Functionally graded hydroxyapatite-alumina-zirconia biocomposite: Synergy of toughness and biocompatibility

Functionally Gradient Materials (FGM) are considered as a novel concept to implement graded functionality that otherwise cannot be achieved by conventional homogeneous materials. For biomedical applications, an ideal combination of bioactivity on the material surface as well as good physical property (strength/toughness/hardness) of the bulk is required in a designed FGM structure. In this perspective, the present work aims at providing a smooth gradation of functionality (enhanced toughening of the bulk, and retained biocompatibility of the surface) in a spark plasma processed hydroxyapatite-alumina-zirconia (HAp-Al2O3-YSZ) FGM bio-composite. In the current work HAp (fracture toughness similar to 1.5 MPa.m(1/2)) and YSZ (fracture toughness similar to 62 MPa.m(1/2)) are coupled with a transition layer of Al2O3 allowing minimum gradient of mechanical properties (especially the fracture toughness similar to 3.5 MPa.m(1/2)).The in vitro cyto-compatibilty of HAp-Al2O3-YSZ FGM was evaluated using L929 fibroblast cells and Saos-2 Osteoblast cells for their adhesion and growth. From analysis of the cell viability data, it is evident that FGM supports good cell proliferation after 2, 3, 4 days culture. The measured variation in hardness, fracture toughness and cellular adhesion across the cross section confirmed the smooth transition achieved for the FGM (HAp-Al2O3-YSZ) nanocomposite, i.e. enhanced bulk toughness combined with unrestricted surface bioactivity. Therefore, such designed biomaterials can serve as potential bone implants. (C) 2012 Elsevier B.V. All rights reserved.

On the toughness enhancement in hydroxyapatite-based composites

Among various biologically compatible materials, hydroxyapatite (HA) has excellent bioactivity/osteointegration properties and therefore has been extensively investigated for biomedical applications. However, its inferior fracture toughness limits the wider applications of monolithic HA as a load-bearing implant. To this end, HA-based biocomposites have been developed to improve their mechanical properties (toughness and strength) without compromising biocompatibility. Despite significant efforts over last few decades, the toughness of HA-based composites could not be enhanced beyond 1.5-2 MPa m(1/2), even when measured using indentation techniques. In this perspective, the present work demonstrates how spark plasma sintering can be effectively utilized to develop hydroxyapatite titanium (HA-Ti) composites with varying amounts of Ti (5, 10 and 20 wt.%) with extremely high single edge V-notch beam fracture toughness (4-5 MPa m(1/2)) along with a good combination of elastic modulus and flexural strength. Despite predominant retention of HA and Ti, the combination of critical analysis of X-ray diffraction and transmission electron microscopy investigation confirmed the formation of the CaTi4(PO4)(6) phase with nanoscale morphology at the HA/Ti interface and the formation of such a phase has been discussed in reference to possible sintering reactions. The variations in the measured fracture toughness and work of fracture with Ti addition to the HA matrix were further rationalized using the analytical models of crack bridging as well as on the basis of the additional contribution from crack deflection. The present work opens up the opportunity to further enhance the toughness beyond 5 MPa m(1/2) by microstructural designing with the desired combination of toughening phases. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cryogenically cured hydroxyapatite-gelatin nanobiocomposite for bovine serum albumin protein adsorption and release

This research paper presents the first results on the protein adsorption and release kinetics and in vitro biodegradability of cryogenically cured hydroxyapatite-gelatin based micro/macroporous scaffolds (CHAMPS). While the adsorption and release of bovine serum albumin (BSA) protein exhibits steady state behavior over an incubation period of up to 10 days, Fourier transform infrared (FT-IR) analysis importantly confirms the absence of any change in the secondary structure of BSA proteins due to interaction with the CHAMPS scaffold. The compression properties of the CHAMPS scaffold with interconnected porosity (pore size similar to 50-200 mm) is characterized by a non-linear stress-strain response with a strength close to 5 MPa and a maximum strain of up to 24%. The slow but systematic increase in weight loss over a period of 7 days as well as apatite layer formation indicates its good bioactivity. The extensive micro-computed tomography (micro-CT) analysis establishes cancellous bone-like highly interconnected and complex porous architecture of the CHAMPS scaffold. Importantly, the excellent adsorption (up to 50%) and release (up to 60% of adsorbed protein) of BSA has been uniquely attributed to the inherent porous microstructure of the CHAMPS scaffold. Overall, the present study provides an assessment of the interaction of protein with the gelatin-hydroxyapatite macroporous scaffold in vitro, as well as reporting for the first time the efficacy of such scaffolds to release 60% of BSA loaded onto the scaffold in vitro, which is significantly higher than earlier literature reports.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-beta (TGF-beta) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-beta-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

Fretting wear behaviour of hydroxyapatite-titanium composites in simulated body fluid, supplemented with 5 g l(-1) bovine serum albumin

Damaged articulating joints can be repaired or replaced with synthetic biomaterials, which can release wear debris due to articulation, leading to the osteolysis. In a recent work, it has been shown that it is possible to achieve a better combination of flexural strength/fracture toughness as well as in vitro bioactivity and cytocompatibility properties in spark plasma sintered hydroxyapatite-titanium (HA-Ti) composites. Although hydroxyapatite and titanium are well documented for their good biocompatibility, nanosized hydroxyapatite (HA) and titanium (Ti) particles can cause severe toxicity to cells. In order to address this issue, fretting wear study of HA-Ti composites under dry and wet (1x SBF, supplemented with 5 g l(-1) bovine serum albumin (BSA)) condition was performed to assess the wear resistance as well as wear debris formation, in vitro. The experimental results reveal one order of magnitude lower wear rate for HA-10 wt% Ti (7.5 x 10(-5) mm(3) N-1 m(-1)) composite than monolithic HA (3.9 x 10(-4) mm(3) N-1 m(-1)) in simulated body fluid. The difference in the tribological properties has been analyzed in the light of phase assemblages and mechanical properties. Overall, the results suggest the potential use of HA-Ti composites over existing HA-based biocomposites in orthopedic as well as dental applications.

Hydroxyapatite-titanium bulk composites for bone tissue engineering applications

The research work on bulk hydroxyapatite (HA)-based composites are driven by the need to develop biomaterials with better mechanical properties without compromising its bioactivity and biocompatibility properties. Despite several years of research, the mechanical properties of the HA-based composites still need to be enhanced to match the properties of natural cortical bone. In this regard, the scope of this review on the HA-based bulk biomaterials is limited to the processing and the mechanical as well as biocompatibility properties for bone tissue engineering applications of a model system that is hydroxyapatite-titanium (HA-Ti) bulk composites. It will be discussed in this review how HA-Ti based bulk composites can be processed to have better fracture toughness and strength without compromising biocompatibility. The advantages of the functionally gradient materials to integrate the mechanical and biocompatibility properties is a promising approach in hard tissue engineering and has been emphasized here in reference to the limited literature reports. On the biomaterials fabrication aspect, the recent results are discussed to demonstrate that advanced manufacturing techniques, like spark plasma sintering can be adopted as a processing route to restrict the sintering reactions, while enhancing the mechanical properties. Various toughening mechanisms related to careful tailoring of microstructure are discussed. The in vitro cytocompatibilty, cell fate processes as well as in vivo biocompatibility results are also reviewed and the use of flow cytometry to quantify in vitro cell fate processes is being emphasized. (C) 2014 Wiley Periodicals, Inc.

Effects of Withania somnifera and Tinospora cordifolia Extracts on the Side Population Phenotype of Human Epithelial Cancer Cells: Toward Targeting Multidrug Resistance in Cancer

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

Strength reliability and in vitro degradation of three-dimensional powder printed strontium-substituted magnesium phosphate scaffolds

Strontium ions (Sr2+) are known to prevent osteoporosis and also encourage bone formation. Such twin requirements have motivated researchers to develop Sr-substituted biomaterials for orthopaedic applications. The present study demonstrates a new concept of developing Sr-substituted Mg-3(PO4)(2) - based biodegradable scaffolds. In particular, this work reports the fabrication, mechanical properties with an emphasis on strength reliability as well as in vitro degradation of highly biodegradable strontium-incorporated magnesium phosphate cements. These implantable scaffolds were fabricated using three-dimensional powder printing, followed by high temperature sintering and/or chemical conversion, a technique adaptable to develop patient-specific implants. A moderate combination of strength properties of 36.7 MPa (compression), 242 MPa (bending) and 10.7 MPa (tension) were measured. A reasonably modest Weibull modulus of up to 8.8 was recorded after uniaxial compression or diametral tensile tests on 3D printed scaffolds. A comparison among scaffolds with varying compositions or among sintered or chemically hardened scaffolds reveals that the strength reliability is not compromised in Sr-substituted scaffolds compared to baseline Mg-3(PO4)(2). The micro-computed tomography analysis reveals the presence of highly interconnected porous architecture in three-dimension with lognormal pore size distribution having median in the range of 17.74-26.29 mu m for the investigated scaffolds. The results of extensive in vitro ion release study revealed passive degradation with a reduced Mg2+ release and slow but sustained release of Sr2+ from strontium-substituted magnesium phosphate scaffolds. Taken together, the present study unequivocally illustrates that the newly designed Sr-substituted magnesium phosphate scaffolds with good strength reliability could be used for biomedical applications requiring consistent Sr2+-release, while the scaffold degrades in physiological medium. Statement of significance The study investigates the additive manufacturing of scaffolds based on different strontium-substituted magnesium phosphate bone cements by means of three-dimensional powder printing technique (3DPP). Magnesium phosphates were chosen due to their higher biodegradability compared to calcium phosphates, which is due to both a higher solubility as well as the absence of phase changes (to low soluble hydroxyapatite) in vivo. Since strontium ions are known to promote bone formation by stimulating osteoblast growth, we aimed to establish such a highly degradable magnesium phosphate ceramic with an enhanced bioactivity for new bone ingrowth. After post-processing, mechanical strengths of up to 36.7 MPa (compression), 24.2 MPa (bending) and 10.7 MPa (tension) could be achieved. Simultaneously, the failure reliability of those bioceramic implant materials, measured by Weibull modulus calculations, were in the range of 4.3-8.8. Passive dissolution studies in vitro proved an ion release of Mg2+ and PO43- as well as Sr2+, which is fundamental for in vivo degradation and a bone growth promoting effect. In our opinion, this work broadens the range of bioceramic bone replacement materials suitable for additive manufacturing processing. The high biodegradability of MPC ceramics together with the anticipated promoting effect on osseointegration opens up the way for a patient-specific treatment with the prospect of a fast and complete healing of bone fractures. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Spark plasma sintering of TiNi nano-powders for biological application

Nano-sized TiNi powder with an average size of 50nm was consolidated using spark plasma sintering (SPS) at 800 °C for 5min. A layer of anatase TiO 2 coating was formed on the sintered TiNi by chemical reaction with a hydrogen peroxide (H2O2) solution at 60 °C followed by heat treatment at 400 °C to enhance the bioactivity of the metal surface. Cell culture using osteoblast cells and a biomimetic test in simulated body fluid proved the biocompatibility of the chemically treated SPS TiNi. © IOP Publishing Ltd.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

Generating artificial homologous proteins according to the representative family character in molecular mechanics properties - an attempt in validating an underlying rule of protein evolution

The molecular mechanics property is the foundation of many characters of proteins. Based on intramolecular hydrophobic force network, the representative family character underlying a protein’s mechanics property is described by a simple two-letter scheme. The tendency of a sequence to become a member of a protein family is scored according to this mathematical representation. Remote homologs of the WW-domain family could be easily designed using such a mechanistic signature of protein homology. Experimental validation showed that nearly all artificial homologs have the representative folding and bioactivity of their assigned family. Since the molecular mechanics property is the only consideration in this study, the results indicate its possible role in the generation of new members of a protein family during evolution.

Detection of hepatitis B virus markers using a biosensor based on imaging ellipsometry

A biosensor based on imaging ellipsometry (BIE) has been developed and validated in 169 patients for detecting five markers of hepatitis B virus (HBV) infection. The methodology has been established to pave the way for clinical diagnosis, including ligand screening, determination of the sensitivity, set-up of cut-off values (CoVs) and comparison with other clinical methods. A matrix assay method was established for ligand screening. The CoVs of HBV markers were derived with the help of receiver operating characteristic curves. Enzyme-linked immunosorbent assay (ELISA) was the reference method. Ligands with high bioactivity were selected and sensitivities of 1 ng/mL and 1 IU/mL for hepatitis B surface antigen (HBsAg) and surface antibody (anti-HBs) were obtained respectively. The CoVs of HBsAg, anti-HBs, hepatitis B e antigen, hepatitis B e antibody and core antibody were as follows: 15%, 18%, 15%, 20% and 15%, respectively, which were the percentages over the values of corresponding ligand controls. BIE can simultaneously detect up to five markers within 1 h with results in acceptable agreement with ELISA, and thus shows a potential for diagnosing hepatitis B with high throughput.

rhegf-Loaded Plga-Alginate Microspheres Enhance The Healing Of Full-Thickness Excisional Wounds In Diabetised Wistar Rats

[EN] Diabetic foot ulcers (DFUs) represent a major clinical challenge in the ageing population. To address this problem, rhEGF-loaded Poly-Lactic-co-Glycolic-Acid (PLGA)-Alginate microspheres (MS) were prepared by a modified w/o/w-doubleemulsion/ solvent evaporation method. Different formulations were evaluated with the aim of optimising MSs properties by adding NaCl to the surfactant solution and/or the solvent removal phase and adding alginate as a second polymer. The characterization of the developed MS showed that alginate incorporation increased the encapsulation efficiency (EE) and NaCl besides increasing the EE also became the particle surface smooth and regular. Once the MS were optimised, the target loading of rhEGF was increased to 1% (PLGA-Alginate MS), and particles were sterilised by gamma radiation to provide the correct dosage for in vivo studies. In vitro cell culture assays demonstrated that neither the microencapsulation nor the sterilisation process affected rhEGF bioactivity or rhEGF wound contraction. Finally, the MS were evaluated in vivo for treatment of the full-thickness wound model in diabetised Wistar rats. rhEGF MS treated animals showed a statistically significant decrease of the wound area by days 7 and 11, a complete re-epithelisation by day 11 and an earlier resolution of the inflammatory process. Overall, these findings demonstrate the promising potential of rhEGF-loaded MS (PLGA-Alginate MS) to promote faster and more effective wound healing, and suggest its possible application in DFU treatment.

Solid-state NMR study of bioactive binary borosilicate glasses

A series of binary borosilicate glasses prepared by the sol-gel method are shown to be bioactive. Tetraethyl orthosilicate (TEOS) and trimethylborate (TMB) in acidic medium are used to prepare xB(2)O(3)center dot(1-x)SiO2 glass systems for x = 0.045-0.167. The formation of a layer of apatite-like mineral on the glass surface becomes apparent after soaking in simulated body fluid for 48 h. We have measured the B-11-B-11 homonuclear second moments of the borosilicate glasses and inferred that no macroscopic phase separation occurred in our glasses. The B-11 chemical shift data also show that the formation of clustered boroxol rings is negligible in our glass system. Although the bioactivity of our borosilicate glasses is less than that of CaO-SiO2 sol-gel glasses, these simple binary systems could be taken as reference glass systems for the search of new bioactive borosilicate glasses. (C) 2008 Elsevier Ltd. All rights reserved.

Avaliação de propriedades mecânicas e térmicas de compósito à base de polietileno de alta densidade e hidroxiapatita deficiente de cálcio

No presente trabalho, foram processados compósitos de polietileno de alta densidade (PEAD) com hidroxiapatita deficiente de cálcio (HA), com o objetivo de obter materiais com melhores propriedades mecânicas e bioatividade. A adição da HA deficiente de cálcio proporcionou um aumento no módulo de elasticidade (maior rigidez), menor resistência ao impacto e decréscimo do grau de cristalinidade do PEAD, proporcionando uma maior bioatividade ao material. A análise térmica exploratória (sistema não isotérmico) foi realizada por meio da técnica de calorimetria exploratória diferencial (DSC) e foram avaliados os teores de fosfato de cálcio e a velocidade de rotação da rosca no processamento dos materiais. No estudo da cristalização não-isotérmica observou-se uma diminuição da temperatura de cristalização com o aumento da taxa de resfriamento para todos os materiais sintetizados. A energia de ativação (Ea) da cristalização dos materiais foi avaliada por meio dos métodos Kissinger e Ozawa. A amostra com 5% de HA deficiente de cálcio e velocidade de processamento de 200 rpm foi a que apresentou menor valor de energia de ativação, 262 kJ/mol, menor desvio da linearidade e a que mais se assemelhou à matriz de PEAD sem HA. O teor de hidroxiapatita deficiente de cálcio não favorece o processo de cristalização devido à alta energia de ativação determinada pelos métodos descritos. Provavelmente, a velocidade de rotação, favorece a dispersão da carga na matriz de PEAD, dificultando o processo de cristalização. Na aplicação do método de Osawa-Avrami, os coeficientes de correlação indicaram perda na correlação linear. Estas perdas podem estar associadas a uma pequena percentagem de cristalização secundária e/ou à escolha das temperaturas utilizadas para determinar a velocidade de cristalização. Na determinação dos parâmetros pelo método de Mo, as menores percentagens de cristalização apresentaram um grande desvio da linearidade, com coeficiente de correlação bem menor que 1 e com o aumento da percentagem de cristalização, o desvio da linearidade diminui, ficando próximo de 1. Os resultados obtidos mostraram que o modelo de Mo e de Osawa-Avrami não foram capazes de definir o comportamento cinético dos materiais produzidos neste trabalho.