From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

Nuclear Deformability of Breast Cells Analyzed from Patients with Malignant and Benign Breast Diseases.

Breast cancer is a heterogeneous and dynamic disease, in which cancer cells are highly responsive to alterations in the microenvironment. Today, conventional methods of detecting cancer give a rather static image of the condition of the disease, so dynamic properties such as invasiveness and metastasis are difficult to capture. In this study, conventional molecular-level evaluations of the patients with breast adenocarcinoma were combined with in vitro methods on micropatterned poly(methyl methacrylate) (PMMA) biomaterial surfaces that deform cells. A correlation between deformability of the nuclei and cancer stemness, invasiveness, and metastasis was sought. Clinical patient samples were from regions of the breast with different proximities to the tumor. Responses at the single-cell level toward the micropatterned surfaces were studied using CD44/24, epithelial cell adhesion marker (EpCAM), MUC1, and PCK. Results showed that molecular markers and shape descriptors can discriminate the cells from different proximities to the tumor center and from different patients. The cells with the most metastatic and invasive properties showed both the highest deformability and the highest level of metastatic markers. In conclusion, by using a combination of molecular markers together with nuclear deformation, it is possible to improve detection and separation of subpopulations in heterogenous breast cancer specimens at the single-cell level.

A Cell Culture Chip with Transparent, Micropillar-Decorated Bottom for Live Cell Imaging and Screening of Breast Cancer Cells.

In the recent years, microfabrication technologies have been widely used in cell biology, tissue engineering, and regenerative medicine studies. Today, the implementation of microfabricated devices in cancer research is frequent and advantageous because it enables the study of cancer cells in controlled microenvironments provided by the microchips. Breast cancer is one of the most common cancers in women, and the way breast cancer cells interact with their physical microenvironment is still under investigation. In this study, we developed a transparent cell culture chip (Ch-Pattern) with a micropillar-decorated bottom that makes live imaging and monitoring of the metabolic, proliferative, apoptotic, and morphological behavior of breast cancer cells possible. The reason for the use of micropatterned surfaces is because cancer cells deform and lose their shape and acto-myosin integrity on micropatterned substrates, and this allows the quantification of the changes in morphology and through that identification of the cancerous cells. In the last decade, cancer cells were studied on micropatterned substrates of varying sizes and with a variety of biomaterials. These studies were conducted using conventional cell culture plates carrying patterned films. In the present study, cell culture protocols were conducted in the clear-bottom micropatterned chip. This approach adds significantly to the current knowledge and applications by enabling low-volume and high-throughput processing of the cell behavior, especially the cell-micropattern interactions. In this study, two different breast cancer cell lines, MDA-MB-231 and MCF-7, were used. MDA-MB-231 cells are invasive and metastatic, while MCF-7 cells are not metastatic. The nuclei of these two cell types deformed to distinctly different levels on the micropatterns, had different metabolic and proliferation rates, and their cell cycles were affected. The Ch-Pattern chips developed in this study proved to have significant advantages when used in the biological analysis of live cells and highly beneficial in the study of screening breast cancer cell-substrate interactions in vitro.

In vitro two-step granuloma formation model for testing innate immune response to implants and coatings.

The extensive innate immune response to implanted biomaterials contributes significantly to their sub-par performance and failure. Granuloma formation is one of such reactions which results in multi-cell type clusters in line with the immune reaction to implanted materials. However, currently no in vitro model of granuloma formation exists that takes into account the arrival of multiple cell types (immune cells and connective tissue cells) to the implant insertion site. In this study, we developed a two-step model based on stimulated macrophage seeding followed by fibroblast introduction after a physiologically relevant time period for mimicking initial steps of immune reaction to biomaterials and inducing granuloma like behavior. Both LPS and TNF-α induction resulted in granuloma like formations which persisted longer than the control conditions. Introduction of human fibroblasts resulted in the colonization of the surfaces where the cell numbers and the collagen secretion were dependent on the microenvironment. In order to demonstrate the capacity of our model system to monitor the reaction to a given coating, a validated antimicrobial coating (Polyarginine (PAR)/Hyaluronic acid (HA)) was used as a testing bed. The coating prevented the adhesion of macrophages while allowing the adhesion of the fibroblast at the time of their arrival. Similar to its antimicrobial activity, macrophage metabolic activity and M2 differentiation in the presence of PAR was dependent to its chain length. The incorporation of fibroblasts resulted in decreased TNF-α and increased IL-1RA secretion especially in stimulation conditions. The pro- and anti-inflammatory cytokine secretions were low for PAR/HA coatings in line with the decreased number of macrophage presence. In the presence of complex PBMC population, the coating resulted in slightly less cellular attachment, without any significant cytokine secretion; the absence of inflammatory reaction was also demonstrated in vivo in a mouse model. The described in vitro granuloma testing system can control the macrophage reaction as a function of stimulation. It can also be used for testing new biomaterials for the potential innate immune responses and also for validation of implant coatings beyond their primary function from the immune response point of view.

The role of biomaterials and scaffolds in immune responses in regenerative medicine: macrophage phenotype modulation by biomaterial properties and scaffold architectures.

Scaffolds are an integral part of the regenerative medicine field. The contact of biomaterials with tissue, as was clearly observed over the years, induces immune reactions in a material and patient specific manner, where both surface and bulk properties of scaffolds, together with their 3D architecture, have a significant influence on the outcome. This review presents an overview of the reactions to the biomaterials with a specific focus on clinical complications with the implants in the context of immune reactions and an overview of the studies involving biomaterial properties and interactions with innate immune system cells. We emphasize the impact of these studies on scaffold selection and upscaling of microenvironments created by biomaterials from 2D to 3D using immune cell encapsulation, seeding in a 3D scaffold and co-culture with relevant tissue cells. 3D microenvironments are covered with a specific focus on innate cells since a large proportion of these studies used innate immune cells. Finally, the recent studies on the incorporation of adaptive immune cells in immunomodulatory systems are covered in this review. Biomaterial-immune cell interactions are a critical part of regenerative medicine applications. Current efforts in establishing the ground rules for such interactions following implantation can control immune response during all phases of inflammation. Thus, in the near future for complete functional recovery, tissue engineering and control over biomaterials must be considered at the first step of immune modulation and this review covers these interactions, which have remained elusive up to now.

Micropatterned Surfaces Expose the Coupling between Actin Cytoskeleton-Lamin/Nesprin and Nuclear Deformability of Breast Cancer Cells with Different Malignancies.

Mechanotransduction proteins transfer mechanical stimuli through nucleo-cytoskeletal coupling and affect the nuclear morphology of cancer cells. However, the contribution of actin filament integrity has never been studied directly. It is hypothesized that differences in nuclear deformability of cancer cells are influenced by the integrity of actin filaments. In this study, transparent micropatterned surfaces as simple tools to screen cytoskeletal and nuclear distortions are presented. Surfaces decorated with micropillars are used to culture and image breast cancer cells and quantify their deformation using shape descriptors (circularity, area, perimeter). Using two drugs (cytochalasin D and jasplakinolide), actin filaments are disrupted. Deformation of cells on micropillars is decreased upon drug treatment as shown by increased circularity. However, the effect is much smaller on benign MCF10A than on malignant MCF7 and MDAMB231 cells. On micropatterned surfaces, molecular analysis shows that Lamin A/C and Nesprin-2 expressions decreased but, after drug treatment, increased in malignant cells but not in benign cells. These findings suggest that Lamin A/C, Nesprin-2 and actin filaments are critical in mechanotransduction of cancer cells. Consequently, transparent micropatterned surfaces can be used as image analysis platforms to provide robust, high throughput measurements of nuclear deformability of cancer cells, including the effect of cytoskeletal elements.

A two-compartment bone tumor model to investigate interactions between healthy and tumor cells.

We produced a novel three-dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor microenvironment, the migration tendency of the tumor cells, and the efficacy of an anticancer drug, Doxorubicin, on the cancer cells. The model consisted of two compartments: (a) a healthy bone tissue mimic, made of poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate (ß-TCP) sponge seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, made of lyophilized collagen sponge seeded with human osteosarcoma cells (Saos-2). The tumor mimic component was placed into a central cavity created in the healthy bone mimic and together they constituted the complete 3D bone tumor model (3D-BTM). The porosities of both sponges were higher than 85% and the diameters of the pores were 199 ± 52 µm for the PLGA/TCP and 50-150 µm for the collagen scaffolds. The compression Young's modulus of the PLGA/TCP and the collagen sponges were determined to be 4.76 MPa and 140 kPa, respectively. Cell proliferation, morphology, calcium phosphate forming capacity and alkaline phosphatase production were studied separately on both the healthy and tumor mimics. All cells demonstrated cellular extensions and spread well in porous scaffolds indicating good cell-material interactions. Confocal microscopy analysis showed direct contact between the cells present in different parts of the 3D-BTM. Migration of HUVECs from the healthy bone mimic to the tumor compartment was confirmed by the increase in the levels of angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, and interleukin 8 in the tumor component. Doxorubicin (2.7 µg.ml-1) administered to the 3D-BTM caused a seven-fold decrease in the cell number after 24 h of interaction with the anticancer drug. Caspase-3 enzyme activity assay results demonstrated apoptosis of the osteosarcoma cells. This novel 3D-BTM has a high potential for use in studying the metastatic capabilities of cancer cells, and in determining the effective drug types and combinations for personalized treatments.

Amplification of nuclear deformation of breast cancer cells by seeding on micropatterned surfaces to better distinguish their malignancies.

Information about the mechanical properties of cancer cells leads to new insights about their malignancy levels. The more flexible the cancer cells and their nuclei are, the more aggressive and invasive they are. Flexibility is a result of composition and properties of molecular constituents of cells and its extent is expressed by deformation. Differences in the mechanical properties could be modulated by topography and chemistry of the substrate. In this study, the main hypothesis is that the difference in the mechanical properties of malignant and benign breast cancer cells could be used as a discriminator of these cells and reflected by the extent of nuclear deformation on micropatterned substrates. We compared benign (MCF10A), malignantnoninvasive (MCF7), and malignant highly invasive (MDAMB231) breast cancer cell lines using their nuclear deformability on micropatterned surfaces designed with square prism-shaped micropillars of poly(methyl methacrylate) (PMMA) (8 µm high, 4 × 4 µm2 area, 4 µm gap). Several shape descriptors (circularity, solidity, roundness, aspect ratio) were used to analyze nuclear deformation. We were able to discriminate the three cells when the descriptor circularity and hydrophobic micropatterned surfaces were used. The cells showed nuclear deformability in the order following the extent of their malignancies. The most aggressive cell, MDAMB231, had the lowest circularity value, 0.37, whereas the noninvasive malignant, MCF7, and benign, MCF10A, cells had higher values 0.47 and 0.77, respectively. Mechanism of the deformation was shown at the molecular level that the expression of Lamin A/C and Nesprin-2 genes decreased with increased nuclear deformation. In summary, biomechanical properties of cells can provide useful information about their cancer state and they can be reflected in the biological markers.

Engineered natural and synthetic polymer surfaces induce nuclear deformation in osteosarcoma cells.

Cell-substrate interactions involve constant probing of microenvironment by cells. One of the responses of cells to environmental cues is to change the conformation of their cytoplasm and nucleus. We hypothesized that surface chemistry and topography could be engineered to make these differences significant enough. When designing the substrates that would accentuate these differences, we prepared surfaces carrying cell adhesive biological cues arranged in specific patterns. Collagen type I and poly(lactic acid-co-glycolic acid) (PLGA) were used to represent substrates with biological cues and those without, and these materials were decorated with four square prism micropillars with different dimensions. The nuclear deformations were analyzed using some descriptors. Nucleus area and solidity were the best descriptors in distinguishing the substrates in terms of biological cues, while nucleus area, solidity, and circularity were more sensitive to the interpillar distances. Another distinguishing factor tested was the duration of contact. Nucleus area was the only descriptor sensitive to nuclear deformation change with time. PLGA was more suitable in nuclear conformation analysis while collagen was better in cell adhesion and proliferation. These deformations lead to changes in the molecular processes and further studies are needed to better understand cell mechanobiology. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 366-376, 2019.

Micro and Nanofabrication methods to control cell-substrate interactions and cell behavior: A review from the tissue engineering perspective.

Cell-substrate interactions play a crucial role in the design of better biomaterials and integration of implants with the tissues. Adhesion is the binding process of the cells to the substrate through interactions between the surface molecules of the cell membrane and the substrate. There are several factors that affect cell adhesion including substrate surface chemistry, topography, and stiffness. These factors physically and chemically guide and influence the adhesion strength, spreading, shape and fate of the cell. Recently, technological advances enabled us to precisely engineer the geometry and chemistry of substrate surfaces enabling the control of the interaction cells with the substrate. Some of the most commonly used surface engineering methods for eliciting the desired cellular responses on biomaterials are photolithography, electron beam lithography, microcontact printing, and microfluidics. These methods allow production of nano- and micron level substrate features that can control cell adhesion, migration, differentiation, shape of the cells and the nuclei as well as measurement of the forces involved in such activities. This review aims to summarize the current techniques and associate these techniques with cellular responses in order to emphasize the effect of chemistry, dimensions, density and design of surface patterns on cell-substrate interactions. We conclude with future projections in the field of cell-substrate interactions in the hope of providing an outlook for the future studies.