The interplay between tissue growth and scaffold degradation in engineered tissue constructs

In vitro tissue engineering is emerging as a potential tool to meet the high demand for replacement tissue, caused by the increased incidence of tissue degeneration and damage. A key challenge in this field is ensuring that the mechanical properties of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a mathematical model (based upon a multiphase continuum framework) to investigate the interplay between tissue growth and scaffold degradation during tissue construct evolution in vitro. Our model accommodates a cell population and culture medium, modelled as viscous fluids, together with a porous scaffold and ECM deposited by the cells, represented as rigid porous materials. We focus on tissue growth within a perfusion bioreactor system, and investigate how the predicted tissue composition is altered under the influence of (1) differential interactions between cells and the supporting scaffold and their associated ECM, (2) scaffold degradation, and (3) mechanotransduction-regulated cell proliferation and ECM deposition. Numerical simulation of the model equations reveals that scaffold heterogeneity typical of that obtained from $\mu $ CT scans of tissue engineering scaffolds can lead to significant variation in the flow-induced mechanical stimuli experienced by cells seeded in the scaffold. This leads to strong heterogeneity in the deposition of ECM. Furthermore, preferential adherence of cells to the ECM in favour of the artificial scaffold appears to have no significant influence on the eventual construct composition; adherence of cells to these supporting structures does, however, lead to cell and ECM distributions which mimic and exaggerate the heterogeneity of the underlying scaffold. Such phenomena have important ramifications for the mechanical integrity of engineered tissue constructs and their suitability for implantation in vivo.     

A validated model of GAG deposition,cell distribution,and growth of tissue engineered cartilage cultured in a rotating bioreactor

In this work a new phenomenological model of growth of cartilage tissue cultured in a rotating bioreactor is developed. It represents an advancement of a previously derived model of deposition of glycosaminoglycan (GAG) in engineered cartilage by (i) introduction of physiological mechanisms of proteoglycan accumulation in the extracellular matrix (ECM) as well as by correlating (ii) local cell densities and (iii) tissue growth to the ECM composition. In particular, previously established predictions and correlations of local oxygen concentrations and GAG synthesis rates are extended to distinguish cell secreted proteoglycan monomers free to diffuse in cell surroundings and outside from the engineered construct, from large aggrecan molecules, which are constrained within the ECM and practically immovable. The model includes kinetics of aggregation, that is, transformation of mobile GAG species into immobile aggregates as well as maintenance of the normal ECM composition after the physiological GAG concentration is reached by incorporation of a product inhibition term. The model also includes mechanisms of the temporal evolution of cell density distributions and tissue growth under in vitro conditions. After a short initial proliferation phase the total cell number in the construct remains constant, but the local cell distribution is leveled out by GAG accumulation and repulsion due to negative molecular charges. Furthermore, strong repulsive forces result in expansion of the local tissue elements observed macroscopically as tissue growth (i.e., construct enlargement). The model is validated by comparison with experimental data of (i) GAG distribution and leakage, (ii) spatial‐temporal distributions of cells, and (iii) tissue growth reported in previous works. Validation of the model predictive capability—against a selection of measured data that were not used to construct the model—suggests that the model successfully describes the interplay of several simultaneous processes carried out during in vitro cartilage tissue regeneration and indicates that this approach could also be attractive for application in other tissue engineering systems. Biotechnol. Bioeng. 2010. 105: 842–853. © 2009 Wiley Periodicals, Inc.     

A novel population balance model to investigate the kinetics of in vitro cell proliferation: part II. Numerical solution, parameters' determination, and model outcomes

Based on the general theoretical model developed in Part I of this work, a series of numerical simulations related to the in vitro proliferation kinetics of adherent cells is here presented. First the complex task of assigning a specific value to all the parameters of the proposed population balance (PB) model is addressed, by also highlighting the difficulties arising when performing proper comparisons with experimental data. Then, a parametric sensitivity analysis is performed, thus identifying the more relevant parameters from a kinetics perspective. The proposed PB model can be adapted to describe cell growth under various conditions, by properly changing the value of the adjustable parameters. For this reason, model parameters able to mimic cell culture behavior under microgravity conditions are identified by means of a suitable parametric sensitivity analysis. Specifically, it is found that, as the volume growth parameter is reduced, proliferation slows down while cells arrest in G0/G1 or G2/M depending on the initial distribution of cell population. On the basis of this result, model capabilities have been tested by means of a proper comparison with literature experimental data related to the behavior of synchronized and not-synchronized cells under micro- and standard gravity levels.     

Pectin-based injectable biomaterials for bone tissue engineering

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.     

Geometric control of myogenic cell fate

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells.     

Cell Proliferation and Oxygen Diffusion in a Vascularising Scaffold

The supply of oxygen to proliferating cells within a scaffold is a key factor for the successful building of new tissue in soft tissue engineering applications. A recent in vivo model, where an arteriovenous loop is placed in a scaffold, allows a vascularising network to form within a scaffold, establishing an oxygen source within, rather than external, to the scaffold. A one-dimensional model of oxygen concentration, cell proliferation and cell migration inside such a vascularising scaffold is developed and investigated. In addition, a vascularisation model is presented, which supports a vascularisation front which moves at a constant speed. The effects of vascular growth, homogenous and heterogenous seeding, diffusion of cells and critical hypoxic oxygen concentration are considered. For homogenous seeding, a relationship between the speed of the vascular front and a parameter defining the rate of oxygen diffusion relative to the rate of oxygen consumption determines whether a hypoxic region exists at some time. In particular, an estimate of the length of time that a fixed point in the scaffold will remain under hypoxic conditions is determined. For heterogenous seeding, a Fisher-like travelling wave of cells is established behind the vascular front. These findings provide a fundamental understanding of the important interplay between the parameters and allows for a theoretical assessment of a seeding strategy in a vascularising scaffold.     

Inferring Growth Control Mechanisms in Growing Multi-cellular Spheroids of NSCLC Cells from Spatial-Temporal Image Data

We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue organization processes. Importantly, calibrating the model with two nutriment-rich growth conditions, the outcome for two nutriment-poor growth conditions could be predicted. As the final model is however quite complex, incorporating many mechanisms, space, time, and stochastic processes, parameter identification is a challenge. This calls for more efficient strategies of imaging and image analysis, as well as of parameter identification in stochastic agent-based simulations.     

Direct,dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.     

Identification of the heparin-binding determinants within fibronectin repeat III1: role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1.     

Modeling spatial distribution of oxygen in 3d culture of islet beta‐cells

Three‐dimensional (3D) scaffold culture of pancreatic β‐cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β‐cells is that β‐cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β‐cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell‐laden collagen culture on β‐cell proliferation, a culture model with encapsulation of an oxygen‐generator was established. The oxygen‐generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β‐cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial‐temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation‐aided 3D culture would augment the oxygen supply required for the β‐cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell‐laden scaffold cultures with an in situ oxygen supply significantly improved the β‐cells' biological function. These β‐cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β‐cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221–228, 2017     

Simulation model of doxorubicin activity in islets of human breast cancer cells

During cytotoxic chemotherapy, cancer cells are exposed to a dynamic concentration-versus-time curve. Besides the area under this curve, the shape of this curve may determine the cytotoxic effect. This report describes the concept that cell damage is determined by the molar drug accumulation history inside the tumor cells. Cell numbers of large populations of human MCF-7 cells exposed to three different doxorubicin concentration-versus-time profiles were recorded for 31 days. The drug accumulation history in the cells was calculated using cellular drug transport parameters derived from doxorubicin uptake and efflux measurements on MCF-7 cells attached to culture dishes. Recovery of the proliferation rate of a cell population after drug exposure was described using a mathematical model of cell damage. The model fitted well to the proliferation assays. It allowed for comparison of the effects of changes in doxorubicin concentration-versus-time profiles in vitro. The model was then used to predict the effect of the changes in the doxorubicin concentration profile in vivo, in tumor islets, after a bolus injection of doxorubicin. In the model doxorubicin exposure resulted in less cell damage inside the tumor islets than at the rim.     

The effect of substrate and adsorbed proteins on adhesion, growth and shape of CaCo-2 cells

Extracellular matrix (ECM) proteins play a critical role in many cellular functions, from spreading, migration and proliferation to apoptosis. This role can be altered when proteins of the native ECM are adsorbed to different substrates which cause structural modifications that can influence their biological function. The effects on CaCo-2 cells of laminin-1, fibronectin, collagen-1 and ECM gel adsorbed to glass and to tissue culture polystyrene (PS) were compared in terms of adhesion, proliferation, shapes and spreading of cells in culture. Significant differences between glass and PS surfaces were observed for proliferation and cell shape. Protein surfaces prepared on PS substrates had, in most cases, more pronounced effects on cells than uncoated PS, especially if coated by collagen-1. Adsorbed ECM gel was the most adhesive for cells, but its effect on cell proliferation was not notably different from the controls (glass or PS). These findings indicate that the choice of the substrate can have a significant effect on experimental results and should be taken into consideration when comparing results obtained on different surfaces.     

A cellular automaton model for the proliferation of migrating contact-inhibited cells.

A cellular automaton is used to develop a model describing the proliferation dynamics of populations of migrating, contact-inhibited cells. Simulations are carried out on two-dimensional networks of computational sites that are finite-state automata. The discrete model incorporates all the essential features of the cell locomotion and division processes, including the complicated dynamic phenomena occurring when cells collide. In addition, model parameters can be evaluated by using data from long-term tracking and analysis of cell locomotion. Simulation results are analyzed to determine how the competing processes of contact inhibition and cell migration affect the proliferation rates. The relation between cell density and contact inhibition is probed by following the temporal evolution of the population-average speed of locomotion. Our results show that the seeding cell density, the population-average speed of locomotion, and the spatial distribution of the seed cells are crucial parameters in determining the temporal evolution of cell proliferation rates. The model successfully predicts the effect of cell motility on the growth of isolated megacolonies of keratinocytes, and simulation results agree very well with experimental data. Model predictions also agree well with experimentally measured proliferation rates of bovine pulmonary artery endothelial cells (BPAE) cultured in the presence of a growth factor (bFGF) that up-regulates cell motility.     

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.     

Preparation and characterization of a VEGF-Fc fusion protein matrix for enhancing HUVEC growth

To enhance vascularization of hydrophobic implants in vivo, a VEGF-Fc fusion protein consisting of vascular endothelial growth factor (VEGF) fused to the immunoglobulin G Fc domain was prepared as an artificial extracellular matrix (ECM). VEGF-Fc was stably immobilized on a polystyrene plate due to the hydrophobicity of the Fc domain, and significantly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). Additionally, the use of VEGF-Fc as an ECM markedly promoted the proliferation of HUVECs longer than 72?h and induced the reorganization of actin filaments into larger stress fibers within these cells. The VEGF-Fc fusion protein may be a promising artificial ECM for enhancing endothelial cell growth.     

Dental pulp stem cell-derived extracellular matrix: autologous tool boosting bone regeneration

Background aimsTo facilitate artificial bone construct integration into a patient's body, scaffolds are enriched with different biologically active molecules. Among various scaffold decoration techniques, coating surfaces with cell-derived extracellular matrix (ECM) is a rapidly growing field of research. In this study, for the first time, this technology was applied using primary dental pulp stem cells (DPSCs) and tested for use in artificial bone tissue construction.MethodsRat DPSCs were grown on three-dimensional-printed porous polylactic acid scaffolds for 7 days. After the predetermined time, samples were decellularized, and the remaining ECM detailed proteomic analysis was performed. Further, DPSC-secreated ECM impact to mesenchymal stromal cells (MSC) behaviour as well as its role in osteoregeneration induction were analysed.ResultsIt was identified that DPSC-specific ECM protein network ornamenting surface-enhanced MSC attachment, migration and proliferation and even promoted spontaneous stem cell osteogenesis. This protein network also demonstrated angiogenic properties and did not stimulate MSCs to secrete molecules associated with scaffold rejection. With regard to bone defects, DPSC-derived ECM recruited endogenous stem cells, initiating the bone self-healing process. Thus, the DPSC-secreted ECM network was able to significantly enhance artificial bone construct integration and induce successful tissue regeneration.ConclusionsDPSC-derived ECM can be a perfect tool for decoration of various biomaterials in the context of bone tissue engineering.     

Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures

Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line. ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index. Cells on ECM were heterogeneous in size and insulin content. They showed extended areas of confluence. Cultures on plastic demonstrated an organisation in clusters and low mitotic activity. However, ECM did not allow for reconstitution of an islet-like structure. When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture. Glucagon and somatostatin secretion were similar on both substrates. These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.     

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage-dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover, because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen-dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.