In Vitro and In Vivo Biocompatibility of Bacterial Cellulose.

Bacterial cellulose is a unique biomaterial produced by various species of bacteria that offers a range of potential applications in the biomedical field. To provide a cost-effective alternative to soft-tissue implants used in cavity infills, remodeling, and subdermal wound healing, in vitro cytotoxicity and in vivo biocompatibility of native bacterial cellulose were investigated. Cytotoxicity was assessed using a metabolic assay on Swiss 3T3 fibroblasts and INS-1832/13 rat insulinoma. Results showed no cytotoxicity, whether the cells were seeded over or under the bacterial cellulose scaffolds. Biocompatibility was performed on Sprague-Dawley rats (males and females, 8 weeks old) by implanting bacterial cellulose membranes subcutaneously for 1 or 12 weeks. The explanted scaffolds were then sliced and stained with hematoxylin and eosin for histological characterization. The first series of results revealed acute and chronic inflammation persisting over 12 weeks. Examination of the explants indicated a high number of granulocytes within the periphery of the bacterial cellulose, suggesting the presence of endotoxins within the membrane, confirmed by a Limulus amebocyte lysate test. This discovery motivated the development of non-pyrogenic bacterial cellulose scaffolds. Following this, a second series of animal experiments was done, in which materials were implanted for 1 or 2 weeks. The results revealed mild inflammation 1 week after implantation, which then diminished to minimal inflammation after 2 weeks. Altogether, this study highlights that unmodified, purified native bacterial cellulose membranes may be used as a cost-effective biomedical device provided that proper endotoxin clearance is achieved.

Optimizing Antioxidant Potential: Factorial Design-Based Formulation of Fucoidan and Gallic Acid-Conjugated Dextran Blends.

The role of oxidative stress in health and homeostasis has generated interest in the scientific community due to its association with cardiovascular and neurodegenerative diseases, cancer, and other diseases. Therefore, extensive research seeks to identify new exogenous antioxidant compounds for supplementation. Polysaccharides are recognized for their antioxidant properties. However, polysaccharide chemical modifications are often necessary to enhance these properties. Therefore, dextran was conjugated with gallic acid (Dex-Gal) and later combined with fucoidan A (FucA) to formulate blends aimed at achieving superior antioxidant activity compared to individual polysaccharides. A factorial design was employed to combine FucA and Dex-Gal in different proportions, resulting in five blends (BLD1, BLD2, BLD3, BLD4, and BLD5). An analysis of surface graphs from in vitro antioxidant tests, including total antioxidant capacity (TAC), reducing power, and hydroxyl radical scavenging, guided the selection of BLD4 as the optimal formulation. Tests on 3T3 fibroblasts under various conditions of oxidative stress induced by hydrogen peroxide revealed that BLD4 provided enhanced protection compared to its isolated components. The BLD4 formulation, resulting from the combination of Dex-Gal and FucA, showed promise as an antioxidant strategy, outperforming its individual components and suggesting its potential as a supplement to mitigate oxidative stress in adverse health conditions.

Cancer-associated fibroblasts barrier breaking via TGF-ß blockade paved way for docetaxel micelles delivery to treat pancreatic cancer.

TGF-ß is a crucial regulator in tumor microenvironment (TME), especially for myofibroblastic cancer-associated fibroblasts (myCAFs). The myCAFs can be motivated by TGF-ß signaling to erect pro-tumor TME, meanwhile, myCAFs overexpress TGF-ß to mediate the crosstalk between tumor and stromal cells. The blockade of TGF-ß can break cancer-associated fibroblasts barrier, consequently opening the access for drugs into tumor. The TGF-ß is a promising target in anti-tumor therapy. Herein, we introduced a two-stage combination therapy (TC-Therapy), including TGF-ß receptor I inhibitor SB525334 (SB) and cytotoxicity agent docetaxel micelle (DTX-M). We found that SB and DTX-M synergistically inhibited myCAFs proliferation and elevated p53 protein expression in BxPC-3/3T3 mixed cells. Gene and protein tests demonstrated that SB cut off TGF-ß signaling via receptor blockade and it did not arouse TGF-ß legend compensated internal autocrine. On the contrary, two agents combined decreased TGF-ß secretion and inhibited myCAFs viability marked by α-SMA and FAPα. TC-Therapy was applied in BxPc-3/3T3 mixed tumor-bearing mice model. After TC-Therapy, the α-SMA+/ FAPα+ myCAFs faded increasingly and collagenous fibers mainly secreted by myCAFs decreased dramatically as well. More than that, the myCAFs barrier breaking helped to normalize micro-vessels and paved way for micelle penetration. The TGF-ß protein level of TC-Therapy in TME was much lower than that of simplex DTX-M, which might account for TME restoration. In conclusion, TGF-ß inhibitor acted as the pioneer before nano chemotherapeutic agents. The TC-Therapy of TGF-ß signaling inhibition and anti-tumor agent DTX-M is a promising regimen without arising metastasis risk to treat pancreatic cancer. The therapeutic regimen focused on TGF-ß related myCAFs reminds clinicians to have a comprehensive understanding of pancreatic cancer.

Characterization and biocompatibility of a bilayer PEEK-based scaffold for guiding bone regeneration.

BACKGROUND: Polyetheretherketone (PEEK) is well known for its excellent physical-chemical properties and biosafety. The study aimed to open up a new method for clinical application of PEEK to reconstruct large-scale bone defects. METHODS: A bilayer scaffold for bone regeneration was prepared by combining a sulfonated PEEK barrier framework (SPEEK) with a hydrogel layer loaded with aspirin (ASA) and nano-hydroxyapatite (nHAP) by the wet-bonding of Polydopamine (PDA). RESULTS: The hydrogel was successfully adhered to the surface of SPEEK, resulting in significant changes including the introduction of bioactive groups, improved hydrophilicity, and altered surface morphology. Subsequent tests confirmed that the bilayer scaffold exhibited enhanced compression resistance and mechanical compatibility with bone compared to a single hydrogel scaffold. Additionally, the bilayer scaffold showed stable and reliable bonding properties, as well as excellent biosafety verified by cell proliferation and viability experiments using mouse embryo osteoblast precursor (MC3T3-E1) cells. CONCLUSION: The bilayer bone regeneration scaffold prepared in this study showed promising potential in clinical application for bone regeneration.

New Insights Into Application Relevant Properties of Cu2+-Doped Brushite Cements.

Doping of brushite cements with metal ions can entail many positive effects on biological and physicochemical properties. Cu2+ ions are known to exhibit antibacterial properties and can additionally have different positive effects on cells as trace elements, whereas high Cu2+ concentrations are cytotoxic. For therapeutical applications of bone cement, a combination of good biocompatibility and sufficient mechanical properties is required. Therefore, the aim of this study was to investigate different physicochemical and biological aspects, relevant for application, of a brushite cement with Cu2+-doped ß-tricalcium phosphate, monocalcium phosphate monohydrate and phytic acid as setting retarder. Additionally, the ion release was compared with a cement with citric acid as setting retarder. The investigated cements showed good injectability coefficients, as well as compressive strength values sufficient for application. Furthermore, no antibacterial effects were detected irrespective of the Cu2+ concentration or the bacterial strain. The cell experiments with eluate samples showed that the viability of MC3T3-E1 cells tended to decrease with increasing Cu2+ concentration in the cement. It is suggested that these biological responses are caused by the difference in the Cu2+ release from the hardened cement depending on the solvent medium. Furthermore, the cements showed a steady release of Cu2+ ions to a lesser extent in comparison with a cement with citric acid as setting retarder, where a burst release of Cu2+ was observed. In conclusion, despite the anticipated antibacterial effect of Cu2+-doped cements was lacking and mammalian cell viability was slightly affected, Cu2+-concentrations maintained the physicochemical properties as well as the compressive strength of cements and the slow ion release from cements produced with phytic acid is considered advantageous compared to citric acid-based formulations.

A comparison of several separation processes for eggshell membrane powder as a natural biomaterial for skin regeneration.

BACKGROUND: Numerous studies have focused on skin damage, the most prevalent physical injury, aiming to improve wound healing. The exploration of biomaterials, specifically eggshell membranes (ESMs), is undertaken to accelerate the recovery of skin injuries. The membrane must be separated from the shell to make this biomaterial usable. Hence, this investigation aimed to identify more about the methods for membrane isolation and determine the most efficient one for usage as a biomaterial. METHODS AND MATERIALS: For this purpose, ESM was removed from eggs using different protocols (with sodium carbonate, acetic acid, HCl, calcium carbonate, and using forceps for separation). Consequently, we have examined the membranes' mechanical and morphological qualities. RESULTS: According to the analysis of microscopic surface morphology, the membranes have appropriate porosity. MTT assay also revealed that the membranes have no cytotoxic effect on 3T3 cells. The results indicated that the ESM had acquired acceptable coagulation and was compatible with blood. Based on the obtained results, Provacol 4 (0.5-mol HCl and neutralized with 0.1-mol NaOH) was better than other methods of extraction and eggshell separation because it was more cell-compatible and more compatible with blood. CONCLUSION: This study demonstrates that ESMs can be used as a suitable biomaterial in medical applications.

Versatile cell cultivation on injectable poly(butylene adipate-co-terephthalate) microcarriers: Impact of surface properties across different cell types.

Injectable cell therapies offer several advantages compared with traditional open surgery, including less trauma to the patient, shorter recovery time, and lower risk of infection. However, a significant problem is the difficulty in developing effective cell delivery carriers that are cyto-compatible and maintain cell viability both during and after injection. In the presented study, it was aimed to develop poly(butylene adipate-co-terephthalate) (PBAT) microcarriers using the emulsion preparation-solvent evaporation technique. The optimized diameter of the PBAT microcarriers was determined as 104 ± 15 µm at 700 rpm and there would be no blockage after injection due to the nonswelling feature of microcarriers. Furthermore, the cellular activities of PBAT microcarriers were evaluated in static culture for 7 days using L929 mouse fibroblasts, MC3T3-E1 mouse pre-osteoblasts, and rat adipose-derived mesenchymal cells (AdMSCs). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide results and Sscanning electron microscope images showed that PBAT microcarriers increased the adhesion and proliferation properties of pre-osteoblasts and stem cells, while L929 fibroblasts formed aggregates by adhering to certain regions of the microcarrier surface and did not spread on the surface. These results emphasize that PBAT microcarriers can be used as injectable carriers, especially in stem cell therapies, but their surface properties need to be modified for some cells.

Emulsion electrospun epigallocatechin gallate-loaded silk fibroin/polycaprolactone nanofibrous membranes for enhancing guided bone regeneration.

Guided bone regeneration (GBR) membranes play an important role in oral bone regeneration. However, enhancing their bone regeneration potential and antibacterial properties is crucial. Herein, silk fibroin (SF)/polycaprolactone (PCL) core-shell nanofibers loaded with epigallocatechin gallate (EGCG) were prepared using emulsion electrospinning. The nanofibrous membranes were characterized via scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, water contact angle (CA) measurement, mechanical properties testing, drug release kinetics, and 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) free radical scavenging assay. Mouse pre-osteoblast MC3T3-E1 cells were used to assess the biological characteristics, cytocompatibility, and osteogenic differentiation potential of the nanofibrous membrane. Additionally, the antibacterial properties againstStaphylococcus aureus (S. aureus)andEscherichia coli (E. coli)were evaluated. The nanofibers prepared by emulsion electrospinning exhibited a stable core-shell structure with a smooth and continuous surface. The tensile strength of the SF/PCL membrane loaded with EGCG was 3.88 ± 0.15 Mpa, the water CA was 50°, and the DPPH clearance rate at 24 h was 81.73% ± 0.07%. The EGCG release rate of membranes prepared by emulsion electrospinning was reduced by 12% within 72 h compared to that of membranes prepared via traditional electrospinning.In vitroexperiments indicate that the core-shell membranes loaded with EGCG demonstrated good cell compatibility and promoted adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells. Furthermore, the EGCG-loaded membranes exhibited inhibitory effects onE. coliandS. aureus. These findings indicate that core-shell nanofibrous membranes encapsulated with EGCG prepared using emulsion electrospinning possess good antioxidant, osteogenic, and antibacterial properties, making them potential candidates for research in GBR materials.

Zinc phosphate glass microspheres promoted mineralization and expression of BMP2 in MC3T3-E1 cells.

Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc-titanium-calcium-sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 µm, while the larger ones ranged from 106 to 150 µm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3-E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3-E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26-fold, p < .001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27-fold, p < .001; larger: 35-fold, p < .001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3-E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3-5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3-E1 cells.

The adaptation of 3T3 cells to simulated microgravity by retrieving the major cell cycle-related protein expression during long-term in vitro proliferation.

The present study aimed to assess the effects of simulated microgravity (SMG) on 3T3 cell proliferation and the expression of cell cycle regulators. 3T3 cells were induced to SMG by Gravite® for 8 days, while the control group was treated with 1G condition. The result showed that the SMG condition causes a decrease in proliferative activity in 3T3 cells. In the SMG group, the expression of cell cycle-related proteins was lower than the control on day 3. However, these proteins were upregulated in 3T3 cells of the SMG group on day 5, suggesting that these cells were rescued from the arrest and retrieved a higher proliferation. A down-regulation of cell cycle-related proteins was observed in 3T3 cells of both SMG and control groups on day 7. In conclusion, SMG results in the attenuation of cell proliferation during the initial exposure to SMG, but the cells will adapt to this condition and retrieve normal proliferation by increasing the expression of cell cycle regulators.

The role of TGF-ß in the electrotactic reaction of mouse 3T3 fibroblasts in vitro.

Endogenous electric fields (EFs) serve as a crucial signal to guide cell movement in processes such as wound healing, embryonic development, and cancer metastasis. However, the mechanism underlying cell electrotaxis remains poorly understood. A plausible hypothesis suggests that electrophoretic or electroosmotic forces may rearrange charged components of the cell membrane, including receptors for chemoattractants which induce asymmetric signaling and directional motility. This study aimed to explore the role of Transforming Growth Factor Beta (TGFß) signaling in the electrotactic reaction of 3T3 fibroblasts. Our findings indicate that inhibiting canonical and several non-canonical signaling pathways originating from the activated TGF-ß receptor does not hinder the directed migration of 3T3 cells to the cathode. Furthermore, suppression of TGF-ß receptor expression does not eliminate the directional migration effect of 3T3 cells in the electric field. Additionally, there is no observed redistribution of the TGF-ß receptor in the electric field. However, our studies affirm the significant involvement of Phosphoinositide 3-Kinase (PI3K) in electrotaxis, suggesting that in our model, its activation is likely associated with factors independent of TGFß action.

The SPI1/SMAD5 cascade in the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells: a mechanism study.

BACKGROUND: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation. METHODS: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays. RESULTS: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells. CONCLUSION: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells.

l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

Novel shish-kebab structured nanofibrous decorating chitosan unidirectional scaffolds to mimic extracellular matrix for tissue engineering.

Electrospun nanofibrous scaffolds are renowned for their ability to mimic the microstructure of the extracellular matrix (ECM). However, they often fail to replicate the geometry of target tissues, and the biocompatibility of these scaffolds those made from synthetic polymers is always limited due to the lack of cell binding sites. To address these issues, we proposed an innovative approach that combined unidirectional freeze-drying and electrospinning. During this process, electrospun polycaprolactone (PCL) nanofibers were chopped into nanofibrils, which range in size up to several hundred micrometers, and were incorporated into the chitosan scaffolds via unidirectional freeze-drying. In these scaffolds, the chitosan phase was responsible for maintaining the structural integrity at the macroscale, while the embedded nanofibers enhanced the surface topography at the microscale. The resulting scaffolds exhibited a high porosity of 90% and an impressive water uptake capacity of 2500%. Furthermore, 3T3 fibroblast cells showed strong interactions with the scaffolds, characterized by high rates of cell proliferation and viability. The cells also displayed significant orientation along the direction of the pores, suggesting that the scaffolds effectively guided cellular growth.

3D Melt-Extrusion Printing of Medium Chain Length Polyhydroxyalkanoates and Their Application as Antibiotic-Free Antibacterial Scaffolds for Bone Regeneration.

In this work, we investigated, for the first time, the possibility of developing scaffolds for bone tissue engineering through three-dimensional (3D) melt-extrusion printing of medium chain length polyhydroxyalkanoate (mcl-PHA) (i.e., poly(3-hydroxyoctanoate-co-hydroxydecanoate-co-hydroxydodecanoate), P(3HO-co-3HD-co-3HDD)). The process parameters were successfully optimized to produce well-defined and reproducible 3D P(3HO-co-3HD-co-3HDD) scaffolds, showing high cell viability (100%) toward both undifferentiated and differentiated MC3T3-E1 cells. To introduce antibacterial features in the developed scaffolds, two strategies were investigated. For the first strategy, P(3HO-co-3HD-co-3HDD) was combined with PHAs containing thioester groups in their side chains (i.e., PHACOS), inherently antibacterial PHAs. The 3D blend scaffolds were able to induce a 70% reduction of Staphylococcus aureus 6538P cells by direct contact testing, confirming their antibacterial properties. Additionally, the scaffolds were able to support the growth of MC3T3-E1 cells, showing the potential for bone regeneration. For the second strategy, composite materials were produced by the combination of P(3HO-co-3HD-co-HDD) with a novel antibacterial hydroxyapatite doped with selenium and strontium ions (Se-Sr-HA). The composite material with 10 wt % Se-Sr-HA as a filler showed high antibacterial activity against both Gram-positive (S. aureus 6538P) and Gram-negative bacteria (Escherichia coli 8739), through a dual mechanism: by direct contact (inducing 80% reduction of both bacterial strains) and through the release of active ions (leading to a 54% bacterial cell count reduction for S. aureus 6538P and 30% for E. coli 8739 after 24 h). Moreover, the composite scaffolds showed high viability of MC3T3-E1 cells through both indirect and direct testing, showing promising results for their application in bone tissue engineering.

FFAR-mediated signaling drives migration of pancreatic cancer cells in hypoxic fibroblast co-cultures.

The tumor microenvironment (TME) comprises cancer and non-cancerous stromal cells, including fibroblasts. Free fatty acids (FFAs) regulate various biological responses by binding to G protein-coupled FFA receptors (FFARs). In this study, we examined the impact of FFAR1 and FFAR4 on the cell migration of pancreatic cancer PANC-1 cells co-cultured with 3T3 fibroblast cells under hypoxic conditions. PANC-1 cells cultured at 1 % O2 exhibited elevated FFAR1 expression and decreased FFAR4 expression compared to those at 21 % O2. Cell migration of PANC-1 cells was reduced under 1 % O2 conditions. FFAR1 knockdown enhanced PANC-1 cell migration, whereas FFAR4 knockdown inhibited it. Co-culture of PANC-1 cells with 3T3 cells at 1 % O2 significantly increased FFAR4 expression, while FFAR1 expression remained unchanged. To evaluate the effects of FFAR1 and FFAR4 on PANC-1 cell migration in co-culture with 3T3 cells, we conducted a wound healing assay using the Culture-Insert 2 Well. PANC-1 and 3T3 cells were individually seeded into the two wells and incubated at both 21 % and 1 % O2 for 13 h. The cell migration of PANC-1 cells co-cultured with 3T3 cells at 1 % O2 was notably higher compared to 21 % O2. TUG-770 reduced and TUG-891 enhanced the cell migration of PANC-1 cells co-cultured with 3T3 cells under both 21 % and 1 % O2 conditions. These findings suggest that FFAR1 and FFAR4 play important roles in regulating the cell migration of PANC-1 cells co-cultured with 3T3 cells under hypoxic conditions.

Cytocompatibility, cell-material interaction, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts in contact with engineered Mg/PLA composites.

Bioabsorbable Mg wire-reinforced poly-lactic acid (PLA) matrix composites are potential candidate for load-bearing orthopedic implants offering tailorable mechanical and degradation properties by stacking sequence, volume fraction and surface modification of Mg wires. In this study, we investigated the cytocompatibility, cell-material interaction, and bone differentiation behavior of MC3T3-E1 pre-osteoblast cells for medical-grade PLA, Mg/PLA, and PEO-Mg/PLA (having PEO surface modification on Mg wires) composites. MTT and live/dead assay showed excellent biocompatibility of both composites while cell-material interaction analysis revealed that cells were able to adhere and proliferate on the surface of composites. Cells on the longitudinal surface of composites showed a high and uniform cell density while those on transversal surfaces initially avoided Mg regions but later migrated back after the formation of the passivation layer. Bone differentiation tests showed that cells in extracts of PLA and composites were able to initiate the differentiation process as osteogenesis-related gene expressions, alkaline phosphatase protein quantity, and calcium mineralization increased after 7 and 14 days of culture. Interestingly, the bone differentiation response of PEO-Mg/PLA composite was found to be similar to medical-grade PLA, proving its superiority over Mg/PLA composite.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

Disaccharide-assisted inkjet freezing for improved cell viability.

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.

A promising eco-sustainable wound dressing based on cellulose extracted from Spartium junceum L. and impregnated with Glycyrrhiza glabra L extract: Design, production and biological properties.

Glycyrrhiza glabra extract is widely known for its antioxidant and anti-inflammatory properties and can improve the wound healing process. The aim of this work was to shorten the time of the healing process by using an eco-sustainable wound dressing based on Spanish broom flexible cellulosic fabric by impregnation with G. glabra extract-loaded ethosomes. Chemical analysis of G. glabra extract was performed by LC-DAD-MS/MS and its encapsulation into ethosomes was obtained using the ethanol injection method. Lipid vesicles were characterized in terms of size, polydispersity index, entrapment efficiency, zeta potential, and stability. In vitro release studies, biocompatibility, and scratch test on 3T3 fibroblasts were performed. Moreover, the structure of Spanish broom dressing and its ability to absorb wound exudate was characterized by Synchrotron X-ray phase contrast microtomography (SR-PCmicroCT). Ethosomes showed a good entrapment efficiency, nanometric size, good stability over time and a slow release of polyphenols compared to the free extract, and were not cytotoxic. Lastly, the results revealed that Spanish broom wound dressing loaded with G. glabra ethosomes is able to accelerate wound closure by reducing wound healing time. To sum up, Spanish broom wound dressing could be a potential new green tool for biomedical applications.