Cytoskeleton systems contribute differently to the functional intrinsic properties of chondrospheres

Abstract

Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time.

Introduction

CSs represent three-dimensional structures organized by densely packed chondrocytes in physiological environment, including in particular naturally synthesized specific ECM proteins, collagen type II and aggrecan. These self-organized structures retain the architectural and functional characteristics typical for the tissue of origin, the cartilage. CSs are the promising tool for cartilage repair, upon transplantation they are able to adhere and to integrate into native hyaline cartilage thus meeting the essential requirement for tissue reconstitution [1]. This approach is known as autologous chondrocyte implantation (ACI), in which articular cartilage biopsy is taken to obtain primary chondrocytes, to culture and multiply them in vitro and to produce CSs to be implanted then into the chondral defect [2]. Additionally, this method overcome the possibility of contamination, inflammation, and immune response since autologous сells without any additional synthetic scaffold are used for implantation. Despite the encouraging potential of CSs in clinical practice, the mechanisms guiding their fundamental functional characteristics such as intrinsic capacity to fuse, attach and spread on the adhesive surface are still poorly understood. Furthermore, CSs biomechanical properties which strongly influence the tissue regeneration and repair, need further elucidation since to form a healthy mature tissue cells must be organized both by spatial and temporal manners.

The development of a three-dimensional configuration involves cell migration, polarization, shape change, the establishment of specific intercellular adhesions and redistribution of cell-cell and cell-surface adhesion forces, aggregation and compaction. These processes are intimately guided by cytoskeleton systems. In self-assembled systems each cell has to organize its cytoskeletal structure in accordance with the cytoskeletal arrangement of the neighboring cells. There is a wealth of evidence to suggest that three cytoskeletal systems, namely the actin cytoskeleton, MTs and IFs network regulate all or part of fore mentioned processes. Several studies have demonstrated the engagement of cytoskeleton systems in course of spheroids formation [3,4], though their precise role in regulating the intrinsic functional capacity of cell aggregates to fuse, to disassemble on adhesive surface and maintain specific morphology has not been thoroughly explored.

Tissue fusion is a fundamental principle in developmental biology [5,6] and is highly relevant to tissue engineering methodology. When spheroids are placed in close contact with each other, they spontaneously coalesce to form one larger structure following the self-assembly principle. Fusion of spheroids is one of the approaches to study how tissues and organs are formed in tissue engineering [7], [8], [9], [10], and CSs are commonly used to study spheroid fusion for cartilage engineering [11], [12], [13], [14]. The intrinsic property of spheroids to fuse offers the exciting possibility of fabricating tissue constructs with a complex cytoarchitecture by directed assembly of numerous cell aggregates. Previous studies analyzing the fusion of spheroids revealed that their fusion could be described by a model of the coalescence of highly viscous liquid drops [5,6,[15], [16], [17], [18]]. According to this concept, the viscosity and surface tension determine the fusion [19]. In agreement with the differential adhesion hypothesis (DAH) [6,16,17], spheroids fuse over time to a more energetically favorable configuration of one larger spheroid. Cell aggregates, however, are much more complicated than liquid drops and the mechanisms involved in spheroids fusion needs further elucidation. Another possible approach in tissue engineering is biofabrication of tissue sheets by attachment and spreading of patterned spheroids on the biocompatible adhesive matrix or scaffold followed by gradual fusion of disassembling cell aggregates [20,21]. Spreading of spheroids could be also used for biocompatibility tests of various biomaterials and scaffolds.

The biomechanical properties of cells guide their ultimate three-dimensional organization in tissue. In gastrulating zebrafish embryos, actin- and myosin-mediated tension and differential adhesion among cells drive the sorting of germ-layer progenitor cells [22]. Cell proliferation within spheroids is affected not only by spacial gradients of soluble factors such as oxygen, nutrients, and regulatory macromolecules, but by external forces also. Spheroids from murine mammary carcinoma cells generated within an agarose gel, were exposed to compressive loads during growth, the cells proliferated more slowly at zones of high stress and induced apoptotic cell death in regions of significantly higher mechanical stress [23]. As more examples of cells within three-dimensional aggregates responding to mechanical cues through the cytoskeleton systems are found, the questions of exact role of each component, namely actin microfilaments, microtubules, intermediate filaments, are becoming central.

Applying the classic perturbants of cytoskeleton, Cyto D, Noc and WFA, we assessed the potential role of actin microfilaments, MTs and IFs respectively in CSs fusion and spreading dynamics, in course of maintaining their biomechanical properties. Cyto D was used to demonstrate that an intact F-actin network is required for CSs fusion, spreading and supports biomechanical properties. According to the classic study by Cooper [24], low concentration of 0.2μM Cyto D inhibits membrane ruffling, higher concentrations of 2-20μM Cyto D are necessary to remove stress fibers. To evaluate the effect of stress fibers on cardiovascular spheroid composition, the concentration of 5μM Cyto D for 5 days incubation was used [25]. Cyto D and Noc concentrations used in this study were reported to disrupt the specific cytoskeletal elements without compromising the cell viability [26,27]. Noc is commonly used to study MT-dependent processes because of its ability to depolymerize rapidly MTs when administered at micromolar concentrations [28], while low concentrations of nocodazole inhibit microtubule dynamic instability in vitro (4 nM-12μM) and in cells (4-400 nM) [29]. Noc didn't affect СS fusion, but influenced essentially CSs spreading and biomechanical properties. WFA which is known to influence a variety of IFs, has shown promise as a potent vimentin inhibitor. At concentrations between 250 nM and 1 μM, increased serine-38 phosphorylation of vimentin causes the retraction of VIFs from the cell periphery and its juxtanuclear localization [30]. Concentrations at and above 1 μM WFA show dose-dependent effects on VIFs causing perinuclear condensation [31,32]. Exceeding 2 μM, WFA causes apoptosis and secondary vimentin cleavage within 2–4 h [30,31,33]. According to our data, WFA has affected spheroids spreading only. In our study we deliberate the mechanisms underlying observed effects of cytoskeleton perturbants on CSs intrinsic properties.