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- ItemMelt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork(Washington, DC : ACS Publ., 2022) Włodarczyk-Biegun, Małgorzata K.; Villiou, Maria; Koch, Marcus; Muth, Christina; Wang, Peixi; Ott, Jenna; del Campo, AranzazuThe permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.
- ItemToward light‐regulated living biomaterials(Hoboken, NJ : Wiley, 2018) Sankaran, Shrikrishnan; Zhao, Shifang; Muth, Christina; Paez, Julieta; Del Campo, AránzazuLiving materials are an emergent material class, infused with the productive,adaptive, and regenerative properties of living organisms. Property regulation in living materials requires encoding responsive units in the living components to allow external manipulation of their function. Here, an optoregulated Escherichia coli (E. coli)-based living biomaterial that can be externally addressed using light to interact with mammalian cells is demonstrated. This is achieved by using a photoactivatable inducer of gene expression and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. Hydrogel surfaces functionalized with the bacteria can expose cell adhesive molecules upon in situ light-activation, and trigger cell adhesion. Surface immobilized bacteria are able to deliver a fluorescent protein to the mammalian cells with which they are interacting, indicating the potential of such a bacterial material to deliver molecules to cells in a targeted manner.
- ItemMelt Electrowriting of Scaffolds with a Porosity Gradient to Mimic the Matrix Structure of the Human Trabecular Meshwork(New York : Cold Spring Harbor Laboratory, 2022) Włodarczyk-Biegun, Małgorzata K.; Villiou, Maria; Koch, Marcus; Muth, Christina; Wang, Peixi; Ott, Jenna; del Campo, AranzazuThe permeability of the Human Trabecular Meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases like glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues, and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa, and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffolds design, i.e., density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW to reconstruct complex morphological features of natural tissues.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(Hoboken, NJ : Wiley, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; Del Campo, AránzazuCollagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
- ItemDatasets from a vapor diffusion mineral precipitation protocol for Dictyostelium stalks(Amsterdam : Elsevier, 2016) Eder, Magdalena; Muth, Christina; Weiss, Ingrid M.Datasets from a slow carbonate vapor diffusion and mineral precipitation protocol for Dictyostelium ECM and cellulose stalks show examples for composite materials obtained by an in vitro approach, which differs substantially from the in vivo approach reported in The Journal of Structural Biology, doi: 10.1016/j.jsb.2016.03.015 [1]. Methods for obtaining the datasets include bright field transmitted light microscopy, fluorescence microscopy, LC-PolScope birefringence microscopy, variable pressure scanning electron microscopy (VP-SEM/ESEM), and Raman imaging spectroscopy.
- ItemThermal decomposition of the amino acids glycine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and histidine(London : BioMed Central, 2018) Weiss, Ingrid M.; Muth, Christina; Drumm, Robert; Kirchner, Helmut O.Background: The pathways of thermal instability of amino acids have been unknown. New mass spectrometric data allow unequivocal quantitative identification of the decomposition products. Results: Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185 °C and 280 °C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70% volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H2O, some NH3 and no CO2. Cysteine produces CO2 and little else. The reactions are described by polynomials, AA→a NH3+b H2O+c CO2+d H2S+e residue, with integer or half integer coefficients. The solid monomolecular residues are rich in peptide bonds. Conclusions: Eight of the 20 standard amino acids decompose at well-defined, characteristic temperatures, in contrast to commonly accepted knowledge. Products of decomposition are simple. The novel quantitative results emphasize the impact of water and cyclic condensates with peptide bonds and put constraints on hypotheses of the origin, state and stability of amino acids in the range between 200 °C and 300 °C.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(ChemRxiv, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; del Campo, AránzazuHsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.