Soumyaranjan Mohanty

Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds.

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive.

Fabrication of scalable tissue engineering scaffolds with dual-pore microarchitecture by combining 3D printing and particle leaching.

Limitations in controlling scaffold architecture using traditional fabrication techniques are a problem when constructing engineered tissues/organs. Recently, integration of two pore architectures to generate dual-pore scaffolds with tailored physical properties has attracted wide attention in tissue engineering community. Such scaffolds features primary structured pores which can efficiently enhance nutrient/oxygen supply to the surrounding, in combination with secondary random pores, which give high surface area for cell adhesion and proliferation. Here, we present a new technique to fabricate dual-pore scaffolds for various tissue engineering applications where 3D printing of poly(vinyl alcohol) (PVA) mould is combined with salt leaching process. In this technique the sacrificial PVA mould, determining the structured pore architecture, was filled with salt crystals to define the random pore regions of the scaffold. After crosslinking the casted polymer the combined PVA-salt mould was dissolved in water. The technique has advantages over previously reported ones, such as automated assembly of the sacrificial mould, and precise control over pore architecture/dimensions by 3D printing parameters. In this study, polydimethylsiloxane and biodegradable poly(ϵ-caprolactone) were used for fabrication. However, we show that this technique is also suitable for other biocompatible/biodegradable polymers. Various physical and mechanical properties of the dual-pore scaffolds were compared with control scaffolds with either only structured or only random pores, fabricated using previously reported methods. The fabricated dual-pore scaffolds supported high cell density, due to the random pores, in combination with uniform cell distribution throughout the scaffold, and higher cell proliferation and viability due to efficient nutrient/oxygen transport through the structured pores. In conclusion, the described fabrication technique is rapid, inexpensive, scalable, and compatible with different polymers, making it suitable for engineering various large scale organs/tissues.

Electrosynthesis of acetate from CO2 by a highly structured biofilm assembled with reduced graphene oxide–tetraethylene pentamine

Microbes can reduce CO2 into multicarbon chemicals with electrons acquired from the cathode of a bioelectrochemical reactor. This bioprocess is termed microbial electrosynthesis (MES). One of the main challenges for the development of highly productive MES reactors is achieving efficient electron transfer from the cathode to microbes. Here, carbon cloth cathodes modified with reduced graphene oxide functionalized with tetraethylene pentamine (rGO-TEPA) were readily self-assembled in the cathodic chamber of a MES reactor. Electroactive biofilms with unique spatial arrangement were subsequently formed with Sporomusa ovata at the surface of rGO-TEPA-modified electrodes resulting in a more performant MES process. The acetate production rate from CO2 was increased 3.6 fold with the formation of dense biofilms when wild type S. ovata was combined with rGO-TEPA. An improvement of 11.8 fold was observed with a highly structured biofilm including multiple spherical structures possibly consisting of bioinorganic networks of rGO-TEPA and bacterial cells from a novel strain of S. ovata adapted to reduce CO2 faster. The three dimensional biofilms observed in this study enabled highly effective electric interactions between S. ovata and the cathode, demonstrating that the development of dense cathode biofilms is an effective approach to improve MES productivity.

3D Printed Silicone–Hydrogel Scaffold with Enhanced Physicochemical Properties

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

Engineering complex tissue-like microgel arrays for evaluating stem cell differentiation

Development of tissue engineering scaffolds with native-like biology and microarchitectures is a prerequisite for stem cell mediated generation of off-the-shelf-tissues. So far, the field of tissue engineering has not full-filled its grand potential of engineering such combinatorial scaffolds for engineering functional tissues. This is primarily due to the many challenges associated with finding the right microarchitectures and ECM compositions for optimal tissue regeneration. Here, we have developed a new microgel array to address this grand challenge through robotic printing of complex stem cell-laden microgel arrays. The developed microgel array platform consisted of various microgel environments that where composed of native-like cellular microarchitectures resembling vascularized and bone marrow tissue architectures. The feasibility of our array system was demonstrated through localized cell spreading and osteogenic differentiation of human mesenchymal stem cells (hMSCs) into complex tissue-like structures. In summary, we have developed a tissue-like microgel array for evaluating stem cell differentiation within complex and heterogeneous cell microenvironments. We anticipate that the developed platform will be used for high-throughput identification of combinatorial and native-like scaffolds for tissue engineering of functional organs.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

Hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions and to perform functional comparisons with fresh human precision‐cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis in the hiPSC‐derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels comparable with hPCLS indicating excellent future perspectives for the development of a bioartificial liver system for toxicity testing or as liver support device for patients.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Impedance-based monitoring for tissue engineering applications

Impedance is a promising technique for sensing the overall process of tissue engineering. Different electrode configurations can be used to characterize the scaffold that supports cell organization in terms of hydrogel polymerization and degree of porosity, monitoring cell loading, cell proliferation as well as the spatial distribution of cell aggregates in 3D. We have previously shown that impedance measurements allow accurate determination of conductivity in physiological solutions independent of validation and analysis of a specific equivalent circuit. Similarly to a physiological solution, cell culture medium conductivity, and hence the measured impedance, can respond to proliferating or dying cells populating the scaffold. Impedance may therefore be a key parameter for monitoring the biochemical dynamics that modulate 3D mammalian cell cultures over time. Furthermore, the conductivity of the medium filling the pores of the scaffold can serve as the basis for porosity determination using Archie’s law. Different networks of structured or random channels and degree of porosity can be detected. In addition, by combining a number of two-, three- and four-terminal (2T, 3T, 4T) configurations, it is possible to obtain complementary information on spatial distribution of cells in a 3D scaffold. 2T- and 3T configurations also reflect the impedance at the interface between an electrode and cell-loaded scaffold (polarization impedance, Zp), which may convey a further degree of information about the biochemical phenomena taking place in that sub-volume.

Hepatic differentiation of human induced pluripotent stem cells in a perfused 3d porous polymer scaffold for liver tissue engineering

B design and 3-D printing of scaffold with heterogeneous internal geometry is essential for cell distribution, blood vessel in growth and biomaterial degradation in bone tissue engineering. This study was designed to demonstrate the heterogeneous pores and channels in 3-D printed scaffolds for bone tissue engineering. Scaffolds were prepared using ceramic particles through 3-D printing. Pores and connecting channels with diameters of 200mm-500mm were designed for facilitating cell seeding and cell distribution. Internal pores of 50mm-200mm were designed for bone regeneration. Nano-sized surface topography was designed for enhanced degradation of scaffold. The fabricated scaffolds were evaluated using scanning electronic microscopy. SEM of fabricated scaffolds revealed that 400mm500mm inter-connecting channels crossed over the entire scaffold, that ~200 mm internal pores were scattered over the scaffold and connected to each other and to the interconnecting channels, and that ~200 nm pores showed on the surfaces of inter-connecting channels and internal pores, which would play an important role in increasing the surface ratio of materials and facilitating material degradation. A heterogeneous profile of connecting channels and internal pores was evident in these 3-D printed biomimetic scaffolds. As a conclusion, the biomimetic design and fabrication of scaffolds for bone tissue engineering can be fulfilled by a 3-D printing process. Heterogeneous profiles of inter-connecting channels, internal pores, and nano-sized surface topography can be generated to provide a biomimetic environment suitable for bone tissue engineering.Background: Myocardial infarction (MI) was the leading cause of death in worldwide. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of biological processes, including cell viability, proliferation, development and differentiation. The purpose of this study was to investigate difference of miRNA profiles between infarct zone and border zone in post-MI remodeling using the second generation sequencing.Neia Naldaiz-Gastesi1, Patricia Garcia-Parra1, Maria Goicoechea1, Sonia Alonso-Martin2, Ana Aiastui1, Macarena Lopez-Mayorga3, Paula Garcia-Belda4, Jaione Lacalle1,5, Veronique Le Berre6, Ander Matheu1, Jose Manuel Garcia-Verdugo4, Jaime J. Carvajal3, Frederic Relaix2, Adolfo Lopez de Munain1 and Ander Izeta1 1Instituto Biodonostia, Spain 2Myology Research Center, France 3Centro Andaluz de Biologia del Desarrollo, Spain 4Instituto Cavanilles, Universidad de Valencia, Spain 5University of the Basque Country (UPV-EHU), Spain 6UMR INSA, FranceOur knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONSS cells have been recognized as a potential tool to restore cells damaged by cerebral ischemic injury. Key functions such as the replacement of neural cells have been recently challenged by intrinsic bystander capacities of undifferentiated donor cells. One of opportunity for neurological disorder treatment is the transplantation of mesenchymal stem cells (MSCs) which have neuroprotective, neuroregenerative and anti-inflamatory properties. However, a comprehensive knowledge how transplanted MSCs exert their therapeutic achievements is still lacking. The aim of the project was to analyze the presence, distribution and quantity of human bone marrow mesenchymal stem cells (hBM-MSCs) transplanted into focal brain ischemic rats. The experiments were performed in adult male Wistar rats withbrain focal ischemiainduced with 1μl/50nmol ouabain(sodium-potassium pump inhibitor) injection into right stratium. Then 5x105 hBM-MSC (Lonza) stained with iron nanoparticles and rhodamine (Molday, BioPAL) were transplanted into internal carotid artery, 48 hours after brain insult. At 1, 3, 7 and 14 days rat brains were removed. Immunocytochemical analysis of human markers using different antibodies anti: CD44, STEM121and Ku80 were performed. The preliminary results showed that after intra-arterially injection of hBM-MSC, the donor cells were present in the ipsilateral rat hemisphere between cortical cortex and stratium near the ischemic lesion. The positive staining for Molday particles and human antigens were observed at 1, 2, 3 and 7 days after hBM-MSC transplantation. The further studies relating to the function of transplanted cells are in progress.M stem cells (MSCs) represent a population of multipotent stem cells with immunomodulatory, antiapoptotic and cytoprotective capabilities and thus hold a great promise for treatment of many inflammatory diseases and for use in a regenerative medicine. Numerous studies have shown that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone. However, the exact mechanism of such synergism has not yet been described.