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Carol M Cleetus,1 Fabian Alvarez Primo,1 Gisel Fregoso,2 Nivedita Lalitha Raveendran,3 Juan C Noveron,4 Charles T Spencer,5 Chinatalapalle V Ramana,3 Binata Joddar1 1Department of Metallurgical, Materials and Biomedical Engineering, The University of Texas at El Paso, El Paso, TX 79968, USA; 2Department of Electrical and Computer Engineering, The University of Texas at El Paso, El Paso, TX 79968, USA; 3Department of Mechanical Engineering, The University of Texas at El Paso, El Paso, TX 79968, USA; 4Department of Chemistry and Biochemistry, The University of Texas at El Paso, El Paso, TX 79968, USA; 5Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX 79968, USACorrespondence: Binata Joddar Email bjoddar@utep.eduIntroduction: In this in-vitro study, we designed a 3D printed composite of zinc oxide (ZnO) nanoparticles (NPs) with photocatalytic activities encapsulated within hydrogel (alginate) constructs, for antibacterial purposes applicable towards wound healing. We primarily sought to confirm the mechanical properties and cell compatibility of these ZnO NP infused scaffolds.Methods: The antibacterial property of the ZnO NPs was confirmed by hydroxyl radical generation using ultraviolet (U.V.) photocatalysis. Titanium dioxide (TiO2), a well-known antibacterial compound, was used as a positive control (1% w/v) for the ZnO NP-based alginate constructs and their antibacterial efficacies compared. Among the ZnO group, 3D printed gels containing 0.5% and 1% w/v of ZnO were analyzed and compared with manually casted samples via SEM, swelling evaluation, and rheological analysis. Envisioning an in-vivo application for the 3D printed ZnO NP-based alginates, we studied their antibacterial properties by bacterial broth testing, cytocompatibility via live/dead assay, and moisture retention capabilities utilizing a humidity sensor.Results: 3D printed constructs revealed significantly greater pore sizes and enhanced structural stability compared to manually casted samples. For all samples, the addition of ZnO or TiO2 resulted in significantly stiffer gels in comparison with the alginate control. Bacterial resistance testing on Staphylococcus epidermidis indicated the addition of ZnO NPs to the gels decreased bacterial growth when compared to the alginate only gels. Cell viability of STO-fibroblasts was not adversely affected by the addition of ZnO NPs to the alginate gels. Furthermore, the addition of increasing doses of ZnO NPs to the alginate demonstrated increased humidity retention in gels.Discussion: The customization of 3D printed alginates containing antibacterial ZnO NPs leads to an alternative that allows accessible mobility of molecular exchange required for improving chronic wound healing. This scaffold can provide a cost-effective and durable antibacterial treatment option.Keywords: zinc oxide nanoparticles, chronic wound healing, 3D printing, antibacterial, hydroxyl radicals, alginate hydrogels/scaffolds

Parkinson's disease (PD), a progressive central nervous system disorder marked by involuntary movements, poses a significant challenge in neurodegenerative research due to the gradual degeneration of dopaminergic (DA) neurons. Early diagnosis and understanding of PD's pathogenesis could slow disease progression and improve patient management. In vitro modeling with DA neurons derived from human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) offers a promising approach. These neurons can be cultured on electrospun (ES) nanofibrous polycaprolactone (PCL) scaffolds, but PCL's hydrophobic nature limits cell adhesion. We investigated the ability of ES PCL scaffolds coated with hydrophilic extracellular matrix-based biomaterials, including cell basement membrane proteins, Matrigel, and Fibrin, to enhance NPC differentiation into DA neurons. We hypothesized that fibrin-coated scaffolds would maximize differentiation based on fibrin's known benefits in neuronal tissue engineering. The scaffolds both coated and uncoated were characterized using scanning electron microscopy (SEM), transmission electron microscopy, Fourier transform infrared spectroscopy-attenuated total reflectance, and dynamic mechanical analysis to assess their properties. NPCs were seeded on the coated scaffolds, differentiated, and matured into DA neurons. Immunocytochemistry targeting tyrosine hydroxylase (TH) and SEM confirmed DA neuronal differentiation and morphological changes. Electrophysiology via microelectrode array recorded their neuronal firing. Results showed enhanced neurite extension, increased TH expression, and active electrical activity in cells on fibrin-coated scaffolds. Diluted fibrin coatings particularly promoted more pronounced neuronal differentiation and maturation. This study introduces a novel tissue-on-a-chip platform for neurodegenerative disease research using DA neurons., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Stereolithography (SLA) 3D printing is a rapid prototyping technique and reproducible manufacturing platform, which makes it a useful tool to develop advanced microfluidic devices for bioanalytical applications. However, limited information exists regarding the physical, chemical, and biological properties of the photocured polymers printed with SLA. This study demonstrates the characterization of a commercially available SLA 3D printed polymer to evaluate the potential presence of any time-dependent changes in material properties that may affect its ability to produce functional, capillary-action microfluidic devices. The printed polymer was analyzed with Fourier transform infrared-attenuated total reflectance, contact angle measurements, tensile test, impact test, scanning electron microscopy, and fluid flow analysis. Polymer biocompatibility was assessed with propidium iodide flow cytometry and an MTT assay for cell viability. The material characterization and biocompatibility results were then implemented to design and fabricate a self-driven capillary action microfluidic device for future use as a bioanalytical assay. This study demonstrates temporally stable mechanical properties and biocompatibility of the SLA polymer. However, surface characterization through contact angle measurements shows the polymer's wettability changes over time which indicates there is a limited postprinting period when the polymer can be used for capillary-based fluid flow. Overall, this study demonstrates the feasibility of implementing SLA as a high-throughput manufacturing method for capillary action microfluidic devices.

This study implemented the application of microcomputed tomography (micro-CT) as a characterization technique for the study and investigation of the microstructure of 3D scaffold structures produced via three-dimensional bioprinting (3DBP). The study focused on the preparation, characterization, and cytotoxicity analysis of gold nanoparticles (Au-NPs) incorporated into 3DBP hydrogels for micro-CT evaluation. The Au-NPs were characterized by using various techniques, including UV-vis spectrometry, dynamic light scattering (DLS), zeta potential measurement, and transmission electron microscopy (TEM). The characterization results confirmed the successful coating of the Au-NPs with 2 kDa methoxy-PEG and revealed their spherical shape with a mean core diameter of 66 nm. Cytotoxicity analysis using live-dead fluorescent microscopy indicated that all tested Au-NP solutions were nontoxic to AC16 cardiomyocytes in both 2D and 3D culture conditions. Scanning electron microscopy (SEM) showed distinguishable differences in image contrast and intensity between samples with and without Au-NPs, with high concentrations of Au-NPs displaying nanoparticle aggregates. Micro-CT imaging demonstrated that scaffolds containing Au-NPs depicted enhanced imaging resolution and quality, allowing for visualization of the microstructure. The 3D reconstruction of scaffold structures from micro-CT imaging using Dragonfly software further supported the improved visualization. Mechanical analysis revealed that the addition of Au-NPs enhanced the mechanical properties of acellular scaffolds, including their elastic moduli and complex viscosity, but the presence of cells led to biodegradation and reduced mechanical strength. These findings highlight the successful preparation and characterization of Au-NPs, their nontoxic nature in both 2D and 3D culture conditions, their influence on imaging quality, and the impact on the mechanical properties of 3D-printed hydrogels. These results contribute to the development of functional and biocompatible materials for tissue engineering and regenerative medicine applications.

In this study, we designed a tissue-engineered neurocardiac model to help us examine the role of neuronal regulation and confirm the importance of neural innervation techniques for the regeneration of cardiac tissue. A three-dimensional (3D) bioprinted neurocardiac scaffold composed of a mixture of gelatin-alginate and alginate-genipin-fibrin hydrogels was developed with a 2:1 ratio of AC16 cardiomyocytes (CMs) and retinoic acid-differentiated SH-SY5Y neuronal cells (NCs) respectively. A unique semi-3D bioprinting approach was adopted, where the CMs were mixed in the cardiac bioink and printed using an anisotropic accordion design to mimic the physiological tissue architecture in vivo. The voids in this 3D structure were methodically filled in using a NC-gel mixture and crosslinked. Confocal fluorescent imaging using microtubule-associated protein 2 (MAP-2) and anticholine acetyltransferase (CHAT) antibodies for labeling the NCs and the MyoD1 antibody for the CMs revealed functional coupling between the two cell types in the final crosslinked structure. These data confirmed the development of a relevant neurocardiac model that could be used to study neurocardiac modulation under physiological and pathological conditions.

Heart diseases cause over 17.9 million total deaths globally, making them the leading source of mortality. The aim of this review is to describe the characteristic mechanical, chemical and cellular properties of human cardiac tissue and how these properties can be mimicked in 3D bioprinted tissues. Furthermore, the authors review how current healthy cardiac models are being 3D bioprinted using extrusion-, laser- and inkjet-based printers. The review then discusses the pathologies of cardiac diseases and how bioprinting could be used to fabricate models to study these diseases and potentially find new drug targets for such diseases. Finally, the challenges and future directions of cardiac disease modeling using 3D bioprinting techniques are explored.

Doxorubicin (DOX) is a highly effective anthracycline chemotherapy agent effective in treating a broad range of life-threatening malignancies but it causes cardiotoxicity in many subjects. While the mechanism of its cardiotoxic effects remains elusive, DOX-related cardiotoxicity can lead to heart failure in patients. In this study, we investigated the effects of DOX-induced cardiotoxicity on human cardiomyocytes (CMs) using a three-dimensional (3D) bioprinted cardiac spheroidal droplet based-system in comparison with the traditional two-dimensional cell (2D) culture model. The effects of DOX were alleviated with the addition of N -acetylcysteine (NAC) and Tiron. Caspase-3 activity was quantified, and reactive oxygen species (ROS) production was measured using dihydroethidium (DHE) staining. Application of varying concentrations of DOX (0.4 μM-1 μM) to CMs revealed a dose-specific response, with 1 μM concentration imposing maximum cytotoxicity and 0.22 ± 0.11% of viable cells in 3D samples versus 1.02 ± 0.28% viable cells in 2D cultures, after 5 days of culture. Moreover, a flow cytometric analysis study was conducted to study CMs proliferation in the presence of DOX and antioxidants. Our data support the use of a 3D bioprinted cardiac spheroidal droplet as a robust and high-throughput screening model for drug toxicity. In the future, this 3D spheroidal droplet model can be adopted as a human-derived tissue-engineered equivalent to address challenges in other various aspects of biomedical pre-clinical research., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Hyperglycemia-mediated cardiac dysfunction is an acute initiator in the development of vascular complications, leading to cardiac fibrosis. To investigate the effects of hyperglycemia-mediated changes in cardiomyocytes, cells were cultured in-vitro under normoglycemic (5 mM or 25 mM D-glucose) and hyperglycemic (5 → 50 mM or 25 → 50 mM D-glucose) conditions, respectively. After 24-h of hyperglycemic exposure, cells were collected for RNA-sequencing (RNA-seq) studies to further investigate the differentially expressed genes (DEG) related to inflammation and fibrosis in samples cultured under hyperglycemic-in comparison with normoglycemic-conditions. Western Blotting was done to evaluate the protein expression of YAP1/TAZ under hyperglycemia induced stress conditions, as it is known to be involved in fibrotic and vascular inflammatory-mediated conditions. RNA-seq revealed the DEG of multiple targets including matrix metalloproteinases and inflammatory mediators, whose expression was significantly altered in the 5 → 50 mM in comparison with the 25 → 50 mM condition. Western Blotting showed a significant upregulation of the protein expression of the YAP1/TAZ pathway under these conditions as well (5 → 50 mM). To further probe the relationship between the inflammatory extracellular-signal-regulated kinase (ERK 1/2) and its downstream effects on YAP1/TAZ expression we studied the effect of inhibition of the ERK 1/2 signaling cascade in the 5 → 50 mM condition. The application of an ERK 1/2 inhibitor inhibited the expression of the YAP1/TAZ protein in the 5 → 50 mM condition, and this strategy may be useful in preventing and improving hyperglycemia associated cardiovascular damage and inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

3D bioprinting offers a simplified solution for the engineering of complex tissue parts for in-vitro drug discovery or, in-vivo implantation. However, significant amount of challenges exist in 3D bioprinting of neural tissues, as these are sensitive cell types to handle via extrusion bioprinting techniques. We assessed the feasibility of bioprinting human neural progenitor cells (NPCs) in 3D hydrogel lattices using a fibrinogen-alginate-chitosan bioink, previously optimized for neural-cell growth, and subsequently modified for structural support during extrusion printing, in this study. The original bioink used in this study was made by adding optimized amounts of high- and medium-viscosity alginate to the fibrinogen-chitosan-based bioink and making it extrudable under shear pressure. The mechanically robust 3D constructs promoted NPC cluster formation and maintained their morphology and viability during the entire culture period. This strategy may be useful for co-culturing of NPCs along with other cell types such as cardiac, vascular, and other cells during 3D bioprinting., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Cardiac organoids are 3-dimensional (3D) structures composed of tissue or niche-specific cells, obtained from diverse sources, encapsulated in either a naturally derived or synthetic, extracellular matrix scaffold, and include exogenous biochemical signals such as essential growth factors. The overarching goal of developing cardiac organoid models is to establish a functional integration of cardiomyocytes with physiologically relevant cells, tissues, and structures like capillary-like networks composed of endothelial cells. These organoids used to model human heart anatomy, physiology, and disease pathologies in vitro have the potential to solve many issues related to cardiovascular drug discovery and fundamental research. The advent of patient-specific human-induced pluripotent stem cell-derived cardiovascular cells provide a unique, single-source approach to study the complex process of cardiovascular disease progression through organoid formation and incorporation into relevant, controlled microenvironments such as microfluidic devices. Strategies that aim to accomplish such a feat include microfluidic technology-based approaches, microphysiological systems, microwells, microarray-based platforms, 3D bioprinted models, and electrospun fiber mat-based scaffolds. This article discusses the engineering or technology-driven practices for making cardiac organoid models in comparison with self-assembled or scaffold-free methods to generate organoids. We further discuss emerging strategies for characterization of the bio-assembled cardiac organoids including electrophysiology and machine-learning and conclude with prospective points of interest for engineering cardiac tissues in vitro., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this study, we developed three-dimensional (3D) printed annular ring-like scaffolds of hydrogel (gelatin-alginate) constructs encapsulated with a mixture of human cardiac AC16 cardiomyocytes (CMs), fibroblasts (CFs), and microvascular endothelial cells (ECs) as cardiac organoid models in preparation for investigating the role of microgravity in cardiovascular disease initiation and development. We studied the mechanical properties of the acellular scaffolds and confirmed their cell compatibility as well as heterocellular coupling for cardiac tissue engineering. Rheological analysis performed on the acellular scaffolds showed the scaffolds to be elastogenic with elastic modulus within the range of a native in vivo heart tissue. The microstructural and physicochemical properties of the scaffolds analyzed through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy-attenuated total reflectance (ATR-FTIR) confirmed the mechanical and functional stability of the scaffolds for long-term use in in vitro cell culture studies. HL-1 cardiomyocytes bioprinted in these hydrogel scaffolds exhibited contractile functions over a sustained period of culture. Cell mixtures containing CMs, CFs, and ECs encapsulated within the 3D printed hydrogel scaffolds exhibited a significant increase in viability and proliferation over 21 days, as shown by flow cytometry analysis. Moreover, via the expression of specific cardiac biomarkers, cardiac-specific cell functionality was confirmed. Our study depicted the heterocellular cardiac cell interactions, which is extremely important for the maintenance of normal physiology of the cardiac wall in vivo and significantly increased over a period of 21 days in in vitro . This 3D bioprinted "cardiac organoid" model can be adopted to simulate cardiac environments in which cellular crosstalk in diseased pathologies like cardiac atrophy can be studied in vitro and can further be used for drug cytotoxicity screening or underlying disease mechanisms.

In this study, three types of electrospun scaffolds, including furfuryl-gelatin (f-gelatin) alone, f-gelatin with polycaprolactone (PCL) in a 1:1 ratio, and coaxial scaffolds with PCL (core) and f-gelatin (sheath), were developed for tissue engineering applications. Scaffolds were developed through single nozzle electrospinning and coaxial electrospinning, respectively, to serve as scaffolds for cardiac tissue engineering. Uniform fibrous structures were revealed in the scaffolds with significantly varying average fiber diameters of 760 ± 80 nm (f-gelatin), 420 ± 110 nm [f-gelatin and PCL (1:1)], and 810 ± 60 nm (coaxial f-gelatin > PCL) via scanning electron microscopy. The distinction between the core and the sheath of the fibers of the coaxial f-gelatin > PCL electrospun fibrous scaffolds was revealed by transmission electron microscopy. Thermal analysis and Fourier transformed infrared (FTIR) spectroscopy revealed no interactions between the polymers in the blended electrospun scaffolds. The varied blending methods led to significant differences in the elastic moduli of the electrospun scaffolds with the coaxial f-gelatin > PCL revealing the highest elastic modulus of all scaffolds (164 ± 3.85 kPa). All scaffolds exhibited excellent biocompatibility by supporting the adhesion and proliferation of human AC16 cardiomyocytes cells. The biocompatibility of the coaxial f-gelatin > PCL scaffolds with superior elastic modulus was assessed further through adhesion and functionality of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, thereby demonstrating the potential of the coaxially spun scaffolds as an ideal platform for developing cardiac tissue-on-a-chip models. Our results demonstrate a facile approach to produce visible light cross-linkable, hybrid, biodegradable nanofibrous scaffold biomaterials, which can serve as platforms for cardiac tissue engineered models., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Purpose: To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested., Methods: Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and the applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample., Results: Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases., Conclusion: This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.

Myocardial fibrosis is one of the major complications of long-term diabetes. Hyperglycemia induced cardiomyocyte atrophy is a frequent pathophysiological indicator of diabetic heart. The objective of this study was to investigate the cardioprotective effect of glycyrrhizin (GLC) on myocardial damage in diabetic rats and assess the anti-inflammatory and anti-fibrotic effect of GLC. Our study demonstrates that hyperglycemia can elevate cardiac atrophy in diabetic animals. Type 2 diabetic fatty and the lean control rats were evaluated for cardiac damage and inflammation at 8-12 weeks after the development of diabetes. Western blot and immunohistochemical studies revealed that gap junction protein connexin-43 (CX43), cardiac injury marker troponin I, cardiac muscle specific voltage gated sodium channel Na V 1.5 were significantly altered in the diabetic heart. Furthermore, oxidative stress mediator receptor for advanced glycation end-products (RAGE), as well as inflammatory mediator phospho-p38 MAPK and chemokine receptor CXCR4 were increased in the diabetic heart whereas the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), the antioxidant proteins that protect against oxidative damage was reduced. We also observed an increase in the expression of the pleiotropic cytokine, transforming growth factor beta (TGF-β) in the diabetic heart. GLC treatment exhibited a decrease in the expression of phospho-p38 MAPK, RAGE, Na V 1.5 and TGF-β and it also altered the expression of CX43, CXCR4, Nrf2 and troponin I. These observations suggest that GLC possesses cardioprotective effects in diabetic cardiac atrophy and that these effects could be mediated through activation of Nrf2 and inhibition of CXCR4/SDF1 as well as TGF-β/p38MAPK signaling pathway.

Bone tissue engineering (BTE) aims to develop strategies to regenerate damaged or diseased bone using a combination of cells, growth factors, and biomaterials. This article highlights recent advances in BTE, with particular emphasis on the role of the biomaterials as scaffolding material to heal bone defects. Studies encompass the utilization of bioceramics, composites, and myriad hydrogels that have been fashioned by injection molding, electrospinning, and 3D bioprinting over recent years, with the aim to provide an insight into the progress of BTE along with a commentary on their scope and possibilities to aid future research. The biocompatibility and structural efficacy of some of these biomaterials are also discussed., Competing Interests: Conflict of Interest Statement William Weiss acknowledges he has no financial conflicts of interest, but serves as an editor for Canadian Orthopedic Association Bulletin, is on the editorial board for Arthroscopy: Journal of Arthroscopic and Related Surgery; Bone & Joint 360, and is on the social media board for Arthroscopy Association of North America (AANA). All other authors acknowledge no conflict of interest.

Since conventional human cardiac two-dimensional (2D) cell culture and multilayered three-dimensional (3D) models fail in recapitulating cellular complexity and possess inferior translational capacity, we designed and developed a high-throughput scalable 3D bioprinted cardiac spheroidal droplet-organoid model with cardiomyocytes and cardiac fibroblasts that can be used for drug screening or regenerative engineering applications. This study helped establish the parameters for bioprinting and cross-linking a gelatin-alginate-based bioink into 3D spheroidal droplets. A flattened disk-like structure developed in prior studies from our laboratory was used as a control. The microstructural and mechanical stability of the 3D spheroidal droplets was assessed and was found to be ideal for a cardiac scaffold. Adult human cardiac fibroblasts and AC16 cardiomyocytes were mixed in the bioink and bioprinted. Live-dead assay and flow cytometry analysis revealed robust biocompatibility of the 3D spheroidal droplets that supported the growth and proliferation of the cardiac cells in the long-term cultures. Moreover, the heterocellular gap junctional coupling between the cardiomyocytes and cardiac fibroblasts further validated the 3D cardiac spheroidal droplet model., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this article.

Ligation of the left anterior descending (LAD) coronary artery has been commonly employed to induce myocardial infarction (MI) in animals; however, it is known to pose setbacks in the form of cardiac arrhythmias and unpredictable areas of necrotic damage. Cryo-infarction is an alternate method that has been adopted to create a reproducible model of a myocardial injury. In this study, Sprague-Dawley rats were subjected to thoracotomy followed by cryo-induced infarction of the heart, while the control-sham group was only subjected to thoracotomy following which the heart was collected from all animals. Tissue sections were stained with hematoxylin and eosin and analyzed to determine cardiac muscle density, fiber length, and fiber curvature. Observations revealed reduced muscle density, cardiac fiber length, and distorted fibers in infarcted tissue sections. Gomori's Trichrome staining was performed on tissue sections to study the effects of post MI on collagen, which showed enhanced intensity of collagen staining indicating fibrosis for the experimental models as compared to the sham models, an established consequence to myocardial injury. Immunohistochemical staining of the tissue sections with DAPI and connexin-43 (Cx-43) revealed that there was reduced DAPI staining and a less pronounced expression of Cx-43 in the experimental samples as compared to the sham samples. Results implied significant cell damage resulting from the cryo-infarction, subsequently disrupting and disaggregating the functional Cx-43 junction in cardiac myocytes, which is essential for normal and healthy cardiac physiology and function. This quantitative histological study of cryo-induced MI in a rat model can aid others attempting to optimize MI models in rats via cryo-injury, to study cardiac disease progression, and to aid in the construction of engineered cardiac tissues., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66 ± 0.84 kPa), physiologic (8.98 ± 1.29 kPa) and fibrotic (18.27 ± 3.17 kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.

In this study, we used an alginate-gelatin bioink to design and print 3D constructs with lattice, honeycomb and fibrous bundle patterns. These designs were printed using a small-scale laboratory printer, at first and later translated to a larger scale, high throughput-printing platform. A comparative analysis of the structures printed using two dissimilar platforms using gross morphologic evaluation, scanning electron microscopy and swelling assay confirmed our hypothesis that a design printed using a small-scale laboratory bioprinter for optimization of bioink composition and printing parameters can be successfully translated into a large scale-printing platform for high throughput printing of constructs. Since the designs for printing were implemented using a software which was common across both printers, this endpoint was feasible. The only difference in printing parameters resulted from variation in extrusion pressure which was due to a significant difference in barrel size used across both printers (3 ml versus 30 ml), while all other parameters stayed the same. Although the scaffolds were not bioprinted with cells, in future we will investigate how cell viability can be differentially regulated by the variation of extrusion pressure across both platforms., Competing Interests: The authors have no conflicts of interests to declare for this study. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

In this study, we designed, synthesized, and characterized ultrahigh purity single-walled carbon nanotube (SWCNT)-alginate hydrogel composites. Among the parameters of importance in the formation of an alginate-based hydrogel composite with single-walled carbon nanotubes, are their varying degrees of purity, their particulate agglomeration and their dose-dependent correlation to cell viability, all of which have an impact on the resultant composite's efficiency and effectiveness towards cell-therapy. To promote their homogenous dispersion by preventing agglomeration of the SWCNT, three different surfactants-sodium dodecyl sulfate (SDS-anionic), cetyltrimethylammonium bromide (CTAB-cationic), and Pluronic F108 (nonionic)-were utilized. After mixing of the SWCNT-surfactant with alginate, the mixtures were cross-linked using divalent calcium ions and characterized using Raman spectroscopy. Rheometric analysis showed an increase in complex viscosity, loss, and storage moduli of the SWCNT composite gels in comparison with pure alginate gels. Scanning electron microscopy revealed the presence of a well-distributed porous structure, and all SWCNT-gel composites depicted enhanced electrical conductivity with respect to alginate gels. To characterize their biocompatibility, cardiomyocytes were cultured atop these SWCNT-gels. Results comprehensively implied that Pluronic F108 was most efficient in preventing agglomeration of the SWCNTs in the alginate matrix, leading to a stable scaffold formation without posing any toxicity to the cells.

In this study, fibrin was added to a photo-polymerizable gelatin-based bioink mixture to fabricate cardiac cell-laden constructs seeded with human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) or CM cell lines with cardiac fibroblasts (CF). The extensive use of platelet-rich fibrin, its capacity to offer patient specificity, and the similarity in composition to surgical glue prompted us to include fibrin in the existing bioink composition. The cell-laden bioprinted constructs were cross-linked to retain a herringbone pattern via a two-step procedure including the visible light cross-linking of furfuryl-gelatin followed by the chemical cross-linking of fibrinogen via thrombin and calcium chloride. The printed constructs revealed an extremely porous, networked structure that afforded long-term in vitro stability. Cardiomyocytes printed within the sheet structure showed excellent viability, proliferation, and expression of the troponin I cardiac marker. We extended the utility of this fibrin-gelatin bioink toward coculturing and coupling of CM and cardiac fibroblasts (CF), the interaction of which is extremely important for maintenance of normal physiology of the cardiac wall in vivo. This enhanced "cardiac construct" can be used for drug cytotoxicity screening or unraveling triggers for heart diseases in vitro., Competing Interests: The authors declare no competing financial interest.

Cardiovascular tissue engineering endeavors to repair or regenerate damaged or ineffective blood vessels, heart valves, and cardiac muscle. Current strategies that aim to accomplish such a feat include the differentiation of multipotent or pluripotent stem cells on appropriately designed biomaterial scaffolds that promote the development of mature and functional cardiac tissue. The advent of additive manufacturing 3D bioprinting technology further advances the field by allowing heterogenous cell types, biomaterials, and signaling factors to be deposited in precisely organized geometries similar to those found in their native counterparts. Bioprinting techniques to fabricate cardiac tissue in vitro include extrusion, inkjet, laser-assisted, and stereolithography with bioinks that are either synthetic or naturally-derived. The article further discusses the current practices for postfabrication conditioning of 3D engineered constructs for effective tissue development and stability, then concludes with prospective points of interest for engineering cardiac tissues in vitro. Cardiovascular three-dimensional bioprinting has the potential to be translated into the clinical setting and can further serve to model and understand biological principles that are at the root of cardiovascular disease in the laboratory., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Three-dimensional bioprinting is an innovative technique in tissue engineering, to create layer-by-layer structures, required for mimicking body tissues. However, synthetic bioinks do not generally possess high printability and biocompatibility at the same time. So, there is an urgent need for naturally derived bioinks that can exhibit such optimized properties. We used furfuryl-gelatin as a novel, visible-light crosslinkable bioink for fabricating cell-laden structures with high viability. Hyaluronic acid was added as a viscosity enhancer and either Rose Bengal or Riboflavin was used as a visible-light crosslinker. Crosslinking was done by exposing the printed structure for 2.5 min to visible light and confirmed using Fourier transform infrared spectroscopy and rheometry. Scanning electron microscopy revealed a highly porous networked structure. Three different cell types were successfully bioprinted within these constructs. Mouse mesenchymal stem cells printed within monolayer and bilayer sheets showed viability, network formation and proliferation (∼5.33 times) within 72 h of culture. C2C12 and STO cells were used to print a double layered structure, which showed evidence of the viability of both cells and heterocellular clusters within the construct. This furfuryl-gelatin based bioink can be used for tissue engineering of complex tissues and help in understanding how cellular crosstalk happens in vivo during normal or diseased pathology. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 314-323, 2019., (© 2018 Wiley Periodicals, Inc.)

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cell-based therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulin-producing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research., Competing Interests: Conflict-of-interest statement: The authors have no conflicts to declare.

3D bioprinting holds great promise in the field of regenerative medicine as it can create complex structures in a layer-by-layer manner using cell-laden bioinks, making it possible to imitate native tissues. Current bioinks lack both high printability and biocompatibility required in this respect. Hence, the development of bioinks that exhibit both properties is needed. In our previous study, a furfuryl-gelatin-based bioink, crosslinkable by visible light, was used for creating mouse mesenchymal stem cell-laden structures with a high fidelity. In this study, lattice mesh geometries were printed in a comparative study to test against the properties of a traditional rectangular-sheet. After 3D printing and crosslinking, both structures were analysed for swelling and rheological properties, and their porosity was estimated using scanning electron microscopy. The results showed that the lattice structure was relatively more porous with enhanced rheological properties and exhibited a lower degradation rate compared to the rectangular-sheet. Further, the lattice allowed cells to proliferate to a greater extent compared to the rectangular-sheet, which initially retained a lower number of cells. All of these results collectively affirmed that the lattice poses as a superior scaffold design for tissue engineering applications.

The implantation of stem cells in vivo is the ideal approach for the restoration of normal life functions, such as replenishing the decreasing levels of affected dopaminergic (DA) neurons during neurodegenerative disease conditions. However, combining stem cells with biomaterial scaffolds provides a promising strategy for engineering tissues or cellular delivery for directed stem cell differentiation as a means of replacing diseased/damaged tissues. In this study, mouse mesenchymal stem cells (MSCs) were differentiated into DA neurons using sonic hedgehog, fibroblast growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor, while they were cultured within collagen-coated 3D graphene foams (GF). The differentiation into DA neurons within the collagen-coated GF and controls (collagen gels, plastic) was confirmed using β -III tubulin, tyrosine hydroxylase (TH), and NeuN positive immunostaining. Enhanced expression of β -III tubulin, TH, and NeuN and an increase in the average neurite extension length were observed when cells were differentiated within collagen-coated GF in comparison with collagen gels. Furthermore, these graphene-based scaffolds were not cytotoxic as MSC seemed to retain viability and proliferated substantially during in vitro culture. In summary, these results suggest the utility of 3D graphene foams towards the differentiation of DA neurons from MSC, which is an important step for neural tissue engineering applications.

Adult stem cells such as mesenchymal stem cells (MSC) are known to possess the ability to augment neovascularization processes and are thus widely popular as an autologous source of progenitor cells. However there is a huge gap in our current knowledge of mechanisms involved in differentiating MSC into endothelial cells (EC), essential for lining engineered blood vessels. To fill up this gap, we attempted to differentiate human MSC into EC, by culturing the former onto chemically fixed layers of EC or its ECM, respectively. We expected direct contact of MSC when cultured atop fixed EC or its ECM, would coax the former to differentiate into EC. Results showed that human MSC cultured atop chemically fixed EC or its ECM using EC-medium showed enhanced expression of CD31, a marker for EC, compared to other cases. Further in all human MSC cultured using EC-medium, typically characteristic cobble stone shaped morphologies were noted in comparison to cells cultured using MSC medium, implying that the differentiated cells were sensitive to soluble VEGF supplementation present in the EC-medium. Results will enhance and affect therapies utilizing autologous MSC as a cell source for generating vascular cells to be used in a variety of tissue engineering applications.

Stem cells offer tremendous promise for regenerative medicine as they can become a variety of cell types. They also continuously proliferate, providing a renewable source of cells. Recently, it has been found that 3D printing constructs using stem cells, can generate models representing healthy or diseased tissues, as well as substitutes for diseased and damaged tissues. Here, we review the current state of the field of 3D printing stem cell derived tissues. First, we cover 3D printing technologies and discuss the different types of stem cells used for tissue engineering applications. We then detail the properties required for the bioinks used when printing viable tissues from stem cells. We give relevant examples of such bioprinted tissues, including adipose tissue, blood vessels, bone, cardiac tissue, cartilage, heart valves, liver, muscle, neural tissue, and pancreas. Finally, we provide future directions for improving the current technologies, along with areas of focus for future work to translate these exciting technologies into clinical applications., (© Biomedical Engineering Society 2018.)

Gastroparesis (GP) is associated with depletion of interstitial cells of Cajal (ICCs) and enteric neurons, which leads to pyloric dysfunction followed by severe nausea, vomiting and delayed gastric emptying. Regenerating these fundamental structures with mesenchymal stem cell (MSC) therapy would be helpful to restore gastric function in GP. MSCs have been successfully used in animal models of other gastrointestinal (GI) diseases, including colitis. However, no study has been performed with these cells on GP animals. In this study, we explored whether mouse MSCs can be delivered from a hydrogel scaffold to the luminal surfaces of mice stomach explants. Mouse MSCs were seeded atop alginate-gelatin, coated with poly-l-lysine. These cell-gel constructs were placed atop stomach explants facing the luminal side. MSCs grew uniformly all across the gel surface within 48 h. When placed atop the lumen of the stomach, MSCs migrated from the gels to the tissues, as confirmed by positive staining with vimentin and N -cadherin. Thus, the feasibility of transplanting a cell-gel construct to deliver stem cells in the stomach wall was successfully shown in a mice stomach explant model, thereby making a significant advance towards envisioning the transplantation of an entire tissue-engineered 'gastric patch' or 'microgels' with cells and growth factors., Competing Interests: The authors declare no conflict of interest.

A 34-amino acid long collagen-like peptide rich in proline, hydroxyproline, and glycine, and with four photoreactive N-acyl-7-nitroindoline units incorporated into the peptide backbone was synthesized by on-resin fragment condensation. Its circular dichroism supports a stable triple helix structure. The built-in photochemical function enables the decomposition of the peptide into small peptide fragments by illumination with UV light of 350 nm in aqueous solution. Illumination of a thin film of the peptide, or a thin film of a photoreactive amino acid model compound containing a 5-bromo-7-nitroindoline moiety, with femtosecond laser light at 710 nm allows for the creation of well-resolved micropatterns. The cytocompatibility of the peptide was demonstrated using human mesenchymal stem cells and mouse embryonic fibroblasts. Our data show that the full-length peptide is cytocompatible as it can support cell growth and maintain cell viability. In contrast, the small peptide fragments created by photolysis are somewhat cytotoxic and therefore less cytocompatible. These data suggest that biomimetic collagen-like photoreactive peptides could potentially be used for growing cells in 2D micropatterns based on patterns generated by photolysis prior to cell growth.

In this study, a hybrid system consisting of 3D printed polycaprolactone (PCL) filled with hydrogel was developed as an application for reconstruction of long bone defects, which are innately difficult to repair due to large missing segments of bone. A 3D printed gyroid scaffold of PCL allowed a larger amount of hydrogel to be loaded within the scaffolds as compared to 3D printed mesh and honeycomb scaffolds of similar volumes and strut thicknesses. The hydrogel was a mixture of alginate, gelatin, and nano-hydroxyapatite, infiltrated with human mesenchymal stem cells (hMSC) to enhance the osteoconductivity and biocompatibility of the system. Adhesion and viability of hMSC in the PCL/hydrogel system confirmed its cytocompatibility. Biomineralization tests in simulated body fluid (SBF) showed the nucleation and growth of apatite crystals, which confirmed the bioactivity of the PCL/hydrogel system. Moreover, dissolution studies, in SBF revealed a sustained dissolution of the hydrogel with time. Overall, the present study provides a new approach in bone tissue engineering to repair bone defects with a bioactive hybrid system consisting of a polymeric scaffold, hydrogel, and hMSC., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

A persistent theme in biomaterials research comprises of surface engineering and modification of bare metallic substrates for improved cellular response and biocompatibility. Graphene Oxide (GO), a derivative of graphene, has outstanding chemical and mechanical properties; its large surface to volume ratio, ease of surface modification and processing make GO an attractive coating material. GO-coatings have been extensively studied as biosensors. Further owing to its surface nano-architecture, GO-coated surfaces promote cell adhesion and growth, making it suitable for tissue engineering applications. The need to improve the long-term durability and therapeutic effectiveness of commercially available bare 316L stainless steel (SS) surfaces led us to adopt a polymer-free approach which is cost-effective and scalable. GO was immobilized on to 316L SS utilizing amide linkage, to generate a strongly adherent uniform coating with surface roughness. GO-coated 316L SS surfaces showed increased hydrophilicity and biocompatibility with SHSY-5Y neuronal cells, which proliferated well and showed decreased reactive oxygen species (ROS) expression. In contrast, cells did not adhere to bare uncoated 316L SS meshes nor maintain viability when cultured in the vicinity of bare meshes. Therefore the combination of the improved surface properties and biocompatibility implies that GO-coating can be utilized to overcome pertinent limitations of bare metallic 316L SS implant surfaces, especially SS neural electrodes. Also, the procedure for making GO-based protective coatings can be applied to numerous other implants where the development of such protective films is necessary., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca(2+) ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering.

The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs. Biological activity was monitored by colony formation of iPS cells. Treatment with a low concentration of FA sustained the membrane fluidity and biological activity, which were comparable to those of mitomycin C-treated MEFs. The biological activity was further confirmed by sustained expression of alkaline phosphatase, SSEA-1, and other pluripotency markers in iPS cells after 3-5 days of culture on FA-fixed MEFs. Chemical fixation of feeder cells has several advantages such as providing ready-to-use culture substrates without contamination by proliferating feeder cells. Therefore, our results provide an important basis for the development of chemically fixed culture substrates for pluripotent stem cell culture as an alternative to conventional treatment by mitomycin C or x-ray irradiation.

Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO2), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO2. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO2 using an established organ culture model. Saphenous veins cultured with arterial pO2 developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO2 or internal mammary arteries cultured at arterial pO2 did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO2 is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO2 in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO2. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO2 relative to those cultured at venous pO2. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO2 stimulates IH via a ROS-dependent pathway.

Conventional culture of human induced pluripotent (hiPS) cells requires feeder cell preparation that is time consuming and labour intensive. Alternatively, feeder-free culture of hiPS cells requires high cell seeding densities, specialized culture medium, and growth supplements, which raise the cost. Furthermore, recombinant systems require a long time for colony formation. Here, we employed chemically fixed autologous feeder cells for hiPS cell culture without additional time needed for colony formation. Treatment of (2.5% glutaraldehyde or formaldehyde for 10 min) autologous human dermal fibroblasts allowed hiPS cells to adhere and grow as undifferentiated colonies characterized by the expression of pluripotency markers such as alkaline phosphatase, Oct-3/4, and stage-specific embryonic antigen-4. Furthermore, hiPS cells cultured on chemically fixed feeders formed teratomas in vivo, characterized by all three germ layers. The chemically fixed autologous feeders may be used as a substitute for large scale culture of hiPS cells as a convenient in-house and a cost-effective method.

There have been great efforts to develop cell culture systems to regulate stem cell functions. Development of cell culture substrates is one of the important approaches for stem cell culture because substrates influence stem cell functions such as attachment, proliferation, self-renewal, and induction of differentiation. Stem cells are surrounded by their specific microenvironments in vivo, composed of cells, cytokines, and an extracellular matrix (ECM), which may dynamically change and affect cellular activities accordingly. To mimic such microenvironments, cell culture substrates can be prepared by coating bioactive proteins such as ECM proteins and signaling molecules as ligands for cell surface receptors. Compared with protein-coated substrates, cell- and cell-formed ECM-derived substrates have shown great progress and attracted significant attention as functional and prospective biomaterials for stem cell culture and regenerative medicine. In this review, we summarize the latest progress of these new substrates derived from cells and cell-formed ECMs.

In this study, we investigated the effect of varying localized concentration gradients of NGF and Sema3A on the axonal outgrowth of embryonic chick DRG explants and primary neurons in vitro. Immobilized 2D NGF or Sema3A micropatterns were produced using photolithography on tissue culture cover slips. Two distinct regions were identified: slow, with little or no change in concentration of chemotropic factor; and steep, with a transition from low to high. The direction of axonal outgrowth was defined as proximal or distal, with proximal growing towards the higher concentration of immobilized NGF/Sema3A and vice versa for distal. Axons grew preferentially in the proximal direction when explants were seeded onto steep NGF, and distally in response to steep Sema3A. On slow NGF, or on slow Sema3A there was no difference in the directional specificity of axonal outgrowth. DRG primary neurons seeded onto steep NGF migrated proximally, whereas neurons seeded onto slow NGF migrated in all directions. Conversely, neurons seeded onto steep or slow Sema3A did not extend any axons. Our 2D immobilized micropatterns of chemotropic factors show promise for further development of in vitro nerve tissue engineering studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Stem cells possess the ability to self-renew and differentiate into other cell types. In vivo, stem cells reside in their own anatomic niches in a defined physiological environment, from which they are released to differentiate into a required cell type when deemed appropriate. While a resident within the niche, the stem cell receives signals that in turn maintain the cell in a pluripotent state. In addition, the niche also provides nourishment to the cell. Physically, the niche also serves to anchor the cell via various ECM components and cell-adhesion molecules. Therefore, in vitro models that replicate the in vivo niche will lead to a better understanding of stem cell fate and turnover. In turn, this will help inform attempts to culture stem cells in vitro on artificial niche-like substrates. In this review, we have highlighted recent studies describing artificial niche-like substrates used to culture embryonic and induced pluripotent stem cells in vitro., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Dopamine, an adhesive protein can be covalently deposited onto biomaterials. In this study, we evaluated the ability of dopamine-coated surfaces for small interfering RNA (siRNA) immobilization and release. Dopamine was deposited onto 316L stainless steel discs either as a monolayer at acidic pH or as polydopamine at alkaline pH, following which siRNA was immobilized onto these discs. To investigate the RNA interference ability of immobilized siRNA, reduction of luciferase expression in HeLa, and reduction of Egr-1 expression and cell proliferation in human aortic smooth muscle cells (HAoSMCs) were determined. Dopamine treatment of 316L stainless steel discs under both the acidic and alkaline conditions resulted in the deposition of amino (NH2) groups, which enabled electrostatic immobilization of siRNA. The immobilized siRNA was released from both types of coatings, and enhanced the percent suppression of firefly luciferase activity of HeLa significantly up to ~96.5% compared to HeLa on non-dopamine controls (18%). Both the release of siRNA and the percent suppression of firefly luciferase activity were sustained for at least 7 days. In another set of experiments, siRNA sequences targeting to inhibit the activity of the transcription factor Egr-1 were eluted from dopamine-coated surfaces to HAoSMCs. Egr-1 siRNA eluted from dopamine-coated surfaces, significantly reduced the proliferation of HAoSMCs and their protein expression of Egr-1. Therefore, this method of surface immobilization of siRNA onto dopamine-coated surfaces might be effective for nucleic acid delivery from stents., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We investigated the in vitro effects of the molecular weight (MW) of hyaluronic acid (HA) on the maintenance of the pluripotency and proliferation of murine embryonic stem (ES) cells. High (1000 kDa) or low (4-8 kDa) MW HA was derivatized using an ultraviolet-reactive compound, 4-azidoaniline, and the derivative was immobilized onto cell culture cover slips. Murine ES cells were cultured on these HA surfaces for 5 days. High-MW HA interacted with murine ES cells via CD44, whereas low-MW HA interacted with these cells mostly via CD168. ES cells grown on both high- and low-MW HA appeared undifferentiated after 3 days. However, more cells adhered, proliferated, and exhibited greater amounts of phospho-p42/44 mitogen-activated-protein-kinase on low- compared with high-MW HA. Expression of Oct-3/4 and phosphorylation of STAT3 were enhanced by ES cells on low-MW HA, not on high-MW HA. After release from HA, cells cultured on low-MW HA in the presence of differentiating medium showed enhanced expression of α-SMA or CD31 compared with cells cultured on high-MW HA. It was concluded that low-MW HA substrates were effective in maintaining murine ES cells in a viable and undifferentiated state, which favors their use in the propagation of ES cells for tissue engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ex vivo culture of arteries and veins is an established tool for investigating mechanically induced remodeling. Porcine saphenous veins (PSV) cultured ex vivo with a venous mechanical environment, serum-supplemented cell-culture medium and standard cell-culture conditions (5% CO₂ and 95% balance air ~140 mmHg pO₂) develop intimal hyperplasia (IH), increased cellular proliferation, decreased compliance and exhibit inward eutrophic remodeling thereby suggesting that nonmechanical factors stimulate some changes observed ex vivo. Herein we explore the contribution of exposure to greater than venous pO₂ and serum to these changes in cultured veins. Removing serum from culture medium did not inhibit development of IH, but did reduce cellular proliferation and inward eutrophic remodeling. In contrast, veins perfused using reduced pO₂ (75 mmHg) showed reduced IH. Among the statically cultured vessels, veins cultured at arterial pO₂ (95 mmHg) and above showed IH as well as increase in proliferation and vessel weight compared to fresh veins; veins cultured at venous pO₂ did not. Taken together, these data suggest that exposure of SV to arterial pO₂ stimulates IH and cellular proliferation independent of changes in the mechanical environment, which might provide insight into the etiology of IH in SV used as arterial grafts.

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~3-fold increase in proliferation and ~3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O(2)(•-)) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Crosslinked gels (hylans) containing long-chain (MW>1 x 10(6)Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20 kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI-TOF showed the HA digests to contain HA 4-8mers with a 75+/-0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9+/-3 amine groups/nm(2) and 0.57+/-0.44 microg/cm(2), respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell-surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior.

Prior studies suggest that hyaluronan (HA), a glycosaminoglycan, may upregulate innately poor elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). HA scaffolds could thus be useful to regenerate damaged vascular elastin. In an earlier study, we established that the elastogenic effects of non-oligomeric HA are fragment size- and/or dose-specific. We currently investigate the pro-elastogenic effects of exogenous HA oligomers on rat aortic smooth muscle cells (RASMCs). RASMCs were cultured with pure HA oligomers (4-mers) and mixtures (4-8mers) obtained by enzymatic digestion of long-chain HA (MW approximately 2000kDa). Polyacrylamide gel electrophoresis (PAGE)/Matrix Assisted Laser Desorption/Ionization Spectroscopy Time-Of-Flight Analysis (MALDI-TOF) showed HA digestates to contain a mixture of 4-8mers with a predominance of 4-mers (75+/-0.4% w/w). Cell layers supplemented with both pure HA 4-mers or oligomer mixtures showed proliferation levels similar to non-HA controls over 21 days of culture. Pure 4-mers and oligomer mixtures enhanced DNA-normalized output of tropoelastin by 1.6 and 1.8 times, respectively, and that of matrix elastin by approximately 2.7 times relative to controls. Sodium dodecyl sulfate (SDS)-PAGE/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. HA oligomers induced enhanced synthesis of the elastin crosslinker, desmosine, and appeared to stabilize the elastin matrix by suppression of elastin-laminin receptor (ELR) activity relative to controls. Transmission electron micrographs (TEMs) showed elastin deposits within oligomer-supplemented cultures to be distinct, longitudinally oriented, aggregating fibrils, and clumps, and to be less abundant and mostly amorphous in controls. HA oligomers preserved normal fibrillin-mediated elastin matrix deposition. Results suggest that HA oligos are highly pro-elastogenic, promote elastin fibril formation, and stabilize elastin matrix and may thus be usefully incorporated into scaffolds for guided elastin regeneration.

Tissue engineering of vascular elastin matrices disrupted by mechanical injury, disease, or congenitally absent, is among other factors, limited by the lack of suitable cell scaffolds to up-regulate and guide innately poor elastin synthesis by adult vascular smooth muscle cells (SMCs). Evidence suggests that scaffolds based on hyaluronan (HA), a glycosaminoglycan, may be useful to elicit elastogenic cell responses, although these effects appear to be dictated by HA fragment size and/or dose. This study investigates the efficacy of a simple, frequently adopted exogenous HA supplementation model to test this hypothesis. Rat aortic SMCs were cultured with HA (2 x 10(6) Da (HMW) > or = MW < or = 2.2 x 10(4) Da) supplemented at doses between 0.2 and 200 microg/ml. Cell layers were biochemically assayed for DNA, elastin and collagen content. Fragmented, but not high molecular weight (HMW) HA, stimulated cell proliferation in inverse correlation fragment size while the opposite effect was observed for synthesis of soluble and matrix elastin; almost no dose effects were observed within any group. SDS-Page/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. Quantitative differences in elastin deposition were mirrored in TEM micrographs. Elastin was mostly deposited in the form of amorphous clumps but fibers were increasingly present in cell layers cultured with HMW HA. HA and its fragments did not disrupt normal fibrillin-mediated mechanisms of elastin matrix deposition. While the current outcomes confirm that the effects of HA on elastin synthesis are fragment size-specific, this study shows that an exogenous supplementation model does not necessarily simulate cellular matrix synthesis responses to HA-based biomaterial scaffolds.