Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Long-term maintenance of a microfluidic 3D human liver sinusoid.

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPA's initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies.

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

A novel low-volume two-chamber microfabricated platform for evaluating drug metabolism and toxicity.

To evaluate drug and metabolite efficacy on a target organ, it is essential to include metabolic function of hepatocytes, and to evaluate metabolite influence on both hepatocytes and the target of interest. Herein, we have developed a two-chamber microfabricated device separated by a membrane enabling communication between hepatocytes and cancer cells. The microscale environment created enables cell co-culture in a low media-to-cell ratio leading to higher metabolite formation and rapid accumulation, which is lost in traditional plate cultures or other interconnected models due to higher culture volumes. We demonstrate the efficacy of this system by metabolism of tegafur by hepatocytes resulting in cancer cell toxicity.

Organs-on-a-chip: a new tool for drug discovery.

INTRODUCTION: The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as 'organs-on-a-chip', have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue. AREAS COVERED: The aim of this article is to provide an overview of the organs-on-a-chip systems that have been recently developed. First, the authors introduce single-organ platforms, focusing on the most studied organs such as liver, heart, blood vessels and lung. Later, the authors briefly describe tumor-on-a-chip platforms and highlight their application for testing anti-cancer drugs. Finally, the article reports a few examples of other organs integrated in microfluidic chips along with preliminary multiple-organs-on-a-chip examples. The article also highlights key fabrication points as well as the main application areas of these devices. EXPERT OPINION: This field is still at an early stage and major challenges need to be addressed prior to the embracement of these technologies by the pharmaceutical industry. To produce predictive drug screening platforms, several organs have to be integrated into a single microfluidic system representative of a humanoid. The routine production of metabolic biomarkers of the organ constructs, as well as their physical environment, have to be monitored prior to and during the delivery of compounds of interest to be able to translate the findings into useful discoveries.

In vivo comparison between laser-treated and grit blasted/acid etched titanium.

OBJECTIVE: Laser profiling of titanium has been of considerable interest in the field of oral implantology. However, very few pre-clinical and clinical studies have been performed with laser-treated implants, especially focusing on isotropic roughness topography. The aim of the study was to compare the cortical bone response of Ti-implants discs treated with pico-sec pulsed laser (LAS) and conventional grit-blasted/acid-etched (GAE) method. MATERIALS AND METHODS: Prior to the in vivo experiment, in vitro cell viability testing of the LAS surface treatment was preformed. Then, 5 mm diameter Titanium (Ti) discs treated with LAS and GAE method were implanted in a pre-validated rabbit tibia cortical bone model and assessed with histology and histomorphometric measurements. In total, eight New Zealand White adult female rabbits were used. RESULTS: The in vitro cell viability testing with osteoblast-like cells confirmed cytocompatibility of the LAS surface treatment. Further, the rabbit experiment demonstrated a bone-to-implant contact of 68% (±17) for the laser-treated discs and 49% (±21) for the GAE discs 8 weeks after the implantation, which was statistically not different. CONCLUSION: Laser surface treatment gives the same results to the grit-blasting/acid-etched method and thus is a valid alternative to conventional roughening for dental implant materials.

Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions.

Cells in situ are surrounded with defined structural elements formed by the nanomolecular extracellular matrix (ECM), and at the same time subjected to different mechanical stimuli arising from variety of physiological processes. In this study, using a nanotextured substrate mimicking the structural elements of the ECM and simulated microgravity, we wanted to develop a multifactorial model and understand better what guides cells in determining the morphological cell response. In our set-up, bone precursor cells from rat bone marrow were isolated and cultured on nanotextured polystyrene substrate (pitch 200 nm, depth 50 nm). Simulated microgravity was applied to the cells, using a random positioning machine (RPM). The results demonstrated that cells cultured on nanotextured substrate align parallel to the grooves and re-align significantly, but not completely, when subjected to simulated microgravity. The nanotextured substrate increased cell number and alkaline phosphatase (ALP) activity, whereas simulated microgravity decreased cells number and ALP activity. When the nanotextured substrate and simulated microgravity were combined together, the negative effect of the simulated microgravity ALP and cell number was reversed. In conclusion, absence of mechanical load in simulated microgravity has a negative effect on initial osteoblastogenesis, and nanotextured surfaces can partly reverse such a process.

The effect of nanometric surface texture on bone contact to titanium implants in rabbit tibia.

Designing biomaterial surfaces to control the reaction of the surrounding tissue is still considered to be a primary issue, which needs to be addressed systematically. Although numerous in vitro studies have described different nano-metrically textured substrates capable to influence bone cellular response, in vivo studies validating this phenomenon have not been reported. In this study, nano-grooved silicon stamps were produced by laser interference lithography (LIL) and reactive ion etching (RIE) and were subsequently transferred onto the surface of 5 mm diameter Titanium (Ti) discs by nanoimprint lithography (NIL). Patterns with pitches of 1000 nm (500 nm ridge and groove, 150 nm depth), 300 nm (150 nm ridge and groove, 120 nm depth; as well as a 1:3 ratio of 75 nm ridge and 225 nm groove, 120 nm depth) and 150 nm (75 nm ridge and groove, 30 nm depth) were created. These samples were implanted in a rabbit tibia cortical bone. Histological evaluation and histomorphometric measurements were performed, comparing each sample to conventional grit-blasted/acid-etched (GAE) titanium controls. Results showed a significantly higher bone-to-implant contact at 4 weeks for the 300 nm (1:3) specimens, compared to GAE (p = 0.006). At 8 weeks, there was overall more bone contact compared to 4 weeks. However, no significant differences between the nano-textured samples and the GAE occurred. Further studies will need to address biomechanical testing and the use of trabecular bone models.

Understanding the role of nano-topography on the surface of a bone-implant.

Bone-implant material development is proceeding at a high pace, and has shifted from straightforward biomaterial testing to more advanced cell-targeted approaches for surface modification and design. It has been long known that cells can recognize and respond to topographical features by changing their morphology and behavior. The progress in surface analytical devices, as well as in techniques for production of topographical features on the nanometer scale allow for the characterization of natural tissues and the reproduction of biomimetic nanofeatures in material surfaces. In this review some of the most common surface-characterization and surface-manufacturing techniques will be addressed and results from in vitro and in vivo studies will be presented. Knowledge on biomaterial nanotopography can be exploited for active stimulation and control of cellular behavior like attachment, migration, spreading, gene expression, proliferation, differentiation and secretion of matrix components.

Substrate nanotexture and hypergravity through centrifugation enhance initial osteoblastogenesis.

Mimicking the structural nanomolecular extracellular matrix with synthetically designed nanosized materials is a relatively new approach, which can be applied in the field of bone tissue engineering. Likewise, bone tissue-engineered constructs can be aided in their development by the use of several types of mechanical stimuli. In this study, we wanted to combine nanotextured biomaterials and centrifugation in one multifactorial system. Mesenchymal stem cells were isolated from rat bone marrow, and cultured on a nanogrooved polystyrene substrate (200-nm-wide pitch with a depth of 50 nm). Constant centrifugation of 10 g was applied to cells up to 7 days. Results showed that on a nanogrooved substrate osteoblast-like cells align parallel to the groove direction. Centrifugation of 10 g also affected cell morphology on a smooth surface. Moreover, cell alignment was significantly reduced for cells grown on nanogrooved substrates, which were subsequently subjected to centrifugation. Independently, both stimuli increased the number of cells after 7 days of culture. However, when both stimuli were combined, an additive effect on cell number was observed, followed by an enhanced effect on osteocalcin mRNA expression and matrix mineralization. In conclusion, biomaterial surface modification as well as centrifugation are effective means to enhance bone cell behavior, moreover, readily available to many tissue engineers.

Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture.

BACKGROUND: Periodontal ligament (PDL) cells play an important role in regulating osseous remodeling and ligament formation. Mechanical loading and the specific cellular environment are involved in these processes, regulating cell behavior. However, most in vitro experimental setups investigate mechanical loading or substrate texture separately and thus do not fully represent the PDL microenvironment. Therefore, the authors investigated the influence of combined mechano-topographical stimuli on PDL cell morphology, proliferation, and osteogenic and ligament differentiation. METHODS: Human PDL cells were subjected to nanometric substrate patterning and cyclic tensile stress for 2 days. Cell morphology was assessed by fluorescent staining. Further, DNA content and messenger RNA expression of osteogenic (Runx2, OCN) and ligament-related (scleraxis transcription factor (SCXA), ELN) genes were determined. RESULTS: PDL cells adapted to the topography of nanometric groove patterns, aligning parallel with the texture. When subjected to mechanical stress, cells lost their initial orientation to the nanopattern. When subjected to dual stimuli, total DNA amounts were increased at 3 days of culture. Moreover, a significant synergistic effect on upregulation of Runx2 was observed in the combined group. For ligament-related markers, SCXA and elastin expression increased with mechanical loading and decreased on nanopatterned surfaces. CONCLUSION: These results suggest that mechanical stimulation is crucial in regulating periodontal cell behavior, through modulation of osteogenic and ligament gene activity, while extracellular matrix-resembling structures induce different responses from PDL cells in morphology and gene expression.

In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates.

The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1ß and TNF-α cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1ß, TNF-α, TGF-ß and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical Editor: The authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1ß and TNF-α cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

The interaction between nanoscale surface features and mechanical loading and its effect on osteoblast-like cells behavior.

Osteoblasts respond to mechanical stimulation by changing morphology, gene expression and matrix mineralization. Introducing surface topography on biomaterials, independently of mechanical loading, has been reported to give similar effects. In the current study, using a nanotextured surface, and mechanical loading, we aimed to develop a multi-factorial model in which both parameters interact. Mechanical stimulation to osteoblast-like cells was applied by longitudinal stretch in parallel direction to the nanotexture (300 nm wide and 60 nm deep grooves), with frequency of 1 Hz and stretch magnitude varying from 1% to 8%. Scanning electron microscopy showed that osteoblast-like cells subjected to mechanical loading oriented perpendicularly to the stretch direction. When cultured on nanotextured surfaces, cells aligned parallel to the texture. However, the parallel cell direction to the nanotextured surface was lost and turned to perpendicular when parallel stretch to the nanotexture, greater than 3% was applied to the cells. This phenomenon could not be achieved when a texture with micro-sized dimensions was used. Moreover, a significant synergistic effect on upregulation of fibronectin and Cfba was observed when dual stimulation was used. These findings can lead to a development of new biomimetic materials that can guide morphogenesis in tissue repair and bone remodeling.