The micromechanical environment of intervertebral disc cells determined by a finite deformation,anisotropic, and biphasic finite element model

Cellular response to mechanical loading varies between the anatomic zones of the intervertebral disc. This difference may be related to differences in the structure and mechanics of both cells and extracellular matrix, which are expected to cause differences in the physical stimuli (such as pressure, stress, and strain) in the cellular micromechanical environment. In this study, a finite element model was developed that was capable of describing the cell micromechanical environment in the intervertebral disc. The model was capable of describing a number of important mechanical phenomena: flow-dependent viscoelasticity using the biphasic theory for soft tissues; finite deformation effects using a hyperelastic constitutive law for the solid phase; and material anisotropy by including a fiber-reinforced continuum law in the hyperelastic strain energy function. To construct accurate finite element meshes, the in situ geometry of IVD cells were measured experimentally using laser scanning confocal microscopy and three-dimensional reconstruction techniques. The model predicted that the cellular micromechanical environment varies dramatically between the anatomic zones, with larger cellular strains predicted in the anisotropic anulus fibrosus and transition zone compared to the isotropic nucleus pulposus. These results suggest that deformation related stimuli may dominate for anulus fibrosus and transition zone cells, while hydrostatic pressurization may dominate in the nucleus pulposus. Furthermore, the model predicted that micromechanical environment is strongly influenced by cell geometry, suggesting that the geometry of IVD cells in situ may be an adaptation to reduce cellular strains during tissue loading.     

Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease treatment and cultured for 5 days. Cellular adhesion of each hepatic cell type, gene expression and extracellular matrix deposition were analyzed by immunohistochemistry and immunoblotting. Immunohistochemical analysis demonstrated that the primary culture of fetal liver cells contained at least hepatoblasts, mesenchymal cells, endothelial cells, hemopoietic cells and Kupffer cells. Although hepatoblasts, mesenchymal cells, and endothelial cells aggregated separately in the initial step, they then formed a spheroid together, adhering to the glass slide, which led to the formation of flattened hepatic organoids. Hepatoblasts more preferentially adhered to mesenchymal cells than endothelial cells. Several extracellular matrix depositions were seen in aggregates consisting of at least hepatoblasts and mesenchymal cells within 12 h, but were poor in those lacking hepatoblasts. These data show that the primary culture of fetal liver cells contains most cell types constituting fetal livers, and may be useful for studying cell–cell interactions during liver development.     

Epidermal morphogenesis in an <Emphasis Type="BoldItalic">in-vitro</Emphasis> model using a fibroblasts-embedded collagen scaffold

Summary A novel culture system included a self-designed bi-layer 3-D collagen scaffold with different pore size on both sides and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-β1), in the cell–cell and cell–matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air–liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10 ng per ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10 ng per ml of EGF addition in the air-lifted stage resulted in uneven cell–matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-β1).     

A numerical study to determine pericellular matrix modulus and evaluate its effects on the micromechanical environment of chondrocytes

Chondrocyte biosynthesis is highly sensitive to mechanical strain. A thin pericellular matrix (PCM) surrounds the cell and plays an important role in mechanotransduction. PCM material properties are difficult to measure directly because of its size and connectivity to both cell and extracellular matrix (ECM). The purpose of this study was to develop a method of calculating linear elastic properties of the PCM using an inverse finite element approach with experimental properties of cell and chondron taken from the literature. Finite element models were constructed of both the equivalent chondron case and the chondrocyte-PCM structure, and a Fibonacci search obtained PCM moduli that matched the ECM strain field between the two cases. The most important result was that ECM strain adjacent to a chondron inclusion was sensitive to the chondron properties and may be used to calculate PCM mechanical properties, consistent with our strain field hypotheses. PCM moduli obtained through this method range from 43 to 240 kPa, were significantly higher than previously published but resulted in only a 0.5-21% decrease in relative effective cell strain. Similarities between effective strain ratios led to the conclusion that matching experimental techniques used to measure cell and PCM properties was more important than absolute values of the properties.     

A finite element model of cell-matrix interactions to study the differential effect of scaffold composition on chondrogenic response to mechanical stimulation

Mechanically induced cell deformations have been shown to influence chondrocyte response in 3D culture. However, the relationship between the mechanical stimulation and cell response is not yet fully understood. In this study a finite element model was developed to investigate cell-matrix interactions under unconfined compression conditions, using a tissue engineered encapsulating hydrogel seeded with chondrocytes. Model predictions of stress and strain distributions within the cell and on the cell boundary were shown to exhibit space-dependent responses that varied with scaffold mechanical properties, the presence of a pericellular matrix (PCM), and the cell size. The simulations predicted that when the cells were initially encapsulated into the hydrogel scaffolds, the cell size hardly affected the magnitude of the stresses and strains that were reaching the encapsulated cells. However, with the inclusion of a PCM layer, larger cells experienced enhanced stresses and strains resulting from the mechanical stimulation. It was also noted that the PCM had a stress shielding effect on the cells in that the peak stresses experienced within the cells during loading were significantly reduced. On the other hand, the PCM caused the stresses at the cell-matrix interface to increase. Based on the model predictions, the PCM modified the spatial stress distribution within and around the encapsulated cells by redirecting the maximum stresses from the periphery of the cells to the cell nucleus. In a tissue engineered cartilage exposed to mechanical loading, the formation of a neo-PCM by encapsulated chondrocytes appears to protect them from initially excessive mechanical loading. Predictive models can thus shed important insight into how chondrocytes remodel their local environment in order to redistribute mechanical signals in tissue engineered constructs.     

The contribution of adhesion signaling to lactogenesis

The mammary gland undergoes hormonally controlled cycles of pubertal maturation, pregnancy, lactation, and involution, and these processes rely on complex signaling mechanisms, many of which are controlled by cell–cell and cell–matrix adhesion. The adhesion of epithelial cells to the extracellular matrix initiates signaling mechanisms that have an impact on cell proliferation, survival, and differentiation throughout lactation. The control of integrin expression on the mammary epithelial cells, the composition of the extracellular matrix and the presence of secreted matricellular proteins all contribute to essential adhesion signaling during lactogenesis. In vitro and in vivo studies, including the results from genetically engineered mice, have shed light on the regulation of these processes at the cell and tissue level and have led to increased understanding of the essential signaling components that are regulated in temporal and cell specific manner during lactogenesis. Recent studies suggest that a secreted matricellular protein, CTGF/CCN2, may play a role in lactogenic differentiation through binding to β1 integrin complexes, enhancing the production of extracellular matrix components and contributions to cell adhesion signaling.     

Influence of power-law rheology on cell injury during microbubble flows

The reopening of fluid-occluded pulmonary airways generates microbubble flows which impart complex hydrodynamic stresses to the epithelial cells lining airway walls. In this study we used boundary element solutions and finite element techniques to investigate how cell rheology influences the deformation and injury of cells during microbubble flows. An optimized Prony–Dirichlet series was used to model the cells’ power-law rheology (PLR) and results were compared with a Maxwell fluid model. Results indicate that membrane strain and the risk for cell injury decreases with increasing channel height and bubble speed. In addition, the Maxwell and PLR models both indicate that increased viscous damping results in less cellular deformation/injury. However, only the PLR model was consistent with the experimental observation that cell injury is not a function of stress exposure duration. Correlation of our models with experimental observations therefore highlights the importance of using PLR in computational models of cell mechanics/deformation. These computational models also indicate that altering the cell’s viscoelastic properties may be a clinically relevant way to mitigate microbubble-induced cell injury.     

Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90

Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEO_av2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEO_av2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEO_av2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEO_av2.0 dehydrogenated NPEOs, NPEO_av2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.English edition: The paper was edited by a native speaker through KN international ()     

Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage

The pericellular matrix (PCM) is a narrow region of tissue that completely surrounds chondrocytes in articular cartilage. Previous theoretical models of the "chondron" (the PCM with enclosed cells) suggest that the structure and properties of the PCM may significantly influence the mechanical environment of the chondrocyte. The objective of this study was to quantify changes in the three-dimensional (3D) morphology of the chondron in situ at different magnitudes of compression applied to the cartilage extracellular matrix. Fluorescence immunolabeling for type-VI collagen was used to identify the boundaries of the cell and PCM, and confocal microscopy was used to form 3D images of chondrons from superficial, middle, and deep zone cartilage in explants compressed to 0%, 10%, 30%, and 50% surface-to-surface strain. Lagrangian tissue strain, determined locally using texture correlation, was highly inhomogeneous and revealed depth-dependent compressive stiffness and Poisson's ratio of the extracellular matrix. Compression significantly decreased cell and chondron height and volume, depending on the zone and magnitude of compression. In the superficial zone, cellular-level strains were always lower than tissue-level strains. In the middle and deep zones, however, tissue strains below 25% were amplified at the cellular level, while tissue strains above 25% were decreased at the cellular level. These findings are consistent with previous theoretical models of the chondron, suggesting that the PCM can serve as either a protective layer for the chondrocyte or a transducer that amplifies strain, such that cellular-level strains are more homogenous throughout the tissue depth despite large inhomogeneities in local ECM strains.     

Finite Element Modeling Predictions of Region-specific Cell-matrix Mechanics in the Meniscus

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of ~0.07 for the rounded inner cells, as compared to levels of 0.02–0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.     

Application of the u-p finite element method to the study of articular cartilage.

The finite element method using the principle of virtual work was applied to the biphasic theory to establish a numerical routine for analyses of articular cartilage behavior. The matrix equations that resulted contained displacements of the solid matrix (mu) and true fluid pressure (p) as the unknown variables at the element nodes. Both small and large strain conditions were considered. The algorithms and computer code for the analysis of two-dimensional plane strain, plane stress, and axially symmetric cases were developed. The u-p finite element numerical procedure demonstrated excellent agreement with available closed-form and numerical solutions for the configurations of confined compression and unconfined compression under small strains, and for confined compression under large strains. The model was also used to examine the behavior of a repaired articular surface. The differences in material properties between the repair tissue and normal cartilage resulted in significant deformation gradients across the repair interface as well as increased fluid efflux from the tissue.     

An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue

Introduction  

The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.     

Multilevel finite element modeling for the prediction of local cellular deformation in bone

The underlying mechanisms by which bone cells respond to mechanical stimuli or how mechanical loads act on osteocytes housed in lacunae in bone are not well understood. In this study, a multilevel finite element (FE) approach is applied to predict local cell deformations in bone tissue. The local structure of the matrix dictates the local mechanical environment of an osteocyte. Cell deformations are predicted from detailed linear FE analysis of the microstructure, consisting of an arrangement of cells embedded in bone matrix material. This work has related the loads applied to a whole femur during the stance phase of the gait cycle to the strain of a single lacuna and of canaliculi. The predicted bone matrix strains around osteocyte lacunae and canaliculi were nonuniform and differed significantly from the macroscopically measured strains. Peak stresses and strains in the walls of the lacuna were up to six times those in the bulk extracellular matrix. Significant strain concentrations were observed at sites where the process meets the cell body.     

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds

Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG) for the outer annulus fibrosus (AF) phase and pig acellular cartilage ECM (ACECM) for the inner nucleus pulposus (NP) phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4±13.1 μm and 231.6±57.2 μm, respectively). PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence) were distributed in the scaffold, with no dead cells (red fluorescence) being found. The cell—scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell—scaffold constructs in vivo.     

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Effect of endplate calcification and mechanical deformation on the distribution of glucose in intervertebral disc: a 3D finite element study

The intervertebral disc (IVD) is avascular, receiving nutrition from surrounding vasculature. Theoretical modelling can supplement experimental results to understand nutrition to IVD more clearly. A new, 3D finite element model of the IVD was developed to investigate effects of endplate calcification and mechanical deformation on glucose distributions in IVD. The model included anatomical disc geometry, non-linear coupling of cellular metabolism with pH and oxygen concentration and strain-dependent properties of the extracellular matrix. Calcification was simulated by reducing endplate permeability (～79%). Mechanical loading was applied based on in vivo disc deformation during the transition from supine to standing positions. Three static strain conditions were considered: supine, standing and weight-bearing standing. Minimum glucose concentrations decreased 45% with endplate calcification, whereas disc deformation led to a 4.8-63% decrease, depending on the endplate condition (i.e. normal vs. calcified). Furthermore, calcification more strongly affected glucose concentrations in the nucleus compared to the annulus fibrous region. This study provides important insight into nutrient distributions in IVD under mechanical deformation.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

A Riemannian manifold analysis of endothelial cell monolayer impedance parameter precision

Endothelial cell adhesion and barrier function play a critical role in many biological and pathophysiological processes. The decomposition of endothelial cell adhesion and barrier function into cell–cell and cell–matrix components using frequency dependent cellular micro-impedance measurements has, therefore, received widespread application. Few if any studies, however, have examined the precision of these model parameters. This study presents a parameter sensitivity analysis of a representative cellular barrier function model using a concise geometric formulation that includes instrumental data acquisition settings. Both model state dependence and instrumental noise distributions are accounted for within the framework of Riemannian manifold theory. Experimentally acquired microimpedance measurements of attached endothelial cells define the model state domain, while experimentally measured noise statistics define the data space Riemannian metric based on the Fisher information matrix. The results of this analysis show that the sensitivity of cell–cell and cell–matrix impedance components are highly model state dependent and several well defined regions of low precision exist. The results of this study further indicate that membrane resistive components can significantly reduce the precision of the remaining parameters in these models. This work was supported by a National Science Foundation CAREER Award (AE), BES-0238905, and in part by the American Heart Association under Grant 0265029B (AE).     

Cell death in intervertebral disc degeneration

Degeneration of intervertebral disc (IVD) is mainly a chronic process of excessive destruction of the extracellular matrix (ECM), and also is thought to be the primary cause of low back pain. Presently, however, the underlying mechanism of IVD degeneration is still not elucidated. Cellular loss from cell death has been believed to contribute to the degradation of ECM and plays an important role in the process of IVD degeneration, but the mechanisms of cell death in degenerated IVD remain unclear. Apoptosis, a very important type of IVD cell death, has been considered to play a crucial role in the process of degeneration. Autophagy, a non-apoptosis death type of programmed cell death, has been considered extensively involved in many pathological and physiological processes, including the degenerative diseases. Thus, the research on cell death in IVD degeneration has become a new focus recently. In this review, by analyzing the available literature pertaining to cell death in IVD and discussing the inducing factors of IVD degeneration, NP cells and ECM in IVD degeneration, apoptotic signal transduction pathways involved in IVD cell death, the relationship of cell death with IVD degeneration and potential therapeutic strategy for IVD degeneration by regulating cell death, we conclude that different stimuli induce cell death in IVD via various signal transduction pathways, and that cell death may play a key role in the degenerative process of IVD. Regulation of cell death could be a potential and attractive therapeutic strategy for IVD degeneration.     

Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen–glycosaminoglycan–chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63ΔNα, and the expression of keratin 3 and 14-3-3σ in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell–matrix interactions on epithelial cell morphogenesis.