Biology subjects

Freudenberg, U.

Publications and source records attributed to Freudenberg, U..

7 recordsLinked to original sources

bioRxiv2025

Fabrication of Short Polymeric μFibers as Building Blocks for Anisotropic High-Throughput Compatible 3D Tissue Models

Modeling the 3D microenvironment and cell growth of natively anisotropic human tissues in vitro constitutes a significant challenge in tissue engineering and biofabrication. Short polymeric fibers gain growing attention in this field due to their applicability as pipettable or injectable anisometric building blocks in 3D hydrogel-based cell culture systems or bioinks. However, the production of monodisperse short fibers with high production rates suitable for screening remains challenging. In this study, short, quasi-monodisperse, magneto-responsive, fluorescent poly({varepsilon}-caprolactone) {micro}fibers with variable dimensions in the micrometer range are produced in a scalable, semi-continuous two-step fabrication process combining controlled wet-dry spinning with subsequent cryosectioning. Influences of the spinning process parameters on fiber properties and process features, as well as boundary spinning conditions and upscaling potential, are explored using Design of Experiments approaches. Further, magnetic alignment of the {micro}fibers in a weak magnetic field and incorporation of nile red as fluorescent dye for facile analysis in 3D are demonstrated. Implementation of aligned {micro}fibers into a hydrogel-based 3D vasculogenesis model, produced in a high-throughput automated manner, is shown to stimulate oriented cell growth. This highlights the potential of our {micro}fibers as guiding elements inside tissue and disease models and their suitability for automated high-throughput applications.

bioengineering↗

bioRxiv2025

Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2025

Mechanical control of tissue growth during limb regeneration

The axolotl is a highly regenerative species, capable of restoring full limbs, regardless of the amputation site. However, the regeneration rate is adjusted with the plane of amputation along the proximo-distal (PD) axis, leading to equivalent regeneration times regardless of the extent of tissue removal. We hypothesized that this phenomenon could be partly explained by differences in tissue mechanical properties. In this work, we describe tissue growth mathematically and evaluate cell cycle parameters of regenerating limbs amputated at different levels along the PD axis, demonstrating a linear correlation between the cell cycle length and the amputation site during early regeneration phases. We show as well, that blastema cells require their endogenous context to retain such proliferation differences. We measured mechanical properties in regenerating limbs with in vivo optical and standard indentation-based techniques and demonstrated that distal blastema cells are stiffer than proximal ones. Accordingly, we demonstrated that axolotl cells decrease their proliferation with increased extracellular matrix stiffness in vitro. Next, we evaluated the activity of the mechanotransducers YAP/TAZ in vivo by using a GTIIC-based reporter line combined with target gene expression data, which indicated that their activity peaks during the blastema stage, with higher activity after proximal amputations. Hence, our findings strongly suggest a mechanical dependence for the position-dependent regulation of cell proliferation during axolotl limb regeneration, where YAP/TAZ likely plays a role in the mechanotransduction mechanism.

biophysics↗

bioRxiv2024

A precision microgel platform to direct vascular morphogenesis in vitro

The dynamic organization of tissue development is reciprocally controlled by localized gradients of morphogens emanating from distinct clusters of cells that act as signaling centers1. While microgels2,3 have shown promise to recapitulate this process in engineered tissue constructs, their capacity to tailor morphogen distribution in space and time remained limited4-7. Here, we introduce a library of sulfated glycosaminoglycan (sGAG)-based microgels that offer unprecedented control over morphogen affinity (GUIDe, Gel Units to Instruct Development), thus enabling precise formation of concentration gradients. Multiparametric adjustment of the microgel charge patterns resulting from sGAG ionization was key to programmable morphogen release. The potential of our microgel system to guide tissue morphogenesis is demonstrated through the local administration of VEGF gradients in a microgel-in-gel in vitro vasculogenesis model and in hiPSC-derived kidney organoid cultures. Our micromaterials-based methodology offers valuable new options to mimic and modulate morphogen signaling centers, thereby advancing tissue/organ development research.

bioengineering↗

bioRxiv2019

3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.

bioengineering↗

bioRxiv2017

Interleukin-4 restores neurogenic plasticity of the primary human neural stem cells through suppression of Kynurenic acid production upon Amyloid-β42 toxicity

The immune response is an important determinant of the plasticity and neurogenic capacity of neural stem cells (NSCs) upon amyloid-beta42 (A{beta}42) toxicity in Alzheimers disease (AD). However, the direct effects of individual immuno-modulatory effectors on NSC plasticity remain to be elucidated and are the motivation for reductionist tissue-mimetic culture experiments. Using starPEG-Heparin hydrogel system that provides a defined 3D cell-instructive neuro-microenvironment culture system, sustains high levels of proliferative and neurogenic activity of human NSCs, and recapitulates the fundamental pathological consequences of Amyloid toxicity upon A{beta}42 administration, we found that the anti-inflammatory cytokine interleukin-4 (IL4) restores the plasticity and neurogenic capacity of NSCs by suppressing the A{beta}42-induced kynurenic acid-producing enzyme kynurenine aminotransferase 2 (KAT2), which we also found to be upregulated in the brains of the AD model, APP/PS1dE9 mouse. Our transcriptome analyses showed that IL4 treatment restores the expression levels of NSC and cortical subtype markers. Thus, our dissective neuro-microenvironment culture revealed IL4-mediated neuroinflammatory crosstalk for human NSC plasticity and predicted a new mechanistic target for therapeutic intervention in AD.

neuroscience↗

bioRxiv2017

Instructive starPEG-Heparin biohybrid 3D cultures for modeling human neural stem cell plasticity, neurogenesis, and neurodegeneration

Three-dimensional models of human neural development and neurodegeneration are crucial when exploring stem-cell-based regenerative therapies in a tissue-mimetic manner. However, existing 3D culture systems are not sufficient to model the inherent plasticity of NSCs due to their ill-defined composition and lack of controllability of the physical properties. Adapting a glycosaminoglycan-based, cell-responsive hydrogel platform, we stimulated primary and induced human neural stem cells (NSCs) to manifest neurogenic plasticity and form extensive neuronal networks in vitro. The 3D cultures exhibited neurotransmitter responsiveness, electrophysiological activity, and tissue-specific extracellular matrix (ECM) deposition. By whole transcriptome sequencing, we identified that 3D cultures express mature neuronal markers, and reflect the in vivo make-up of mature cortical neurons compared to 2D cultures. Thus, our data suggest that our established 3D hydrogel culture supports the tissue-mimetic maturation of human neurons. We also exemplarily modeled neurodegenerative conditions by treating the cultures with A{beta}42 peptide and observed the known human pathological effects of Alzheimers disease including reduced NSC proliferation, impaired neuronal network formation, synaptic loss and failure in ECM deposition as well as elevated Tau hyperphosphorylation and formation of neurofibrillary tangles. We determined the changes in transcriptomes of primary and induced NSC-derived neurons after A{beta}42, providing a useful resource for further studies. Thus, our hydrogel-based human cortical 3D cell culture is a powerful platform for studying various aspects of neural development and neurodegeneration, as exemplified for A{beta}42 toxicity and neurogenic stem cell plasticity.\n\nSignificanceNeural stem cells (NSC) are reservoir for new neurons in human brains, yet they fail to form neurons after neurodegeneration. Therefore, understanding the potential use of NSCs for stem cell-based regenerative therapies requires tissue-mimetic humanized experimental systems. We report the adaptation of a 3D bio-instructive hydrogel culture system where human NSCs form neurons that later form networks in a controlled microenvironment. We also modeled neurodegenerative toxicity by using Amyloid-beta4 peptide, a hallmark of Alzheimers disease, observed phenotypes reminiscent of human brains, and determined the global gene expression changes during development and degeneration of neurons. Thus, our reductionist humanized culture model will be an important tool to address NSC plasticity, neurogenicity, and network formation in health and disease.

neuroscience↗