Cytocompatibility of two coating materials, amorphous alumina and silicon carbide, using human differentiated cell cultures.

The cytocompatibility of two coating materials, amorphous alumina and silicon carbide deposited by radio-frequency sputtering, was studied using alveolar bone osteoblasts and gingival fibroblasts from human healthy tissues. Cytocompatibility was assessed at the level of both the basic (attachment, proliferation and cell protein content) and the specific features (intracellular alkaline phosphatase activity and the cytoskeleton) of the cells in direct contact with the coating. Titanium was used as the reference material. The results showed that both silicon carbide and amorphous alumina are cytocompatible for human fibroblasts and osteoblasts, whereas titanium appears the least cytocompatible of all the three substrates. Moreover, the amorphous alumina coating seems slightly bioactive. It seems that these coatings, particularly amorphous alumina, could be used to protect alloys against corrosion, and consequently combine the good mechanical properties of the alloys with the good biocompatibility of the coatings. These coatings seem to perform more suitably than titanium if the strength of the bond between the coating and the underlying alloys is strong enough to give a stable composite material.     

Phenotypic expression of bone-related genes in osteoblasts grown on calcium phosphate ceramics with different phase compositions

Calcium phosphate ceramics with different hydroxyapatite (HA) and tricalcium phosphate (TCP) ratios have different chemical properties. Does the difference in phase composition affect osteoblast behavior? In this study, osteoblasts were cultured on 4 kinds of calcium phosphate ceramics, i.e. pure (HA), HT1 (HA/TCP, 70/30), HT2 (HA/TCP, 35/65), and pure TCP. Cell proliferation of SaOS-2 cells together with bone-related genes' mRNA expression and protein production in osteoblasts cultured on different calcium phosphate ceramics were detected at different time points. Data suggested that cell proliferation rate on TCP ceramics was lower than that on the other substrates tested. Generally, mRNA expressions for osteonectin and osteocalcin were similar among the four kinds of ceramics in most circumstances, whereas at six days, alkaline phosphatase mRNA expression was higher on HA and HT1 surfaces than on the other two materials. Collagen I mRNA expression was also affected by the phase composition of substrates. Osteocalcin and bone sialoprotein production in SaOS-2 cells was very similar no matter which ceramic surface the cells were grown upon. This study revealed that calcium phosphate ceramics substrate could support osteoblast growth and bone-related gene expression and its gene expression pattern explained the basis of the biocompatibility and bioactivity for calcium phosphate ceramics.     

Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone

ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.     

In vitro characterisation of zirconia coated by bioactive glass

An in vitro evaluation of a biomedical device, which combines the mechanical properties of zirconia substrates with the bioactivity of two different glass layers (AP40 and RKKP), was performed. In this work, data on different kinds of analysis were reported both on as-sintered zirconia samples and on RKKP- and AP40-coated zirconia substrates. Structure, composition and morphology of the apatite layer growth on the coated samples after 30 days of soaking in an acellular simulated body fluid, serum protein adsorption, fibroblasts and human osteoblast-like cells adhesion, growth, morphology and biochemical aspects were studied. Results of soaking test in SBF, revealed the growth of an apatite layer on the surface of the glass-coated samples. Proteins adsorbed to the materials were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and results evidenced that the two glass-coated materials bound a higher amount of total protein than did the zirconia substrate. Fibroblasts and osteoblast-like cells cultured on RKKP- and AP40-coated zirconia showed a higher proliferation rate, leading to confluent cultures with higher cell density and a generally better expression of osteoblast alkaline phosphatase activity in comparison with zirconia substrate. In conclusion, our results indicate that the surface chemical characteristics of the two glass coatings AP40 and RKKP, with no great differences between them, substantially enhance zirconia integration with bone cells at least in vitro. This effect may be of significance in the stability of glass-coated zirconia orthopaedic and dental implants.     

纳米级二氧化锆增韧羟基磷灰石生物复合陶瓷对兔骨髓基质干细胞增殖、分化的影响

背景：课题组拟对纳米级二氧化锆增韧的羟基磷灰石生物陶瓷进行一些初步研究,主要集中在生物力学匹配性,化学稳定性,以及生物相容性实验,其中体外细胞培养实验具有可控性,可重复性,能很好地反映材料的生物相容性。 目的：比较纳米级二氧化锆增韧的羟基磷灰石、纯羟基磷灰石两种材料对兔骨髓基质干细胞增殖、分化的影响。 方法：将骨髓基质干细胞置于含体积分数为20%胎牛血清的DMEM培养基中培养,传代后改用含β-甘油磷酸钠,地塞米松和维生素C的条件培养基培养。取传至第3代的成骨细胞,以1.0×108 L-1浓度接种于放有材料块的细胞培养板中,培养第1~10天倒置相差显微镜观察细胞生长情况,绘制细胞生长曲线,并进行碱性磷酸酶活性检测。培养第6天的细胞和材料复合物用多聚甲醛固定进行扫描电镜观察。 结果与结论：MTT法测得两种材料培养的细胞生长曲线无显著差异。复合培养的兔骨髓基质干细胞能够保持正常分泌碱性磷酸酶的功能。电镜照片也同样证实了两种材料表面均有细胞的附着。说明纳米级二氧化锆增韧的羟基磷灰石、纯羟基磷灰石均不影响成骨细胞增长分化,具有优良的成骨细胞相容性。     

Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation

Being very useful in the analysis of bone cell differentiation and activity, osteoblast cultures are also used in the in vitro biocompatibility study of new materials. The aim of this work was to evaluate sheep osteoblast cultures derived from normal and ovariectomized animals, and then to assess the in vitro biomaterial behavior on these cultures, taking into account the quality of bone where orthopedic devices are clinically used. For this purpose, we characterized sheep osteoblast cultures, isolated from iliac crest bone of normal (NB osteoblast culture) and osteopenic after ovariectomy (OB osteoblast culture) sheep. Moreover, we studied cell behavior when cultured on different biomaterials (titanium and two biological glasses, RKKP and AP40). Cell characterization at baseline demonstrated that both cultures (NB and OB) showed normal osteoblastic behavior. On the contrary, osteoblasts derived from osteopenic bone and cultivated on AP40 for 6 days revealed a different behavior in terms of both cell morphology and metabolic activity. Statistical analysis (one-way analysis of variance and Scheffé's post hoc multiple-comparison tests) revealed significant differences in Ca level (p<0.0005), MTT test (p<0.0005) and OC production (p<0.05). These in vitro tests demonstrated that sheep osteoblast cultures can be useful when determining biocompatibility and osteointegration of orthopedic materials, and also when evaluating for the presence of osteoporosis.     

Pasteurella multocida toxin is a mitogen for bone cells in primary culture.

The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage.     

硼硅酸盐对成骨细胞体外生物活性的影响

背景：硼硅酸盐不仅可通过矿化作用形成羟基碳酸盐磷灰石层,而且具有强化学反应活性,可促进骨细胞再生。 目的：通过体外培养实验观察硼硅酸盐生物玻璃对兔成骨细胞生长行为的影响。 方法：根据 ISO10993-12：2007 的要求制备硼硅酸盐生物玻璃初次浸提液与二次浸提液。分离培养兔骨髓间充质干细胞,取第2代细胞诱导生成成骨细胞。取第5-15代成骨细胞,分别以硼硅酸盐生物玻璃初次浸提液、硼硅酸盐生物玻璃二次浸提液与α-MEM培养基培养,观察硼硅酸盐生物活性玻璃对成骨细胞增殖、蛋白合成、碱性磷酸酶活性、细胞凋亡及细胞横向与纵向迁移的影响。 结果与结论：初次浸提液组与二次浸提液组成骨细胞增殖优于α-MEM培养基组(P < 0.05),且初次浸提液组成骨细胞增殖优于二次浸提液组(P < 0.05)。初次浸提液组成骨细胞总蛋白含量高于二次浸提液组与α-MEM培养基组(P < 0.05)。3组间成骨细胞碱性磷酸酶活性、凋亡率、横向迁移距离及Transwell 中穿膜细胞数比较差异均无显著性意义。表明硼硅酸盐生物玻璃具有良好的细胞相容性,对成骨细胞增殖有一定的良性调节作用。  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion

Titanium (Ti) and its alloys are used extensively in orthopedic implants due to their excellent biocompatibility and mechanical properties. However, titanium-based implant materials have specific complications associated with their applications, such as the loosening of implant-host interface owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections. Hence, a surface which displays selective biointeractivity, i.e. enhancing beneficial host cell responses but inhibiting pathogenic microbial adhesion, would be highly desirable. This present study aims to improve biocompatibility and confer long-lasting antibacterial properties on Ti via polyelectrolyte multilayers (PEMs) of hyaluronic acid (HA) and chitosan (CH), coupled with surface-immobilized cell-adhesive arginine-glycine-aspartic acid (RGD) peptide. The HA/CH PEM-functionalized Ti is highly effective as an antibacterial surface but the adhesion of bone cells (osteoblasts) is poorer than on pristine Ti. With additional immobilized RGD moieties, the osteoblast adhesion can be significantly improved. The density of the surface-immobilized RGD peptide has a significant effect on osteoblast proliferation and alkaline phosphatase (ALP) activity, and both functions can be increased by 100-200% over that of pristine Ti substrates while retaining high antibacterial efficacy. Such substrates can be expected to have good potential in orthopedic applications.     

Interaction of an ordered mesoporous bioactive glass with osteoblasts,fibroblasts and lymphocytes,demonstrating its biocompatibility as a potential bone graft material

Ordered mesoporous 85SiO₂–10CaO–5P₂O₅ bioactive glass (MBG85) is an excellent candidate as a graft for bone tissue regeneration, owing to its excellent textured properties, structural characteristics and crystalline apatite rate formation. To assess MBG85 biocompatibility, different parameters have been evaluated (cell morphology, size/complexity, proliferation, viability, cell cycle, reactive oxygen species content, lactate dehydrogenase release) using human Saos-2 osteoblasts after treatment with either MBG85 extracts or 1% MBG85 directly added to cells. The osteoblast response to MBG85 was compared with L929 fibroblast behaviour after the same treatment. The high cell viability observed and the absence of signs of cell damage in both cell types demonstrates MBG85 biocompatibility. Only a cytostatic effect was observed through the reduction of cell proliferation, related with the initial Ca elution, whereas Si leaching did not result into any negative effect. In vitro lymphocytic proliferation analysis was also carried out with SR.D10 clone after treatment with either MBG85 extracts or culture supernatants of L929 fibroblasts previously treated with 1% MBG85 (cell-conditioned extracts). The absence of modification of in vitro T-cell response underlines the biocompatibility of MBG85 and its potential application in the field of bone and dental grafting.     

Advanced nanocomposite materials for orthopaedic applications. I. A long-term in vitro wear study of zirconia-toughened alumina

The use of ceramic-on-ceramic (alumina- and zirconia-based) couplings in hip joint prostheses has been reported to produce lower wear rates than other combinations (i.e., metal-on-polyethylene and ceramic-on-polyethylene). The addition of zirconia into an alumina matrix (zirconia-toughened alumina, ZTA) has been reported to result in an enhancement of flexural strength, fracture toughness, and fatigue resistance. The development of new processing routes in nonaqueous media has allowed to obtain high-density ZTA nanocomposites with a very homogeneous microstructure and a significantly smaller and narrower particle-size distribution of zirconia than conventional powder mixing methods. The aim of the present study was to set up and validate a new ZTA nanocomposite by testing its biocompatibility and wear behavior in a hip-joint simulator in comparison with commercial alumina and experimental alumina specimens. The primary osteoblast proliferation onto ZTA nanocomposite samples was found to be not significantly different from that onto commercial alumina samples. After 7 million cycles, no significant differences were observed between the wear behaviors of the three sets of cups. In this light, it can be affirmed that ZTA nanocomposite materials can offer the option of improving the lifetime and reliability of ceramic joint prostheses.     

Sol-gel derived aluminosilicate coatings on alumina as substrate for osteoblasts

Rat bone marrow stromal cell differentiation on aluminosilicate 3Al₂O₃–2SiO₂ coatings was investigated. Thin ceramic coatings were prepared on -alumina substrates by the sol–gel process and calcined in order to establish an amorphous aluminosilicate ceramic phase with and without nanosized transitional mullite crystals. In addition, coatings of thermally sprayed aluminosilicate and diphasic γ-alumina–silica nanosized colloids were prepared. Cell culture testing by rat osteoblasts showed good biocompatibility for aluminosilicates with sustained normal osteoblast functions. Despite mutual disparities in physical and chemical nanostructures, the culture findings suggested fairly similar osteoblast response to all tested coatings. The results suggest that topographical frequency parameters and chemical uniformity are important parameters in determining the best conditions for osteoblasts on sol–gel derived aluminosilicate materials.     

Characterization and cell behavior of titanium surfaces with PLL/DNA modification via a layer-by-layer technique

This study describes the fabrication of two types of multilayered films onto titanium by layer-by-layer (LBL) self-assembly, using poly-L-lysine (PLL) as the cationic polyelectrolyte and deoxyribonucleic acid (DNA) as the anionic polyelectrolyte. The assembling process of each component was studied using atomic force microscopy (AFM) and quartz crystal balance (QCM). Zeta potential of the LBL-coated microparticles was measured by dynamic light scattering. Titanium substrates with or without multilayered films were used in osteoblast cell culture experiments to study cell proliferation, viability, differentiation, and morphology. Results of AFM and QCM indicated the progressive build-up of the multilayered coatings. The surface morphology of three types of multilayered films showed elevations in the nanoscale range. The data of zeta potential showed that the surface terminated with PLL displayed positive charge while the surface terminated with DNA displayed negative charge. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control (p < 0.05) after 3 and 7 days culture, respectively. Alamar blue measurement showed that the PLL/DNA-modified films have higher cell viability (p < 0.05) than the control. Still, the alkaline phosphatase activity assay revealed a better differentiated phenotype on three types of multilayered surfaces compared to noncoated controls. Collectively our results suggest that PLL/DNA were successfully used to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility.     

Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides).

In vitro cell biocompatibility models are crucial in the study of any newly synthesized material. Our focus has been on the development of a new class of biocompatible, degradable, high-strength polymeric materials, the poly(anhydride-co-imides), for use in bone regeneration. This study examined osteoblast cell adherence, proliferation, viability, and phenotypic preservation on the surface of the poly(anhydride-co-imide) poly[pyromellitylimidoalanine (PMA-ala):1,6-bis(carboxyphenoxy) hexane (CPH)] over a period of time. Cell proliferation on PMA-ala:CPH degradable matrices over 21 days was examined. Throughout the 21-day period of study, osteoblast proliferation was similar on PMA-ala:CPH and on tissue culture polystyrene controls. Osteoblasts maintained their characteristic morphology as demonstrated by both scanning electron microscopy and immunofluorescence studies. Alkaline phosphatase activity for cells grown on PMA-ala:CPH was confirmed. Retention of the osteoblastic phenotype was demonstrated using immunofluorescence techniques and staining with antibodies against osteocalcin (an extracellular matrix protein of bone) and osteopontin (a marker of cell adhesion). Radioimmunoassay results provided evidence that levels of osteocalcin production by osteoblasts were similar when cells were cultured on PMA-ala:CPH and on tissue culture polystyrene controls. The present study provided evidence of normal osteoblast function on PMA-ala:CPH surfaces. PMA-ala:CPH may therefore be useful as a synthetic material for orthopedic applications.     

Biocompatibility studies on biodegradable polyester-based composites of human osteoblasts: a preliminary screening.

A series of biodegradable composites with natural hydroxyapatite, designed for possible use in orthopedics applications, were preliminarily screened for biocompatibility by employing primary cultures of human osteoblasts in a direct contact method. The cells were seeded at low density onto the materials under investigation and allowed to grow for 2 weeks. They then were analyzed for morphology, proliferation, viability, alkaline phosphatase activity (AP), osteocalcin (OC) production, and extracellular matrix mineralization. The results showed that all materials have good biocompatibility. Cell viability tests demonstrated that in all cases the values were comparable to the control, and the addition of hydroxyapatite always resulted in an enhancement of performance with respect to the plain polymer. AP and OC analysis confirmed that all composites allowed the expression of phenotypic markers. Scanning electron microscopy provided direct evidence of intense cell adhesion and proliferation on the tested materials.     

Potential of FeAlCr intermetallics reinforced with nanoparticles as new biomaterials for medical devices

Novel FeAlCr oxide dispersion strengthened intermetallics that are processed by powder metallurgy have been developed as potential biomaterials. The alloys exhibit a small grain size and a fine dispersion of yttria provides the material with a high yield strength and depending on the alloy composition good ductility (up to 5%). The biocompatibility of the alloy was assessed in comparison with commercial alumina. Saos-2 osteoblast-like cells were either challenged with mechanically alloyed particles, or seeded onto solid samples. Viability and proliferation of cells were substantially unaffected by the presence of a high concentration of particles (1 mg/mL). Solid samples of novel FeAlCr intermetallic have shown a good biocompatibility in vitro, often approaching the behavior of materials well known for their biological acceptance (e.g. alumina). It has been found that osteoblasts are able to produce ALP, a specific marker of cells with bone-forming activity. In this respect, ALUSI alloys hold the promise to be suitable substrate for bone integration. The finding of no cytotoxic effect in the presence of the alloy particles is a reliable proof of the absence of acute toxicity of the material.     

Enhanced functions of osteoblasts on nanophase ceramics

Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was significantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was significantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was significantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the first evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties.     

Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography

Modification of the chemistry and surface topography of nanophase ceramics was used to provide biomaterial formulations designed to direct the adhesion and proliferation of human mesenchymal stem cells (HMSCs). HMSC adhesion was dependent upon both the substrate chemistry and grain size, but not on surface roughness or crystal phase. Specifically, cell adhesion on alumina and hydroxyapatite was significantly reduced on the 50 and 24 nm surfaces, as compared with the 1500 and 200 nm surfaces, but adhesion on titania substrates was independent of grain size. HMSC proliferation was minimal on the 50 and 24 nm substrates of any chemistry tested, and thus significantly lower than the densities observed on either the 1500 or 200 nm surfaces after 3 or more consecutive days of culture. Furthermore, HMSC proliferation was enhanced on the 200 nm substrates, compared with results obtained on the 1500 nm substrates after 7 or more days of culture. HMSC proliferation was independent of both substrate surface roughness and crystal phase. Rat osteoblast and fibroblast adhesion and proliferation exhibited similar trends to that of HMSCs on all substrates tested. These results demonstrated the potential of nanophase ceramic surfaces to modulate functions of HMSCs, which are pertinent to biomedical applications such as implant materials and devices.