Biology subjects

van Griensven, M.

Publications and source records attributed to van Griensven, M..

10 recordsLinked to original sources

bioRxiv2026

Highlighting strain rate dependent vibrational behavior of electrospun bundles for tendon/ligament and enthesis fascicle tissue regeneration

Enthesis tissue engineering aims to develop scaffolds that replicate the mechanical and structural gradients of the tendon/ligament-bone interface. Among the different biofabrication techniques, electrospinning is surely one of the most promising to fabricate morpho-mechanically relevant enthesis fascicle-inspired scaffolds. An interesting and totally unexplored characteristic of these nanofibrous scaffolds is their ability, when mechanically tested, to produce/transmit strain rate and nanofiber fracture-dependent mechanical vibrations, which can potentially influence surrounding tissues and cells. This study develops a method to investigate how scaffold geometry and material affect vibrational behavior under mechanical stimulation. Electrospun bundles of poly(L-lactic) acid/collagen type I (PLLA/Coll) were fabricated to mimic the fibrocartilage, the enthesis junction, and the tendon/ligament regions, while block copolymer poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT-PBT) bundles represented only the tendon/ligament. Scaffolds were morphologically and mechanically characterized, including strain rate-dependent vibrational response. Scanning electron microscopy confirmed distinct fiber architectures. Under monotonic tensile tests to failure, scaffolds exhibited strain rate-dependent mechanical behavior, with PLLA/Coll bundles showing dominant vibrational frequencies up to 4.2 {+/-} 0.9 Hz with a scaffolds geometry-dependent manner. PEOT-PBT scaffolds instead, displayed higher vibration attenuation, with dominant frequencies peaking at 0.539 {+/-} 0.063 Hz. They also showed lower tensile properties, reflecting a different mechanical and vibrational profile respect to PLLA/Coll bundles. Integrating vibrational characterization with mechanical testing offers a novel framework for designing scaffolds that more accurately reproduce the gradient mechanical environment of fibrous musculoskeletal tissues such as tendons/ligaments and their entheses. These findings highlight the potential of this combined approach to further increase the mechanical comprehension of electrospun scaffolds. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/699441v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1e4f38dorg.highwire.dtl.DTLVardef@31e658org.highwire.dtl.DTLVardef@9c590org.highwire.dtl.DTLVardef@18996e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Understanding the structure and mechanics of the sheep calcaneal enthesis: a relevant animal model for tissue engineering applications

Tendon/enthesis injuries are a worldwide clinical problem. Along the enthesis, collagen fibrils show a progressive loss of anisotropy and an increase in mineralization reaching the bone. This causes gradients of mechanical properties. The design of scaffolds to regenerate these load-bearing tissues requires of being validated in vivo in relevant large animal models. The sheep tendon of triceps surae muscle is an optimal animal model for this scope with limited knowledge about its structure and mechanics. We decided to understand in-depth its structure and full-field mechanics. Collagen fibrils morphology was investigated via scanning electron microscopy revealing a marked change in orientation/dimensions passing from tendon to enthesis. Backscatter electron images and nanoindentation at the enthesis/bone marked small gradients of mineralization at the mineralized fibrocartilage reaching 27%wt and indentation modulus around 17-30 GPa. The trabecular bone instead had indentation modulus around 15-22 GPa. Mechanical tensile tests with digital image correlation confirmed the typical non-linear behavior of tendons (failure strain = 8.2{+/-}1.0%; failure force = 1369{+/-}187 N) with maximum principal strains reaching mean values of {varepsilon}p1[~]7%. The typical auxetic behavior of tendon was highlighted by the minimum principal strains ({varepsilon}p2[~]5%), progressively dampened at the enthesis. Histology revealed that this behavior was caused by a local thickening of the epitenon. Cyclic tests showed a force loss of 21{+/-}7 % at the last cycle. These findings will be fundamental for biofabrication and clinicians interested in designing the new generation of scaffolds for enthesis regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/630234v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@143f0a8org.highwire.dtl.DTLVardef@16cb394org.highwire.dtl.DTLVardef@181be61org.highwire.dtl.DTLVardef@f9f2d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceTendon and enthesis lesions are a clinical problem. To validate scaffolds for these applications large animal models are needed. Sheep tendon of triceps surae muscle is an optimal site for this scope. However, little is known about its extracellular matrix structure and mechanical properties. This work investigates the structure and mechanics of this tissue from different points of view. Scanning electron microscopy and histology studied its extracellular matrix morphology and composition. Backscattered electron images and nanoindentation assessed gradients of mineralization and stiffness at the enthesis. Mechanical tensile and cyclic tests coupled with digital image correlation elucidated its mechanics and superficial strain distribution. These findings will be fundamental for biofabrication and clinician experts to design innovative scaffolds to regenerate the enthesis.

bioengineering↗

bioRxiv2024

MiR-125b differentially impacts mineralization in dexamethasone and calcium-treated human mesenchymal stem cells

Bone metabolism is highly regulated, and microRNAs (miR) can contribute to this process. Among them, miR-125b is well-known to enhance osteoporosis and reduce osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this work we aim to evaluate and understand how miR-125b modulates mineralization of hMSCs in two different in vitro models. Cells were cultured in dexamethasone or calcium medium and transfected with miR-125b mimic. Exposure to dexamethasone or calcium medium increased mineralization of hMSCs and was associated with decreased miR-125b expression. Transfection of miR-125b mimic in dexamethasone-treated cells increased mineralization while it decreased it in calcium-treated cells. Levels of osteogenic markers presented the same difference. We identified STAT3, p53 and RUNX2 as direct targets of miR-125b in hMSCs. While these targets remained identical in both treatments, their modulation after transfection was different. We showed that miR-125b mimicking differentially modulated the expression of the miR-199a/214 cluster, probably via STAT3/miR-199a/214, and p53/miR-214 pathways. In conclusion, miR-125b affinity for targets implicated in bone remodeling changed depending on the in vitro models used to induce mineralization and led to opposite physiological effect. This works shows the complexity of drugs such as dexamethasone and opens the door for new in vitro models of mineralization.

molecular biology↗

bioRxiv2024

FGF9 treatment reduces off-target chondrocytes from iPSC-derived kidney organoids

Renal failure due to drug nephrotoxicity or disease is frequently observed in patients. The development of in vitro models able to recapitulate kidney biology offers new possibilities to study drug toxicity or model diseases. Induced pluripotent stem cell-derived kidney organoids already show promise, but several drawbacks must be overcome to maintain them in culture, among which is the presence of non-renal cell populations such as cartilage. We modified the culture protocol and maintained kidney organoids in medium containing FGF9 for one additional week compared to the control protocol (Takasato). In comparison to the control, the FGF9-treated kidney organoids had reduced cartilage at day 7+25 and diminished chondrocyte marker expression. Importantly, the renal structures assessed by immunofluorescence were unaffected by the FGF9 treatment. This reduction of cartilage produces a higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work. HighlightsO_LIKidney organoids develop cartilage between days 7+18 and 7+25 C_LIO_LIExtending the FGF9 supplementation reduces cartilage and chondrocyte markers C_LIO_LIFGF9-treated organoids present less EMT marker expression than control organoids C_LIO_LIRenal structures are not affected by the extended FGF9 treatment C_LI eTOC blurbLaPointe and colleagues showed that a one-week extension of FGF9 supplementation in iPSC-derived kidney organoids leads to a reduction of off-target cartilage. The renal structures are not impacted by FGF9 treatment and EMT markers are reduced. This modified organoid protocol will enable longer culture periods, a benefit for the use of organoids for screening or therapies.

cell biology↗

bioRxiv2024

Revealing the Auxetic Behavior of Biomimetic Multi-material and Region-specific Nanofibrous Scaffolds via Synchrotron Multiscale Digital Volume Correlation: Innovative Building Blocks for the Enthesis Regeneration

Enthesis lesions are one of the prevalent causes of injuries in the tendon tissue. The gradient of mineralization, extracellular matrix organization and auxetic mechanical properties, make enthesis regeneration challenging. Innovative electrospun fascicle-inspired nanofibrous poly(L-lactic)acid/collagen type I blend scaffolds were developed. Specifically, a mineralized fibrocartilage-inspired region (with/without nano-mineralization with hydroxyapatite), where random and aligned nanofibers coexist, is connected to a tendon-like region made of aligned nanofibers, through a conical non-mineralized fibrocartilage-inspired junction. Scanning electron microscopy and synchrotron nano-tomography show the morphological biomimicry of scaffolds with the natural tendon fascicles. Human mesenchymal stromal cells spheroids cultures confirm a balanced expression of both tendon, cartilage and bone markers on the non-mineralized scaffolds compared with the mineralized ones. Mechanical tests, at different physiological strain-rates, reveal a biomimetic mechanical behavior of scaffolds and the ability of junctions to tune the mechanics of their surrounding sites. Multiscale synchrotron in situ tensile tests, coupled with Digital Volume Correlation, elucidate the full-field strain distribution of scaffolds from the structural down to the nanofiber level, highlighting the auxetic mechanical behavior of junctions typical of the natural enthesis. The findings and cutting-edge investigations of our study suggest the suitability of these enthesis-inspired fascicles as innovative scaffolds for enhanced enthesis regeneration.

bioengineering↗

bioRxiv2023

Ultrastructural comparison of human kidney organoids and human fetal kidneys reveals features of hyperglycemic culture

Induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential for a large variety of applications. However, they do not persist long in culture, for which reasons are still unclear. Furthermore, their morphological maturation, an essential feature for kidney function, has not been sufficiently assessed. Kidney organoids are transcriptionally much alike end-of-first-trimester fetal kidneys and present many of the same cell types. From large transmission electron microscopy tilescans of specific regions of interest, we compared the ultrastructures of iPSC-derived kidney organoids at various timepoints to human fetal kidneys of the first trimester. Unlike healthy fetal kidneys, large glycogen deposits developed over time in all organoid cell types, but particularly in podocytes and in chondrocytes, one of the off-target populations that contaminate the culture. Deeper investigation showed that glycogen synthase kinase 3b (GSK3{beta}) levels and activation were diminished over time, correlated with the accumulation of glycogen. Activated YAP was strongly expressed and large lipid droplets accumulated over time in proximal tubules. Accordingly, EGFR signaling increased significantly over time. Mitochondria in glomeruli and tubules contained few or no cristae, indicating mitochondrial damage. Together these features are known for hyperglycemic cultures and diabetic nephropathy. Measuring the glucose concentration in the kidney organoid culture medium showed a concentration of 2.94 g/mL, which is considered an elevated, pre-diabetic-like concentration in vitro. In summary, our ultrastructural assessment of iPSC-derived kidney organoids using an age-matched fetal human reference allowed the evaluation of cellular morphology, and we identified intracellular features that can inform the cellular state, which is particularly important while physiological testing of organoids is limited. Translational StatementKidney organoids hold promise as a future treatment for patients with end-stage kidney disease. The engineering of kidney organoids with correct and healthy morphology in vitro is therefore essential, to guarantee functionality after transplantation. The present study provided deeper insights into the structural organization and ultrastructure of cells in kidney organoids compared to age-matched human fetal kidneys. Accordingly, we found several features in the regular kidney organoid culture, which are known to occur in hyperglycemic cultures and diabetic nephropathy, indicating that the current medium composition may be inducing pathological cellular phenotypes. This study therefore creates a better understanding of current limitations in the kidney organoid culture, increases knowledge of their function and cellular organization, and sets the foundation for further research to create advanced organoids.

bioengineering↗

bioRxiv2023

Structural development of the human fetal kidney: new stages and cellular dynamics in nephrogenesis

Research on the ultrastructural development of the kidney is limited, and research on rodent kidneys prevails. Yet, large differences between rodent and human nephrogenesis exist and therefore translation between species is not desirable. At the same time, there is an increasing demand for human research, in addition to assessing the potential of novel therapies such as renal organoids. We therefore generated an interactive atlas of large transmission electron microscopy tile scans of first trimester human kidneys, specifically Carnegie stage 20 until post-conceptional week 12. Analysis identified key ultrastructural features of proximal and distal progenitor cells such as cell shape, microvilli and luminal budding in the renal vesicle. Regarding glomerular development, we identified a new W-shaped body stage and three distinct sub-stages of the well-known capillary loop stage. Chromatin organization, nuclear shape and location were used to describe tubule cell identity and maturity, indicating a specific order of tubular maturation. The greatest congruence with adult tissue was seen in proximal tubules and the least in distal tubules. Finally, cytoplasmic glycogen depositions in collecting duct cells, which are absent in adult tissue, were found to be an early feature distinguishing distal tubules from collecting ducts as well as differentiating cortical from medullary collecting ducts. The findings of this research provide new fundamental insights for researchers who aim to understand and recreate kidney development.

developmental biology↗

bioRxiv2022

COMPUTATIONAL EVIDENCE FOR MULTI-LAYER CROSSTALK BETWEEN THE CADHERIN-11 AND PDGFR PATHWAYS

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell-cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR- binding to extracellular cadherin-11 regions increases the PDGFR- activity, whereas the interaction between PDGFR-{beta} and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between {beta}-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR- and -{beta}. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.

systems biology↗

bioRxiv2021

Enhanced microvasculature formation and patterning in iPSC-derived kidney organoids cultured in physiological hypoxia

Functional kidney organoids have the potential to be used in implantable kidney grafts for patients with end-stage kidney disease, because they have been shown to self-organize from induced pluripotent stem cells into most important renal structures. To date, however, long-term kidney organoid culture has not succeeded, as nephrons lose their phenotype after approximately 25 days. Furthermore, the renal structures remain immature with diminishing endothelial networks with low connectivity and limited organoid invasion. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, VEGF-A expression and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of proangiogenic VEGF-A189 mRNA and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids. Significance statementCulturing kidney organoids in a hypoxic environment improved patterning of the endothelial network and improved vascularization. These findings may help improve the quality of kidney organoids, and could eventually improve the kidney graft for transplantation in patients with end-stage kidney disease. Furthermore, the organoids will be more suitable for drug testing and in developmental biology. The findings might also be translatable to other organoid models containing endothelial cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/473849v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@d5cde7org.highwire.dtl.DTLVardef@c0ff46org.highwire.dtl.DTLVardef@b76191org.highwire.dtl.DTLVardef@166823d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Mechanical and possible auxetic properties of human Achilles tendon during in vitro testing to failure

The Achilles tendon is the strongest tendon in the human body, but the basis of its high tensile strength has not been elucidated in detail. Here we have loaded healthy, human, Achilles tendons to failure in an anatomically authentic fashion while studying the local three-dimensional deformation and strains in real time, with very high precision, using digital image correlation (DIC). These studies identified a remarkable degree of anisotropic, medio-lateral auxetic behavior, with Poissons ratios not exceeding minus 1 in any part of the tendon at any time; under certain loads, discrete areas within the tendon had a Poissons ratio below minus 6. Early in the loading cycle, the proximal region of the tendon accumulated high lateral strains while longitudinal strains remained low. This behavior shielded the mid-substance of the tendon, its weakest part, from high longitudinal strains until immediately before rupture. These new insights are of great relevance to understanding the material basis of tendon injuries, designing improved prosthetic replacements, and developing regenerative strategies.

bioengineering↗