MTHFR gene with maternal risk for DS.Down syndrome (DS) is one of the most common chromosomal abnormalities among live-born babies and one of the best-known intellectual disability disorders in humans. Errors leading to trisomy 21 are primarily arising from defects in chromosomal segregation during maternal meiosis (about 88% of cases), and the focus of many investigations has been to identify maternal risk factors favoring chromosome 21 malsegregation during oogenesis. Maternal polymorphisms of genes required for folate metabolism are the most investigated risk factors for the birth of children with DS. Through this review, we sought to investigate the association of the polymorphisms “C677T” and “A1298C” of the We will use the databases PubMed, Embase, Scopus and Web of Science to search for case-control studies published from 1999 up to September 2021 without language restriction. Results will be presented as relative risks and 95% confidence intervals for dichotomous outcomes and mean differences, or standardized mean differences along with 95% confidence intervals, for continuous outcomes. The all data synthesis will be analyzed on the Review Manager 5.2 version software.This study will be able to clarify all the doubts we seek and that it will be able to provide accurate data that will be able to describe how these polymorphisms can act to increase the predisposition for the birth of children with DS in different populations and under different dietary conditions.MTHFR gene with increased the maternal risk for Down syndrome.This study will clarify the relationship between C677T and A1298C polymorphisms This systematic review and meta-analysis protocol has been registered on the Prospective Registry of International Systematic Review and Meta-analyses: CRD42021269338. Several subsequent studies carried out for more than 15 years support a potential contribution of polymorphisms of genes required for folate metabolism as maternal risk factors for the birth of a child with DS.Folate metabolism provides one-carbon units required for DNA synthesis and methylation, thus playing a pivotal role in maintaining genome stability during cell divisions.MTHFR) is one of the most important enzymes in folate metabolism. It converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, an active form of folic acid acting as methyl donor for the remethylation of homocysteine to methionine in a reaction catalyzed by methionine synthase. The methionine is then converted into S-adenosyl-methionine (SAM), which is the main intracellular methyl donor compound for methylation reactions.,8Methylenetetrahydrofolate reductase  is the result of a transition mutation from C to T at nucleotide 677, causing an amino acid substitution  in the catalytic domain of the protein.The most well known polymorphisms of the MTHFR enzymatic activity. The MTHFR A1298C polymorphism (rs1801131) is another common missense mutation leading to a Glu429Ala aminoacidic change in the regulatory domain of the protein and the homozygous genotype (C/C) results in about 40% reduction in enzymatic activity. Noteworthy, the MTHFR C677T and A1298C polymorphisms are in strong linkage disequilibrium (LD), and the 677T allele has been nearly always observed in cis with the 1298A one. LD is not complete, but very low frequencies have generally been reported for the rare 677T-1298C haplotype.The homozygous genotype (T/T) is responsible for the reduction in up to 70% in MTHFR polymorphisms was proposed considering the fact that MTHFR works as a dimer protein in which monomers associate head to tail, and the stability of the dimer depends on what aminoacid is present at position 222 and what at position 429, resulting from the combined MTHFR 677/1298 genotype. Consequently, the overall activity of the MTHFR protein is better explained by the combined MTHFR 677/1298 genotype, rather than by each single polymorphism alone.A biological explanation for the LD existing between the two MTHFR 677T allele renders the enzyme thermolabile, particularly in homozygous (T/T) individuals that are prone to dimer destabilization under conditions of reduced folate bioavailability. In summary, combinations of genetic and dietary factors contribute to MTHFR protein stability and activity. In addition, dietary folates and related metabolic factors can also regulate MTHFR gene methylation and expression levels.,13Physiological levels of folate stabilize the MTHFR dimer, but the  supporting the original hypothesis by James et al and suggesting that MTHFR polymorphisms promote changes in global DNA methylation during maternal meiosis leading to chromosomal non-disjunction and increasing the maternal risk to have children with DS.A recent study revealed that the fission yeast methylenetetrahydrofolate reductase protein (Met11) is important for maintaining the pericentromeric heterochromatin structure to ensure mitotic and meiotic chromosome segregation fidelity,MTHFR C677T and A1298C polymorphisms have been linked to an increased risk of chromosome malsegregation in lymphocytes of mothers of DS children. Following the original study by James and coworkers, several investigators performed case–control studies to evaluate MTHFR C677T and/or A1298C polymorphisms as maternal risk factors for the birth of a child with DS, but the small sample-size of these studies coupled to differences in allele frequencies, lifestyles and dietary habits among different populations, yielded conflicting results.–22In addition, both MTHFR C677T and/or A1298C polymorphisms are associated with the maternal risk for having a child with DS, and despite that significant associations emerged for the C677T one in the overall population, the different inclusion criteria selected by the authors yielded conflicting or inconclusive results after subgroup stratification according to ethnicity.–25At least three different meta-analyses of those studies, performed between 2013 and 2014, addressed whether MTHFR C677T/A1298C polymorphisms.,26–28 Moreover, congenital heart defects (CHD) are frequently observed in DS babies and owing to the role of folate metabolism in cell division and methylation, there is increasing interest in searching whether maternal MTHFR polymorphisms could represent risk factors for CHD in DS newborns.,17,21More recent case–control studies have highlighted a different effect size of these polymorphisms in the maternal risk for a DS birth according to ethnicity, maternal age at conception, errors occurring at maternal meiosis I or II, gene-nutrient interactions, and combined MTHFR C677T and/or A1298C polymorphisms as maternal risk factors for DS addressing not only the overall contribution of each of the two polymorphisms to DS risk, but performed in such a way to extract data on combined genotypes, ethnicity, serum folate levels, maternal age at conception, errors occurred at maternal meiosis I or II, as well as data on CHD in the DS children, in order to shed light on the different role of the MTHFR genotype in the various subgroups. Therefore, the aim this review is to evaluate the association between frequency of the C677T and/or A1298C polymorphisms of the MTHFR gene with maternal risk for DS.What is missing is an updated systematic review and meta-analysis of the studies investigating 2MTHFR gene and the maternal risk for DS.The aim of this systematic review and meta-analysis is to clarify the relationship between the C677T and A1298C polymorphisms 3 This protocol was registered in the prospective international registry of systematic reviews and the trail registration is number CRD42021269338 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=269338).The elaboration of this study will be conducted according to Preferred Reporting Items for Systematic Review and Meta-analysis Protocol Guidelines.3.13.1.1Will be included studies have to meet the following criteria:(1)articles should be published from 1999 up to September 2021 without language restriction;(2)article should have sufficient data on both allele and genotype frequencies to calculate the odds ratio with 95% confidence intervals (CI);(3)article should be case–control studies; and(4)authors should describe the genotyping protocols.MTHFR C677T and/or A1298C allele and genotype frequencies will be excluded as well as Review articles, editorials and/or commentaries, meeting abstracts and any other kind of article not published in a peer-reviewed journal.Case–control studies not showing tabular data with 3.1.2Studies shall include women who gave birth to at least one child with DS in this systematic review and meta-analyses. Women/mothers of children who do not have DS or other diagnosed syndromes, and without history of spontaneous miscarriages will be the control group.3.1.3Maternal risk for the birth of a child with DS.3.1.4The role of polymorsphisms as risk factors for CHD in DS newborns.3.23.2.1We will use four databases: PubMed, EMBASE, Scopus and Web of Science to search for articles of studies of case control published from 1999 up to September 2021 without language restriction.3.2.2The search terms (MESH) that will be used to conduct the research are: (mothers OR pregnancy) AND (methylenetetrahydrofolate reductase OR MTHFR OR genetic polymorphism OR gene polymorphism) AND (down syndrome OR trisomy 21). The studies considered eligible will also be selected from the reference lists of the retrieved articles.3.33.3.1Two authors, CTAG and JRDL, will independently examine the results of the research using the titles and abstracts. Duplicate studies and reviews will be excluded, and the studies by the same authors will be controlled for a possible overlapping of included patients. The reviewers will review the full text to determine whether the studies meet the inclusion criteria. Various characteristics of the eligible studies will be extracted: title, surnames of the first authors, year of publication, study location (country), study design, primary objective, population, sample size, follow-up period, inclusion/exclusion criteria, genotyping method, type of control and primary results. The process of selection of the studies is summarized in Figure 3.3.2The assess the risk of bias in eligible studies using the Newcastle-Ottawa Scale 9-star tool. The quality of each study will be judged in three broad categories – namely: selection of the study population, comparability of the groups and determination of the exposure or the result of interest. The Newcastle-Ottawa Scale score vary from 0 to 9, in which (score 0–5) was low quality study and (score 6–9) was high quality study.3.42 ≥ 50%), the random effects model will be used to combine the studies to calculate the OR and 95% CI, using the DerSimonian-Laird algorithm in the meta for package, which provides functions to conduct meta-analysis in R. Other characteristics and results of the study will be summarized narratively if the meta-analysis cannot be performed for all or for some of the included studies. Sensitivity analyses will be used to explore the robustness of the results in relation to the quality of the study and the sample size. Sensitivity analyses will be shown in a summary table. If meta-analysis cannot be performed for all or some of the included studies, the results will be presented qualitatively.The data will be entered into the Review Manager 5.2 version software . For dichotomous results, the odds ratio (OR) and 95% CI for each study will be extracted or calculated. In case of heterogeneity (I3.52 test with a significance level of P < .1. To assess heterogeneity, we plan to calculate the I2 statistic, which is a quantitative measure of inconsistency between studies. A value of 0% indicates no heterogeneity, whereas I2 values ≥50% indicate a substantial level of heterogeneity; however, heterogeneity will be assessed only if it is appropriate to conduct a meta-analysis.The heterogeneity between the results of the study will be assessed using a standard χ3.6MTHFR C677T and A1298C polymorphism. Standardized data extraction forms will be created specifically for this review and the results will be later inserted into a database. All data entries will be double rechecked.Will be perform subgroup analyses to investigate the effect of ethnic differences in allele frequencies, combined genotypes, circulating folate levels and maternal age at conception to analyze the type of meiotic error in modulating the maternal risk for DS resulting from 3.7The analysis of the evidence for all outcomes will be assessed using the Grading of Recommendations Assessment, Development and Evaluation Working Group methodology by classifying the evidence as high, moderate, low, or very low.3.8Ethical approval is not necessary because it is a systematic review article that will use data published in the literature. The findings of this manuscript will be published in a peer-reviewed journal and will be updated if there is enough new evidence to alter our conclusions.4 Folate is an important nutrient required for DNA synthesis and methylation, and several in vitro studies have shown that its depletion causes chromosome damage and malsegregation in cultured cells.,33 The folic acid, nothing else is a synthetic form of folate, a water-soluble vitamin whose active form is 5-methyltetrahydrofolate.Studies published since 1999 support a relationship between genetic polymorphisms of folate metabolic genes and the maternal risk for having a child with DS, albeit with varying effect size in different populations and under different nutrient bioavailability. MTHFR is one of the major enzymes in folate metabolism, linking the folate and the methionine cycles, thus providing one-carbon moieties for DNA synthesis and methylation reactions.Indeed, the rationale for the association between maternal polymorphisms of folate metabolic genes and chromosome 21 non-disjunction is an altered chromatin structure of chromosome 21 favoring its malsegregation during meiosis and resulting from the impaired folate metabolism caused by the polymorphisms.MTHFR polymorphisms, namely C677T and A1298C, impair MTHFR stability and activity overall leading to hyperhomocysteinemia and reduced SAM production, and have been largely investigated as maternal risk factors for spontaneous abortion and DS risk.,16–28,34Common MTHFR C677T polymorphism and the risk of birth of a child with DS,–25 but many questions are still unsolved. Particularly, it is becoming evident that combined MTHFR C677T/A1298C genotypes give rise to more or less stable MTHFR dimers, and that intracellular levels of folate, SAM, and metabolic cofactors regulate MTHFR protein stability and activity, as well as the promoter methylation and expression levels of the MTHFR gene itself,,12,28 also the frequency of MTHFR polymorphisms and their combinations varies among different populations, likely as a result of complex gene-nutrient interactions over time that might have favored certain haplotypes.Previous literature meta-analyses support an association between the maternal  It is therefore time to perform an updated systematic review and meta-analysis of the literature concerning the association of the polymorphisms C677T and A1298C of the maternal MTHFR gene with DS risk in attempt to address all these points.There is also evidence that genetic factors predisposing to chromosome 21 non-disjunction in maternal meiosis I might be different from those predisposing to errors occurring at maternal meiosis II.Studies conducted over the years have some limitations regarding the sample size of these studies, the differences in allele frequencies and how the lifestyle and eating habits of mothers of children with DS could influence the measurement of this risk. Furthermore, data resulting from the analysis of ethnicity in these groups, when not properly conducted, generate conflicting and inconclusive results.With this protocol of systematic review and meta-analysis, we wish to identify the association of the polymorphisms C677T and A1298C of the MTHFR gene with the maternal risk for DS, as well as to perform subgroup analyses to investigate the effect of ethnic differences in allele frequencies, combined genotypes, circulating folate levels, maternal age at conception and type of meiotic error in modulating the maternal risk for DS resulting from these polymorphisms, and investigate their role as risk factors for CHD in DS newborns.We hope that our study will be able to clarify all the doubts we seek and that it will be able to provide accurate data that will be able to describe how these polymorphisms can act to increase the predisposition for the birth of children with DS in different populations and under different dietary conditions. This could help to identify individuals and populations at higher risk and seek nutritional strategies that can be implemented to reduce the maternal risk for DS and associated congenital complications.We would like to thank to doctoral student, Medeiros, KS and Sarmento, ACA from Post-graduation Program in Health Sciences, University Federal of Rio Grande do Norte for the contribution for the preparing this protocol systematic review and meta-analysis.Conceptualization: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz, Saulo Victor e Silva.Methodology: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz, Fabio Coppedè, Maria das Graças Almeida.Resources: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz.Supervision: Fabio Coppedè, Maria das Graças Almeida.Writing – original draft: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz.Writing – review & editing: Fabio Coppedè, Maria das Graças Almeida.


The 3D-printed scaffolds were seeded with Mus musculus calvaria cells (MC3T3). After the cells attained confluence, osteogenesis was induced with and without the addition of calcitonin receptor fragment peptide (CRFP) and the bone matrix production was analyzed. Mechanical compression testing was carried out to measure compressive strength, stiffness, and elastic modulus. Results: For the ABS scaffolds, there was a 9.8% increase in compressive strength (p < 0.05) in the scaffolds with no pre-coating and the treatment with CRFP, compared to non-treated scaffolds. Similarly, MED610 scaffolds treated with CRFP showed an 11.9% (polylysine pre-coating) and a 20% (no pre-coating) increase (p < 0.01) in compressive strength compared to non-treated scaffolds. Conclusions: MED610 scaffolds are excellent BGS as they support osteoblast growth and show enhanced bone growth with enhanced compressive strength when augmented with CRFP.Background: Currently used synthetic bone graft substitutes (BGS) are either too weak to bear the principal load or if metallic, they can support loading, but can lead to stress shielding and are unable to integrate fully. In this study, we developed biocompatible, 3D printed scaffolds derived from µCT images of the bone that can overcome these issues and support the growth of osteoblasts. Methods: Cylindrical scaffolds were fabricated with acrylonitrile butadiene styrene (ABS) and Stratasys The development of bone graft substitutes (BGS) is an essential field of orthopedic research . BGS areSynthetic BGS are being developed to deal with the complications of autografts and allografts . One exaThree-dimensional (3D) printing or additive manufacturing (AM) is an exciting new advance that has helped in improving ease and accuracy in the fabrication of complex and customized shapes. Owing to the ability to control the porosity and permeability in the design of the BGS, many studies have used 3D printing in developing implants ,30,31. SHelguero et.al. developed 2D acrylonitrile butadiene styrene (ABS) scaffolds fabricated using FDM printing, pre-treated with calcitonin receptor fragment peptide (CRFP) and showed enhanced biomechanical characteristics and bone matrix proliferation . The ABS®’s MED610 material is also a non-biodegradable material which has shown biocompatibility compliance with ISO 10993 [StratasysSO 10993 . MED610 SO 10993 ,48. The In this study, we developed and evaluated the biocompatibility, osteoconductivity, and biomechanics of ABS and MED610 bone scaffolds inspired by natural bone’s structural physiology, seeded with MC3T3 stem cells, and treated with CRFP.® DesignX software. Regions of interest were identified to extract the cortical bone structure (R1) from the diaphysis and the trabecular structure (R2) at the metaphysis of the femur assuming a segmental defect model, as illustrated in ® software. We designed a cylindrical scaffold (5 mm height × 5 mm diameter) with internal structures created from the trabecular patterns extracted from R2 and four pillar structures with equal spacing on the outer rim of the cylinder, having dimensions (1 mm thickness × 1.5 mm length × 5 mm height) derived from the cortical bone thickness in R1. We carried out many reiterations by extracting different trabecular features and porosity and finalized our design based on the structural consistency of the 3D-printed result. Our final design had a porosity of 50% to facilitate penetration of the cells into the scaffold. Three-dimensional printing gives us the flexibility to change the number of pillars or the density of the trabecular structures based on the location and the strength requirements in the implantation site. Using the finalized design, we 3D-printed ABS scaffolds using FDM fabrication , and MED610 scaffolds were fabricated using polyjet 3D printing  [A femur of a skeletally mature rat was computerized tomography (CT)-scanned  at an isotropic voxel resolution of 10 μm. The resulting CT imaging files (DICOM files) were converted into a 3D stereolithography model (STL file) by thresholding and combining the DICOM files using InVesalium software . This 3D representation was processed in GeomagicMN, USA) . We alsoThe ABS scaffolds were washed with deionized water for 24 h, followed by sterilization in an increasing concentration of ethanol solution  for 15 min and air-drying in a cell culture hood. The MED610 scaffolds were also washed in deionized water. However, sterilization was done by autoclaving at 132 °C for 4 min as per the manufacturer’s recommendation.We pre-coated the scaffolds by treating them with 0.01% polylysine solution for 2 h, rinsing them with sterile Dulbecco’s phosphate buffer saline (DPBS), and air-drying for 1 h in a sterile cell culture hood. Polylysine, a known contributor to osteoconduction, has been shown to help the cells to attach to the scaffold surface ,50.3 cells per chamber were seeded onto the scaffolds and cultured in media (MEMAfter the sterilization and pre-coating (in some cases), the scaffolds were attached to the bottom surface of the cell culture plate using sterilized vacuum grease to prevent them from floating during the cell culture process. MC3T3 cells extracted from the C3 vertebra of a mouse with a density of 1 × 10tomycin) . The culAfter the cells reached 80% confluency, we added 4 mM β-glycerol phosphate (G6P) and 0.05 he cells ,53. The ® and the pixel values were normalized with ‘0’ being the brightest measure (100% white) and ‘1’ being the darkest measure (100% black). We calculated the average of these normalized pixel intensities for each ROI as a measure to quantify dark spots on the scaffold . A von Kossa image with bone matrix deposition on the scaffold surface will have darker pixels in the ROI, and average dark pixel intensities would be higher in comparison with the scaffold surface without bone matrix deposition. Comparative t-tests at a significance level of 0.05, were carried out to determine the difference in average dark pixel intensity of ROIs of unseeded and seeded scaffold surfaces.The MED610 material is a USP Class VI standard biocompatible material and is certified not to cause an inflammatory response with implantation . To inveAs the cylindrical scaffolds we designed were inspired by the cortical bone and trabecular bone (R1 and R2) in a segmental defect model , ideallyisotropy . From prisotropy . This reAs explained earlier, ideal BGS need to have similar strength characteristics to that of the natural bone, be osteoconductive , and be biocompatible (accepted by the host). To achieve this, we seeded MC3TC cells onto the cylindrical scaffolds and induced osteogenesis to generate bone matrix. This layer of bone matrix has been shown to improve the osteoconductivity of the BGS after implantation and helps in bone penetration into the implant . It has CRFP, which is known to enhance the bone matrix production and contribute to osteoinduction, was actively used for inducing osteogenesis along with other osteogenic agents (ascorbic acid and glycerol phosphate). For the ABS and MED610 scaffolds, the different combinations for pre-coating and osteoinduction are listed in t-test to compare the original scaffold to the scaffolds with no pre-coating (Types A1 and A4).For the ABS material, we carried out a one-way analysis of variance (ANOVA) at a significance level of 0.05 to compare the changes in mechanical properties of scaffolds pre-treated with polylysine to the original scaffolds . In addition, we carried out a Student’s t-tests at a significance level of 0.017 (Bonferroni correction).For MED610 material, we carried out two one-way ANOVA tests at a significance level of 0.05. The first one-way ANOVA test involved scaffolds pre-treated with polylysine being compared to the original strength of the scaffold . In the second test, we compared the scaffolds with no pre-coating . We followed up the one-way ANOVA tests with a pairwise comparison of the groups by carrying out Student’s t-test.Even though the 3D structure and porosity of the ABS and MED 610 scaffolds are similar, the intrinsic mechanical properties of these materials differ, which leads to varying results in compression testing of both materials. To compare the performance of ABS with MED 610 scaffolds, we evaluated the structural contribution of bone matrix in enhancing the strength of the scaffolds in both materials by subtracting the strength of seeded and differentiated scaffolds with the mean strength of original unseeded scaffolds. We compared similar types in ABS and MED 610 by carrying out a Student’s t-tests at significance level of 0.05 were carried out to compare the calcium deposition in different types of ABS scaffolds.Post-compression, ABS scaffolds were stained with 2% Alizarin red (Lifeline Cell Technologies) for validating calcium deposits (a mark of bone deposition) and imaged (Zeiss LSM-510). Imaging studies were carried out as explained in Similarly, in MED610 scaffolds, the von Kossa staining method was applied to validate the bone matrix deposition on the scaffold surface and an imaging study was carried out as explained in p = 0.001), demonstrating significant mineralization on the surface of MED610 scaffolds seeded with bone cells in comparison to unseeded scaffolds, showing that it is an osteoconductive material and allows for bone matrix deposition  showed us that the p = 0.009) when CRFP is used to induce osteogenesis ,  a. There p = 0.0015) in the t-test, we found that the strength increased significantly (p = 0.002) by 11.9% for scaffolds treated with CRFP (p < 0.001) in scaffolds with CRFP treatment (p = 0.05). There was no significant increase in the stiffness and elasticity modulus even though increasing trends were observed. This is illustrated in p = 0.0011 and p < 0.001, respectively). The results of microscopy are illustrated in In the case of MED 610 scaffolds, we found that the data collected on the compression testing follows normal distribution from the Shapiro–Wilk test. The first one-way ANOVA study showed significant differences (p = 0.0085) higher contribution of strength from the bone matrix deposition . We attribute this increase in strength to the formation of bone matrix deposition (shown using Alizarin red staining). The increases in the strength of other combinations of ABS scaffolds (Type A2 and Type A3) was not found to be significant.In our initial studies on cylindrical ABS scaffolds, we observed bone growth, osteogenesis, and osteointegration by evaluating the effect of the increase in the bone matrix on the mechanical strength of the implants. In some cases, we coated the scaffolds with polylysine before cell seeding, as polylysine is known to promote cell adherence to plastic surfaces . In someIn the studies with MED 610, we observed that the scaffolds showed a significant increase in compressive strength compared to the (Type M1) dry scaffolds (9.6% for Type M3 and 19.8% for Type M5) when they were treated with CRFP along with the osteogenic reagents. This shows that the treatment with CRFP itself plays a significant role in generating the bone matrix and improving the mineralization and biomechanical properties of the scaffolds. The stiffness and elasticity modulus for both ABS and MED610 scaffolds revealed that there was no significant increase in these properties in all of the scaffold combinations. This shows that the scaffolds treated with CRFP, which are not pre-coated, give quantitively better results.When we compared the performance of ABS with MED610, we observed that in the case where the scaffold was pre-coated with polylysine, contribution of strength from the bone matrix decreased. However, we see that in the case where CRFP was used along with the osteoinduction reagents (G6P and AA) in both pre-coated and non-coated scaffolds, there was no significant difference between the performances of the two materials; This shows us that the bone matrix deposition on both ABS and MED610 scaffolds is nearly similar when CRFP is used during osteoinduction.One of the limitations in the design of our scaffolds is that the cylindrical scaffold was designed by mimicking the cross-section of a femur and a cylindrical design pattern was selected for ease in our in vitro studies. Eventually, when biomimetic scaffolds are to be used for in vivo studies to replace bone stock, the scaffolds will be designed using the shape and contour of the bone surface. In this study, the pillars in the scaffolds were designed to be as thin as possible to increase the porosity of the scaffold. This pattern was selected to have a better field of view in the imaging studies and also to observe how far cells can penetrate within the grooves of the scaffolds. In the future, the thickness of the pillars can be changed to adjust for the axial stiffness of the scaffold so that it uniformly distributes stress when integrated with the healthy bone.We successfully demonstrated that artificial 3D-printed scaffolds developed by seeding cells perform better biomechanically and produce more bone matrix when CRFP is used in combination with osteogenic reagents. We also found that scaffolds without pre-coating perform better in comparison to the scaffolds coated with polylysine before cell seeding. These results are essential for developing scaffolds for our future in vivo animal studies.


Anxiety is frequently observed in the preoperative setting. The negative impact of preoperative anxiety is well known. In the context of gynaecological surgery, anxiety is exacerbated by the fact that the intervention can have catastrophic repercussions on a woman’s body image, sexuality, and psycho-affective well-being. Music listening is increasingly used as an alternative therapy for minimizing preoperative anxiety. Personal preferences, familiarity, and popularity may be key elements for an optimal relaxation response to music.This study aimed to determine whether listening to self-selected music decreases preoperative anxiety in women scheduled to undergo gynaecologic surgery compared with predetermined music from an application (MUSIC CARE®).The MUANX study was a single-blind, monocentric, parallel, superiority, randomized controlled trial. A total of 174 women were included and randomized in two groups between August 2017 and September 2018. Patients in the intervention group listened to the personal music playlist that they had created before being hospitalized. Patients in the control group listened to the predetermined playlist on the MUSIC CARE® application. All patients received standard nursing care and listened to 20 min of music 1 h before surgery. Anxiety scores were assessed before and after the music session using Spielberger’s State-Trait Anxiety Inventory (STAI).The mean age of the 171 evaluated patients was 41.5 years (SD = 10.0 years). Before the music session, the STAI state anxiety score was similar in the control group  and the intervention group . After the music session, this score had significantly decreased in both the control group  and the intervention group , with no significant difference in score reduction between groups. Physiological parameters were unchanged after the music session. No significant differences in postoperative measurements  were observed between the two groups.Self-selected music is as effective as predetermined music for reducing patient anxiety before gynaecological surgery. As it has no side effects and is easily applicable in gynaecological surgical services, this non-drug intervention may be proposed by healthcare professionals in the management of preoperative anxiety.ClinicalTrials.gov) #NCT03226834. Registered on 24 July 2017. https://clinicaltrials.gov/ct2/show/NCT03226834?term=muanx&draw=2&rank=1The MUANX trial (MUsic therapy on ANXiety) is registered at the US National Institutes of Health ( Surgery and preoperative waiting are anxiety-provoking situations , 2. It iThis is especially the case in the context of gynaecological surgery, which most often results in the partial or total removal of an organ that is necessarily linked to maternity or femininity. Sexuality can be damaged . For exaHigh levels of anxiety result in the activation of the autonomic nervous system and in negative physiological manifestations , 11–13. High levels of anxiety can complicate the administration of preoperative drugs, while negatively interfering with the induction of anaesthesia and delaying recovery .In view of the above, reducing preoperative anxiety appears to be essential. Traditionally, the reduction of preoperative anxiety was based on the use of sedatives or anti-anxiety drugs administered prophylactically in the preoperative phase. However, these treatments can have negative side effects like drowsiness and respiratory depression. They can also interact with anaesthetic agents by prolonging the recovery phase and the duration of hospitalization . In receIn surgical context, listening to music has an effect on blood pressure and pulse rate, and the classic music resulted in lowered blood pressure and heart rate . In addiTo date, however, most published controlled randomized trials have evaluated the effects of music interventions on preoperative anxiety by comparing patients who listen to researcher-selected or predetermined music with patients who receive standard care alone. Few studies have compared the impact of different styles of music on preoperative anxiety . This isOur study seeks to fill this gap in the literature by letting patients create their own, culturally adapted music playlist. Our hypothesis was that music therapy programmes, which use specific rhythm sequences to potentiate the anxiolytic effects of music, could be optimized by adapting them to the preferences and cultural contexts of patients.The aim of our study was to determine whether listening to self-selected music decreases preoperative anxiety in women scheduled to undergo gynaecologic surgery as compared with predetermined music from a music therapy software programme (MUSIC CARE®). Physiological parameter evolutions after the music session, post-operative pain intensity, and duration of post-surgical hospitalization were also compared.https://link.springer.com/article/10.1186/s13063-018-3093-6 [Complete information on research methods may be found in study protocol, which is freely available at 8-3093-6 .The MUANX study was a single-blind, monocentric, parallel, superiority, randomized controlled trial conducted in the gynaecological surgery department in French hospital. Patients were randomly allocated to one of two groups: the intervention group listened to a self-selected playlist and the control group listened to a predetermined playlist selected from among those available on the MUSIC CARE® application. Healthcare team members were blinded to patient allocation.The criteria for inclusion in the study were being aged 18 to 70 years; being scheduled to undergo gynaecological surgery under general anaesthesia or spinal anaesthesia at the gynaecological surgery department of the South Reunion Island University Hospital between August 2017 and September 2018; and not receiving anti-anxiety drugs prior to surgery. Exclusion criteria were having generalized anxiety disorder, depression, dementia, or neuromotor disabilities. All eligible patients received standard nursing care and were asked to create a 20-min music playlist based on their personal tastes before being hospitalized.Eligible adults were informed about this study by gynaecologists during their operative consultation or anaesthesiologists during pre-operative screening.Patients in the intervention group listened to the personal music playlist that they had created before being hospitalized. They were allowed to listen to the playlist on the device of their choice: smartphone, tablet, computer, or CD player.Patients in the control group listened to the predetermined playlist that they had selected before randomization from among those available on the MUSIC CARE® application . MUSIC CThe objective of MUSIC CARE® is to promote wellbeing and to induce a relaxed state in patients by loosening their muscles and reducing their level of anxiety or pain. Patients were able to choose from among 25 MUSIC CARE® playlists, which include reverie, Cuban night, guitar ballad, film scores, folk guitar, etc. Sega and Maloya were not included in this playlist.A department nurse or care provider with training in MUSIC CARE® was in charge of explaining the application to study participants.Each patient was asked to create a personal music playlist based on her personal tastes  and to choose a predetermined playlist from those available on the MUSIC CARE® application (before randomization).The music intervention started within the hour preceding surgery. The planned duration of the music session was 20 min .STAI form Y-A).The primary outcome was the difference between the preoperative anxiety score assessed 15 to 20 min before the music-listening session and the preoperative anxiety score assessed straight after the session, as measured with the STAI (State-Trait Anxiety Inventory) for state anxiety. The mean change score from baseline to post music intervention was used to compare groups. The state anxiety score was measured using the second part of the Spielberger’s STAI questionnaire  the difference in the Visual Analogue Scale (VAS) anxiety score between before and after the music session . The VASThe trait anxiety score was measured based on the answers given by study participants to the first part of the STAI questionnaire (French version: STAI form Y-B). The latter was filled during the inclusion visit one day before surgery.The 2013 Cochrane systematic review found that music listening resulted, on average, in a 6-point decrease in the preoperative STAI state anxiety score of patients compared to standard care alone [The randomization of eligible patients was carried out on the day of surgery using Ennov Clinical®. Randomization was single-blinded and balanced between the control group and the intervention group, with a 1:1 allocation ratio and without stratification. Patients were aware of the group to which they had been allocated, but care team members were not. The minimization method programmed in the CSRandomization module of Ennov Clinical® was used to allocate patients. The predictive side of this method was reduced with the two tools available in the CSRandomization module , 36.On the day of surgery, randomization was performed by the research team interviewer directly via the electronic case report form (eCRF). Standard care providers and care team members in charge of outcome assessment were blinded to patients’ group allocation. Only research team members were aware of the group to which patients had been allocated. ‘Do Not Disturb’ signs were placed on patient room doors during the music session. Patients were warned not to mention their group allocation to care team members.n (percentages) or means, standard deviation (SD). The two groups were compared using the Mann-Whitney U test for quantitative variables and the chi-square test or Fisher’s exact test or the Fisher-Freeman-Halton test for categorical variables, as appropriate. Measures before and after the music session were compared using the Wilcoxon signed-rank tests. The evolution of the STAI state anxiety score was evaluated according to the STAI trait anxiety score level using the Kruskal-Wallis test. Multiple 2 to 2 comparisons of the different components of the STAI trait anxiety score were performed using the Mann-Whitney U test with an alpha level of 0.83% (Bonferroni correction: 5%/6). Spearman’s correlation coefficient between the STAI state anxiety score and the STAI trait anxiety score was calculated.Data were expressed as One patient with missing data on the primary outcome was excluded from the analysis. Except for the multiple 2 to 2 comparisons, the significance level was set to 5%. Analyses were performed using SAS® version 9.4 .A total of 174 women were included and randomized in the MUANX trial between 1 August 2017 and 20 September 2018. Of these, 89 (51%) were allocated to the control group (predetermined music group) and 85 (49%) to the intervention group (self-selected music group). In the control group, two patients were excluded because they refused to listen to music. One patient had postponed surgery after she received music therapy. For this patient, we analysed the primary outcome evaluation (but data were missing on postoperative measurements). In the self-selected music group, one patient was excluded due to missing data on the primary outcome and one patient included in analysis had an interruption during the music listening. The flow chart describing the process of participant selection is presented in Fig. n = 119) and world music . As regards medical history, 143 (84.6%) women had a history of hospitalization, 136 (81.4%) had a history of anaesthesia, 133 (78.2%) had a history of surgery, and 145 (84.8%) had a history of pregnancy. During surgery, 170 (99.6%) had general anaesthesia and 1 (0.6%) had spinal anaesthesia. The patients were mainly operated for uterine surgery . Baseline socio-demographic characteristics, musical habits, medical history, and types of surgery are shown in Table Baseline socio-demographic characteristics, musical habits, medical history, and types of surgery were similar in the two groups Table . Women wp = 0.857). Nineteen (11.1%) women had a high/very high initial STAI state anxiety score (≥ 56). The initial mean VAS anxiety score for the total sample was 3.4, SD = 2.5 . As regards physiological parameters, 53 (31%) patients had a high initial pulse rate and 37 (21.8%) had a high initial blood pressure. No significant differences in initial anxiety scores or initial physiological parameters were found between the two groups (p > 0.05). Anxiety scores and physiological parameters before the music session are presented in Table Before the music session, the mean STAI state anxiety score for the total sample was 38.9 SD = 12.4). The initial STAI state anxiety score was similar in the control group and the intervention group . Likewise, no significant difference in surgical procedure duration was observed between the two groups . Surgery was initiated 92.2 min (SD = 60.1 min) after the music session had ended, with no significant difference between the two groups (p = 0.286).No significant difference in music session duration was found between the two groups  was observed in the total sample. The STAI state anxiety score had also decreased significantly between before and after the music session in each group . There was no significant difference in score reduction between the two groups (p = 0.215). The evolution of the STAI state anxiety score between before and after the music session is shown in Table After the music session, a significant decrease in the STAI state anxiety score  was observed in the total sample. The VAS anxiety score had also decreased significantly in each group . There was no significant difference in score reduction between the two groups (p = 0.798). The evolution of the VAS anxiety score between before and after the music session is shown in Table After the music session, a significant decrease in the VAS anxiety score , systolic blood pressure , or diastolic blood pressure  was observed in the total sample. There was no significant difference in the reduction of physiological parameters between the two groups (p > 0.05). The evolution of physiological parameters between before and after the music session is shown in Table No significant reduction in pulse rate . Likewise, no significant difference in the duration of hospitalization was found between the two groups (p = 0.250). The mean postoperative pain intensity score was 2.5 (SD = 2.5) and the mean duration of hospitalization was 3.6 days (SD = 1.7 days). Postoperative measurements are shown in Table No significant difference in the postoperative pain intensity score was observed between the two groups (Participants had a mean STAI trait anxiety score of 42.5 (SD = 9.8). Twenty (11.8%) women had a high/very high STAI trait anxiety score (≥ 56).p < 0.001). The reduction of the STAI state anxiety score between before and after the music session was inversely correlated to the STAI trait anxiety score . On average, the STAI state anxiety score decreased by 4.2 points (SD = 4.7 points) in patients with a very low STAI trait anxiety score (≤ 35), by 5.5 (SD = 7.6 points) in patients with a low STAI trait anxiety score (36–45), by 7.7 points (SD = 8.6 points) in patients with a middle STAI trait anxiety score (46–55), and by 11.8 (SD = 10.1 points) in patients with a high/very high STAI trait anxiety score (≥ 56) (p = 0.012). Comparisons 2 to 2 showed a significant difference in the reduction of the STAI state anxiety score between patients with a very low STAI trait anxiety score and patients with a high/very high STAI trait anxiety score (p = 0.003). The evolution of the STAI state anxiety score according to the STAI trait anxiety score is shown in Fig. The STAI state anxiety score before the music session was correlated to the STAI trait anxiety score . The decrease in the STAI state anxiety score observed in patients who had included local music in their personal playlist was not significantly different from the decrease observed in patients who listened to predetermined music .In the self-selected music group, 23 (27.4%) patients had included local music in their personal playlist. After the music session, the STAI state anxiety score had decreased by 7.4 points (SD = 7.4 points) in these patients, compared with a decrease of 4.8 (SD = 6.2 points) in the 61 (72.6%) patients who had not included local music in their personal playlist . Our main finding is that preoperative anxiety scores, between after and before music session, decrease significantly in patients who receive music therapy, regardless of whether they listen to self-selected music or predetermined music. No significant difference in anxiety reduction was observed between the predetermined music group and the self-selected music group. By contrast, physiological parameters (pulse rate and blood pressure) had not decreased in either group after the music session. Our finding did not confirm the research hypothesis as measured by the primary outcome. We can consider the quality of music playlist created by patients. In fact, several factors are important for an anxiolytic and positive effect of music on anxiety: a high degree of periodicity, a catch melody line, a key that is experience as pleasant, few changes in volume or rhythm, harmony sequences that are rousing, and an absence of song words . Each paAnother possible reason for these results is that 80% patients included in the study has history of anaesthesia and history of surgery, so the patients are experienced in hospital and then had lower level of anxiety. The baseline STAY score in our study was 38.9 ± 12.4 points whereas the average pre-surgical score found in the literature is 43 ± 8 points [Our results are in line with the published literature, including the 2013 Cochrane systematic review which found that music listening can have a beneficial effect on preoperative anxiety , 25, 37.In our study, we expected that patients who listened to self-selected music would have included local music, also Creole music  , 29, in It should be noted that our patients did not have high levels of anxiety prior to the music session. Indeed, only 19 (11.1%) of our patients showed high/very high STAI state anxiety scores at the beginning of the study. On the one hand, these results confirm those of a 2012 study on the prevalence of anxiety in patients scheduled to undergo gynaecological surgery . On the Our study has several limitations that must be acknowledged. First, all patients were women. This may have led to a selection bias, which makes it difficult to extrapolate our results to the entire surgical population. Indeed, our study focused on gynaecological surgery; it might be interesting to extend it to other types of surgery and in a male population. Second, our study was single-blind: while care team members were unaware of the group to which patients had been allocated, patients themselves were evidently aware of this. Third, all patients in the control group used the provided headphones to listen to the predetermined playlist, whereas patients in the intervention group were allowed to listen to their personal playlist without headphones if they wished. Our study did not explore the differential effects of music listening on the anxiety of patients depending on whether or not they used headphones. However, it should be noted that a 2011 study found no difference in the efficacy of music therapy between patients who listened to music on headphones and patients who listened to broadcast music .Our study shows that listening to self-selected music is as effective as listening to predetermined music for reducing patient anxiety prior to gynaecological surgery. A 20-min music session was found to significantly reduce anxiety in gynaecological patients. Music listening is a simple anti-anxiety therapy that has no side effects and that can be proposed by nurses in the gynaecological preoperative context to complement classical therapy.