21211
2021
eng
doctoralthesis
1
2020-09-28
--
2020-09-15
A first step to an integral biointerface design for the early phase of regeneration
Ein erster Schritt zur Etablierung eines integralen biologischen Grenzflächendesigns für die frühe Phase der Regeneration
The implantation of any foreign material into the body automatically starts an immune reaction that serves as the first, mandatory step to regenerate tissue. The course of this initial immune reaction decides on the fate of the implant: either the biomaterial will be integrated into the host tissue to subsequently fulfill its intended function (e.g., tissue regeneration), or it will be repelled by fibrous encapsulation that determines the implant failure. Especially neutrophils and macrophages play major roles during this inflammatory response and hence mainly decide on the biomaterial's fate. For clinically relevant tissue engineering approaches, biomaterials may be designed in shape and morphology as well as in their surface functionality to improve the healing outcome, but also to trigger stem cell responses during the subsequent tissue regeneration phase.
The main focus of this thesis was to unravel the influence of scaffold characteristics, including scaffold morphology and surface functionality, on primary human innate immune cells (neutrophils and macrophages) and human mesenchymal stromal cells (hMSCs) to assess their in vitro immune response and tissue regeneration capacity, respectively. The fiber-based constructs were produced either via melt electrowriting (MEW), when the precise control over scaffold morphology was required, or via solution electrospinning (ES), when the scaffold design could be neglected. All the fiber-based scaffolds used throughout this thesis were composed of the polymer poly(ε caprolactone) (PCL).
A novel strategy to model and alleviate the first direct cell contact of the immune system with a peptide-bioactived fibrous material was presented in chapter 3 by treating the material with human neutrophil elastase (HNE) to imitate the neutrophil attack. The main focus of this study was put on the effect of HNE towards an RGDS-based peptide that was immobilized on the surface of a fibrous material to improve subsequent L929 cell adhesion. The elastase efficiently degraded the peptide-functionality, as evidenced by a decreased L929 cell adhesion, since the peptide integrated a specific HNE-cleavage site (AAPV-motif). A sacrificial hydrogel coating based on primary oxidized hyaluronic acid (proxHA), which dissolved within a few days after the neutrophil attack, provided an optimal protection of the peptide-bioactivated fibrous mesh, i.e, the hydrogel alleviated the neutrophil attack and largely ensured the biomaterial's integrity. Thus, according to these results, a means to protect the biomaterial is required to overcome the neutrophil attack.
Chapter 4 was based on the advancement of melt electrowriting (MEW) to improve the printing resolution of MEW scaffolds in terms of minimal inter-fiber distances and a concomitant high stacking precision. Initially, to gain a better MEW understanding, the influence of several parameters, including spinneret diameter, applied pressure, and collector velocity on mechanical properties, crystallinity, fiber diameter and fiber surface morphology was analyzed. Afterward, innovative MEW designs (e.g., box-, triangle-, round , and wall-shaped scaffolds) have been established by pushing the printing parameters to their physical limits. Further, the inter-fiber distance within a standardized box-structured scaffold was successfully reduced to 40 µm, while simultaneously a high stacking precision was maintained. In collaboration with a co-worker of my department (Tina Tylek, who performed all cell-based experiments in this study), these novel MEW scaffolds have been proven to facilitate human monocyte-derived macrophage polarization towards the regenerative M2 type in an elongation-driven manner with a more pronounced effect with decreasing pore sizes.
Finally, a pro-adipogenic platform for hMSCs was developed in chapter 5 using MEW scaffolds with immobilized, complex ECM proteins (e.g., human decellularized adipose tissue (DAT), laminin (LN), and fibronectin (FN)) to test for the adipogenic differentiation potential in vitro. Within this thesis, a special short-term adipogenic induction regime enabled to more thoroughly assess the intrinsic pro-adipogenic capacity of the composite biomaterials and prevented any possible masking by the commonly used long-term application of adipogenic differentiation reagents. The scaffolds with incorporated DAT consistently showed the highest adipogenic outcome and hence provided an adipo-inductive microenvironment for hMSCs, which holds great promise for applications in soft tissue regeneration.
Future studies should combine all three addressed projects in a more in vivo-related manner, comprising a co-cultivation setup of neutrophils, macrophages, and MSCs. The MEW-scaffold, particularly due to its ability to combine surface functionality and adjustable morphology, has been proven to be a successful approach for wound healing and paves the way for subsequent tissue regeneration.
Die Implantation eines Biomaterials löst stets eine Immunreaktion im Körper aus, die den ersten zwingenden Schritt zur Geweberegeneration darstellt. Der Verlauf dieser anfänglichen Immunreaktion entscheidet über das Schicksal des Implantats: Entweder wird das Biomaterial in das Wirtsgewebe integriert, um anschließend seine vorgesehene Funktion (z.B. Geweberegeneration) zu erfüllen, oder aber es findet eine Abstoßungsreaktion durch Einkapselung des Implantats statt. Insbesondere Neutrophile und Makrophagen spielen für die Immunantwort eine wichtige Rolle und entscheiden daher hauptsächlich über das Schicksal des Biomaterials. Für klinisch relevante Ansätze der Gewebezüchtung können Biomaterialien sowohl in ihrer Morphologie als auch in ihrer Oberflächenfunktionalität so gestaltet werden, dass sie zum einen die Wundheilung verbessern, zum anderen auch Stammzellreaktionen während der anschließenden Geweberegenerationsphase auslösen.
Der Fokus dieser Doktorarbeit lag auf der Beurteilung des Einflusses von Morphologie und Oberflächenfunktionalität fasriger Scaffolds auf die frühe Phase der Geweberegeneration. Insbesondere wurde die in vitro-Immunantwort von primären humanen Immunzellen (Neutrophile und Makrophagen) sowie die Geweberegenerationskapazität von humanen mesenchymalen Stromazellen (hMSCs) untersucht. Die hierfür verwendeten faserbasierten Poly(ε-Caprolacton) (PCL) Scaffolds wurden entweder mittels Solution Electrospinning (ES) oder Melt Electrowriting (MEW) hergestellt. Während ES eine zufällig orientierte Faserablage zur Folge hat, erlaubt MEW eine präzise Kontrolle der Scaffold-Morphologie.
Zunächst wurde eine neue Strategie zur Nachahmung und Abmilderung des ersten direkten Zellkontakts während der Immunreaktion vorgestellt. Dabei wurde die Interaktion zwischen Neutrophilen mit einem Peptid-bioaktivierten Fasermaterial untersucht (Kapitel 3), wobei der sog. Neutrophilen-Angriff mittels des Enzyms Neutrophilen Elastase (HNE) nachgeahmt wurde. Das an der Faseroberfläche immobilisierte CGGGAAPVGGRGDS-Peptid verfügte über eine spezifische HNE-Schnittstelle (AAPV-Motiv), an welcher die Elastase das Peptid effizient degradieren konnte. Das Degradationsverhalten des Enzyms wurde anschließend über L929 Zelladhärenz analysiert, welche über das RGDS-Motiv im Peptid vermittelt wurde. Im Rahmen der Arbeit konnte nachgewiesen werden, dass der Neutrophilen-Angriff und die damit einhergehende Verringerung des RGDS-Motivs zu einer reduzierten Zelladhärenz führte. Die Einbettung des Scaffolds in ein Hydrogel auf der Basis von Aldehyd-haltiger Hyaluronsäure (proxHA) bot während des Neutrophilen-Angriffs einen optimalen Schutz der Peptidfunktionalität. Um diese wiederum anschließend für Adhäsionsversuche verfügbar zu machen, konnte das Hydrogelsystem derartig eingestellt werden, dass sich dieses innerhalb weniger Tage auflöste. Auf diese Weise konnte das Hydrogel den Neutrophilen-Angriff abmildern und so die Integrität des Biomaterials weitestgehend gewährleisten.
Kapitel 4 behandelt die Präzisierung der Faserablage, insbesondere die Verringerung des Faserabstands, während des MEW-Prozesses. Zunächst wurde der Einfluss verschiedener Parameter (Spinndüsendurchmesser, angelegter Luftdruck und Kollektorgeschwindigkeit) auf die mechanischen Eigenschaften, die Kristallinität, den Faserdurchmesser und die Faseroberflächenmorphologie analysiert. Durch Optimierung der Druckparameter konnten innovative MEW-Designs (u.a. mit runder Porengeometrie) gedruckt werden. Der Abstand zwischen den Fasern in einem Scaffold mit standardisierter kastenförmiger Porengeometrie wurde erfolgreich auf 40 µm reduziert, während gleichzeitig eine hohe Stapelpräzision gewährleistet wurde. In Zusammenarbeit mit einer Kollegin am Lehrstuhl (Tina Tylek, die alle zellbasierten Experimente in dieser Studie durchführte) wurde nachgewiesen, dass diese innovativen MEW-Scaffolds die Polarisierung menschlicher Makrophagen in Richtung des regenerativen M2-Typs förderten. Die Makrophagen-Polarisierung ging einher mit einer Zellelongation, wobei dieser Effekt verstärkt für kleinere Porengrößen auftrat.
Abschließend stand die Untersuchung der pro-adipogenen Wirkung von faserfunktionalisierten MEW-Scaffolds im Fokus (Kapitel 5), welche mit ECM-Proteinen, wie beispielsweise dezellularisiertes Fettgewebe (DAT), beschichtet wurden. Das pro-adipogene Potential dieser Materialien wurde mit Hilfe einer adipogenen Kurzzeitinduktion näher analysiert, da eine Langzeitapplikation der Differenzierungsreagenzien diesen Effekt überdeckte. Die Scaffolds mit der DAT-Beschichtung zeigten durchweg die höchste adipogene Differenzierung und boten somit für Stammzellen eine adipo-induzierende Mikroumgebung, weshalb sie für die Anwendung in der Weichgeweberegeneration sehr vielversprechend sind.
An diese Arbeit anschließende Experimente sollten alle drei Projekte in einem Co-Kulturansatz von Neutrophilen, Makrophagen und MSCs kombinieren, um so einen stärkeren in vivo-Bezug herzustellen. Hierfür erweist sich das MEW-Scaffold insbesondere durch seine Kombinationsfähigkeit der Oberflächenfunktionalität und Morphologie als Ansatz für einen erfolgreichen Wundheilungsprozess und ebnet damit den Weg für eine bestmögliche Geweberegeneration.
urn:nbn:de:bvb:20-opus-212117
10.25972/OPUS-21211
X 129509
Deutsches Urheberrecht mit Print on Demand
Carina Blum
deu
swd
Scaffold <Tissue Engineering>
deu
swd
Biomaterial
eng
uncontrolled
tissue regeneration
eng
uncontrolled
melt electrowriting
deu
uncontrolled
Scaffold
Chemie und zugeordnete Wissenschaften
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/21211/Blum_Carina_Dissertation.pdf
25401
2020
eng
2
12
article
1
--
--
--
Precisely defined fiber scaffolds with 40 μm porosity induce elongation driven M2-like polarization of human macrophages
Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.
Biofabrication
10.1088/1758-5090/ab5f4e
urn:nbn:de:bvb:20-opus-254012
2022-01-27T00:21:25+00:00
sword
swordwue
attachment; filename=deposit.zip
47fff767a0138a89738c727290f311ff
Biofabrication 2020, 12(2):025007. DOI: 10.1088/1758-5090/ab5f4e
617989
false
true
Tina Tylek
Carina Blum
Andrei Hrynevich
Katrin Schlegelmilch
Tatjana Schilling
Paul D Dalton
Jürgen Groll
eng
uncontrolled
cell elongation
eng
uncontrolled
human macrophages
eng
uncontrolled
melt electrowriting (MEW)
eng
uncontrolled
macrophage polarization
eng
uncontrolled
3D scaffolds
Medizin und Gesundheit
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
OpenAIRE
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/25401/bf_12_2_025007.pdf
25638
2021
eng
33
33
article
1
2022-02-12
--
--
Translation of collagen ultrastructure to biomaterial fabrication for material-independent but highly efficient topographic immunomodulation
Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of “melt electrofibrillation” is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment.
Advanced materials
10.1002/adma.202101228
urn:nbn:de:bvb:20-opus-256381
publish
Advanced materials (2021) 33:33, 2101228. https://doi.org/10.1002/adma.202101228
617989
true
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Matthias Ryma
Tina Tylek
Julia Liebscher
Carina Blum
Robin Fernandez
Christoph Böhm
Wolfgang Kastenmüller
Georg Gasteiger
Jürgen Groll
eng
uncontrolled
biofabrication
eng
uncontrolled
extracellular matrix
eng
uncontrolled
immunomodulation
eng
uncontrolled
macrophages
eng
uncontrolled
melt electrofibrillation
eng
uncontrolled
melt electrowriting
Medizin und Gesundheit
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
OpenAIRE
Institut für Systemimmunologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/25638/Advanced_Materials_2021_Ryma_Translation_of_Collagen.pdf
31854
2022
eng
11
7
article
1
--
--
--
Correlative Analysis of Intra– Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification
In recent decades, hybrid characterization systems have become pillars in the study of cellular biomechanics. Especially, Atomic Force Microscopy (AFM) is combined with a variety of optical microscopy techniques to discover new aspects of cell adhesion. AFM, however, is limited to the early-stage of cell adhesion, so that the forces of mature cell contacts cannot be addressed. Even though the invention of Fluidic Force Microscopy (FluidFM) overcomes these limitations by combining the precise force-control of AFM with microfluidics, the correlative investigation of detachment forces arising from spread mammalian cells has been barely achieved. Here, a novel multifunctional device integrating Fluorescence Microscopy (FL) into FluidFM technology (FL-FluidFM) is introduced, enabling real-time optical tracking of entire cell detachment processes in parallel to the undisturbed acquisition of force-distance curves. This setup, thus, allows for entailing two pieces of information at once. As proof-of-principle experiment, this method is applied to fluorescently labeled rat embryonic fibroblast (REF52) cells, demonstrating a precise matching between identified force-jumps and visualized cellular unbinding steps. This study, thus, presents a novel characterization tool for the correlated evaluation of mature cell adhesion, which has great relevance, for instance, in the development of biomaterials or the fight against diseases such as cancer.
Advanced Materials Technologies
2365-709X
10.1002/admt.202200195
urn:nbn:de:bvb:20-opus-318544
@articleWeigl.2022, author = Weigl, Franziska and Blum, Carina and Sancho, Ana and Groll, Jürgen, year = 2022, title = Correlative Analysis of Intra– Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification, pages = 2200195, volume = 7, number = 11, issn = 2365-709X, journal = Advanced Materials Technologies, doi = 10.1002/admt.202200195
md5:727f7a4d2c13f0f91252dd3b03448fb4
2023-06-06T13:38:03+00:00
/tmp/php2XZXph
bibtex
647f36bb4ed733.28516577
Advanced Materials Technologies 2022, 7(11):2200195. DOI: 10.1002/admt.202200195
false
true
CC BY-NC: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell 4.0 International
Franziska Weigl
Carina Blum
Ana Sancho
Jürgen Groll
eng
uncontrolled
Fluorescence Microscopy
eng
uncontrolled
FluidFM technology
eng
uncontrolled
detachment force quantification
Medizin und Gesundheit
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/31854/Weigl_Advanced.pdf
25769
2021
eng
13
17
article
1
--
--
--
Appreciating the First Line of the Human Innate Immune Defense: A Strategy to Model and Alleviate the Neutrophil Elastase-Mediated Attack toward Bioactivated Biomaterials
Biointerface engineering is a wide-spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano-bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide-modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.
Small
10.1002/smll.202007551
urn:nbn:de:bvb:20-opus-257691
publish
Small 2021, 17(13):2007551. DOI: 10.1002/smll.202007551
false
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Carina Blum
Mehmet Berat Taskin
Junwen Shan
Tatjana Schilling
Katrin Schlegelmilch
Jörg Teßmar
Jürgen Groll
eng
uncontrolled
solution electrospinning
eng
uncontrolled
human neutrophil elastase (HNE)
eng
uncontrolled
peptide immobilization
eng
uncontrolled
polymeric matrix
Medizin und Gesundheit
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/25769/Blum_small.pdf
17104
2017
eng
558
7
article
1
2018-11-02
--
--
Phytic acid as alternative setting retarder enhanced biological performance of dicalcium phosphate cement in vitro
Dicalcium phosphate cement preparation requires the addition of setting retarders to meet clinical requirements regarding handling time and processability. Previous studies have focused on the influence of different setting modifiers on material properties such as mechanical performance or injectability, while ignoring their influence on biological cement properties as they are used in low concentrations in the cement pastes and the occurrence of most compounds in human tissues. Here, analyses of both material and biological behavior were carried out on samples with common setting retardants (citric acid, sodium pyrophosphate, sulfuric acid) and novel (phytic acid). Cytocompatibility was evaluated by in vitro tests with osteoblastic (hFOB 1.19) and osteoclastic (RAW 264.7) cells. We found cytocompatibility was better for sodium pyrophosphate and phytic acid with a three-fold cell metabolic activity by WST-1 test, whereas samples set with citric acid showed reduced cell number as well as cell activity. The compressive strength (CS) of cements formed with phytic acid (CS = 13 MPa) were nearly equal to those formed with citric acid (CS = 15 MPa) and approximately threefold higher than for other setting retardants. Due to a proven cytocompatibility and high mechanical strength, phytic acid seems to be a candidate replacement setting retardant for dicalcium phosphate cements.
Scientific Reports
10.1038/s41598-017-00731-6
28373697
urn:nbn:de:bvb:20-opus-171047
Scientific Reports 2017, 7:558. DOI: 10.1038/s41598-017-00731-6
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Susanne Meininger
Carina Blum
Martha Schamel
Jake E. Barralet
Anita Ignatius
Uwe Gbureck
eng
uncontrolled
implants
eng
uncontrolled
biomedical materials
eng
uncontrolled
dicalcium phosphate cement
eng
uncontrolled
phytic acid
Medizin und Gesundheit
open_access
Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17104/086_Gbureck_SCIENTIFIC-REPORTS.pdf