Engineering a Novel Stem Cells from Apical Papilla-Macrophages Organoid for Regenerative Endodontics.

INTRODUCTION: A 3-dimensional (3D) tissue construct with a heterogeneous cell population is critical to understand the interactions between immune cells and stem cells from the apical papilla (SCAPs) in the periapical region for developing treatment strategies in regenerative endodontics. This study aimed to develop and characterize a 3D tissue construct with a binary cell system for studying the interactions between SCAPs and macrophages in the presence of lipopolysaccharide (proinflammatory) and interleukin 4 (anti-inflammatory) environments. METHODS: SCAPs and macrophages were seeded in the 3D-printed dumbbell-shaped molds to generate tissue constructs with a binary cell population. Two experimental (lipopolysaccharide and interleukin 4) and control (non-stimulation) conditions were applied to the tissue constructs to determine the characteristics of the tissue construct, the volume of viable cells, and their morphology using confocal laser scanning microscopy from a 0- to 7-day period. Experiments were conducted in triplicate, and data were analyzed with trend analysis and 2-way analysis of variance at a significance of P < .05. RESULTS: The tissue constructs revealed distinct SCAP-macrophage interaction in pro/anti-inflammatory environments. SCAPs displayed characteristic self-organization as a cap-shaped structure in the tissue construct. The growth of cells and cell-to-cell and cell-to-matrix interactions resulted in 70% and 30% decreased dimension of the tissue graft on the SCAP side and macrophage side, respectively, at day 7 (P < .0001). The tissue environments influenced SCAP-macrophage interactions, resulting in an altered viable cell volume (P < .05), morphology, and structural organization. CONCLUSIONS: This study developed and characterized an apical papilla organoid in a 3D collagen-based tissue construct for studying SCAP-macrophage crosstalk in tissue regeneration as well as repair.

Engineering Murine Adipocytes and Skeletal Muscle Cells in Meat-like Constructs Using Self-Assembled Layer-by-Layer Biofabrication: A Platform for Development of Cultivated Meat.

Global meat consumption has been growing on a per capita basis over the past 20 years resulting in ever-increasing devotion of resources in the form of arable land and potable water to animal husbandry which is unsustainable and inefficient. One approach to meet this insatiable demand is to use biofabrication methods used in tissue engineering in order to make skeletal muscle tissue-like constructs known as cultivated meat to be used as a food source. Here, we demonstrate the use of a scaffold-free biofabrication method that forms cell sheets composed of murine adipocytes and skeletal muscle cells and assembles these sheets in parallel to create a 3D meat-like construct without the use of any exogenous materials. This layer-by-layer self-assembly and stacking process is fast (4 days of culture to form sheets and few hours for assembly) and scalable (stable sheets with diameters >3 cm are formed). Tissues formed with only muscle cells were equivalent to lean meat with comparable protein and fat contents (lean beef had 1.5 and 0.9 times protein and fat, respectively, as our constructs) and incorporating adipocyte cells in different ratios to myoblasts and/or treatment with different media cocktails resulted in a 5% (low fat meat) to 35% (high fat meat) increase in the fat content. Not only such constructs can be used as cultivated meat, they can also be used as skeletal muscle models.

A Pumpless Microfluidic Neonatal Lung Assist Device for Support of Preterm Neonates in Respiratory Distress.

Premature neonates suffer from respiratory morbidity as their lungs are immature, and current supportive treatment such as mechanical ventilation or extracorporeal membrane oxygenation causes iatrogenic injuries. A non-invasive and biomimetic concept known as the "artificial placenta" (AP) would be beneficial to overcome complications associated with the current respiratory support of preterm infants. Here, a pumpless oxygenator connected to the systemic circulation supports the lung function to relieve respiratory distress. In this paper, the first successful operation of a microfluidic, artificial placenta type neonatal lung assist device (LAD) on a newborn piglet model, which is the closest representation of preterm human infants, is demonstrated. This LAD has high oxygenation capability in both pure oxygen and room air as the sweep gas. The respiratory distress that the newborn piglet is put under during experimentation, repeatedly and over a significant duration of time, is able to be relieved. These findings indicate that this LAD has a potential application as a biomimetic artificial placenta to support the respiratory needs of preterm neonates.

A 3D Self-Assembled In Vitro Model to Simulate Direct and Indirect Interactions between Adipocytes and Skeletal Muscle Cells.

The molecular mechanisms of the development and progression of diabetes and obesity involve complex interactions between adipocytes and skeletal muscle cells. Although 2D in-vitro models are the gold standard for the mechanistic study of such behaviors, they do not recreate the complexity and dynamics of the interactions between the cell types involved. Alternatively, animal models are used but are expensive, difficult to visualize or analyze, are not completely representative of human physiology or genetic background, and have associated ethical considerations. 3D co-culture systems can be complementary to these approaches. Here, using a newly developed 3D biofabrication method, adipocytes and myoblasts are positioned precisely either in direct physical contact or in close proximity such that the paracrine effects could be systematically studied. Suitable protocols for growth and differentiation of both cells in the co-culture system is also developed. Cells show more restrained lipid and protein production in 3D systems compared to 2D ones and adipocytes show more lipolysis in indirect contact with myoblasts as response to drug treatment. These findings emphasize importance of physical contact between cells that have been overlooked in co-culture systems using transwell inserts and can be used in studies for the development of anti-obesity drugs.

π-SACS: pH Induced Self-Assembled Cell Sheets Without the Need for Modified Surfaces.

The ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration. Newly emerging bioprinting methods sometimes lack the high cellular density needed to provide biophysical cues to orchestrate cellular behavior to recreate tissue architecture and function. Alternate methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of spheroids, organoids, or cell sheets. Cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues. Until now, the preparation of these sheets has involved culturing on specialized substrates that can be triggered by temperature or phase change (hydrophobic to hydrophilic) to release cells growing on them and form sheets. Here a new technique is proposed that allows delamination of cells and secreted ECM and rapid self-assembly into a cell sheet using a simple pH trigger and without the need to use responsive surfaces or applying external stimuli such as electrical and magnetic fields, only with routine tissue culture plates. This technique can be used with cells that are capable of syncytialization and fusion such as skeletal muscle cells and placenta cells. Using C2C12 myoblast cells we show that the pH trigger induces a rapid delamination of the cells as a continuous layer that self-assembles into a thick dense sheet. The delamination process has little effect on cell viability and maturation and preserves the ECM components that allow sheets to adhere to each other within a short incubation time enabling formation of thicker constructs when multiple sheets are stacked (double- and quadruple-layer constructs are formed here). These thick grafts can be used for regeneration purposes or as in vitro models.

A rapid biofabrication technique for self-assembled collagen-based multicellular and heterogeneous 3D tissue constructs.

Although monolayer cell culture models are considered as gold standard for in vitro modeling of pathophysiological events, they cannot reconstruct in vivo like gradient of gases and nutrients and lack proper cell-cell and cell-matrix interactions. Spherical cellular aggregates, otherwise known as multicellular spheroids, are widely used as three-dimensional in vitro models to mimic natural in vivo cellular microenvironment for applications such as drug screening. Although very useful, the previously established techniques are limited to low cell numbers, their processes are usually slow, and sometimes show limitations in terms of the cell type that can be used. Here, a versatile technique based on rapid self-assembly of cells and extracellular matrix material in different shapes using microfabricated molds is introduced to form multicellular tissue constructs. The self-assembly process takes less than 6 h and produces a mechanically robust tissue construct that could be handled easily. We demonstrate that a variety of shapes including spherical, cuboidal, dumbbell- and cross-like shapes could be fabricated using this approach. Interestingly, the structures formed with non-spherical shapes were able to retain that shape even after removal from the molds and during long term cell culture. This versatile approach is applicable to a variety of cell types (breast cancer cell lines MCF-7, MDA-MB-321, Hs-578T; osteosarcoma cell line SaOS-2; endothelial cell line HUVEC) as well as a range of cell numbers (104-106). Furthermore, we also show that the constructs could be spatially patterned to position various cell types in a precisely controlled way. Such heterogeneous constructs that are formed provide physiologically relevant cell densities, 3D structure as well as close positioning of multiple types of cells that are not possible using other fabrication approaches. This fabrication approach will find significant applications in developing 3D cell culture models for drug discovery as well as tissue grafts for implantation. STATEMENT OF SIGNIFICANCE: In this manuscript we describe a method for rapid formation of tissue constructs (6 h as opposed to several days for current state of art methods). We also identify the essential factors needed for such a rapid consolidation into a construct. We demonstrate the ability to form non-spherical constructs of various shapes that retain their shape over long term as opposed to those formed with current state of art that lose their shape during long time cell culture. We also show the ability to form precise heterogeneous constructs consisting of multiple cell types and with well-defined interfaces that are not possible with current state of art methods. This method could be used with a wide variety of cell types and are mechanically robust within 6 h to be handled with tweezers. We believe that such multicellular, heterogeneous constructs would be of significant use to biologists and drug discovery researchers investigating mechanisms involved in diseases processes or the effect of drug on them.

ExCeL: combining extrusion printing on cellulose scaffolds with lamination to create in vitro biological models.

Bioprinting is rapidly developing into a powerful tool in tissue engineering, for both organ printing and the development of in vitro models that can be used in drug discovery, toxicology and in vitro bioreactors. Nevertheless, the ability to create complex 3D culture systems with different types of cells and extracellular matrices integrated with perfusable channels has been a challenge. Here we develop an approach that combines the xurography of a scaffold material (cellulose) with extrusion printing of bioinks onto it, followed by assembly in a layer-by-layer fashion to create complex 3D culture systems that could be used as in vitro models of biological processes. This new method, termed ExCeL, can recapitulate the complexities of natural tissues with a proper 3D distribution of cells, extracellular matrices, and different molecules, while providing the whole structure with mechanical stability, and direct and easy access to the cells, even the ones that are positioned deep in the bulk of the structure, without the need to fix or section the samples. Briefly, the bioprinting of predefined patterns with a feature size of ∼1 mm has been made possible by treating paper with the hydrogel's crosslinker and printing cell-embedded hydrogel that will solidify immediately upon contact with the paper. These impregnated layers can be used as single layers or in a layer-by-layer approach by stacking them (here up to four layers) for applications such as cell migration and proliferation in 3D structures composed of collagen or alginate. Cells are generally sensitive to the amount of FBS in their culture media and we have shown how FBS amount will effect cell migration. By cutting the paper in certain patterns, printing hydrogel on the remaining parts of it, and stacking the paper in layers, features like embedded channels are formed that will provide cells will better mass transfer in thick structures. This technique provides biologists with a unique tool to perform sophisticated in vitro assays.

A Bioprinted In Vitro Model for Osteoblast to Osteocyte Transformation by Changing Mechanical Properties of the ECM.

Osteocytes are key contributors to bone remodeling. During the remodeling process, trapped osteoblasts undergo a phenotypic change to become osteocytes. The specific mechanisms by which osteocytes work are still debatable and models that exist to study them are sparse. This work presents an in vitro, bioprinted model based on the previously developed technique, ExCeL, in which a cell-embedded hydrogel is printed and immediately crosslinked using paper as a crosslinker-storing substrate. This process mimics the phenotypical change of osteoblast to osteocyte by altering the mechanical properties of the hydrogel. By printing Saos-2, osteosarcoma cells, embedded in the alginate hydrogel with differing mechanical properties, their morphology, protein, and gene expression can be changed from osteoblast-like to osteocyte-like. The stiffer gel is 30 times stiffer and results in significantly smaller cells with reduced alkaline phosphatase activity and expression of osteoblast-marker genes such as MMP9 and TIMP2. There is no change in viability between cells despite encapsulation in gels with different mechanical properties. The results show that the phenomenon of osteoblasts becoming encapsulated during the bone remodeling process can be replicated using the ExCeL bioprinting technique. This model has potential for studying how osteocytes can interact with external mechanical stimuli or drugs.

Mechanical, material, and biological study of a PCL/bioactive glass bone scaffold: Importance of viscoelasticity.

Microsphere sintering method was used to fabricate bone tissue engineering scaffolds made of polycaprolactone (PCL)/bioactive glass 58S5Z (58S modified with 5 wt% Zinc). First, the effect of PCL/58S5Z ratio on the mechanical properties (elastic modulus and yield strength) was investigated. It was found that samples with 5 wt% 58S5Z (named 5%BG) had the highest elastic modulus and yield strength among all samples, i.e., with 0, 5, 10, and 20 wt% bioactive glass. Then, considering the importance of viscoelastic properties of bone, the viscoelastic behavior of 0%BG (scaffold with only PCL) and 5%BG samples was determined by performing compressive stress relaxation test and subsequently a Generalized Maxwell model was developed. Findings indicated a similar amount and pattern of predicted storage and loss moduli and loss factor of the composite scaffolds to those of the bone. In the next step, the analysis of biological behavior of the scaffolds using MTT assay, DAPI and Alizarin red staining demonstrated that 5%BG scaffolds had higher in vitro cell viability and bone formation compared to 0%BG ones. Furthermore, in vivo study employing H&E staining of the scaffolds implanted in rats' calvarium for 50 days, confirmed the earlier findings and showed that 5%BG-filled defects had higher and more uniform bone formation compared to both 0%BG-filled and empty defects.

Poly(lactic-co-glycolic acid)(PLGA)/TiO2 nanotube bioactive composite as a novel scaffold for bone tissue engineering: In vitro and in vivo studies.

The aim of this study was to synthesize and characterize novel three-dimensional porous scaffolds made of poly (lactic-co-glycolic acid)/TiO2 nanotube (TNT) composite microspheres for bone tissue engineering applications. The incorporation of TNT greatly increases mechanical properties of PLGA/TNT microsphere-sintered scaffold. The experimental results exhibit that the PLGA/0.5 wt% TNT scaffold sintered at 100 °C for 3 h showed the best mechanical properties and a proper pore structure for tissue engineering. Biodegradation test ascertained that the weight of both PLGA and PLGA/PLGA/0.5 wt% TiO2 nanotube composites slightly reduced during the first 4 weeks following immersion in SBF solution. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and alkaline phosphatase activity (ALP activity) results represent increased cell viability for PLGA/0.5%TNT composite scaffold in comparison to the control group. In vivo studies show the amount of bone formation for PLGA/TNT was approximately twice of pure PLGA. Vivid histologic images of the newly generated bone on the implants further supported our test results. Eventually, a mathematical model showed that both PLGA and PLGA/TNT scaffolds' mechanical properties follow an exponential trend with time as their degradation occurs. By a three-dimensional finite element model, a more monotonous distribution of stress was present in the scaffold due to the presence of TNT with a reduction in maximum stress on bone.