Initial Cell Viability Research Articles

The immobilization of a cyclo-trimer of acetonitrile onto poly (lactic acid) (PLA) surface: Improvement of attachment and differentiation of osteoblast-like cells

Poly lactic acid (PLA) has been commercialized as guided bone regeneration membranes. However, PLA membranes are associated with their inherent shortcomings such as insufficient osteoinductive ability. Herein, a cyclo-trimer of acetonitrile (CTA) was covalently immobilized onto PLA films, which would combine the bioactive functions of CTA and good degradability of PLA. The immobilization of CTA enhanced the initial attachment, cell viability, and osteogenic differentiation of osteoblast-like cells. The BMP-SMAD signaling pathway may be involved in the enhancement of osteoinductive capacity of CTA immobilized PLA films. Overall, PLA-CTA films shows a potential for guided bone regeneration.

Bilayer regenerated cellulose/quaternized chitosan-hyaluronic acid/collagen electrospun scaffold for potential wound healing applications

In this study, a bilayer electrospun scaffold has been prepared using regenerated cellulose (RC)/quaternized chitosan (CS) as the primary layer and collagen/hyaluronic acid (HA) as the second layer. An approximate 48 mol% substituted (estimated from 1H NMR) quaternized CS was used in this study. Both layers were crosslinked with EDC/NHS, reflecting an increase in UTS (2.29 MPa for the bilayer scaffold compared to 1.82 MPa for the RC scaffold). Initial cell viability, cell adhesion and proliferation, FDA staining for live cells, and hydroxyproline release rate from cells were evaluated with L929 mouse fibroblast cells. Also, detailed in vitro studies were performed using HADF cells, which include MTT Assay, Live/Dead imaging, DAPI staining, gene expression of PDGF, VEGF-A, and COL1 in RT-PCR, and cell cycle analysis. The collagen/HA-based bilayer scaffold depicted a 9.76-fold increase of VEGF-A compared to a 2.1-fold increase for the RC scaffold, indicating angiogenesis and vascularization potential. In vitro scratch assay was performed to observe the migration of cells in simulated wounds. Antimicrobial, antioxidant, and protease inhibitory activity were further performed, and overall, the primary results highlighted the potential usage of bilayer scaffold in wound healing applications.

One Year Follow-up on the First Patient Treated with Nula-Cel: An Autologous CRISPR/Cas9 Gene Corrected CD34+ Cell Product to Treat Sickle Cell Disease

Introduction Sickle cell disease (SCD) is the most common serious genetic disease in the world with over 500,000 births per year globally. It is estimated that there are >100,000 patients in the US with SCD and at least 20% have severe disease. Patients with SCD have at least one HBB gene with an adenine to thymidine variant at position 6 of the coding region resulting in a glutamic acid to valine amino acid change ( HbS). The success of allogeneic hematopoietic stem cell transplantation demonstrates that reconstitution of the hematopoietic compartment with HSCs that contain at least one non- HbS allele can cure the disease. Homology directed repair gene editing (HDR-editing) has achieved high frequencies of conversion of the thymidine to the non-pathologic adenine in CD34+ hematopoietic stem and progenitor cells from patients with SCD. Nulabeglogene autogedtemcel (nula-cel) is an investigational drug product in which HDR-editing of plerixafor-mobilized CD34+ HSPCs corrects the underlying variant that causes SCD. This is the first drug to directly correct a disease-causing variant. We report on the 1 year follow up of the first and only patient to receive nula-cel. Results Patient 1 is now a 23 year old female with homozygous SCD. She was consented and enrolled on a Phase I//II trial to test the safety and efficacy of nula-cel. In the 2 years prior to enrollment, the patient averaged 6 VOC's and 4 hospitalizations per year. Two aphereses of plerixafor-mobilized CD34+ cells were performed followed by fresh CD34+ cell purification. The purified CD34+ cells were pooled and cryopreserved. A 5-day manufacturing process starting with 9.3 x 10 6 cells/kg of cryopreserved CD34+ cells resulted in a yield of 8.75 x 10 6 CD34/kg with an on-target allele correction frequency of 33%. In August, 2022 the thawed drug product was infused after the patient received AUC-adjusted busulfan myeloablative conditioning chemotherapy. The initial cell viability was 77% giving a viable infused cell dose of 6.74 x 10 6 CD34/kg. Follow-up exploratory apoptois studies, however, showed the viable CD34+cell dose may have been as low as 3.5 x 10 6 CD34+/kg. Neutrophil engraftment occurred at transplant Day +40. Because of the lack of platelet recovery and continued platelet and RBC transfusion requirements, the patient was started on eltrombopag on day +106 (marrow cellularity of 5%). Since eltrombopag usage was not part of the protocol, its use prompted the reporting of an SAE but the trial was never put on FDA hold. The last dose of GCSF was D+140, the last platelet transfusion was D+181 and the last RBC transfusion was D+263. With rising platelet counts and hemoglobin and a stable ANC >1500, eltrombopag was discontinued on D+322. The blood counts have continued to slowly increase after discontinuation of the eltrombopag. On D+307, with a Hgb of 8.5 g/dl, hemoglobin electrophoresis demonstrated HgbA=12.5%, HgbF >78% and HgbS=4.5%. On D+349 the blood tests show Hgb=9.1 g/dl, Platelet=77,000/ul, ANC=1788/ul with an absolute reticulocyte count of 119,130/ul. There are no signs or symptoms of hemolysis. Table 1 shows the time course of gene marking with stable allele gene correction and INDEL frequencies since D+49. No change in off-target INDEL frequency nor change in on-target INDEL spectrum has occurred and no evidence of oligoclonal or clonal hematopoiesis has been observed. Clinically, the patient has shown marked improvement in quality of life with zero VOE's or other manifestations of SCD. The patient still has not reached a steady state and updated results will be presented. Conclusion We describe the clinical results of the first patient treated with an autologous cell product (nula-cel) in which the pathologic variant was directly corrected. The mechanism of action is fundamentally different than other clinical gene editing and gene therapy programs as the level of the pathologic HgbS is directly decreased thereby removing the dominant negative effect that HgbS has on red blood cells. The high HgbF is an unexpected but potentially effective mechanism of action for the clinical benefit and is being further studied. Longer follow up to assess the durability of the curative clinical impact is underway. Improvements in cell manufacturing are being implemented to shorten the duration of transfusion needs and increase the level of gene correction of engrafted cells. Plans to treat the next patients with an improved nula-cel product are in place.

Investigation of TiO2 nanoparticle interactions in the fibroblast NIH-3T3 cells via liquid-mode atomic force microscope.

Long before we recognized how significant they were, nanoparticles were already all around in the environment. Since then, an extensive number of synthetic nanoparticles have been engineered to improve our quality of life through rigorous scientific research on their uses in practically every industry, including semiconductor devices, food, medicine, and agriculture. The extensive usage of nanoparticles in commodities that come into proximity with human skin and internal organs through medicine has raised significant concerns over the years. TiO2 nanoparticles (NPs) are widely employed in a wide range of industries, such as cosmetics and food packaging. The interaction and internalization of TiO2 NPs in living cells have been studied by the scientific community for many years. In the present study, we investigated the cell viability, nanomechanical characteristics, and fluorescence response of NIH-3T3 cells treated with sterile DMEM TiO2 nanoparticle solution using a liquid-mode atomic force microscope and a fluorescence microscope. Two different sorts of response systems have been observed in the cells depending on the size of the NPs. TiO2 nanoparticles smaller than 100nm support its initial stages cell viability, and cells internalize and metabolize NPs. In contrast, bigger TiO2 NPs (> 100nm) are not completely metabolized and cannot impair cell survival. Furthermore, bigger NPs above 100nm could not be digested by the cells, therefore hindering cell development, whereas below 100nm TiO2 stimulated uncontrolled cell growth akin to cancerous type cells. The cytoskeleton softens as a result of particle internalization, as seen by the nanomechanical characteristics of the nanoparticletreated cells. According to our investigations, TiO2 smaller than 100nm facilitates unintended cancer cell proliferation, whereas larger NPs ultimately suppress cell growth. Before being incorporated into commercial products, similar effects or repercussions that could result from employing different NPs should be carefully examined.

Assessment of Biocompatibility of 3D‐Printed Cardiovascular Stent

AbstractThis study aims to evaluate the biocompatibility of the 3D‐printed stent and its efficiency on reducing the oxidized‐low‐density lipoprotein (ox‐LDL) damage in endothelial cells using the zebrafish embryos model. The human umbilical vein endothelial cells (HUVECs) significantly (p‐value <0.001) lose their viability upon exposure to ox‐LDL treatment (100 µg/ml). An exposure of HUVECs to resveratrol (RSL)‐unloaded 3D stent slightly lowers the cell viability as compared with untreated control group. On the contrary, RSL‐loaded 3D stent exposure groups maintain initial cell viability. The amounts of TNF‐α and IL‐β released from zebrafish embryos (1.82 ± 0.75 and 1.87 ± 0.24) in the group treated with the RSL‐loaded 3D stents are significantly lower (p < 0.001) than those from the LPS alone treated group (8.13 ± 2.29 and 23.79 ± 3.82, respectively). It is found that RSL‐loaded 3D stent at a RSL dose of 1 mm shows no mortality during the developmental stages, but RSL‐loaded 3D stent at a dose of 2 mm shows a lowered survival rate (93.3 ± 3.3%), shorter length of larvae, pericardial and yolk sac edemas of 53.3 ± 23.3% and 3.3 ± 3.3%, respectively. The RSL‐loaded 3D stents efficiently downregulate the proinflammatory cytokines, and protect an organism against oxidative stress, while producing minimal developments defects in zebrafish embryos.

Colchicine as a novel drug for the treatment of osteosarcoma through drug repositioning based on an FDA drug library.

BackgroundColchicine is a traditional medication that is currently approved to treat gout and familial Mediterranean fever (FMF). However, colchicine has a wide range of anti-inflammatory activities, and several studies have indicated that it may be useful in a variety of other conditions, such as rheumatic disease, cardiac disease, and cancer. Osteosarcoma, the most common type of bone sarcoma, is derived from primitive bone-forming mesenchymal cells. In this study, we investigated whether colchicine could be used to treat osteosarcoma through the regulation of cell cycle signaling.MethodsTwo human osteosarcoma cell lines, U2OS and Saos-2, were used. A clonogenic assay was used to determine the antiproliferative effects of colchicine on osteosarcoma cells. Reactive oxygen species (ROS) production and apoptosis were measured by flow cytometry. Migration and invasion assays were performed to investigate the inhibitory effects of colchicine. The signaling pathways related to colchicine treatment were verified by GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.ResultsColchicine was selected as the lead compound based on the results of initial screening and cell viability assays conducted in Saos-2 and U2Os cells. Colchicine reduced the viability of Saos-2 and U2OS cells in a concentration-dependent manner. It also significantly inhibited colony-forming ability and induced ROS production and apoptosis. It also inhibited the migration and invasion of both Saos-2 and U2OS cells. GOBP and KEGG enrichment analyses indicated the involvement of microtubule-based processes and cancer-related pathways.ConclusionsThese findings suggest that colchicine has therapeutic potential in osteosarcoma.

What Happens on the Back Table? Viability and Osteogenic Potential of Reamed Autogenous Bone Graft as a Function of Time and Temperature-A Pilot Study.

To provide guidelines for reamed autogenous bone graft (R-ABG) in nonunion surgery to best manage modifiable intraoperative factors, like storage temperature and time, for optimal graft viability. Our hypothesis was that R-ABG graft will show decreased cell viability as a function of increased time and storage temperature. R-ABG samples were obtained using an RIA system from 10 patients undergoing harvest for the treatment of a nonunion. A 10 cm3 sample of cancellous bone graft was divided into 10 samples. A control sample was processed immediately and analyzed using fluorescence activated cell sorting to determine its cell viability. The remaining samples were stored at 0, 15, and 37°C and processed similarly after 60 and 120 minutes. A paired t test was used to compare the mean change in percent viability. The mean percent cell viability for the experimental conditions was not significantly different from the initial percent cell viability (P ≥ 0.08). There were no significant differences in the change in mean percentage of viable cells (P ≥ 0.07). After 60 and 120 minutes of storage, there were no significant differences in mean cell viability based on storage temperature (P ≥ 0.53 and P ≥ 0.68, respectively). A power analysis estimated that a sample size of 52 patients would be needed to detect a difference. The results of our pilot study indicate that storage conditions may have less effect on cell viability than previously hypothesized. Further research with more patients is needed to confirm these preliminary results. Level II.

Combinations of photoinitiator and UV absorber for cell-based digital light processing (DLP) bioprinting

Digital light processing (DLP) bioprinting, which provides predominant speed, resolution, and adaptability for fabricating complex cell-laden three-dimensional (3D) structures, requires a combination of photoinitiator (PI) and UV absorber (UA) that plays critical roles during the photo-polymerization of bioinks. However, the PI and UA combination has not been highlighted for cell-based DLP bioprinting. In this study, the most used PIs and UAs in cell-based bioprinting were compared to optimize a combination that can ensure the maximum DLP printability, while maintaining the cellular activities during the process. The crosslinking time and printability of PIs were assessed, which are critical in minimizing the cell damage by the UV exposure during the fabrication process. On the other hand, the UAs were evaluated based on their ability to prevent the over-curing of layers beyond the focal layer and the scattering of light, which are required for the desirable crosslinking of a hydrogel and high resolution (25–50 µms) to create a complex 3D cell-laden construct. Lastly, the cytotoxicity of PIs and UAs was assessed by measuring the cellular activity of 2D cultured and 3D bioprinted cells. The optimized PI and UA combination provided high initial cell viability (>90%) for up to 14 days in culture and could fabricate complex 3D structures like a perfusable heart-shaped construct with open vesicles and atriums. This combination can provide a potential starting condition when preparing the bioink for the cell-based DLP bioprinting in tissue engineering applications.

The additive effect of iloprost on the biological properties of Mineral trioxide aggregate on mesenchymal stem cells

Background/purposeIloprost has been proposed as a potential biomaterial owing to angiogenic and odontogenic properties. However, the liquid form can limit its use during clinical applications. Mineral trioxide aggregate (MTA) has been used for various dental applications in which cell–material interaction is essential. This study aimed to investigate additive effects of iloprost on the biological properties of MTA on the viability, attachment, migration and differentiation of human mesenchymal stem cells (hMSCs). Materials and methodsStandardized human dentin disks were prepared. MTA was prepared by mixing distilled water or iloprost solution, and the lumen of the disks was filled with MTA or MTA-iloprost. hMSCs on disk alone and hMSCs on culture plates were used as controls. Cell viability and attachment were measured after 1, 7 and 14 days using AlamarBlue assay and scanning electron microscopy (SEM). Cell migration in MTA or MTA-iloprost extracts was determined using a wound-healing model.Osteogenic differentiation was evaluated by real-time reverse transcriptase polymerase chain reaction for alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OSP) gene expressions after 7 and 14 days of osteogenic induction. ResultsCells on MTA-iloprost surface showed similar viability with MTA at 1 and 14 days but enhanced cellular viability and cell spreading compared to MTA at 7 days (p < 0.05). Cell migration was similar by MTA-iloprost and MTA extracts (p > 0.05). MTAiloprost significantly upregulated BSP, OCN and OSP expressions compared to MTA (p < 0.05). ConclusionThe addition of iloprost to MTA improved the initial cell viability and osteogenic potential of hMSCs.

Preparation of Gelatin/Polycaprolactone Electrospun Fibers Loaded with Cis-Platinum and Their Potential Application for the Treatment of Prostate Cancer

Although the development of nanomaterials for cancer therapy has received much attention in recent years, prostate cancer still remains one of the most troubling cancers in males. In this work, biomimetic polycaprolactone (PCL)/gelatin (GT)/cis-platinum (CDDP) (PG@CDDP) fibrous films were developed via electrospinning technology. The microstructure and chemical properties of PG@CDDP fibers are investigated. It is found that the microscopic morphology and diameter of PG@CDDP fibers are changed compared to those of the PG fibers. The Fourier transform-infrared spectroscopy and wide-angle X-ray diffraction demonstrate that CDDP is successfully incorporated into the fibers. The human prostate cancer cells in vitro to electrospun films (PG and PG@CDDP) were evaluated based on initial cell response and cell viability. The density, elongation, and viability of adhered cancer cells significantly reduce with an increased concentration of CDDP in PG fibers. Thus, the developed PG@CDDP fiber matrix has great potential as candidate scaffolds for the treatment of prostate cancer.

Assessment of albumin and aspartate levels as a simple indicator of the efficacy of cryopreservation

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; The process of hepatocytes cryopreservation is standardised by most of the laboratories. However there is a variation with respect to the Protocols, media and equipments used amongst the laboratories. Similarly, the tests available to evaluate the efficacy also varies. They are expensive and sometimes might not measure the parameter required for a particular research study. Hence we propose a methodology to study the few basic parameters like cell viability, synthetic function of the cell and cell stability. We have also used a simple percentile calculation to know the efficacy of cryopreservation. This shall help in functional validation of the cell after cryopreservation. The same can also be used to compare the quality of hepatocytes between different batches. The objective of the study was to characterisation of the cells to determine the efficacy of cryopreservation.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Two step collagen isolation method was used to isolate the hepatocytes. Initial cell viability was calculated. A sample of cells were taken for characterisation and the remaining cells cryopreserved. The sample cells were divided into two batches one for pre cryopreservation culture and the other for post cryopreservation. The pre cryopreservation culture was done on monolayer using collagen coated 6 well plate. The other sample was placed in the cryovials for cryopreservation for 1week. After 1 week the cryopreserved cells were thawed and the post cryopreservation viability calculated, followed by post cryopreservation culture. During the process of culture (both pre cryopreservation and post cryopreservation) for 5days Albumin was measured daily and average calculated, peak Aspartate (AST) at 24 hours was recorded. The percentile difference of the obtained values between the pre cryopreservation and post cryopreservation culture was calculated.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; A total of 12 specimen were enrolled for the study. The mean pre cryopreservation viability of the cells was 66.58%. The post cryopreservation, viability of the cells was 36.43%. The mean difference was -30.170%. The pre cryopreservation albumin values had a mean of 150ng/ml. The post cryopreservation albumin values had a mean of 135.83ng/ml. The mean difference was -14.170ng/ml. The pre cryopreservation peak Aspartate values had a mean of 234.17 IU/ml. The post cryopreservation peak aspartate values had a mean of 230 IU/ml. The mean difference was -4.176 IU/ml.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; This simple method can validate the cells after cryopreservation by measurement of cell viability, synthetic function of the cell and cell stability.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Functionalized peptide hydrogels as tunable extracellular matrix mimics for biological applications

AbstractThe development of tailorable and biocompatible three‐dimensional (3D) substrates or molecular networks that reliably mimic the extracellular matrix (ECM) and influence cell behavior and growth in vitro is of increasing interest for cell‐based applications in the field of tissue engineering and regenerative medicine. In this context, we present a novel coiled coil‐based peptide that self‐assembles into a 3D‐α‐helical fibril network and functions as a self‐supporting hydrogel. By functionalizing distinct coiled‐coil peptides with cellular binding motifs or carbohydrate ligands (mannose), and by utilizing the multivalency and modularity of coiled‐coil assemblies, tailored artificial ECMs are obtained. Fibrillar network and ligand density, as well as ligand composition can readily be adjusted by changes in water content or peptide concentrations, respectively. Mesoscopic structure of these networks was assessed by rheology and small‐angle neutron scattering experiments. Initial cell viability studies using NIH/3T3 cells showed comparable or even superior cell viability using the presented artificial ECMs, compared to commercially available 3D‐cell culture scaffold Matrigel. The herein reported approach presents a reliable (low batch‐to‐batch variation) and modular pathway toward biocompatible and tailored artificial ECMs.

Copper-doped 3D porous coating developed on Ti-6Al-4V alloys and its in vitro long-term antibacterial ability

In this study, 3D porous anodic coatings containing 1.92 wt% copper (Cu-1.92 wt%) are developed on Ti-6Al-4V alloys via micro-arc oxidation (MAO). The in vitro long-term antibacterial activity of the MAO-treated samples is systematically evaluated. The results show that after 14 days’ immersion in Hanks’ solution, the antibacterial rate of Cu-1.92 wt% against Staphylococcus aureus (S. aureus) remains at 100%, while that against Escherichia coli (E. coli) decreases to 92% and 12% after 8 and 14 days, respectively, suggesting that Cu-1.92 wt% still exhibits good long-term antibacterial properties even after 14 days. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis and reactive oxygen species (ROS) fluorescence imaging reveal that the antibacterial efficiency of Cu-1.92 wt% is higher than those of Cu-1.92 wt%-8D, Cu-1.92 wt%-14D, and the untreated control. The enhanced antibacterial properties of the MAO samples are attributed to ROS-mediated DNA damage prompted by the presence of Cu in the coatings. In addition, Cu-1.92 wt% favors good initial cell adhesion and high cell viability. These results reveal that the fabricated biocompatible copper-doped 3D porous coatings exhibit good in vitro long-term antibacterial properties and have potential to be applied in the orthopaedics clinic in the future.

Collagen/bioceramic-based composite bioink to fabricate a porous 3D hASCs-laden structure for bone tissue regeneration

To successfully achieve the porous cell-blocks, a bioink is a prerequisite requirement. However, although various hydrogel-based bioinks have been applied, a hydrogel/bioceramic-based composite bioink consisting of cells has not been actively investigated owing to its poor printability and low initial cell-viability. In this study, a new bioink consisting of fibrillated collagen, cells, and bioceramic (β-TCP) is suggested to attain a 3D porous cell-laden composite structure with high cellular responses, in aspects of initial cell viability, proliferation, and differentiation using preosteoblasts (MC3T3-E1) and human adipose stem cells (hASCs). By manipulating the processing conditions and weight fractions of the ceramic in the bioink, a 3D porous cell-laden composite structure can be fabricated successfully. The cell-laden composite structure revealed that the printed structure was mechanically stable, the laden cells were satisfactorily viable, and even cell proliferation/differentiation was well performed. Moreover, the cells in the composite structure exhibited significant osteogenic activities compared to the pure collagen bioink (control), and higher levels of osteogenic gene expression of the hASC-laden composite structure were observed without using an osteogenic medium than those of the control using an osteogenic medium, indicating that the laden β-TCP triggered osteogenic differentiation of the hASCs.

Collagen Coating Effects on Fe-Mn Bioresorbable Alloys.

Bioresorbable iron-manganese alloys (Fe-30%Mn) are considered as one of the next-generation resorbable materials for orthopedic applications. Previous in vitro study showed that Fe30Mn scaffolds with 10% porosity displayed strong mechanical properties and adequate degradation rate without severe cytotoxicity effect. However, the cellular compatibility of these alloys in terms of cell-to-cell and alloy-to-cell interactions is not ideal. Collagen is the most abundant protein in human bone, providing structural support beneficial to bone healing. We hypothesized that coating collagen on Fe30Mn can improve osteointegration or activities promoting cell adhesion, migration, and proliferation, as the alloy degrades. After preparing collagen coating on Fe-30Mn via spin coating, we conducted a corrosion test and a direct cytotoxicity test on four Fe30Mn groups: non-porous and 10% porosity, with and without collagen coating. Furthermore, we evaluated and compared the morphologies of cells over a period of 7 days. Results showed that there was no significant difference between the collagen-coated and non-coated groups in corrosion rates, yet a significant decrease from the porous non-coated group to the porous collagen-coated group in cytotoxicity level was found. Cell morphology on the porous non-coated group displayed round shape, whereas that on the porous collagen-coated group displayed flattened spreading. The study showed that the collagen coating significantly increased the initial cell viability and adhesion for both the porous and non-porous groups without impeding their degradation rates. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:523-535, 2020.

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, 1H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440 nm upon excitation around 380 nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by 1H NMR study. The binding constants are calculated to be 5.3 × 107 and 6.8 × 106 M−1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC50 in the order of 0.023 mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.Communicated by Ramaswamy H. Sarma

Bioviability of Mangrove Leaves Extract (Sonneratia alba) on Fibroblast BHK-21 Cell Culture

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Dental caries is a major oral health problem which is most commonly found in Indonesia. One of the main factors in role of causing dental caries is Streptococcus mutans which is the primary target of the prevention of caries. Leaf extract of Sonneratia alba has&lt;br /&gt;been proven to have an antimicrobial effect towards Streptococcus mutans, but its cytotoxic activity on normal cells is still unknown. The initial cell viability in this study was conducted using cell line BHK-21. &lt;strong&gt;Purpose:&lt;/strong&gt; The aim of this study is to determine the cell viability of&lt;br /&gt;fibroblast cells line BHK-21 by given Sonneratia alba’s leaf extract. &lt;strong&gt;Materials and Methods:&lt;/strong&gt; The experiment was conducted using post test only control group design. BHK-21 fibroblast cell culture on 96 wells were divided into cell control group (n=9), media control group (n=9) and test groups (n=9). The test groups were given various concentration of Sonneratia alba’s leaf extract, they were: 5%; 10%; and 20%. The cell cultures were incubated for 24 hours before and after treatment using Sonneratia alba’s leaf extract. After dripping MTT into the microplates, OD were read using ELISA reader and the viability were counted. The data that was acquired from the viability calculation was statistically analyzed using One Way ANOVA and LSD test. &lt;strong&gt;Results:&lt;/strong&gt; Data showed the increase of viability cell in all the test groups. The highest viability cell showed by the test group with 10% concentration of Sonneratia alba (153,7122%±7,42458), and the lowest found in the test group with 20% concentration of leaf extract (141,4682%±7,48752). &lt;strong&gt;Conclusion:&lt;/strong&gt; In this experiment, the leaf extract of Sonneratia alba proved to be nontoxic on fibroblast cells line BHK-21, the extract increased the viability and also caused proliferation of fibroblast cells.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Keywords:&lt;/strong&gt; Dental caries, Sonneratia alba, fibroblast, viability, cell culture&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;br /&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Correspondence:&lt;/strong&gt; Aprilia, Department of Conservation, Faculty of Dentistry, Hang Tuah Univesity, Arif Rahman Hakim 150, Surabaya, Phone 031-5945864, Email: &lt;span style="text-decoration: underline;"&gt;drg.aprilia.spkg@gmail.com&lt;/span&gt;&lt;/p&gt;

Surface stabilization treatment enhances initial cell viability and adhesion for biodegradable zinc alloys

Zinc-based alloys hold promise as next generation biodegradable implants. Although zinc implants exhibit excellent biocompatibility in vivo, they have been found to inhibit cell attachment and viability under in vitro conditions. In order to clarify the cell response disparity and improve the biocompatibility of zinc implant materials, Zn-1Mg and Zn-1Mg-0.5Ca alloys underwent surface stabilization treatment in cell culture medium. Surface processing resulted in a stable surface oxide film of ∼300 nm thickness. The stability of the oxide layer substantially increased the viability of cells in both direct and indirect contact assays. It is consequently believed that the surface film characteristics of zinc implants may be an important determinant of biocompatibility.

Cytocompatible carbon nanotube reinforced polyethylene glycol composite hydrogels for tissue engineering

Hydrogels are attractive materials for stimulating 3D cell growth and tissue regeneration, and they provide mechanical support and physical cues to guide cell behavior. Herein, we developed a robust methodology to increase the stiffness of polyethylene glycol (PEG) hydrogels by successfully incorporating carbon nanotubes (CNTs) within the polymer matrix. Interestingly, hydrogels containing pristine CNTs showed a higher stiffness (1915 ± 102 Pa) than both hydrogels without CNTs (1197 ± 125 Pa) and hydrogels incorporating PEG-grafted CNTs (867 ± 103 Pa) (p < 0.005). The swelling ratio was lower for hydrogels with pristine CNTs (45.4 ± 3.5) and hydrogels without CNTs (46.7 ± 5.1) compared to the hydrogels with PEG-grafted CNTs (62.8 ± 2.6). To confirm that the CNT-reinforced hydrogels were cytocompatible, the viability, proliferation, and morphology of encapsulated L929 fibroblasts was investigated. All hydrogel formulations supported cell proliferation, and the addition of pristine CNTs increased initial cell viability (83.3 ± 10.7%) compared to both pure PEG hydrogels (51.9 ± 8.3%) and hydrogels with PEG-CNTs (63.1 ± 10.9%) (p < 0.005). Altogether, these results demonstrate that incorporation of CNTs could effectively reinforce PEG hydrogels and that the resulting cytocompatible nanocomposites are promising scaffolds for tissue engineering.

ENDOTHELIAL PROGENITOR CELLS ENCAPSULATED IN MATRIX-SUPPLEMENTED MICROGEL IMPROVES CELL RETENTION AND THERAPEUTIC EFFICACY IN PULMONARY ARTERIAL HYPERTENSION

Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary single-cell microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury. We hypothesize that microencapsulation of L-EPC with an agarose hydrogel supplemented with integrin-binding proteins will increase survival and retention of L-EPCs injected into the jugular vein and result in greater therapeutic benefits compared to non-cocooned cells in a rat monocrotaline (MCT) model of PAH. L-EPCs were encapsulated by vortex-emulsion using various concentrations of agarose, together with fibronectin and fibrinogen, and capsule size and initial cell viability were assessed. Encapsulated and non-encapsulated L-EPCs were transduced with luciferase and administered to SD rats three days after injection of MCT. L-EPCs were tracked in vivo by bioluminescence imaging (BLI) to assess cell persistence and bio-distribution for up to three weeks post cell injection. At end-study, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were assessed for therapeutic efficacy. The initial BLI signals at 15 minutes after delivery were similar in non-cocooned and cocooned L-EPCs; however, only cocooned cells could be detected by BLI after four and 24 hours (28±12% and 12±8% of baseline signal, respectively; p< 0.0001 and 0.05, n=11). Microencapsulation of L-EPCs led to significant improvement in RVSP three weeks after delivery compared to MCT alone (56±24 vs. 80±7 mmHg, respectively; p<0.05), whereas no improvement in pulmonary hemodynamics was seen with delivery of non-encapsulated cells. These results demonstrate that single-cell cocooning can significantly increase retention of L-EPCs within the lungs. Furthermore, even a modest increase in L-EPC persistence over 24 hours can provide an important therapeutic benefit, not seen with non-encapsulated L-EPCs in the rat MCT model of PAH.