Mage = 67.4, SDage = 4.4) were assigned to a control condition, a neutral condition and a negative stereotype condition during three separate visits . In each physical activity session, participants performed the same training. Objective physical activity level was the time spent at moderate to vigorous intensity measured by accelerometry and subjective physical activity level was measured with the RPE-session method. Inactivity time was also objectively assessed. Results revealed no effect of the different conditions on objective physical activity level, but subjective physical activity level and inactivity time were lower in the neutral condition and in the negative stereotype condition compared to the control condition. It was suggested that when a negative stereotype is induced, participants perceived the task as less intense compared to the control condition, which result in less inactivity time, suggesting that the negative stereotype had a positive influence on physical activity. Another interesting result was that effects were similar in the negative stereotype condition and in the neutral condition, revealing that the neutral condition might not be a control condition. While these results are not in line with the stereotype threat literature, they echo previous recent studies also showing a positive effect of a negative stereotype induction, calling into question the stereotype threat theory.The present research examined the effect of a negative stereotype induction on older adults' physical activity level, measured objectively and subjectively. Twenty older adults (18 women and two men;  With the increase of age, a decrease in physical activity (PA) participation is mostly observed accompanied by a physical decline , which According to the ST theory, when a negative stereotype toward one group is induced, in a context where this stereotype can be applied, a decreased performance is expected . A largeAll studies conducted in the physical domain have the common point that they only focused on isolated tasks measuring physical capacities , and mostly in non-ecological situations. For example, in Marquet et al. , particiIn the literature, different methods have been used to quantify subjective and objective PA level. A subjective method commonly used is the Session-RPE method  based onPrevious studies have shown that PA level can be influenced by psychological factors such as personality traits, social comparison, or even stereotypes e.g., , 34, 35], 3534, 3Therefore, the main aim of the present research was to observe the effect of a negative stereotype induction on older adults' PA level measured objectively through accelerometry  as well as subjectively using the Session-RPE method. Based on the mixed results observed in the literature, we did not make hypotheses.Mage = 67.4, SDage = 4.4) were recruited. A sensitivity power analysis, based on a within-subject design with repeated measures, assuming an α of 0.05 and power of 0.80, indicated that our sample size allows us to detect moderate-to-large effects of f = 0.30. Participants were directly contacted during their sports class and provided informed consent. The inclusion criteria included: more than 60 years old, being identified with the physical activity domain, being affiliated to a physical activity group. The exclusion criteria included: any bone, muscle or tendon trauma of the limbs . According to the ST theory  and were consequently added in this study.Twenty older adults  ranging from 60 to 76 years old  it has greater statistical power  and (ii)Objective PA level and IT were measured with tri-axis accelerometers GT3X . Participants had to wear the accelerometer on the right side of the hip, adjusted with an elastic belt, during all training sessions. Actilife v-6.13.4 Lite Pro software was used to extract PA and IT values. Data were downloaded in 1 second epoch to measure PA and IT. According to Freedson algorithm, we defined IT as 0–99 counts per minute and MVPA > 1,952 per min .Subjective PA level was evaluated according to the Session-RPE method . After tDuring the three visits, separated by 7 days, participants performed the same physical activity session, lasting ~45–50 min at the same time of day. The session content is provided as During the first session , participants were greeted by a female experimenter. After giving consent, all participants responded individually to the domain identification questionnaire. Prior to the session, each participant was given an accelerometer. The protocol was then initiated and participants performed the PA lesson. At the end of each session, participants reported their perceived intensity. Participants came back to the fitness class 1 week later, were randomly assigned to another condition  and realized the same training lesson. Then, participants came back a last time to perform the final training session 1 week later in the condition to which they had not yet been assigned . During each session, as a stereotype manipulation check, participants were asked: “For this training session, your PA level will be compared to:” with three answers options: “younger people,” “people of the same age” or “older people.” All participants correctly answered the question in each session. Finally, they were thanked and debriefed.Given the within-subject nature of the design, we did not activate stereotypes by manipulating task diagnosticity, as is classically done in ST research . IndeedF-test. Since normality was assumed (ps > 0.05) for objective PA level and IT, a one-way ANOVA with repeated measures was conducted with Conditions  as a within-subject factor. Paired sample t-tests were used for comparison within groups and Bonferroni correction was used to adjust for multiple comparisons. For subjective PA level, normality was not assumed (ps < 0.05). A non-parametric Friedman ANOVA was thus conducted and Conover's post-hoc tests were used for comparisons within groups. Effect sizes were expressed as the eta-squared  for ANOVA analysis, as Cohen d  for t-tests and as Kendall's W index  for Friedman ANOVA.Variable data normality was checked using the Shapiro–Wilk test and homogeneity of variance with the To the best of our knowledge, no study has investigated the durability of the effect of the stereotype immediately after its induction. In other words, does the stereotype influence participants during the entire session or does its effect dissipate over time? Therefore, each session was also split into two times: T1 corresponding to the first 50% of the training session and T2 referring to the last 50% of the training session and a 3 (Conditions) x 2 (Time of measurement) ANOVA was conducted with Conditions and Time of measurement as within-subject factors.SDs for the study variables are presented in Means and F = 0.65; p = 0.53; η2 = 0.04), suggesting that the different conditions had no effect on PA level  = 9.29; p < 0.001; η2 = 0.34). Specifically, IT was higher in the control condition  compared to the neutral condition  and to the negative stereotype condition . There was no significant difference in IT between the neutral condition and the negative stereotype condition  = 17.23; p < 0.001; Kendall's W = 0.58). Subjective PA level was higher in the control condition  compared to the neutral condition  and to the negative stereotype condition . There was no significant difference in IT between the neutral condition and the negative stereotype condition  = 0.65; p = 0.53; η2 = 0.04) but a Time of measurement main effect  = 37.47; p < 0.001; η2 = 0.68). More importantly, there was no significant Condition x Time of measurement main effect  = 1.51; p = 0.24; η2 = 0.08).For objective PA level, the ANOVA revealed no Condition main effect  = 9.29; p < 0.001; η2 = 0.34), a Time of measurement main effect  = 69.11; p < 0.001; η2 = 0.79), as well as a Condition x Time of measurement interaction effect  = 3.3; p = 0.05; η2 = 0.16). Specifically, at T1, participants' IT was higher in the control condition  compared to the neutral condition  and to the negative stereotype condition . There was no significant difference in IT between the neutral condition and the negative stereotype condition (p = 1.0). At T2, there was no significant difference between the three conditions concerning IT . This complementary analysis revealed that the effects on IT were present only during the first part of the session.For IT, results revealed a Condition main effect , but subjective PA level  and IT were lower in the neutral condition and in the negative stereotype condition compared to the control condition. Overall, these results are not in line with the ST theory and are worthy of discussion.Firstly, as evoked, there was no significant difference between control and negative stereotype conditions concerning objective PA level. In other words, the time spent at moderate to vigorous PA intensities was similar between the two conditions. Even if this result is not in line with the ST theory, it seems however in line with most recent studies conducted in older adults showing no effect of the negative stereotype induction on objective variables in the physical domain e.g., , 16, 18], 1816, 1However, subjective PA level was impacted. Subjective participants' PA level was significantly lower in the negative stereotype condition compared to the control condition. This result suggests that participants in the negative stereotype condition felt that their PA level was lower even if it was objectively similar to the control condition. This mixed result for the same variable measured with different tools is not that surprising. Indeed, past studies have also shown that the effects of negative stereotypes could be different according to the tools used e.g., , 33]. Fo. Fo33)].Subjective PA level was measured with the “Rate of Perceived Exertion”  and previous studies have found that this variable can be influenced by a ST situation e.g., , 38]. Gr. Gr38)].In sum, we found no effect on objective PA level but a positive effect on subjective PA level and on objective IT. These positive results echo previous recent studies also showing a positive effect of the induction of a negative stereotype, calling into question the ST theory e.g., , 43, 47], 4743, 4A last interesting result is that effects observed in the negative stereotype condition were also found in the neutral condition. Specifically, even if objective PA level was not affected, participants in the neutral condition had a lower subjective PA level and were less inactive than in the control condition. These results suggest that the neutral condition may be not a control condition. Some previous studies conducted among older adults investigated the effects of ST by only comparing the neutral condition and the negative stereotype condition , 15. ChaDespite interesting results, the current study presents some limits and perspectives. Firstly, even if the sample size is consistent with previous studies conducted in older adults e.g., , 19], it, it19)],To conclude, the present research found that a negative stereotype induction had no influence on objective PA level but have a positive effect on IT and subjective PA level during a training session, which is contrary to the ST theory. Moreover, it appears necessary in future research to add a control condition when investigating ST since the neutral condition seems not to be a control condition.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.MD: conceptualization, methodology, formal analysis, writing—original draft, supervision, and data curation. AP: conceptualization, methodology, and investigation. KK: methodology and formal analysis. AGP: conceptualization, methodology, formal analysis, and writing—original draft. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exosomes, as nano-sized extracellular vehicles acting as cell-to-cell communicators, are novel promising therapeutics in the area of bone tissue engineering. Moreover, magnetic nanoparticles, whose integration with other appropriate components is viewed as an intriguing approach to strengthen bone tissue engineering efficacy. We investigated the effect of magnetic enriched with exosomes on osteogenic differentiation.Exosomes were isolated from human adipose-derived mesenchymal stem cells by Exo-spin™ kit (MSC-EX). Alginate (Alg) scaffold containing 1% (w/w) cobalt ferrite nanoparticles (CoFe2O4) was produced. MSC-EX were gently loaded onto Alg and Alg-cobalt ferrite (Alg-CF) scaffolds yielding Alg-EX and Alg-CF-EX scaffolds. The effects of MSC-Ex and magnetic hydrogel composite under an external static magnetic field (SMF) on proliferation and differentiation of MSCs were evaluated by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and energy dispersive X-ray (EDX) analysis.Our results showed that Alg and Alg-CF scaffolds were not only cytotoxic but also supported AdMSCs proliferation. MSC-EX loading of the scaffolds enhanced AdMSCs proliferation significantly. According to the results, Alg-CF-EX scaffolds under magnetic stimulation exhibited the most potent effect on osteogenic differentiation of cultured AdMSCs as evidenced by higher ALP activity and mineralization.We provided evidence that the combination of Alg hydrogel, CFNPs, and MSC-EX resulted in the construction of a bone tissue-engineering scaffold that highly supports the osteogenic commitment of MSCs.  Mesenchymal stem cells (MSCs) have been extensively investigated for tissue repair and regeneration , 2. DireRecently, magnetic nanoparticles (MNPs) have attracted rising interest in the field of biomedicine. For instance, MNPs have been widely applied as drug carriers to facilitate targeted drug delivery  as contrin vitro. Indeed, we prepared a novel bone regeneration scaffold combining a hydrogel scaffold with MNPs and MSC-EX, as three different elements stimulating bone tissue regeneration. Furthermore, we will investigate the synergistic effect between the magnetic scaffold and exosome osteogenic differentiation. In the present study, we aimed to develop a cell-free bone engineering scaffold comprising alginate hydrogel, cobalt ferrite nanoparticles (CFNPs), and exosomes isolated from adipose-derived MSCs (AdMSC-EX) and investigate its osteoinductive potential Isolation of adipose-derived mesenchymal stem cells (AdMSCs)2. culture media were changed every 3–4 days until the cells grew about 90% in confluency. The cells were passaged at the confluence of 90% using 0.25% trypsin-EDTA (Ethylenediaminetetraacetic acid). Characterization of the obtained AdMSCs was performed by a combination of morphologic, flow cytometric, and differentiation analysis (adipogenic and osteogenic). For flow cytometric characterization, AdMSCs were evaluated for cell surface expression of different markers including cluster of differentiation . The cells at passage 3 (P3) were analyzed for their ability to differentiate into osteoblasts and adipocytes. For this, the cells were cultured with the induction medium in 6-well plates. For osteogenic differentiation, stem cells were first seeded in each well of a 6-well plate at a density of 50000 cells/well. When the cells were 90% in confluency, the culture medium was replaced with osteogenic induction medium (DMEM supplemented with 10% FBS+ 7-10 M dexamethasone+ 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid-2-phosphate (Sigma). After 21 days, mineral deposition was assessed by Alizarin red staining (Sigma). For adipogenic differentiation, cells were cultured with the adipogenic medium (containing 50 µg/ml ascorbic-2-phosphate+ 7-10 M dexamethasone and 50 µg/ml indomethacin (Sigma) for 21 days. Finally, the cells were stained by Oil Red O (Sigma) to assess adipogenesis.In this experimental study, to isolate adipose-derived MSCs (AdMSCs), adipose tissue samples were collected under sterile conditions from patients 16– 45 years old, undergoing general abdominal operation in Shahid Sadoughi Hospital, Yazd, Iran, after obtaining informed consent. All procedures were approved by Shahid Beheshti University of Medical Sciences Ethical Committee, Tehran, Iran (Ethic code “IR.SBMU.REC.1399.020”). The samples in sterile phosphate-buffered saline (PBS) were transferred to the research laboratory of Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences. Tissue samples were first ground with a sterile surgical blade. Then, they were treated with collagenase I  and incubated for 20 min at 37 °C to yield a homogenous suspension. Afterward, the resultant suspensions were centrifuged at 2000 RPM for 20 min. Cell pellets were cultured in Dulbecco’s Modified Eagle Medium high glucose  supplemented with 20% fetal bovine serum , 100 µg/ml streptomycin and 100 IU/ml penicillin in a standard incubator with 37 °C humidified air and 5% COExosome isolation and characterizationMSCs were cultured in T-75 flasks. When the cells grew about 80% in confluency, culture media were replaced with DMEM media supplemented with 10% exosome-free FBS. The conditioned media were collected after 48 hr and stored at -20 °C. Before exosome isolation, the collected conditioned media were concentrated using Amicon Ultra centrifugal filters. Exosomes were isolated using an Exo-spin kit  in accordance with the manufacturer’s instructions. Transmission electron microscopy (TEM) analysis was carried out to observe the morphology of the isolated exosomes. Exosome suspension was first fixed using 1% glutaraldehyde and a drop was placed on a carbon-coated grid and left at RT to dry. The grids were then washed with sterile PBS twice, followed by staining with 1% uranyl acetate for 10 min. The samples were analyzed using an Em208s 100kv . In the next step of exosome characterization, dynamic light scattering (DLS) was used for determining the size distribution of the isolated exosomes by means of a Malvern Zeta sizes Nano ZS . Isolated exosomes were quantified based on their total protein concentration by Bradford assay protocol using bovine serum albumin (BSA) as standard . Scaffold construction3)3) was drop-wisely added to cobalt nitrate (Co (NO3)2) aqueous solution. The pH of the resultant solution was adjusted to 11 by addition of ammonia solution. Then, polyvinyl pyrrolidone (PVP) solution was added followed by stirring for 15 min. Finally, the obtained solution was placed in a 180 °C oven for 3 hr. To construct alginate (Alg) scaffolds, a 1% aqueous solution of sodium alginate (Sigma) was first prepared and stirred. Then, this solution was gently added to calcium chloride solution (3%) and the mixture was left at RT for 15 min until gel formation. The remaining calcium chloride solution was removed and the prepared hydrogels (Alg scaffold) were freeze-dried. For construction of Alg-CF scaffolds, different concentrations of CFNPs were added prior to freeze-drying  of ferric nitrate (Fe (NOe-drying . Scaffold characterizationScanning electron microscopy (SEM), TEM, and DLSTo observe the cross-sectional morphology of the prepared scaffolds, SEM analysis was conducted . The samples were coated with gold and the analysis was accomplished at an accelerating voltage of 30 kV. Also, the synthesized nanoparticles were analyzed for their morphology and size distribution by TEM and DLS, respectively.Fourier transform infrared spectroscopy (FTIR)-1 using an FTIR spectrometer .To evaluate the chemical integrity of the prepared scaffolds, freeze-dried scaffolds were first ground and the powder was used for FITR analysis. FTIR estimations were recorded within the wavelength range of 400–4000 cmX-ray diffraction (XRD) analysisXRD is a useful technique to confirm the crystalline or amorphous nature of materials. XRD patterns were registered  in the 2θ range of 5–50°.Vibrating sample magnetometer (VSM) analysisMagnetic evaluation of Alg-CF scaffolds with different concentrations of CFNPs  was carried out on a magnetometer at the physiological temperature (310 K) at Kashan University, Iran. Porosity measurementS. A 10 ml centrifuge tube was filled with ethanol and weighed as W1. Specimens of each scaffold were immersed in the ethanol-containing tubes and the total weights were recorded as W2. Then the scaffolds were taken out and the remaining mass of ethanol and 10 ml tubes after scaffold removal were designated as W3. Finally, porosity measurement (%) was performed using the following equation: Porosity (%) = (W2 – W3 – WS) ∕ (W1 – W3) × 100Dry weights of prepared Alg and Alg-CF scaffolds were designated as WContact angle measurementThe hydrophilicity of the scaffold network is evaluated by measuring the angle (θ) between the surface of the scaffold with the line tangent to the water droplet. To do this, one droplet of distilled water (5 µl) was placed on the surface of specimens of each scaffold and after 3 sec, photographs were taken. In order to measure contact angles, the Digimizer software (Version 5.4.6) was utilized. Compressive strength testThe mechanical fate of the scaffolds was analyzed by the compressive strength test. The compressive strength of the scaffolds was measured according to the ASTM F2150-7 standard by means of a Zwick/Roel machine . The elastic modulus size of the scaffold was calculated from the elastic area line slope of the obtained curve. Scaffold specimens were loaded at the speed of 1 mm/min.AdMSCs monitoring in the structure of scaffoldsAlg and Alg-CF scaffolds were washed with 70% alcohol three times and then exposed to UV light for one hour. Then, AdMSCs  were gently seeded on the surfaces of Alg and Alg-CF scaffolds and incubated at 37 °C in DMEM supplemented with 10% FBS. After culture, scaffolds were fixed using glutaraldehyde solution (2.5% in PBS) for 2 hr followed by dehydration in serial alcohol concentration. The scaffolds were characterized by SEM. Also, 4′, 6-diamidino-2-phenylindole (DAPI) staining was carried out on days 7 and 14 to confirm the adhesion and presence of cells on the scaffolds. DAPI solution was added to the cultures and after that, the scaffolds were observed by a fluorescent microscope. Cell viability assayTo evaluate the cytocompatibility of the scaffolds, an MTT assay was performed(18). To this end, Alg and Alg-CF scaffolds with different concentrations of CFNPs  were prepared. To investigate the effect of a magnetic field on AdMSC proliferation, some of the wells were stimulated by a static magnetic field (SMF) with a magnetic field strength of 125 mT. After scaffold sterilization, in every well, a total number of 50,000 AdMSCs were gently seeded on the scaffolds. Cell viability was assessed after 4, 7, and 14 days of incubation in a standard cell culture incubator. To examine the effects of MSC-EX on the viability and proliferation of the cells, 100 µl volumes of the isolated MSC-EX were loaded onto Alg and Alg-CF scaffolds to yield Alg-EX and Alg-CF-EX scaffolds. Similar to the previous section, an MTT assay was conducted., these experiments were performed under both non-induced and induced conditions. In wells considered as the negative control, only AdMSCs were cultured. All experiments were repeated at least in triplicates.Alkaline phosphatase activityTM (Sigma-Aldrich). After centrifugation at 20,000 g for 10 min, supernatants were transferred to another tube. Then, Alkaline Phosphatase Yellow (pNPP) Liquid Substrate (Sigma-Aldrich), as ALP substrate solution, was added at a 1:1 ratio and the tube was incubated in the dark at room temperature for 30 min. Afterward, NaOH solution (3 M) at an ALP substrate was added and optical density (OD) was read at 405 nm.Alkaline phosphatase (ALP) is involved in the catalysis of p-nitrophenolphosphate hydrolysis into p-nitrophenol and phosphate. ALP activity in different groups was estimated on days 7, 14, and 21 after addition of osteogenic media. To measure ALP activity at every time interval, the adherent cells were lysed for 15 min with CelLyticExtracellular matrix mineralizationAlizarin red is an organic compound capable of specifically staining the mineralized matrix of the cultured cells. The intensity of red staining corresponds to the level of cell-matrix mineralization and osteoblast formation. The test was performed on day 21 after osteoinduction. After removal of osteogenic media and washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 1 hr. Subsequently, 1 ml of alizarin red solution  was added to each well. After 1 hr, the wells were rinsed with PBS. Finally, after the addition of acetic acid, the ODs of the wells were read at 405 nm.Dispersive x-ray (EDX) analysisSEM-EDX analysis was performed on day 21 to detect and confirm mineralization  following osteogenic differentiation. Cultures were first fixed in 4% paraformaldehyde at room temperature. SEM-related images were taken using an energy-dispersive X-ray spectrometer  which was attached to the JEOL JSM-6500F microscope. Surfaces were analyzed at 15kV for 5 min.Statistical analysispost hoc test was used. For all tests, statistically significant differences were accepted for P-values less than 0.05The acquired data were expressed as Mean ± standard deviation (SD). The data of each test were obtained from three independent experiments. Analysis of the data was performed using GraphPad Prism software (Version 8.0.1). To make comparisons between the groups, one-way ANOVA followed by Tukey’s Characterization of isolated AdMSCsThe isolated AdMSCs at passage 3 (P3) exhibited a spindle-shaped morphology similar to fibroblast cells indicating morphologic characteristics of undifferentiated MSCs . This obMSC-EX isolation and characterizationTM kit. The results of TEM analysis showed that the vast majority of the isolated particles possessed membranous cup-shaped structures similar to the previously reported morphological structure of exosomes . This broadband was related to O–H stretching of H2O molecules attributed to the chemical adsorption of molecules of water at the surface of CFNPs  in comparison with the Alg scaffold and the control group. Based on the obtained results, an Alg-CF scaffold containing 0.1 gr CFNPs was selected for further experiments. To investigate the impact of MSC-EX on cellular proliferation on the scaffolds, MTT assays were again performed. MSC-EX was loaded on both Alg and Alg-CF (0.1 gr) scaffolds and proliferation of the cultured AdMSCs on the respective scaffolds under both non-induced and induced conditions were assessed after cell seeding. Based on The viability and proliferation of AdMSCs on the synthesized scaffolds on days 4, 7, and 14 after cell seeding were evaluated by MTT assay. The results elucidated that in all groups, AdMSCs were alive at each time interval evaluated. As demonstrated in ALP assayP<0.0001). In both types of scaffolds, that is Alg and Alg-CF, ALP activity increased in a time-dependent manner. As shown in P<0.0001). Also, MSC-EX loading promoted ALP activity. Comparison of ALP activity in the scaffolds with and without EX showed that at all time intervals, MSC-EX loading significantly showed increased ALP activity (P<0.0001). These results show synergistic effects of CFNPs and MSC-EX on the enhancement of ALP activity and hence the osteogenesis process so that the scaffolds containing both CFNPs and EX (Alg-CF-EX) exhibited the highest level of ALP activity under induced conditions . Also, EX loading was found to enhance matrix mineralization, so that calcium deposition reached its maximum level in the Alg-CF-EX group. According to P<0.0015), Alg-EX, and Alg-CF-EX (P<0.0001) wells. In the alizarin red staining analyses, we could apprehend the mentioned synergy in the Alg-CF-EX group. This analysis was conducted on day 21 post osteoinduction to stain calcium deposits produced by differentiated cells. Both Alg and Alg-CF scaffolds under non-induced and induced conditions showed calcium deposition. This indicates that the cultured cells had successfully differentiated into osteoblast-like cells. mineralization was significantly higher in Alg-CF, Alg-EX, and Alg-CF-EX wells compared with Alg and negative control wells , confirmed the presence of higher calcium deposits in Alg-CF compared with Alg scaffolds) In the current study, we assessed the potential of an alginate-based bone engineering scaffold containing CFNPs and MSC-EX. To the best of our knowledge, this is the first study to examine the osteogenic differentiation ability of a synthesized tissue-engineered scaffold incorporating three different components of alginate hydrogel, CFNPs, and MSC-EX. Cell proliferation was found to be higher on Alg-EX and Alg-CF-EX when compared with Alg and Alg-CF, respectively, bringing to mind the notion that AdMSC-EX might be a more potent inducer of cell proliferation than CFNP. Then, we investigated the Osteogenic differentiation properties of the scaffolds by ALP assay, alizarin red staining, and SEM-EDX analysis. Based on our results the highest activity of ALP was observed in the Alg-CF-EX scaffold indicating the positive synergistic effects of CFNPs and MSC-EX on the ALP activity and hence the osteogenesis process. The alizarin red test comparison between the potential of MSC-EX and CFNPs suggests that MSC-EX induces a higher calcium deposition as we found a higher calcium deposition in the Alg-EX group compared with Alg-CFNP group even in the presence of the magnetic field. Finally, the results of SEM-EDX analysis showing higher levels of calcium and phosphorus in the Alg-CF-EX group were in line with the alizarin red test and corroborated the highest level of osteogenic induction when the three different elements of hydrogel, MNP, and exosome are combined .in vitro osteogenic differentiation of AdMSCs. Another arm of our synthesized scaffold is related to MNP and magnetic field. Previous studies have focused mainly on iron oxide MNPs. These MNPs have some limitations including weak magnetic properties under physiological conditions and at small sizes. In the present study, we utilized CFNPs. In comparison with iron oxide MNPs, CFNPs have excellent mechanical hardness and physiochemical characteristics with higher stability and colloidal dispersibility at physiological conditions under both in vitro and in vivo conditions. Their findings indicated that SMF-stimulated cell-laden hydrogels exhibited higher mineralization as well as faster vascularization (which is needed for bone reconstruction) compared with MNPs or SMF alone(3O4) by a three-dimensional (3D) bioplotter. It was shown that this nanocomposite scaffold significantly induced the proliferation of human bone marrow-derived stem cells (BMSCs), ALP activity, ECM mineralization, and expression of osteogenic genes . Iron oxide nanoparticles have been shown to promote osteogenic differentiation of BMSCs through induction of the mitogen-activated protein kinase (MAPK) pathway , and polylactic acid (PLA), it was demonstrated that addition of MNPs and the use of a pulsed electromagnetic field (PEMF) did not affect the proliferation rate while MNPs combined with PEMF exerted a positive effect on osteogenic differentiation of BMSCs  scaffolds were not only cytotoxic but also supported the viability/proliferation of the cultured AdMSCs. We provided evidence that magnetic composites containing exosomes can be more effective. In the current study, we developed a novel cell-free bone engineering scaffold composed of three distinct components of Alg hydrogel, CFNPs, and MSC-EX and evaluated FS Contributed to all experimental work, drafting, and statistical analysis. SH and MS Provided scientific consulting and contributed to concept and design. AJ Contributed to the experimental work and data collection. HN Provided scientific consulting, English and scientific review and editing, and final approval of the manuscript. AKH was responsible for overall supervision. All authors read and approved the final manuscript.The authors have no conflicts of interest.


This study comparatively analyzed the hemodynamic changes resulting from various simulated stent-assisted embolization treatments to explore an optimal treatment strategy for intracranial vertebral artery fusiform aneurysms. An actual vertebral fusiform aneurysm case treated by large coil post-stenting (PLCS) was used as a control.A single case of an intracranial vertebral artery fusiform aneurysm underwent a preoperative and eight postoperative finite element treatment simulations: PLCS [single and dual Low-profile Visualized Intraluminal Support (LVIS)], Jailing technique  with or without large coils (LCs). Qualitative and quantitative assessments were performed to analyze the most common hemodynamic risk factors for recurrence.Jailing technique and PED-only had a high residual flow volume (RFV) and wall shear stress (WSS) on the large curvature of the blood flow impingement region. Quantitative analysis determined that PLSC and PED had a lower RFV compared to preoperative than did the jailing technique [PED+LC 2.46% < PLCS 1.2  4.75% < PLCS 1.1 (single LVIS) 6.34% < PED 6.58% < Jailing 2.2 12.45% < Jailing 1.2 12.71% < Jailing 1.1 14.28% < Jailing 2.1 16.44%]. The sac-averaged flow velocity treated by PLCS, PED and PED+LC compared to preoperatively was significantly lower than the jailing technique [PED+LC = PLCS 1.2  17.5% < PLCS 1.1 (single LVIS) = PED 27.5% < Jailing 1.2 = Jailing 2.2 32.5% < Jailing 1.1 37.5% < Jailing 2.1 40%]. The sac-averaged WSS for the PLCS 1.2  model was lower than the PED+LC, while the high WSS area of the Jailing 1 model was larger than for Jailing 2 [PLCS 1.2 38.94%  < PED+LC 41% < PLCS 1.1 43.36% (single LVIS) < PED 45.23% < Jailing 2.1 47.49% < Jailing 2.2 47.79% < Jailing 1.1 48.97% < Jailing 1.2 49.85%].For fusiform aneurysms, post large coil stenting can provide a uniform coil configuration potentially reducing the hemodynamic risk factors of recurrence. Flow diverters also may reduce the recurrence risk, with long-term follow-up required, especially to monitor branch blood flow to prevent postoperative ischemia. Fusiform aneurysms are more prone to occur in the posterior circulation . IntracrHemodynamically, wall shear stress (WSS) is an important risk factor for aneurysm rupture. Low WSS induces destructive remodeling caused by inflammatory cells, resulting in aneurysm instability. Higher than normal WSS also can result in the enlargement and rupture of aneurysms based on other mechanisms . This stHowever, no CFD mechanism-related studies exist on the potential recurrence risk among different reconstruction techniques for intracranial vertebral artery fusiform aneurysms. This present study modeled an actual case of vertebral artery fusiform aneurysm treated with PLCS without considering thrombosis, simulating and comparing the preoperative hemodynamic effects and eight post-operative finite element treatment simulations– PLCS x 2 ), Jailing technique x 4 (single and double LVIS both simulated twice with different coil configurations), and 2x Pipeline Embolization Device (PED) with or without large coils (LCs) – to analyze the most common hemodynamic risk factors for recurrence.A man in his 40 s with an intracranial fusiform aneurysm in the dominant vertebral artery experienced a sudden headache once 2 months ago. The left vertebral artery fusiform aneurysm diagnosed on MRI in a local hospital was treated with PLCS techniques  using Siemens equipment Axiom Artis Zeego, Siemens Medical Solutions, Erlangen, Germany). The acquired raw data were reconstructed in Mimics 17 software  to generate STL files subsequently imported into Geomagic 12 software for model repair, trimming, and smoothing .A two-step finite element simulation of stent deployment was devised . Firstly−8kg/m3, Young's modulus of 10,000 Pa, and Poisson's ratio of 0.39 (The simulation of the coil insertion process was carried out in ABAQUS . The pro of 0.39 . The thr*20 mm) was determined by two neuro-interventionists with over 10-year experience.The simulated hemodynamics of the preoperative untreated model was adopted as baseline parameters. The jailing technique with coiling was conducted twice, to simulate both separated and connected coils. A total of eight postoperative treatment options were simulated : 1. PED 3 and a viscosity of.0035 kg/m·s . A grid independence test was performed to determine the appropriate grid size for the stability of the calculation outcomes and the efficiency calculation. Due to the different geometric dimensions of vessels, stents, and coils, the mesh sizes of different object surfaces are determined to various values. The grid size for the stent wire surface was finally set to 1/6 of the circumference of the wire. The artery and coil surface were 0.16 mm along with the 0.03 mm LVIS surface and the 0.015 mm PED surface grid. A three-layer boundary mesh was added to improve the accuracy of the simulation results in the near-surface region of the model. Final mesh calculations were generated as follows: 3 million for the preoperative model; 96 million for PED and PED with large coil; 45 million for Jailing1.1, 1.2, and PLCS1.1; 65 million for Jailing 2.1, 2.2, and PLCS1.2. The hemodynamic simulations were fitted with the Navier-Stokes equations for steady-state simulations using ANSYS CFX version 2019 . The blood was designed as an incompressible, laminar flow, Newtonian fluid with a density of 1,056 kg/m5 kg/m·s . The ves5 kg/m·s . Outlet 5 kg/m·s . A steadQualitative and quantitative methods were used to analyze and compare the flow velocity, WSS, and RFV (v >0.03 m/s)  in the aRFV was discerned in the sac after PED implantation alone. According to the simulated projection and down-the-barrel view, the Jailing technique had a higher RFV on the large curved side due to the non-uniformity of the coil embolization, more similar to PED-only implantation than the RFV values of the PED+LC and PLCS techniques , 5.Both PLCS and PED with or without large-coils had lower residual percentages of RFV than the Jailing technique compared with pre-operation. The residual percentage of RFV of for dual stents in PLCS and Jailing are smaller than for the single stent [PED + LC 2.46% < PLCS 1.2  4.75% < PLCS 1.1 (single LVIS) 6.34% < PED 6.58% < Jailing 2.2 12.45% < Jailing 1.2 12.71% < Jailing 1.1 14.28 % < Jailing 2.1 16.44%; The streamline diagram and quantitative analysis showed that the averaged flow velocity in the aneurysm sac after PLCS for PED with and without large-coils decreased significantly more than for the Jailing technique. The sac-averaged flow velocity for dual stents in PLCS and Jailing technique are smaller than single stent [PED+LC = PLCS1.2 (double LVIS) 17.5% < PLCS1.1 (single LVIS) = PED 27.5% < Jailing1.2 = Jailing2.2 32.5% < Jailing1.1 37.5% < Jailing2.1 40%; WSS values of all postoperative models decreased significantly compared with pre-operation. Jailing 1 experienced a larger high WSS region than Jailing 2 .WSS from the PLCS 1.2 (double LVIS) had the largest decline [PLCS1.2  38.94% < PED+LC 41% < PLCS1.1 (Single LVIS) 43.36% < PED 45.23% < Jailing2.1 47.49% < Jailing 2.2 47.79% < Jailing 1.1 48.97% < Jailing1.2 49.85 %; The main conclusion of this study is that PLCS and PED with or without large-coils can significantly decrease the hemodynamic risk factors of recurrence in the treatment of fusiform vertebral aneurysm compared with the jailing technique. Hemodynamic studies have shown that high WSS, large RFV, and high-velocity areas after interventional treatment are risk factors for aneurysmal recurrence , 19, 30.Quantitative analysis showed hemodynamic risk factors for recurrence after jailing was also confirmed higher than PLCS and PED techniques due to incomplete embolization from the micro-catheter fixed by the stent . Hong etProcedure critical points of the PLCS technique on coil, stent, and embolizing microcatheter selection should be emphasized for fusiform aneurysms. Coil packing has some inherent randomness in clinical practice. Coil configuration for PLCS should achieve a more even distribution than the jailing technique. The framing coils (diameter ≥ aneurysm width) often require repeated adjustment contributing to uniform coil distribution. While protecting the patency of the involved branches, semi-deployment of the stent can assist in framing the large coils. Soft coils with a smaller primary helix diameter were preferred to result in stent well-apposition. The proper amount of large-coils –4 is norThis study has the following limitations. This proof-of-concept study was a single case. However, it effectively demonstrates hemodynamic effects after treatment for different stent-assisted techniques. Comprehensive intracranial stents such as Solitaire and Neuroform were not tested. In addition, not all treatment strategy combinations were simulated such as Jailing + Pipeline. Since a Murray flow outlet was employed in this study, the flow rate change of the PICA branch was inapplicable. The pressure outlet should be a feasible way to evaluate the influence on the flow rate of PICA. Flow change may be affected by thrombus and stent endothelialization. This study did not address the degree of vascular curvature which can affect the different stent-assisted strategies. The coil distribution from the PLCS technique is somewhat random and the simulation cannot be completely consistent with actual placement. This CFD study used common assumptions such as rigid walls and a lack of specific inflow and outflow tract flow conditions.For fusiform aneurysms of the intracranial vertebral artery, the PLCS technique can more uniformly pack aneurysm sacs and may reduce the hemodynamic risk factors of recurrence similar to flow diverters, though long-term follow-up is still required. Attention should be paid to the impact on any perforators or branches to lessen the ischemic risk.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institution Review Board of Huashan Hospital affiliated to Fudan University approved this retrospective study and waived the requirement for informed consent. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.XZ and JX conceived and designed the research and handled the funding and supervision. YJ acquired the data and drafted the manuscript. YJ, GLu, LG, GLi, and RZ analyzed and interpreted the data. YJ, XL, and HW performed the statistical analysis. All authors made critical revisions to the manuscript for important intellectual content and reviewed the final version of the manuscript.This work was partially supported by the National Natural Science Foundation of China (No. 81771242).GLi, RZ, XL, and JX were employed by the company ArteryFlow Technology Co., Ltd. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A series of difunctional epoxy methacrylate resins (EAs) containing at least one epoxy and at least one methacrylate group were synthesized by means of an addition reaction between epoxy-terminated diglycidyl ethers and methacrylic acid. In order to investigate the impact of polymer architecture on the course of addition reactions and further coating properties, several different types of diglycidyl ethers, i.e., linear, containing aliphatic or aromatic rings, with a short or polymeric backbone, were employed in the synthesis. The carboxyl–epoxide addition esterification reactions have been found to, in a relatively straightforward manner, control the extent of acrylation depending on the substrate feed ratio and reaction time. The structure of obtained pre-polymers was evaluated by FT-IR and NMR methods. At the same time, the extent of addition reactions was validated via quantitative analysis, including non-volatile matter content (NV), acid value (PAVs), and epoxy equivalent value (EE) analysis. The modification was carried out in a manner likely to create a compound with one epoxy and one carbon–carbon pendant group. Hence, due to the presence of both functionalities, it is possible to crosslink compositions based on synthesized EAs via two distinct mechanisms: (i) cationic polymerization or (ii) free-radical polymerization. Synthesized epoxy methacrylate pre-polymers were further employed for use in formulate photocurable coating compositions by the cationic or radical process. Furthermore, the photopolymerization behavior and properties of cured coatings were explored regarding some structural factors and parameters. The investigated polymeric materials cure in a short time to obtain coatings with good properties, which is why they can be successfully used to produce protective and decorative coatings for many industries. Light-induced polymerization is used primarily for the production of coatings, adhesives, printing inks, dental fillings, and hydrogels, as well as 3D-printed elements ,5,6,7. PThere are two main types of photopolymerization, depending on the type of reactive particles formed during the decay of the photoinitiator : radicalThe chemical structure of UV-curable resin can be roughly divided into four groups: epoxy acrylates, polyester acrylates, urethane acrylates, and silicone acrylates. Among these, the epoxy acrylates, so-called vinyl ester resins (VERs), have been found especially desirable in applications such as UV-curable paints and varnishes . ConventIn contrast to the numerous papers on VERs, only a few works dealing with multi-functional monomers/resins having two types of photopolymerizable groups in the structure have appeared until now ,20,21,22It cannot be overlooked that EA pre-polymers with both epoxy and vinyl end-groups can polymerize via two different polymerization mechanisms—cationic or radical ,26,27,28Our previous study  presenteFirst, the diglycidyl ethers were modified with methacrylic acid, and then the reaction products were used to produce varnish coatings. The as-synthesized products were characterized in terms of non-volatile-matter content (NV), partial acid numbers (PAVs), MAA conversion (MAAC), epoxy equivalent (EE), conversion of epoxy group (EGC), and viscosity (η). The basic physicochemical properties are presented in −1 and 1633 cm−1 at MAA can be attributed to C=O stretching and C=C vibrations, respectively. As shown in −1 at the DGEBA-MAA spectrum. This phenomenon can be reasonably ascribed to the formation of ester bonds due to the reaction of carboxyl and epoxide groups of MAA and DGEBA, respectively , where EE0 is the EE (g/mol) determined for diglycidyl ethers’ prior modification; EEmeasured—EE value at a specific time.The epoxy equivalent (EE) was determined using the colorimetric titration method . The epoThe viscosity was measured at room temperature using a cone-plate-type viscometer (LAMY REOLOGY RM-100 plus CP 2000).2, Excelitas Technologies, Waltham, MA, USA). The weight of samples 15 ± 0.1 mg was placed into an open aluminum liquid DSC pan. The measurements were carried out under identical conditions. The sample was maintained at a prescribed temperature for 0.5 min before each measurement run began. The polymerization solution was composed of epoxy methacrylate resin and 3 wt% of photoinitiator. The reaction heat released in the polymerization is directly proportional to the number of epoxide (C) or methacrylate (R) groups reacted in the system. By integrating the area under the exothermic peak, the conversion of photoreactive groups or the extent of reaction was determined according to eqn: p (%) = (ΔHt/ΔH0) * 100, where ΔHt is the reaction heat evolved at time t, and ΔH0 is the theoretical heat for complete conversion. A reaction heat (ΔH0) for an epoxide groups polymerization of 110 kJ mol−1 and methacrylate double bond polymerization of 58 kJ mol−1 was used. The rate of polymerization is directly related to the heat flow (dH/dt) as in the following equation: Rp (%/s) = (dH/dt)/ΔH0.The UV-curing process was isothermally monitored (25 °C) for 10 min using a photo-DSC apparatus  equipped with UV light emitter Omnicure S2000 , pendulum hardness test, cross-cut adhesion test, gloss, and yellowness index. The hardness of coatings was evaluated using a Persoz pendulum on the glass substrate  according to ISO 1522 standard. The gloss was measured by means of the spectrometer method  according to ASTM D523. The yellowness index was measured by means of the spectrometer method according to ASTM E313 using a precision colorimeter NH-145 .In this article, the syntheses of six epoxy methacrylic compounds were made based on diglycidyl ethers of various structures  were presented. The synthesis’s essence was adding methacrylic acid to epoxy groups of ethers in such a way that hybrid structures, containing both epoxy groups and double bonds, remained in the post-reaction mixture. The modification progress was monitored by titration , and the structure of the expected products was validated by infrared spectroscopy and nuclear magnetic resonance. The conducted modification allowed us to obtain products that can polymerize both cationic and radical mechanisms. Moreover, the obtained products (apart from the compound based on bisphenol A diglycidyl ether—DGEBA) are characterized by low viscosity (100–500 Pa∙s), which makes them extremely useful for the production of coating materials. Hence, the research presents the influence of different chain architectures of synthesized epoxy methacrylate resins (EAs) based on low-viscous diglycidyl ethers on the course of the photocuring process according to the cationic and radical mechanisms. It was shown that the epoxy methacrylate pre-polymers with shorter chain lengths and features of chemical structure such as aromatic, cycloaliphatic, or some branched structures, i.e., DGEBA, CHDMDE, and NPDE, polymerize faster and obtain higher conversion of epoxy or methacrylic groups in contrast to epoxy methacrylates based on diglycidyl ethers containing a linear carbon chain structure, i.e., HDE, PDODE, and PTMODE. The obtained cured coatings have a wide range of properties, which makes them potentially valuable for applications as protective coatings, varnishes, or films in various industries.


We compared the TAT-splitting in different phases , pure hexahedral (H), (T + H)-phase, and (H + T)-phase) of the Pr3+:YPO4 crystal. The TAT-splitting in the (H + T)-phase was observed to be stronger than that in other phases, while the Pr3+ ion had stronger dressing than the Eu3+ ion in the host material of YPO. Furthermore, we observed that the ratio of primary and secondary TAT-splitting can be controlled by the single and double dressing effect using the power and detuning of employed laser fields. In our experiment, we observed that secondary splitting from secondary dressed levels can only be observed at the resonance wavelength in the three-level system. Based on the results, we proposed a model for a multi-channel optical router and logic NAND gate. The routing action results from primary and secondary TAT-splitting, while the NAND gate was realized by the primary dressed states.For the first time, we investigated the electric-dipole transition dependent primary and secondary temporal Autler–Townes (TAT)-splitting of a hybrid signal (fluorescence and Stokes) in Pr 3+:YPO4.For the first time, we investigated the electric-dipole transition dependent primary and secondary temporal Autler–Townes (TAT)-splitting of a hybrid signal (fluorescence and Stokes) in Pr In Pr3+-doped yttrium orthophosphate (YPO4), i.e., Pr3+:YPO4, the “atom-like” properties of the dopant in which the atomic coherence can be induced when interacting with multiple laser beams is similar to other rare-earth-doped crystals.4–6 Unlike atomic gases, Pr3+:YPO4 can be used in integrated quantum circuits due to their solid crystal structure and induced coherence. Coherence excitation has been explored mostly in doped crystals such as Eu3+:YPO4, Pr3+:YPO4, and Pr3+:Y2SiO5, for improving lifetime, coherence time, spectral bandwidth, and the induction of non-classic behaviour. The Autler–Townes (AT) splitting of multi-order fluorescence (FL) has been investigated in numerous atomic-like media.7,8 In atomic-like media, the lifetime of FL processes can be controlled by the dressing effect, which can be adjusted by the power or detuning of laser fields.9 Recent progress has widely explored solid-state atomic coherent materials with EIT,10 reduction of optical velocity,11 optical storage based on all-optical routing,12 coherent storage of light pulses,13 and optical read and write information.14Physicists have achieved incredible progress in understanding and controlling the quantum coherence excitation and coherence transfer in atomic gases. These processes lead to numerous famous and intriguing physical phenomena, such as electromagnetically induced transparency (EIT)3+ and Pr3+ ions are more sensitive to the site symmetry and the surrounding crystal-field of the host material than other crystal ions,15,16 which makes them an attractive material for important applications such as in scintillation detectors, medical imaging, display devices17,18 while demonstrating a high chemical and thermal stability.19,20 In this regard, a double-cascade dressed optical metal oxide semiconductor field-effect transistor is realized by exploiting the enhancement and suppression in different phases of Eu3+:YPO4 and Pr3+:YPO4 crystals.21 YPO4 crystallizes with the zircon structure (xenotime-type) with a tetragonal symmetry (a = b = 0.6894 nm and c = 0.6027 nm) and space group I41/amd,22 where the site symmetry for Y3+ ions is the D2d point-group.23 The structure can be described as chains parallel to the c-axis of the corner-sharing structural units built of (YO8) dodecahedron and a (PO4) tetrahedron linked together by an edge.24 The YPO4 matrix has excellent optical and physical properties such as a large indirect bandgap (∼8.6 eV), high dielectric constant (∼7 eV), refractive index (∼1.72), high melting point (∼160 °C), and phonon energy (∼1080 cm−1).25Eu3+:YPO4 crystal. We observed that the secondary TAT-splitting is very sensitive to the electric dipole transitions, which can be controlled through the wavelength and power of laser beams. Based on our results, we proposed a model of a multi-channel optical router and logic NAND gate controlled by a laser power.In this study, we investigated the relationship between temporal AT-splitting (TAT-splitting) and excitation spectra of the mixed-phase  + less T) Pr3+:YPO4 crystal was held in a cryostat (CFM-102) maintained at 77 K by flowing liquid nitrogen. 3+:YPO4. ES) and fluorescence (FL) hybrid signal under phase-matched four-wave mixing. Two dye lasers (narrow scan with a line width of 0.04 cm−1) were pumped by an injection-locked single-mode Nd:YAG laser , which were used to generate the pumping fields E1  and E2  with frequency detuning of Δi = Ωmn − ωi, where Ωmn is the corresponding atomic transition frequency between levels |m〉 and |n〉. ωi  is the laser frequency. In this experiment, the sample of PrE1, ES was generated in a two-level system with phase matching condition . The density matrix elements of ES from a mixed (H + T)-phase Pr3+:YPO4via perturbation chain can be written asGi = μiEi/ℏ is the Rabi frequency of field Ei, with the electric dipole matrix elements μij of levels |i〉 and |j〉 and Γij = (Γi + Γj)/2 is the transverse decay rate, where, Γi/j = Γpop + Γion–spin + Γion–ion + Γphonon − Γdressing + Γnon-rad. The density matrix of the accompanying FL can be written asd1 = Γ10 + iΔ1. The lifetime of FL is given as ΓFL = Γ10 + Γ11. The temporal intensity of FL is given as I(t) = ρ(2)11 exp(−ΓFLt). By opening E1 and E2 in a ∧-type three-level system  measured from the (H + T)-phase Pr3+:YPO4 crystal in two-level and three-level ∧-type systems, respectively. The excitation spectrum in E1 from 585 nm to 610 nm by fixing the gate position (gate width = 100 ns) at a certain point  → 3H4 (δ0), 1D2 (γ1) → 3H4 (δ0), 1D2 (γ2) → 3H4 (δ2), and 1D2 (γ1) → 3H4 (δ2), respectively. Under the action of the crystal field of YPO and site symmetry (D2 + D2d) of the (H + T)-phase, the terms 3H4 (ground state) and 1D2 (excited state) under dipole-allowed transition can split into seven and four fine structure levels, as shown in 26 In our experiment, we observed only four peaks in (much hand less T)-phase YPO4 (D2 + D2d) is not strong enough to completely lift the 2J + 1 degeneracy of the levels. It is worth mentioning here that each electric-dipole transition corresponds to different lifetimes and dressing effects. ES) without the adiabatic population transfer. If we set |1〉 as the frequency reference point, the Hamiltonian for primary TAT-splitting can be written using .27 From the equation H|±〉 = λ±|±〉, we can obtain λ± = [Δ1 ± (Δ12 + 4|G1|2)1/2]/2. The splitting distance between |+〉 and |−〉 is Δ± = λ+ − λ− = (Δ12 + 4|G1|2)1/2. The primary splitting distance Δ± is directly proportional to |G1|2. In the primary TAT-splitting, the left and right peaks of the time-intensity signal correspond to the dressed states |+〉 and |−〉, respectively, as shown in E1 and E2 are turned on, the primary dressed level |−〉 is further split into two secondary dressed levels |−+〉 and |−−〉, as shown in  (where ) and from the equation H′|−±〉 = λ−±|−±〉, we can obtain Δ−± = λ−+ − λ−− = [Δ2 ± (Δ22 + 4|G2|2)1/2]/2. At Δ2 ≡ 0, we can know that the increase in the |G2|2 can lead to an increase in the |λ−|. Using the above equations, we can derive the formula for TAT-splitting8tAT ∝ 1/Γnon-rad + A exp(Δ±/KBT). The secondary splitting distance of the right peak in 3+:YPO4 also plays a critical role in the TAT-splitting. When E1 was fixed at 595 nm, the particle transfer occurs between 1D2 (γ2) → 3H4 (δ0) and level |1〉 splits into |+〉 and |−〉, and primary TAT-splitting is observed in G1|2/d1 in E1 and when E2 was turned on, |−〉 will further split into |−+〉 and |−−〉 with the dressing effect of |G2|2/d2. Due to further splitting of |−〉, the secondary TAT-splitting would appear in E2 to 596.7 nm (particle transition between 1D2 (γ1) → 3H4 (δ0)), primary splitting becomes strong and the intensity of secondary peak decreases, while the splitting distance Δ−± increases  in a two-level system by changing the detuning of E1 with blocked E2. When E1 is fixed at far off-resonant (Δ1 > 0), the temporal intensity signal has a very weak amplitude and has no AT splitting  → 3H4 (δ0) due to the resonant excitation of E1. At the resonance wavelength (Δ1 ≡ 0), a strong primary TAT-splitting is observed modelled by |G1|2/d1E1 is further increased to the off-resonant wavelength, the primary TAT-splitting reduces  → 3H4 (δ2). As detuning is further increased to off-resonant, the TAT-splitting disappears due to a very weak dressing effect, as shown in plitting . As Δ1 g reduces . The weaA” and “B” are two inputs on the time-intensity signal corresponding to either logic 0 or logic 1. When the wavelength of the input beam is changed to resonance, the logic input  satisfies the logical condition  of the NAND gate, so the output of MOSFET is observed as OFF state  in A and B signal satisfies the logical condition  of the NAND gate, and MOSFET output is observed to be in the ON state  in C = (Ioff − Ion)/(Ioff + Ion), where Ioff is the light intensity at the OFF-state and Ion is the light intensity at the ON-state.18 The switching contrast C is measured to be about 88%  in a ∧-type three-level system. The temporal intensity signal in E1 at resonance (Δ1 ≡ 0) and changing the detuning of E2. To observe the maximum TAT-splitting, the powers of both E1 and E2 were fixed at a high value (5 mW). Even when E2 is fixed at far off-resonance, very weak AT splitting is observed , double TAT-splitting (both primary and secondary) is observed due to the dipole-allowed transitions between 1D2 (γ2) → 3H4 (δ0), as shown in Δ± = λ+ − λ− = (Δ12 + 4|G1|2)1/2) between |+〉 and |−〉 and secondary splitting distance (Δ−± = λ−+ − λ−− = [Δ2 ± (Δ22 + 4|G2|2)1/2]/2) between the dressed levels |−+〉 and |−−〉 increases. Due to this, very clear double TAT-splitting is observed in E2 moves towards the peak position of the second spectral peak (1D2 (γ1) → 3H4 (δ0)), secondary splitting distance (Δ−±) reduces and becomes very weak /(tAT + t), where (tAT− t) is the splitting between two neighbour peaks then, higher the contrast index, greater accuracy of information and less crosstalk between channels. From our experiment, we measured η = 87% , and the average power of our routing can be operated at 1–2.0 μW.The multi-channel optical routing was realized by the primary and secondary TAT-splitting results observed in p to 91%  as the dE1 with the increase in the splitting distance from bottom to top peaks as the power of E1 is increased from low to high in (H + T)-phase Pr3+:YPO4. At first, the power of E1 is too little (1 mW) to split |1〉 into dressed energy levels, so we cannot see the right peak appearing in E1 (≈4 mW), the energy level |1〉 splits into |±〉, and primary AT-splitting appears in Δ± = λ+ − λ− = (Δ12 + 4|G1|2)1/2) between |+〉 and |−〉 is directly proportional to the power of E1. With an increase in E1, |G1|2 keeps increasing, which leads to an increase in the splitting distance, and prominent primary TAT-splitting was observed in E1 and E2 at resonant wavelengths and changing P2 of E2 in a ∧-type three-level system. In a ∧-type system, when power was changed from 1 mW (E2 (≈8 mW), the primary dressed level |−〉 further split into two secondary dressed levels |−+〉 and |−−〉, as shown in Δ−± = λ−+ − λ−− = [Δ2 ± (Δ22 + 4|G2|2)1/2]/2) between the primary and secondary peak in the time-domain is only determined by E2. By increasing the power of E2, |G2|2 increases, which leads to an increase in Δ−±. The observation of the primary and secondary dressing results from the exhibition of the strong atomic-like behavior of the mixed-phase Pr3+:YPO4 due to a strong transfer probability of T-phase and low symmetry of H-phase. Hence, the prominent secondary peak is observed at high power -phase Pr3+:YPO4, and T-phase Pr3+:YPO4, respectively. The time-domain signal is obtained under the same experiment condition as defined for 3+:YPO4  site symmetry, which combines the strong transfer probability and dipole moment of the T-phase with strong atomic-like behavior of the H-phase Pr3+:YPO4. Hence, strong TAT-splitting was observed in (much-T + less-H)-phase, as shown in 3+:YPO4 -phase Pr3+:YPO4  with  of the NAND gate, and the output of the MOSFET Y performs OFF-state as a spectral peak in Y satisfies the logical 0 condition. The temporal intensity input spectral signal satisfies the logical condition  of the NAND gate, and the output of the MOSFET Y performs ON-state as a spectral peak in Y satisfies the logical 1 condition, where the switching contrast C is about 85% from P) increases from 15%  increases to 93%  had a strong dressing effect than that from the pure T-phase or H-phase. Also, we discussed the dressing effect dependency on the site symmetry of different ions (Eu3+ and Pr3+) in the host material YPO4. We also observed that the Pr3+ ion had stronger TAT-splitting than Eu3+ ion in the host material of YPO. Further, the TAT-splitting depends on the distance between bright states caused by the dressing effect. The splitting distance increases, if laser power increases, and the time delay becomes larger. The primary and secondary AT-splitting were caused by single and double dressing, respectively, and mainly depends upon the electric dipole transition and power of laser beams. The multi-channel optical router and logic NAND gate were also realized from the TAT-splitting. The channel equalization ratio, contrast index, and switching contrast were controlled by laser detuning and the power of input beams.In summary, we have demonstrated the primary and secondary TAT-splitting from different phases of the PrThere are no conflicts to declare.


In this study, we have employed Digital Light Processing (DLP) printing technology for the fabrication of solid microneedle (MN) arrays. Several arrays with various geometries, such as cones, three-sided pyramids and four-sided pyramids, with different height to aspect ratios of 1:1, 2:1 and 3:1, were printed. Post-processing curing optimizations showed that optimal mechanical properties of the photocurable resin were obtained at 40 °C and 60 min. Ex vivo skin studies showed that piercing forces, penetration depth and penetration width were affected by the MN geometry and height to aspect ratio. Cone-shaped MNs required lower applied forces to penetrate skin and showed higher penetration depth with increasing height to aspect ratio, followed by three-sided and four-sided printed arrays. Cytotoxicity studies presented 84% cell viability of human fibroblasts after 2.5 h, suggesting the very good biocompatibility of the photocurable resin. Overall, DLP demonstrated excellent printing capacity and high resolution for a variety of MN designs. Three-dimensional (3D) printing, usually recognized as additive manufacturing, is a layer-by-layer fabrication process in which different materials, such as metals, polymers and ceramics, can be processed to produce complex 3D objects . Three-dNowadays, several 3D printing techniques, such as vat photopolymerization, for example, Laser Stereolithography (SLA) and/or Digital Light Processing (DLP), are utilized to allow the fabrication of 3D physical objects. SLA cures and solidifies photo-polymeric resins using an ultraviolet (UV) laser and has been recognized as one of the most used photopolymerization 3D printing techniques due to its high precision and accuracy . NeverthTherefore, the vat photopolymerization printing concept has revolutionized several fields, such as pharmaceutical and biomedical, with significant research outcomes ,5,6. In The first MN array was introduced in the 1970s and was made of silicone. Since then, MNs have been developed and fabricated with different materials, such as metal, polymer, glass and ceramic . In addiFurthermore, MN types can be divided according to the various methods of drug delivery as solid, hollow, coated, porous, hydrogel-forming and polymer or dissolvable arrays ,10,11,12As previously stated, MNs’ material composition is one of the most important aspects regarding their application and fabrication process. It should ensure that MNs are biocompatible and present suitable mechanical strength. However, most photosensitive resins have demonstrated poor biocompatibility , causingThe aim of this work was to evaluate the capacity of DLP using a photocurable microfluidic resin for the printing of microneedle arrays of various geometries and height to aspect ratios and investigate their effects on the mechanical properties and piercing capacity of the fabricated arrays. The cytotoxicity of the resin was also investigated to verify that it is safe for skin penetration and non-toxic to the skin. ® Cell Viability Imaging Kit (Blue/Green) was purchased from Fisher Scientific™ . The porcine skin was purchased freshly from a local slaughterhouse .The MNs were 3D-printed using microfluidic resin, MiiCraft BV-007A . Human dermal fibroblasts (HDFs), human dermal fibroblast growth medium and trypsin EDTA solution (1×) were purchased from Sigma Aldrich . The ReadyProbesThree MN geometries were designed using the SolidWorks CAD software . Based on the needle shapes of each MN patch design, they were named ‘cone’, ‘3-sided pyramid’ and ‘4-sided pyramid’. The MNs’ dimensions are represented in The MNs were designed on a square patch of 15 mm × 15 mm × 0.5 mm symmetrically, comprising 7 rows and 7 columns, with a total number of 49 needles per design. The interspacing between the needles was adjusted at 1.85 mm. On the other hand, cylinders were also designed and printed, respectively, for compression testing. The cylinder design was in accordance with ASTM D695-15 standards, where the cylinders had a diameter of ø12.7 mm and a length of 25.4 mm2 and a layer exposure time of 3.3 s accordingly. The first layer exposure time was set to 112 s with identical UV intensity to ensure the appropriate adherence of the MNs to the printing platform. The generated printing files were then exported to the printer for fabrication. For the printing of the MNs and the compression test specimens, a MAX X27 DLP  printer, equipped with an LED wavelength of 385 nm and a vat of uncured microfluidic resin, was used. The printer software Asiga Composer  was applied to slice the models and to generate the printing files. The MNs’ printing orientation was set to 45° on the printing platform with the aid of a support, which was selected based on our previous studies [Once printed, the MNs were removed from the printing platform gently and immersed in an isopropanol bath for 10 min to wash out any uncured resin residues. The support structures were then gently removed and the MNs were then left to dry in an ambient environment for 60 min. Then, the printed MNs were cured for 60 min and at 40 °C, which were the optimized curing settings obtained, in a UV-A heated chamber  with a wavelength of 405 nm. In order to evaluate the curing settings’ effect on the mechanical properties of the microfluidic resin, several solid cylinders were designed, printed and cured at different times and temperatures. The predetermined curing times used were 30 min, 45 min and 60 min and were combined with pre-set chamber temperatures of 40 °C, 50 °C and 60 °C. A total of 45 samples were analyzed, 5 samples for each respective time and temperature. The cylinders were subjected to a compression test using the Tinius Olsen testing machine , set up with two flat test plates and a 25 kN load cell, following the standard test method for compressive properties of rigid plastic, ASTM D695. Each cylindrical specimen was placed between the two plates and a compression force was applied alongside their longitudinal axis at a constant speed of 1.5 mm/min. For each sample, the applied force value and position were recorded and presented by the Horizon software . The obtained data were analyzed and, therefore, true stress and true strain values were calculated. The compressive modulus, as well as the yield strength of each sample, was calculated according to the 0.5% offset curve method.Scanning electron microscopy  was utilized to investigate the quality of the 3D-printed MNs. MNs were kept secured on an aluminum stub with a conductive carbon adhesive tape . The MNs were then examined via SEM and images of the MNs were captured by an electron beam accelerating voltage of 1.0 kV and magnification of 80×.To achieve the maximum force value that could be applied to the MNs, fracture tests were carried out. The MNs were subjected to an axial force test using the Tinius Olsen testing machine , with the same set-up as the compression tests and following the identical standard test method. The MN’s base was glued to the moving plate using double-sided tape and pressed against the static plate until the needles fractured, at a constant speed of 1.5 mm/min. A total of 5 samples, cured with the optimized settings, were analyzed, and for each one, the applied force value and position were recorded and presented by the Horizon software. TM Sciences Corporation, Princeton, NJ, USA). After removing a thick fatty layer of tissue, full thickness was then transferred in a buffer saline of pH 7.4 to the texture analyzer to perform the piercing test. The skin samples were collected freshly from a local slaughterhouse  and any hair removed using a razor blade. For the skin piercing test, full-thickness skins (1 mm) were excised using a Dermatome . The compression type of application force included the pre-test, test speed and post-test speeds of 1.00 mm/s, 0.1 mm/s, 1 mm/s, respectively. Double-sided adhesive tape was used to fix the MNs onto the probe, while full-thickness skins were placed onto the petri dishes. Each experiment was repeated 6 times. ® software . A single 3D-printed MN array (7 × 7) was used for the OCT analysis. OCT  was utilized to investigate the penetration capability of the 3D-printed MNs into the freshly excised mouse (2–3 weeks old) dorsal skin. Skin hair was removed using an electric razor  in order to avoid any penetration issues of the laser beam into the deeper tissue parts (2 mm). Then, 3D-printed MNs were inserted into the skin tissue manually. The penetration depths and widths of each dimension of the 3D-printed MNs were determined using a microscope attached to the OCT instrument. False color was used to distinguish the penetrating layers  and 3D-printed MNs using Adobe Photoshop CS65 cells/well was added to each well containing disks. The cells were then incubated for 2.5 h and stained with blue and green stain  according to manufacturer guidelines. Briefly, 10 mL of a mixture containing an equal number of drops of each dye per milliliter was added to each well. The plate was then incubated at 37 °C and 5% CO2 for 30 min before taking images using a ZOE Fluorescent Cell Imager  at 4× magnification. Cells colored in blue represented the total amount of cells while those in green the damaged cells. The experiments were performed in triplicate. Cell viability was calculated as follows: To evaluate the cytotoxicity of the MNs, circular disks were printed using the respective resin. The disks were designed with a diameter of 6 mm and 0.6 mm thickness in order to fit in a 96-well plate. Each side of the disks was sterilized under UV light for 1 h and subsequently soaked in fibroblast growth medium for 48 h before placing them into a 96-well plate. Human dermal fibroblast cells were harvested from the main culture at low passage (<10) at confluency of 80–90% and >90% viability. They were suspended in fresh media and 100 mL total volume with a concentration of 1 × 102 and the curing of resin layers took place at an exposure time of 3.3 s. The layer thickness identified as Z axis resolution (slice thickness) was defined as 25 µm. The X-Y resolution of the printing refers to the pixel size of the DLP projector at the exposure location of the UV light and the resin. Compared to our pervious study, the Z and X-Y resolutions were enhanced from 50 and 50 µm to 25 and 27 µm, respectively. Such changes in resolution resulted in the fabrication of finer structures and, most importantly, more defined and sharper tips for the MNs. As shown in In the current study, DLP was selected as the printing technology due to the higher-resolution prototyping, fast processing and excellent surface finishing. For the optimization of the printing process, the applied UV laser (385 nm) intensity was set at 3 mW/cmDuring the application on the skin, the MN patches are subjected to compressive forces along their longitudinal axis to pierce the skin and, therefore, they must be strong enough to be applied safely and to reduce the chances of breakage. Thus, to prevent failure during application, their mechanical properties need to be evaluated. As mentioned in previous studies , the posThroughout the compression testing of the specimens, it was concluded that the maximum load cell (25 kN) of the testing machine was not sufficient to achieve the value of the ultimate strength for some of the specimens, since some of the specimens did not appear to have any fracture in their structure. Due to this reason, the maximum elastic point of the material, the yield strength, was determined. Yield strength was evaluated via the following equation.The findings suggested that the exposure time has a significant influence on the properties of the resin, rather than the curing chamber temperature. Therefore, increasing the exposure time can enhance the mechanical properties since it increases the degree of cross-linking. Furthermore, the influence of chamber temperature becomes weaker with increasing exposure time. This is possible to conclude since the yield strength values for the lower and higher temperature, obtained for 60 min, have a smaller gap compared with other values. As shown in According to the obtained results, the post-printing curing process influences the mechanical properties of the resins and thus a systematic evaluation is required. This step played a significant role in the photopolymerization of the printed MNs since it enhanced their mechanical properties, which lowered the possibilities of failure during application.All MN arrays could successfully pierce the porcine skin without being fractured or broken during the testing. This was attributed to the production of microchannels without damaging the geometry of the needles ,18. HoweAs shown in Furthermore, the penetration force was affected by the design, where the cones required lesser forces to penetrate the skin in comparison to three-sided pyramids. The four-sided pyramids required relatively higher forces. Nevertheless, for all the designs, the penetration forces varied from 30 to 43 mN per needle of each array. Further evaluation of the piercing capacity of 3D-printed MNs involved skin penetration studies via OCT. It is known that OCT is considered to be a non-destructive and non-invasive method that can capture high-resolution images from the deeper tissues ,20. It hAs can be seen in The penetration capacity varied according to the MN designs, where cone shapes showed better skin penetration than three- and four-sided pyramid shapes, respectively. Similar findings were obtained for the penetration width, which increased with increasing HAR. However, the penetration width was higher for four-sided compared to three-sided and cone geometries. This was attributed to the higher volume of the four-sided MNs in comparison to the other shapes, which results in higher volumetric expansion of the skin . The finThe piercing capacity of 3D-printed MN designs in this work appears to be far better when compared to similar studies for dissolvable MN patches. For example, Lau et al. (2017) developed multi-layered pyramidal dissolvable patches with a total length of 500 μm, but the penetration capacity was limited to 30%, with only 150 μm depth in animal skin . In anotIn order to assess the security and confidence level of the MN patches, the fracture force was evaluated to estimate the failure probability during the course of application. All MN designs were tested to compare their mechanical behavior and evaluate the effect of needle geometry and the HAR.The data recorded from the axial force measurements are displayed in A careful look at The margin of safety can be a useful index to evaluate the MNs’ safety for application. The index was calculated using the values obtained from previous studies and the following equation:As presented in The biocompatibility of MNs is a critical factor that must be considered and investigated prior to any human trials. According to our knowledge, there are no biocompatibility/cytotoxicity studies on the specified resin used in this study. Therefore, the cytotoxicity of the microfluidic resin was investigated using HDFs, where cell viability was evaluated through fluorescence microscopy. As shown in Microneedle arrays of various geometries, such as cone, three-sided and four-sided, with different height to aspect ratios varying from 1:1, 2:1 and 3:1, were printed using Digital Light Processing (DLP). The MNs’ mechanical properties were affected by the curing temperature and duration. The shape of the MN designs and the height to aspect ratio were found to be critically important for the piercing capacity, penetration depth and penetration width of the arrays. The biological evaluation of the photocurable resin showed excellent biocompatibility in HDFs. Such mechanical integrity and biocompatibility may introduce a new field of research on the 3D printing of solid and, particularly, hollow MNs using microfluidic resin. Overall, DLP was proven to be an ideal technology for the future design and optimization of 3D-printed MNs.