Key Engineering Materials Vols. 284-286

Abstract: Our experiments of mouse osteoblast-like MC3T3-E1 cells cultured on a glass substrate showed that as surface roughness of a substrate increased, cell proliferation, cell differentiation and subsequent mineralization were reduced.

Abstract: Fluoridated hydroxyapatite (FHA) discs with various fluorine contents have been used to study the effect of fluorine content on early-stage cell behavior. FHA powders with fluorine content in the range 0-0.577 (mol F /mol apatite) were pressed into discs and sintered at 1200°C for 1 hour. SAOs-2 rat osteosarcoma cells were cultured on each FHA disc and tissue culture polystyrene (control) with the same seeding density for 4 hours. The cell count was conducted using Alamarblue, and the morphology of cell attachment was observed using environmental scanning electron microscopy. It was apparent that the fluorine content in FHA had significant impact on the early cell behavior.

Abstract: The aim of this study was to test the in vitro cytotoxicity of wood-based biomorphic Silicon Carbide (SiC) ceramics, using MG-63 human osteoblast-like cells. This innovative material has been recently developed and it exhibits unique mechanical properties towards their application in biomedical technology. In the solvent extraction test the SiC ceramic extracts had almost no effect on cellular activity even at 100% concentration. A similar behaviour was found for Ti6Al4V and bioactive glass, used as reference materials. The results of the cell morphology and the cellular attachment response have also demonstrated that the in vitro performance of these biomorphic SiC ceramics is qualitatively comparable to that produced by titanium alloy and bioactive glass, which seems very promising.

Abstract: Cytotoxicity test was essential for the pre-clinical evaluation of bioceramics. Proliferation assays such as MTT, XTT and WST-1 were commonly used for measuring biocompatibility. WST-1 was more convenient than MTT because of its water-solubility and storage condition. The calcium phosphate glass and β-TCP have been used for bone substitute, and some magnetic ferrites have been used for hyperthermic treatment. L929, mouse fibroblast cell, was the representative cell-line for in vitro biocompatibility test. The extracts of test samples were prepared by ISO10993-12:2002. The biocompatibilities of the extracts were measured by MTT and WST-1 assay and their pH were measured with pH meter. The cellular survival rate of CPG was the lowest and the results of the WST-1 test showed results similar to those of the MTT test. Thus, proliferation assays using WST-1 may be conveniently and routinely applicable to pre-clinical evaluation of bioceramics.

Abstract: Hybrid bioactive glass-polyvinyl alcohol foams for use as scaffolds in tissue engineering were developed through the sol-gel route. Hybrids produced by this route present a high acidic character due to the catalysts added during processing and may also contain residual organics after the drying step. Therefore, an additional cleaning step is necessary to produce biocompatible materials. In this study hybrid PVA/bioactive glass foams were cleaned using various procedures and cytotoxicity evaluation was conducted. All the cleaning methods used increased the cell viability levels compared to samples not subjected to a cleaning procedure. The most effective cleaning procedure used was the immersion in NH4OH solution. The cleaning procedure changed the composition and pore structure of the final material.

Abstract: Sol-Gel coatings are a good choice for protection and bioactivation of metals used as dentistry and standard surgical implant materials. These films should both prevent degradation of the substrates by wear or corrosion, and bioactivate the material for inducing the formation of a hydroxyapatite (HA) rich layer onto the material surface, thereby permitting a natural bonding to living tissues. The aim of this work was to estimate the clastogenicity in vitro by Single Cell Gel lectrophoresis Assay (SCGE) or “comet” assay of coatings of TiN applied by magnetron sputtering and of hybrid layers obtained by Sol-Gel containing glass, glass-ceramic and HA particles on stainless steel AISI 304. Six test specimens were prepared: AISI 304 Stainless Steel coated with an hybrid silica single film (SF), applied by sol-gel process, AISI 304 SS coated with double film with bioactive glass (DFG), glass-ceramics (DFGC) and HA (DFHA) particles, AISI 304 SS coated with TiN multi films (MFTiN) applied by PVD and bare AISI 304 SS (304SS). Significantly lower DNA migration (p>0.005) was observed in the cells of the cultures corresponding to the samples coated with SF, DFG, DFGC, DFHA and MFTiN respect to the bare 304 SS. The comparison between negative control and the same coated samples did not reveal any statistically significant difference (p>0.005) in clastogenicity in vitro evaluated by SCGE.

Abstract: Calcium phosphate-based glasses (PG) are of interest as both scaffold and delivery materials for tissue rebuilding because of their chemical similarity to bone. Since it is essential that these materials exhibit local and systemic biocompatibility and do not adversely affect host tissues, the present study was undertaken to examine the effects of PG containing different amounts of Ca on human T lymphocytes in vitro. This was carried out by measuring the effects of extracts of the PG on the direct and mitogen-induced activation of T cells from human peripheral blood, as well as assessing CD4 and CD8, surface antigens which define T-helper and T-suppressor cells, respectively. The results showed that DNA synthesis by resting T lymphocytes was unaffected by all the PG. However, extracts of the PG containing 24 mol% of Ca caused a very marked inhibition of mitogen-induced T cell activation. This PG also reduced both the resting CD4+ and CD8+ T cells, as well as activated CD8+ cells. In contrast, high Ca-PG significantly augmented DNA synthesis by mitogen-activated T cells. These experiments show that PG containing differing levels of Ca can have pronounced and differential effects on the activation and function of T lymphocytes in vitro.

Abstract: Alumina ceramics have excellent mechanical and biocompatible properties, but are bioinert and hence have no bone-bonding properties. We took a tissue engineering approach in an attempt to modify the ceramic surface and so provide an osteogenic/osteoconductive milieu. We used fresh human bone marrow cells obtained from the iliac crest by needle aspiration for culture expansion of mesenchymal stem cells (MSC) followed by in vitro osteogenic differentiation on both tissue culture polystyrene (TCPS) and alumina ceramics. We have succeeded in expanding the number of MSC from all 35 cases and compared the differentiation capability of selected MSC on alumina ceramics to that on TCPS. The cells on both substrata showed extensive alkaline phosphatase staining and mineralization as evidenced by calcein uptake. Biochemical analyses revealed high levels of alkaline phosphatase activity, osteocalcin expression, and calcium content. These data indicate that an alumina ceramic surface can support a differentiation cascade of MSC resulting in osteoblastic phenotype expression of the cells. Based on these results, we have performed clinical applications of tissue engineered total joint replacements for osteoarthritic patients.

Abstract: Due to its intrinsically poor repair potential, injuries to articular cartilage do not heal and clinical intervention is required. Osteochondral grafts may improve healing while promoting integration with host tissue. We report here the development of an osteochondral graft based on a hybrid of a hyrogel and a polymer-bioactive glass composite (PLAGA-BG) microsphere scaffold. This novel osteochondral construct consists of three regions: gel-only, gel/composite interface, and a composite-only-region. The three phases differ in calcium phospate (Ca-P) or BG content. The objective of the current study is to investigate the effects of scaffold composition on chondrocyte response, and to evaluate the effects of co-culture on osteoblasts and chondrocyte growh and differentiation on the hybrid scaffold. The PLAGA-BG microsphere scaffold supported the growth of chondrocytes and initial results indicate that in the presence of BG, chondrocyte-mediated mineralization may be stimulated. Co-culture of osteoblasts and chondrocytes on the multi-phased scaffold with varied Ca-P content facilitated the formation of multiple matrix zones: a GAGrich chondrocyte region, an interfacial matrix rich in GAG+collagen, and a mineralized collagen matrix with osteoblasts. In summary, chondrocyte response has been optimized as a function of scaffold composition and the novel osteochondral graft has the potential to support the simultaneous formation of multiple types of tissue in vitro.

Abstract: Autograft, allograft, and biomaterials had been developed for bone regeneration. In recent year, a tissue engineering technique has been paid much attention for next generation implant. A problem of bone tissue engineering to be solved is a development of the substrate that is suitable for cell adhesion, proliferation, and differentiation. A biomimic scaffold for tissue culture was proposed, and then a cell response on the scaffold was estimated. The scaffold composed by a calcium deficient apatite with an adsorbed serum protein was formed on a ceramic hydroxyapatite (HAp) and surface-modified titanium by a soaking in cell-culture medium supplemented with fetal bovine serum. Excellent results on cell proliferation and cell adhesion were obtained only on osteoblast-like cells (MC3T3-E1). An actin filament in narrow filopodium of the spindle-shaped MC3T3-E1 cells on the ceramic HAp had a regular course. On the other hand, ends of the actin filament of the widely spread cells on the apatite layer with serum protein were scattering. It was suggested that the scattering of the actin end showed an existence of fibronectin, and then tight adhesion would be obtained by the many focal adhesion. Accordingly, the effectiveness of the biomimic scaffold containing serum protein on cell growth was confirmed.