Symposium F.SM06—Biofabrication for Emulating Biological Tissues

With the advancement of 3D printing in the 21st century, prospective treatments for regenerative medicine, cosmetology, and material science are actively being explored^1,2. This research aims to investigate the feasibility of 3D bioprinting technology in producing viable skin tissue. In order to achieve this, a novel procedure for the construction of dermal and epidermal organotypics was developed. This evaluation was performed by comparing organotypic skin samples consisting of non-printed epidermal and dermal layers (NN), non-printed epidermal and printed dermal layers (NP), printed epidermal and non-printed dermal layers (PN), and printed epidermal and dermal layers (PP). The assessment of claudin and filaggrin immunohistochemical staining of the samples suggest a revision to the methodology in which printing the epidermal layer at a lower pressure while printing the dermal layer at a relatively higher pressure may lead to improved cellular function and overall integrity of the organotypic skin-equivalent. These results demonstrate the potential of bioprinting as a novel technique for producing a skin equivalent product, which may serve as an alternative to conventional tissue engineering methods.

1. Chang R, Nam J, Sun W: Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication-based direct cell writing. Tissue Eng Part A 2008, 14(1):41-48.
2. Chien S: Effects of disturbed flow on endothelial cells. Ann Biomed Eng 2008, 36(4):554-562.

Microgels of biopolymers such as alginate are widely used to encapsulate cells and other biological payloads. Alginate is an attractive material for cell encapsulation because spherical gels are easily created by contacting aqueous droplets of sodium alginate with divalent cations such as Ca²⁺. Alginate chains in the gel become crosslinked by Ca²⁺ cations into a 3-D network. When alginate gels are placed in a buffer, however, the Ca²⁺ crosslinks are eliminated by exchange with Na⁺, thereby weakening and degrading the gels. With time, encapsulated cells are released into the external solution.

Here, we describe a simple solution to the above problem, which involves forming alginate gels enveloped by a thin shell of a covalently crosslinked gel. The shell is formed via free-radical polymerization using conventional monomers such as acrylamide (AAm) or acrylate derivatives, including polyethylene glycol-diacrylate (PEGDA). The entire process is performed in a single step at room temperature (or 37°C) under mild, aqueous conditions. It involves combining the alginate solution with a radical initiator, which is then introduced as droplets into a reservoir containing Ca²⁺ and monomers. Within minutes, the droplets are converted into alginate-polymer microcapsules with a core of alginate and a shell of the polymer (AAm or PEGDA). The stability of these microcapsules under physiological conditions is compared with that of conventional alginate/Ca²⁺ microgels. The latter swell and degrade when placed in buffer or in chelators like sodium citrate, whereas the former remain stable under all conditions.

We also show that a various types of cells can be safely encapsulated in these hybrid beads. We encapsulate both mammalian cell and different strains of genetically engineered bacteria in these microcapsules and find that the cells remain viable and functional over time. In particular, engineered 'reporter' bacterial cells respond to different molecular signals from the external solution such as IPTG, AI-1 and hydrogen peroxide. The cells are retained in the hydrogel capsules throughout the study which is a crucial aspect for studying cell's response to molecular cues in a robust manner. Moreover, multiple sets of capsules each containing different strains can be synthesized and co-cultured to study cell-cell communication, providing an ideal platform for studying in vitro signaling events. Lastly, a variation of the synthesis technique is performed to generate multilayered microcapsules with a liquid core surrounded by concentric layers of alginate and AAm gels.

The use of multiple materials has helped in the development of tissue engineering. The use of polymeric matrices and biomaterials added to particular materials offers specific solutions both due to the interaction of the biomaterials and the mechanical properties of the generated biomaterial. In this work, the mechanical properties of compression of a three-component scaffolds for tissue engineering composed of polycaprolactone (PCL), hydroxyapatite (HA), and graphene oxide (GO) are shown. Scaffolds were subjected to compression on the Cell Scale™ brand Univert™ testing machine. Once the experimental data had been obtained, the stress-strain curve was obtained, also the compression mechanical behavior model was performed using the Levenberg-Marquardt least-squares algorithm in the Comsol Multiphisics™ software. To explain the compression phenomenon, scanning electron microscopy analyzes are performed where scaffolds are observed before being compressed and also after compression.

Nopal is a cactus that is distributed in different agroclimatic zones in a wild and cultivated way, due to its high forage potential, it has been destined to be used in the food, agricultural and pharmaceutical industry, however, when the cladode nopal reaches certain maturity, its use in the previously mentioned scopes is no longer viable, due to its high availability of fiber and lignin; this content of fiber in nopal could be implemented in the cosmetic and medical scopes as a biopolymer. The present study shows the physical and mechanical properties of nopal fiber components, and its thermographic analysis as an approach to determine its suitability and availability to fulfill the function of a biopolymer. An acid/alkaline extraction with potassium hydroxide (KOH; 1%) at 50° C was performed on the nopal sample previously dried. After the extraction, the sample was dried in a vacuum oven and its size was reduced by grinding. Mechanical tests were performed on nopal fibers in the Cell Scale Univert testing machine to obtain stress-strain curves. From the thermographic analysis, the kinetic drying model describing the process necessary for finite element modeling was proposed. The evaluation of the physical and mechanical characteristics allows us to determine the use and viability of nopal fiber as a biomaterial.

To engineer and build tissues, one requires an understanding of key relationships between cell behavior and the underlying substrate. In native tissues, extracellular matrix proteins are in the form of fibrous networks, sheets, and fibers. Our laboratory has been developing methods to form extracellular matrix fiber alloys and cell sheets. Over the past few years, we have been investigating the role of composition and processing conditions of various extracellular matrix fibers and alloy fibers on mechanical properties and biological properties. These fibers are also critical components of tissues such as blood vessels. We have also determined the role of mechanical strain on binding of different antibodies and peptide-functionalized contrast agents to these fibers. Importantly, we discovered that composition can be tuned to stabilize extracellular matrix proteins in fiber forms, which is especially useful with compositions where it is difficult to process the proteins into fibers. Through an integrated computational and experimental approach, we have discovered relationships between hydrophobicity and protein fiber forming capability and stability. Moreover, we have verified that the protein alloy fibers support cell adhesion and allow one to tune the mechanical properties of the fiber. This is of significance as the mechanical properties of the substrate play an important role in modulating cell behavior, e.g. cell migration and proliferation. We have also developed several methods to generate a layered tissue patch that mimics the cellular organization of native vessels and a bioMEMS device that can be used to assess physiological function of these tissue-engineered constructs with patient data input.

Hybrid bioelectronic systems offer unique advantages to achieve two-way electronic communication with living cells and tissues. Bioelectronic devices are readily multiplexed and form seamless, stable interfaces with surrounding cells and tissues, representing a distinct advantage over conventional systems such as patch clamp and optical dyes. First, we will present an overview of our recent heart-on-a-chip platform which integrated both extra- and intracellular devices for monitoring cardiac electrophysiology during episodes of acute hypoxia. This system allowed us to monitor not only cell-cell communication (e.g., wavefront propagation) but also action potentials at several spatially-distinct regions simultaneously. Next, we will discuss prospects for flexible bioelectronic scaffolds for achieving hybrid, 3D cardiac or brain systems. We developed a Photo-crosslinkable Silk Fibroin (PSF) derivative which was compatible with conventional photolithography processes and enabled flexible scaffolds with well-defined geometries and cm-scale uniformity. Our freestanding PSF-based scaffolds supported bioelectronic devices, provided excellent electrical passivation, and adhered both cardiac and neuron model cells. Finally, we will discuss recent work to develop stimulation elements for spatially-selective neural activation. Taken together, these research directions open new avenues for engineered, bioelectronics-innervated cardiac and brain systems. We will discuss prospects for merging our bioelectronic devices with state-of-the-art tissue engineering techniques.

Ion pumps are devices that use external power to introduce a net ionic flux. For example, in the photosynthetic process, light is used to pump protons up a concentration gradient which will be used in sequential steps to generate fuels. Although every cell membrane is essentially an ion pump, an artificial ion pump that can operate in steady state with no associated redox reactions is yet to be demonstrated. In this contribution we report a first of its kind “all electric”, ion pump based on a ratcheting mechanism.
Electronic ratchets are devices that utilize modulation in a spatially varying electric field to drive steady state current. Similar to peristaltic pumps, where the pump mechanism is not in direct contact with the pumped fluid, electronic ratchets induce net ionic current with no direct charge transport between the power source and the pumped charge carriers. Thus, electronic ratchets can be used to pump ions in steady state with no electrochemical reactions between the power source and the pumped ions resulting in an “all electric” ion pump.
Porous capacitor based ion pumps were fabricated by coating the two surfaces of nano-porous alumina wafers with gold. The electric field within the nano-pores is modulated by oscillating the capacitors voltage. Thus, when immersed in solution, ions within the pores experience a modulating electric field resulting in ratchet based ion pumping. The device pumping performance was studied both theoretically and experimentally for various input signals, geometries and solutions. It is shown that different ions can be pumped selectively by tuning the input signal frequency.

The advantages of droplet-based microfluidics are evident: multiple lab functions can be integrated on a single chip, sample consumption is minimal and objects like cells can be studied in an isolated environment, thus offering high precision at high throughput. A key point for end-user applications is the availability of powerful but easy-to-implement methods for real-time analysis and automatized manipulation of the droplets. We developed an optical device, consisting of a fast camera with an integrated data processing unit that uses smart algorithms for label-free real-time monitoring and active manipulation of passing droplets. The device continuously analyzes up to 10000 frames per second in real-time. In bright-field images parameters like size, brightness, granularity, circumference and speed of the droplets and their content are analyzed and can be used to manipulate the passing droplets based on their properties. We demonstrate the capabilities of our device by measuring different droplet production parameters and the label-free detection of cells encapsulated in droplets. Furthermore, we performed field-mediated, label-free sorting of cell containing droplets from empty droplets. Therefore, peripheral electronics consisting of a function generator and high-voltage amplifier that are connected to a sorting electrode are controlled by our device. Decision making is based on predefined parameter ranges that are compared to the measurement results of the droplets right before the sorting gate. Similarly, in another experiment we demonstrate efficient separation of droplets of different size. We envision that our novel device will be a useful tool for the online monitoring of passing droplets, enhancing the possibilities and dissemination of label-free methods for screening and manipulation in lab-on-a-chip systems.

3D cell cultures to create biomimetic environments are finding numerous applications in drug discovery, regenerative medicine, and cancer research, among many other fields. 3D cultures often rely on scaffolds, typically made of polymeric and biopolymeric materials, which support cell attachment and favour tissue development. These scaffolds possess certain structural and mechanical properties, such as porosity and stiffness, that mimic the extracellular matrix providing a similar microenvironment to that found in vivo. However, these scaffolds are typically passive, merely providing a templated support. We have been focusing on electroactive scaffolds capable of electrically monitoring cellular processes (eg. cell growth, migration) by following a synthetic biology approach. Porous conducting polymer scaffolds made of PEDOT (poly(3,4-ethylenedioxythiophene): HA (Hyaluronic acid) and/or collagen dispersions were obtained by the freeze-drying method, fine-tuning the chemicophysical and mechanical properties of the scaffold to render the system suitable for a variety of biological applications. We demonstrate that these scaffolds, apart from being a cell growth support, are capable of electrically monitoring cell growth. Current work is focusing on understanding the role of cancer cell migration through the scaffolds, in addition to the typical mechanical and biochemical cues present in 3D scaffolds.

A major challenge in the application of regenerative medicine strategies is the replication of the complexity and functionality of living tissues. Biofabrication techniques, such as inkjet, extrusion, or light-assisted printing, can result in living structures that further replicate the 3D architecture and organization of native tissue. Recent developments have resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques However, it remains largely unknown which materials and technical parameters are essential for the fabrication of intrinsically hierarchical cell-material constructs that truly mimic biological function of tissues. Moreover, it is unlikely that any of these technologies used individually would be able to replicate this complexity and functionality. Hence, we previously urged the field to shift its focus from materials and technologies toward the biological development of the resulting constructs.
This is illustrated by an example from the field of orthopaedics, in particular the treatment of osteochondral defects in a large (equine) animal model. Driven by the observations that the collagen structure provides articular cartilage with its extraordinary mechanical properties and that this structure does not recover once damaged, we evaluated the performance of a biofabricated composite osteochondral implant designed to provide long-term mechanical resistance, comparing implants cell-seeded with cell-free implants. The implants were composed of a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electro-written polycaprolactone micrometer-scale fibers and displaying a zonal, cartilage-mimetic architecture. This structure was firmly integrated with a room temperature-setting calcium phosphate cement-based bone anchor via a converged melt electrowriting-extrusion-based printing approach. Cell-containing implants were pre-cultured in vitro and samples were then implanted orthotopically in ponies for 6 months. The reinforcement of the cartilage phase enhanced both the compressive and shear properties of the implants considerably, closer to native values than achieved before.
Abundant deposition of glycosaminoglycan- and type II collagen-rich tissue was observed in vitro, which was maintained during the 6-month in vivo implantation period, showing that the composite architecture, comprising multi-scale printing of hydrogel, microfibers and ceramic materials had provided the required mechanical stability and integration of the implant within the subchondral bone to allow survival of the implant in the mechanically challenging environment of the equine knee joint over an extensive period of time while retaining its mechanical characteristics. Our findings underscore the hypothesis that the mechanical stability is more determining for the success of the implant than the presence of cells. This observation is of great translational importance and highlights the aptness of advanced (bio)fabrication and 3D printing technologies for functional tissue restoration in the harsh articular environment.

The recent development of bioengineering enables to create human tissues by integrating various native microenvironments, including tissue-specific cells, biochemical and biophysical cues. A significant transition of 3D bioprinting technology into the biomedical field helps to improve the function of engineered tissues by recapitulating physiologically relevant geometry, complexity, and vascular network. Bioinks, used as printable biomaterials, facilitate dispensing of cells through a dispenser as well as support their viability and function by providing engineered extracellular matrix. The successful construction of functional human tissues requires accurate environments that are able to mimic the biochemical and biophysical properties of the target tissue. This talk will cover my research interests in building 3D human tissues and organs to understand, diagnose, and treat various intractable diseases, particularly for cardiovascular diseases and diebetes. A development of tissue-derived decellularized extracellular matrix bioink will be mainly discussed as a straightforward strategy to provide biological and biophysical phenomena into engineered tissues. I will also discuss the development of a 3D vascularized stem cell patch that is generated by integrating the concept of tissue engineering and the developed platform technologies. Combined with recent advances in human pluripotent stem cell technologies, printed human tissues could serve as an enabling platform for studying complex physiology in tissue and organ contexts of individuals.

Today, tissue engineering evolves exponentially and provides real solutions to create biological models and solve clinical problems. However, we still face challenges that hinder its full potential to generate complex and large-size tissues and organs. Here we present advances in chaotic bioprinting, an additive manufacturing technique that enables the creation of complex biological structures at an unprecedented level of resolution and throughput.
Continuous chaotic printing uses chaotic advection for deterministic and continuous extrusion of fibers with internal multilayered structures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min^-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (~10² cm² cm^-3) at high resolution (~10 µm). This creates a new opportunity to produce structures with extremely high surface area to volume (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications.
Here, we illustrate the application of continuous chaotic bioprinting into the fabrication of complex multi-layered bacterial microcosmoi. We demonstrate that the degree of resolution achieved within these constructs can be finely controlled up to the range of a few microns of separation between layers and that the degree of interface between bacterial layers greatly matters for competition.
We also demonstrate the application of tissue-like structures in which living layers of muscle cells evolve into a coherent segment of muscle. Lumina can be easily built into these constructs by using a combination of sacrificial and permanent inks and a multi-port modification to the chaotic printhead.
We are currently exploring more applications of chaotic printing into tissue engineering such as the fabrication of vascularized cancer models, and multi-cell type tissue-like architectures where distinctive mammalian cell layers share a high amount of common interface.
Different fields, outside biosciences and technologies, are also in pursuit of simple and robust ways to create multilayered and multi-material structures to attain functionalities that monolithic materials do not exhibit. We envision that chaotic printing may be applied to technological scenarios such as the fabrication of batteries, superconductors, super catalytic surfaces, and 3D microfluidic reactors.

Bone defects are commonly caused by traumatic injuries and tumor removal and critically sized defects overwhelm the regenerative capacity of the native tissue. Reparative strategies such as auto, xeno, and allografts have proven to be insufficient to cure these defects. For the first time, we introduce the use of handheld melt spun three dimensional printers that can deposit scaffolding materials directly within the defect site to properly fill the cavity. Engineered composite filaments were generated from poly(caprolactone) doped with zinc oxide nanoparticles and hydroxyapatite microparticles. The use of PCL-based materials allowed low-temperature printing to avoid overheating of the surrounding tissues. The in situ printed scaffolds showed moderate adhesion to the bone tissue, which can prevent scaffold dislocation. The printed scaffolds showed to be osteoconductive and supported the osteodifferentiation of mesenchymal stem cells. Biocompatibility of the scaffolds upon in situ printing subcutaneously in mice showed promising results.

In patients with severe kidney disease, renal clearance is compromised resulting in the accumulation of a plethora of endogenous waste molecules that cannot be removed by current dialysis techniques, the most often applied treatment. These uremic retention solutes, also named uremic toxins, are a heterogeneous group of organic compounds of which many are too large to be filtered and/or are protein-bound. Their renal excretion depends largely on renal tubular secretion by which the binding is shifted towards the free fraction that can be eliminated. To facilitate this process, kidney proximal tubule cells are equipped with a range of transport proteins that cooperate in cellular uptake and urinary excretion. In recent years, we and others have invested in the development of bioengineered kidney tubules that could potentially restore this essential function. For this, well characterized human kidney proximal tubule cells were combined with functionalized, living membranes. This presentation addresses these developments in the context of functional kidney tubule engineering. Furthermore, hurdles to take before a safe implementation of bioengineered kidneys in clinics becomes a realistic option will be discussed.

Neurulation is a key developmental process that gives rise to the neural tube (NT), the precursor structure of the central nervous system (CNS). Neurulation is initiated after the gastrulation by a neural induction process, during which the dorsal ectoderm is specified into a patterned tissue containing the neural plate (NP) and the non-neural ectoderm (NNE) separated by the neural plate border (NPB). After neural induction, the NP folds and fuses to form the tubular NT. The development of the NT continues with differentiation of distinct classes of neuronal progenitor cells located at defined positions within the NT along both the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this talk, I will discuss the recent research activities in my group to leverage the developmental potential and self-organization property of human pluripotent stem cells (hPSCs) in conjunction with 2D and 3D bioengineering tools to achieve the development of spatially patterned multicellular tissues that mimic certain aspects of early human neurulation process, including neural induction, AP and DV patterning of the NT. These stem cell-derived models offer promising trackable systems to study neural development and disease.

The ovaries contain a finite resource of potential eggs and sex hormone-producing cells, and therefore, life-saving cancer treatments that irradiate or chemically induce cell death within the ovaries will likely result in premature ovarian insufficiency (POI) with reduced ovarian hormones and infertility. The option to have biological children, is a key quality of life measure for cancer survivors. Women with POI will experience co-morbidities associated with loss of ovarian hormones and a shorter life expectancy. The only method for fertility preservation for pediatric patients, who do not yet make eggs, is ovarian tissue cryopreservation (OTC). This tissue can then be transplanted back to restore fertility and hormone function. However, only 20 – 30% of transplants result in livebirth and it produces an average of 2 – 5 years of hormone restoration, leaving many without biological children and decades of post-cancer survival without essential hormone production. One major contributor to the shortened function of transplanted ovarian tissue is the significant spike in activation of the ovarian reserve (primordial follicles, containing the oocytes and hormone-producing cells) and subsequent depletion, at least in part, due to the disruption in the microenvironment. Additionally, some patients have metastatic disease within the ovary and therefore cannot use that tissue in its current form. Therefore, a safe, long-term solution for fertility and hormone restoration would involve isolating the ovarian cells that are essential for function from potential cancer cells and housing them in a microenvironment that maintains the bank of potential eggs and prolongs hormone production. We have defined the architectures of a 3D printed scaffold that support the necessary cell-cell contacts between the oocyte and the surrounding hormone producing support cells. This bioprosthetic ovary of the 3D printed gelatin scaffold with isolated ovarian follicles restored fertility and hormone function and supported livebirths and rearing of offspring in mice. To advance this research through the next steps toward translation to the clinic, we must define the follicular microenvironment that will enable a regulated recruitment of eggs from the ovarian reserve and support a safe, effective and long-term transplant. We have mapped the matrisome composition across the ovarian compartments and the stiffness of tissue components in ovaries of large mammals. Our novel proteomics screen and analysis has enabled us to identify new matrisome proteins within the ovary and pinpoint proteins of interest that may control primordial follicle activation or quiescence. We have additionally defined the physical properties of the ovarian compartments, where the cortical (outer) region is denser and stiffer than the medullary (inner) region. We have developed decellularized ovary extracellular matrix inks and are investigating the roles of specific biochemical and physical cues on primordial follicle quiescence and activation. A bioprosthetic ovary, defined by these microenvironment properties, would improve current options for fertility and hormone restoration for women.

Lyophilized silk fibroin (silk) scaffolds have been used to develop 3D models for multiple disease states.¹ Silk is a natural, biocompatible, FDA approved biomaterial that allows a high degree of control over physical properties.² Lyophilization of different silk solutions produces porous scaffolds with varying pore sizes and Young’s moduli.³ However, the effect of varying the drying time and temperature during lyophilization on pore size, pore shape, and bulk modulus is not well described. Understanding the impact of drying time and temperature conditioning on silk scaffold structural properties will allow better definition of lyophilization protocols and tailoring of silk scaffolds to specific disease states.
Silk fibroin was extracted from B. Mori silkworm cocoons as previously described.¹ Solutions of 3, 6, 9, and 12% silk (w/v) were created by concentrating silk solution or diluting with water. Rate-controlled lyophilization cycles were used to create porous scaffolds (15.6 mm diameter). Primary drying times (18 h – 1 week) and drying temperatures (-45°C to -15°C) were explored to determine the impact on pore size, pore shape, and Young’s modulus. Shelf and sample temperatures were recorded during lyophilization. Pores were evaluated through fluorescent and scanning electron microscopy of scaffolds cut into 200 µm thick sections. An ImageJ protocol was developed to evaluate pore size and shape. Scaffolds were hydrated (impact on secondary structures was examined through FTIR), biopsied (6 mm) and compressed between two parallel plates. The Young’s modulus of the scaffolds was determined through uniaxial compression testing on an Instron (2mm/min to 50% strain). Young’s modulus was calculated from the stress-strain curve.
Not all lyophilization runs produced viable scaffolds from all silk concentrations. Higher silk percentages (9 and 12%) were shown to require longer drying times than lower silk percentages (3 and 6%). A gradient of pore sizes was seen throughout scaffolds with smaller pores being present in the outermost regions. Pore size was non-normally distributed when imaged at low magnifications (4x) but was normally distributed at high magnifications (500x) suggesting multiple scales may be important for pore evaluation. Due to low average circularity values, Feret diameter, area, and perimeter were used to compare scaffolds instead of treating pores as spheres. Preliminary data suggests silk concentration had a greater impact on pore size and shape than drying time and temperature. Young’s moduli values increased as silk concentration increased in agreement with previous literature.³ Young’s moduli values for each silk concentration were significantly different from each other silk concentration (p < 0.01). Scaffolds fabricated from the same silk concentration but with stepwise temperature drying had significantly lower Young’s moduli (6%, p<0.001; 12%, p < 0.0001) than scaffolds dried at a constant temperature.
The impact of lyophilization parameters on Young’s modulus, and pore size and shape for four different silk formulations were evaluated. The Young’s moduli of scaffolds increased as silk concentration increased. Drying temperature significantly affected the Young’s moduli of scaffolds with the same silk concentration. Changes in silk concentration had a larger impact on pore size and shape than lyophilization parameters, although analysis is still ongoing. These results begin to demonstrate the impact of drying time and temperature on silk scaffold properties. This knowledge can be used to tailor lyophilization protocols to produce silk scaffolds with specific properties, such as a soft matrix with a desired pore range for tissue engineering applications.
1.D.N. Rockwood, Nat Protoc.6,1612-31 (2011). 2. J.E. Brown, Adv Healthc Mater.6, 1600762(2017) 3. M. Amirikia, Biologicals.57,1-8(2019)

Methods now exist to create single- or multi-organ engineered living systems that can be used to model a variety of disease processes. Aside from providing new insights into disease mechanisms, these systems can also be used to screen new drugs to treat the disease, or even to identify different therapeutic strategies for sub-populations possessing different genetic profiles. Here we present several models that require a microvascular network, modeling different stages of metastatic cancer. In one set of studies, circulating tumor cells are introduced via side channels into the microvascular bed, where they arrest, become activated and undergo transendothelial migration. Model systems include the brain, muscle, placenta and bone marrow, each showing distinctive characteristics. These or others can be used to model drug delivery to a developing tumor, either primary or metastatic.
Microfluidic devices used in these studies are, for the most part, simple, single layer systems that can be cast in a single piece of PDMS, forming channels and gel compartments by bonding to a coverslip. Alternatively, systems can be injection molded in tissue culture plastics. Microphysiological systems MPS) can be ether ‘engineered’ or self-organize using the same capabilities of developing organisms. Our approach is to rely, as much as possible, on the innate capabilities of the cells to interact to form the structures of a particular organ. Cells or cell assemblies/organoids are mixed in hydrogel solution and injected into one or more adjacent compartments allowing for direct signaling or physical contact between the multiple different cell types. Many of these systems contain microvascular networks that can be perfused via side channels, with pumping by simple low-pressure diaphragm pumps, directly connected to the MPS to minimize media volumes. The role of circulating immune cells can also be studied, as these, too, arrest in the vascular beds and extravasate into the surrounding gel matrix.
Results will be shown first demonstrating the form and function of the microvascular beds. Then several examples will be taken from the systems mentioned above to illustrate different applications, and to demonstrate the flexibility of the underlying platform. Future directions will be discussed including the challenges ahead in generating perfusable organoids.

In biological research, the interaction of biomolecules, cells and materials is an eternal topic. Many technologies have been employed to construct three-dimensional micro-and nano- hierarchical structure of materials. However, it is still a challenge to combine the artificially controllable biomaterials and the complicated biomolecular interaction/cell living unit, and thus to realize the regulation and control of the cell culture by using these materials. Moreover, it is necessary to develop a biotechnology for cell research to real-time and accurately monitor the interaction of the biomolecules/cells and the biomaterials, which could effectively reveal the biological mechanism of the cell behavior. In this point, we proposed to generate the desired functional materials by using microfluidic methods. Based on this technology, we have created a series of novel biomaterials, which showed demonstrated values in cells culture and organs-on-chips construction.

Cardiovascular disease is the leading cause of death in the US, with many cases attributed to cardiac ischemia. Cardiac ischemia occurs when an artery supplying blood to the heart is partially or fully blocked. This results in reduced blood flow to downstream tissue, gradually transforming normoxic blood (21% O₂) into hypoxic blood (<5% O₂). If hypoxic conditions are present for an extended period of time, a myocardial infarction (MI) may occur, leading to abnormal cardiac electrophysiology, loss of function, and tissue death. Despite the effects of ischemic stresses on cardiac electrophysiology, there are few tools that enable multiplexed, real-time studies of cardiac function under ischemia-like conditions.

Herein, we present a heart-on-a-chip model with integrated bioelectronics for studying the effects of acute hypoxia on cardiac function. This model consisted of a PDMS microfluidic channel, cell culture area, and extra- and intracellular recording elements that provided complementary electrophysiological readouts from cardiac model cells. The microfluidic channel enabled rapid modulation of medium oxygenation within the reservoir, which mimicked oxygenation regimes induced by an onset or removal of a coronary occlusion. Basic functionality of the microfluidic channel was confirmed with immunohistochemistry; reversibly-activated hypoxia-related transduction pathways were activated and visualized within the cardiomyocytes.

Our platform provided multiplexed bioelectronic readouts from up to 16 spatially-distinct locations. Extracellular recording elements consisted of 30-micron Au pads and provided stable, continuous readouts of wavefront propagation. Intracellular recording elements penetrated the cytosol following highly-localized electroporation, providing readouts that followed the action potential. A hypoxic regime applied over a 6-hour window caused an initial period of tachycardia followed by a reduction in beat rate and eventually arrhythmia; disruption of wavefronts and continuous reduction in propagation speed from 19 ± 5 mm/s to < 13 ± 8 mm/s; and narrowing of the AP. Moreover, multiplexed recordings of APs allowed us to observe heterogeneities between cells under both normoxic and hypoxic conditions, highlighting a clear advantage of our platform over tradtional techniques such as patch clamp.

The noninvasive nature of our platform enables a wide range of studies relevant to MI and could also be extended to incorporate other elements of ischemia including acidosis, hyperkalemia, nutrient deprivation, and waste accumulation. Moreover, our multiplexing capabilities could be expanded to larger and denser arrays for more complex biological systems including hypoxic gradients. Ultimately, our platform could yield fundamental insights into cardiac signaling pathways and/or provide a high-throughput platform for assessing therapeutics.

The International Agency for Research on Cancer (IARC) of the World Health Organization reports 1,276,106 incidences and 358,989 fatalities resulting from prostate cancer world wide in 2018. Prostate cancer is the second leading cause of cancer death among men in the United States. Prostate cancer has the propensity to migrate to or metastasize to bone. Although curative treatments are available for primary prostate cancer tumors, advanced-stage prostate cancer that has metastasized to bone often results in a poor prognosis. The primary cause of morbidity due to prostate cancer is the metastasis to bone. The effects of various biochemical factors in the bone microenvironment have been demonstrated in the disease progression to metastasis; however, the role of physical factors such as mechanical cues induced by the interstitial fluid flow around the bone is poorly explored. It has been established that interstitial flow has a pro-migratory and biomechanical stimulatory effect on cancer cell invasion. To address this issue, we designed a 3D in-vitro model to evaluate the progression of prostate cancer cells at metastasis condition, colonizing the bone site under dynamic conditions. In this study, we developed a 3D dynamic in-vitro model employing nanoclay-polycaprolactone-hydroxyapatite scaffolds and a specially designed perfusion bioreactor. Initially, we differentiated human mesenchymal stem cells (hMSCs) on scaffolds under dynamic culturing conditions to duplicate an accurate bone-like microenvironment. Next, we utilized these bone mimetic scaffolds to grow prostate cancer cells under identical dynamic conditions to recapitulate prostate cancer metastasis to bone. Finally, we performed various cellular assays and immunocytochemistry studies to establish the feasibility of dynamic culture. We observed a significant increase in bone growth under dynamic culture conditions evaluated by bone-related biomarkers (ALP, RUNX2, and OCN) compared to static culture. We also noticed upregulation in E-cadherin and downregulation of vimentin levels under dynamic conditions that confirms the occurrence of mesenchymal to epithelial (MET) transition of prostate cancer cells at the metastatic bone site. We further examined the influence of fluid-induced mechanical cues on cells by monitoring alteration in orientation and morphology of hMSCs and prostate cancer cells. Overall, we observed significant differences in cellular morphology and gene expressions of bone cells and prostate tumors grown under flow conditions. The established perfusion bioreactor based 3D dynamic in-vitro model can be further utilized to elucidate the mechanisms of prostate cancer metastasis and screen novel drug therapies geared towards bone metastasis prostate cancer.

The ability to harness the processes by which complex tissues arise during embryonic development would improve our ability to engineer complex tissue-like constructs in vitro—a long-standing goal of tissue engineering and regenerative medicine. In embryos, uniform populations of stem cells are exposed to spatial gradients of diffusible extracellular signaling proteins, known as morphogens. Varying levels of these signaling proteins induce stem cells to differentiate into distinct cell types at different positions along the gradient, thus creating spatially patterned tissues.

Here, we describe two straightforward and easy-to-adopt microfluidic strategies to expose human pluripotent stem cells (hPSCs) in vitro to spatial gradients of desired differentiation-inducing extracellular signals. Both approaches afford a high degree of control over the distribution of extracellular signals while preserving the viability of the cultured stem cells. The first microfluidic platform is commercially available and entails static culture, whereas the second microfluidic platform requires fabrication and dynamic fluid exchange. In each platform, we first computationally modeled the spatial distribution of differentiation-inducing extracellular signals. Then, we used each platform to expose hPSCs to a gradient of these signals (in this case, inducing a cell type known as the primitive streak), resulting in a regionalized culture with differentiated primitive streak (PS) cells predominately localized on one side and undifferentiated stem cells at the other side of the device. By combining this approach with a fluorescent reporter for differentiated cells and live-cell fluorescence imaging, we characterized the spatial and temporal dynamics of primitive streak differentiation within the induced signaling gradients. We envision that microfluidic approaches to create precisely controlled morphogen gradients will add to the stem cell and developmental biology toolkit, and may eventually pave the way to create increasingly spatially patterned tissue-like constructs in vitro.

A major challenge in the application of regenerative medicine strategies is the replication of the complexity and functionality of living tissues. Biofabrication techniques, such as inkjet, extrusion, or light-assisted printing, can result in living structures that further replicate the 3D architecture and organization of native tissue. Recent developments have resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques However, it remains largely unknown which materials and technical parameters are essential for the fabrication of intrinsically hierarchical cell-material constructs that truly mimic biological function of tissues. Moreover, it is unlikely that any of these technologies used individually would be able to replicate this complexity and functionality. Hence, we previously urged the field to shift its focus from materials and technologies toward the biological development of the resulting constructs.
This is illustrated by an example from the field of orthopaedics, in particular the treatment of osteochondral defects in a large (equine) animal model. Driven by the observations that the collagen structure provides articular cartilage with its extraordinary mechanical properties and that this structure does not recover once damaged, we evaluated the performance of a biofabricated composite osteochondral implant designed to provide long-term mechanical resistance, comparing implants cell-seeded with cell-free implants. The implants were composed of a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electro-written polycaprolactone micrometer-scale fibers and displaying a zonal, cartilage-mimetic architecture. This structure was firmly integrated with a room temperature-setting calcium phosphate cement-based bone anchor via a converged melt electrowriting-extrusion-based printing approach. Cell-containing implants were pre-cultured in vitro and samples were then implanted orthotopically in ponies for 6 months. The reinforcement of the cartilage phase enhanced both the compressive and shear properties of the implants considerably, closer to native values than achieved before.
Abundant deposition of glycosaminoglycan- and type II collagen-rich tissue was observed in vitro, which was maintained during the 6-month in vivo implantation period, showing that the composite architecture, comprising multi-scale printing of hydrogel, microfibers and ceramic materials had provided the required mechanical stability and integration of the implant within the subchondral bone to allow survival of the implant in the mechanically challenging environment of the equine knee joint over an extensive period of time while retaining its mechanical characteristics. Our findings underscore the hypothesis that the mechanical stability is more determining for the success of the implant than the presence of cells. This observation is of great translational importance and highlights the aptness of advanced (bio)fabrication and 3D printing technologies for functional tissue restoration in the harsh articular environment.

In patients with severe kidney disease, renal clearance is compromised resulting in the accumulation of a plethora of endogenous waste molecules that cannot be removed by current dialysis techniques, the most often applied treatment. These uremic retention solutes, also named uremic toxins, are a heterogeneous group of organic compounds of which many are too large to be filtered and/or are protein-bound. Their renal excretion depends largely on renal tubular secretion by which the binding is shifted towards the free fraction that can be eliminated. To facilitate this process, kidney proximal tubule cells are equipped with a range of transport proteins that cooperate in cellular uptake and urinary excretion. In recent years, we and others have invested in the development of bioengineered kidney tubules that could potentially restore this essential function. For this, well characterized human kidney proximal tubule cells were combined with functionalized, living membranes. This presentation addresses these developments in the context of functional kidney tubule engineering. Furthermore, hurdles to take before a safe implementation of bioengineered kidneys in clinics becomes a realistic option will be discussed.

To engineer and build tissues, one requires an understanding of key relationships between cell behavior and the underlying substrate. In native tissues, extracellular matrix proteins are in the form of fibrous networks, sheets, and fibers. Our laboratory has been developing methods to form extracellular matrix fiber alloys and cell sheets. Over the past few years, we have been investigating the role of composition and processing conditions of various extracellular matrix fibers and alloy fibers on mechanical properties and biological properties. These fibers are also critical components of tissues such as blood vessels. We have also determined the role of mechanical strain on binding of different antibodies and peptide-functionalized contrast agents to these fibers. Importantly, we discovered that composition can be tuned to stabilize extracellular matrix proteins in fiber forms, which is especially useful with compositions where it is difficult to process the proteins into fibers. Through an integrated computational and experimental approach, we have discovered relationships between hydrophobicity and protein fiber forming capability and stability. Moreover, we have verified that the protein alloy fibers support cell adhesion and allow one to tune the mechanical properties of the fiber. This is of significance as the mechanical properties of the substrate play an important role in modulating cell behavior, e.g. cell migration and proliferation. We have also developed several methods to generate a layered tissue patch that mimics the cellular organization of native vessels and a bioMEMS device that can be used to assess physiological function of these tissue-engineered constructs with patient data input.

The ovaries contain a finite resource of potential eggs and sex hormone-producing cells, and therefore, life-saving cancer treatments that irradiate or chemically induce cell death within the ovaries will likely result in premature ovarian insufficiency (POI) with reduced ovarian hormones and infertility. The option to have biological children, is a key quality of life measure for cancer survivors. Women with POI will experience co-morbidities associated with loss of ovarian hormones and a shorter life expectancy. The only method for fertility preservation for pediatric patients, who do not yet make eggs, is ovarian tissue cryopreservation (OTC). This tissue can then be transplanted back to restore fertility and hormone function. However, only 20 – 30% of transplants result in livebirth and it produces an average of 2 – 5 years of hormone restoration, leaving many without biological children and decades of post-cancer survival without essential hormone production. One major contributor to the shortened function of transplanted ovarian tissue is the significant spike in activation of the ovarian reserve (primordial follicles, containing the oocytes and hormone-producing cells) and subsequent depletion, at least in part, due to the disruption in the microenvironment. Additionally, some patients have metastatic disease within the ovary and therefore cannot use that tissue in its current form. Therefore, a safe, long-term solution for fertility and hormone restoration would involve isolating the ovarian cells that are essential for function from potential cancer cells and housing them in a microenvironment that maintains the bank of potential eggs and prolongs hormone production. We have defined the architectures of a 3D printed scaffold that support the necessary cell-cell contacts between the oocyte and the surrounding hormone producing support cells. This bioprosthetic ovary of the 3D printed gelatin scaffold with isolated ovarian follicles restored fertility and hormone function and supported livebirths and rearing of offspring in mice. To advance this research through the next steps toward translation to the clinic, we must define the follicular microenvironment that will enable a regulated recruitment of eggs from the ovarian reserve and support a safe, effective and long-term transplant. We have mapped the matrisome composition across the ovarian compartments and the stiffness of tissue components in ovaries of large mammals. Our novel proteomics screen and analysis has enabled us to identify new matrisome proteins within the ovary and pinpoint proteins of interest that may control primordial follicle activation or quiescence. We have additionally defined the physical properties of the ovarian compartments, where the cortical (outer) region is denser and stiffer than the medullary (inner) region. We have developed decellularized ovary extracellular matrix inks and are investigating the roles of specific biochemical and physical cues on primordial follicle quiescence and activation. A bioprosthetic ovary, defined by these microenvironment properties, would improve current options for fertility and hormone restoration for women.

Today, tissue engineering evolves exponentially and provides real solutions to create biological models and solve clinical problems. However, we still face challenges that hinder its full potential to generate complex and large-size tissues and organs. Here we present advances in chaotic bioprinting, an additive manufacturing technique that enables the creation of complex biological structures at an unprecedented level of resolution and throughput.
Continuous chaotic printing uses chaotic advection for deterministic and continuous extrusion of fibers with internal multilayered structures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min^-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (~10² cm² cm^-3) at high resolution (~10 µm). This creates a new opportunity to produce structures with extremely high surface area to volume (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications.
Here, we illustrate the application of continuous chaotic bioprinting into the fabrication of complex multi-layered bacterial microcosmoi. We demonstrate that the degree of resolution achieved within these constructs can be finely controlled up to the range of a few microns of separation between layers and that the degree of interface between bacterial layers greatly matters for competition.
We also demonstrate the application of tissue-like structures in which living layers of muscle cells evolve into a coherent segment of muscle. Lumina can be easily built into these constructs by using a combination of sacrificial and permanent inks and a multi-port modification to the chaotic printhead.
We are currently exploring more applications of chaotic printing into tissue engineering such as the fabrication of vascularized cancer models, and multi-cell type tissue-like architectures where distinctive mammalian cell layers share a high amount of common interface.
Different fields, outside biosciences and technologies, are also in pursuit of simple and robust ways to create multilayered and multi-material structures to attain functionalities that monolithic materials do not exhibit. We envision that chaotic printing may be applied to technological scenarios such as the fabrication of batteries, superconductors, super catalytic surfaces, and 3D microfluidic reactors.

Methods now exist to create single- or multi-organ engineered living systems that can be used to model a variety of disease processes. Aside from providing new insights into disease mechanisms, these systems can also be used to screen new drugs to treat the disease, or even to identify different therapeutic strategies for sub-populations possessing different genetic profiles. Here we present several models that require a microvascular network, modeling different stages of metastatic cancer. In one set of studies, circulating tumor cells are introduced via side channels into the microvascular bed, where they arrest, become activated and undergo transendothelial migration. Model systems include the brain, muscle, placenta and bone marrow, each showing distinctive characteristics. These or others can be used to model drug delivery to a developing tumor, either primary or metastatic.
Microfluidic devices used in these studies are, for the most part, simple, single layer systems that can be cast in a single piece of PDMS, forming channels and gel compartments by bonding to a coverslip. Alternatively, systems can be injection molded in tissue culture plastics. Microphysiological systems MPS) can be ether ‘engineered’ or self-organize using the same capabilities of developing organisms. Our approach is to rely, as much as possible, on the innate capabilities of the cells to interact to form the structures of a particular organ. Cells or cell assemblies/organoids are mixed in hydrogel solution and injected into one or more adjacent compartments allowing for direct signaling or physical contact between the multiple different cell types. Many of these systems contain microvascular networks that can be perfused via side channels, with pumping by simple low-pressure diaphragm pumps, directly connected to the MPS to minimize media volumes. The role of circulating immune cells can also be studied, as these, too, arrest in the vascular beds and extravasate into the surrounding gel matrix.
Results will be shown first demonstrating the form and function of the microvascular beds. Then several examples will be taken from the systems mentioned above to illustrate different applications, and to demonstrate the flexibility of the underlying platform. Future directions will be discussed including the challenges ahead in generating perfusable organoids.

Neurulation is a key developmental process that gives rise to the neural tube (NT), the precursor structure of the central nervous system (CNS). Neurulation is initiated after the gastrulation by a neural induction process, during which the dorsal ectoderm is specified into a patterned tissue containing the neural plate (NP) and the non-neural ectoderm (NNE) separated by the neural plate border (NPB). After neural induction, the NP folds and fuses to form the tubular NT. The development of the NT continues with differentiation of distinct classes of neuronal progenitor cells located at defined positions within the NT along both the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this talk, I will discuss the recent research activities in my group to leverage the developmental potential and self-organization property of human pluripotent stem cells (hPSCs) in conjunction with 2D and 3D bioengineering tools to achieve the development of spatially patterned multicellular tissues that mimic certain aspects of early human neurulation process, including neural induction, AP and DV patterning of the NT. These stem cell-derived models offer promising trackable systems to study neural development and disease.

Hybrid bioelectronic systems offer unique advantages to achieve two-way electronic communication with living cells and tissues. Bioelectronic devices are readily multiplexed and form seamless, stable interfaces with surrounding cells and tissues, representing a distinct advantage over conventional systems such as patch clamp and optical dyes. First, we will present an overview of our recent heart-on-a-chip platform which integrated both extra- and intracellular devices for monitoring cardiac electrophysiology during episodes of acute hypoxia. This system allowed us to monitor not only cell-cell communication (e.g., wavefront propagation) but also action potentials at several spatially-distinct regions simultaneously. Next, we will discuss prospects for flexible bioelectronic scaffolds for achieving hybrid, 3D cardiac or brain systems. We developed a Photo-crosslinkable Silk Fibroin (PSF) derivative which was compatible with conventional photolithography processes and enabled flexible scaffolds with well-defined geometries and cm-scale uniformity. Our freestanding PSF-based scaffolds supported bioelectronic devices, provided excellent electrical passivation, and adhered both cardiac and neuron model cells. Finally, we will discuss recent work to develop stimulation elements for spatially-selective neural activation. Taken together, these research directions open new avenues for engineered, bioelectronics-innervated cardiac and brain systems. We will discuss prospects for merging our bioelectronic devices with state-of-the-art tissue engineering techniques.

The recent development of bioengineering enables to create human tissues by integrating various native microenvironments, including tissue-specific cells, biochemical and biophysical cues. A significant transition of 3D bioprinting technology into the biomedical field helps to improve the function of engineered tissues by recapitulating physiologically relevant geometry, complexity, and vascular network. Bioinks, used as printable biomaterials, facilitate dispensing of cells through a dispenser as well as support their viability and function by providing engineered extracellular matrix. The successful construction of functional human tissues requires accurate environments that are able to mimic the biochemical and biophysical properties of the target tissue. This talk will cover my research interests in building 3D human tissues and organs to understand, diagnose, and treat various intractable diseases, particularly for cardiovascular diseases and diebetes. A development of tissue-derived decellularized extracellular matrix bioink will be mainly discussed as a straightforward strategy to provide biological and biophysical phenomena into engineered tissues. I will also discuss the development of a 3D vascularized stem cell patch that is generated by integrating the concept of tissue engineering and the developed platform technologies. Combined with recent advances in human pluripotent stem cell technologies, printed human tissues could serve as an enabling platform for studying complex physiology in tissue and organ contexts of individuals.

In biological research, the interaction of biomolecules, cells and materials is an eternal topic. Many technologies have been employed to construct three-dimensional micro-and nano- hierarchical structure of materials. However, it is still a challenge to combine the artificially controllable biomaterials and the complicated biomolecular interaction/cell living unit, and thus to realize the regulation and control of the cell culture by using these materials. Moreover, it is necessary to develop a biotechnology for cell research to real-time and accurately monitor the interaction of the biomolecules/cells and the biomaterials, which could effectively reveal the biological mechanism of the cell behavior. In this point, we proposed to generate the desired functional materials by using microfluidic methods. Based on this technology, we have created a series of novel biomaterials, which showed demonstrated values in cells culture and organs-on-chips construction.