Philipp S. Lienemann

Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

In situ cell manipulation through enzymatic hydrogel photopatterning

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)--a key ECM crosslinking enzyme--is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

A Versatile Approach to Engineering Biomolecule‐Presenting Cellular Microenvironments

By combining a novel protein-capture hydrogel with state-of-the-art mammalian recombinant protein production, cellular microenvironments are fabricated that locally instruct in-vitro cell behavior through selective presentation of the expressed proteins.

Decrease in VEGF Expression Induces Intussusceptive Vascular Pruning

Objective—The concept of vascular pruning, the “cuting-off” of vessels, is gaining importance due to expansion of angio-modulating therapies. The proangiogenic effects of vascular endothelial growth factor (VEGF) are broadly described, but the mechanisms of structural alterations by its downregulation are not known. Methods and Results—VEGF165-releasing hydrogels were applied onto the chick chorioallantoic membrane on embryonic day 10. The hydrogels, designed to completely degrade within 2 days, caused high-level VEGF presentation followed by abrupt VEGF withdrawal. Application of VEGF resulted in a pronounced angiogenic response within 24 hours. The drastic decrease in level of exogenous VEGF-A within 48 hours was corroborated by enzyme-linked immunosorbent assay. Following this VEGF withdrawal we observed vasculature adaptation by means of intussusception, including intussusceptive vascular pruning. As revealed on vascular casts and serial semithin sections, intussusceptive vascular pruning occurred by emergence of multiple eccentric pillars at bifurcations. Time-lapse in vivo microscopy has confirmed the de novo occurrence of transluminal pillars and their capability to induce pruning. Quantitative evaluation corroborated an extensive activation of intussusception associated with VEGF withdrawal. Conclusion—Diminution of VEGF level induces vascular tree regression by intussusceptive vascular pruning. This observation may allude to the mechanism underlying the “normalization” of tumor vasculature if treated with antiangiogenic drugs. The mechanism described here gives new insights into the understanding of the processes of vasculature regression and hence provides new and potentially viable targets for antiangiogenic and/or angio-modulating therapies during various pathological processes.

Modular Poly(ethylene glycol) Matrices for the Controlled 3D-Localized Osteogenic Differentiation of Mesenchymal Stem Cells

The in vitro formation of physiologically relevant engineered tissues is still limited by the availability of adequate growth-factor-presenting cell-instructive biomaterials, allowing simultaneous and three-dimensionally localized differentiation of multiple tissue progenitor cells. Together with ever improving technologies such as microfluidics, printing, or lithography, these biomaterials could provide the basis for generating provisional cellular constructs, which can differentiate to form tissue mimetics. Although state-of-the-art biomaterials are endowed with sophisticated modules for time- and space-controlled positioning and release of bioactive molecules, reports on 3D arrangements of differentiation-inducing growth factors are scarce. This paper describes the stable and localized immobilization of biotinylated bioactive molecules to a modular, Factor XIII-cross-linked poly(ethylene glycol) hydrogel platform using a genetically engineered streptavidin linker. Linker incorporation is demonstrated by Western blot, and streptavidin functionality is confirmed by capturing biotinylated alkaline phosphatase (ALP). After optimizing bone morphogenetic protein 2 (BMP-2) biotinylation, streptavidin-modified hydrogels are able to bind and present bioactive BMP-2-biotin. Finally, with this immobilization scheme for BMP-2, the specific osteogenic differentiation of mesenchymal stem cells is demonstrated by inducing ALP expression in confined 3D areas. In future, this platform together with other affinity-based strategies will be useful for the local incorporation of various growth factors for engineering cell-responsive constructs.

Enzyme responsive GAG-based natural-synthetic hybrid hydrogel for tunable growth factor delivery and stem cell differentiation.

We describe an enzymatically formed chondroitin sulfate (CS) and poly(ethylene glycol) (PEG) based hybrid hydrogel system, which by tuning the architecture and composition of modular building blocks, allows the application-specific tailoring of growth factor delivery and cellular responses. CS, a negatively charged sulfate-rich glycosaminoglycan of the extracellular matrix (ECM), known for its growth factor binding and stem cell regulatory functions, is used as a starting material for the engineering of this biomimetic materials platform. The functionalization of CS with transglutaminase factor XIII specific substrate sequences is utilized to allow cross-linking of CS with previously described fibrin-mimetic TG-PEG hydrogel precursors. We show that the hydrogel network properties can be tuned by varying the degree of functionalization of CS as well as the ratio and concentrations of PEG and CS precursors. Taking advantage of TG-PEG hydrogel, compatible tagged bio-functional building blocks, including RGD peptides or matrix metalloproteinase sensitive domains, can be incorporated on demand allowing the three-dimensional culture and expansion of human bone marrow mesenchymal stem cells (BM-MSCs). The binding of bone morphogenetic protein-2 (BMP-2) in a CS concentration dependent manner and the BMP-2 release mediated osteogenic differentiation of BM-MSCs indicate the potential of CS-PEG hybrid hydrogels to promote regeneration of bone tissue. Their modular design allows facile incorporation of additional signaling elements, rendering CS-PEG hydrogels a highly flexible platform with potential for multiple biomedical applications.

Electrochemical Control of the Enzymatic Polymerization of PEG Hydrogels: Formation of Spatially Controlled Biological Microenvironments

Control of pH gradient profile at the electrode-electrolyte interfaces allows the control of the enzymatic PEG-hydrogel polymerization. By tuning the solution pH, buffer capacity, and the applied current, the extent of the local inhibition and confinement of the Factor XIII-mediated polymerization of PEG are controlled. This technology opens new perspectives for the production of 3D-structured biological microenvironments.

Longitudinal in vivo evaluation of bone regeneration by combined measurement of multi-pinhole SPECT and micro-CT for tissue engineering

Over the last decades, great strides were made in the development of novel implants for the treatment of bone defects. The increasing versatility and complexity of these implant designs request for concurrent advances in means to assess in vivo the course of induced bone formation in preclinical models. Since its discovery, micro-computed tomography (micro-CT) has excelled as powerful high-resolution technique for non-invasive assessment of newly formed bone tissue. However, micro-CT fails to provide spatiotemporal information on biological processes ongoing during bone regeneration. Conversely, due to the versatile applicability and cost-effectiveness, single photon emission computed tomography (SPECT) would be an ideal technique for assessing such biological processes with high sensitivity and for nuclear imaging comparably high resolution (<1 mm). Herein, we employ modular designed poly(ethylene glycol)-based hydrogels that release bone morphogenetic protein to guide the healing of critical sized calvarial bone defects. By combined in vivo longitudinal multi-pinhole SPECT and micro-CT evaluations we determine the spatiotemporal course of bone formation and remodeling within this synthetic hydrogel implant. End point evaluations by high resolution micro-CT and histological evaluation confirm the value of this approach to follow and optimize bone-inducing biomaterials.

Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity

The precise manipulation of growth factor signaling is central to the progress of tissue engineering. Methods for direct time-resolved activation of signaling pathways through controlled receptor dimerization have been reported; however, these suffer from the risks associated with gene transfer. Here we present an alternative gene transfer-free approach in the form of a protein switch featuring pharmacologically controlled ON-OFF regulation of growth factor activity. The reversible operation of the switch enables stimulation of target processes within a defined period of time. The protein switch provides a means for both studying and manipulating signaling processes, and is thus believed to be a valuable tool for basic research as well as tissue engineering and biomedical applications.