Michael Gasik

Reduction of Biofilm Infection Risks and Promotion of Osteointegration for Optimized Surfaces of Titanium Implants

Titanium-based implants are widely used in modern clinical practice; however, complications associated with implants due to bacterial-induced infections arise frequently, caused mainly by staphylococci, streptococci, Pseudomonas spp. and coliform bacteria. Although increased hydrophilicity of the biomaterial surface is known to be beneficial in minimizing the biofilm, quantitative analyses between the actual implant parameters and bacterial development are scarce. Here, the results of in vitro studies of Staphylococcus aureus and Staphylococcus epidermidis proliferation on uncoated and coated titanium materials with different roughness, porosity, topology, and hydrophilicity are shown. The same materials have been tested in parallel with respect to human osteogenic and endothelial cell adhesion, proliferation, and differentiation. The experimental data processed by meta-analysis are indicating the possibility of decreasing the biofilm formation by 80-90% for flat substrates versus untreated plasma-sprayed porous titanium and by 65-95% for other porous titanium coatings. It is also shown that optimized surfaces would lead to 10-50% enhanced cell proliferation and differentiation versus reference porous titanium coatings. This presents an opportunity to manufacture implants with intrinsic reduced infection risk, yet without the additional use of antibacterial substances.

Finite element analysis of the residual thermal stresses on functionally gradated dental restorations

The aim of this work was to study, using the finite element method (FEM), the distribution of thermal residual stresses arising in metal-ceramic dental restorations after cooling from the processing temperature. Three different interface configurations were studied: with conventional sharp transition; one with a 50% metal-50% ceramic interlayer; and one with a compositionally functionally gradated material (FGM) interlayer. The FE analysis was performed based on experimental data obtained from Dynamic Mechanical Analysis (DMA) and Dilatometry (DIL) studies of the monolithic materials and metal/ceramic composites. Results have shown significant benefits of using the 50% metal-50% ceramic interlayer and the FGM interlayer over the conventional sharp transition interface configuration in reduction of the thermal residual stress and improvement of stress profiles. Maximum stresses magnitudes were reduced by 10% for the crowns with 50% metal-50% ceramic interlayer and by 20% with FGM interlayer. The reduction in stress magnitude and smoothness of the stress distribution profile due to the gradated architectures might explain the improved behavior of these novel dental restorative systems relative to the conventional one, demonstrated by in-vitro studies already reported in literature.

Novel process concept for the production of H 2 and H 2 SO 4 by SO 2 -depolarized electrolysis

Outotec open cycle (OOC) is a new low-energy process linking together production of hydrogen and sulfuric acid. While sulfuric acid is the world’s most widely produced chemical by mass at approximately 200 Mt/a, the OOC gives the potential for making 4 Mt/a of hydrogen gas as a by-product. H2SO4 manufacture requires a source of sulfur dioxide. 30% of world production of H2SO4 is from the SO2 by-product of pyrometallurgical processing of sulfur containing concentrates of metals such as copper, nickel and zinc. SO2 can also be made by direct combustion of sulfur. In OOC, a divided electrochemical cell is used for SO2-depolarized electrolysis of water. SO2 is fed to the anolyte and converted to H2SO4, while hydrogen gas is produced at the cathode. On the industrial scale, the equipment will be in the form of a membrane electrolyzer assembly or stack. A case is described where the OOC would be connected to a pyrometallurgical plant smelting 1 Mt/a of nickel and copper concentrate, producing 1 Mt/a of H2SO4 and 20 kt/a of hydrogen.

Graphene-augmented nanofiber scaffolds demonstrate new features in cells behaviour.

Three-dimensional (3D) customized scaffolds capable to mimic a native extracellular matrix open new frontiers in cells manipulation and advanced therapy. The major challenge is in a proper substrate for in vitro models on engineered scaffolds, capable to modulate cells differentiation. Here for the first time we demonstrate novel design and functionality of the 3D porous scaffolds of aligned, self-assembled ceramic nanofibers of ultra-high anisotropy ratio (~107), augmented into graphene shells. This unique hybrid nano-network allows an exceptional combination of selective guidance stimuli of stem cells differentiation, immune reactions variations, and local immobilization of cancer cells, which was not available before. The scaffolds were shown to be able to direct human mesenchymal stem cells (important for stimulation of neuronal and muscle cells) preferential orientation, to suppress major inflammatory factors, and to localize cancer cells; all without additions of specific culture media. The selective downregulation of specific cytokines is anticipated as a new tool for understanding of human immune system and ways of treatment of associated diseases. The effects observed are self-regulated by cells only, without side effects, usually arising from use of external factors. New scaffolds may open new horizons for stem cells fate control such as towards axons and neurites regeneration (Alzheimer’s disease) as well as cancer therapy development.

SO2 carry-over and sulphur formation in a SO2-depolarized electrolyser

SO2-depolarized electrolysis (SDE) is considered as one of the most efficient hydrogen production methods. To maximize H2 production in SDE, the phenomena occurring in the cell need to be understood and controlled. In this work, electrochemically driven SO2 carry-over and elemental sulphur formation in cathode space are analysed by various methods: electrochemical, titration and photon correlation spectroscopy (PCS) under different conditions during SDE operation. The results indicate that SO2 carry-over is a fast process in an operating SDE and is difficult to control with only working parameter optimization. The PCS method provides SO2 carry-over information at early stages compared to other methods. Moreover, PCS might be further implemented as an in situ method for SDE system control.

Silica-gentamicin nanohybrids: Synthesis and antimicrobial action

Orthopedic applications commonly require the administration of systemic antibiotics. Gentamicin is one of the most commonly used aminoglycosides in the treatment and prophylaxis of infections associated with orthopedic applications, but gentamicin has a short half-life. However, silica nanoparticles (SiO2 NPs) can be used as elegant carriers for antibiotics to prolong their release. Our goal is the preparation and characterization of SiO2-gentamicin nanohybrids for their potential antimicrobial administration in orthopedic applications. In vitro gentamicin release profile from the nanohybrids (gentamicin-conjugated SiO2 NPs) prepared by the base-catalyzed precipitation exhibited fast release (21.4%) during the first 24 h and further extension with 43.9% release during the five-day experiment. Antimicrobial studies of the SiO2-gentamicin nanohybrids versus native SiO2 NPs and free gentamicin were performed against Bacillus subtilis (B. subtilis), Pseudomonas fluorescens (P. fluorescens) and Escherichia coli (E. coli). SiO2-gentamicin nanohybrids were most effective against B. subtilis. SiO2 NPs play no antimicrobial role. Parallel antimicrobial studies for the filter-sterilized gentamicin were performed to assess the effect of ultraviolet (UV)-irradiation on gentamicin. In summary, the initial fast gentamicin release fits the need for high concentration of antibiotics after orthopedic surgical interventions. Moreover, the extended release justifies the promising antimicrobial administration of the nanohybrids in bone applications.

Hybrid graphene–Ceramic nanofibre network for spontaneous neural differentiation of stem cells

A challenge in regenerative medicine is governed by the need to have control over the fate of stem cells that is regulated by the physical and chemical microenvironment in vitro and in vivo. The differentiation of the stem cells into specific lineages is commonly guided by use of specific culture media. For the first time, we demonstrate that human mesenchymal stem cells are capable of turning spontaneously towards neurogenic lineage when seeded on graphene-augmented, highly anisotropic ceramic nanofibres without special differentiation media, contrary to commonly thought requirement of ‘soft’ substrates for the same purpose. Furthermore, pro-inflammatory gene expression is simultaneously suppressed, and expression of factors promoting focal adhesion and monocytes taxis is upregulated. This opens new possibilities of using local topo-mechanical cues of the ‘graphenized’ scaffold surfaces to guide stem cell proliferation and differentiation, which can be used in studies of neurological diseases and cell therapy.

Understanding biomaterial-tissue interface quality: combined in vitro evaluation

Abstract One of the greatest challenges in the development of new medical products and devices remains in providing maximal patient safety, efficacy and suitability for the purpose. A ‘good quality’ of the tissue-implant interface is one of the most critical factors for the success of the implant integration. In this paper this challenge is being discussed from the point of view of basic stimuli combination to experimental testing. The focus is in particular on bacterial effects on tissue-implant interaction (for different materials). The demonstration of the experimental evaluation of the tissue-implant interface is for dental abutment with mucosal contact. This shows that testing of the interface quality could be the most relevant in controlled conditions, which mimic as possible the clinical applications, but consider variables being under the control of the evaluator.

A new approach for modelling lattice energy in finite crystal domains

Evaluation of internal energy in a crystal lattice requires precise calculation of lattice sums. Such evaluation is a problem in the case of small (nano) particles because the traditional methods are usually effective only for infinite lattices and are adapted to certain specific potentials. In this work, a new method has been developed for calculation of lattice energy. The method is a generalisation of conventional geometric probability techniques for arbitrary fixed lattices in a finite crystal domain. In our model, the lattice energy for wide range of two- body central interaction potentials (including long-range Coulomb potential) has been constructed using absolutely convergent sums. No artificial cut-off potential or periodical extension of the domain (which usually involved for such calculations) have been made for calculation of the lattice energy under this approach. To exemplify the applications of these techniques, the energy of Coulomb potential has been plotted as the function of the domain size.

New BEST - biomaterials enhanced simulation test

H cartilage is a critical tissue to normal articular function. Natural restoring of this tissue in patients with rheumatoid arthritis, osteoarthritis or traumas is extremely limited considering its low regenerative capacity. These conditions cause an enormous constraint to the daily routine and, consequently, lower quality of life and they are managed, according with severity, through the use of pharmaceutics, surgery, transplant or prosthetic procedures. Tissue Engineering (TE) emerges as a new promising tool to provide long lasting regenerating solutions. In this work we used the combination of 3D extruded-based scaffolds made of poly (e-caprolactone) (PCL) and human bone marrow-derived mesenchymal stem cells (MSC) to provide initial higher cell densities, followed by differentiation in chondrocytes. The aim of this study is to understand the influence in cell behavior of scaffolds manufactured by layer-by-layer extrusion with a pore size (190 – 390 μm) and fiber alignments (045o and 0-90o), as well as of two different atmospheric conditions, normoxia (21% O2) and hypoxia (5% O2). Results obtained showed that pore size and fiber alignment do not pose a limitation for cell adhesion and proliferation within the range of pore sizes studied. However, cell distribution and dimension of chondrocyte aggregates strongly depend on fiber alignment. The results obtained in this study point out that higher chondrocyte population is obtained, when previously to differentiation stage, MSC are expanded in the PCL under hypoxia condition, rather than normoxia.O group at the Translational Centre for Regenerative Medicine in Leipzig developed a method for melanocyte cultivation from the Outer Root Sheath(ORS) by expanding stem cells, precursors and dedicated cells and differentiating them into melanocytes. Based on this methodology, we are developing an autologous, transplantation-based, causal treatment of depigmentation disorders such as Vitiligo. In the scope of cultivation, we are tracking shifts in gene expression profiles, from pluripotency in undifferentiated cells to melanotic status in late differentiation stages of the ORS melanocyte culture.Introduction: Spermidine is a naturally occurring polyamine involved in multiple biological processes, including DNA metabolism, autophagy and aging. Spermidine induces autophagy in cultured yeast and mammalian cells, as well as in nematodes and flies. Genetic inactivation of genes essential for autophagy abolishes the life span-prolonging effect of spermidine in yeast, nematodes and flies. These findings complement expanding evidence that autophagy mediates cytoprotection against a variety of noxious agents and can confer longevity when induced at the whole-organism level. In an acute burn injury the zone of stasis is initially vital but may progress to coagulation necrosis with time. In this experimental study, we hypothesized that apoptosis, autophagy induced by spermidine plays a role in burn wound progression.M clinical trials are underway to evaluate the use of adipose tissue-derived stromal cells (hASC) for regenerative medicine. Nevertheless, prior to its clinical application, hASC need ex vivo expansion in compliance with GMP guidelines in order to obtain a suitable amount of cells. Fetal bovine serum (FBS) is the most widely used serum in standard culture conditions, but the use of animal origin components bears disadvantages and is not an option in clinical cell therapy due to several safety issues. So our aim was to show the effectiveness of pooled allogeneic human serum (aHS) as a supplement for hASC culturing. In this study, the hASC were expanded in DMEM supplemented with aHS and the immunophenotype and differentiation potential of the cells were evaluated. The first difference observed between FBS and aHS cultures was that hASC in aHS proliferated markedly faster than in FBS, which was confirmed by Ki-67 expression, the cumulative population doubling, and by a growth curve. To better understand this hASC expansion pattern, the transcription factors C-myc and C-fos as well as mRNA levels of hTERT, and pluripotency genes (OCT-4 and NANOG) were assessed. No significant expression was observed and the hASC cultured in aHS underwent replicative senescence with normal karyotype. These cells assayed were not able to generate a teratoma. Therefore, this study confirms and extends the benefits of rapid expansion of hASC in culture media supplemented with aHS, with the cells maintaining their phenotype and with no spontaneous cell transformation.A is characterized by an impairment of muscle regenerative potential and a progressive loss of skeletal muscle. This condition, called sarcopenia, has important health care implications for humans, as it contributes to frailty, functional loss and premature death. The ability of skeletal muscle to regenerate is owed to a population of myogenic stem cells called satellite cells (SCs). In previous papers of our research group we demonstrated an age-related decrease in the antioxidant capacity of human SCs that may negatively affect the ageing SCs ability to repair muscle. Despite the involvement of caspases in muscle wasting, the real role of these enzymes is still controversial. To test the possibility of caspases involvement in SCs death in human ageing muscles, we cultured in vitro primary cells derived from vastus lateralis of young and aged subjects. We analysed both initiator caspases and effector caspases in the presence or absence of specific or broad pharmacologic inhibitors. Furthermore, we evaluated the expression of various genes that play a critical role in oxidative stress and cell death. Our findings highlighted an increased rate of spontaneous apoptosis and an up-regulation of CASP2, 6, 7, 9 and other cell death genes in aged SCs, supporting the hypothesis of an intrinsic ageing of SCs and previous reports demonstrating an increased susceptibility to apoptosis of SCs from old animals. These results suggest that a greater proportion of SCs from old subjects might undergo programmed cell death in vivo in response to stressful stimuli, thereby impairing skeletal muscle regeneration.P migration of stem cells, either endogenous or transplanted, is crucially important for embryonic development, homeostasis in adults, and tissue repair after injury. However, the detailed mechanisms of the directed migration of these cells are not clear. During the past few years, our study showed that the differentiating mesenchymal stem cells (MSCs) possess different migratory capacity and the chemotactic responses of these cells correlates closely with their differentiation states. Accordingly, the formation and the asymmetrical distribution of focal adhesions (FAs) between the leading lamella and the cell rear, the phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as the turnover of FAs varied greatly in differentiating MSCs, leading to the most effective chemotactic responses of MSCs in certain differentiation states. Further, we demonstrated that signaling through PI3K/Akt and MAPKs are involved in regulating the directed migration of MSCs. More importantly, we found that beta-catenin signaling is prerequisite for the chemotactic migration of MSCs. In this talk, I will summarize our data regarding the regulatory effects of beta-catenin signaling on MSCs that undergo chemotaxis.Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.T is a pressing global medical need for the development of tissue-engineered alternatives to supplement the scarce supply of human donor corneal tissue for transplantation and vision restorative therapy. There are currently over 10 million people blind due to corneal scarring or disease, with 1-2 million new cases of corneal blindness arising annually. To meet this enormous global demand, our research has focused on developing transparent, tissue-engineered collagen-based hydrogels for implantation in the cornea. In preclinical studies, designs have evolved with respect to collagen composition and use of various synthetic cross-linking agents. Utilizing advanced in vivo imaging techniques such as optical coherence tomography and in vivo confocal microscopy, we have developed methods to visualize and longitudinally quantify bio-integration, wound healing, and cellular and neural compatibility of the biomaterial implants in rabbit and pig models, in real time. The implantable biomaterials and imaging techniques have also been extended into the clinic, where 10 patients received the first tissueengineered corneal substitutes, and were followed and documented extensively for over six years postoperatively. In more recent work, our clinical experiences have resulted in refined designs of biomaterials and surgical implantation techniques, with a focus on robustness of materials in the operating room and optimization of the degradation profile of biomaterials in vivo, to facilitate host cell invasion and regeneration of tissue. Considerations of cost, raw material availability, and safety for human use are also becoming increasingly important as future efforts are aiming towards large-scale production of tissueengineered corneas to meet an increasing global demand.