Preparation and Characterization of Plasma-Derived Fibrin Hydrogels Modified by Alginate di-Aldehyde

Fibrin hydrogels are one of the most popular scaffolds used in tissue engineering due to their excellent biological properties. Special attention should be paid to the use of human plasma-derived fibrin hydrogels as a 3D scaffold in the production of autologous skin grafts, skeletal muscle regeneration and bone tissue repair. However, mechanical weakness and rapid degradation, which causes plasma-derived fibrin matrices to shrink significantly, prompted us to improve their stability. In our study, plasma-derived fibrin was chemically bonded to oxidized alginate (alginate di-aldehyde, ADA) at 10%, 20%, 50% and 80% oxidation, by Schiff base formation, to produce natural hydrogels for tissue engineering applications. First, gelling time studies showed that the degree of ADA oxidation inhibits fibrin polymerization, which we associate with fiber increment and decreased fiber density; moreover, the storage modulus increased when increasing the final volume of CaCl₂ (1% w/v) from 80 µL to 200 µL per milliliter of hydrogel. The contraction was similar in matrices with and without human primary fibroblasts (hFBs). In addition, proliferation studies with encapsulated hFBs showed an increment in cell viability in hydrogels with ADA at 10% oxidation at days 1 and 3 with 80 µL of CaCl₂; by increasing this compound (CaCl₂), the proliferation does not significantly increase until day 7. In the presence of 10% alginate oxidation, the proliferation results are similar to the control, in contrast to the sample with 20% oxidation whose proliferation decreases. Finally, the viability studies showed that the hFB morphology was maintained regardless of the degree of oxidation used; however, the quantity of CaCl₂ influences the spread of the hFBs.     

Alginate core–shell microcapsule reduces the DMSO addition-induced osmotic damage to cells by inhibiting cellular blebs

In cryopreservation, the addition of cryoprotectant can change the intra- and extra-cellular osmotic pressure, affect the cell morphology, and induce blebs on the plasma membrane. In this study, the blebs of cells microencapsulated in the alginate microsphere induced by osmotic shock were studied, and the effects of microencapsulation on bleb size and cell viability were determined. Firstly, a coaxial co-flow focusing device was applied to generate cell-laden microcapsules using alginate hydrogel in this paper. Then, cellular blebs induced by DMSO with various concentrations under microencapsulation were com-pared with that when non-encapsulated, and the dynamic process of cellular bleb was investigated. Finally, the qualitative relationship between bleb size and cell viability in the presence of DMSO was built, and thus the effects of microencapsulation on bleb size and viability were evaluated. The results show that the bleb size is smaller and the cell viability is higher, and cell microencapsulation can signif-icantly inhibit the excessively large blebs generated on the cell membrane and reduce the osmotic dam-age to cells when loading cryoprotectant and then to improve cell viability during cryopreservation. This work can provide insights for optimizing cryoprotectant-loading protocols, offer a new avenue to study cell blebbing, and advance future research on cryopreservation of rare cells and biomaterials.     

Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis

Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.     

Hydrogen Peroxide-Preconditioned Human Adipose-Derived Stem Cells Enhance the Recovery of Oligodendrocyte-Like Cells after Oxidative Stress-Induced Damage

Oxidative stress associated with neuroinflammation is a key process involved in the pathophysiology of neurodegenerative diseases, and therefore, has been proposed as a crucial target for new therapies. Recently, the therapeutic potential of human adipose-derived stem cells (hASCs) has been investigated as a novel strategy for neuroprotection. These cells can be preconditioned by exposing them to mild stress in order to improve their response to oxidative stress. In this study, we evaluate the therapeutic potential of hASCs preconditioned with low doses of H₂O₂ (called HC016 cells) to overcome the deleterious effect of oxidative stress in an in vitro model of oligodendrocyte-like cells (HOGd), through two strategies: i, the culture of oxidized HOGd with HC016 cell-conditioned medium (CM), and ii, the indirect co-culture of oxidized HOGd with HC016 cells, which had or had not been exposed to oxidative stress. The results demonstrated that both strategies had reparative effects, oxidized HC016 cell co-culture being the one associated with the greatest recovery of the damaged HOGd, increasing their viability, reducing their intracellular reactive oxygen species levels and promoting their antioxidant capacity. Taken together, these findings support the view that HC016 cells, given their reparative capacity, might be considered an important breakthrough in cell-based therapies.     

天然椰子油提取物对H2O2诱导的HSF细胞氧化损伤的保护作用

采用倒置生物显微镜观察人皮肤成纤维细胞(HSF)的形态,通过MTT法检测细胞存活率,并通过检测细胞内活性氧(ROS)水平、总抗氧化能力和抗氧化酶活性探讨天然椰子油提取物对H_2O_2诱导的HSF细胞氧化损伤的保护作用。结果表明,天然椰子油提取物在质量浓度为12.5~50 mg/L范围内对H_2O_2诱导的HSF细胞氧化损伤具有保护作用,且呈浓度依赖性。天然椰子油提取物可以抑制H_2O_2引起的细胞形态损伤,提高细胞存活率,还可以通过降低细胞内ROS水平、提高细胞总抗氧化能力以及提高细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性,进而起到保护HSF细胞氧化应激损伤的作用。     

Fabrication and characterization of natural origin chitosan‐ gelatin‐alginate composite scaffold by foaming method without using surfactant

A natural origin tripolymer scaffold from chitosan, gelatin, and alginate was fabricated by applying foaming method without adding any foam stabilizing surfactant. Previously, in foaming method of scaffold fabrication, toxic surfactants were used to stabilize the foam, but in this work, the use of surfactant has been avoided strictly, which can provide better environment for cellular response and viability. In foaming method, stable foam is produced simply by agitating the polymer (alginate‐gelatin) solution, and the foam is crosslinked with CaCl₂, glutaraldehyde, and chitosan to produce tripolymer alginate‐gelatin‐chitosan composite scaffold. Microscopic images of the composite scaffold revealed the presence of interconnected pores, mostly spread over the entire surface of the scaffold. The scaffold has a porosity of 90% with a mean pore size of 57 μm. Swelling and degradation studies of the scaffold showed that the scaffold possesses excellent properties of hydrophilicity and biodegradability. In vitro cell culture studies by seeding L929 mouse fibroblast cells on scaffold revealed excellent cell viability, proliferation rate and adhesion as indicated by MTT assay, DNA quantification, and phase contrast microscopy of cell‐scaffold construct. The natural origin composite scaffold fabricated by the simplest method i.e., foaming method, but without adding any surfactant, is cheap, biocompatible, and it might find potential applications in the field of tissue engineering. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

A hybrid system of hydrogel/frog egg-like microspheres accelerates wound healing via sustained delivery of RCSPs

In this article, a hybrid system of hydrogel/frog egg-like microspheres (H-FMS) formed by the combination of coaxial electrostatic spraying and freeze-drying was introduced for enhancing wound healing efficiency through the sustained release of Rana chensinensis skin peptides (RCSPs). The porous PVA/gelatin hydrogel were obtained and frog egg-like microspheres (FMS) of sodium alginate (SA), shaping uniform and smooth, were embedded into hydrogel. Based on PVA/gelatin hydrogel, the FMS addition increased the water absorption of hydrogel to 1,105%. RCSPs were more effectively encapsulated into FMS than solid microspheres (MS). Not only does the H-FMS act as good “depots” for sustained release of RCSPs over 9 days, without exhibiting obvious burst release, but also show good biocompatibility in vitro. In vivo studies on wound healing as well as the histology of fibroblasts, re-epithelialization, inflammation, and hair follicles indicated that the structure of H-FMS released RCSPs continuously and promoted wound healing in rats significantly.     

The inclusion of fetal bovine serum in gelatin/PCL electrospun scaffolds reduces short‐term osmotic stress in HEK 293 cells caused by scaffold components

Components of gelatin/polycaprolactone (PCL) electrospun scaffolds are released to surrounding media and cause osmotic changes that adversely affect cell viability and proliferation. In this study, the physiological properties of gelatin/PCL scaffolds were investigated by qRT‐PCR and by performing cellular studies on HEK 293 cells. Components released from gelatin/PCL scaffolds were found to induce osmotic stress response in these cells. However, osmotic stress was inhibited by adding fetal bovine serum (FBS) to scaffolds. In addition, focal adhesion related genes were found to be up‐regulated in HEK 293 cells on gelatin/PCL/20% FBS scaffolds, and this induced the down‐regulations of cell‐death related genes. Furthermore, the inclusion of 20% FBS improved the viabilities of HEK 293 cells on gelatin/PCL scaffolds. This study indicates that adding FBS to gelatin/PCL scaffolds improves scaffold bio‐affinity. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Amylopectin Multiple Aldehyde Crosslinked Hydrogel as an Injectable and Self‐Healing Cell Carrier for Bone Tissue Engineering

Despite excellent processing and biological properties of gelatin for use as a cell carrier, none of the gelatin‐based hydrogel cell carriers reported to date offer all characteristics including quick formation, injectability, self‐healing, and durability, which are simultaneously required for an ideal system. Here, a gelatin‐based hydrogel with dynamic Schiff base linkages, so‐called “dynamic hydrogel,” as an injectable cell carrier consisting of gelatin and amylopectin multiple aldehyde (AMPA), with all the required characteristics is reported. Biocompatibility and osteoinductivity of the hydrogel are verified through the culture of human bone marrow‐derived mesenchymal stem cells (hBMSCs). As live/dead results show, hBMSCs are alive and highly viable ≈85–90% within the hydrogel after 5 days. According to bromodeoxyuridine cell proliferation assay, a significant increase in the number of the cells seeded in the hydrogel confirms its clinical significance for cell therapy. Most importantly, histological visualization using Mason's Trichrome staining indicates secretion of extracellular matrix around the cells loaded in the hydrogel and also expression level evaluation of the crucial osteogenic markers, confirms that the hydrogel can provide osteoinductive support for osteocyte differentiation of hBMSCs after 14 days. Therefore, this hydrogel provides more progress on the path toward bone tissue engineering and further treatment of bone diseases.     

Triolein and Trilinolein Ameliorate Oxidized Low-Density Lipoprotein-Induced Oxidative Stress in Endothelial Cells

Uptake of oxidized low-density lipoprotein by endothelial cells is a critical step for the initiation of atherosclerosis. Triacylglycerol uptake in these cells is understood to be a part of the process. The present investigation, comparison among the effects of simple acylglycerol, including tristearin, triolein, and trilinolein, upon oxidized low-density lipoprotein -induced oxidative stress was undertaken. Results indicated that trilinolein (78 % ± 0.02) and triolein (90 % ± 0.01) increased cell viability of endothelial cells exposed to oxidized low-density lipoprotein, whereas tristearin decreased the cell viability (55 % ± 0.03) (P < 0.05). Oxidized low-density lipoprotein treatment significantly increased apoptosis (23 %), compared to cells simultaneously exposed to trilinolein (19 %) or triolein (16 %), where apoptosis was reduced (P < 0.05). On the other hand, exposure to tristearin further increased oxidized low-density lipoprotein -induced cell apoptosis (34 %). Treatment with trilinolein or triolein on oxidized low-density lipoprotein -stimulated endothelial cells inhibited the expression of ICAM-1 and E-selectin mRNA. Moreover, both trilinolein and triolein demonstrated a strong antioxidant response to oxidative stress caused by oxidized low-density lipoprotein. Taken together, the results indicate trilinolein and triolein possess anti-inflammatory properties, which are mediated via the antioxidant defense system.     

Role of Autophagy in Lysophosphatidylcholine-Induced Apoptosis of Mouse Ovarian Granulosa Cells

Lysophosphatidylcholine (LPC), also known as lysolecithin, is one of the major components of oxidized low-density lipoproteins (ox-LDL). In the pathogenetic process of diverse diseases, LPC acts as a significant lipid mediator. However, no evidence shows that LPC can affect the female reproductive system. In our study, we found that LPC inhibited the cell viability of primary mouse ovarian granulosa cells. Meanwhile, LPC was shown to induce apoptosis, which is accompanied by an increase in apoptosis-related protein levels, such as cleaved caspase-3, cleaved caspase-8 and Bax, as well as a decrease in Bcl-2. The total numbers of early and late apoptotic cells also increased in the LPC-treated cells. These results indicated that LPC could induce apoptosis of mouse ovarian granulosa cells. Furthermore, the increase in autophagy-related protein levels and the number of autophagic vesicles suggested that LPC could induce autophagy. The inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the induction of apoptosis and autophagy by LPC, which indicated that oxidative stress was involved in LPC-induced apoptosis and autophagy. Interestingly, the inhibition of autophagy by 3-MA could reserve the inhibition of cell viability and the induction of apoptosis by LPC. In conclusion, oxidative stress was involved in LPC-induced apoptosis, whileautophagy of mouse ovarian granulosa cells and the inhibition of autophagy could alleviate LPC-induced apoptosis.     

A Biomimetic Hybrid Hydrogel Based on the Interactions between Amino Hydroxyapatite and Gelatin/Gellan Gum

In this work, a gelatin (Gel)‐oxidized gellan gum (OG)/amino hydroxyapatite (mHap) hybrid hydrogel with Schiff base linkages is reported. The mHap is obtained by modifying hydroxyapatite with tetraethyl orthosilicate and 3‐aminopropyl‐triethoxysilane. The effects of different mHap contents on the structure, morphology, and properties of hydrogels are particularly investigated. Scanning electron microscopy coupled with energy dispersion spectroscopy reveals that mHap of around 100 nm is uniformly distributed inside the hydrogel with interconnected porous structures. Notably, the hydrogel with 1 wt% mHap possesses the highest compressive stress (2.01 ± 0.10 MPa) at 90% strain, as well as the lowest equilibrium swelling ratio (97% ± 5%) and degradation rate than other hydrogels. Besides, an ultra‐high compressive stress equivalent to 91% of the initial stress can be obtained by this hydrogel after 50 loading‐unloading cycles (85% strain). Meanwhile, after being swollen, this improved hydrogel also exhibits better structural stability than Gel‐OG hydrogel. The in vitro 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay further shows that all hydrogels are nontoxic against mouse fibroblasts. This work provides a biomimetic strategy to construct the organic/inorganic hydrogels with excellent interactions, elasticity, reversibility, and biocompatibility, which is of great importance for the practical applications in cartilage tissue engineering.     

Influence of Process Variables on the Physical Properties of Gelatin/SA/HYA Composite Hydrogels

A novel composite hydrogel based on gelatin, sodium alginate (SA) and hyaluronic acid (HYA) was fabricated by freeze-drying method using 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linker. The effects of chemical cross-linking, including cross-linker content and cross-linking time, on the morphology, swelling ratio and compressive strength of the gelatin/SA/HYA hydrogel were investigated. The influence of pH value of the swelling medium on the swelling ratio of the gelatin/SA/HYA composite hydrogel was also studied. The results showed that the gelatin/SA/HYA composite hydrogel had a three-dimensional interconnected structure and the pore size decreased with increasing EDC concentration. The IR absorption peak intensity of the gelatin/SA/HYA hydrogel has no obvious variety with increasing EDC content. The swelling ratio of the gelatin/SA/HYA hydrogel decreased with increasing cross-linker content and cross-linking time; however, the compressive strength increased with increasing EDC content and cross-linking time. The hydrogel swelling peak reached at pH 7. Therefore, the architecture and the physical properties of the gelatin/SA/HYA composite hydrogel can be adjusted by controlling the chemical cross-linking conditions and pH value of swelling medium.     

High Concentrations of Genistein Decrease Cell Viability Depending on Oxidative Stress and Inflammation in Colon Cancer Cell Lines

Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H₂O₂) production were studied. The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G₂/M arrest, increased H₂O₂, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H₂O₂ production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.     

Investigations of cell immobilization in alginate: rheological and electrostatic extrusion studies

In this study, the process of electrostatic extrusion as a method for cell immobilization was investigated. We have assessed the effects of concentrations of yeast cells (as a model cell type) and Na alginate on the size of the resulting microbeads and attempted to rationalize the obtained findings by rheological characterization of the cell–alginate suspensions. Under the investigated conditions, microbeads, 50–600 µm in diameter, were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with their sizes having higher standard deviations. Rheological characterization revealed non‐Newtonian, pseudoplastic behavior of cell–alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5 × 10⁸ and 1 × 10⁹ cells mL^?1) did not significantly affect the rheological properties of the Na alginate solution. Finally, we have investigated the kinetics of alginate gelation with respect to the quantity of Ca²⁺ ions and the presence of cells. The molar ratio of α‐L ‐guluronic acid units to Ca²⁺ ions of 4:1 provided complete crosslinking. The presence of cells decreased the rate of network formation as well as the strength of the obtained Ca alginate hydrogel. Copyright © 2006 Society of Chemical Industry     

Three‐dimensional hydrogel encapsulated embryonic stem and carcinoma cells as culture platforms for cytotoxicity studies

The utility of alginate hydrogels for three‐dimensional (3‐D) culture of mouse embryonic stem cells (mESCs) and future development of 3‐D stem cell culture‐based in vitro screens of toxicity is described. Using alginate hydrogels of various stiffness, we first evaluated the impact of substrate modulus on mESC viability, proliferation, as well as expression of pluripotency and germ‐layer markers and observed that low concentration alginate hydrogels (0.5% and 1% alginate) were most suitable for long‐term culture of mESCs. These results were not unique to mESCs; long‐term viability and proliferation of mouse embryonic carcinoma cells (mECCs) was also best supported by similar conditions. Finally, we determined cytotoxic responses of alginate encapsulated cells to commercially available chemicals and interestingly observed similar responses for mESCs and mECCs, thereby suggesting that mECCs can predict stem cell responses to chemicals. These studies will facilitate future design of optimal stem cell‐based platforms of organ‐specific and developmental toxicity. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3180–3184, 2015     

Flexible Polysaccharide Hydrogel with pH‐Regulated Recovery of Self‐Healing and Mechanical Properties

A self‐healable hydrogel with recoverable self‐healing and mechanical properties is reported. The hydrogel (coded as ACSH) crosslinked by Schiff base linkage contains two polysaccharides of acrylamide‐modified chitosan (AMCS) and oxidized alginate (ADA). Self‐healing and mechanical properties are heavily influenced by the crosslinking time. The hydrogel crosslinked for 2 h possesses better mechanical and self‐healing properties than hydrogel crosslinked for 24 h. Macroscopic test shows that hydrogel without self‐healing ability can recover the self‐repair and mechanical properties by adjusting the pHs. The recovery of self‐healing and mechanical properties relies on the pH sensitivity of the Schiff base linkage. Adjusting the pH to acid, the Schiff base linkage becomes unstable and breaks. Regulating the pH to neutral, reconstruction of Schiff base linkage leads to recovery of the self‐repair and mechanical properties. The recoverable self‐healing property can be cycled once breakage and reconstruction of the Schiff base linkage can be conducted. In addition, this study demonstrates that the hydrogel can be remodeled into different shapes based on self‐healing property of the hydrogel. It is anticipated that this self‐healable hydrogel with recoverable self‐healing and mechanical properties may open a new way to investigate self‐healing hydrogel and find potential applications in different biomedical fields.     

Metformin Attenuates UVA-Induced Skin Photoaging by Suppressing Mitophagy and the PI3K/AKT/mTOR Pathway

Ultraviolet (UV) radiation is a major cause of photoaging that can induce DNA damage, oxidative stress, and cellular aging. Metformin (MF) can repair DNA damage, scavenge reactive oxygen species (ROS), and protect cells. However, the mechanism by which MF inhibits cell senescence in chronic skin damage induced by UVA is unclear. In this study, human foreskin fibroblasts (HFFs) treated with UVA were used as an in vitro model and UVA-induced skin photoaging in Kunming mice was used as an in vivo model to investigate the potential skin protective mechanism of MF. The results revealed that MF treatment attenuated UVA-induced cell viability, skin aging, and activation of the PI3K/AKT/mTOR signaling pathway. Furthermore, MF treatment alleviated the mitochondrial oxidative stress and decreased mitophagy. Knockdown of Parkin by siRNA increased the clearance of MF in senescent cells. The treatment of Kunming mice with MF at a dose of 10 mg/kg/day significantly reduced UVA-induced skin roughness, epidermal thinning, collagen degradation, and skin aging. In conclusion, our experimental results suggest that MF exerts anti-photoaging effects by inhibiting mitophagy and the PI3K/AKT/mTOR signaling pathway. Therefore, our study improves the current understanding of the protective mechanism of MF against photoaging.     

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin,Tested Over a Wide Range of Concentrations

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10⁻¹¹–10⁻² M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10⁻⁹–10⁻⁵ M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high—but not low—melatonin concentrations, although the mechanism of action still remains to be elucidated.     

Pre-Crosslinking with Hydrogel Microparticles Enhances the Printability of Alginate-Based Inks

The main challenge in extrusion-based bioprinting is to develop inks which must comprise a manifold of characteristics before, during, and after printing. To tackle the challenge of good shape fidelity and printability of low concentration inks, in this study hydrogel microparticles (HMPs) are proposed to produce internally pre-crosslinked inks. Alginate (Alg) and oxidized alginate (OA)-based HMPs are fabricated and used as Ca²⁺-releasing reservoirs. OA HMPs are used to demonstrate the versatility of this approach and to show its suitability also for chemically modified alginate. Embedded in either fresh Alg or OA solution, HMPs are used to pre-crosslink the inks. Rheological measurements revealed that HMP pre-crosslinking increases the yield stress and viscosity while reducing the loss angle of bioinks. Moreover, printing experiments reveal that being able to tailor rheological properties is an effective tool to improve printability. Furthermore, pre-crosslinking significantly alters the hydogel internal microstructure. In vitro studies show that NIH/3T3 cells proliferate in HMP pre-crosslinked bioinks modified with gelatin. Altogether, a low-cost and easy to use setup to prepare HMPs is presented and for the first time, the possibility of using such HMPs as pre-crosslinking agent to tailor the printability of alginate-based bioinks is demonstrated.