3D Bioprinting of Model Tissues That Mimic the Tumor Microenvironment.

The tumor microenvironment (TME) influences cancer progression. Therefore, engineered TME models are being developed for fundamental research and anti-cancer drug screening. This paper reports the biofabrication of 3D-printed avascular structures that recapitulate several features of the TME. The tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells (106 cells/mL); it is embedded in the same type of hydrogel containing primary cells-tumor-associated fibroblasts isolated from the peritumoral environment and peripheral blood mononuclear cells. Hoechst staining of cryosectioned tissue constructs demonstrated that cells remodeled the hydrogel and remained viable for weeks. Histological sections revealed heterotypic aggregates of malignant and peritumoral cells; moreover, the constituent cells proliferated in vitro. To investigate the interactions responsible for the experimentally observed cellular rearrangements, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods. Although unable to replicate the complexity of the TME, the approach presented here enables the self-assembly and co-culture of several cell types of the TME. Further studies will evaluate whether the bioprinted constructs can evolve in vivo in animal models. If they become connected to the host vasculature, they may turn into a fully organized TME.

Using Sacrificial Cell Spheroids for the Bioprinting of Perfusable 3D Tissue and Organ Constructs: A Computational Study.

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.

Evaluation of the influence of patient positioning on the reliability of lateral cephalometry.

Two-dimensional cephalometry is widely used for monitoring orthodontic treatments and for quantifying the outcome of maxillofacial surgery. Despite careful use of a cephalostat, successive radiographs might differ due to slight differences in patient posture. This study evaluates the reliability of lateral cephalometric measurements and estimates the impact of patient positioning on this reliability. We studied cephalograms of 104 patients; 31 of them had two radiographs because the first was deemed unsuitable for cephalometric analysis. Using AudaxCeph 3.0 (Audax, Ljubljana, Slovenia), two observers traced each cephalogram twice, one month apart. We evaluated intra- and interobserver agreement via Bland-Altman analysis, intraclass correlation coefficient (ICC), standard error of measurement, and smallest detectable difference (SDD). First, we studied the reliability of the hard tissue part of the Tweed-Merrifield analysis for 73 single cephalograms and for the better ones of patients with two exposures. Then, we studied 31 unsatisfactory cephalograms, and the ones recorded at improved patient posture. Although intraobserver bias was less than 0.5° or 0.3 mm, interobserver bias was significant for most measurements. Intraobserver reliability was high (ICC > 0.9), whereas interobserver reliability was good (ICC > 0.83) except for FMPA, FMIA and OP. Head rotations and inclinations had little impact on reliability (e.g., interobserver SDD decreased for 3 of 11 measurements). We conclude that averaging the positions of bilateral structures enables a reliable cephalometric analysis in spite of imprecise patient posture. Retaking cephalograms is ethically questionable in such cases.

Computational study of the interplay of adhesion and chemotaxis in the cell seeding of tissue engineering scaffolds with incorporated chemoattractants.

Cell migration is important in embryogenesis, metastasis and wound healing. Often, morphogenesis involves chemotactic cell movement up or down chemical gradients. We have developed a computational model of a cell suspension in the vicinity of a porous scaffold that incorporates a chemoattractant substance. In order to study the interplay of adhesion and chemotaxis on cell seeding , we developed a computational model of the system and simulated its evolution using a Metropolis Monte Carlo algorithm. We varied the chemotactic strengths of the cells in order to identify the optimal conditions for a rapid and uniform seeding.

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems.

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Computational modeling of epithelial-mesenchymal transformations.

An epithelial-mesenchymal transformation (EMT) involves alterations in cell-cell and cell-matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell-substrate adhesion than by a decrease in cell-cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.

Spatial distribution of VEGF isoforms and chemotactic signals in the vicinity of a tumor.

We propose a mathematical model that describes the formation of gradients of different isoforms of vascular endothelial growth factor (VEGF). VEGF is crucial in the process of tumor-induced angiogenesis, and recent experiments strongly suggest that the molecule is most potent when bound to the extracellular matrix (ECM). Using a system of reaction-diffusion equations, we study diffusion of VEGF, binding of VEGF to the ECM, and cleavage of VEGF from the ECM by matrix metalloproteases (MMPs). We find that spontaneous gradients of matrix-bound VEGF are possible for an isoform that binds weakly to the ECM (i.e. VEGF(165)), but cleavage by MMPs is required to form long-range gradients of isoforms that bind rapidly to the ECM (i.e. VEGF(189)). We also find that gradient strengths and ranges are regulated by MMPs. Finally, we find that VEGF molecules cleaved from the ECM may be distributed in patterns that are not conducive to chemotactic migration toward a tumor, depending on the spatial distribution of MMP molecules. Our model elegantly explains a number of in vivo observations concerning the significance of different VEGF isoforms, points to VEGF(165) as an especially significant therapeutic target and indicator of a tumor's angiogenic potential, and enables predictions that are subject to testing with in vitro experiments.