Biology subjects

Rouwkema, J.

Publications and source records attributed to Rouwkema, J..

7 recordsLinked to original sources

bioRxiv2025

Long-Term Functional 3D Human Skeletal Muscle Constructs from Human Induced Pluripotent Stem Cells Enable Electrically Controlled Remodelling Studies

Studying skeletal muscle remodelling across large temporal scales is difficult because most 3D tissue-engineered skeletal muscle (3D-TESM) models cannot sustain stable development over prolonged culture. Skeletal muscle adaptations in vivo are strongly influenced by changes in tissue length, but most TESM models are mechanically restrictive, limiting elongation and thereby constraining structural remodelling. The stiffness of the pillars that anchor the tissues is a key determinant of how much they can shorten or elongate during development, thereby shaping both their morphology and force-generating capacity. To address this limitation, we investigated how TESMs derived from human induced pluripotent stem cells (hiPSCs) develop over two weeks when suspended between pillars with distinct mechanical properties, ranging from mechanically compliant to mechanically stiff boundary conditions. We focused on both passive and active force-generating properties. With compliant pillars, tissues compacted primarily along their length (~66% reduction), followed by progressive elongation (~50% increase) from day 5 onward. In contrast, with stiff pillars, tissues compacted across their width, with no longitudinal shortening. Despite these morphological differences, projected tissue area decreased similarly across setups. Spontaneous contractions emerged from day 7 and continued until day 14, typically occurring in bursts at 0.6-0.8 Hz. Passive force peaked early with compliant pillars and declined thereafter, while remaining relatively stable with stiff pillars. Under stepwise electrical stimulation, maximal contractility wasconsistently observed at ~50 Hz. Active force rose sharply between day 12 and day 14, with the steepest increase (up to 2000%) in compliant pillars. Although sample size was limited, the consistency of trends across stiffness conditions and surface treatments provides valuable insights into how boundary mechanics shape 3D-TESM development. These findings offer a foundation for designing next-generation muscle culture systems that support dynamic remodelling, force maturation, and training-responsive behaviours. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/672872v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@4caa3borg.highwire.dtl.DTLVardef@13c421dorg.highwire.dtl.DTLVardef@11881a1org.highwire.dtl.DTLVardef@1e673a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Spatiotemporally Programmed Release of Aptamer Tethered Dual Angiogenic Growth Factors

In tissue extracellular matrix (ECM), multiple growth factors (GFs) are sequestered through affinity interactions and released as needed by proteases, establishing spatial morphogen gradients in a time-controlled manner to guide cell behavior. Inspired by these ECM characteristics, we developed an "intelligent" biomaterial platform that spatially controls the combined bioavailability of multiple angiogenic GFs, specifically vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB). Utilizing aptamer affinity interactions and complementary sequences within a GelMA matrix, our platform achieves on-demand, triggered release of individual GFs which can be "programmed" in temporally-controlled, repeatable cycles. The platform features stable incorporation of dual aptamers specific for both GFs, functional aptamer-CS molecular recognition in a 3D microenvironment with long-term stability of at least 15 days at physiological temperature, and spatially localized sequestration of individual GFs. Additionally, the system allows differential amounts of GFs to be released from the same hydrogels at different time-points, mimicking dynamic GF presentation in a 3D matrix similar to the native ECM. This flexible control over individual GF release kinetics opens new possibilities for dynamic GF presentation, with adjustable release profiles to meet the spatiotemporal needs of growing engineered tissue. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/607163v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@15f9db4org.highwire.dtl.DTLVardef@6a9834org.highwire.dtl.DTLVardef@2e896borg.highwire.dtl.DTLVardef@13fe883_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Development of an Organ-on-a-Chip for Correlative Microscopy: Visualizing Early Osteogenesis in 3D with High Resolution

Correlative microscopy approaches offer powerful means to study tissue development across spatial scales, but combining 3D light and electron imaging remains technically challenging. Here, we present a practical workflow that integrates organ-on-a-chip culture with longitudinal fluorescence imaging and volume electron microscopy. By modifying an existing chip platform designed for aligned tissue growth, we demonstrate the feasibility of extended 3D live imaging and subsequent high-pressure freezing of intact microtissues. Fluorescence-guided targeting enables focused ion beam/scanning electron microscopy (FIB/SEM) of selected regions, revealing ultrastructural features such as cellular organization, collagen alignment, and matrix mineralization. While not aimed at new biological discoveries, this study highlights the compatibility and potential of this pipeline for future high-resolution, multiscale studies of tissue morphogenesis and pathology in controlled microenvironments.

bioengineering↗

bioRxiv2024

Shape that matters: Yolk geometry spatially modulates developing vascular networks within chick chorioallantoic membrane

Controlling the multiscale organization of vasculature within diverse geometries is essential for shaping tissue-specific and organ-specific architectures. Nevertheless, how geometrical characteristics of surrounding tissues influence vessel morphology and blood flow remains unclear. Where the regulation of vascular organization by mechanical signals associated with fluid flow is well known, this study postulates that the organization of developing vasculature can also be regulated by mechanical signals connected to the confinement and thus the deformation of surrounding tissues. To test the Shape-Induced Vascular Adaptation (SIVA) concept, fertilized chicken egg contents containing developing vasculature were cultured within engineered eggshell platforms of different shapes. Our findings demonstrate that the vascularized chick chorioallantoic membrane (CAM) adapts to the shape of engineered eggshell, long before reaching its boundaries. This adaptation affects the organization of the vascular network within the CAM, affecting parameters such as vessel area, branching, orientation, length, diameter and endpoints. Specifically, we observed that sharp corners in the engineered eggshell led to more elongated vascular structures. To further explore the dynamic nature of this phenomenon, a proof-of-concept experiment was performed using a shape-shifting engineered eggshell that deforms the egg content from circle to square shape. Using this shape-shifting prototype, we observed a direct effect of eggshell deformation on the vessel morphology and flow dynamics in a time-dependent manner. Overall, our exovo experimental platform provides a unique opportunity to study how mechanical stimuli such as shape influence the spatial and temporal organization of developing vascularized tissues. By subjecting these tissues to various static and dynamic conditions, we induced both local and global changes in their organization. This class of perturbation provides us with an additional tool which can be used for shaping vascular organization within developing tissues and to engineer tissues with geometrically tunable vessel structures.

bioengineering↗

bioRxiv2024

Bioprinting of aptamer-based programmable bioinks to modulate multiscale microvascular morphogenesis in 4D

Dynamic growth factor presentation influences how individual endothelial cells assemble into complex vascular networks. Here, we developed programmable bioinks that facilitate dynamic VEGF presentation to guide vascular morphogenesis within 3D-bioprinted constructs. We leveraged aptamers high affinity for rapid VEGF sequestration in spatially confined regions and utilized aptamer-complementary sequence (CS) hybridization to tune VEGF release kinetics temporally, days after bioprinting. We show that spatial resolution of programmable bioink, combined with CS-triggered VEGF release, significantly influences alignment, organization, and morphogenesis of microvascular networks in bioprinted constructs. The presence of aptamer-tethered VEGF and the generation of instantaneous VEGF gradients upon CS-triggering restricted hierarchical network formation to the printed aptamer regions at all spatial resolutions. Network properties improved as the spatial resolution decreased, with low-resolution designs yielding the highest network properties. Specifically, CS-treated low-resolution designs exhibited significant vascular network remodeling, with increase in vessel density(1.35-fold), branching density(1.54-fold), and average vessel length(2.19-fold) compared to non-treated samples. Our results suggests that CS acts as an external trigger capable of inducing time-controlled changes in network organization and alignment on-demand within spatially localized regions of a bioprinted construct. We envision that these programmable bioinks will open new opportunities for bioengineering functional, hierarchically self-organized vascular networks within engineered tissues.

bioengineering↗

bioRxiv2024

Switching to external flows: perturbations of developing vasculature within chicken chorioallantoic membrane

The impact of fluid flow shear stresses, generated by the movement of blood through vasculature, on the organization and maturation of vessels is widely recognized. Nevertheless, it remains uncertain whether external fluid flows outside of the vasculature in the surrounding tissue can similarly play a role in governing these processes. In this research, we introduce an innovative technique called superfusion-induced vascular steering (SIVS). SIVS involves the controlled imposition of external fluid flow patterns onto the vascularized chick chorioallantoic membrane (CAM), allowing us to observe how this impacts the organization of vascular networks. To investigate the concept of SIVS, we conducted superfusion experiments on the intact chick CAM cultured within engineered eggshell system, using phosphate buffered saline (PBS). To capture and analyze the effects of superfusion, we employed a custom-built microscopy setup, enabling us to image both superfused and non-superfused regions within the developing CAM. This study provides valuable insights into the practical application of fluid superfusion within an in vivo context, shedding light on its significance for understanding tissue development and manipulation in an engineering setting.

bioengineering↗

bioRxiv2020

Aptamer based spatiotemporally controlled growth factor patterning for tunable local microvascular network formation in engineered tissues

Spatiotemporally controlled growth factor availability is of crucial importance for achieving hierarchically organized vascular network formation within engineering tissues. Even though current growth factor delivery systems can provide sustained release and growth factor delivery on demand, they generally do not facilitate temporal control over the release rates and thus adaptation in accordance with the needs of growing engineered tissue. Additionally, with conventional growth factor loading methods, growth factors are often subjected to organic solvents or harsh conditions, leading to lower bioactivity and denaturation of the proteins. To overcome these limitations, this manuscript reports on the development of VEGF specific 5 acrydite modified aptamer functionalized GelMA hydrogels. The covalently incorporated aptamers can selectively bind to proteins with high affinity and specificity, and can thus sequester the target protein from the surrounding environment. The manuscript shows that this not only provides temporal control over the growth factor release via complementary sequence hybridization, but also enables local control of microvascular network formation in 3D.

bioengineering↗