SAXS imaging reveals optimized osseointegration properties of bioengineered oriented 3D-PLGA/aCaP scaffolds in a critical size bone defect model

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca$^{2+}$ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca$^{2+}$ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell-material interaction, the material-medium interaction was also important for the stem cell commitment here, affecting the cell-medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge

Catalyst and Process Design for the Continuous Manufacture of Rare Sugar Alcohols by Epimerization– Hydrogenation of Aldoses

Sugar alcohols are applied in the food, pharmaceutical, poly- mer, and fuel industries and are commonly obtained by reduc- tion of the corresponding saccharides. In view of the rarity of some sugar substrates, epimerization of a readily available monosaccharide has been proposed as a solution, but an effi- cient catalytic system has not yet been identified. Herein, a mo- lybdenum heteropolyacid-based catalyst is developed to trans- form glucose, arabinose, and xylose into less-abundant man- nose, ribose, and lyxose, respectively. Adsorption of molybdic acid onto activated carbon followed by ion exchange to the cesium form limits leaching of the active phase, which greatly improves the catalyst stability over 24 h on stream. The hydro- genation of mixtures of epimers is studied over ruthenium cat- alysts, and it is found that the precursor to the desired polyol is advantageously converted with faster kinetics. This is ex- plained by density functional theory on the basis of its more favorable adsorption on the metal surface and the lower energy barrier for the addition of a hydrogen atom to the pri- mary carbon atom. Finally, different designs for a continuous process for the conversion of glucose into mannitol are stud- ied, and it is uncovered that two reactors in series with one containing the epimerization catalyst and the other containing a mixture of the epimerization and hydrogenation catalysts in- creases the mannitol/sorbitol ratio to 1.5 from 1 for a single mixed-bed reactor. This opens a prospective route to the effi- cient valorization of renewables to added-value chemicals

Methanol-essential growth of Escherichia coli

Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically coupled to the metabolization of gluconate and the designed strain was subjected to laboratory evolution experiments. Evolved strains incorporate up to 24% methanol into core metabolites under a co-consumption regime and utilize methanol at rates comparable to natural methylotrophs. Genome sequencing reveals mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.ISSN:2041-172

Knowledge Transfer in Support of the Development of Oxygen Concentrators in Emergency Settings During the COVID-19 Pandemic

The COVID-19 pandemic simultaneously disrupted supply chains and generated an urgent demand in medical infrastructure. Among personal protective equipment and ventilators, there was also an urgent demand for chemical oxygen. As devices to purify oxygen could not be manufactured and shipped rapidly enough, a simple and accessible oxygen concentrator based on pressure swing adsorption was developed at ETH Zurich in spring 2020. Instead of building devices locally and shipping them, it was decided to educate others in need of oxygen. The implementation encompassed education on process chemistry, material choice, and assembly and optimization of the concentrator and was realized using synchronous teaching tools, such as video call, and asynchronous ones, such as a website and video streaming. The project gained traction and interaction with engineering teams from universities and non-Governmental Organizations (Red Cross and the UN Development Program) in developing countries and emerging market economies, including Ecuador, Mexico, Somalia, and Peru. At the end of the project, the teams were surveyed regarding their experience in the educative knowledge transfer. It was reported that the learning experience prepared these groups well to build the device and to teach others as well. Major challenges were accessing some parts of the device and optimizing its performance. While synchronous communication is expected to be a very effective teaching method, the survey results showed that explanations via a website and video streaming have contributed the most to the implementation of the oxygen concentrator and thereby provide autonomous and sustainable education tools.ISSN:0021-9584ISSN:1938-132

3D microtissue–derived human stem cells seeded on electrospun nanocomposites under shear stress: Modulation of gene expression

Objective Different microenvironments trigger distinct differentiation of stem cells. Even without chemical supplementation, mechanical stimulation by shear stress may help to induce the desired differentiation. The cell format, such as three-dimensional (3D) microtissues (MTs), MT-derived cells or single cells (SCs), may have a pivotal impact as well. Here, we studied modulation of gene expression in human adipose–derived stem cells (ASCs) exposed to shear stress and/or after MT formation. Materials and methods Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) at a weight ratio of 60:40 were seeded with human ASCs as MTs or as SCs and cultured in Dulbecco's modified Eagle's medium without chemical supplementation. After 2 weeks of static culture, the scaffolds were cultured statically for another 2 weeks or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm−2 min−1. Stiffness of the scaffolds was assessed as a function of time. After 4 weeks, minimum stem cell criteria markers and selected markers of osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time polymerase chain reaction. Additionally, cell distribution within the scaffolds and the allocation of the yes-associated protein (YAP) in the cells were assessed by immunohistochemistry. Results MTs decayed completely within 2 weeks after seeding on PLGA/aCaP. The osteogenic marker gene alkaline phosphatase and the endothelial cell marker gene CD31 were upregulated in MT-derived ASCs compared with SCs. Shear stress realised by fluid flow perfusion upregulated peroxisome proliferator–activated receptor gamma 2 expression in MT-derived ASCs and in SCs. The nuclear-to-cytoplasmic ratio of YAP expression was doubled under perfusion compared with that under static culture for MT-derived ASCs and SCs. Conclusions Osteogenic and angiogenic commitments were more pronounced in MT-derived ASCs seeded on bone biomimetic electrospun nanocomposite PLGA/aCaP than in SCs seeded without induction medium. Furthermore, the static culture was superior to the perfusion regimen used here, as shear stress resulted in adipogenic commitment for MT-derived ASCs and SCs, although the YAP nuclear-to-cytoplasmic ratio indicated higher cell tensions under perfusion, usually associated with preferred osteogenic differentiation

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge

Hybrid nanocomposite as a chest wall graft with improved vascularization by copper oxide nanoparticles

Chest wall repair can be necessary after tumor resection or chest injury. In order to cover or replace chest wall defects, autologous tissue or different synthetic materials are commonly used, among them the semi-rigid gold standard Gore-Tex® and prolene meshes. Synthetic tissues include composite materials with an organic and an inorganic component. On the basis of previously reported hybrid nanocomposite poly-lactic-co-glycolic acid amorphous calcium phosphate nanocomposite (PLGA/aCaP), a CuO component was incorporated to yield (60%/35%/5%). This graft was tested in vitro by seeding with murine adipose-derived stem cells (ASCs) for cell attachment and migration. The graft was compared to PLGA/CaCO$_{3}$ and PLGA/hydroxyapatite, each providing the inorganic phase as nanoparticles. Further characterization of the graft was performed using scanning electron microscopy. Furthermore, PLGA/aCaP/CuO was implanted as a chest wall graft in mice. After 4 weeks, total cell density, graft integration, extracellular matrix components such as fibronectin and collagen I, the cellular inflammatory response (macrophages, F4/80 and lymphocytes, CD3) as well as vascularization (CD31) were quantitatively assessed. The nanocomposite PLGA/aCaP/CuO showed a good cell attachment and cells migrated well into the pores of the electrospun meshes. Cell densities did not differ between PLGA/aCaP/CuO and PLGA/CaCO$_{3}$ or PLGA/hydroxyapatite, respectively. When applied as a chest wall graft, adequate stability for suturing into the thoracic wall could be achieved. Four weeks post-implantation, there was an excellent tissue integration without relevant fibrotic changes and a predominating collagen I matrix deposition within the graft. Slightly increased inflammation, reflected by increased infiltration of macrophages could be observed. Vascularization of the graft was significantly enhanced when compared with PLGA/aCaP (no CuO). We conclude that the hybrid nanocomposite PLGA/aCaP/CuO is a viable option to be used as a chest wall graft. Surgical implantation of the material is feasible and provides stability and enough flexibility. Proper tissue integration and an excellent vascularization are characteristics of this biodegradable material