Effect of surface modification of graphene oxide with a reactive silane coupling agent on the mechanical properties and biocompatibility of acrylic bone cements.

Carbon allotrope materials (i.e. carbon nanotubes (CNTs), graphene, graphene oxide (GO)), have been used to reinforce acrylic bone cement. Nevertheless, the intrinsic incompatibility among the above materials produces a deficient interphase. Thus, in this work, the effect of the content of functionalized graphene oxide with a reactive silane on the mechanical properties and cell adhesion of acrylic bone cement was studied. GO was obtained by an oxidative process on natural graphite; subsequently, GO was functionalized with 3-methacryloxypropyltrimethoxysilane (MPS) to enhance the interphase between the graphenic material and acrylic polymeric matrix. Pristine GO and functionalized graphene oxide (GO-MPS) were characterized physicochemically (XPS, XRD, FTIR, and Raman) and morphologically (SEM and TEM). Silanized GO was added into the acrylic bone cement at different concentrations; the resulting materials were characterized mechanically, and their biocompatibility was also evaluated. The physicochemical characterization results showed that graphite was successfully oxidized, and the obtained GO was successfully functionalized with the silane coupling agent (MPS). SEM and TEM images showed that the GO is composed of few stacked layers. Compression testing results indicated a tendency of increasing stiffness and toughness of the acrylic bone cements at low concentration of functionalized GO. Additionally, the bending testing results showed a slightly increase in bone cement strain with the incorporation of GO-MPS. Finally, all samples exhibited cell viability higher than 70%, which means that materials are considered non-cytotoxic, according to the ISO 10993-5 standard.

Silanized graphene oxide as a reinforcing agent for acrylic bone cements: physicochemical, mechanical and biological characterization.

Recently, different carbon-based nanomaterials have been used as reinforcing agents in acrylic bone cement formulations. Among them, graphene oxide (GO) has attracted the attention of scientific community since it could improve not only the mechanical properties but also the biocompatibility characteristics of these materials. However, using GO presents some drawbacks, such as its poor dispersion and lack of interaction with polymeric matrices, which should be prior resolved to achieve its optimal performance in acrylic bone cement. Thus, in this work, GO was treated with 3-methacryloxy propyl trimethoxy silane at various concentrations (1, 3 and 5 wt.%) to improve the interaction between the nanofiller and the poly (methyl methacrylate) matrix. Modified GO was incorporated at different percentages (0.1, 0.5 and 0.75 wt.%) into acrylic bone cement formulations and some properties were evaluated. The silanization process of the GO was confirmed by FTIR, TGA and EDX. The improvement in the mechanical performance was monitored on the compression properties whereas those related with biological properties were evaluated by osteoblast cell viability and hemocompatibility tests. Results suggest that using a 1 wt.% of the silane coupling agent, during surface treatment of GO, yields the best mechanical performance in this type of materials. It was also found that the presence of neat GO or silanized GO does not compromise the cytocompatibility and hemocompatibility of acrylic bone cement formulations.

Cytokines secretion from human mesenchymal stem cells induced by bovine bone matrix.

BACKGROUND: Bovine bone matrix is a natural material that has been used in the treatment of bone lesions. In this study, bovine bone matrix Nukbone® (NKB) was investigated due its osteoconductive and osteoinductive properties. This biomaterial induces CBFA-1 activation and osteogenic differentiation, although the cytokines involved in these processes is still unknown. OBJECTIVE: The aim of this work was to determine the influence of NKB on the pro-osteoblastic and anti-osteoblastic cytokines secretion from human mesenchymal stem cells (hMSCs). METHODS: The hMSCs were cultured onto NKB and cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were analized at 0-14 days by immunoassay. In addition, hemocompatibility of NKB and characterization of hMSCs were evaluated. RESULTS: NKB induces an increase on pro-osteoblastic cytokine secretion IL-4 and a decrease on anti-osteoblastic cytokine IL-6 secretion, at days 7 and 14 of cell culture. Interestingly, there was no statistical difference between secretion profiles of others cytokines analized. CONCLUSIONS: The up-regulation of IL-4 and down-regulation of IL-6, and the secretion profiles of other cytokines examined in this work, are findings that will contribute to the understanding of the role of NKB, and similar biomaterials, in bone homeostasis and in the osteoblastic differentiation of hMSCs.

Cell-free scaffold from jellyfish Cassiopea andromeda (Cnidaria; Scyphozoa) for skin tissue engineering.

Disruption of the continuous cutaneous membrane in the integumentary system is considered a health problem of high cost for any nation. Several attempts have been made for developing skin substitutes in order to restore injured tissue including autologous implants and the use of scaffolds based on synthetic and natural materials. Current biomaterials used for skin tissue repair include several scaffold matrices types, synthetic or natural, absorbable, degradable or non-degradable polymers, porous or dense scaffolds, and cells capsulated in hydrogels or spheroids systems so forth. These materials have advantages and disadvantages and its use will depend on the desired application. Recently, marine organisms such as jellyfish have attracted renewed interest, because both its composition and structure resemble the architecture of human dermic tissue. In this context, the present study aims to generate scaffolds from Cassiopea andromeda (C. andromeda), with application in skin tissue engineering, using a decellularization process. The obtained scaffold was studied by infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), differential scanning calorimetry analysis (DSC), and scanning electron microscopy (SEM). Crystal violet staining and DNA quantification assessed decellularization effectiveness while the biocompatibility of scaffold was determined with human dermic fibroblasts. Results indicated that the decellularization process reduce native cell population leading to 70% reduction in DNA content. In addition, SEM showed that the macro and microstructure of the collagen I-based scaffold were preserved allowing good adhesion and proliferation of human dermic fibroblasts. The C. andromeda scaffold mimics human skin and therefore represents great potential for skin tissue engineering.

Antibacterial activity of a glass ionomer cement doped with copper nanoparticles.

Copper nanoparticles (NCu) were synthetized and added to commercial glass ionomer cement, to evaluate in vitro its antibacterial activity against oral cavity strains. The NCu were synthesized by copper acetate reduction with L-ascorbic acid and characterized by FTIR, Raman, XPS, XRD and TEM. Then, commercial glass ionomer cement (GIC) was modified (MGIC) with various concentrations of NCu and physicochemically characterized. Cell viability was tested against human dental pulp fibroblasts (HDPFs) by Alamar-Blue assay and antibacterial test was performed against S. mutans and S. sanguinis by colony forming unit (CFU) growth method. Synthesized NCu rendered a mixture of both metallic copper and cuprous oxide (Cu2O). HDPF viability reduces with exposure time to the extracts (68-72% viability) and MGIC with 2-4 wt% NCu showed antimicrobial activity against the two tested strains.

Effect of the rigid segment content on the properties of segmented polyurethanes conjugated with atorvastatin as chain extender.

Segmented polyurethanes were prepared with polycaprolactone diol as soft segment and various amounts of 4,4´-Methylenebis(cyclohexyl isocyanate) and atorvastatin, a statin used for lowering cholesterol, in order to obtain SPU with different content of rigid segments. Polyurethanes with 35% or 50% of rigid segment content were physicochemically characterized and their biocompatibility assessed with L929 fibroblasts. High concentrations of atorvastatin were incorporated by increasing the content of rigid segments as shown by FTIR, Raman, NMR, XPS and EDX. Thermal and mechanical characterization showed that polyurethanes containing atorvastatin and 35% of rigid segments were low modulus (13 MPa) semicrystalline polymers as they exhibited a glass transition temperature (Tg) at -38 °C, melting temperature (Tm) at 46 °C and crystallinity close to 35.9% as determined by DSC. In agreement with this, X-ray diffraction showed reflections at 21.3° and 23.6° for PCL without reflections for atorvastatin suggesting its presence in amorphous form with higher potential bioavailability. Low content of rigid segments led to highly degradable polymer in acidic, alkaline and oxidative media with an acceptable fibroblast cytotoxicity up to 7 days possibly due to low atorvastatin content.

Electrospun polycaprolactone/chitosan scaffolds for nerve tissue engineering: physicochemical characterization and Schwann cell biocompatibility.

Electrospun polycaprolactone (PCL)/chitosan (CH) blend scaffolds with different CH weight ratios were prepared to study the effect of scaffold composition on its physicochemical and biological properties. Scanning electron microscopy showed bead-free homogeneous randomly arranged nanofibers whose average diameter decreased from 240 to 110 nm with increasing CH content. The infrared spectra of the PCL/CH blends were very similar to the neat PCL scaffold. Energy-dispersive x-ray spectroscopy analysis confirmed the presence of carbon, oxygen and nitrogen in the scaffolds, although fluorine-from chemicals used as solvent-was also detected. The water contact angle decreased from 113° (for PCL) to 52° with increasing chitosan content. The biocompatibility was evaluated using fibroblasts and Schwann cell (SC) cultures. Cytotoxicity assays using fibroblasts demonstrated that electrospun scaffolds could be considered as non-cytotoxic material. Biocompatibility tests also revealed that the SCs adhered to scaffolds with different CH content, although the formulation containing CH at 5 wt% exhibited the highest proliferation on days 1 and 3. A better cell distribution was observed in the CH/PCL blends than in the neat PCL or CH scaffolds, where the cells were clustered. Immunochemistry analysis confirmed that SCs expressed the specific p75 cell marker on the scaffolds, suggesting that PCL/CH scaffolds would be good candidates for peripheral nerve tissue engineering.

Preparation and bioactive properties of nano bioactive glass and segmented polyurethane composites.

Composites of glutamine-based segmented polyurethanes with 5 to 25 wt.% bioactive glass nanoparticles were prepared, characterized, and their mineralization potential was evaluated in simulated body fluid. Biocompatibility with dental pulp stem cells was assessed by MTS to an extended range of compositions (1 to 25 wt.% of bioactive glass nanoparticles). Physicochemical characterization showed that composites retained many of the matrix properties, i.e. those corresponding to semicrystalline elastomeric polymers as they exhibited a glass transition temperature (Tg) between -41 and -36℃ and a melting temperature (Tm) between 46 and 49℃ in agreement with X-ray reflections at 23.6° and 21.3°. However, with bioactive glass nanoparticles addition, tensile strength and strain were reduced from 22.2 to 12.2 MPa and 667.2 to 457.8%, respectively with 25 wt.% of bioactive glass nanoparticles. Although Fourier transform infrared spectroscopy did not show evidence of mineralization after conditioning of these composites in simulated body fluid, X-ray diffraction, scanning electron microscopy, and energy dispersive X-ray microanalysis showed the formation of an apatite layer on the surface which increased with higher bioactive glass concentrations and longer conditioning time. Dental pulp stem cells proliferation at day 5 was improved in bioactive glass nanoparticles composites containing lower amounts of the filler (1-2.5 wt.%) but it was compromised at day 9 in composites containing high contents of nBG (5, 15, 25 wt.%). However, Runx2 gene expression was particularly upregulated for the dental pulp stem cells cultured with composites loaded with 15 and 25 wt.% of bioactive glass nanoparticles. In conclusion, low content bioactive glass nanoparticles and segmented polyurethanes composites deserve further investigation for applications such as guided bone regeneration membranes, where osteoconductivity is desirable but not a demanding mechanical performance.

Human mesenchymal stem cell behavior on segmented polyurethanes prepared with biologically active chain extenders.

The development of elastomeric, bioresorbable and biocompatible segmented polyurethanes (SPUs) for use in tissue-engineering applications has attracted considerable interest because of the existing need of mechanically tunable scaffolds for regeneration of different tissues, but the incorporation of osteoinductive molecules into SPUs has been limited. In this study, SPUs were synthesized from poly (ε-caprolactone)diol, 4,4'-methylene bis(cyclohexyl isocyanate) using biologically active compounds such as ascorbic acid, L-glutamine, ß-glycerol phosphate, and dexamethasone as chain extenders. Fourier transform infrared spectroscopy (FTIR) revealed the formation of both urethanes and urea linkages while differential scanning calorimetry, dynamic mechanical analysis, X-ray diffraction and mechanical testing showed that these polyurethanes were semi-crystalline polymers exhibiting high deformations. Cytocompatibility studies showed that only SPUs containing ß-glycerol phosphate supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation, rendering them potentially suitable for bone tissue regeneration, whereas other SPUs failed to support either cell growth or osteogenic differentiation, or both. This study demonstrates that modification of SPUs with osteogenic compounds can lead to new cytocompatible polymers for regenerative medicine applications.

Towards optimization of the silanization process of hydroxyapatite for its use in bone cement formulations.

The aim of this work was to provide some fundamental information for optimization of silanization of hydroxyapatite intended for bone cement formulations. The effect of 3-(trimethoxysilyl) propyl methacrylate (MPS) concentration and solvent system (acetone/water or methanol/water mixtures) during HA silanization was monitored by X-ray diffraction (XRD), FTIR spectroscopy and EDX analysis. The effect of silanized HA on the mechanical properties of acrylic bone cements is also reported. It was found that the silanization process rendered hydroxyapatite with lower crystallinity compared to untreated HA. Through EDX, it was observed that the silicon concentration in the HA particles was higher for acetone-water than that obtained for methanol-water system, although the mechanical performance of cements prepared with these particles exhibited the opposite behavior. Taking all these results together, it is concluded that methanol-water system containing MPS at 3wt.% provides the better results during silanization process of HA.

Comparative study on the mechanical and fracture properties of acrylic bone cements prepared with monomers containing amine groups.

In this work, the effect of the incorporation of comonomers containing amine groups on the mechanical and fracture properties of acrylic bone cements was studied. Cements were prepared with either diethyl amino ethyl acrylate (DEAEA), dimethyl amino ethyl methacrylate (DMAEM) or diethyl amino ethyl methacrylate (DEAEM) as comonomer in the liquid phase. It was found that strength and modulus decreased with increasing comonomer content in the bending and compressive tests. It was also observed that fracture toughness (K(IC)) and the critical strain energy release rate (G(IC)) increase with increasing comonomer concentration and are significantly higher compared to the control formulation. The mechanical and fracture properties of cements were also evaluated after soaking the specimens in Simulated Body Fluid (SBF) for 3 and 6 months. It was found that the mechanical properties of cements decreased when the samples were stored in SBF, although the impact strength increased in the first 3 months and then decreased. SEM micrographs were in agreement with the results obtained during mechanical characterization as the increase in toughness was confirmed by the appearance of ductile tearing pattern which is associated with plastic deformation.

Combined influence of barium sulfate content and co-monomer concentration on properties of PMMA bone cements for vertebroplasty.

In this work, the combined influence of barium sulfate content and co-monomer concentration on the properties of acrylic bone cement for percutaneous vertebroplasty (PVP) was investigated using a response surface methodology. Cements were prepared with methyl methacrylate (MMA) and either diethyl amino ethyl methacrylate (DEAEM) or dimethyl amino ethyl methacrylate (DMAEM) as co-monomer in the liquid phase, while variable amounts of barium sulfate were incorporated to the solid phase in order to improve the radiopacity of cements. It was found that various properties such as peak temperature, setting time, residual monomer content, mechanical properties and injectability, had an effect on the occurrence of interactions (combined effect) between the barium sulfate and DEAEM in bone cements formulations when independent variables were at their maximum.

Comparative study on the properties of acrylic bone cements prepared with either aliphatic or aromatic functionalized methacrylates.

Bone cements prepared with methacrylic acid (MAA) and diethyl amino ethyl methacrylate (DEAEM) were compared with formulations employing 4-methacryloyloxybenzoic acid (MBA) and 4-diethyaminobenzyl methacrylate (DEABM) as comonomer. The influence of these new aromatic monomers on various physicochemical, setting and mechanical properties was assessed. Surface characterization demonstrated that bone cements prepared with any of the functionalized monomers exhibited increasing hydrophilicity with monomer concentration and that the aromatic monomers provided more hydrophilic cements than their aliphatic counterparts for low concentrations of the functional monomer. It was also found that bone cements prepared with high amounts of the acidic aliphatic monomer provided the highest exotherm of reaction and their setting times were shorter than MBA based cements. On the other hand, DEABM containing bone cements exhibited shorter setting times than DEAEM formulations and slightly higher peak temperatures. In general, it was found that the glass transition temperature increased with the presence of acidic comonomer and decreased when alkaline comonomers were present, especially aliphatic ones. When aromatic methacrylates were used at 0.05 molar fraction, the highest tensile and compressive strength were achieved i.e. 46 and 118 MPa for MBA and 51 and 108 MPa for DEABM formulations. A further increase in the aromatic monomer concentration led to cements of low mechanical properties due to solubility problems as revealed by SEM.