Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Photopatterning of Synthetic Hydrogels

Katarzyna Mosiewicz

In recent years sophisticated biomaterials have made a significant impact on our understanding of cellular behavior, particularly in the fields of stem cell research and tissue engineering. Nevertheless, the promising potential of stem cells for regenerative medicine has remained largely unmet. One reason for such limitations could be that there is still incomplete understanding of stem cell behavior due to a lack of advanced in vitro tools suitable to achieve adequate control over cellular processes. By mimicking the dynamic and complex biophysical and biochemical interactions between cells and their natural extracellular matrixes (ECMs), it is possible that we may bridge this gap between current limited in vitro models and complex in vivo organisms. In this thesis, an innovative class of biomaterials is presented that enables the flexible in vitro recapitulation of the dynamic biochemical and biophysical signaling involved in controlling cell fate. Such systems of increasing fidelity to real physiological processes could bring new insights into fundamental cell biology as well as bring us to closer towards cell-based therapeutics applications. In order to achieve a dynamic artificial ECM (aECM), we designed chemical schemes which would allow us to control the biochemical and biophysical properties of the hydrogel in space, time and intensity. The chosen concept is built on a synthetic hydrogel providing static physicochemical support, while spatiotemporal control over a single or multiple signals is achieved by spatiotemporal photoactivation reactions within this hydrogel. Indeed, by modulating the duration, location and intensity of illumination, desired heterogeneities in biochemical and biophysical signal can be introduced in the otherwise homogeneous and static hydrogel network. To implement the designed hydrogel photo-patterning tool, new chemical components were synthesized. Synthetic hydrogels based on poly (ethylene glycol) (PEG) were utilized due to their inert properties, cell biocompatibility and lack of unspecific protein binding. Biochemical signal tethering was rendered spatiotemporally controllable by adding photosensitivity to the existing enzyme-based crosslinking scheme. This was achieved by functionalizing one of enzymatic substrate with a photo-labile “caging” moiety, thereby preventing crosslinking to occur until light illumination. This caged substrate was incorporated into the hydrogel network and served as a light-controllable and specific ligand-binding site. Importantly, it was possible to photo-pattern proteins, which are much complex than peptide-based biomolecules, and their bioactivity was fully preserved with this site-specific immobilization scheme. It was also possible to pattern biophysical cues by making the Michael-type (MT) addition reaction for crosslinking PEG-based hydrogel photosensitive. In this case, the thiol moieties of one of the reactive PEG macromers undergoing crosslinking, were equipped with a caging group which prevented its susceptibility to the counter-reactive functionality, the vinyl sulfone group. Thus, the crosslinking density of the hydrogel backbone could be controlled by light, which was directly translated into differential patterns of hydrogel stiffness. Using this approach, user-defined biochemical and biophysical patterns and even gradients could be obtained in the spatiotemporal manner within the same hydrogel. The crosslinking reaction is the critical parameter in the success of hydrogel photo-patterning, and must rely on a well-controlled and highly selective chemical scheme. Therefore, we also developed a novel enzyme-based cross-linking scheme for synthetic PEG hydrogels. The reaction originates from the naturally occurring post-translational modification of proteins by phosphopantetheine (Ppant) group performed by a family of enzymes known as Ppant transferases (PPTases). By synthesizing biochemical components of this reaction, we implemented a PPTase-catalyzed reaction to form and bio-functionalize PEG hydrogel. The utility of the developed hydrogel platforms was determined in the context of 2D and 3D cell culture. Photo-directed patterns of a cell-adhesive fibronectin fragment triggered adhesion and spreading of muscle progenitor cells, a phenomenon which occurred only within the areas of patterned signaling protein. A photo-patterned elasticity gradient was used to direct mesenchymal stem cell spreading and migration. Finally, the PPTase-based hydrogel was proven to be suitable for 3D culture, as shown by encapsulation of primary human fibroblasts. In this thesis, we demonstrated that a combination of chemical schemes could be assembled to achieve a functional materials toolbox which would begin to reconstruct the complexity of the dynamic in vivo ECM. Such a tool is expected to contribute to the field of cell biology by allowing us to study questions which previously could not be addressed in vitro, and could ultimately contribute to cell-based regenerative medicine.

EPFL2012

Functional polypeptide and hybrid materials: Precision synthesis via alpha-amino acid N-carboxyanhydride polymerization and emerging biomedical applications

Harm-Anton Klok

Polypeptides derived from naturally occurring a-amino acids have emerged as a unique and versatile family of bio-inspired biomaterials that can be tailor-made for varying biomedical applications such as controlled drug release, gene delivery, tissue engineering and regenerative medicine. In contrast to traditional biodegradable polymers such as aliphatic polyesters and polycarbonates, polypeptides are inherently functional, allow precise control over polarity and charges, show excellent stability against hydrolysis, and are prone to rapid biodegradation in vivo by specific enzymes. Ring-opening polymerization (ROP) of alpha-amino acid N-carboxyanhydrides (NCAs) is the most straightforward and practical approach for large-scale production of high molecular weight polypeptides. In the past decade, a remarkable progress has been made in controlled NCA polymerization, which offers an unprecedented access to precision polypeptide and hybrid materials by combining with living radical polymerization, click chemistry, and/or post-polymerization modification. Notably, several micellar anti-cancer drugs based on poly(ethylene glycol)-polypeptides have been already advanced to the clinical evaluation. In this review paper, we give an overview on de novo design, controlled synthesis and emerging biomedical applications of functional polypeptide and hybrid materials. (C) 2013 Elsevier Ltd. All rights reserved.

Elsevier2014

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.