Key Engineering Materials Vol. 631

Abstract: Several tests for the biological evaluation of bioceramic materials and medical devices are provided in specific international standards, where in vitro tests have a major role. Tests involving exposure of cells in culture require the use of validated positive controls, which, in the same preparation and treatment conditions, present a substantial and well-known cytotoxicity. The present work aimed to test and validate 3 different sources of low cost, commercially available latex, as positive controls in cytotoxicity tests for bioceramic materials performed by indirect exposure. The tested origins for latex samples were: surgical gloves without powder, 100% pure amber latex hospital-grade tourniquets and 60 % latex White tubing. MC3T3-E1 murine pre-osteoblasts in culture were exposed to conditioned media (extracts) of each material tested, along with sintered stoichiometric hydroxyapatite bioceramics, and polystyrene beads as negative control. Cell viability was determined by XTT and Crystal Violet Exclusion tests. Concentration curves of the extracts were performed to obtain the DC₅₀. Only the 100% pure amber latex tubing was proven to be cytotoxic, with cell survival less than 5%. This material did not affected neighboring groups at the same experimental system. Moreover, latex samples showed great repeatability in different tests against latex and biomaterials, with consistent toxicity under 20% cell survival as shown in 3 different cell viability parameters. We conclude that fragments of latex ambar tubing are suited as effective positive controls in tests of medical bioceramic materials.

Abstract: @font-face { font-family: "ＭＳ 明朝"; }@font-face { font-family: "Cambria Math"; }@font-face { font-family: "@ＭＳ 明朝"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0mm 0mm 0.0001pt; font-size: 10pt; font-family: "Times New Roman"; }.MsoChpDefault { font-size: 10pt; }div.WordSection1 { page: WordSection1; } Bioresorbable materials may be advantageous for use in bone regeneration applications because they do not leave residues of foreign material, improving the long-term success of implant restoration. The purpose of this study was to investigate the osteoclastogenesis and bioresorption of synthesized calcium phosphate ceramic materials for orthopaedic and dental biomaterial applications. Differentiation into mature human osteoclasts on carbonated hydroxyapatite (CA) was significantly enhanced compared to hydroxyapatite (HA). Osteoclasts derived from human peripheral mononuclear blood cells adhered and differentiated into giant multinuclear TRAP- positive cells on every type of synthesized sample based on the histological analysis. Morphological observations using fluorescence and quantitative analysis revealed that the actin rings of osteoclasts on CA were thick and small in diameter, similar to the rings found on bone slices. Scanning electron microscopic images and quantitative analysis indicated that the resorption pits on CA were significantly deeper than those on HA due to the enhanced tight sealing ability between osteoclasts and their substrate.

Abstract: The influence of surface chemistry, roughness and hidrophilicity on the protein adhesion onto the biomaterial modulates the cell attachment, proliferation and differentiation. β-TCP is a synthetic bone substitute with bioactive, osteoconductive properties. Insulin is a polypeptide hormone that acts as a growth-stimulating factor for some cells types in culture. Few studies have been discussing this hormone role in bone remodeling and cells metabolism in vitro. The aim of this work was to evaluate the cell responses when insulin is adsorbed on β-TCP commercial discs surfaces. FTIR detected the characteristics bands of insulin adsorbed on sintered powder surface. The insulin adsorption on discs surface increase its wettability and favored growth and differentiation of the pre-osteoblastics cells. Although no addition of differentiation factors was present, it was observed deposits of extracellular matrix and collagen at 7 days in culture. The increase on affinity and cellular activity to the adsorbed insulin β-TCP substract was remarkable. It was quite clear that the insulin presents a role on activate and speed up the bone cells response while adhered to β-TCP substract. However, the insulin application on bone grafts for clinical purposes must be considered after detailed studies on animal models, which are required to fully evaluate the safe use of hormone containing grafts in clinical trials.

Abstract: Osteoporosis (OP) is a systemic metabolic disease identified with decrease of bone mineral density and deterioration of microstructure leading to fragility fractures in elderly. Boron (B) is assumed to stimulate osteoblasts. Hydroxyapatite (HAp) is clinically used to conduct bone regeneration and improves implant integration. Nano(n)-HAp expands the surface area for cell adhesion and may improve bone regeneration and tissue integration. The objective of this study was to examine the adhesion, proliferation and differentiation of B-n-HAp with mesenchymal stem cells (MSC’s). Human bone marrow derived MSC’s phenotype was assessed using scanning and transmission electron microscopy after combining with B-n-HAp and n-HAp. Cell adhesion and proliferation potential of these ceramics was examined with the real time cell analysis (xCELLigence, Roche Applied Science and ACEA Bioscience, USA) system and adipogenic-osteogenic differentiation was analyzed with morphological and quantitative methods. MSC’s adhesion and proliferation rates (cell index, 4.50) were higher than controls (cell index, 4.00). Adipogenic and osteogenic differentiation potential of MSC’s remained unchanged in the presence of B-n-HAp ceramics. In conclusion, B-n-HAp stimulates MSC’s adhesion, proliferation and differentiation and has a potential to regenerate bone tissue.

Abstract: Similar to other glucocorticoids, dexamethasone (DEX) induces osteoblast differentiation. At high concentrations, glucocorticoids may induce osteoporosis as a side effect. However, the exact mechanism of these two opposing effects has not been elucidated. To understand the mechanism of DEX-induced osteoblast differentiation, we developed a real-time osteoblast differentiation detection system using dual labeling of cells with fluorescent proteins. The promoter sequences of type I collagen and osteocalcin were ligated with mCherry and green fluorescent protein (GFP), respectively. Type I collagen is an early marker of osteoblast differentiation, and osteocalcin is a terminal differentiation marker. We investigated the effects of DEX on cell proliferation and differentiation using cells transformed with both constructs. Low DEX concentrations (<10 μM) induced calcification, as determined by alizarin-red staining, whereas calcification was inhibited at higher concentrations (>100 μM). Consistent with these results, mCherry-associated red fluorescence as an early marker was evident under both conditions, whereas green fluorescence associated with terminal differentiation was evident only at lower DEX concentrations. The level of green fluorescence diminished in a DEX-concentration-dependent manner. Thus, DEX does not inhibit the early stages of osteoblast differentiation but instead inhibits terminal differentiation.

Abstract: Silver halides represent a yet unexplored avenue for imparting antimicrobial activity in calcium phosphates. Silver halide colloids were added to calcium phosphate. Concurrent melting of silver halides and crystallization of carbonated apatite was achieved by heating to increase the silver halide surface area available to bacteria.Pseudomonas aeruginosa were more sensitive to silver iodide and silver bromide than Staphylococcus aureus. Silver iodide demonstrated greater activity than silver bromide. Silver chloride did not produce an antibacterial response. Both amorphous calcium phosphate and carbonated apatite displayed similar antibacterial activity when accompanied by silver halides. It is thought that amorphous calcium phosphate dissolves more readily and increases the bioavailability of the silver halide particles. Silver iodide displays a greater antibacterial response of all silver halides, with a response that is improved in a more resorbable matrix.

Abstract: We reported the apatite-forming ability of 30CaO∙70SiO₂ scaffolds with 0~100 ppm Ag ions by soaking in simulated body fluid (SBF). This study was to evaluate the effects of the concentrations of Ag ions in the 30CaO∙70SiO₂ gels on in-vitro biocompatibility of osteoblasts (MC3T3). After seeding cells on the surface of Ag-30CaO∙70SiO₂ gels scaffold, cellular behaviors were evaluated by an assay of cell counting kit-8. Cytotoxicity of the scaffold samples was evaluated by employing the extract solutions of the scaffold samples by the assays of neutral red, MTT and BrdU. In addition, live & dead assay was performed by using a gel covering method, which the scaffolds have been directly contacted with the incubated cells on the well plate. According to the results of CCK-8 assay, the optical density value of the absorbance of the resulting solution decreased as the concentration of Ag ions in the scaffolds increased. Moreover, their cell viability was measured to be less than 50% at the Ag concentrations of 50 ppm or more, and dead cells were observed in the experiment results of both the cytotoxicity and gel covering tests. From these experimental results, we concluded that the Ag-30CaO∙70SiO₂ scaffolds with less than 50 ppm Ag ion concentration were considered as biocompatible.

Abstract: Novel bioresorbable calcium-phosphate cement (CPC) with anti-washout property was developed by adding thermally cross-linked gelatin particles as pore generator into a CPC. The CPC was composed of α-tricalcium phosphate (α-TCP) and surface-modified hydroxyapatite (HAp) with inositol phosphate as a chelating agent (IP6-HAp). The bioresorbable CPC hybridized with gelatin particles was successfully fabricated by mixing the aqueous sodium chondroitin sulfate solution including Na₂HPO₄ and the pre-mixed powders composed of α-TCP (72 mass%), IP6-HAp (18 mass%), and the gelatin particles (10 mass%). The hybridized CPC paste showed initial setting time (IST) of 5 minutes and exhibited anti-washout property. Compressive strength after setting for 24 h reached to 4.2 MPa. An in vivo preliminary study using pig’s tibia model demonstrated that the hybridized CPC could be easily injected and set promptly without washout. In addition, no fragmentation in the specimens was observed after 8 weeks implantation. Moreover, a histological observation (Villanueva bone stain) revealed that almost 80% of the hybridized CPC specimens were resorbed and that immature bones were formed inside the specimens.

Abstract: Hydroxyapatite (HAp) is one of components of bone and teeth, and has an osteoconductivity. Thus, the HAp has been used as biomaterials for bone graftings. We have succeeded in development of the novel chelate-setting calcium-phosphate cement (CPC) using pure HAp particles surface-modified with inositol phosphate (IP6). While, biological apatite presented in bone and teeth of mammals contains various ions: Na⁺, K⁺, Mg²⁺, Cl^-, F^- and CO₃^2-, in addition to Ca²⁺ and PO₄^3- ions. In this work, in order to create the chelate-setting CPC with enhanced osteoconductivity, the above-mentioned biological apatite powder (hereafter, bone HAp), instead of pure HAp, was used as a starting powder for fabrication of the chelate-setting cement. The biocompatibility of the resulting chelate-setting bone HAp cement (hereafter, IP6-bone HAp cement) was examined using a rabbit’s tibia model. When the living reaction to hard tissue was histologically examined after 4 weeks implantation, we could observe that newly-formed bone directly bonded to the surface of the specimen. The newly-formed bone was also present around the cement specimen. The amounts of newly-formed bone around IP6-bone HAp cement was about 1.5 times those around IP6-pure HAp cement without bone minerals. The above findings demonstrate that the present IP6-bone HAp cements are one of the promising candidates as novel CPC with enhanced osteoconductivity.

Abstract: The testing of novel biomaterials for percutaneous vertebroplasty depends on suitable animal models. The aim of this study was to develop ex vivo a reproducible and feasible model of percutaneous vertebroplasty, for ulterior application in vivo. A large animal model was used (Merino sheep), due to its translational properties. Vertebroplasty was performed under tactile and fluoroscopic control, through a bilateral modified parapedicular access in lumbar vertebrae (n=12). Care was taken in order to avoid disruption of the vertebral foramen. The average defect volume was 1234±240 mm³. This mean volume ensures practical defects to test novel injectable biomaterials. 6 vertebrae were injected with a commercial cement (Cerament®, Bone Support, Sweden). Adequate defect filling was observed in all vertebrae. All vertebrae were assessed by microCT, prior to and post defect creation and after biomaterial injection. All vertebrae were mechanical tested. No mechanical failure was observed under loads higher than the physiological. Ultimately, this model is considered suitable for pre-clinical in vivo studies, mimicking clinical application.