning electron microscopy (SEM). SaOs-2 proliferation was determined by counting cells on six different and independent fields at days 1, 3, and 6. Videos showed the capacity of cells to displace the particles. Dynamic follow-up showed active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 and peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long-term culture under videomicroscopy allowed a better understanding of the colonization process of β-TCP particles by osteoblastlike cells and macrophages. Data obtained from long-term videomicroscopy are in agreement with in vivo observations confirming the interest of β-TCP to promote osteogenesis

Bone tissue formation with human mesenchymal stem cells and biphasic calcium phosphate ceramics: The local implication of osteoclasts and macrophages

Human mesenchymal stem cells (hMSC) have immunomodulative properties and, associated with calcium phosphate (CaP) ceramics, induce bone tissue repair. However, the mechanisms of osteoinduction by hMSC with CaP are not clearly established, in particular the role of osteoclasts and macrophages. Biphasic calcium phosphate (BCP) particles were implanted with or without hMSC in the paratibial muscles of nude mice. hMSC increased osteoblastic gene expression at 1 week, the presence of macrophages at 2 and 4 weeks, osteoclastogenesis at 4 and 8 weeks, and osteogenesis at 4 and 8 weeks. hMSC disappeared from the implantation site after 2 weeks, indicating that hMSC were inducers rather than effectors of bone formation. Induced blockage of osteoclastogenesis by anti-Rankl treatment significantly impaired bone formation, revealing the pivotal role of osteoclasts in bone formation. In summary, hMSC positively influence the body foreign reaction by attracting circulating haematopoietic stem cells and inducing their differentiation into macrophages M1 and osteoclasts, thus favouring bone formation

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

EPIdemiology of Surgery-Associated Acute Kidney Injury (EPIS-AKI) : Study protocol for a multicentre, observational trial

More than 300 million surgical procedures are performed each year. Acute kidney injury (AKI) is a common complication after major surgery and is associated with adverse short-term and long-term outcomes. However, there is a large variation in the incidence of reported AKI rates. The establishment of an accurate epidemiology of surgery-associated AKI is important for healthcare policy, quality initiatives, clinical trials, as well as for improving guidelines. The objective of the Epidemiology of Surgery-associated Acute Kidney Injury (EPIS-AKI) trial is to prospectively evaluate the epidemiology of AKI after major surgery using the latest Kidney Disease: Improving Global Outcomes (KDIGO) consensus definition of AKI. EPIS-AKI is an international prospective, observational, multicentre cohort study including 10 000 patients undergoing major surgery who are subsequently admitted to the ICU or a similar high dependency unit. The primary endpoint is the incidence of AKI within 72 hours after surgery according to the KDIGO criteria. Secondary endpoints include use of renal replacement therapy (RRT), mortality during ICU and hospital stay, length of ICU and hospital stay and major adverse kidney events (combined endpoint consisting of persistent renal dysfunction, RRT and mortality) at day 90. Further, we will evaluate preoperative and intraoperative risk factors affecting the incidence of postoperative AKI. In an add-on analysis, we will assess urinary biomarkers for early detection of AKI. EPIS-AKI has been approved by the leading Ethics Committee of the Medical Council North Rhine-Westphalia, of the Westphalian Wilhelms-University Münster and the corresponding Ethics Committee at each participating site. Results will be disseminated widely and published in peer-reviewed journals, presented at conferences and used to design further AKI-related trials. Trial registration number NCT04165369

Biomimétisme, Cytocompatibilité et Cytodynamique

The influence of RDG coating on Ti was tested on osteoblasts behaviors. This coating allowed an earlier mineralization and an increase of surface mineralized. Functionalized CNTs have been used to promote calcification to mimic the in vivo mineralization of collagen fibers. After 14 days of incubation, functionalized CNTs promoted the calcospherites formation, which were similar to those in woven bone. This in vitro calcification model developed in our laboratory was used to study the Sr2+ incorporation in mineral and its released. Sr2+ was incorporated in mineral during calcification without inducing any change in crystal parameters or crystallinity. Sr2+ released was rapid at the begging and then slower to reach 30% of Sr2+ released after 61 days. In a last study, cell behaviors in direct contact with β-TCP particles using long term culture under videomicroscopy were observed. Video sequences realized after 8 days of culture showed an active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 did not proliferate but came in direct contact with the particles. Peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long term culture under videomicroscopy allowed a better understanding of the colonization process of β-TCP particles by osteoblast-like cells and macrophages. Data obtained from long term videomicroscopy are in agreement with in vivo observations confirming the interest of β-TCP to promote osteogenesis.L'influence d'un revêtement de type RGD sur du Ticp sur l'adhésion, l'étalement et la minéralisation des ostéoblastes a été étudié. Ce revêtement accélérait la vitesse de minéralisation et augmentait la surface des zones de minéralisation. Des CNTs fonctionnalisés ont été utilisés pour induire la calcification dans le but de mimer in vitro la minéralisation des fibres de collagène dans le tissu osseux. Après 14 jours d'incubation dans un liquide biosynthétique, les CNTs fonctionnalisés ont induit la formation de calcosphérites, similaires à ceux formés dans le woven bone. Ce modèle de calcification in vitro biomimétique, mis au point sur des polymères dans notre laboratoire, a permis d'étudier l'incorporation du Sr2+ dans le minéral osseux et sa désorption. Sr2+ a été incorporé dans le minéral lors de la calcification sans induire de changement des paramètres du cristal ou de la cristallinité quelque soit la concentration initiale en Sr2+. La désorption du Sr2+ a été rapide initialement avec libération de 20 à 25 % en 16 jours, puis plus lente pour atteindre 30 % après 61 jours. Enfin, les observations en cytodynamique ont permis de voir les premières étapes d'adhésion cellulaire et de colonisation du β-TCP. Les séquences vidéo réalisées sur 8 jours de culture, ont permis d'observer la prolifération des ostéoblastes (SaOs-2) qui sembla se produire en direction des particules de β-TCP pour venir en contact direct avec les particules. Les macrophages (J774-2 et macrophages péritonéaux de souris) ne proliféraient pas mais venaient au contact direct des particules pour les dégrader. Les observations MEB ont permis de constater que les cellules émettaient des prolongements cytoplasmiques et s'étiraient pour venir se fixer et ensuite s'étaler à la surface du biomatériau. Les données obtenues grâce aux observations faites avec la vidéomicroscopie sur long terme nous ont permis de confirmer la cytocompatibilité et la biodégradabilité du β-TCP

Prévention des risques lors de la grossesse durant l'exercice officinal

TOULOUSE3-BU Santé-Centrale (315552105) / SudocSudocFranceF

Biomimétisme, cytocompatibilité et cytodynamique

L'influence d'un revêtement de type RGD sur du Ticp sur l'adhésion, l'étalement et la minéralisation des ostéoblastes a été étudié. Ce revêtement accélérait la vitesse de minéralisation et augmentait la surface des zones de minéralisation. Des CNTs fonctionnalisés ont été utilisés pour induire la calcification dans le but de mimer in vitro la minéralisation des fibres de collagène dans le tissu osseux. Après 14 jours d'incubation dans un liquide biosynthétique, les CNTs fonctionnalisés ont induit la formation de calcosphérites, similaires à ceux formés dans le woven bone. Ce modèle de calcification in vitro biomimétique, mis au point sur des polymères dans notre laboratoire, a permis d'étudier l'incorporation du Sr2+ dans le minéral osseux et sa désorption. Sr2+ a été incorporé dans le minéral lors de la calcification sans induire de changement des paramètres du cristal ou de la cristallinité quelque soit la concentration initiale en Sr2+. La désorption du Sr2+ a été rapide initialement avec libération de 20 à 25 % en 16 jours, puis plus lente pour atteindre 30 % après 61 jours. Enfin, les observations en cytodynamique ont permis de voir les premières étapes d'adhésion cellulaire et de colonisation du b-TCP. Les séquences vidéo réalisées sur 8 jours de culture, ont permis d'observer la prolifération des ostéoblastes (SaOs-2) qui sembla se produire en direction des particules de b-TCP pour venir en contact direct avec les particules. Les macrophages (J774-2 et macrophages péritonéaux de souris) ne proliféraient pas mais venaient au contact direct des particules pour les dégrader. Les observations MEB ont permis de constater que les cellules émettaient des prolongements cytoplasmiques et s'étiraient pour venir se fixer et ensuite s'étaler à la surface du biomatériau. Les données obtenues grâce aux observations faites avec la vidéomicroscopie sur long terme nous ont permis de confirmer la cytocompatibilité et la biodégradabilité du b-TCP.The influence of RDG coating on Ti was tested on osteoblasts behaviors. This coating allowed an earlier mineralization and an increase of surface mineralized. Functionalized CNTs have been used to promote calcification to mimic the in vivo mineralization of collagen fibers. After 14 days of incubation, functionalized CNTs promoted the calcospherites formation, which were similar to those in woven bone. This in vitro calcification model developed in our laboratory was used to study the Sr2+ incorporation in mineral and its released. Sr2+ was incorporated in mineral during calcification without inducing any change in crystal parameters or crystallinity. Sr2+ released was rapid at the begging and then slower to reach 30% of Sr2+ released after 61 days. In a last study, cell behaviors in direct contact with b-TCP particles using long term culture under videomicroscopy were observed. Video sequences realized after 8 days of culture showed an active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 did not proliferate but came in direct contact with the particles. Peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long term culture under videomicroscopy allowed a better understanding of the colonization process of b-TCP particles by osteoblast-like cells and macrophages. Data obtained from long term videomicroscopy are in agreement with in vivo observations confirming the interest of b-TCP to promote osteogenesis.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF