Fighting preterm birth with novel surgical tools and biomaterials engineering

With advances in fetal diagnosis and therapy, fetoscopy has become a good option to treat a series of life threatening diseases like twin-to-twin transfusion syndrome or severe congenital diaphragmatic hernia. The efficacy of fetoscopy for those types of disorders is not debatable; however, it comes with a daunting limitation: iatrogenic preterm premature rupture of the fetal membrane (iPPROM). The injury created by the instruments to access the amniotic cavity is the reason for fetal membrane rupture in almost one third of all interventions. The consequences of very early iPPROM are death or very preterm birth leading to physiological complications for the baby, and psychological burdens for the family. A solution preventing iPPROM is necessary to make fetoscopy a safer procedure.

We first concentrated on developing a minimally invasive medical device to stabilize the fetal membrane defect caused by fetoscopic instruments. Our approach consists in placing bioadhesives at the wounded site from the inside of the amniotic cavity following the intervention. To do so, we designed and produced an umbrella-shape receptor whose mechanical properties enabled its crimping inside the accession catheter and ensured its automatic deployment inside the cavity. The umbrella is then pulled against the membrane and glued by gathering injectable bioadhesives. We demonstrated evidence of the functionality of the method by applying existing biocompatible glues on an ex vivo model replicating physiological conditions.

While the application method is a requirement for a reliable delivery of injectable materials, questions remain about the nature of those materials. Restauration of the native tissue might provide functional advantages over the simple sealing of it. Hence, the development of platforms enabling the in vitro study of cell response to biological cues was the focus of the next investigations. First, we took advantage of the tunability of polyethylene glycol (PEG) synthetic hydrogels to locally bind growth factors (GFs) by affinity. We established a GF gradient in 3D and assessed multiple methods to quantify cell behaviour in response to those gradients.

Next, we utilized a method to pre-fabricate hydrogel gradients in a full 96-well plate and established scripts to automate image acquisition, image processing and data analysis. This platform enabled a fast quantification of the influence of GF concentration on the recruitment of cells in large number of samples in parallel. We then reproduced a wound in a tissue model in vitro, injected PEG hydrogels carrying GF and quantified cell recruitment into the healing hydrogel. Finally, we used a proteomics approach to investigate the effect of bioactive signals on inducing a differential production of extracellular matrix (ECM) by the cells. This investigation was motivated by the critical role of ECM proteins in tissue functionality and thus by the importance of faithfully reconstituting the native structure of the tissue.

To summarize, our efforts contributed to the advancement of fetal therapy by bringing innovative solutions in two distinct fields. On one side, by the development of a technology to apply materials at the site of defect; and on the other side, by the establishment of platforms allowing the exploration of cell behaviour. Combined, they carry a real potential to engineer methods for precise delivery of healing-inducing biomaterials and consequently to reduce the risks of iPPROM.

New Tools for Hydrogel-Based 3D Bioprinting

Andrea Negro

The in vitro recapitulation of tissue and organ function represents one of the main objectives of tissue engineering. Developments in this field have numerous applications, from alleviating the shortage of donor organs to providingmore representative platforms for drug testing. As such, there is an increasingly significant body of literature devoted to the development of better strategies to reconstruct living matter outside of the body. The three-dimensional (3D) architecture of a tissue, its histoarchitecture, is a key aspect to consider when engineering a tissue. Indeed, tissue function not only arises from individual elements that compose the tissue, its different cell types and their extracellular matrices (ECM), but also fromtheir precise spatial arrangement and interactions. Bioprinting is considered as one of the most promising techniques to recapitulate the complexity of in vivo histoarchitectures. Early bioprinting examples have shown successful application of inkjet printing to geometrically arrange cells and biomolecules in 2D and, to a much lesser extent, in 3D. However, no group has shown yet a technological platformable to print a living tissue construct whose make-up could even closelymimic the histoarchitecture of a native tissue. In this thesis the most significant challenges in using inkjet printers for tissue engineering were tackled. Perhaps the most critical bottleneck of state-of-the-art bioprinting is the lack of suitable bioinks; precursor liquids with distinct physico-chemical properties that very rapidly transforminto solid hydrogels and also possess the necessary bioactive characteristics to guide cell development into a functional tissue. To this end, a novel double network hydrogel systemwas conceived which combines ultra-rapid cross-linking of alginate, a biologically inert polysaccharide network, with the slower enzymatic crosslinking of a synthetic but highly bioactive poly(ethylene glycol) (PEG) gel. The latter could be tailored virtually on demand tomimic a desired ECM in a tissue. Based on this bioink concept it was demonstrated that complex multicomponent 3D shapes could be printed. Importantly, using primary human fibroblasts as model system, the bioinks showed outstanding characteristics as cellular microenvironments, facilitating rapid 3D cell migration and self-organization into multicellular networks. To mimic the spatial complexity of living tissues, it is crucial to master the deposition of multiple, cell-containing bioinks into user-defined 3D structures. Each bioink could thus mimic a specific cell/ECMcomponent present in a native tissue. To be able to reproduce an appropriate histoarchitecture, bioinks have to be deposited precisely enough to approximate their natural 3D spatial arrangement down to a cellular scale. Technically, this implies that the dispensing units must be extremely well aligned and synchronized to one another in order to pattern according to a specified design. In order to address these challenges, several novel bioprinting concepts were developed, which allowed the patterning of multiple bioinks containing living cells into 3D tissue-like geometries. In the last part of this thesis, an important first step towards the engineering of macroscopic tissue models was taken. Accordingly, a fundamental requirement for the viability and maturation of a printed tissue construct is the supply of nutrients and growth factors through a microfluidic network, similar to the vascularization in a native tissue. To this end, a novel approach for the printing of 3D constructs with an interconnected channel network enabling rapid nutrient transport was devised. This was achieved by exploiting the multicomponent printing developed earlier. A channel network inside the construct was patterned using a degradable hydrogel that could be selectively removed to leave behind the perfusable microfluidic system. Moreover, it was possible to combine this sacrificial layer approach with cell-containing bioinks to generate a cellularized perfusable tissue model. Encouraging preliminary long-termculture experiments revealed that dynamic cell culture conditions could be maintained, leading to viable cell growth over more than one week under perfusion conditions. Taken together, this thesis presents several important steps toward an inkjet-based platform for in vitro tissue engineering. It is expected that the strategies developed here will provide a solid basis for future advances in the emerging bioprinting field.

EPFL2014

Tissue engineering for prenatal applications

Anna-Sofia Johanna Kiveliö

Fetal therapies have become available for a restricted number of life-threatening clinical conditions. Harnessing tissue engineering for prenatal applications has not been widely pursued even though isolating cells from fetal and extraembryonic tissues has been routinely done for years. The objectives of this thesis were twofold: i) the development of materials based and tissue engineering based strategies to prevent iatrogenic preterm prelabour rupture of fetal membranes (iPPROM) after diagnostic or therapeutic interventions into the amniotic cavity and ii) the generation of tissue substitutes from patient derived fetal cells, which can be employed for prenatal or perinanal transplantation to restore or replace defective tissues. For the prevention of iPPROM, mussel-mimetic tissue adhesive (mussel glue) a biomaterial which was recently described to not compromise cell viability and to have good tissue sealing capability was compared to fibrin glue. In this in vivo study we assessed whether in a mid-gestational rabbit model punctured fetal membranes could be efficiently sealed with mussel glue. Mussel glue showed comparable in vivo performance to fibrin glue in sealing fetal membranes though no apparent healing of the membranes could be observed in any of the samples. The limited ability of naturally derived scaffolds to promote fetal membrane healing inspired the engineering of synthetic plugging material with specifically tailored biological and mechanical properties which could activate the cells in the amnion and induce a healing response. In this thesis the modularly designed biomimetic poly(ethylene glycol) (PEG)-based hydrogel platform (called TG-PEG from here on) was used together with fetal cells to demonstrate that upon presentation of appropriate biological cues in 3D tissue mimicking environment, mesenchymal progenitor cells from amnion can be mobilized, induced to proliferate and supported in maintaining their native extracellular matrix production, thus creating a suitable environment for healing to take place. These data provide the basis for future engineering of materials with defined mechanical and biochemical properties and the ability to present migration and proliferation inducing factors, namely PDGF, bFGF, or EGF which could be key in resolving the clinical problem of iPPROM and allowing the field of fetal surgery to move forward. Cleft palate, where the bones of the palatal halves fail to fuse properly, is one of the most common birth defects. Tissue engineering has been envisioned as a treatment option but current approaches have been limited by the lack of i) appropriate autologous cell sources and ii) structural organization and vascularization. Tissue engineering of cleft palates using of autologous amniocentesis-derived and thus ethically unproblematic fetal amniotic fluid cells (AFCs) could be done parallel to the ongoing pregnancy with living reconstruction material being ready when the child is born. We describe using TG-PEG hydrogels to first evaluate 3D osteogenic differentiation of AFCs and creation of vascular structures from AFC derived endothelial cells (enAFC) and undifferentiated AFC either in random or organized channel cocultures in vitro and in vivo. Next, these approaches were combined in an osteogenic matrix with a channel perfused with enAFC and finally the integration and functional properties of these fetal bone constructs was tested in ectopic mouse model.

EPFL2015

La0.5Sr0.5Fe1-yMyO3-[delta] (M = Ti, Ta) perovskite oxides for oxygen separation membranes

Defne Bayraktar

Mixed ionic electronic conducting perovskite materials have been receiving considerable attention for the application of oxygen separation membranes. These membranes have potential to be integrated in industrial processes that require pure oxygen and to provide advantages considering economical and environmental aspects. The used perovskite materials can accommodate a high concentration of disordered oxygen vacancies and provide high oxygen permeation flux in the presence of an oxygen partial pressure gradient. However, the materials with high oxygen flux are observed to have low chemical and mechanical stability, originating from oxygen vacancy formation and reduction of the B-site transition metal ion. Based on the fact that the stability of the materials at reducing pO2 is dependent on the redox stability of the B-site cation, a partial substitution of the B-site transition metal with a more stable cation is considered in this thesis to improve the stability of the base perovskite oxide. The approach used for the identification of the suitable material based on the host composition La0.5Sr0.5FeO3-δ (LSF) was to first explore the B-site substituting elements in terms of their effect on the thermal expansion and the isothermal expansion measured during the atmosphere change from air to argon. The elements Al, Cr, Zr, Ga, Ti, Sn, Ta, In, V, and Mg were used for 10 or 20% substitution of Fe. The isothermal expansion of the host material was decreased by substitution, which was attributed to decreased amount of oxygen vacancy formation as it was evidenced later by TGA of chosen compositions. As a result, the compositions substituted with higher valence ions (particularly, 20% Ti4+, LSFTi2, and 10% Ta5+, LSFTa1) were identified as the least expanding materials in isothermal conditions. The effect of substitution was further characterized extensively including structural, mechanical, and oxygen transport properties. The oxygen vacancy (VO••) concentration changes induced by substitution of Fe were reflected in unit cell sizes of heat-treated samples under argon atmosphere. The increase in the unit cell size was attributed to the isothermal expansion due to B-site reduction and lowered to half in case of substituted LSFTi2 and LSFTa1 compared to the host material LSF. Similar trends were observed for interrelated properties such as the coefficient of thermal expansion, isothermal expansion, and the actual amount of oxygen vacancies formed under argon at 900°C, all of which were higher for LSF. The mechanical properties of LSF, LSFTi2, and LSFTa1 were characterized at room temperature. The Young's modulus and the bending strength of the materials were in the same range (141-147 GPa and ∼120 MPa, respectively) while the fracture toughness of the LSF sample was improved by Ti and especially Ta substitution. The fractography of the samples provided evidence that the several LSF samples showed extended cracks possibly related to residual stresses forming during cooling, due to non-equilibrated oxygen stoichiometry across the sample. The potential step measurements showed that the chemical diffusion and the surface exchange coefficients measured during oxidation of the samples were higher than the ones measured during reduction of the samples. The tendency was reversed at lowered pO2 along with the decrease of both coefficients. The influence of B-site substitution on both coefficients was not substantial. The oxygen permeation flux of LSF, LSFTi2, and LSFTa1 was measured under air/argon gradient on planar membranes. The permeation rates of the membranes with thicknesses close to 1 mm were considered to be limited by surface exchange kinetics at temperatures above 875°C. The permeation rate of LSF was decreased (from 0.2 µmolcm-2s-1) to its third by Ti and Ta additions. The permeation rate of Ti and Ta-substituted samples showed a drop around 875°C, keeping a similar activation energy at lower and higher temperature regions. Oxygen vacancy ordering in the perovskite structure was given as a possible explanation, which provided explanation for the slow equilibrium in the lower temperature region. Tubular membranes of LSF and LSFTi2 (0.47 mm and 0.36 mm thick, respectively) and a planar LSFTa1 membrane were characterized using an air/(Ar-CH4) gradient. The measurements conducted using pure Ar on the lean-side provided the possibility to compare the thickness dependence of the permeation. In agreement with the previous observations, the permeation of the materials at and above 900°C was independent of the thickness, therefore, surface exchange limited. Stability under reducing atmospheres was increased by substitution. The LSF membrane failed shortly after the introduction of CH4 to the system, while LSFTi2 survived 5% CH4 and its oxygen permeation was improved substantially by a factor of 14. LSFTa1 membrane was measured with pure CH4 and the permeation was increased by a factor of 9, reaching 0.7 µmolcm-2s-1 at 900°C. The membrane operated stably over 2000h with pure CH4, 1000h. A slow degradation of 3.7% per 1000h was observed at 1000°C, most probably due to cation migration. The compositions identified as a result of B-site substitution screening were effective in improving the stability of the materials without extensive loss of the oxygen permeation rate. Ta-substituted material was shown to be promising with its long-term stability as a partial oxidation membrane.

EPFL2008