Fabrication and characterization of silk/forsterite composites for tissue engineering applications

In this research, novel composite scaffolds consisting of silk fibroin and forsterite powder were prepared by a freeze-drying method. In addition, the effects of forsterite powder contents on the structure of the scaffolds were investigated to provide an appropriate composite for bone tissue engineering applications. The morphology studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques showed that the forsterite ceramic was well distributed throughout the structures of SF/forsterite scaffolds. Furthermore, the forsterite powder (up to 40 wt%) was homogenously distributed within the silk fibroin as a matrix.     

Design and fabrication of novel chitin hydrogel/chitosan/nano diopside composite scaffolds for tissue engineering

In this research, novel porous composite scaffolds consisting of chitin, chitosan and nano diopside powder were prepared using the freeze-drying method. The prepared nanocomposite scaffolds were characterized by SEM, XRD, BET, TGA and FT-IR techniques. In addition, swelling, degradation and biomineralization capability, cell viability and cell attachment of the composite scaffolds were evaluated. The results indicated better swelling and degradation properties of such scaffolds their ability to become bioactive. Cytocompatibility of the scaffolds were assessed by MTT assay and cell attachment studies using Human Gingival Fibroblast cells. Cell viability studies demonstrated no sign of toxicity and cells were found to be attached to the pore walls within the scaffolds. These results suggested that the developed composite scaffolds could be a potential candidate for tissue engineering.     

Novel 3D scaffolds of chitosan-PLLA blends for tissue engineering applications: Preparation and characterization

This work addresses the preparation of 3D porous scaffolds of blends of chitosan and poly(l-lactic acid), CHT and PLLA, using supercritical fluid technology. Supercritical assisted phase-inversion was used to prepare scaffolds for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage regeneration. On the other hand, PLLA is a synthetic biodegradable polymer widely used for biomedical applications. Supercritical assisted phase-inversion experiments were carried out in samples with different polymer ratios and different polymer solution concentrations. The effect of CHT:PLLA ratio and polymer concentration and on the morphology and topography of the scaffolds was assessed by SEM and Micro-CT. Infra-red spectroscopic imaging analysis of the scaffolds allowed a better understanding on the distribution of the two polymers within the matrix. This work demonstrates that supercritical fluid technology constitutes a new processing technology, clean and environmentally friendly for the preparation of scaffolds for tissue engineering using these materials.     

A macroscopic model for species transport during in vitro tissue growth obtained by the volume averaging method

In this work, we study the possibility of deriving a macroscopic model to describe reaction and transport by diffusion and convection of two species within a porous medium as encountered during in vitro tissue growth. The starting point is a boundary value problem of diffusion-advection and reaction in a three-phase system, the two species being identified as the nutrient for cell growth and the metabolic product within the framework of tissue culture. The method of volume averaging is applied to the set of microscopic equations. Under the local mass equilibrium assumption and a series of constraints on the parameters of the system that are identified, one obtains a one-equation macroscopic model corresponding to a dispersion-reaction equation. Associated closure problems allowing the computation of effective coefficients that appear in this macroscopic model are provided.     

Fabrication of Porous TiO<Subscript>2</Subscript> Hollow Spheres and Their Application in Gas Sensing

In this work, porous TiO₂ hollow spheres with an average diameter of 100 nm and shell thickness of 20 nm were synthesized by a facile hydrothermal method with NH₄HCO₃ as the structure-directing agent, and the formation mechanism for this porous hollow structure was proved to be the Ostwald ripening process by tracking the morphology of the products at different reaction stages. The product was characterized by SEM, TEM, XRD and BET analyses, and the results show that the as-synthesized products are anatase phase with a high surface area up to 132.5 m²/g. Gas-sensing investigation reveals that the product possesses sensitive response to methanal gas at 200°C due to its high surface area.     

A novel strategy for cartilage tissue engineering: Collagenase-loaded cryogel scaffolds in a sheep model

In this study, we developed a novel strategy, through which cartilage tissue pieces were placed in a sheep cartilage defect model and covered with a collagenase incorporated cryogel scaffold (in vivo cartilage tissue engineering, IVCTE group). While applying this strategy, the chondrocytes could be isolated inside the body and the treatment could be accomplished in one session. To compare our strategy, to another group, in which we used cultured cells and Chondro-gide, standard matrix-induced autologous chondrocyte implantation (MACI) was applied. Although the MACI applied group demonstrated better healing than IVCTE, the type II collagen synthesis was better in the IVCTE group compared to MACI applied group. Collagenase did not have detrimental effect on surrounding cartilage in IVCTE group. The preliminary results of the novel strategy applied group (IVCTE) were promising.     

In situ grown fibrous composites of poly(dl-lactide) and hydroxyapatite as potential tissue engineering scaffolds

In situ grown hydroxyapatite (HA) within electrospun poly(dl-lactide) (PDLLA) fibers were initially investigated as potential tissue engineering scaffolds with respect to the mechanical performances, biomineralization capability, degradation behaviors, cell growth and differentiation profiles. The tensile strength and Young’s moduli of in situ grown composites (IGC) were 8.2 ± 1.1 and 63.5 ± 5.6 MPa, respectively, which were significantly higher than those of blend electrospun composites (BEC) with 25.2% of HA inoculation. The interactions between HA and matrix polymers were approved by the red-shifts of CO stretching and OH- stretching modes and the increases in glass transition temperatures of fibrous composites. The localization of apatite phase on the fiber surface improved the biomineralization capability and enhanced the morphological stability of the fibers and fibrous mats even when the degradation of matrix polymers was detected. The cell viability and alkaline phosphatase levels were significantly higher for composites IGC, indicating favorable scaffolds for cell proliferation and osteogenic differentiation.     

The potential of unsaturated polyesters in biomedicine and tissue engineering: Synthesis,structure-properties relationships and additive manufacturing

The success of Tissue Engineering (TE) based approaches is strongly dependent on the development of novel biomaterials for the design of 3D matrices with tailored biomechanical properties to promote the regeneration of human tissues and organs.This review covers the critical aspects related with the preparation of new unsaturated polyester (UP) resin formulations with suitable biological, chemical, thermal and morphological properties for the additive manufacturing (AM) of TE constructs. In this context, the basic principles of available AM technologies, with a special focus on novel stereolithography processes such as microstereolithography (micro-SLA), stereo-thermal-lithography (STLA), two-photon polymerization (TPP) and nanostereolithography (nano-SLA), are also presented and discussed. Ultimately, the present review will provide a better insight into the limitations and potential of combining UP and AM towards the rationale design/fabrication of complex artificial tissue substitutes.     

Incorporating life cycle assessment and ecodesign tools for green chemical engineering: A case study of competences and learning outcomes assessment

Chemical engineers assume a broad range of roles in industry, spanning the development of new process designs, the maintenance and optimization of complex systems, and the production of intermediate materials, final products and new technologies. The technical aptitude that enables chemical engineers to fulfill these various roles along the value chain makes them compelling participants in the environmental assessment of the product in question. Therefore, the introduction of life cycle assessment (LCA) and ecodesign concepts into the chemical engineering curriculum is essential to help these future professionals to face design problems with a holistic view of the technical, economic, social and environmental impacts of their solutions. The teaching of these and other disciplines by means of student-centered methods, based on a holistic structure, have demonstrated better teamwork and communication skills. For that reason, this paper proposes a Micro (Assess-Analyze-Act) (M-3A) model of assessment mainly focused on closing the loop of the learning activities. This model has been applied to an ecodesign case study of the “University master’s Degree in chemical engineering” of the University of Cantabria/University of the Basque Country, with positive feedback of the students. They felt that the approach has allowed them to utilize their analytical skills in quantifying a situation before applying other subjective measures, and that the public discussion of the results was a satisfactory element for improving their communication skills. Moreover, the students found that the workload was nicely adjusted, highlighting the acquisition of 4 competences preferentially: teamwork, creativity; relevance of environmental issues and initiative and entrepreneurship. Finally, the students suggest that the application of this methodology into their degree could motivate future students improving their performance.     

Development of 3D in Vitro Technology for Medical Applications

In the past few years, biomaterials technologies together with significant efforts on developing biology have revolutionized the process of engineered materials. Three dimensional (3D) in vitro technology aims to develop set of tools that are simple, inexpensive, portable and robust that could be commercialized and used in various fields of biomedical sciences such as drug discovery, diagnostic tools, and therapeutic approaches in regenerative medicine. The proliferation of cells in the 3D scaffold needs an oxygen and nutrition supply. 3D scaffold materials should provide such an environment for cells living in close proximity. 3D scaffolds that are able to regenerate or restore tissue and/or organs have begun to revolutionize medicine and biomedical science. Scaffolds have been used to support and promote the regeneration of tissues. Different processing techniques have been developed to design and fabricate three dimensional scaffolds for tissue engineering implants. Throughout the chapters we discuss in this review, we inform the reader about the potential applications of different 3D in vitro systems that can be applied for fabricating a wider range of novel biomaterials for use in tissue engineering.     

High-Throughput Screening in Protein Engineering: Recent Advances and Future Perspectives

Over the last three decades, protein engineering has established itself as an important tool for the development of enzymes and (therapeutic) proteins with improved characteristics. New mutagenesis techniques and computational design tools have greatly aided in the advancement of protein engineering. Yet, one of the pivotal components to further advance protein engineering strategies is the high-throughput screening of variants. Compartmentalization is one of the key features allowing miniaturization and acceleration of screening. This review focuses on novel screening technologies applied in protein engineering, highlighting flow cytometry- and microfluidics-based platforms.     

Effect of irradiation on the proteinase inhibitor activity and digestibility (in vitro) of safflower oilcake

Trypsin and chymotrypsin inhibitor activities of safflower oilcake were studied before and after irradiation. The various doses to which samples were exposed ranged from 7 Gy to 10 kGy. The trypsin inhibitor is inactivated at 42 Gy, whereas the chymotrypsin inhibitor remains active, even at the much higher dose of 10 kGy. Thein vitro digestibility values also showed a significant improvement after irradiation. Exposure to a low dose of 42 Gy is sufficient to improve the nutritional value of the oilcake.     

Determination of cytocompatibility and osteogenesis properties of in situ forming collagen-based scaffolds loaded with bone synthesizing drug for bone tissue engineering

Bone tissue engineering using in situ forming 3D scaffolds can be an alternative to surgically treated scaffolds. This work aimed to develop in situ forming scaffolds using poly (lactic-co-glycolic acid) and a bone synthesizing drug (risedronate) with or without the porogenic agent (collagen). Hybrid scaffolds were formed through solvent-induced phase inversion technique and were morphologically evaluated using scanning electron microscopy (SEM). The effect of scaffolds on Saos-2 cell line viability using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test besides their effect on cell growth using fluorescence microscope was assessed. Furthermore, alkaline phosphatase (ALP) activity as well as Ca²⁺ deposition on the scaffolds was evaluated. SEM images revealed the porous structure for collagen-based scaffolds. Saos-2 cell proliferation was significantly enhanced with risedronate-loaded scaffolds compared to those lacking the drug. Porous collagen-based scaffolds were more favorable for both the cell growth and the promotion of ALP activity. Furthermore, collagen-based scaffolds promoted the Ca²⁺ deposition compared to their counterparts without collagen. Such results suggest that collagen-based scaffolds offer excellent biocompatibility for bone regeneration, where this biocompatible nature of scaffold leads to the proliferation of cells that lead to the deposition of mineral on the scaffold. Such in situ forming 3D scaffolds provide a promising noninvasive approach for bone tissue engineering.     

Budesonide-Loaded Guar Gum Microspheres for Colon Delivery: Preparation,Characterization and in Vitro/in Vivo Evaluation

A novel budesonide (BUD) colon delivery release system was developed by using a natural polysaccharide, guar gum. The rigidity of the microspheres was induced by a chemical cross-linking method utilizing glutaraldehyde as the cross-linker. The mean particle size of the microspheres prepared was found to be 15.21 ± 1.32 µm. The drug loading and entrapment efficiency of the formulation were 17.78% ± 2.31% and 81.6% ± 5.42%, respectively. The microspheres were spherical in shape with a smooth surface, and the size was uniform. The in vitro release profiles indicated that the release of BUD from the microspheres exhibited a sustained release behavior. The model that fitted best for BUD released from the microspheres was the Higuchi kinetic model with a correlation coefficient r = 0.9993. A similar phenomenon was also observed in a pharmacokinetic study. The prolongation of the half-life (t_1/2), enhanced residence time (mean residence time, MRT) and decreased total clearance (CL) indicated that BUD microspheres could prolong the acting time of BUD in vivo. In addition, BUD guar gum microspheres are thought to have the potential to maintain BUD concentration within target ranges for a long time, decreasing the side effects caused by concentration fluctuation, ensuring the efficiency of treatment and improving patient compliance by reducing dosing frequency. None of the severe signs, like the appearance of epithelial necrosis and the sloughing of epithelial cells, were detected.     

Zeta-potential and local filtration properties: Constitutive relationships for TiO2 from experimental filtration measurements

The use of filtration to separate solid from liquids is a commonly-used unit operation employed in a range of different applications. The development of accurate models is therefore important in facilitating e.g. design, scale-up and troubleshooting. Accurate constitutive relationships between local filtration properties need to be formulated in order to form precise models of the filtration process. One important parameter that is seldom considered explicitly in filtration models is the inter particle forces, e.g. electrostatic forces. The aim of this study is to investigate local filtration properties, under controlled conditions, for the model material TiO₂ where the ζ-potential, and thereby one important parameter affecting the inter-particle interactions, is altered. In this study the local solidosity is measured during filtration by γ-attenuation, the local pressure is measured using capillary tubes. These data are used to calculate the local specific filtration resistance. The compressibility of the filtration cake was changed substantially by altering the ζ-potential. Several published constitutive relationships between local data and specific filtration resistance were applied; they yielded very similar parameters that were in good accordance with the characterization of the material.     

Novel polyurethane‐based thermosensitive hydrogels as drug release and tissue engineering platforms: design and in vitro characterization

Poloxamer P407 (P407) is a Food and Drug Administration approved triblock copolymer; its hydrogels show fast dissolution in aqueous environment and weak mechanical strength, limiting their in vivo application. In this work, an amphiphilic poly(ether urethane) (NHP407) was synthesized from P407, an aliphatic diisocyanate (1,6‐hexanediisocyanate) and an amino acid derived diol (N‐Boc serinol). NHP407 solutions in water‐based media were able to form biocompatible injectable thermosensitive hydrogels with a lower critical gelation temperature behavior, having lower critical gelation concentration (6% w/v versus 18% w/v), superior gel strength (G′ at 37 °C about 40 000 Pa versus 10 000 Pa), faster gelation kinetics (<5 min versus 15–30 min) and higher stability in physiological conditions (28 days versus 5 days) compared to P407 hydrogels. Gel strength and PBS absorption at 37 °C increased whereas dissolution rate (in phosphate‐buffered saline (PBS) at 37 °C) and permeability to nutrients (studied using fluorescein isothiocyanate–dextran model molecule) decreased as a function of NHP407 hydrogel concentration from 10% to 20% w/v. By varying the concentration, NHP407 hydrogels were thus prepared with different properties which could suit specific applications, such as in situ drug/cell delivery or bioprinting of scaffolds. Moreover, deprotected amino groups in NHP407 could be exploited for the grafting of bioactive molecules obtaining biomimetic hydrogels. © 2016 Society of Chemical Industry     

Creep-resistant dextran-based polyurethane foam as a candidate scaffold for bone tissue engineering: Synthesis,chemico-physical characterization,and in vitro and in vivo biocompatibility

A highly crosslinked composite dextran-based scaffold (named DexFoam) was tailored to overcome specific deficiencies of polymeric and ceramic bone scaffolds and to guarantee a bone-mimicking microenvironment for the proliferation of human mesenchymal stem cells in vitro. The creep resistance for up to 90% compressive stain, the capability to regain the original shape after deformation, and the good thermal stability in both physiological and “body limit” conditions make DexFoam a valid alternative to the currently available bone scaffolds. Histopathological evaluation for host reaction and tissue colonization of DexFoam scaffold, implanted subcutaneously in mice, demonstrated its in vivo biocompatibility and biodegradability.     

Quercetin and epigallocatechin gallate induce in vitro a dose-dependent stiffening and hyperpolarizing effect on the cell membrane of human mononuclear blood cells

The bioactivity of polyphenols is closely linked to their ability to interact with biological membranes. The study evaluates the in vitro effect of quercetin and epigallocatechin on the membrane anisotropy and transmembrane potential of peripheral blood mononuclear cells (PBMCs) isolated from 26 type 2 diabetes mellitus patients compared to 25 age matched controls. The in vitro assays were analyzed in correlation with the biochemical and inflammatory profile of the subjects and with insulin resistance parameters (HOMA-IR, plasma resistin) as well. For type 2 diabetes patients, the increase of HOMA-IR and resistin concentration was associated with a significant decrease of the PBMCs membrane anisotropy. The two tested polyphenols induced a dose-dependent hyperpolarizing effect and stiffening of the cell membranes for all tested subjects. Physiological levels of quercetin and epigallocatechin gallate had the tendency to normalize the PBMCs membrane anisotropy of the cells isolated from diabetes patients, bringing it to the level of cells isolated from normoglycemic ones. Epigallocatechin gallate induced higher effects compared to quercetin on the membranes isolated from subjects with higher cardiovascular risk. The decrease of membrane fluidity and the hyperpolarizing effect could explain the cardiovascular protective action of the tested compounds.     

3,4,5-Trichloroaniline Nephrotoxicity in Vitro: Potential Role of Free Radicals and Renal Biotransformation

Chloroanilines are widely used in the manufacture of drugs, pesticides and industrial intermediates. Among the trichloroanilines, 3,4,5-trichloroaniline (TCA) is the most potent nephrotoxicant in vivo. The purpose of this study was to examine the nephrotoxic potential of TCA in vitro and to determine if renal biotransformation and/or free radicals contributed to TCA cytotoxicity using isolated renal cortical cells (IRCC) from male Fischer 344 rats as the animal model. IRCC (~4 million cells/mL; 3 mL) were incubated with TCA (0, 0.1, 0.25, 0.5 or 1.0 mM) for 60–120 min. In some experiments, IRCC were pretreated with an antioxidant or a cytochrome P450 (CYP), flavin monooxygenase (FMO), cyclooxygenase or peroxidase inhibitor prior to incubation with dimethyl sulfoxide (control) or TCA (0.5 mM) for 120 min. At 60 min, TCA did not induce cytotoxicity, but induced cytotoxicity as early as 90 min with 0.5 mM or higher TCA and at 120 min with 0.1 mM or higher TCA, as evidenced by increased lactate dehydrogenase (LDH) release. Pretreatment with the CYP inhibitor piperonyl butoxide, the cyclooxygenase inhibitor indomethacin or the peroxidase inhibitor mercaptosuccinate attenuated TCA cytotoxicity, while pretreatment with FMO inhibitors or the CYP inhibitor metyrapone had no effect on TCA nephrotoxicity. Pretreatment with an antioxidant (α-tocopherol, glutathione, ascorbate or N-acetyl-l-cysteine) also reduced or completely blocked TCA cytotoxicity. These results indicate that TCA is directly nephrotoxic to IRCC in a time and concentration dependent manner. Bioactivation of TCA to toxic metabolites by CYP, cyclooxygenase and/or peroxidase contributes to the mechanism of TCA nephrotoxicity. Lastly, free radicals play a role in TCA cytotoxicity, although the exact nature of the origin of these radicals remains to be determined.     

Stimulations of strontium-doped calcium polyphosphate for bone tissue engineering to protein secretion and mRNA expression of the angiogenic growth factors from endothelial cells in vitro

The vascularization of tissue-engineered bone is the key problem needed solving before application of tissue-engineered bone in clinical practice. Meanwhile, endothelial cells are the major and important source of seed cells in bone tissue engineering, and significant on promoting vascularization in tissue-engineered bone. Vascularization (namely angiogenesis) is a process mainly controlled by several angiogenic growth factors (VEGF, bFGF and MMP-2) which can be secreted by endothelial cells. Therefore, the research on the stimulations of SCPP to the secretion of the angiogenic growth factors from endothelial cells is very important. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the protein secretion levels and mRNA expression of VEGF, bFGF and MMP-2 from cultured endothelial cells. As a control, we also researched the effect of HA on the mRNA expressions and protein secretion of angiogenic growth factors from cultured endothelial cells. We cultured endothelial cells with SCPP scaffolds containing various concentration of strontium and HA. The results obtained in the MTT and SEM tests indicated that endothelial cells on SCPP scaffold exhibited higher proliferation rate and were easy to get a good spread than them on CPP, the best state of growth and proliferation of cells could be observed on 8%SCPP. The results of ELISA demonstrated that the protein levels of VEGF, bFGF and MMP-2 from cultured endothelial cells increased with the increasing Sr doped in calcium polyphosphate in SCPP groups, the peaks appeared on 8%SCPP. All SCPP groups showed a better ability to stimulate the protein secretion of VEGF, bFGF and MMP-2 from endothelial cells relative to CPP group and HA group. The results of RT-PCR suggested that the 8%SCPP group exhibited a significantly higher mRNA expression of VEGF, bFGF and MMP-2 relative to CPP group and HA group. In conclusion, the results of this study demonstrated that 8%SCPP had obvious promotion for secretion and mRNA expression of angiogenic growth factors from cultured endothelial cells.