Visualization and manipulation of repair and regeneration in biological systems using light

Tissue repair after an injury is a fundamental process in biomedicine. It can involve regeneration, which uses new growth to restore tissue function. The interest in repair and regeneration is motivated by the desire to treat injuries and diseases and has attracted researchers for centuries. In the last decades, it evolved in the field of regenerative medicine, which has the ultimate goal of providing strategies for regenerating human cells, tissues, or even organs, for instance, via engineering principles. Already since the first experiments on regeneration by Abraham Trembley, novel findings in biomedicine, repair, and regeneration have been enabled or accompanied by research in optics, for example, on the development of novel microscopy techniques. Nowadays, novel optical techniques are advancing, which allow to understand the role of single cells in tissue repair processes. Moreover, repair processes within cells can be visualized and manipulated. Ultimately, optics can provide enabling techniques for regenerative therapies. This habilitation thesis aims to present several of these advances. On a single cell level, femtosecond laser nanosurgery was used to target specific intracellular structures during concurrent imaging in vitro. The relation of femtosecond laser nanosurgery to the cell state and cellular staining was investigated. Manipulation of single Z-discs in cardiomyocytes using a femtosecond oscillator laser system was accomplished, which allows to better elucidate the role of a single Z-disc in cardiomyocyte function. In particular, measurements on cell survival, (calcium-) homeostasis, and morphology yielded only minor deviations from control cells after single Z-disc ablation. A reduction in force generation was elucidated via traction force microscopy and gene expression level changes, for instance, an upregulation of -actinin were examined. Additionally, light-based systems to influence single cells in their alignment or to trigger single cells, for example, to activate other cells via optogenetics were applied. On the tissue scale, imaging via confocal microscopy or multiphoton microscopy has been applied for various contexts of regenerative approaches. Furthermore, a fiber-based imaging approach, which could later be used for longitudinal imaging in vivo and builds upon a fluorescence microscope system and an imaging fiber bundle in combination with reconstruction via a neural network, was developed. As another imaging strategy, an abdominal imaging window served to image the mouse liver in vivo via multiphoton microscopy in successive imaging sessions. Manipulation in tissue was applied in colonoids, which resemble the structure of the colon on an in vitro scale, and revealed different cell dynamics dependent on the location of the damage. In particular, activation of the Wnt signaling pathway after crypt damage was observed. Cell ablation via a femtosecond laser amplifier system during concurrent two-photon microscopy was also established during in vivo liver imaging to study micro-regenerative processes. Furthermore, laser-based delivery processes with novel materials or in the context of genome editing using CRISPR/Cas9 technology were investigated as enabling technologies for regenerative medicine. In conclusion, this thesis addresses the question of how optics can help to illuminate future directions in research on tissue repair and regeneration, as well as, regenerative therapies by addressing (longitudinal) imaging in a complex environment, sophisticated cell-manipulation strategies, and the application of novel materials for laser-based delivery

Parameters for Optoperforation-Induced Killing of Cancer Cells Using Gold Nanoparticles Functionalized With the C-terminal Fragment of Clostridium Perfringens Enterotoxin

Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10−11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells

Probing interneuronal cell communication via optogenetic stimulation

This study uses an all-optical approach to probe interneuronal communication between spiral ganglion neurons (SGNs) and neurons of other functional units, in this case cortex neurons (CNs) and hippocampus neurons (HNs), for the first time. We combined a channelrhodopsin variant (CheRiff) with a red genetically encoded calcium indicator (jRCaMP1a), enabling simultaneous optical stimulation and recording from spatially separated small neuronal populations. Stimulation of SGNs was possible with both optogenetic manipulated HNs and CNs, respectively. Furthermore, a dependency on the pulse duration of the stimulating light in regard to the evoked calcium response in the SGNs was also observed. Our results pave the way to enable innovative technologies based on “biohybrid” systems utilizing the functional interaction between different biological (eg, neural) systems. This can enable improved treatment of neurological and sensorineural disorders such as hearing loss

Evaluation of laser induced sarcomere microdamage: Role of damage extent and location in cardiomyocytes

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laserbased nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6-7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms. Copyright

Characterization of tissue engineered endothelial cell networks in composite collagen-agarose hydrogels

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either Matrigel™ and rat tail collagen I (Matrigel™/rCOL) or human collagen (hCOL) with SeaPlaque™ agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in Matrigel™/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of Matrigel™/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Gold nanoparticle-mediated laser stimulation causes a complex stress signal in neuronal cells

Gold nanoparticle mediated laser stimulation of neuronal cells allows for cell activation on a single-cell level. It could therefore be considered an alternative to classical electric neurostimulation. The physiological impact of this new approach has not been intensively studied so far. Here, we investigate the targeted cell's reaction to a laser stimulus based on its calcium response. A complex cellular reaction involving multiple sources has been revealed. © 2017 SPIE-OSA

Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue’s native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids’ response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids

Biodistribution, biocompatibility and targeted accumulation of magnetic nanoporous silica nanoparticles as drug carrier in orthopedics

Background: In orthopedics, the treatment of implant-associated infections represents a high challenge. Especially, potent antibacterial effects at implant surfaces can only be achieved by the use of high doses of antibiotics, and still often fail. Drug-loaded magnetic nanoparticles are very promising for local selective therapy, enabling lower systemic antibiotic doses and reducing adverse side effects. The idea of the following study was the local accumulation of such nanoparticles by an externally applied magnetic field combined with a magnetizable implant. The examination of the biodistribution of the nanoparticles, their effective accumulation at the implant and possible adverse side effects were the focus. In a BALB/c mouse model (n = 50) ferritic steel 1.4521 and Ti90Al6V4 (control) implants were inserted subcutaneously at the hindlimbs. Afterwards, magnetic nanoporous silica nanoparticles (MNPSNPs), modified with rhodamine B isothiocyanate and polyethylene glycol-silane (PEG), were administered intravenously. Directly/1/7/21/42 day(s) after subsequent application of a magnetic field gradient produced by an electromagnet, the nanoparticle biodistribution was evaluated by smear samples, histology and multiphoton microscopy of organs. Additionally, a pathohistological examination was performed. Accumulation on and around implants was evaluated by droplet samples and histology. Results: Clinical and histological examinations showed no MNPSNP-associated changes in mice at all investigated time points. Although PEGylated, MNPSNPs were mainly trapped in lung, liver, and spleen. Over time, they showed two distributional patterns: early significant drops in blood, lung, and kidney and slow decreases in liver and spleen. The accumulation of MNPSNPs on the magnetizable implant and in its area was very low with no significant differences towards the control. Conclusion: Despite massive nanoparticle capture by the mononuclear phagocyte system, no significant pathomorphological alterations were found in affected organs. This shows good biocompatibility of MNPSNPs after intravenous administration. The organ uptake led to insufficient availability of MNPSNPs in the implant region. For that reason, among others, the nanoparticles did not achieve targeted accumulation in the desired way, manifesting future research need. However, with different conditions and dimensions in humans and further modifications of the nanoparticles, this principle should enable reaching magnetizable implant surfaces at any time in any body region for a therapeutic reason. © 2020 The Author(s)

CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10-/-, Cd14-/-, and Alpk1-/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14-/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1-/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge. © 2020 Pascal Brooks et al