A Study of the Printability of Alginate-Based Bioinks by 3D Bioprinting for Articular Cartilage Tissue Engineering

Three-dimensional bioprinting combined with natural hydrogels is a promising technology for the treatment of several pathologies and different tissue regeneration. One of the most studied tissues is cartilage, a complex and avascular tissue that displays a limited self-repair capacity after injuries. Herein, the development of alginate-based hydrogels and scaffolds containing different microstructure is presented and the printability of alginate by 3D bioprinting is studied. Rheological characterization was performed for the determination of viscosity and viscoelastic properties of hydrogels and mechanical characterization was carried out for the determination of compressive modulus of alginate hydrogels. All these characteristics were correlated with alginate behaviour during 3D bioprinting process. For the printability evaluation filament diameter, perimeter of the pores, area of the pores and shrinkage of alginate scaffolds were measured. The results demonstrate that alginate microstructure has a great influence on its printability and on hydrogels’ physicochemical properties. Molecular weight of alginate determines its viscosity while M/G ratio determines cross-linking conditions and mechanical properties that vary with cross-linking density. These results suggest the importance of an exhaustive control of the viscoelastic and mechanical properties of alginate hydrogels to obtain structures with high resolution and precision

Effects of Folinic Acid Administration on Lower Limb Ischemia/Reperfusion Injury in Rats

Surgery under ischemic conditions, lasting up to 3 h, is routinely performed in orthopedic surgery, causing undesirable injury due to ischemia-reperfusion syndrome, with short and medium-term functional repercussions. To date, there is no established prophylactic treatment. In this work we evaluated folinic acid (FA) in a rodent model of lower limb ischemia-reperfusion (IRI-LL). 36 male WAG rats underwent 3 h of lower limb ischemia. In the saline group, rats received intraperitoneal administration of saline (used as vehicle for treatment). In the experimental group, rats were pretreated with FA (2.5 mg/kg) before the end of ischemia. After ischemia, animals were sacrificed at 3 h, 24 h or 14 days (for biochemical determination (Na+, K+, Cl-, urea, creatinine, CK, LDH, ALP, ALT, and AST), pathological assessment, or functional study using the rotarod test; respectively). Another six animals were used to establish the reference values. The prophylactic administration of FA significantly reduced the elevation of biochemical markers, especially those that most directly indicate muscle damage (CK and LDH). In addition, it also improved direct tissue damage, both in terms of edema, weight, PMN infiltrate and percentage of damaged fibers. Finally, the administration of FA allowed the animals to equal baseline values in the rotarod test; what did not occur in the saline group, where pre-ischemia levels were not recovered. Following 3 h of lower limb ischemia, FA minimizes the increase of CK and LDH, as well as local edema and leukocyte infiltration, allowing a faster recovery of limb functionality. Therefore, it could be considered as a prophylactic treatment when tourniquet is used in clinics.This research received funding from the University of The Basque Country UPV/EHU (grant reference GIU19/088)

Conditioned Medium from H2O2-Preconditioned Human Adipose-Derived Stem Cells Ameliorates UVB-Induced Damage to Human Dermal Fibroblasts

Human skin exposure to ultraviolet B (UVB) radiation can result in acute photodamage through oxidative modifications of cellular components and biomolecules involved in the metabolism of dermal cells. Recently, the therapeutic potential of human adipose-derived stem cells (hASCs) has been investigated as a novel strategy for photoprotection due to their pro-angiogenic properties, protective activity against oxidative stress and paracrine effect on dermal cells. To enhance these therapeutic properties, hASCs can be preconditioned by exposing them to sublethal cellular stressors. In this study, we first analyzed response capacity against UVB-induced oxidative stress in H2O2-preconditioned hASCs (called HC016 cells); and second, we evaluated the photoprotective effect of HC016-conditioned medium (CM) in an in vitro UVB irradiation model in cultured human foreskin fibroblasts (hFFs). The results demonstrated that HC016 cells have a greater capacity to respond efficiently to UVB-induced oxidative stress, evidenced by higher Nrf2 antioxidant system activity and enhanced viability and migration capacity. Further, HC016-CM treatment increased viability, migratory capacity and collagen type I synthesis in hFFs exposed to UVB radiation, as well as reducing their cytotoxicity, apoptosis, senescence and IL-6 secretion. Collectively, these findings support the view that HC016 cells could protect against UVB-induced photodamage via paracrine mechanisms.This research was founded by the University of the Basque Country (UPV/EHU, grant reference numbers: GIU19/088 and PES 21/50)

H2O2-preconditioned human adipose-derived stem cells (HC016) increase theirresistance to oxidative stress byoverexpressing Nrf2 and bioenergetic adaptation

BackgroundMesenchymal stem cells, including those derived from human adipose tissue (hASCs), are currently being widely investigated for cell therapy. However, when transplanted at the site of injury, the survival and engraftment rates of hASCs are low, mainly due to the harsh microenvironment they encounter, characterized by inflammation and oxidative stress. To overcome these therapeutic limitations, cell preconditioning with low-concentration of hydrogen peroxide (H2O2) has been proposed as a plausible strategy to increase their survival and adaptation to oxidative stress. Nonetheless, the underlying mechanisms of this approach are not yet fully understood. In this study, we analyzed molecular and bioenergetic changes that take place in H2O2 preconditioned hASCs.MethodsLong-term exposure to a low concentration of H2O2 was applied to obtain preconditioned hASCs (named HC016), and then, their response to oxidative stress was analyzed. The effect of preconditioning on the expression of Nrf2 and its downstream antioxidant enzymes (HO-1, SOD-1, GPx-1, and CAT), and of NF-kappa B and its related inflammatory proteins (COX-2 and IL-1 beta), were examined by Western blot. Finally, the Seahorse XF96 Flux analysis system was used to evaluate the mitochondrial respiration and glycolytic function, along with the total ATP production.ResultsWe found that under oxidative conditions, HC016 cells increased the survival by (i) decreasing intracellular ROS levels through the overexpression of the transcription factor Nrf2 and its related antioxidant enzymes HO-1, SOD-1, GPx-1, and CAT; (ii) reducing the secretion of pro-inflammatory molecules COX-2 and IL-1 beta through the attenuation of the expression of NF-kappa B; and (iii) increasing the total ATP production rate through the adaption of their metabolism to meet the energetic demand required to survive.ConclusionsH(2)O(2) preconditioning enhances hASC survival under oxidative stress conditions by stimulating their antioxidant response and bioenergetic adaptation. Therefore, this preconditioning strategy might be considered an excellent tool for strengthening the resistance of hASCs to harmful oxidative stress.Partial funding for this project was provided by the Department of Economic Development and Competitiveness of the Basque Government, the European Regional Development Fund (PREMISE IG-2015/0000558), and the University of the Basque Country (UPV/EHU; research grants PES 17/29 and 16/37)

Antioxidant-Based Therapy Reduces Early-Stage Intestinal Ischemia-Reperfusion Injury in Rats

Intestinal ischemia-reperfusion injury (i-IRI) is a rare disorder with a high mortality rate, resulting from the loss of blood flow to an intestinal segment. Most of the damage is triggered by the restoration of flow and the arrival of cytokines and reactive oxygen species (ROS), among others. Inactivation of these molecules before tissue reperfusion could reduce intestinal damage. The aim of this work was to analyze the preventive effect of allopurinol and nitroindazole on intestinal mucosal damage after i-IRI. Wag/RijHsd rats were subjected to i-IRI by clamping the superior mesenteric artery (for 1 or 2 h) followed by a 30 min period of reperfusion. Histopathological intestinal damage (HID) was assessed by microscopic examination of histological sections obtained from injured intestine. HID was increased by almost 20% by doubling the ischemia time (from 1 to 2 h). Nitroindazole reduced HID in both the 1 and 2 h period of ischemia by approximately 30% and 60%, respectively (p < 0.001). Our preliminary results demonstrate that nitroindazole has a preventive/protective effect against tissue damage in the early stages of i-IRI. However, to better understand the molecular mechanisms underlying this phenomenon, further studies are needed.This research received funding from the University of The Basque Country UPV/EHU (grant reference GIU19/088)

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.Partial funding for this project was provided by the Department of Economic Development and Competitiveness of the Basque Government, the European Regional Development Fund (PREMISE IG-2015/0000558) and the University of the Basque Country (UPV/EHU; research grants GIU 19/088 and PES 17/29 and 16/37)

The Prevention of Ischemia-Reperfusion Injury in Elderly Rats after Lower Limb Tourniquet Use

Background: Lower limb ischemia-reperfusion injury (IRI-LL) is a common major complication of orthopedic surgery, especially in elderly patients. It has previously been demonstrated that folinic acid (FA) reduced IRI-LL damage in 3–4-month-old rats. This current work analyses the effect of FA in the prevention of IRI-LL in elderly animals. Methods: Forty-two 18-month-old male WAG/RijHsd rats were subjected to 3 h of ischemia. Eighteen animals received FA (2.5 mg/kg, ip) 20 min before the end of the ischemia period, while the other half received the same volume of saline solution. The animals were sacrificed after 3 h, 24 h, and 14 days of reperfusion for biochemical (tissue damage markers and electrolytes), histopathological studies of the gastrocnemius muscle and the daily assessment of the limb function by the Rota Rod test, respectively. Results: The administration of FA prior to the end of the ischemia period reduced the increase in LDH and CK observed in non-treated animals by 30–40% (p < 0.0001). When the histological sections were analyzed, FA was found to have reduced the number of damaged muscle fibers per field by 20% (60 ± 17.1 vs. 80.7 ± 16.4, p < 0.0001). The functional test revealed that FA also led to an improvement in the muscle function, assessed by the length of time that the animals kept running on the rod, compared to untreated animals. Conclusions: The administration of FA, prior to the end of the ischemic period, decreases the damage induced by IRI-LL, also achieving a faster recovery of mobility.This research was funded by the University of the Basque Country UPV/EHU (grant reference GIU19/088)

Effects of isothermal crystallization on the mechanical properties of a elastomeric medium chain length polyhydroxyalkanoate

In the present study, the relationship between molecular structure and mechanical properties for a medium chain length polyhydroxyalkanoate (mcl-PHA) composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate was elucidated. The mcl-PHA was crystallized from the melt at four different temperatures between its glass transition and melting point (37, 21, 3 and −21 °C) and its molecular structure was analysed by means of differential scanning calorimetry (DSC) and wide-angle X-ray diffractometry (WAXD). The mechanical properties, which were analysed via tensile-tests and dynamic mechanical analysis (DMA), were clearly affected by the selected crystallization temperature and corresponding molecular structure of the polymer. In this sense, samples crystallized at 37, 21 and 3 °C displayed higher secant moduli calculated at 2% (E2% ∼ 20 MPa) than the sample crystallized at −21 °C (E2% ∼ 7 MPa) due to their higher crystallinity. Even if samples crystallized at 37, 21 and 3 °C had very similar degree of crystallinity, their secant moduli calculated at 50, 100 and 200% (E50%, E100% and E200%) and yield strength (σy) were clearly affected by the selected crystallization temperature, showing a positive correlation (i.e., higher crystallization temperatures and corresponding more ordered crystalline domains with narrower crystal distributions resulted in higher E50%, E100% and E200% values).The authors are thankful for funds from the Basque Government, Department of Education, Universities and Research (GIC12/161-IT-632-13) and the Spanish Ministry of Innovation and Competitiveness MINECO (MAT2013-45559-P)

Shape-memory properties of crosslinked biobased polyurethanes

Biobased crosslinked polyurethanes were synthesized and characterized as shape-memory polymers. Both the macrodiol and the diisocyanate were derived from renewable sources; the first one from castor oil, and the second one from l-lysine amino acid. The influence of component molar ratios, crosslink density and maximum elongation on the shape-memory properties was analyzed. The thermal analysis showed that polyurethanes were microphase separated. Though the study of the shape-memory properties, it was seen that shape-memory was influenced by crosslink density. A higher crosslink density led to a greater shape recovery, since crosslinks are the responsible for memorizing the shape of the material. With the increase of maximum elongation, both shape fixity and recovery decreased due to higher amount of crosslink net points were broken. Moreover, the synthesized polyurethanes showed its potential to be used in biomedical applications, according to the preliminary in vitro cytotoxicity assays.Financial support from the Basque Government (IT-776-13) and from the Spanish Ministry of Economy and Competitiveness (MINECO) (MAT2013-43076-R) is gratefully acknowledged. We also wish to acknowledge the ‘‘Macrobehavior-Mesostructure-Nanotechnology” SGIker unit from the University of the Basque Country, for their technical support. T.C.-C. thanks the University of the Basque Country for Ph.D. grant (PIF/UPV/12/200

Furan-containing biobased polyurethane nanofibers: A new versatile and green support clickable via Diels-Alder reaction

A new biobased polyurethane formulation containing furan was synthesized and subsequently functionalized and de-functionalized via Diels-Alder and retro Diels-Alder reactions. Furan was inserted in the backbone polymer using 2,5-bis(hydroxymethyl)furan as chain extender. At the same time, another composition not containing furan was synthesized as reference. Both formulations were produced initially as films and then processed using electrospinning to obtain nanofibers membranes. The absence of catalysts, the high renewable content, the facile synthesis and spinnability of the polymer allow a quantitative and green production of versatile functionalizable platforms. The kinetic and efficiency of the functionalization were assessed using two different compounds: Fluorescein-5-maleimide and 3-Maleimidopropanoic acid. The high hydrophobicity of the membranes permits to perform the functionalization reactions in water, resulting in a process that minimally affects the membrane integrity which is, at the same time, environmental-friendly. Finally, the good biocompatibility opens the way for its use as biomedical tissue, widening the range of possible application of this material.Financial support from the University of the Basque Country (UPV/EHU) (GIU18/216 and GIU 19/088 Research Groups), from the Council of Gipuzkoa (2020-CIEN-000092-01), from the Basque Country Government (PIBA-2020-1-0041, KK-2020-00053) and from Spanish Ministry of Science, Innovation and Universities and European Union (MICINN/EU/FEDER) in the frame of PID2019-105090RB-I00 project and financial support from the Basque Country Government in the frame of Grupos Consolidados (IT-1690-22) are gratefully acknowledged. Moreover, we are grateful to the Macrobehavior-Mesostructure-Nanotechnology, Electronic Microscopy and Material Microanalysis and NMR Services SGIker units of the UPV/EHU. Stefano Torresi wishes to acknowledge the Ministry of Science and Innovation for his PhD grant (PRE2020-092538)