Biological evaluation of partially stabilized zirconia added HA/HDPE composites with osteoblast and fibroblast cell lines

In the present study, the biocompatibility of partially stabilized zirconia (PSZ) added hydroxyapatite (HA)––high density polyethylene (HDPE) composites was evaluated by proliferation and cell attachment assays on two osteoblast cell lines (G-292, Saos-2) and a type of fibroblast cell isolated from bone tissue namely HBF in different time intervals. Cell-material interactions on the surface of the composites were observed by scanning electron microscopy (SEM). The effect of composites on the behavior of osteoblast and fibroblast cells was compared with those of HDPE and Tissue Culture Poly Styrene (TPS) (as negative control) samples. Results showed that the composite samples supported a higher proliferation rate of osteoblast cells in the presence of composite samples as compared to the HDPE and TPS samples after 3, 7 and 14 days of incubation period. It was showed that an equal or in some cases an even higher proliferation rate of G-292 and Saos-2 osteoblast cells on composite samples in compare to negative controls in culture period (P < 0.05). The number of adhered cells on the composite samples was equal and in some cases higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods (P < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division.     

In vitro response of osteoblasts to hydroxyapatite-reinforced polyethylene composites

A primary human cell culture model was used to investigate a range of hydroxyapaptite (HA)-reinforced high-density polyethylene (HDPE) composites (HAPEXTM). These materials are being developed as potential bone-substitute materials. When designing and optimizing a second-generation biomaterial, it is important to achieve a balance between mechanical and biological properties without compromising either. Biochemical and histological parameters have been used to compare the biological response of 20% and 40% volume HA in HDPE. Cellular DNA and incorporation of tritiated thymidine was measured to assess cell proliferation. Alkaline phosphatase (ALP) production was used as a marker of osteoblast phenotype expression. In this preliminary study, osteoblasts cultured on the 20% HAPEXTM showed a greater increase in the rate of proliferation and osteoblast expression as indicated by an increase in ALP activity compared to the 40% HAPEXTM over the time period studied. Osteoblast-like cells showed a flattened morphology on both composites and in some cases a greater covering was observed on the 20% HAPEXTM. These results indicate that the composites may not be identical in terms of bioactivity and that further research on surface topography and physico-chemical properties is required to assess fully the biological response of these composites. © 1998 Kluwer Academic Publishers     

In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite

In vitro performance of hydroxyapatite (HA)-reinforced polyethylene (PE) composite (HAPEX) has been characterized from both mechanical and biological aspects. The mechanical properties of HAPEX, such as tensile strength and Young's modulus, showed little change after immersion in a physiological solution at 37 and 70 degrees C for various periods. In addition, the biological response of primary human osteoblast-like (HOB) cells in vitro on HAPEX was assessed by measuring DNA synthesis and osteoblast phenotype expression. Cell proliferation rate on HAPEX was demonstrated by an increase in DNA content with time. A high tritiated thymidine ([3H]-TdR) incorporation/DNA rate was observed on day 1 for HAPEX, indicating a stimulatory effect on cell proliferation. The alkaline phosphatase (ALP) activity was expressed earlier on HAPEX than on unfilled PE and increased with time, indicating that HOB cells had commenced differentiation. Furthermore, it was found that the HA particles in the composite provided favourable sites for cell attachment. It appears that the presence of HA particles in HAPEX may have the advantage of acting as microanchors for bone bonding in vivo.     

钛表面制备羟基磷灰石/壳聚糖复合涂层研究

通过原位水热合成和溶胶-凝胶浸提涂敷法在碱处理的钛表面制备了HA/CS复合涂层. 接触角检测表明碱处理使钛表面具有超亲水性.X射线衍射分析表明复合涂层成分为HA和CS, 各组分含量由热重分析确定. 用扫描电镜对复合涂层的形貌进行观察,发现不同HA含量的复合涂层具有不同的形貌. 通过培养成骨细胞考察了复合涂层的细胞相容性.Alamar Blue检测表明HA/CS复合涂层表面细胞粘附及增殖能力较好. ALP检测表明HA/CS复合涂层表面的细胞分化能力较好. 综合研究结果表明, 复合涂层有较好的细胞相容性.     

羟基磷灰石/钛合金骨替代材料体外培养成骨细胞的实验研究

实验选用小鼠头盖骨成骨细胞,采用体外细胞培养技术对具有羟基磷灰石涂层的钛合金(HA/Ti)与未经过表面改性的钛合金两种骨替代材料进行细胞相容性评价,动态观察两种骨替代材料对成骨细胞生长、附着的影响。结果表明两种骨替代材料对成骨细胞生长无抑制作用,未发生细胞毒性反应,细胞在两种材料表面均能正常粘附、生长、增殖,均具有良好的细胞附着形态和细胞增殖率,而HA/钛合金材料具有更好的成骨性,是一种骨细胞相容性良好的骨替代材料。     

In vitro cellular response to titanium electrochemically coated with hydroxyapatite compared to titanium with three different levels of surface roughness

The in vitro response of primary human osteoblast-like (HOB) cells to a novel hydroxyapatite (HA) coated titanium substrate, produced by a low temperature electrochemical method, was compared to three different titanium surfaces: as-machined, Al₂O₃-blasted, plasma-sprayed with titanium particles. HOB cells were cultured on different surfaces for 3, 7 and 14 days at 37 °C. The cell morphology was assessed using scanning electron microscopy (SEM). Cell growth and proliferation were assessed by the measurement of total cellular DNA and tritiated thymidine incorporation. Measurement of alkaline phosphatase (ALP) production was used as an indicator of the phenotype of the cultured HOB cells. After three days incubation, the electrochemically coated HA surface produced the highest level of cell proliferation, and the Al₂O₃-blasted surface the lowest. Interestingly, as the incubation time was increased to 7 days all surfaces produced a large drop in tritiated thymidine incorporation apart from the Al₂O₃-blasted surface, which showed a small increase. Cells cultured on all four surfaces showed an increased expression of ALP with increased incubation time, although there was not a statistically significant difference between surfaces at each time point. Typical osteoblast morphology was observed for cells cultured on all samples. The HA coated sample showed evidence of a deposited phase after three days of incubation, which was not observed on any other surface. Cells incubated on the HA coated substrate appeared to exhibit the highest number of cell processes attaching to the surface, which was indicative of optimal cell attachment. The crystalline HA coating, produced by a low temperature route, appeared to result in a more bioactive surface on the c.p. Ti substrate than was observed for the other three different Ti surfaces.     

Preparation of graded zirconia–CaP composite and studies of its effects on rat osteoblast cells in vitro

Hydroxyapatite (HAP) has excellent biocompatibility and bone bonding ability, but it is mechanically weak and brittle. To overcome this problem, we prepared a graded composite with calcium phosphide (CaP, decomposed from HAP during sintering) coating on the surface of zirconia (ZrO₂) ceramics. The mechanical properties and microstructure characteristics were studied with various techniques. The biocompatibility of graded ZrO₂–CaP composite was examined with rat osteoblast cells (OB cells) in vitro. Its effects on the production of alkaline phosphatase (ALP), Interleukin-6 (IL-6) and Growth-transforming Factor-β (TGF-β) by the OB cells were measured. The results showed that the mean tensile strength of the graded ZrO₂–CaP composites was 17.8 MPa, the maximum bending strength was 1112.24 MPa, and K_IC was 7.3–11.4 MPa·m^1/2, indicating that the composite was physically strong for use as an implant material. The ALP activity, IL-6 and TGF-β concentrations of the graded composite treated OB cells were much higher than that of the pure ZrO₂ treated group. There was no significant difference in ALP activity, the IL-6 and TGF-β concentrations between the graded ZrO₂–CaP composite group and HAP. The cytotoxicity of the composite material to rat fibroblast cells was insignificant. The graded zirconia–CaP composite greatly facilitated the proliferation and differentiation of rat OB cells in vitro, demonstrating excellent biocompatibility.     

Hydroxyapatite reinforcement of different starch-based polymers affects osteoblast-like cells adhesion/spreading and proliferation

The aim of this study was to determine which, from a range of the starch-based biomaterials, would be more suitable to be used in orthopaedic applications. This included blends of corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and its composites with increasing percentages of hydroxyapatite (HA). Osteoblast-like cells (SaOs-2) were cultured in direct contact with the polymers and composites and the effect of the incorporation and of increasing percentages of the ceramic in osteoblast adhesion/proliferation was assessed. In the evaluation of cell adhesion and proliferation rate, two variables were considered; cells adhered to the bottom of the tissue culture polystyrene wells (TCPS) and cells adhered to the surface of the materials, in order to distinguish, respectively: (i) the effect of possible degradation products released from the materials to the culture medium and (ii) the effect of the surface properties on the osteoblast-like cells. In addition, the morphology of cells adherent to the surface of the starch-based polymers was analysed and correlated with their topography and with other chemical properties previously evaluated.The proliferation rate was found to differ from blend to blend as well as with the time of culture and with the presence of HA depending on the material. SEVA-C and respective composites systematically presented the higher number of cells comparatively to the other two blends. SPCL composites were found to be less suitable for cell proliferation. The amount of cells quantified after 7 days of culture, both on the surface and on the wells showed a delay in the proliferation of the cells cultured with SPCL composites comparatively to other materials and to TCPS. SCA composites, however, did support cell adhesion but also induce a slight level of toxicity, which results in delayed proliferation on the cells adhered to the wells.Cell morphology on the surface of the materials was also, in almost every case, found to be appropriate. In fact, cells were well adhered and spread on the majority of the surfaces. Thus, starch-based biomaterials can be seen as good substrates for osteoblast-adhesion and proliferation that demonstrates their potential to be used in orthopaedic applications and as bone tissue engineering scaffolds.     

Effect of carbon nanotube and aluminum oxide addition on plasma-sprayed hydroxyapatite coating's mechanical properties and biocompatibility

This study reports on the synthesis of novel bioceramic composite coating of hydroxyapatite (HA) reinforced with carbon nanotubes (CNTs) and aluminum oxide (Al₂O₃) using plasma spray technique. Fracture toughness of HA–20 wt.% Al₂O₃ improved by 158% as compared to HA coating whereas HA–18.4 wt.% Al₂O₃–1.6 wt.% CNT showed an improvement of 300%. Carbon nanotubes provided reinforcement via rebar mechanism. Human fiber osteoblast cell-growth studies showed that biocompatibility of the coating remained unaltered, as Al₂O₃ retained its bio-inertness and CNT, its bioactivity, within the composite coatings. Composite coating showed lower attachment, but higher proliferation rate, for the osteoblast cells, which has been attributed to the surface roughness. An optimized relation between coating composition, its biocompatibility and mechanical properties was established to predict the most suited coating material for orthopedic implants. HA–Al₂O₃–CNT composite coating displayed most improved mechanical properties while retaining its biocompatibility.     

Surface topography and HA filler volume effect on primary human osteoblasts in vitro

HAPEX^TM, a bone analog material, with similar properties to cortical bone, has been studied in vitro with particular reference to the effect of surface topography. The stimulation of a favorable bone response by this composite depends on optimization of the hydroxyapatite (HA) content in relation to the material bioactivity without compromising the mechanical characteristics. In this study we have started to investigate the effects of surface topography on cell attachment and subsequent cellular behavior in relation to proliferation. Different volumes of HA (20% and 40%) were added to a high density polyethylene (HDPE) matrix to produce the test materials. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to examine cell morphology on HAPEX^TM, and the surface characteristics produced by different machining protocols. The measurement of cellular DNA and tritiated thymidine ([³H]-TdR) incorporation has been used to asses cell proliferation upon the materials. The results show that the material surface topography has a large influence on cell proliferation and attachment, and with a controled material topography the 40% vol HA/HDPE composite gives the greater biological response compared to the 20% vol HA/HDPE composite.     

Comparative evaluation of biocompatibility of dense nanostructured and microstructured Hydroxyapatite/Titania composites

This work deals with the biocompatibility of dense nano- and micro-structured Hydroxyapatite/Titania composites prepared by two step and conventional sintering, respectively. By application of two step sintering, it was shown that the final grain size of HA–15 wt.%TiO₂ is maintained lower than 100 nm while by the application of conventional sintering it reaches higher than 100 nm. Biocompatibility of the dense bulks was evaluated by cell attachment and proliferation experiments. Cell morphology, and viability on each nano- and micro-structured Hydroxyapatite/Titania composites were examined at different time points. The nanostructured HA/Titania dense bulk exhibited higher cell viability than a microstructured one. In addition, the effects of ionic products from nano- and micro-structured bulk dissolution on osteoblasts were studied. The MTT test confirmed that the products from nanostructured HA/Titania dense bulk significantly promoted osteoblast proliferation within a certain concentration range.     

Evaluation of in vitro bioactivity and biocompatibility of Bioglass®-reinforced polyethylene composite

The bioactivity and biocompatibility of Bioglass®-reinforced high-density polyethylene composite (Bioglass®/HDPE) have been evaluated in simulated body fluid (SBF) and by in vitro cell culture, respectively. The formation of a biologically active hydroxy-carbonate apatite (HCA) layer on the composite surface after immersion in SBF was demonstrated by thin-film X-ray diffraction, infrared spectroscopy and scanning electron microscopy, indicating the in vitro bioactivity of Bioglass®/HDPE composites. The HCA layer was formed on the 40 vol% composite surface within 3 days immersion in SBF at a formation rate comparable to those on bioactive glass-ceramics, showing that in vitro bioactivity could be obtained in a composite. Furthermore, the composite was biocompatible to primary human osteoblast-like cells. In comparison with unfilled HDPE and tissue culture plastic control, a significant increase in cellular metabolic activity was found on the composite. Therefore, Bioglass®/HDPE composites have a promising biological response as a potential implant material.     

壳聚糖/羟基磷灰石表面修饰聚己内酯多孔骨支架的制备及性能

采用选择性激光烧结技术构建多孔聚己内酯(PCL)骨支架,用原位合成的方法制得壳聚糖/羟基磷灰石(CS/HA)悬浮液,并采用真空浸泡、低速离心和冷冻凝胶的方法使CS/HA黏附在PCL支架的表面,以改善骨支架的生物相容性和细胞增殖活性。通过X射线衍射(XRD)和扫描电子显微镜(SEM)观测复合支架的物相和形貌,测量支架的压缩强度和杨氏模量,测量支架表面的水接触角,并通过体外细胞实验研究复合支架的生物学性能。实验结果表明,原位合成的方法制得了羟基磷灰石(HA);CS/HA凝胶与PCL骨支架表面黏附良好;CS/HA改善了PCL支架表面的亲水性,提升了骨支架的生物相容性和细胞增殖活性。     

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.     

Effect of hydroxyapatite porosity on growth and differentiation of human osteoblast-like cells

To study whether hydroxyapatite (HA) porosity can influence its osteoconductive properties, cell adhesion, proliferation and differentiation were compared in human osteoblast-like cells grown on HA disks of different porosity (A = 20%, B = 40%, C = 60%). Human osteoblast-like cells were isolated and characterized. Proliferation rate and alkaline phosphatase (ALP) activity were assessed at 3, 7, 15, 21, and 28 days. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion studied at 7 and 21 days by scanning electron microscopic analysis. Cell growth on HA was three- to six-fold lower than on polystyrene control disks. At 28 days, 2141 (±350) cells/well grew on the most porous disks (Group C), with highly significant differences from controls (p < 0.005). The ALP production was 2–3 fold lower on HA than on plastic. In the Group C the mean ALP activity was of 2.95 (±0.07) UI/well after 28 days, higher than in the other two groups. At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p < 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks.     

Effect of Porous Activated Charcoal Reinforcement on Mechanical and In-Vitro Biological Properties of Polyvinyl Alcohol Composite Scaffolds

The present work focused on developing an innovative composite material by reinforcing polymer matrix with highly porous activated charcoal. Polyvinyl alcohol-activated charcoal(PVA-AC) composite scaffolds were developed by varying the AC concentrations(0, 0.5, 1, 1.5, 2 and 2.5 wt%) in PVA matrix by freeze drying method. The developed scaffolds were characterized for their physicochemical, mechanical and in-vitro biological properties. In addition, the effect of AC on the attachment, proliferation and differentiation of osteoblast MG 63 cells was evaluated by scanning electron microscopy(SEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, alkaline phosphatase(ALP) activity assay and alizarin red stain-based(ARS) assay. All the PVA-AC composite scaffolds exhibited good bioactivity, hemocompatibility and protein adsorption properties. The scaffolds with high AC concentration(2.5 wt%) showed controlled drug release kinetics that are suitable for long term healing. The mechanical properties of all the PVA-AC composite scaffolds were improved when compared to the pure PVA scaffold. The high porosity, swelling degree and hydrophilicity of PVA-AC composite scaffolds facilitated cell attachment and proliferation. This is due to porous AC present in the sample that supported the osteoblast differentiation and formed mineralized nodules without the addition of any extra agents. From the above studies, it can be concluded that PVA-AC composite scaffolds are promising biomaterials for bone tissue engineering applications.     

Preparation of magnetic and bioactive calcium zinc iron silicon oxide composite for hyperthermia treatment of bone cancer and repair of bone defects

In this paper, a calcium zinc iron silicon oxide composite (CZIS) was prepared using the sol-gel method. X-ray diffraction (XRD) was then employed to test the CZIS composite. The results from the test showed that the CZIS had three prominent crystalline phases: Ca(2)Fe(1.7)Zn(0.15)Si(0.15)O(5), Ca(2)SiO(4), and ZnFe(2)O(4). Calorimetric measurements were then performed using a magnetic induction furnace. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis were conducted to confirm the growth of a precipitated hydroxyapatite phase after immersion in simulated body fluid (SBF). Cell culture experiments were also carried out, showing that the CZIS composite more visibly promoted osteoblast proliferation than ZnFe(2)O(4) glass ceramic and HA, and osteoblasts adhered and spread well on the surfaces of composite samples.     

Enhanced HAPEX topography: Comparison of osteoblast response to established cement

The use of poly(methylmethacrylate) PMMA cement by Charnley in the 1960s revolutionized orthopaedic medicine. Since this time, however, little has changed. The development of bioactive composites, such as HAPEX (a composite of 40% vol hydroxyapatite (HA) in a polyethylene matrix) have potential in orthopaedic applications. The composite has been shown to allow direct bone bonding in vivo, and in vitro studies have shown preferential attachment to HA exposed on the composite surface. In vitro study has also shown that altering the topography HAPEX can enhance osteoblast response. This study uses microscopical investigation of osteoblast cytoskeleton, and biochemical measurement of proliferation (by thymidine incorporation) and phenotype (by alkaline phosphatase activity) to compare primary human osteoblast (HOB) activity on HAPEX and PMMA cement. The study shows large increases in HOB response to the new generation material compared to PMMA, the current implant standard.     

Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/α-tricalcium phosphate composite (Sema3A CMs/SF/α-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/α-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/α-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/α-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/α-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/α-TCP and SF/α-TCP composites. In conclusion, Sema3A CMs/SF/α-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.     

Spark Plasma Sintered Hydroxyapatite/Graphite Nanosheet and Hydroxyapatite/Multiwalled Carbon Nanotube Composites: Mechanical and in Vitro Cellular Properties

Hyroxyapatite (HA) and its nanocomposites reinforced with 0.5, 1, 1.5, and 2 wt% graphite nanosheets (GNs) and multi‐walled carbon nanotubes (MWNTs) are fabricated by means of spark plasma sintering (SPS) process. The effects of MWNT and GN additions on the morphology, mechanical behavior, cell adhesion, and biocompatibility of HA were studied. Three‐point‐bending test shows that the bending strength of MWNT/HA nanocomposites increases with increasing MWNT content. However, the bending strength of GN/HA nanocomposites initially increases by adding 0.5 wt% GN, and then decreases markedly as the filler content increases. Cell culture and viability test results demonstrate that the GNs with diameters of several micrometers retard osteoblast cell adhesion and proliferation on the GN/HA nanocomposite. In contrast, the addition of 2 wt% MWNT to HA is beneficial to promote osteoblast adhesion and proliferation, thereby enhancing the biocompatibility of MWNT/HA nanocomposite.