In recent years, dental implants have become the preferred approach for the restoration of missing teeth. At present, most dental implants are made of pure titanium, and are affected by peri-implantitis and bone resorption, which usually start from the implant neck, due to the complex environment in this region. To address these issues, in this study we modified the surface of titanium (Ti) implants to exploit the antibacterial and osteoinductive effects of single-layer graphene sheets. Chemical vapor deposition (CVD)-grown single-layer graphene sheets were transferred to titanium discs, and a method for improving the adhesion strength of graphene on Ti was developed due to compromised adhesion strength between graphene and titanium surface. A thermal treatment of 2 h at 160 °C was found to enhance the adhesion strength of graphene on Ti to facilitate clinical transformation. Graphene coatings of Ti enhanced cell adhesion and osteogenic differentiation, and imparted antibacterial activity to Ti substrate; these favorable effects were not affected by the thermal treatment. In summary, the present study elucidated the effects of a thermal treatment on the adhesion strength and osteoinductive activity of single-layer graphene sheets on titanium substrates. Since its discovery, graphene has attracted significant attention in various fields, owing to its excellent optical, mechanical, chemical, and electrical properties5. Further extensive studies have started to highlight the unique potential of graphene in the biomedical field, and increasing attention is being paid to its biomedical and biotechnological applications, such as biosensors, drug delivery, and tissue engineering9. Several studies have reported promising effects of graphene and its derivatives on cell adhesion, proliferation, and osteogenic differentiation12. In a previous study, we also confirmed that monolayer graphene could promote the osteogenic differentiation of human adipose-derived stem cells (hASCs) and human bone marrow-derived mesenchymal stem cells (hBMMSCs) in vitro and in vivo, and explored the epigenetic role of graphene in the fate of stem cells13. Moreover, the antibacterial effects of graphene have also been documented16.Graphene, first isolated by Novoselov and Geim in 2004, has a unique two-dimensional (2D) hexagonal structure made of sp18. However, the neck of a dental implant is the weakest area where infection and bone resorption begins. It is a challenged area because it emerges from the bone to the gingiva, and is in contact with a complex bacterial environment of the oral cavity. Therefore, bone integration, gingival attachment and anti-bacteria ability should be realized at the same time. However, better adhesion of osteoblasts, mesenchymal stem cells, or gingival fibroblasts, and less adhesion of bacteria is always contradicted19. It has been well recognized that rough surfaces are better for the cell adhesion24, but on the other hand facilitate the adhesion of bacteria25. On the other hand, smooth surfaces reduce the adhesion of bacteria25, but interfere the attachment of gingival fibroblasts and osteoblasts thus negatively influencing gingival attachment and osteogenesis26. Graphene appears as a possible solution to this contradiction, owing to its potential osteogenic and anti-bacterial ability. Single-layer graphene can be successfully produced in large scale by chemical vapor deposition (CVD) on metal, such as copper and nickel28. However, titanium is the most used material as medical implants30. The modification of the titanium surface with monolayer graphene can be solved by transferration using polymethyl methacrylate (PMMA)33. In our previous study, we found that the adhesion strength of graphene on the Ti surface is not satisfied enough for future clinical application13. Therefore, it is urgent to find a solution to improve the adhesion strength between graphene and titanium.Nowadays, dental implants have been widely used and largely improved the life quality of billions of patients who suffered from missing teethin vitro and in vivo cell adhesion, proliferation, and osteogenic differentiation. In addition, the antibacterial effects of graphene  were also assessed. The incorporation of single-layer graphene is expected to impart a stable antibacterial and osteoinductive properties to the titanium surface.Thermal treatment is the last step of the transferring process to remove PMMA, and we found that prolonged time and higher temperature of thermal treatment improve the adhesion between graphene and Ti substrate. Therefore, in this study, we attempted to improve the adhesion strength of graphene on the surface of a Ti substrate through a thermal treatment. We investigated the effects of the graphene coating  on the The AFM images in Fig. −1 and a sharp and symmetric 2D band at ~2680 cm−1. And this is a typical spectrum for single-layer graphene distinguished from multilayer graphene. Therefore, we can conclude that single-layer graphene was successfully transferred to the Ti surface.Raman spectroscopy Fig.  demonstrMeanwhile, the water contact angle and surface energy Fig.  demonstrDry heating treatment of 80, 100, 160, and 200 °C for 2 h was performed on graphene-coated Ti samples after the mediator-assisted transfer. Scratching tests with the tip of an ultrasonic scaler were performed to evaluate the adhesion of graphene on Ti substrates. Graphene sheets on Ti substrates after thermal treatment of 160 and 200 °C for 2 h remained intact after the scratching, while wrinkles or breakages were observed on graphene sheets after thermal treatment of 80 and 100 °C for 2 h Fig. .Figure 2Furthermore, we performed Raman spectroscopy of several points on each sample . By summarizing the results of these Raman spectroscopy examinations, we found that thermal treatment of 80, 100, 160, and 200 °C for 2 h did not influence the integrity of graphene sheets on Ti substrates, since the typical spectra of single-layer graphene could be observed on the area without scratches. On the other hand, the typical spectra of single-layer graphene still could be observed in the scratched areas of graphene coatings after thermal treatment of 160 °C and 200 °C Fig. , whereasOn the basis of these results, dry heating treatment at 160 °C for 2 h, a lower temperature than 200 °C, was identified as the optimal condition to achieve stronger adhesion between graphene and Ti substrates. Therefore, the following three groups were used in the subsequent experiments: titanium without graphene coating (No-Graphene); graphene-coated titanium (Graphene); graphene-coated titanium subjected to dry heating treatment at 160 °C for 2 h (Graphene-DH). As for surface characterization, there were no significant differences between the Graphene and Graphene-DH groups in roughness, water contact angle, and surface energy, indicating thermal treatment didn’t influence the physical characteristics of graphene Fig. .SEM measurements and confocal microscopy images of FITC-phalloidin staining were used to characterize the morphology and fine structures of the adhered cells. After 12 h of culture, human gingival fibroblasts (hGFs), human adipose-derived stem cells (hASCs), and human bone marrow mesenchymal stem cells (hBMMSCs) on the Graphene and Graphene-DH samples presented obvious lamellipodia, while the pseudopodia of cells on the surfaces of uncoated Ti were relatively short and less evident Fig. . MeanwhiThe CCK-8 cell proliferation tests demonstrated similar logarithmic proliferation curves of hGFs, hASCs, and hBMMSCs on Graphene, Graphene-DH and No-Graphene surfaces Fig. .P < 0.05) on the Graphene and Graphene-DH surfaces compared with the No-Graphene surfaces, whereas no significant differences could be observed between Graphene and Graphene-DH surfaces.Figure P < 0.05), and there were also no significant differences between Graphene and Graphene-DH groups  assays showed that, after 14 or 21 days of osteoinduction (OI), Graphene and Graphene-DH groups demonstrated stronger mineralization staining than No-Graphene for both hASCs and hBMMSCs, and there were no significant differences between Graphene and Graphene-DH groups Fig. . On the ups Fig. .RUNX2, osteocalcin (OCN), and Osterix (OSX), for both hASCs and hBMMSCs, were higher for Graphene and Graphene-DH groups than No-Graphene group (P < 0.05). Meanwhile, no significant differences were found between the Graphene and Graphene-DH groups. Moreover, there were no significant differences among the three groups when the cells were cultured without osteoinduction , whereas no significant differences were observed between the Graphene and Graphene-DH samples  on No-Graphene surfaces than on Graphene and Graphene-DH surfaces, whereas no significant differences were observed between Graphene and Graphene-DH samples Fig. . MeanwhiFour weeks after implantation, ectopic bone formation was evaluated by HE and toluidine blue staining. The bone matrix appeared as uniform acidophilic tissue after HE staining and had a dark blue appearance under toluidine blue staining. As shown in Fig. Increased ectopic bone formation was observed for all groups at eight weeks after implantation, compared with that at four weeks, for both hASCs and hBMMSCs. The relative trends were the same as those observed after four weeks: the Graphene and Graphene-DH groups showed increased ectopic bone formation compared with No-Graphene Fig. .32. In this work, a mediator (PMMA)-assisted transfer technique was used to transfer the single-layer graphene on the surface of smooth titanium discs. Thermal treatment is the last step of the transferring process to remove PMMA by acetone steam bath for 30 min at 57 °C. In our previous research, we found that the adhesion strength between graphene and Ti was not strong enough for future clinical application. But interestingly, we found that prolonged time of thermal treatment improve the adhesion between graphene and Ti substrate. And this is where we got our enlightment to use thermal treatment to improve the adhesion strength between graphene and Ti.Single-layer graphene can now be successfully produced in large scale by chemical vapor deposition on copper foil34.The possible mechanism of enhanced adhesion strength between graphene and Ti after thermal treatment may result from further removal of residual PMMA and acetone, thus reducing the intervals between the graphene coatings and their underlying substrates. Uneven surfaces, such as wrinkles and ripples, are inevitable after transfer of single-layer graphene to other substrates. These areas are vulnerable due to higher density of intervals between the graphene coatings and the substrates, where microcracks will start and extend under the existence of external forcesAs for the settings of temperatures for thermal treatment, we chose 80, 100, 160, and 200 °C, all higher than 56.53 °C (the boiling point of acetone), to remove the potential residual acetone. Meanwhile, the glass transition temperature of PMMA is around 150 °C. Therefore, we speculated that a thermal treatment with higher temperature than 150 °C might further remove the residual PMMA between graphene and titanium substrate, thus reducing the intervals between graphene coatings and Ti substrates. This is why 160 and 200 °C groups were chosen. By reducing residual acetone and PMMA by thermal treatment, a closer contact between graphene and the surface of titanium would be realized.35. Moreover, the thermal conductivity of graphene did not show significant changes between −173 and 127 °C36. Furthermore, Mounet et al. showed that the expansion coefficient of graphene remained substantially unchanged in the range of −23.15 to 127 °C37. The above studies demonstrated that the heat treatment of around 160 °C had no significant effects on the thermal conductivity and expansion coefficient of graphene. In this study, the AFM analysis of surface roughness and the water contact angle measurements demonstrated no differences between the Graphene and Graphene-DH samples. Therefore, thermal treatment at 160 °C is likely to exert no negative effect on the physical and chemical properties of graphene.Scratch tests by a Florida Probe were used to reflect the adhesion strength between graphene sheets and the Ti substrates. With this special probe, the applied force could be kept constantly at 25 g, and therefore the comparability among the groups was assured. Ultrasonic scalers with carbon tips are widely used in periodontal maintenance therapy for dental implants. Therefore, we attached a carbon tip to the Florida Probe to scratch the surface of the materials, mimicking the clinical condition of periodontal probing and periodontal maintenance therapy of dental implants. After scratch tests, graphene coatings of Ti substrates after thermal treatment of 160 and 200 °C remained intact according to Raman spectra. 160 °C was identified as the optimal condition for thermal treatment to improve the adhesion strength of graphene coatings on titanium substrates because of the following reasons. Previous related studies showed that the thermal conductivity of graphene subjected to heat treatment at around 177 °C was similar to that measured around −3 °C38. Compared to autoclaving, dry heating sterilization reduces the rusting of metal medical materials38. Therefore, dry heating treatment at 160 °C for 2 h can not only enhance the adhesion strength of graphene on the Ti surface, but also combines the sterilization process, making it highly beneficial for clinical applications.Meanwhile, dry heating treatment at 160 °C for 2 h is also the same condition as dry heating sterilization, a common and effective sterilization procedure applied in clinical practice39, which may also be a potential transfer method for future clinical application of graphene-coatings on Ti substrates. However, the adhesion strength between graphene and Ti was not examined in the above study. Since the adhesion strength is a critical problem to be solved before the clinical application of graphene-coatings on Ti substrates, the examination of the adhesion strength is an indispensable index to evaluate new transfer methods. The scratch test, as proposed in this study, which used a Florida Probe to keep a constant force of 25 g, mimicking the clinical condition of periodontal probing and periodontal maintenance therapy of dental implants, is likely to be a practical method to evaluate the adhesion strength between graphene-coatings and their substrates.Most recently, a dry transfer technique based on a hot-pressing method was reportedet al. investigated focal adhesion (FA) of hASCs on graphene-related materials and found higher numbers of FAs on graphene40. Furthermore, FAs were found to be more concentrated on the protruding ends of the cells in graphene films than in control groups41, in agreement with the present findings.The present results show that graphene promotes the adhesion of hASCs and hBMMSCs to the substrate. A stronger expression of vinculin was observed for the samples in the graphene-coated groups. Accordingly, the incorporation of graphene may provide a better substrate for the attachment of hASCs and hBMMSCs to titanium. In a previous report, Kim The oral environment around the neck of an implant is complex, and includes soft and bone tissue. Osseointegration determines the function and the survival life of the implants, and the good integrity of the implant neck and of the soft tissue around it is the key to a successful osseointegration. The loose contact between the titanium substrate and the surrounding soft tissue leads to microbial invasion, whose toxic products cause peri-implantitis. Therefore, adhesion and proliferation of hGFs were also examined in our study. Fortunately, we observed extended lamellipodia and higher numbers of vinculin positive tips of cell pseudopodia for hGFs on graphene-coated samples. These results suggest that the graphene coating on Ti substrates might improve the interaction between the material and the soft tissue around it.44. FAs are large protein complexes that regulate the connection between cells and extracellular matrix, and play a key role as mediators of cell adhesion; vinculin is a structural protein of FAs45. Several studies have suggested that the nanoscale structure of the substrate may regulate FAs40. We assumed that the nanoscale structure of graphene might promote the expression of FAs by regulating vinculin expression, thus influencing cell adhesion, and this may also play a role in other cell functions, including proliferation and osteogenic differentiation.Adhesion represents the crucial prerequisite for many cell functions, such as proliferation, synthesis of proteins, and formation of mineral deposits46, and at the same time graphene did not show positive effects on cell proliferation reported in other studies with multilayered graphene and graphene oxide48. This suggests that the introduction of graphene did not affect the physiological cell growth conditions. In addition, the treatment at 160 °C for 2 h did not alter the effect of graphene on cell adhesion and proliferation.No significant differences in cell proliferation on the surfaces of the three types of investigated materials were observed, and it did not affect the growth of hGFs, hASCs, and hBMMSCs. This was consistent with the results of researches on single-layer graphene51. Some of them reported that graphene oxide almost completely suppressed the growth of bacteria, leading to a viability loss up to 80–90%49. However, only few studies examined the antibacterial activity of single-layer graphene. In 2004, Li et al. reported the antibacterial properties against E. coli and S. aureus of single-layer graphene on different substrates including Cu, Ge, and SiO2, and found that graphene on Cu had the best antibacterial ability among these substrates, followed by graphene on Ge and graphene on SiO216. However, Dellieu et al. proposed a different result52. The divergence was mainly resulted from lack of exact viability loss rate of the bacteria. In this study, we tested the antibacterial activity of graphene on a titanium substrate, and demonstrated its in vitro activity against E. coli and S. aureus bacteria. Meanwhile, fluorescence staining demonstrated significantly lower amounts of viable bacteria on the Graphene and Graphene-DH surfaces, indicating that the anti-bacterial activity of graphene was achieved mainly by killing the attached bacteria, rather than preventing the attachment of bacteria. Moreover, thermal treatment of 160 °C for 2 h did not affect the antibacterial activity of graphene on the titanium discs.Since the discovery of the antibacterial effects of a graphene oxide suspension in 2010, a large number of studies have shown that graphene oxide nanosheets and its derivatives have marked antibacterial propertieset al. proposed that the antibacterial activity of graphene is due to the mechanical destruction of the cell membrane caused by the sharp edges of the graphene structure12. Subsequent studies suggested that oxidative stress mediated by superoxide ions might play a role in the antibacterial effects of graphene53. Recently, Tu et al. reported the destructive extraction of phospholipids from the molecular cell membrane upon interaction with graphene validated by the TEM images, which could be one of the mechanisms behind its antibacterial effects54. This strong attraction between graphene and membrane lipids is largely derived from graphene’s unique two-dimensional structure with all sp2 carbons, which facilitates exceptionally strong dispersion interactions between graphene and lipid molecules.Huang Interestingly, the destructive effect of graphene on the membrane of bacterial cells is cell-specific: for instance, graphene has little effects on mammalian cells. This may be because the abundant glycoproteins on the surface of the mammalian cell membrane block out the surface of graphene and protect the phospholipids from adsorption. Basing on the information our tests for antibacterial activity of graphene and recent related studies, knowledge on the exact mechanism is still limited. Since bacterial species involved in the studies are finite, whether graphene has long-term and broad-spectrum antibacterial effects still needs to be further confirmed. Moreover, whether the interaction between single-layer graphene and bacteria is similar to that of graphene oxide or other graphene-related material need to be further explored.in vitro and in vivo, and showed that a heat treatment does not influence these favorable effects. In this research, we found that gene expressions of osteogenic-related genes, such as RUNX2 OSX and OCN, and the protein expression of OCN, were upregulated by graphene under the existence of OM, but the osteogenic differentiation of mesenchymal stem cells on graphene without OM was not significantly promoted compared with Ti substrate. However, some existing studies56 demonstrated the so-called “spontaneous osteogenic differentiation”, referring to the phenomenon that graphene promotes osteogenic differentiation without the existence of osteoinducing factors. These different results were likely to be caused by difference between the substrate under graphene. The substrates used these researches were glass or Si. But we used Ti as substrate in this research. As we know, Ti is the most widely used material is bone implant due to its good biocompatibility and advantages in osteointegration. Therefore, graphene didn’t accelerate the osteogenic differentiation so much compared with Ti surface without the existence of osteoinducing factors. However, under the existence of osteoinducing factors, graphene demonstrated a synergystic effect with OM as we discovered in this article. This phenomenon may result from preconcentration of osteogenic inducers, such as dexamethasone and β-glycerolphosphate, due to graphene’s strong π–π stacking, hydrogen bonding, and electrostatic interactions with proteins.In this study, we demonstrated that graphene-coated titanium can promote the osteogenic differentiation of hASCs and hBMMSCs 46. Meanwhile, multilayer graphene57, graphene oxide58, and graphene-related composites59 can improve the osteogenic differentiation of mesenchymal stem cells (MSCs) and osteoblasts61. Here, we showed that graphene maintained its positive effects on in vitro and in vivo osteogenic differentiation of hASCs and hBMMSCs after being transferred to titanium and heated at 160 °C for 2 h. Meanwhile, as for in vivo study, although the thickness of the new-born bone on the surfaces of Graphene and Graphene-DH groups seemed similar, the quality of the bone on Graphene-DH groups was better than Graphene groups with more acidophilic bone matrix after HE staining and more area of dark blue tissue after toluidine blue staining. This may result from better adhesion strength between graphene-coatings and Ti substrates, which helped to maintain the integrity of graphene-coatings even under sustained mechanical interferences from in vivo environment. Clearly, this conclusion is limited to the animal models employed in our study, and graphene-coated titanium bone implants and in situ osteointegration62 will be a practical in vivo model to illustrate the future application of graphene-coated Ti implants. Basing on the methods to enhance and evaluate the adhesion strength of graphene-coatings on Ti as proposed in this study, we will uncover the exact effect of bone integration of graphene-coated titanium bone implants in vivo by using the in situ osteointegration model in our future experiments.Previously, a number of studies have demonstrated that single-layer graphene can improve the osteogenic differentiation of mesenchymal stem cells (MSCs) and osteoblasts46, mechanical48, biochemical, and molecular aspects60. We previously reported the effect of graphene on cell behavior from the viewpoint of epigenetic regulation, and revealed that graphene promotes osteogenic differentiation of hMSCs by upregulating the methylation level of H3K4 at the promoter regions of osteogenesis-associated genes by inhibiting the RBP2 expression13. Thus, multiple factors may play a role in the graphene-cell interactions, and its internal mechanisms remain to be explored.The mechanism leading to the favorable effect of graphene on osteogenic differentiation has been previously explored from several angles, including morphologicalIn this study, we managed to modify titanium by using the anti-bacterial and osteoinductive effects of single-layer graphene sheet. Despite the satisfactory results obtained a number of issues still need to be solved before its application. First, the stability of the graphene-titanium association during clinical operation remains to be tested. Second, the exact mechanism of graphene-cell and graphene-tissue interactions is still unclear. Finally, further studies are needed to determine the efficiency and range of the graphene antibacterial activity in the complex oral environment.in vitro and in vivo osteogenic differentiation of hASCs and hBMMSCs. In addition, graphene coatings enjoy superior antibacterial effects. Thermal treatment will not influence the favorable effects of graphene on cell adhesion, osteogenic differentiation and antibacterial ability. Therefore, we managed to incorporate both the antibacterial and osteoinductive effects of single-layer graphene sheets on titanium substrates, and facilitate the future clinical application by enhancing their adhesion strength by thermal treatment.Thermal treatment at 160 °C for 2 h could enhance the adhesion strength between graphene coatings and titanium substrates. Graphene coatings enhanced the adhesion of hGFs, hASCs, and hBMMSCs, and promoted the in vivo experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.Our research was approved by the Ethics Committee of the Peking University Health Science Center, Beijing, China (PKUSSIRB-2013023). All 2O)  in an ultrasonic cleaner, respectively, for 15 min. Subsequently, the specimens were dried at room temperature for 1 h. For the transfer process, a thin film of PMMA  was spin-coated on graphene by a Laurell® WS-400BZ-6NPP/LITE spin coater. After treatment at 180 °C for 4 min, the PMMA-coated graphene was transferred to a FeCl3 solution (0.05 g/mL in water) to remove the copper foil substrate, and rinsed with distilled water twice to remove the residues. A smooth Ti disc was then placed in distilled water underneath the film and was picked up from the water with the coating of the film. Finally, the thin PMMA film on the top of graphene was removed by immersion in an acetone steam bath for 30 min at 57 °C. Atomic force microscopy (AFM) measurements and Raman spectra with an excitation laser source of 532 nm were used to confirm the presence of single-layer graphene on the Ti discs. Water contact angles, measured by a SL200 system , were used to examine the surface characteristics of the Ti discs coated with graphene. Before using them for the in vitro and in vivo experiments, the graphene-coated Ti discs were disinfected by soaking in 75% alcohol for 30 min.Single-layer graphene grown on copper foil substrate by CVD  and smooth Ti discs  were cut into appropriate sizes. Ti discs were polished with silicon carbide sandpaper of No. 240, 360, 400, 600, 800, 1000 and 2000 grits in series, and then washed with acetone, absolute alcohol and deionized water  was used to inspect the adhesion of graphene on Ti substrate. With this special probe, the applied force could be kept constant at 25 g. We attached a carbon tip  to the Florida Probe to scratch the surfaces of graphene-coated Ti, mimicking the clinical condition of periodontal probing and periodontal maintenance therapy of dental implants. Both scratched and unscratched areas were analyzed by field-emission scanning electron microscopy  and Raman spectroscopy to evaluate the integrity of graphene coatings.β-glycerophosphate, and 50 μg/mL l-ascorbic acid were added to the medium. Cells were cultured in a controlled environment at 37 °C in an incubator . All subsequent in vitro and in vivo experiments were performed using cells at the third and fourth passage. Moreover, all experiments were carried out in triplicate with cells extracted from three different patients.hASCs and hBMMSCs were purchased from ScienCell Research Laboratories , whereas hGFs were obtained from the attached gingiva of human premolars. The cells were cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin for proliferation. DMEM, FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin were purchased form Gibco . For the osteogenic differentiation, 10 nM dexamethasone, 10 mM The hASCs, hBMMSCs, and hGFs cells were seeded on titanium substrate (No-Graphene), titanium coated with graphene (Graphene), and titanium coated with graphene after dry heating treatment (Graphene-DH), at 10,000 cells/well in a 24-well plate, in a normal stem cell medium. After 12 h of incubation, the cells were washed three times with phosphate buffered saline (PBS) and fixed in cacodylate-buffered 4% glutaraldehyde at of 4 °C for 12 h. Then, the samples were dehydrated under a series of ethanol solution and dried in a professional dryer . After coating with gold, the specimens were observed by SEM . After 24 and 48 h of incubation, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 20 min. Then, after postfixing in 0.1% Triton X-100 for 5 min at room temperature, the cells were incubated in fluorescein isothiocyanate (FITC)-labeled phalloidin for 25 min to stain the cytoskeleton and in 6-diamidino-2-phenylindole (DAPI) solution for 10 min to stain the nucleus. The stained cells were observed by a Confocal Zeiss Axiovert 650 microscope , using excitation laser wavelengths of 488 and 405 nm.63.CCK-8 tests were carried out to monitor the proliferation of hASCs, hBMMSCs, and hGFs. Each day during two weeks of incubation, cells in each group were incubated with the counting reagent for 3 h, according to the manufacturer’s instructions. The relative cell number was determined by measuring the light absorbance  at 450 nm of the formazan dye product in the cultures64.To test the ALP activity, hASCs and hBMMSCs were seeded on different surfaces at 10,000 cells/well in 24-well plates. The ALP activity of each group was determined after osteoinduction (OI) for 7 and 14 days. The ALP levels were normalized to the total protein content, as previously describedhASCs and hBMMSCs were seeded onto different surfaces under the same conditions described above . After 14 and 21 days of osteoinduction, cells on different surfaces were stained with AR-S to monitor the mineralization. The specimens were rinsed three times with PBS and fixed in ice-cold 70% ethanol for 30 min at room temperature, then stained with 0.5% AR-S solution for 1 h to stain the calcium deposits. Finally, the samples were rinsed three times with distilled water.To quantify matrix mineralization, the AR-S-stained samples were incubated in 100 mM cetylpyridinium chloride for 1 h to solubilize and release calcium-bound AR-S into the solution. The absorbance of the released AR-S was measured at 562 nm. The final calcium levels in each group were normalized to the total protein concentrations obtained from duplicate plates.β-actin expression was used as the internal control. The primer sequences were shown in Table 66.hASCs and hBMMSCs were seeded on different surfaces as described above, in 6-well plates. After 7 and 14 days of osteoinduction, the Trizol reagent  was used to isolate the total cellular RNAs of each group. After synthesizing the first strand cDNA using the reverse transcription system , quantification of all gene transcripts was performed by real-time polymerase chain reaction (qPCR) using a Power SYBR Green PCR Master Mix and an ABI PRISM 7500 sequence detection system . The The specimens were rinsed three times in PBS for 5 min and fixed in 4% paraformaldehyde for 20 minutes at room temperature. The samples were washed another three times in PBS and were incubated with specific primary antibodies at 4 °C for 12 h. Anti-vinculin (1:200) and anti-osteocalcin (1:500) primary antibodies  were used to monitor the expression of vinculin and osteocalcin, respectively. The specimens were rinsed another three times in PBS and incubated in 1:500 anti-rabbit or anti-mouse secondary antibodies  for 1 h at room temperature. Afterward, the specimens were stained in DAPI solution for 10 min at 37 °C before being visualized with a Confocal Zeiss Axiovert 650 microscope , using laser wavelengths of 405, 488, and 543 nm.E. coli and Gram-positive S. aureus. Following Li et al.’s approach14, a 104 CFU/mL bacteria solution was introduced to the samples at a density of 80 μL/cm2. After inoculation at 37 °C for 24 h, the dissociated bacteria solution was collected and inoculated into a standard agar culture medium. After incubation at 37 °C for 24 h, the culture plates were photographed and the colony forming units were counted. The samples  inoculated with bacteria were then rinsed three times with PBS, and disinfected in 75% alcohol for 30 min. Later, a same density of 104 CFU/mL bacteria solution was introduced to the samples at a density of 80 μL/cm2 as described above, to examine whether the antibacterial activity of graphene-coated Ti could still exist after bacterial inoculation and disinfection.The antibacterial activity of the samples was assessed using Gram-negative E. coli. In order to visualize the viability of bacteria on the samples, a LIVE/DEADH BacLightTM Bacterial Viability Kit  was used, which contained the green fluorescent DNA-binding stain, Syto 9 and the red fluorescent DNA-binding stain, propidium iodide (PI), SYTO 9 permeated both intact and damaged membranes of the cells, binding to nucleic acids and fluorescing green when excited by a 485 nm wavelength laser, but PI entered only cells with significant membrane damage, which are considered to be non-viable67. The staining procedure was carried out according to the manufacturer’s protocol, bacteria at a concentration of 107 CFU/mL were inoculated on the samples. After incubation at 37 °C for 24 h16, the culture medium was removed and the samples were rinsed with deionized water, then the staining solution was added. After being incubated in the dark at room temperature for 15 min, the samples were observed by Confocal laser scanning microscopy.The antibacterial activity of the graphene samples was evaluated by using Gram-negative in vivo, hASCs and hBMMSCs were cultured on different specimens and then implanted into the dorsal subcutaneous area of eight week-old male BALB/c nude mice, according to the procedure described previously44. For each samples , the following test groups were examined: discs incubated with hASCs in proliferation medium (hASCs without OI), discs incubated with hASCs in osteoinducing medium (hASCs with OI), discs incubated with hBMMSCs in proliferation medium (hBMMSCs without OI), discs incubated with hBMMSCs in osteoinducing medium (hBMMSCs with OI). After four and eight weeks of normal diet, the implants of each group were harvested together with the surrounding tissues. Following fixation with formalin and resin infiltration, the implants were stained with hematoxylin/eosin (HE) and toluidine blue. Bone formation was then observed with a light microscope.To determine the effect of graphene on bone formation post hoc test. For all tests, P-values less than 0.05 were considered indicative of statistically significant differences.All results are presented as mean ± standard deviation; the data were analyzed using the SPSS 19.0 software  by one-way ANOVA followed by a Tukey’s


No significant differences in cell proliferation were observed among DES, endometrial stromal cells of patients with endometriosis (EES) from the proliferative phase (P), EES-S (secretory phase) and EES-M  compared to cells grown on a substrate of the same stiffness at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses. However, cell re-growth of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates at both combined doses. Combination U0126 and MK2206 treatment is more effective than each drug alone in cell growth inhibition of DES. However, further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.Endometriosis is a common gynecological disorder responsible for infertility and pelvic pain. A complete cure for patients with endometriosis awaits new targets and strategies. Here we show that U0126 (a MEK inhibitor) and MK2206 (an AKT inhibitor) synergistically inhibit cell growth of deep endometriotic stromal cells (DES) grown on polyacrylamide gel substrates (PGS) of varying stiffness  or plastic  Endometriosis, a common gynecological disorder responsible for infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sitesin vitroin vitro findings suggest that the AKT and ERK signaling pathways, both of which are important survival pathways, may compensate for each other, resulting in apoptosis resistance in endometriotic stromal cellsWe previously showed that the serine/threonine kinase AKT and extracellular regulated kinase (ERK) signaling pathways may cooperate to support growth of deep endometriotic lesions by enhancing endometriotic stromal cell proliferation and survival in a fibrotic microenvironment in vitro cell culture systems to develop novel strategies for treatment of endometriosis3in vitro experiments have had at least two limitations. First, previous drug screening assays were performed in rigid plastic, which is much stiffer than that occurring in vivo. Studies have shown that matrix stiffness affects responsiveness to cytotoxic drugs in a cell-dependent and drug-dependent manner56in vitroin vivo tissue compliance conditions in vitro. Second, a high recurrence rate after medical treatment with or without surgery is a major clinical problem for patients with endometriosisin vitro study evaluated whether candidate molecules for the treatment of endometriosis could prevent relapse of the disease after discontinuation of treatment. Before validation of the effects of candidate molecules can be performed in animal experiments or clinical trials, it is important to evaluate whether candidate molecules could decrease the number of cells that can survive treatment and consequently prevent re-growth of endometriotic cells in vitro.Until now, the efficacy of numerous molecules has been evaluated in in vitro. We evaluated the effects of U0126 alone and MK2206 alone, as well as the combination of U0126 and MK2206, on inhibition of cell proliferation of DES, endometrial stromal cells with (EES), and/or without (NEES) endometriosis grown on polyacrylamide gel substrates (PGS) of varying stiffness  or plastic. In addition, we evaluated proliferation of viable cells after discontinuation of combined treatment with U0126 and MK2206 in DES, EES, and/or NEES grown on PGS of varying stiffness (2 or 30 kPa) or plastic. For any disease, ideal drugs are those that increasing the probability of the disease cure and decrease normal tissue toxicity. In addition, studies have shown that endometrium of patients with endometriosis may differ biochemically from that of patients without endometriosis. Our previous study showed that levels of phosphorylated AKT and phosphorylated ERK were significantly higher in menstrual endometrium in vivo and in vitro in patients with endometriosis compared to those of patients without endometriosisin vivo tissue compliance of the endometrium or deep infiltrating endometriosis (DIE), respectivelyThe objective of the present study was to investigate whether combined treatment with U0126 and MK2206 can effectively inhibit cell proliferation during and after treatment in DES Combined treatment with U0126 and MK2206 produced a synergic effect in DES grown on substrates of varying stiffness  for ED 95 (effect dose at which 95% growth inhibition occurs), ED 90, and ED 75 See . For ED No significant differences in cell proliferation were observed among DES, EES-P, EES-S, and EES-M compared to cells grown on a substrate of the same stiffness  at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses .No significant differences in cell proliferation were observed between EES and NEES  when compared to cells grown on a substrate of the same stiffness  at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses See .In DES , cell prThe percentage of Annexin V-positive cells treated with U0126 alone was significantly higher in DES, EES-S, and EES-M compared to that in EES-P . When ceSA-βgal activity was observed in DES and EES-P treated with MK2206 alone . Levels When cells were grown on 2-kPa PGS, cell proliferation of EES-M after a 72-h drug discontinuation was significantly higher than that of EES-S at a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose . When ceWhen a lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose was applied, no significant difference in cell proliferation was observed after a 72-h drug discontinuation between EES and NEES  compared to cells grown on a substrate of the same stiffness  See . HoweverCell proliferation of DES was significantly higher in cells grown on 30-kPa PGS and plastic than those grown on 2-kPa PGS after a 72-h drug discontinuation at either a higher (U0126 [30 μM] and MK2206 [9 μM]) or lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose . No signCell proliferation after a 72-h drug discontinuation was significantly increased compared to that after a 48-h treatment in DES, EES  and NEES  grown on substrates of varying stiffness  at a lower combined dose See . At a hiLC3-positive puncta, a marker for autophagy, were observed in DES, EES , and NEES  treated with MK2206 . Cell prThe present study showed a synergic effect of combined treatment with U0126 and MK2206 on DES, whereas an additive or antagonistic effect was observed on EES. The present study supports our speculation that cotargeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways may be effective for treatment of endometriosisIn the present study, we observed that the inhibition of cell proliferation was significantly higher in DES grown on plastic than those grown on 2- or 30-kPa PGS. One study evaluated a total of 18 small-molecule known or suspected inhibitors of cell proliferation in lung fibroblasts grown on soft (1-kPa PGS) or rigid (glass) substratesAkt is well known for its anti-apoptotic activity1015in vitro findings may not support the future clinical use of the combined treatment with U0126 and MK2206 in patients with DIE, because of high cell survival and proliferation after drug discontinuation.The present results showed that cell proliferation after discontinuation of the combined treatment was significantly higher in DES grown on 30-kPa PGS or plastic than those grown on 2-kPa PGS. However, we observed no significant effects of varying stiffness  on cell proliferation of either EES or NEES after drug discontinuation. These findings suggest that DES grown on a rigid substrate may have more potential to relapse than those grown on a soft substrate. Our findings appear to agree with the clinical evidence of a high recurrence rate following medical treatment in endometriosisIn the present study, we further attempted to investigate a potential mechanism underlying cell survival in DES treated with combination U0126 and MK2206. We observed a significantly higher percentage of Annexin V-positive cells in DES than in EES-P, -S, and -M when treated with combination U0126 and MK2206. Nevertheless, we observed higher proliferation of DES after drug discontinuation than of EES-P and EES-S when cells were grown on rigid substrates (30-kPa PGS or plastic). Studies showed that Akt knockdown or inactivation with small-molecule inhibitors markedly increased autophagy171920Another potential explanation is that stem-like cells in endometriosis and menstrual endometrium of patients with endometriosis may be responsible for high cell survival and proliferation after discontinuation of combined treatment with U0126 and MK2206. A growing body of evidence suggests that endometriosis may arise from stem cells2933in vitro model has many limitations: endometriotic tissue and endometrium are composed of multiple cell types and extracellular matrix, but in the present study, only endometriotic and endometrial stromal cells were cultured based on our previous findings. Second, endometriotic tissue and endometrium are three-dimensional (3D), but the present studies used a conventional two-dimensional (2D) culture system. 3D in vitro models have been considered to span the gap between 2D cell cultures and whole-animal systems35in vivo endometriotic tissues.The present in vitro findings may not support the future clinical use of the combined treatment with U0126 and MK2206 in patients with DIE. Further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.In conclusion, the present study showed that combined treatment with U0126 and MK2206 synergistically inhibited cell proliferation of DES. However, cell proliferation of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates. The present Patients aged 20–37 years undergoing laparoscopy for endometriosis were recruited at CHU Clermont-Ferrand, France. None of the women had received hormonal therapy and none used intrauterine contraception for at least 6 months prior to surgery. Recruited patients had regular menstrual cycles (26–32 days) with confirmation of their menstrual history. Endometrial and endometriotic samples from 73 patients who had histological evidence of rectovaginal DIE were used for the present analysis. In addition, endometrial tissues from 21 patients without endometriosis were obtained. The clinical characteristics of patients are shown in DES, EES, and NEES were isolated as previously described 28373839.283738283738et al.Stiffness-controlled 96-well plates were prepared using modifications to the protocol of Syed ® AQueous One Solution Cell Proliferation Assay (MTS) , as previously described37383 cells per well) from the same samples were plated on 2- or 30-kPa PGS or plastic in triplicate in 96-well plates. After 2 h at 37 °C and 5% CO2 to allow cell adhesion and spreading, drugs were added at the indicated concentration with 100 μL culture media , individually or in combinations. U0126  or MK2206  were dissolved in dimethyl sulfoxide (DMSO) (Life Technologies). Chloroquine (Sigma-Aldrich) was dissolved in phenol red-free DMEM/F-12. The Chou-Talalay model calls for cytotoxic agents to be used at a fixed dose ratio42Cell proliferation assays were performed using the CellTiter 966 cells) were seeded onto Primaria flasks (BD Biosciences). After 2 h at 37 °C and 5% CO2 to allow for cell adhesion and spreading, cells were incubated with culture media  containing either U0126 alone (30 μM) (Sigma-Aldrich), MK2206 alone (9 μM) (Sigma-Aldrich), a combination of U0126 (30 μM) and MK2206 (9 μM), or vehicle (DMSO) for 24 h. Cells were stained with Annexin V-FITC and PI  and evaluated for apoptosis by flow cytometry analyses using a BD LSRII flow cytometer (BD Biosciences) according to the manufacturer’s protocol. Both early apoptotic  and late (Annexin V-positive and PI-positive) apoptotic cells were included in cell death determinations.Cells  was performed. Fluorescence histochemical detection of SA-βgal activity was performed according to the protocol published by Debacq-Chainiaux 4 cells per well). After 2 h at 37 °C and 5% CO2 to allow for cell adhesion and spreading, cells were incubated with culture media  containing either U0126 (30 μM) (Sigma-Aldrich), MK2206 (9 μM) (Sigma-Aldrich), U0126 (30 μM) and MK2206 (9 μM), or vehicle (DMSO) only for 24 h. Total RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions  as previously described283738283738WAF1/Cip1 (p21) were measured by quantitative real-time RT-PCR with a Light Cycler  as previously described283738Cells were seeded onto 24-well plates  was used for statistical analysis. Comparisons between different groups were made using one-way analysis of variance following Scheffé’s method, the Mann-Whitney How to cite this article: Matsuzaki, S. et al. Effects of U0126 and MK2206 on cell growth and re-growth of endometriotic stromal cells grown on substrates of varying stiffness. Sci. Rep.7, 42939; doi: 10.1038/srep42939 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Trichinella spiralis, as well as its muscle larvae excretory–secretory products (ES L1), given either alone or via dendritic cells (DCs), induce a tolerogenic immune microenvironment in inbred rodents and successfully ameliorate experimental autoimmune encephalomyelitis. ES L1 directs the immunological balance away from T helper (Th)1, toward Th2 and regulatory responses by modulating DCs phenotype. The ultimate goal of our work is to find out if it is possible to translate knowledge obtained in animal model to humans and to generate human tolerogenic DCs suitable for therapy of autoimmune diseases through stimulation with ES L1. Here, the impact of ES L1 on the activation of human monocyte-derived DCs is explored for the first time. Under the influence of ES L1, DCs acquired tolerogenic (semi-matured) phenotype, characterized by low expression of HLA-DR, CD83, and CD86 as well as moderate expression of CD40, along with the unchanged production of interleukin (IL)-12 and elevated production of IL-10 and transforming growth factor (TGF)-β, compared to controls. The interaction with DCs involved toll-like receptors (TLR) 2 and 4, and this interaction was mainly responsible for the phenotypic and functional properties of ES L1-treated DCs. Importantly, ES L1 potentiated Th2 polarizing capacity of DCs, and impaired their allo-stimulatory and Th1/Th17 polarizing properties. Moreover, ES L1-treated DCs promoted the expansion of IL-10- and TGF-β- producing CD4+CD25hiFoxp3hi T cells in indolamine 2, 3 dioxygenase (IDO)-1-dependent manner and increased the suppressive potential of the primed T cell population. ES L1-treated DCs retained the tolerogenic properties, even after the challenge with different pro-inflammatory stimuli, including those acting via TLR3 and, especially TLR4. These results suggest that the induction of tolerogenic properties of DCs through stimulation with ES L1 could represent an innovative approach for the preparation of tolerogenic DC for treatment of inflammatory and autoimmune disorders. One of the permanent challenges in immunology is overcoming the rising problem of losing the delicate balance, provided by the innate immunity, reflected in responding to foreign antigens while remaining tolerant to self-antigens. If this balance is altered due to an increased inflammatory response and diminished tolerance, it can result in autoimmune diseases such as type I diabetes, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease . Currentin vitro, have turned into phase I clinical trials over time 2 and regulatory response while simultaneously inhibiting Th1 and Th17 response . Muscle larvae were recovered by digestion of the carcasses in pre-warmed gastric juice (2) in complete Dulbecco’s modified Eagle medium (DMEM) (Sigma), for 18 h . Namely, ES L1 antigens were adhered to microtiter plates and their immunoreactivity was analyzed using reference sera with pre-defined titer of anti-T. spiralis specific antibodies, as described previously  and were housed under standard conditions in animal room with access to food and water ic juice , and kepfor 18 h . ES L1 afor 18 h ], as deteviously .+ T cells were isolated from PBMCs by negative MACS, using the Pan T cell isolation kit (Miltenyi Biotec), and they were used as responders in allogeneic coculture experiments with monocyte-derived DCs. The purity of CD14+ monocytes and CD3+ T cell populations was usually higher than 90%, as evaluated by flow cytometry analysis  (data not shown).Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were isolated by density gradient centrifugation on Lymphoprep gradient (Carl Roth). The monocytes were isolated by negative magnetic-activated cell sorting (MACS) from PBMCs, using the Monocyte Isolation kit (Myltenil Biotec), following their cultivation in CellGro DC medium (Cell Genix), supplemented with 100 ng/ml of human recombinant GM-CSF and 20 ng/ml of human recombinant IL-4 (both from R&D Systems) (complete DC growth medium). CD36/well, in the complete DC growth medium for 5 days, refreshing the medium on Day 4. To assess the effect of ES L1 on DCs differentiation, the cells were cultivated during this period in the presence of 10, 50, or 200 µg/ml of ES L1 antigen, or without ES L1 (control DCs). The impact of ES L1 on the maturation of DCs was determined by adding ES L1 antigens to the culture of immature DCs on Day 4, for 48 h. To induce mature DCs, the cells were stimulated with LPS from Escherichia coli  and human recombinant IFN-γ  on Day 5, for the next 24 h. In some experiments, LPS (500 ng/ml) or polyinosininc:polycytidylic acid  were used instead of LPS/IFN-γ, to investigate the stability of phenotypical and functional characteristics of DCs acquired after the pulsing with ES L1. To determine the role of toll-like receptors (TLR)2 and TLR4 on the DCs status induced by ES L1, immature DCs were treated with blocking antibodies against TLR2 and/or TLR4 , or isotype control antibody , 1 h before the treatment with ES L1. The phenotype of DCs was checked using flow cytometry, whereas DC culture supernatants were used for the cytokines analyses. DCs used for functional assays were extensively washed to prevent the transfer of free ES L1 or stimuli to the cocultures.To differentiate immature DCs, monocytes were cultivated in 24-well plate, 0.5 × 10Dendritic cells cultivated with different agents were stained after the culture with the following antibodies/reagents: immunoglobulin (Ig)G1a negative control—peridinin–chlorophyll–protein complex (PerCP), IgG1 negative control—phycoerythrin (PE), IgG1 negative control—fluorescein isothiocyanate (FITC), IgG1a negative control—PE cyanine (Cy)5, IgG1 negative control—allophycocyanin (APC), anti-CD83-FITC, anti-CD86-PE, anti CD40-APC, anti-CCR7-FITC, anti-TGF-β-PeCy5, anti-TLR4:PeCy5 (eBioscience), anti-CD1a-PE (Biolegend), anti-IL12p40/p70-PE, anti-indolamine 2,3 dioxygenase (IDO)-1-APC, anti-Ig-like transcript (ILT)3-PE (R&D Systems), anti-IL-10-FITC (AbD Serotec), anti-CD14-FITC, and anti-HLA-DR-PerCP (Miltenyi). The viability of DCs after the culture with ES L1 antigens was determined by 7-aminoactinomycin D  staining. For T cells staining, the following antibodies were used: anti-forkhead box (Fox)P3-PE, anti-CD4-FITC, anti-CD4-PeCy5, anti-TGF-β-PE, anti-IL-4-PE, anti-IL-10-PE (eBioscience), anti-CD25-PeCy5 (BD Pharmigen), anti-IL-10-FITC (AbD Serotec), IL-17A-PerCP (Biolegend), anti-IFNγ-FITC (R&D Systems), and appropriate negative isotype controls, as indicated.For surface labeling, the cells were washed once in PBS containing 2% FCS and 0.1% Na-azide and then incubated with primary Abs for 30–60 min at 4°C. Intracellular staining was conducted after the surface staining, by using the flow cytometry fixation and permeabilization kit I (R&D). Intracellular staining of IFN-γ, IL-4, IL-10, IL-17, and TGF-β in T cells, was carried out after the 4-h activation of T cells with phorbol-12-myristate-13-acetate  and ionomycin (500 ng/ml) in the presence of monensin (2 µM). For each analysis, more than 5,000 cells were gated according to their specific side-scatter (SSC)/forward-scatter (FSC) properties, thereby avoiding the cells with low SSC/FSC properties (predominantly dead cells), as indicated. Signal overlap between the FL channels was compensated before each experiment using single labeled cells, and non-specific fluorescence was determined by using the appropriate isotype control antibodies and fluorescence minus one controls.l-glutamine, 2-mercaptoethanol , and antibiotics . T cells (1 × 105/well of 96-well plate) were cocultivated with different number of DCs  for 5 days in 96-well round-bottom plates. The blank controls were T cells cultivated separately. To measure the level of T cell proliferation, the cells were pulsed for the last 18 h of coculture with 3H-thymidine . The radioactivity was measured by β-scintillation counting .In mixed leukocyte reaction assays, DCs treated with the different stimuli, as well as non-treated DCs, were cultured with MACS-purified allogenic T cells in complete RPMI 1640 medium containing 10% FCS (Capricorn Scientific), 4/well, 96-well round-bottom plate) were cultivated with naive allogenic T lymphocytes in 1:20 ratio, for 6 days. To detect cytokines in the supernatants of allogenic stimulation assay, DC/T cell cocultures were treated with PMA (20 ng/ml) and Ionomicin (500 ng/ml) for the last 4 h of coculture, followed by the harvesting of cell-free supernatants. The levels of cytokines produced in DCs cultures , as well as in DC/T cell cocultures , were measured in cell-free supernatants by sandwich ELISA Kits (R&D). Additionally, T cells were primed with DCs (2 × 103/well of 96-well round-bottom plate) at a 1:50 DC/T cell ratio for 3 days and then expanded with IL-2  for two more days . In some experiments 1-methylthryptophane , as an IDO-1 inhibitor , in 96-well round-bottom plates. In order to assess the proliferation of responder cells, PBMCs were pre-stained with 1 µM CFSE (Invitrogen) for 20 min, at 37°C, at density of 1 × 107 cells/ml. The proliferation of cells was stimulated with 8 µg/ml phytohemaglutinine (PHA), and after 5 days of coculture, the cells were harvested, stained with 20 µg/ml propidium iodide to exclude dead cells from the analysis, and the proliferation of responder T cells was measured by flow cytometry. The Proliferation Index (PI), i.e., the average number of cells derived from the initial cell, was calculated using proliferation fit statistics in FCS Express 4 .T cells primed with DCs (Tpr.) were extensively washed and then cocultivated with allogeneic PBMCs responder cells. PBMCs, 1 × 10HEK-Blue™ reporter cell lines expressing either TLR2, TLR3, TLR4, TLR5, TLR7, nucleotide-binding oligomerization domain-like receptors (NOD)1, or NOD2   were usel-glutamine  and 10% (v/v) heat-inactivated HyClone FBS (30 min at 56°C) (Thermo Fisher Scientific) at 37°C in 5% CO2/95% air. Cell lines  were cultivated in 150 µl of culture medium. Cells were treated with 50 µl of various concentrations of ES L1 antigens  or PRRs agonists: Pam3CSK4 (10 ng/ml) as TLR2 ligand, Poly (I:C) HMW (1 µg/ml) as TLR3 ligand, LPS (1 ng/ml) E. coli K12 as TLR4 ligand, FLA-ST (50 ng/ml) as TLR5 ligand, Imiquimod (10 µg/ml) as TLR7 ligand, iE-DAP (25 µg/ml) as NOD1 ligand, and MDP (10 µg/ml) as NOD2 ligand . After 22–24 h incubation, supernatants were collected for determination of SEAP activity. QUANTI-Blue™ medium (180 µl) substrate and cell culture supernatant (20 µl) were added per well on a flat-bottom 96-well ELISA plate. After 4 and 24 h of incubation at 37°C in 5% CO2, the levels of SEAP activity were analyzed by measuring the absorbance at 649 nm on a BioTek EL808 microplate reader and Gen5 Data Analysis Software .HEK-Blue™ hTLR2 (hkb-htlr2), HEK-Blue™ hTLR3 (hkb-htlr3), HEK-Blue™ hTLR4 (hkb-htlr4), HEK-Blue™ hTLR5 (hkb-htlr5), HEK-Blue™ hTLR7 (hkb-htlr7), HEK-Blue™ hNOD1 (hkb-hnod1), and HEK-Blue™ hNOD2 (hkb-hnod2) cells were cultured in Dulbecco’s Modified Eagle’s culture medium , supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.3 mg/ml p < 0.05, the differences were considered significant statistically.Data are presented either from representative experiments or as mean ± SD of at least three experiments carried out with different DCs donors. Statistical analysis of the data was conducted using repeated measures ANOVA, followed by a Tukey’s posttest in PRISM5 (Graphpad software), and for in vitro differentiate into immature DCs, by upregulating CD1a and diminishing CD14 expression (+) cells after 4 days of cultivation with ES L1. It was observed that the percentage of 7AAD+ DCs cultivated with 10 or 50 µg/ml was similar to the values obtained in control DCs cultures. In contrast, 200 µg/ml ES L1 increased the percentage of 7AAD+ cells up to 27%  impaired severely the LPS/IFN-γ-induced upregulation of CD83, CD86, CD40, and HLA-DR, whereas the cultivation with 10 µg/ml ES L1 impaired only CD40 upregulation . The data showed that ES L1 impaired significantly the upregulation of HLA-DR, CD40, and IL-12 by DCs, but not CD83 and CD86, upon the Poly (I:C) stimulation. Additionally, ES L1-treated Poly (I:C)-matured DCs produced significantly higher levels of IL-10 compared to control Poly (I:C)-matured DCs . These data suggested that the treatment of DCs with ES L1 impeded full maturation of DCs by different maturation stimuli, showing the stability of ES L1-treated DCs.Since LPS and LPS/IFN-γ stimulation includes a TLR4-dependent induction of DCs maturation , we also+ T cells was performed, and the T cell proliferation was determined based on 3H-thymidine incorporation level. ES L1-treated DCs had a weaker capacity to stimulate T cell proliferation compared to both immature and LPS/IFN-γ-matured DCs  T cell population , and retained that ability even after LPS/IFN-γ challenge  was cocultivated with CFSE-labeled PBMCs (responders) in the presence of PHA. A flow cytometry analysis of the responder cells’ proliferation clearly suggested that T cells primed with ES L1-treated DCs exhibited a stronger capacity to inhibit the proliferation of responder cells in both 1:2 and 1:4 Tpr:responder cell ratios  with ES L1 antigens was investigated. The study was performed on HEK-Blue™ reporter cell lines expressing the individual TLRs , NOD1, or NOD2 receptors. The activation of PRRs was indicated by SEAP activation in culture supernatants of stimulated cells. All used HEK-Blue™ cell lines were treated with ES L1 antigens, while the cells pulsed with corresponding TLR or NOD agonists were used as positive controls, and the cells cultivated only in medium were used as negative control (Figure The data about the receptors that recognize l Figure .Stimulation of HEK-hTLR2 cells with ES L1 induced an increased level of SEAP activity after 4 and 24 h Figures C compareConsidering that ES L1 antigens activate TLR2 and TLR4, the next step was to investigate the relevance of this interaction for the development of semi-mature DCs by ES L1 antigens. For this purpose, TLR2 and TLR4 receptors were blocked individually or simultaneously using specific monoclonal blocking antibodies, prior to DCs treatment with ES L1 antigens. In the presence of the specific blocking antibodies, the expression of DCs surface markers upon ES L1 treatment was significantly changed compared to control DCs treated with ES L1 antigens in the presence of isotype control antibody. Namely, the expression of CD83 was significantly lower, while CD86, HLA-DR, and CD40 expression were significantly higher compared to control ES L1-treated DCs Figures A,B.Additionally, the differences in cytokines production by DCs were analyzed in the presence of specific blocking or isotype control antibodies upon the ES L1 treatment Figure C. In casin vitro generation of DCs from monocytes that can be manipulated to acquire tolerogenic properties (T. spiralis products to induce the development of DCs with tolerogenic properties (T. spiralis ES L1 antigens represent a promising new tool for the generation of human tolerogenic DCs in vitro. In contrast to other protocols for generation of DCs, such as those which include vitamin D3 (A number of studies have dealt with operties , 58–60. operties . Our preoperties , which soperties . Howevertamin D3 , 27 or Itamin D3 , DCs cultamin D3 . Upon cotamin D3 , 61. CCRtamin D3 –64. Sometamin D3 . Also, stamin D3 , which ivia different mechanisms.The stability of tolerogenic DCs and preservation of anti-inflammatory phenotype in inflammatory surrounding is the most important issue when creating cell-based therapies for autoimmune diseases . TLRs wein vivo, compared to models with a polyclonal stimulation of T cells, especially since recent findings suggested that T. spiralis-secreted components are structurally related to some human cell components (via DCs while inhibiting Th1 and Th17 responses (ES L1-treated DCs, even after the challenge with LPS/IFN-γ, exhibited a reduced capacity to induce T cell proliferation in mixed leukocyte reactions, which is in accordance with their observed semi-mature phenotype and tolerogenic functions. The experiments were performed using cells from healthy individuals. Due to HLA-restriction, the allogeneic coculture system applied in our study includes the response of a limited naïve T cell population that are able to respond to allogeneic MHC complexed with self-peptides . This momponents , so onlymponents . The facmponents . Althougmponents . Since Imponents , 76, theesponses , are in esponses .+CD25hiFoxp3hi T regulatory cells. This expansion was reduced upon addition of 1-MT, an IDO-1 inhibitor (via aryl hydrocarbon receptor (Besides the suppression of inflammatory immune response and the enhancement of Th2 type of response, ES L1-treated DCs demonstrated the ability to induce the expansion of CD4nhibitor  during treceptor . An incrreceptor . The resreceptor . Tregs areceptor . The finreceptor , 81. Trereceptor , 82. Thereceptor . Althougreceptor , 84. Thereceptor , 57. We Schistosoma mansoni and lipid of Ascaris lumbricoides reacted with TLR2 on DCs and mediate their differentiation into cells that induce Th2 and regulatory immune responses (N-fucopentaose III of S. mansoni and ES 62 glycoprotein secreted by Acanthocheilonema vitae, were shown to activate TLR4 receptor and lead to consequent polarization of T cell responses toward Th2 (Using HEK-Blue™ reporter cells lines expressing individual TLR or NOD-like receptors, we identified for the first time TLR2 and TLR4 as receptors that interact and induce intacellular signaling after ligation of ES L1 antigens. These results are consistent with the studies that have shown the interaction of TLR2 or TLR4 with other helminth antigens. For example, lipid fractions and lysophosphatidylserine of helminth esponses , 86. Lacward Th2 , 88. EveT. spiralis ES L1 antigens mediate phenotypical and functional maturation of DCs mainly via TLR2 and TLR4. The importance of both TLR2 and TLR4 was also demonstrated for the interaction with S. mansoni antigens (via most TLRs normally results in the production of pro-inflammatory cytokines by DCs (via interaction with TLRs (via suppression of TLR4 expression, since we showed that ES L1 did not alter the expression of TLR4, but rather via modulation of downstream signaling events, and/or engagement of some other receptors expressed on DCs. Since ES L1 antigens are complex mixture of molecules, it is reasonable to assume that other PRRs are also involved in ES L1 recognition and binding. Indeed, it was revealed that ES L1 components are ligands for C-type lectin receptors, mannose receptor (The critical relevance of both TLR2 and TLR4 interaction with ES L1 antigens for the induction of tolerogenic properties in human DCs was demonstrated by blocking these two receptors before adding ES L1 antigens. Tolerogenic properties of ES L1-stimulated DCs were compromised when TLR2 and TLR4 were blocked, indicating, for the first time, that antigens , 89. Sigs by DCs . Howeverith TLRs . It is freceptor , and DC-T. spiralis ES L1 antigens could be a promising new strategy for the development of stable tolerogenic DCs, with an increased capacity to suppress the inflammatory immune response while favoring the expansion of highly potent IL-10- and TGF-β- producing Tregs. Bearing in mind that the major scientific efforts are made to develop cell-based therapies that promote tolerance in humans, and although more investigation on mechanisms underlying the induction of tolerogenic DCs by ES L1 are needed, those tolerogenic DCs may present a potentially new tool for the treatment of inflammatory disorders.In conclusion, this study revealed that the treatment of human monocyte-derived DCs with Animal experiments were performed according to institutional guidelines and were approved by the local Institutional Animal Care and Use Committee of the Institute for the Application of Nuclear Energy. Samples of human peripheral blood were obtained from healthy volunteers after written informed consent in accordance with the Declaration of Helsinki and approval by the Ethical Board of the Institute for the Application of Nuclear Energy.LS-M, MC, NI, AG-M, ST, and EP participated in the design of the study. NI, AG-M, ST, DV, JC, and CA participated in data acquisition and analysis. NI, AG-M, and ST prepared the manuscript. LS-M, MC, ST, DV, and EP participated in data interpretation and manuscript revision. LS-M and EP supervised this study and they have equally contributed. All authors gave final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors LS-M.


LINE)‐1 (L1) is a type of retrotransposon capable of mobilizing into new genomic locations. Often studied in Mendelian diseases or cancer, L1s may also cause somatic mutation in the developing central nervous system. Recent reports showed L1 transcription was activated in brains of cocaine‐treated mice, and L1 retrotransposition was increased in cocaine‐treated neuronal cell cultures. We hypothesized that the predisposition to cocaine addiction may result from inherited L1s or somatic L1 mobilization in the brain.Long interspersed element (mPFC) tissue from 30 CA and 30 control individuals was studied. An Alexafluor488‐labeled NeuN antibody and fluorescence activated nuclei sorting were used to separate neuronal from non‐neuronal cell nuclei. L1s and their 3' flanking sequences were amplified from neuronal and non‐neuronal genomic DNA (gDNA) using L1‐seq. L1 DNA libraries from the neuronal gDNA were sequenced on an Illumina HiSeq2000. Sequences aligned to the hg19 human genome build were analyzed for L1 insertions using custom “L1‐seq” bioinformatics programs.Postmortem medial prefrontal cortex (PCR, were detected in neurons from both CA and control brain samples. Steady‐state L1 mRNA levels in CA and control mPFC were also assessed. Gene ontology and pathway analyses were used to assess relationships between genes putatively disrupted by novel L1s in CA and control individuals. L1 insertions in CA samples were enriched in gene ontologies and pathways previously associated with CA.Previously uncataloged L1 insertions, some validated by mPFC harbor L1 insertions that have the potential to influence predisposition to CA.We conclude that neurons in the  Five toTa subfamily members, remain “competent” to replicate and insert at a new locus ‐1 (L1) is a mobile DNA element that constitutes ~17% of the human genome  L1 gene disruptions might predispose individuals to developing CA. Second, by relieving L1 transcriptional repression, epigenetic changes, caused by chronic cocaine‐taking  because cocaine's rewarding effects are largely due to blockade of dopamine reuptake at ventral tegmental area nerve terminals, some of which synapse on neurons in the mPFC . Specimens of cerebellum from some CA subjects were also obtained.All subjects were judged by a forensic pathologist to have died of cocaine overdose. Subject demographics, postmortem brain characteristics  and DSM‐IV diagnoses are in Table S1. CA diagnosis was verified by interview of family members. Postmortem mPFC  from 30 CA individuals , 15 African‐American (AA)) who died of cocaine intoxication or cocaine‐related cardiovascular toxicity and 30 age, gender, and ethnicity matched controls , who died of heart disease, other natural cause or non‐CNS trauma, were obtained from the University of Miami Miller School of Medicine Brain Endowment Bank2.2n = 84; 50.3% male) and AA  subjects who met DSM‐IV criteria for CA were acquired from the Rutgers University Cell and DNA Repository   through the NIDA Center for Genetic Studies in conjunction with Washington University in Saint Louis and the RUCDR. gDNA was diluted to 20 ng/μl in sterile water before use in genotyping experiments.De‐identified genomic DNA (gDNA) from EBV‐transformed lymphoblastoid cell lines of EA . Pelleted nuclei were suspended in 1× phosphate‐buffered saline containing 3 mM MgCl2, labeled with an AlexaFluor‐488‐conjugated anti‐NeuN antibody , counterstained with diamidino‐2‐phenylindole  and strained through a 30 μm filter‐cap tube  as described , lysed in 1× proteinase K digestion buffer , incubated at 56°C for 16 hr, and gDNA purified by chloroform extraction and ethanol precipitation in the presence of 20 μg molecular biology grade glycogen .Observations in human postmortem tissues suggest L1 retrotransposition events occur more often in neurons than in glia . Previously uncataloged (novel) L1 insertion sites identified by this L1‐seq pipeline were filtered for high quality using Excel . Quality control metrics for a putative, novel L1 insertion were that the reads aligned well to the reference genome , had greater than 98% average percent match (“mean matchpct”>0.98), at least six total reads across pooled populations  and at least two unique reads in a “peak” . Because the new L1‐seq analysis was done at a later time, the annotations of KNR L1s in the databases were more current. This resulted in some of the “novel” L1s detected using the “original” L1‐seq moving into the KNR L1 category.The second L1‐seq bioinformatics analysis used a new version of the L1‐seq bioinformatics program pipeline .We used Primer3  each sample's remaining gDNA before attempting PCR validations. The gDNA input into L1‐seq validation reactions was amplified by multiple displacement amplification performed on 10 ng of each NeuN‐positive or NeuN‐negative gDNA sample using a Repli‐G mini kit . PCR reactions were 25 μl containing 1X GoTaq Hot Start Master Mix , 10 ng WGA gDNA, and 0.2 μmol/L primers. Cycling parameters were 95°C–2 min 30 s, 35 cycles of 95°C–30 s, 50°C–30 s, 72°C–2 min, then 72°C–5 min, 4°C soak. We used two initial primer pairings: FS with ES, and FS with L1HsTAILSP1AP2 , which would amplify the genomic region surrounding or including, respectively, a putative L1 insertion  fluorescence. QuantaSoft v1.7.4 (Bio‐Rad) was used for data analysis and graph generation.We used L1Hs‐ and gene‐specific primers designed to amplify 200–250 bp products. An L1Hs‐specific probe sequence was as described  extracted total RNA (2 μg) from control or cocaine postmortem mPFC with RNA Integrity Numbers ≥6.5, assessed by Agilent RNA 6000 Nanochips with a Bioanalyzer 2100, were heat denatured with random hexamers (50 ng/reaction) and a oligo(dT)25VN primer VN‐3′). Ice‐quenched RNA samples were converted to cDNAs using Superscript III™ (Thermo‐Fisher Scientific) in 20 μl reverse transcription reactions incubated at 25°C–5 min, 42°C–5 min, 50°C–30 min, and then 55°C‐30 min. Reactions were terminated at 70°C–15 min. We diluted the cDNA to 0.5 ng/μl RNA equivalents before ddPCR using 1 ng RNA equivalents as input. We detected L1 mRNA with our double‐quenched, FAM‐labeled L1 probe , FAM‐labeled human ACTB or TBP (in simplex) gene expression primer‐probe assay  were used as reference. Parameters for ddPCR were as described for “Allele Frequency Determination”. Control reactions included cDNA reactions without Superscript III™ reverse transcriptase and no template (water) controls. QuantaSoft v1.7.4 (Bio‐Rad) was used for data analysis.Trizol2.9RRID:SCR_001881; Huang, Sherman, & Lempicki, RRID:SCR_004869; Mi, Poudel, Muruganujan, Casagrande, & Thomas, The L1‐seq bioinformatics analyses generated lists of genomic L1 positions within each studied population. We employed the DAVID . For each list, genes with L1s that overlapped the two populations were excluded or included in the analyses.2.10p‐values  software. Fisher's exact test was done with GraphPad Quickcalcs software at 33.1intragenic novel L1s were found only in the cocaine population (118 vs. 98), but more intergenic novel L1s were found only in the control population (130 vs. 101) such that the total number of novel L1s between the two populations did not differ significantly  than were found only in the control population  .Our rates of L1 detection were consistent with previous estimates for segregating L1Hs elements expected in a population of 60  Fig. , however3.2intragenic L1s detected by the “original” version of the L1‐seq bioinformatics pipeline , CCCTC‐binding factor (CTCF), dachshund family transcription factor 2 (DACH2), DEXD/H‐box helicase 58 (DDX58), macro domain containing 2 (MACROD2), and parathyroid hormone 2 receptor (PTHR2) (data not shown). All of these L1 insertions occur within an intron or promoter region of various gene transcripts (data not shown). Those near intron‐exon junctions  did not interrupt the branch point or splice site (data not shown). Some overlapped DNAseI hypersensitivity sites , were near binding sites for transcription factors  or were near a histone 3 lysine 27 acetylation peak .L1 retrotransposons were confirmed in Williams‐Beuren Syndrome chromosome region 17  or various L1 publications. Studies of L1s in the whole genome sequencing data of phase 1 of the 1000 genomes project have been published . The Sanger sequencing results of the cloned L1 3′ ends are in Appendix S5.DAB1 and TBCK L1s were found in two individuals, one in the control population and one in the CA population (data not shown). The WBSCR17, TET2, and SYBU L1 insertions were found in one individual in the CA population (data not shown). There was no evidence for regional mosaicism of the WBSCR17, TET2, or SYBU L1 insertions in the brains of the respective individuals as each L1 insertion detected in mPFC gDNA was also detected in cerebellar gDNA (data not shown). One caveat to these findings is that single cell neuronal gDNA, which may have revealed cellular somatic mosaicism  resulted in statistically significant GO term enrichments for L1 insertions into genes in the CA population that were not observed in the control population  revealed additional statistically significant GO terms and pathways among genes in the CA population that were not significant in the control population Table . Notably3.5RPPH1) allele assessed by ddPCR would be less than 50% . Both the novel SYBU L1 . This contrasts with the known nonreference L1 in JAK2  are polymorphic in the germline.In theory, if a novel L1 insertion created cellular somatic mosaicism in the mPFC, the ratio of the unknown (L1) to the reference . However, when absolute L1 mRNA levels were corrected for input cDNA quality and quantity by normalization with GAPDH, ACTB, TBP or the geometric mean (GEO mean) of the three different normalizers, relative ratios  were not significantly different between the two populations , 0.149 (ACTB), 0.0785 (TBP), and 0.547  and NeuN‐positive  gDNA of a single CA pool and/or individual . The KLHL1, DAB1, and TBCK L1 insertions were observed in multiple populations and/or individuals , but was subsequently found in the phase 3 dataset of structural variants of the 1000 genomes project  or potentially private L1s (TET2 and WBSCR17), are consistent with the hypothesis that polymorphic germline or early developmental de novo somatic L1s might be risk factors predisposing an individual to developing CA.The confirmed novel L1 insertions in SYBU) showed nominally significant association with disease in a cocaine addiction GWAS , to which cocaine binds, regulates the transporter by suppressing DAT channel activity . Interestingly, cocaine signaling through the σ‐1R receptor induced PDGF in the brain 24 hours after cocaine administration and induction of PDGF led to increased vascular permeability of the blood‐brain barrier  Unknown impact of any L1 on neuronal function; addressable by examining cultured neurons (made heterozygous for the L1 insertion) at baseline and after incubation with cocaine. Blockade of gliotransmission in mice prevents cocaine reinstatement  small sample size; correctable in subsequent studies. With a medium effect size, a population of 60 cocaine addicted persons and 60 control individuals gives 80% power to detect an L1 with a minor allele frequency of 5%. It is likely that L1s with low minor allele frequencies in each population were undetected because our population sizes were 30 individuals per group and we pooled individuals. ii) An unknown number of neurons carrying any a et al.  indicatea et al.  also shode novo mutations acting as antecedent risk factors for CA. Our current data do not indicate that acutely toxic cocaine use or chronic CA increased L1 transcription or retrotransposition in human mPFC. The presence of novel L1s in genes with enrichments in ontologies and pathways previously associated with the effects of cocaine suggests that L1 retrotransposition is a genetic mechanism warranting further study regarding its potential for influencing risk for CA.In summary, although a small fraction of the novel L1s detected by our study may be a molecular consequence of cocaine taking by CA individuals, most are likely germline or developmental HHK is on the scientific board of Transposagen Biopharmaceuticals, Inc. All other authors have no conflicts of interest to declare. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.