Bioactivity in SBF versus trace element effects: The isolated role of Mg2+ and Zn2+ in osteoblast behavior.

The bioactivity assay originally proposed by Kokubo is one of the most commonly used tests to indirectly evaluate the biocompatibility of bioactive glasses. However, extensive evidence has shown that trace elements present in biomaterials may stimulate cellular behavior in different ways even when no apatite formation is observed, i.e., in biomaterials with low or no bioactivity. To further elucidate this topic, we designed three different SiO2-rich bioglass compositions in which CaO was partially replaced by ZnO and MgO, two oxides known to affect bioactivity as well as osteoblastic behavior. The physicochemical changes induced by the presence of oxides and their effects on biological behavior, as well as the adhesion, proliferation and differentiation of human osteoblast-like osteosarcoma cells (MG-63), were followed by a bioactivity assay in simulated body fluid (SBF). The insertion of ZnO or MgO decreased the glass transition (Tg) and crystallization (Tc) temperatures as a function of the increase in nonbonding oxygens, which was directly reflected in the higher solubility. The release of Mg2+ ions from the MgO-containing samples inhibited the bioactivity in SBF, inducing high cell adhesion and proliferation and moderate ALP activity. The release of Zn2+ also inhibited the bioactivity in SBF but, in contrast to the release of Mg2+, induced low cell adhesion and proliferation and high ALP activity compared to the control.

Advanced protocol to functionalize CaP bioceramic surface with peptide sequences and effect on murine pre-osteoblast cells proliferation.

To bring osteoinductive properties to calcium phosphate (CaP) bioceramics, a silicon-substituted hydroxyapatite was functionalized by integrin-adhesive cyclic-pentapeptides (c-(DfKRG)). A new two-step protocol was set up to immobilize peptides at low and controlled density on the ceramic surface and limit contamination by adsorbed molecules. To this aim, a spacer bearing c-(DfKRG)-S-PEG6-NHS molecule was synthesized and bonded to an organosilane previously covalently bonded to the ceramic surface. The functionalized ceramic was tested in vitro for MC3T3-E1 murine pre-osteoblasts. CaP ceramic surface retained good biological properties thanks to low density of bonded molecules preserving part of the bioactive CaP surface free of bioorganic molecules. The final SiHA-T-PEG6-S-c-(DfKRG) was shown to increase cell density and to improve proliferation. Furthermore, the use of a strong and stable covalent bond between inorganic and organic parts prevented early burst release of the peptide and increased the persistence of its bioactivity over time. So, this CaP ceramic associating c-(DfKRG) by covalent grafting could be considered as promising new biomaterials for bone tissue engineering.

Pre-osteoblast cell colonization of porous silicon substituted hydroxyapatite bioceramics: Influence of microporosity and macropore design.

Silicate-substituted hydroxyapatite scaffolds containing multiscale porosity are manufactured. Model parts containing macropores of five cross-sectional geometries (circle, square, rhombus, star and triangle) and two sizes are shaped by microstereolithography. Three open microporosity contents (0.5, 23 or 37 vol%) are introduced in the ceramic. MC3T3-E1 pre-osteoblasts are seeded onto these scaffolds. Analysis of cell colonization inside the macropores after 7 and 14 days of cultivation shows that the cellular filling is proportional to the macropore size and strongly influenced by macropore shape. Straight edges and convex surfaces are detrimental. High aspect ratios, the absence of reentrant angles and the presence of acute angles, by creating concavities and minimizing flat surfaces, facilitate cell colonization. Rhombus and triangle cross-sections are thus particularly favorable, while square and star geometries are the least favored. An increase in the microporosity content strongly impairs cell growth in the macropores. The data are statistically analyzed using a principal components analysis that shows that macro- and microtopographical parameters of scaffolds must be collectively considered with correlated interactions to understand cell behavior. The results indicate the important cell sensing of topography during the initial step of cell adhesion and proliferation and evidence the need for an optimized scaffold design.

Functionalization of phosphocalcic bioceramics for bone repair applications.

This work is devoted to the processing of bone morphogenetic protein (BMP-2) functionalized silicate substituted hydroxyapatite (SiHA) ceramic spheres. The motivation behind it is to develop injectable hydrogel/bioceramic composites for bone reconstruction applications. SiHA microspheres were shaped by spray drying and thoroughly characterized. The silicate substitution was used to provide preferred chemical sites at the ceramic surface for the covalent immobilization of BMP-2. In order to control the density and the release of the immobilized BMP-2, its grafting was performed via ethoxysilanes and polyethylene glycols. A method based on Kaiser's test was used to quantify the free amino groups of grafted organosilanes available at the ceramic surface for BMP-2 immobilization. The SiHA surface modification was investigated by means of X-ray photoelectron spectroscopy, Fourier transformed infrared spectroscopy and thermogravimetry coupled with mass spectrometry. The BMP-2 bioactivity was assessed, in vitro, by measuring the luciferase expression of a stably transfected C3H10 cell line (C3H10-BRE/Luc cells). The results provided evidence that the BMP-2 grafted onto SiHA spheres remained bioactive.

Design of calcium phosphate ceramics for drug delivery applications in bone diseases: A review of the parameters affecting the loading and release of the therapeutic substance.

Effective treatment of critical-size defects is a key challenge in restorative surgery of bone. The strategy covers the implantation of biocompatible, osteoconductive, bioactive and biodegradable devices which (1) well interact with native tissue, mimic multi-dimensional and hierarchical structure of bone and (2) are able to enhance bone repair, treating post implantation pathologies or bone diseases by local delivery of therapeutic agents. Among different options, calcium phosphate biomaterials are found to be attractive choices, due to their excellent biocompatibility, customisable bioactivity and biodegradability. Several approaches have been established to enhance this material ability to be loaded with a therapeutic agent, in order to obtain an in situ controlled release that meets the clinical needs. This article reviews the most important factors influencing on both drug loading and release capacity of porous calcium phosphate bone substitutes. Characteristics of the carrier, drug/carrier interactions, experimental conditions of drug loading and evaluation of drug delivery are considered successively.

Hydroxyapatite microporous bioceramics as vancomycin reservoir: Antibacterial efficiency and biocompatibility investigation.

AbstarctInfections after bone reconstructive surgery are a real therapeutic and economic issue for the modern health care system. As the pathogen (most often Staphylococcus aureus) is able to develop a biofilm inside the bone, local delivery of antibiotics is of interest since high drug concentrations would be delivered directly at the target place. In this context, this study evaluated a porous hydroxyapatite implant as biocompatible bone substitute and vancomycin-delivery system to prevent post-operative infections. A simple method of impregnation with optimised conditions insured a high antibiotic loading (up to 2.3 ± 0.3 mg/m2), with a complete in vitro release obtained within 1-5 days. Additionally, the bacteriostatic and bactericidal effects of vancomycin were retained after loading on hydroxyapatite, as demonstrated after challenge with a Staphylococcus aureus strain. Regarding the biocompatibility, a wound healing assay of pre-osteoblastic MC3T3-E1 cells exposed to various concentrations of vancomycin revealed a dose-dependent reduction in cell migration for antibiotic concentrations higher than 1 mg/mL. Meanwhile, cells were able to proliferate normally on vancomycin-loaded scaffolds, although cell initial adhesion was seriously impaired for scaffolds loaded with 2.3 mg/m2 Loaded scaffolds could be stored up to three months at room temperature without any degradation of the antibiotic. Together, these results demonstrate the efficacy of these hydroxyapatite bone substitutes for local delivery of vancomycin in the context of bone infection.

Quantitative analysis of vascular colonisation and angio-conduction in porous silicon-substituted hydroxyapatite with various pore shapes in a chick chorioallantoic membrane (CAM) model.

UNLABELLED: The development of scaffolds for bone filling of large defects requires an understanding of angiogenesis and vascular guidance, which are crucial processes for bone formation and healing. There are few investigations on the ability of a scaffold to support blood vessel guidance and it this is of great importance because it relates to the quality and dispersion of the blood vessel network. This work reports an analysis of vascularisation of porous silicon-substituted hydroxyapatite (SiHA) bioceramics and the effects of pore shape on vascular guidance using an expedient ex ovo model, the chick embryo chorioallantoic membrane (CAM) assay. Image analysis of vascularised implants assessed the vascular density, fractal dimension and diameter of blood vessels at two different scales (the whole ceramic and pores alone) and was performed on model SiHA ceramics harbouring pores of various cross-sectional geometries (circles, square, rhombus, triangles and stars). SiHA is a biocompatible material which allows the conduction of blood vessels on its surface. The presence of pores did not influence angiogenesis related-parameters (arborisation, fractal dimension) but pore geometry affected the blood vessel guidance and angio-conductive potential (diameter and number of the blood vessels converging toward the pores). The measured angles of pore cross-section modulated the number and diameter of blood vessels converging to pores, with triangular pores appearing of particular interest. This result will be used for shaping ceramic scaffolds with specific porous architecture to promote vascular colonisation and osteointegration. STATEMENT OF SIGNIFICANCE: An expedient and efficient method, using chick embryo chorioallantoic membrane (CAM) assays, has been set up to characterise quantitatively the angiogenesis and the vascular conduction in scaffolds. This approach complements the usual cell culture assays and could replace to a certain extent in vivo experiments. It was applied to silicon-substituted hydroxyapatite porous bioceramics with various pore shapes. The material was found to be biocompatible, allowing the conduction of blood vessels on its surface. The presence of pores does not influence the angiogenesis but the pore shape affects the blood vessel guidance and angio-conductive potential. Pores with triangular cross-section appear particularly attractive for the further design of scaffolds in order to promote their vascular colonisation and osteointegration and improve their performances.

ß-TCP porous pellets as an orthopaedic drug delivery system: ibuprofen/carrier physicochemical interactions.

Calcium phosphate bone substitute materials can be loaded with active substances for in situ, targeted drug administration. In this study, porous ß-TCP pellets were investigated as an anti-inflammatory drug carrier. Porous ß-TCP pellets were impregnated with an ethanolic solution of ibuprofen. The effects of contact time and concentration of ibuprofen solution on drug adsorption were studied. The ibuprofen adsorption equilibrium time was found to be one hour. The adsorption isotherms fitted to the Freundlich model, suggesting that the interaction between ibuprofen and ß-TCP is weak. The physicochemical characterizations of loaded pellets confirmed that the reversible physisorption of ibuprofen on ß-TCP pellets is due to Van der Waals forces, and this property was associated with the 100% ibuprofen release.