Mechanomorphological Guidance of Colloidal Gel Regulates Cell Morphogenesis

Abstract

Microstructural morphology of the extracellular matrix guides the organization of cells in 3D. However, current biomaterials-based matrices cannot provide distinct spatial cues through their microstructural morphology due to design constraints. To address this, colloidal gels are developed as 3D matrices with distinct microstructure by aggregating ionic polyurethane colloids via electrostatic screening. Due to the defined orientation of interconnected particles, positively charged colloids form extended strands resulting in a dense microstructure whereas negatively charged colloids form compact aggregates with localized large voids. Chondrogenesis of human mesenchymal stem cells (MSCs) and endothelial morphogenesis of human endothelial cells (ECs) are examined in these colloidal gels. MSCs show enhanced chondrogenic response in dense colloidal gel due to their spatial organization achieved by balancing the cell-cell and cell-matrix interactions compared to porous gels where cells are mainly clustered. ECs tend to form relatively elongated cellular networks in dense colloidal gel compared to porous gels. Additionally, the role of matrix stiffness and viscoelasticity in the morphogenesis of MSCs and ECs are analyzed with respect to microstructural morphology. Overall, these results demonstrate that colloidal gels can provide spatial cues through their microstructural morphology and in correlation with matrix mechanics for cell morphogenesis.

Keywords: chondrogenic differentiation; colloidal gel; endothelial morphogenesis; mechanomorphology; microstructure; polyurethane.

Figure 1.. Ionic polyurethane (PU) colloids and colloidal aggregation

A) Structure of cationic (CP) and anionic (AP) polycaprolactone based polyurethane and the size and zeta potential of colloidal PU particles in ddH₂O. B) Scanning electron microscopic (scale bar: ~500 nm) and fluorescent images (scale bar: ~50 μm) of colloidal PU particles. C) Change of size of CP and AP colloids due to aggregation mediated by sodium chloride of different strength with respect to time. Rate of aggregation for formation of CPN and APN from CP and AP colloids respectively, measured from size change with respect to time. D) Change of zeta potential of CP and AP colloids due to aggregation mediated by sodium chloride of different strength. E) ‘Dispersibility factor, n’ due to aggregation of CP and AP colloids mediated by sodium chloride of different strength. Variation of ‘n’ with respect to particle fraction in colloidal PU particles (CP and AP) and aggregated CPN and APN system. Kinetics of aggregation measured from change in optical density with time for CPN and APN system.

Figure 2.. Microstructural morphology of colloidal gels.

A) Microstructured morphology of CPN and APN gels from representative scanning electron microscopic images (scale bar: ~1 μm). Inset shows spatial organization of colloidal particles in strands of interconnected particles (scale bar: ~500 nm). B) Confocal scanning fluorescent images of CPN and APN gels showing morphology and spatial distribution of voids in the gels. Morphological features of the microstructure analyzed by measuring porosity and pore of the gels from these images using in ImageJ. (ANOVA, ** P ≤0.01, * P ≤0.05)

Figure 3.. Mechanical characterizations of colloidal gel from rheology.

A) Elastic moduli (G′(ω)), yield stress, yield strain, and tan δ(ω) of CPN and APN gel from 0.05 particle fraction obtained from linear viscoelastic region of strain amplitude strain sweep at constant frequency, ω of 1Hz. Yield measured from the crossover point of elastic (G′) and viscous (G″) moduli B) Variation of elastic moduli (G'), yield stress and yield strain of CPN and APN gels prepared from different particle fractions. C) Dynamic oscillatory response of CPN and APN colloidal gels with 0.05 w/v particle fraction measured from four consecutive cycles of strain ramps by applying a strain of 0.6 followed by recovery to a strain 0.01.

Figure 4.. Time-dependent viscoelastic response of colloidal gels from creep measurement.

A) Deformation of CPN and APN gel with 0.05 w/v particle fraction for 150s at constant stresses of 4 Pa and 40 Pa followed by recovery for 150s. B) Elastic and viscoelastic part of the recovery responses following deformation of CPN and APN gels.

Figure 5.. Human mesenchymal stem cells (MSCs) organization and chondrogenesis in colloidal gels.

A) Representative fluorescent images of 3D organization of MSCs (green: actin and blue: nucleus) in CPN and APN gels formed from 0.05 w/v particle fraction. Morphometric analysis of MSC organization from the size (i.e., area) and shape (i.e., circularity) measurement of cell aggregates and single cells (scale bar: aggregate~50 μm, cells~25 μm). B) Expression of biomolecules by MSCs after chondrogenic differentiation in CPN and APN gels formed from 0.05 w/v particle fraction. Immunohistological staining of sectioned samples for glycosaminoglycans (GAG) from alcian blue stain and collagen from trichrome stain with counterstained by H&E. Quantified expression for synthesis of total amount of GAG and collagen normalized with respect to DNA content. C) Expression of chondrogenic markers by MSCs after chondrogenic differentiation in CPN and APN gels formed from 0.05 w/v particle fraction. Immunohistochemical staining of sectioned sample and semi-quantitative analysis for the normalized expression of collagen II and aggrecan as representative chondrogenic markers. (ANOVA, ** P ≤0.01, * P ≤0.05)

Figure 6.. Endothelial cell (EC) organization and morphogenesis in colloidal gels.

A) Representative H&E-stained images of ECs in CPN and APN colloidal gel prepared from 0.05 w/v particle fraction at 3 days (scale bar: ~100 μm). B) Morphometric analysis of EC organization measured by analyzing the size (area), shape (circularity index) and total length of the cellular aggregates in CPN and APN colloidal gel. C) Representative fluorescent images of ECs in CPN and APN colloidal gel with F-actin and vinculin expression (green = F-actin, red = vinculin, and blue =nucleus) and semi-quantitative analysis for the normalized expression of vinculin. (ANOVA, ** P ≤0.01, * P ≤0.05).