The electrospinning of hybrid polymers is a versatile fabrication technique which takes advantage of the biological properties of natural polymers and the mechanical properties of synthetic polymers. However, the literature is scarce when it comes to comparisons of blends regarding coatings and the improvements offered thereby in terms of cellular performance. To address this, in the present study, nanofibrous electrospun scaffolds of polycaprolactone (PCL), their coating and their blend with gelatin were compared. The morphology of nanofibrous scaffolds was analyzed under field emission scanning electron microscopy (FE-SEM), indicating the influence of the presence of gelatin. The scaffolds were mechanically tested with tensile tests; PCL and PCL gelatin coated scaffolds showed higher elastic moduli than PCL/gelatin meshes. Viability of mouse embryonic fibroblasts (MEF) was evaluated by MTT assay, and cell proliferation on the scaffold was confirmed by fluorescence staining. The positive results of the MTT assay and cell growth indicated that the scaffolds of PCL/gelatin excelled in comparison to other scaffolds, and may serve as good candidates for tissue engineering applications. Electrospinning is a simple, low-cost and versatile method for the production of polymeric nanofibrous scaffolds. These mesh structures offer high porosity and high surface area, thereby enhancing cell behavior, i.e., adhesion, proliferation, etc. ,2,3. SucA broad range of materials can be electrospun into nanofibers. Synthetic polymers have been widely used for different tissue engineering (TE) applications such as bone and cardiac tissue with poly-L-lactide (PLLA) ,7, skin Unfortunately, despite the widespread use of synthetic polymers in the field of TE, these materials are not without their faults. Among their most significant drawbacks is the high hydrophobicity of the raw materials used to create these polymers, which hinders cellular attachment and scaffold infiltration, and ultimately, constrains cellular proliferation .The aforementioned drawback, which is inherent to the surface interaction of synthetic materials, can be overcome via two different approaches: oxygen plasma treatment, that produces changes in the chemical groups of the surface, thereby improving the wettability ,19; or tWhen they are applied as fiber coatings over a synthetic polymer, natural polymers cause an almost negligible change in the mechanical performance of the final mesh, and bring about improvements in its surface properties . HoweverBoth blending and coating are known to enhance scaffold outcomes in terms of cellular fate ,21,22,23PCL pellets  and gelatin powder of porcine skin were purchased from Sigma-Aldrich . Glacial acetic acid and chloroform were purchased from Sigma-Aldrich , and methanol from AppliChem Panreac  and 3- (MTT) were obtained from Roche.v/v), agitating the mixtures with a magnetic stirrer at 600 rpm overnight at room temperature (23 ± 1 °C) [v/v) by stirring the mixture vigorously for 3 h at room temperature. After the preparation of the polymeric solution, PCL (20 wt.%)/gelatin at 80:20 volume ratio was mixed and stirred for 48 h.For electrospinning, first, PCL (10 wt.%) and PCL (20 wt.%) were added to chloroform/methanol  . Gelatin® tube. A high voltage DC power supply  was used to provide the necessary electric field between the needle and the collector.Nanofiber membranes were fabricated on an upright, home-made setup , using a2 piece of flat aluminum foil.For the process of electrospinning, solutions were placed in a 1 mL plastic syringe. For PCL (10 wt.%), a voltage of 13 kV was applied, with a distance of 10 cm between needle and collector. The blended PCL (20 wt.%)/gelatin solution was extruded under 22 kV and a distance of 10 cm. The nanofibers were collected on a 9 × 9 cmThe morphology of the nanofibrous scaffolds was studied with FE-SEM  at an accelerating voltage of 5 kV. Fiber diameters and pore areas of the scaffolds were calculated on the basis on FE-SEM images by using image analysis software  and the Diameter J plugin , respectively.2), and the crosshead displacement speed was set to 100 mm/min for all tests. The thickness was measured with a thickness digital gauge .Mechanical measurements were obtained by applying tensile test loads to specimens prepared from the electrospun nanofibrous mats. In this study, five specimens from each condition were cut with a caliper to widths of 11 mm. Once the samples  had been cut with a microtome blade, the electrospun mats were detached with tweezers from the foil and placed on the traction testing machine  with a load cell of 50 N  at room temperature. The initial distance between grips was 10 mm   were cul2 and Ar respectively) process  in order to increase the hydrophilicity of the surface. Then, electrospun meshes were placed on 24 well plate cell crowns  and sterilized with UV radiation from both sides for 30 min each. Subsequently, the scaffolds were incubated in a 24 well tissue culture plate with MEF cells at a density of 2 × 104 cell per well for 24, 48 and 72 h at 37 °C in 5% CO2 incubator. After the incubation period, the wells were washed with DPBS, and then 50 μL of MTT solution was added to the wells. The media was discarded and 400 μL of DMSO was added to the wells to dissolve the formazan crystals overnight at 37 °C. Finally, the optical density (OD) value was measured at 490 nm using a spectrophotometer. The proliferation of MEFs was determined by OD values.The viability of MEF cells on nanofibrous scaffolds was quantitatively determined using colorimetric MTT assay. Before cell seeding, scaffolds were treated with a plasma  Triton (in DPBS) and 2% bovine serum albumin (BSA) for 15 min. After thorough washing in DBPS (3–4 times), the cells on the scaffolds were stained with phalloidin stain  and incubated for 1 h. The cells were stained with DAPI  for 15 min to visualize their nuclei. Images were then obtained using a fluorescence microscope, at excitation wavelengths of 350 nm and 495 nm for DAPI and AlexaFluor 488, respectively.Cell proliferation on the nanofibrous scaffolds was studied by fluorescence staining. As explained in the previous section, the same sterilization and culture process was carried out. After the incubation period, scaffolds were rinsed three times with DPBS, fixed with 4% paraformaldehyde for 10 min at room temperature and washed with DPBS (3–4 times). The cells were permeabilized with 0.3%  for 5 min each at room temperature. Finally, the scaffolds were air-dried overnight, sputtered with palladium at 18 mA for 75 s and analyzed under FE-SEM to study the morphology of attached cells on scaffolds.n = 3) and a statistical analysis was performed using Wilcoxon test, with p < 0.05 indicating statistical significance.All data presented are expressed as mean ± standard deviations (SD). Cell viability experiments were performed in triplicate . This was confirmed by the work of L. Ghasemi-Mobarakeh et al., where blending PCL with gelatin caused a reduction in mechanical strength [Regarding mechanical characterization, uniaxial tensile tests were carried out. strength  due to tstrength ,34,35. I−1 (asymmetric CH2 stretching), 2867 cm−1 (symmetric CH2 stretching), 1722 cm−1 (carbonyl stretching), 1295 cm−1 (C–O and C–C stretching), and 1167 cm−1 (symmetric C–O–C stretching) [−1 due to N-H stretching of amide bond, C–O stretching at 1637 cm−1 characteristic in gelatin spectra, and N-H bending at 1545 cm−1. Also, the PCL main peaks exhibited lower transmittance. In the PCL/gelatin composite scaffold, all the characteristics bands of PCL and almost all those of gelatin were observed. The characteristic gelatin band at 3378 cm−1 was weakly detected for the PCL/gelatin scaffold. The presence of this band is much less noticeable for PCL/gelatin than for coated gelatin. This may have been due to the fact that in PCL/gelatin, the gelatin amount is lower and further way from the surface because the blending process embeds the it within the scaffold, which ultimately makes it harder to detect via FT-IR [−1 and 1297 cm−1 appeared in the IR spectrum of PCL/gelatin composite scaffolds, corresponding to the characteristic bands of PCL originally situated at 1722 cm−1 and 1295 cm−1. Additionally, the distinctive amide band of gelatin at 1640 cm−1 showed low intensity and shifted to around 1648 cm−1. Another band was detected at 1544 cm−1, characteristic of the amide group. The shifting of original absorption bands toward lower wave numbers in the PCL/gelatin scaffolds indicated that interactions may have occurred, like hydrogen bonds between the ester groups of the PCL and the amine groups of the gelatin molecules within the scaffold [etching) ,39. The scaffold ,40. All p ≤ 0.05)) was observed for blended scaffolds, which achieved better proliferation than PCL or gelatin coated PCL scaffolds. This was because including gelatin within the blend distributed the natural polymer throughout the entire scaffold and positively impacted cell proliferation, especially when compared to scenarios where the presence of gelatin is limited to the scaffold surface (as occurs in coating procedures). Additionally, these results indicate that the PCL, gelatin coated PCL and PCL/gelatin scaffolds did not induce any cytotoxic effects in MEF cells.To verify the affinity of MEF on the scaffolds, a FE-SEM image analysis was performed after 72 h to evaluate cell morphology and visualize attachments . CellulaOur results suggest an increase in cell attachment in the presence of gelatin, regardless of whether it is used as a coating or a blend ,37. HoweThe mechanical microenvironment has previously been shown to affect cell structure ,46,47,48We studied the ways in which gelatin coalescence (coating or blend) enhances the performance of PCL scaffolds in terms of their mechanical properties and cellular response. We also provided a thorough analysis of the differences between using gelatin coated PCL and blended PCL/gelatin scaffolds. PCL and gelatin coated PCL scaffolds showed higher elongation rates and stiffness than the blended PCL/gelatin samples, which had better MEF responses in terms of cell attachment and spreading. Furthermore, our results also indicated that the fiber diameter distribution and pore size were modulated after blending with gelatin. Blended gelatin not only positively influences cellular response, but also improves the mechanical and morphological properties of the fibrous network. Our results suggest that PCL/gelatin blends enhance the performance of polymeric scaffolds, both in cellular and mechanical aspects. All in all, these outcomes provide evidence of the superiority of blended PCL/gelatin scaffolds over their PCL and PCL gelatin coated counterparts under certain circumstances, such as would healing ,33.This work suggests that this methodology could be extended to create other natural and synthetic polymer blended combinations, and that similar results may be expected. The challenges of using polymeric blends usually lie in the creation of stable solutions and the compatibility of the chosen materials. The incorporation of blends in the development of scaffolds for tissue engineering applications undoubtedly shows potential and more promising results than coating protocols for further improvements in scaffold designs.


In this study, we retrospectively evaluated the deviation from the planned axis of 3 Toric intraocular lenses (TIOL). Included in the study 190 eyes, operated by two surgeons using two different manual marking techniques. The patients were implanted with either AcrySof IQ Toric SN6AT (Alcon) (n = 90), POD FT (PhysIOL) (n = 50), or TECNIS Symfony Toric (J&J) (n = 50). At least 1 month postoperatively, the IOL was photographed, and the axis was measured using a designed software. The difference between the planned and actual axis was defined as axis deviation. The effect of IOL type, astigmatism direction, and marking techniques on the average degree and direction of the IOL deviation were evaluated and compared. There was no significant difference in the average deviation between the IOLs , and its direction  deviation, respectively). With the rule, astigmatism had significantly more CW deviation compared with against the rule and oblique astigmatism , but the average deviation was similar. The marking techniques did not influence the degree or direction of the deviation. Hoffer et al.2 reported mean corneal astigmatism of 1.0 D.Nowadays, cataract surgery has become a refractive surgery. With the development of toric intraocular lenses (TIOLs), surgeons can provide satisfactory refractive results as patient’s demand spectacle independence, even in cases of corneal astigmatism. Ferrer-Blasco et al. 3.Optimal astigmatism correction with a TIOL requires accurate IOL alignment and rotational stability. The greater the IOL cylinder power, the greater the importance of correct alignment and stability of the lens. Deviation of a TIOL from its intended orientation decreases its corrective power, with approximately 3.3% loss of cylindrical power for every degree of misorientationOur study aimed to compare the deviation from the planned axis of 3 types of TIOLs, AcrySof IQ Toric , POD FT , TECNIS Symfony Toric, , and to evaluate the effect of the eye characteristics, TIOL type, corneal astigmatism direction and surgeons marking techniques on the amount of deviation from the planned axis and its direction.The medical charts of 190 eyes with a TIOL implantation during cataract surgery, operated by two surgeons using two different manual marking techniques, were retrospectively reviewed. The study adhered to the tenets of the Declaration of Helsinki, and all methods were carried out following the relevant guidelines and regulations. The Meir Medical Centre Ethics Committee approved all experimental protocols. The IRB of the Meir Medical Centre Ethics Committee waived the requirement for informed consent from the study subjects.Three types of TIOLs were compared: SN6AT , POD FT , and Symfony ZXT . The SN6AT AcrySof IQ Toric is monofocal toric, hydrophobic acrylic IOL. It has a 6.0 mm optic diameter, and its overall length is 13.0 mm. The FineVision POD FT is a trifocal toric hydrophilic IOL with an optic body diameter of 6.00 mm and an overall diameter of 11.40 mm. The ZXT Symfony Toric lens is an extended depth of focus, toric, foldable, hydrophobic acrylic 1-piece lens, with an optic diameter of 6 mm and an overall diameter of 13 mm.Preoperatively, all patients underwent measurements by the Lenstar LS 900, IOLMaster 500, Pentacam, and the Atlas topographer.https://rbfcalculator.com/online/index.html). The surgeon chose the implanted TIOL using the Barrett online toric calculator  and the IOL manufactures’ online calculators, . The TIOL power and placement axis were calculated using the Lenstar measurement when agreement between all devices was achieved. When no agreement was achieved between Lenstar and the IOL Master, the measurements that were closer to the axis of the topographer and tomographer were chosen for the IOL calculation. When no agreement was achieved between all devices toric IOL was not implanted. Additional adjustments were done according to the surgeon’s experience and preferences .All biometric measurements were compatible with the strict validation criteria described by Warren Hill  performed all the surgeries at Ein-Tal Eye Center, Tel-Aviv.Ink marks were placed, preoperatively, at the limbus, while the patient's head was positioned behind the Haag-Streit  slit lamps. The two surgeons used different manual marking techniques. One surgeon (EIA), marked the planned axis of the TIOL lens (Primary IOL marking) directly. The second surgeon (GK) marked 0, 180- and 270-degrees (Reference axis marking). The Incision and the IOL axis were marked as the first step of the surgery using the Mendez ring or other similar ring.All surgical procedures performed through a small clear corneal incision (2.2–2.4 mm), using the phacoemulsification technique. After removing the cataract, the IOL was implanted into the capsular bag under an anterior chamber maintainer (ACM) and without an ophthalmic viscoelastic device (OVD). The IOL dialed into the planned axis and pushed gently back against the posterior capsule.7.Follow-up visits were 1 day, 1 week, and 1 month after the surgery. On the 1 month postoperative visits, a complete dilated examination and manifest refraction were performed. The TIOL position was detected, and retroillumination pictures were taken with a slit lamp digital color camera . The Goniotrans software  was used to determine the TIOL axis. This software integrates virtual protractor over an eye picture, by dragging the radial line to the position of the toric lens axis marks on the angles pattern , surgeons marking techniques, and other eye characteristics such as corneal astigmatism (direction and power) axial length, and anterior chamber depth, on the IOL deviation and its direction. TIOL deviation was defined as the difference between the TIOL planned axis and the actual alignment axis measured at least 1 month after surgery.Journal of Cataract and Refractive Surgery on IOL studies4. Continuous variables were compared using a one-way analysis of variance (ANOVA) or the Kruskal–Wallis test, followed by post-hock tests, as indicated by a normality test (Shapiro–Wilk). Categorical variables were compared using the Pearson’s chi-square test. Pearson correlation was used to evaluate the correlation between axial length, anterior chamber depth, preoperative astigmatism, TIOL cylinder power and TIOL spherical power to the degree of rotation. A P value of less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS . Between-subjects design was applied, as specified in the editorial of the The study includes 190 eyes with toric IOL implantation: 90 eyes with SN6AT IOL, 50 with POD FT and 50 with ZXT. The patient’s’ demographics, preoperative biometric data, and the IOL characteristics are summarize in Table The average deviation from the planned axis was similar for the different IOLs . No difference in the misalignment direction was found , Table The corneal astigmatism direction  did not influence on the mean degree of misalignment  Table The deviation of the TIOL from the planned axis divided to subgroup according to the degree of the deviation: < 1° (n = 23), 1°–5° (n = 102), 6°–10° (n = 53), > 10 (n = 12), Table No difference was found in the degree and direction of misalignment between the two surgeons marking techniques, Table The refractive visual outcomes evaluated at least 1 month after the surgery are present in Table 3. In this study, we found similar and low deviations from the planned axis of 3 common TIOL: SN6AT , POD FT , and Symfony ZXT .Toric IOLs designed to correct corneal astigmatism during cataract surgery and increase spectacle independence. It is even more critical with multifocal IOLs, which require minimal residual astigmatism for their total effectivity. Successful astigmatism correction requires accurate measurements and calculation of the power, and axis of corneal astigmatism, precise marking and alignment of the TIOL during the surgery, and excellent rationale stability after the TIOL implantation. Every degree of deviation from the desired axis reduces the effectivity of the TIOL by 3.3%5 found that most of the postoperative TIOL rotation occurs within the first hour. Similarly, Lee and associates6 suggested that postoperative rotation occurs very soon after the surgery since the alignment results for patients examined at least 1 h postoperatively or the following day were identical. Our study evaluated the TIOL position at least 1 month postoperatively using slit-lamp photography and purpose-designed software, assuming no further deviation should be expected. It is important to note that we measured the total deviation from the planned axis, including marking and alignments inaccuracies and postoperative IOL rotation. Our findings were similar to the reports of most studies reporting 5 degrees or less of misalignment 13. We found that the three investigated IOLs had an excellent and similar small rates of deviation from the planned axis . There was also no difference in the deviation CW or CCW direction. The Eye characteristic, axial length, anterior chamber depth, the direction of corneal astigmatism, and the method of the manual marking technique (reference plane or planned IOL position) were found to not influence on the degree of the deviation. The AcrySof SN6AT and the Symfony ZXT are 13.0 mm diameter lenses designed with C-loop haptics, In contrast the FineVision POD FT is an 11.40 mm diameter lens designed with double C-loop 4-point-fixation. One should expect that due to the smaller diameter, a higher rate of rotation is expected14. The stability provided by the unique double C-loop haptics POD FT TIOLs design may compensate for the smaller diameter and explain our finding15.Inoue et al.6 compared the rotational stability of the Acrysof and Tecnis monofocal TIOL . Contrary to our results, they found that the Tecnis TIOL had significantly higher overall rotation, predominantly CCW. As opposed to Lee et al. findings, we found a similar deviation and no predominantly for the deviation direction. CW deviation was found in 56.6% of the eyes implanted with the SN6AT and 55.8% of eyes implanted with the ZXT.Lee et al.16. Ruhswurm et al.17 measured IOL rotation and found significantly greater rotation with the vertical axis.Schartmüller et al. studied the rotational stability of Vivinex XY1 IOL, a single-piece hydrophobic intraocular lens, in 122 eyes. They found significantly higher CW TIOL with no correlation to the corneal astigmatism axisWe found that CW deviation was significantly more common in WTR corneal astigmatism without correlation to the degree of TIOL deviation. The underlying factors for the CW rotation are unclear.18 observed that the oblique axis was the most unstable. In our study, eyes with oblique corneal astigmatism showed better results with more eyes within 5 degrees or less of the planned axis compared with eyes with WRT and ATR corneal astigmatism.Till et al.19. Popp et al. evaluated four different marking techniques: coaxial slit beam, bubble marker, pendular marker, and tonometer marker. All methods except the tonometer marker gave accurate results with rotational misalignment between 1.8° and 2.9°20. We also did not find a significant difference in the TIOL deviation degree and direction when using two different manual marking techniques. Several studies that investigated predictors for TIOL rotation found, contrary to our findings, that longer axial length and lower spherical IOL power are positive predictors for TIOL rotation. These may be due to the larger size of the capsular bag expected in these cases enabling the TIOL to rotate more easily24.Several studies have demonstrated the accuracy of manual marking. The three-step marking method is pretty accurate, with a total error of 4.9° ± 2.1° in toric IOL alignment25. However, the measurement method used in this study is based on software analysis designed for this purpose. It is highly reproducible and similar to methods used in other studies27. It is important to notice that we measured the total deviation from the planned axis, including marking and alignments inaccuracies and postoperative IOL rotation. This provides information on real-life expectations and results of TIOLs.The weaknesses of this study include its retrospective design and lack of randomization. Secondly, PIOL axis measurement technics available today are not free of error. Viestenz et al. demonstrated that slight deviations might be the result of cyclorotation of the eye during standardized photographyIn conclusion, our results suggest that the three investigated TIOL models yield excellent and similar deviations from the planned axis. The IOL design and material had no influence on the magnitude and direction of the toric IOL deviation. We found significantly more CW deviation in WTR corneal astigmatism than in ATR and oblique corneal astigmatism, with no difference in its magnitude. No difference was found when comparing the two surgeons’ manual marking techniques in the degree and direction of the deviation.


The aim of this study was to investigate the interaction of the nanostructured and ground Ti6Al4V titanium alloys with simulated body fluid complemented by the defined precipitation of hydroxyapatite-like coating and to study the cytotoxicity and differentiation capacity of cells with such a modified titanium alloy. Nanostructures were fabricated using electrochemical oxidation. Human mesenchymal stem cells (hMSC) were used to evaluate cell adhesion, metabolic activity and proliferation on the specimens. The differentiation potential of the samples was investigated using PCR and specific staining of osteogenic markers collagen type I and osteocalcin. Our results demonstrate that both pure Ti6Al4V, nanostructured samples, and hydroxyapatite-like coating supported hMSC growth and metabolic activity. Nanostructured samples improved collagen type I synthesis after 14 days, while both nanostructured and hydroxyapatite-like coated samples enhanced collagen synthesis on day 21. Osteocalcin synthesis was the most enhanced by hydroxyapatite-like coating on the nanostructured surfaces. Our results indicate that hydroxyapatite-like coating is a useful tool guiding hMSC osteogenic differentiation.Titanium and its alloys are widely used for substitution of hard tissues, especially in orthopaedic and dental surgery. Despite the benefit of the use of titanium for such applications, there are still questions which must be sorted out. Surface properties are crucial for cell adhesion, proliferation and differentiation. Mainly, micro/nanostructured surfaces positively influence osteogenic differentiation of human mesenchymal stem cells. Ti Titanium and its alloys are widely used for substitution of hard tissues, especially in orthopaedic and dental surgery. Their greatest advantages are low density, high fatigue strength, biocompatibility and excellent resistance to corrosion.2 nanotube layer formation on the surface. It is possible to easily control the diameter and length of the nanotubes by adjusting experimental conditions, such as the electrolyte composition, potential setting or temperature. Diameters of nanotubes ranging between 20–100 nm are the most common.9–12 Selecting the most appropriate nanotube diameter is very important especially for cell interaction. Nanotubes of smaller diameters (15–30 nm) are known to support cell adhesion, while nanotubes of larger diameters (80–100 nm) have been reported to support osteogenic differentiation but can also result in programmed cell death.13,14 So changed nanotopography of the material can improve cell adhesion which is crucial for an implants osseointegration.15 The process of osseointegration is driven by supporting the adsorption of proteins such as vitronectin or fibronectin, which mediate the signals between cells and material surface. It is well known that cell adhesion is not influenced only by surface properties, but also by material chemistry and charge distribution.16–18Another strategy for improving osseointegration is the preparation of nanostructured surface on titanium alloys. Electrochemical oxidation in fluorides containing electrolytes leads to self-organised TiO19 For successful implant integration, biologically effective ions Ca and P are very important for cell adhesion, differentiation and growth of osteogenic cells, and contribute to bone resorption and osteoclastogenesis as well.20–23As mentioned above, the binding of bone and metal implants is mediated by the formation of an apatite layer on the implants surface. Therefore, the presence and morphology of the apatite layer can dramatically influence cell adhesion, differentiation and finally implant integration. Hydroxyapatite coated titanium showed better osteoinductivity and osteoconductivity as compared to untreated titanium surfaces.24 the ion beam assisted vapour deposition method,25 the plasma spraying method,26 the electrochemical deposition method27 and phase-transited lysozyme coating.28 The exposition of titanium in a simulated body fluid (SBF) in order to create Ca and P-containing layer was also investigated. It was revealed, that the presence of the nanostructure enhances calcium and phosphate deposition and titanium surfaces can be spontaneously covered with hydroxyapatite-like layer via exposure to SBF.29Various techniques for the coating of titanium and its alloys with Ca–P layer have been studied, including the sol–gel method,6Al4V alloy. To investigate the biocompatibility effect of nanostructured samples with and without the HAp-like coating, in vitro cultivation using human mesenchymal stem cells (hMSC) was applied, whereas a glass coverslip was used as reference.The purpose of the present work was to study the interaction of human mesenchymal stem cells (hMSC) with HAp-like coating on ground and nanostructured surfaces of the Ti6Al4V alloy with a 16 mm diameter and a 3 mm height were ground using 2500 SiC paper and cleaned with ethanol and demineralised water in an ultrasonic bath. Nanotube preparation was managed by Galvanostat Jaissle IMP 88 PC-200V with the control unit PGU-AUTO Extern. A silver/silver chloride reference electrode with 3 mol L−1 KCl (ACLE) and two graphite rods as counter electrodes were used. The procedure consisted of a potential sweep of 100 mV s−1 to a 30 V potential, followed by a 2200 s delay at this potential. An ammonium sulphate (1 mol L−1) and ammonium fluoride (0.2 wt%) solution was used as the electrolyte. Samples were cleaned again after the exposition . All anodization experiments were carried out at room temperature. For the morphological characterization of samples, a TESCAN VEGA 3 LMU scanning electron microscope (SEM) was used. The length and the inner diameter of the nanotubes were evaluated from the SEM images using ImageJ software. Data from four distant image fields from at least three samples were used for analysis.Disc specimens of a Ti30 Exposures were realized in 90 mL of SBF at a temperature of 37 °C for 168 hours. Experiments were supplemented by electrochemical measurements based on the defined sequence of electrochemical measurements. Measurements were performed using potentiostat Gamry Reference 600 with the Framework 6.25 control program. First, the hourly stabilization of the open circuit potential (OCP) was carried out, followed by repeated impedance monitoring at a constant frequency of 2 kHz and a polarization resistance measurement within ±20 mV/OCP. Once every 24 hours, impedance spectra were scanned at the frequency range 50 kHz to 10 mHz. After exposures, the samples were cleaned again with distilled water in ultrasonic bath. The pH value of SBF was recorded before and after each measurement. Subsequently, the specimens were observed using a TESCAN VEGA 3 LMU scanning electron microscope with an Oxford Instruments EDS analyzer. The results of the elemental analysis were evaluated using the Aztec program. After exposures to simulated body fluid, four sets of specimens were obtained and used for cell tests: ground Ti6Al4V (Ti), nanostructured Ti6Al4V (N), Ti6Al4V incubated in SBF (TiH), nanostructured Ti6Al4V incubated in SBF (NH). Glass was used as a reference group.Specimens with ground and nanostructured surface were exposed to simulated body fluid , prepareAfter the exposure to SBF, the specimens were transferred to a 50 mL volumetric flask filled with a physiological saline solution. The samples were sterilized using a cyclic electron accelerator with Kapitza's resonator Microtron MT25 (Department of Accelerators-Nuclear Physics Institute of the Czech Academy of Sciences). An electron beam of 9.8 meV with a dose of 27 kGy was used.−1 and 100 μg mL−1, Sigma-Aldrich) in CO2 incubator with 5% CO2 at the temperature of 37 °C.Human mesenchymal stem cells  were cultivated in basic medium consisting of: MEM Alpha medium (1X)  supplemented with 10% (v/v) fetal bovine serum , penicillin/streptomycin  the absorbance of the metabolised solution (100 μL) was detected at 490 nm using Tecan Infinite® PRO 200 series reader . The background absorbance (690 nm) was subtracted from the measured data, as well as the absorbance of media with MTS solution.The metabolic activity of the hMSC was determined on days 1, 7, 14 and 21 using the MTS assay . Scaffolds were reloaded into new 12-well plate to avoid the distortion of results by cells occurring outside the scaffolds. To each scaffold, 800 μL of MTS solution in fresh medium were added. After incubation  or using Hoechst 34580 . After two washes with PBS, the cells were visualized using a confocal microscope ZEISS LSM 5 DUO at λexc = 488 nm and λem = 505–550 nm for Alexa Fluor 488, λex = 405 nm, λem = 420–460 nm for Hoechst 34580, λexc = 633 nm and λem > 650 nm for Alexa Fluor 633.On day 14, and 21, the samples were fixed with frozen 70% methanol, washed with phosphate buffered saline (PBS) and incubated in 3% FBS in PBS/0.1% Triton-X for 30 min at RT. Primary monoclonal antibody against type I collagen  or osteocalcin  IgG, T4743, Peninsula Laboratories, USA) was applied overnight at 2–8 °C. After 3 washes with PBS/0.05% Tween, and once in PBS, the samples were incubated with secondary antibody Alexa Fluor 488-conjugated anti-mouse antibody for 45 min, RT  or Alexa Fluor 633-conjugated anti-rabbit antibody . After washing three times with PBS/0.05% Tween, and once in PBS, cell nuclei were stained for 5 minutes with propidium iodide  or vinculin  was applied overnight at 2–8 °C. After washing 3 times with PBS/0.05% Tween  and once in PBS, anti-mouse secondary antibody conjugated with goat anti-mouse IgG/IgA/IgM (H + L) Alexa Fluor 488  was added for 45 min at RT. Subsequently, after same washing procedure, the samples were incubated with phalloidin conjugated with ATTO-633  for 1 hour at RT. Cell nuclei were counterstained with Hoechst 34580  for 30 min at RT and washing twice in PBS. The samples were scanned using confocal microscope ZEISS LSM 5 DUO at wavelengths described above, and Cp−Δ. Light Cycler 480  was used to measure the fluorescence intensity.RNA was isolated from four samples per group in day 0, 7, 14 and 21 using the RNeasy Mini Kit  followed the manufacturer's instructor. RNA content (ng per sample) and ratio of absorbance at 260 nm to 280 nm, indicating RNA purity, was measured using Tecan Infinite® M200 Pro reader. Presence of the RNA in the samples was checked using standard agarose gel electrophoresis. The isolated RNA was processed followed manufacturers instruction. Briefly, osteogenic markers osteocalcin (OCN), collagen type I (COL1A2), integrin binding sialoprotein (ISBP), and EE1  genes were analysed. Detail description of the analysed genes in ESI Table S1.post hoc test. Fluorescence intensity of collagen type I was measured from at least 8 confocal images. The intensity was normalised for glass and statistically evaluated using Multifactorial ANOVA where the level of significance was set as 0.05.The data are presented as mean ± standard deviation. The results from the biological experiments were evaluated statistically using SigmaStat software  by normality test and one-way analysis of variance (ANOVA). The Student–Newman–Keuls, Tukey, or Dunns method was used as 6Al4V alloy was obtained. Due to the presence of both α and β phases in the studied alloy, a non-uniform etching occurred, which led to the formation of cavities (arrows) after β phases  were on glass compared to all titanium samples. However, partial detachment of cells from glass during medium exchange was observed and growing cells might have influenced this effect. No significant differences were observed in mRNA expression of late osteogenic markers osteocalcin (OCN) and integrin-binding sialoprotein (IBSP). The high increase of OCN and IBSP expressions were measured in all groups. On day 0, almost zero expression of IBPS was found (not shown)  .The immunohistochemical staining of osteocalcin, a late marker of osteogenic differentiation, showed that the highest density of osteocalcin was present on nanostructured samples exposed to SBF and lower amount on ground samples exposed to SBF. Other samples showed only faint osteocalcin staining .Our aim was to study the effects of hydroxyapatite layer, formed on different surfaces, on the adhesion, growth and differentiation of the human MSCs. Both nanostructure formation and hydroxyapatite layer formation have already been separately studied, yielding positive effects on both cell differentiation and bone formation.6Al4V with simulated body fluid was investigated, when the two surface treatments were compared – ground and nanostructured surfaces. Many surface treatments of titanium alloys in order to enhance bioactivity and thus the cell differentiation and proliferation have been studied. Electrochemical oxidation under fixed experimental conditions in an electrolyte containing fluorides allowed the preparation of a well-defined and structurally arranged nanomorphology. Based on the previous study by Filova et al.,15 the oxidation process was managed at the potential of 30 V. In that investigation it was observed that nanotubes with the inner diameter of 50–80 nm obtained at the potential of 30 V had the best influence on bone cells adhesion, proliferation and differentiation. Also Oh et al. reported promoted MSC differentiation on nanotubes with diameter 70–100 nm thanks to stem cell elongation leading to cytoskeletal stress and enhanced differentiation into osteoblasts.31 Similarly, Lv et al. found that 70 nm was the optimal nanotube dimension for osteogenic differentiation of human adipose derived MSC in vitro and in vivo.In the first part of this study, the interaction of Ti6Al4V is consists of α and β phases. The β phase is less corrosion stable than α phase because it is enriched with highly soluble vanadium compounds, thus β phase is etched preferentially. The area of the cavities covers approximately 11% of the whole surface as mentioned by Moravec et al.32As mentioned above, TiA wide range of electrochemical methods can be used to study the interaction of biomaterial with the human body environment. Open circuit potential for both ground and nanostructured specimens decreased until its stabilization at the value of −220 mV/ACLE in the case of ground surface and later at the value of −100 mV/ACLE for the nanostructured surface. Increasing polarization resistance values for both surface treatments could be explained by phase interface stabilization. The lower resistance value of the nanostructured surface is caused by the much larger surface of the nanotubes as compared to the ground surface.Another method used was electrochemical impedance spectroscopy, which provides accurate and high-quality information on phase interfaces. The different shape of the impedance spectra is obvious when comparing the ground and nanostructured specimens. In the case of the ground sample, the spectrum from the first day corresponds to the passive layer and the other spectra already showed the change at the phase interface when the layer precipitated. The spectra obtained while exposing the nanostructured specimen have a totally different character; their shape is influenced by the charge transfer through the nanostructured surface.Based on the measurement of impedance spectra over the entire frequency range, a 2 kHz frequency value was selected at which the greatest variances in phase angle and impedance module were detected. From the measurement of these parameters at a constant frequency of 2 kHz, it was possible to accurately evaluate the time of change on phase interface, hydroxyapatite (HAp) layer formation and growth was anticipated. This assumption was confirmed by EDS analyses after the exposure to SBF, when Ca and P were detected at an atomic ratio almost 1.67. The positive effect of the nanotubes is obvious from the single frequency results; the induction period of the nanostructured surface was about 12 hours shorter than that of the ground sample. Higher amount of Ca and P measured by EDS also confirmed the enhanced bioactivity of the nanostructured surface.29,33Also, the SEM microphotographs clearly demonstrated that formation of HAp-like layer was greatly enhanced by nanostructured surface. This outcome is in accordance with previous studies.6Al4V alloy for bone replacements could be occasionally viewed with apprehension, as elements Al and V are known for their possible cytotoxic effects.34–37 In earlier studies dealing with the form of these two elements in the nanotubes15,32 it was confirmed that Al is present in the form of Al2O3, also known as alumina, which is a widely used biomaterial that positively affects the adhesion and growth of osteoblasts,38 especially in the form of nanoparticles. In the case of V, this element was detected as oxide V2O5. Since the effects of V2O5 are more controversial.39,40From the biomedical point of view, the use of Ti41 In collagen composites with HAp, the osteogenic differentiation was influenced by concentrations of collagen, HAp, and pore diameter, with the best results on composite with 50 wt% Hap and 0.05% collagen.42 Similarly, Sun et al. reported better osteogenic differentiation of rat BMCs on collagen/HAp (60 wt% HAp) scaffolds than on β-tricalcium phosphate/Hap or pure HAp scaffold.43As a hydroxyapatite layer is more natural for cells than artificial surfaces, the surface covered by it may have improved biocompatibility and bioactivity. 3D-HAp/poly-d/l-lactide composites showed earlier osteoblast differentiation compared with β-TCP.44,45 As an example, the micropatterned HAp surface showed strong osteoblasts contact guidance effect on 8 μm width microgrooves, whereas on larger patterns the cell alignment in the grooves direction proved to be weak.46 In another study, microgrooved HAp ceramic was applied to direct growth of osteoblasts.47Previous studies shown that micro- and nano-patterned HAp bioceramic surfaces enhance cell attachment and can stimulate osteogenic differentiation or bone regeneration.et al. described the synergic effect of hybrid micro/nanostructured surface of HAp bioceramics on osteogenesic differentiation of hBMSCs (human bone marrow stem cells). In addition, their results also revealed significantly enhanced cell adhesion, proliferation and expression of osteogenic genes on both patterned surfaces, compared to flat control.48It was also shown that micro and nanostructures on bioceramic activate expression of different stem cell integrins. Zhao 6Al4V implants were applied to promote cell adhesion and osteointegration in diabetes mellitus rabbit models.49 The importance of distinct architecture of the biomaterial surface were recently reviewed. Cell fate is guided by the micro and nano patterned substrates, which influences protein adsorption, cell adhesion, migration, growth, differentiation and cell death. Various cell types differently react on the micro or nano patterned surfaces.50 In the study of Özçelik et al. BMSCs preferred smooth surface while osteosarcoma Saos-2 cells preferred nanopillar surfaces. However, the presence of fibronectin improved the BMSCs adhesion on nanopillar surfaces.51 The different behaviour of osteoblasts and myoblasts were strongly influenced by the width of HAp microgrooves prepared by photolithography, and subsequent coating with HAp.46HAp coating with nanorod-structures on Ti13,14 Another approach to promote bone regeneration and new bone formation from the surrounding native bone is surface coating using SBF that create calcium phosphate structures similar to HAp of native bone.52,53 It was supposed that combining these two above mentioned surface modifications could greatly influence osteoinductivity and osteointegration of the Ti6Al4V implants.It is believed that nanotubes offer greater surface area for cell-biomaterial contact, and therefore better mimic bone architecture and function.in vitro. NanoHAp coating of Ti specimen supported growth and osteogenic differentiation of hBMSC, which was enhanced by the addition of growth factors.54For orthopaedic and dental implants success, not only osteoblast cells but also MSC play a key role in bone tissue regeneration. Recently, multipotent cells are becoming preferred model in a tissue engineering studies and hMSC are widely used as an osteogenesis model and for bone-tissue engineering applications. Therefore, in the current study hMSC were chosen to investigate the bioactivity of the created surfaces 55,56 As was shown larger nanotube diameters lead to elongation of the cells and stimulate osteogenic differentiation via cytoskeletal stress.31 Moreover, it was clinically proven, that rough surfaces induce a more rapid osseointegration than the smooth surfaces57–60 and some of the porous Ti-based biomaterials are already in clinical trials.61From the previous studies it can be concluded that by specifically designing the nanotube diameter it is possible to support either hMSC adhesion or osteogenic differentiation.62 As was mentioned, focal adhesion complex consists of many proteins, where the mostly considered are talin, that forms a structural core of the focal adhesion, and vinculin which strengthens and stabilizes the focal adhesion complex.63,64A crucial step for implant stability and subsequent cell differentiation is initial cell adhesion. This process is initiated by the adsorption of proteins from the body fluids to the implant surface that are recognised by the cell integrins, as the first step of the cascade leading to the formation of adhesion plaques. These highly organized protein complexes mediating ECM-cell contact but also act as mechanosensors that regulate cell phenotype.65 Although nanotubes with larger diameters were shown to be osteoinductive without the use of osteogenic supplements,13 other aspects such as cell sources, adsorbed proteins and their conformation, age and even the sex of the donor may influence cell response.66,67This phenomenon can be related to the fact that MSC were shown to adhere better on smaller nanotubes (30 nm), while larger nanotubes (100 nm) induced stem cell differentiation and proliferation.In this study, cell attachment and morphology were evaluated 48 hours after seeding. Well spread polygonal-shaped cells were visible on all type of surfaces. Additionally, the distribution of molecular markers of cell adhesion, talin and vinculin, were evaluated by immunofluorescence staining. As shown in et al. who showed dot-like cytoplasmic localisation of vinculin in osteoblasts on nanostructured Ti6Al4V68 and with our previous study using cpTi.69 We have shown similar dispersal of vinculin and talin in osteoblasts cultured one day on nanotubes with diameters among 24–66 nm.69Distribution of the vinculin positive areas in our work agreed with the study conducted by Saha 6Al4V.15 Costa et al. found that as roughness and complexity of HAp coated PCL matrices increased, the number of focal adhesions decreased.70The study conducted by Filova presented the increase of both talin and vinculin concentrations in osteoblasts on nanotubes created by anodic oxidation at 30 V with an inside diameter around 35–80 nm compared to untreated Ti71 We suggest that less developed focal adhesions on NH group could be caused by the phenomenon that nanotubes were overlapped by the HAp-like layer created by SBF treatment, therefore the simultaneous effect of nanotopography and HAp crystals could not be clearly detected. Our data indicating that talin synthesis or morphology were influenced by presence of the HAp. Talin intensity was lower on TiH and NH samples  compared to both smaller diameters and untreated surface were described.82 Although osteocalcin expression was not significantly affected by hereby used modifications, the numerous signals was visible on NH and less rare on TiH samples, compared to very rare signal on Ti but no signal on N and glass on day 21. The presence of osteocalcin  of nanotubes, but no osteogenic differentiation on nanotube with small diameter about 30 nm. Conversely, Park et al. reported better osteogenic differentiation of hMSC on 15 nm-diameter TiO2 nanotubes.83Oh 6Al4V on bone healing in vivo. Authors suggested that presence of calcium phosphate is important for ectopic bone formation.84 Also Barrere et al. reported enhanced bone integration in case of SBF mediated apatite-coated Ti6Al4V compared to noncoated implants on goat models.85 Cai et al. reported titanium (cpTi) treatment with potassium hydroxide and heat, and subsequent exposure to SBF and its effects on the rat BMSCs. The samples treated with potassium hydroxide, heat and exposed to SBF showed significantly higher metabolic activity, alkaline phosphatase activity and osteocalcin synthesis compared to pure cpTi. Samples treated with potassium and heat but not exposed to SBF did not show positive effect on BMSCs osteogenic differentiation.86The study conducted by Habibovic, showed positive effect of calcium phosphate coated Ti52Considering above mentioned studies and our data, the SBF mediated mineralised surfaces of metal implants and other biomaterials have great future application, as this approach can be used for drug delivery that can dramatically enhance tissue regeneration. For instance, implant surfaces can be functionalised by the incorporation of bioactive molecules, such as antibiotics, growth factor and proteins, even nucleotide acids that can be used in gene therapy.6Al4V exposed to SBF was able to stimulate collagen type I and osteocalcin synthesis in hMSC, together with a high rate of cell growth.In this study, we showed that nanostructured Ti6Al4V. Subsequently, SBF exposition was applied to form HAp-like layer on the ground and nanostructured titanium surface. In this study, SEM microphotographs clearly demonstrated that formation of HAp-like layer was greatly enhanced by nanostructured surfaces and covered almost the whole surface of the specimens. Our data showed that the hereby presented titanium surface modifications supported proliferation and osteogenic differentiation of hMSC. In particular, treatment of ground Ti6Al4V with SBF resulted in enhanced collagen synthesis, similar to nanotubes modified samples after a longer cell culture. In addition, nanotubes exposed to SBF enhanced osteocalcin synthesis better than other surfaces. Our data suggest that Ti6Al4V nanostructuring together with SBF incubation can enhance extracellular matrix formation and thereby can improve bone regeneration.In summary, anodic oxidation at 30 V created nanotubes with inner diameters ranged from 30–120 nm on titanium alloy TiThere are no conflicts to declare.RA-010-C9RA08912H-s001


Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways’ constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body’s repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture. Bone tissue engineering protocols have utilized MSCs differentiated into both osteoblasts and hypertrophic chondrocytes. Overall, engineered bone grafts have performed inconsistently in animal studies of bone regeneration, due, at least in part, to the unknown contributions of the differentiated MSCs. In the current study, we conducted a detailed in vitro and in vivo comparison of bone grafts engineered from human bone marrow MSCs induced into the two ossification pathways. We identified important differences in the gene expression, composition, microstructure and vascularization of the two ossification pathways utilized in bone graft development, which can be used to shape the regenerative engineering strategies for bone. These findings aid our understanding of the relative advantages of replicating the endochondral vs. the intramembranous ossification pathways for bone regeneration.Mesenchymal stromal cells (MSCs) play an integral role in bone formation, maintenance and repair through their ability to differentiate in response to environmental cues, as well as their paracrine activity ,2. In siA majority of the earlier studies aimed to recapitulate the intramembranous ossification pathway, with MSCs differentiating directly into osteoblasts depositing a mineralized bone matrix . CultivaHypertrophic chondrocytes are known to express bone formation genes and deposit significant amounts of mineral ,16. WhenIn recent studies, differentiation into hypertrophic chondrocytes and osteoblasts was evaluated for the ability to promote the healing of critical size calvarial and femoral defects ,19,20. IHowever, the vascularization of the engineered constructs was comparable between the two groups. In a recent study, human bone marrow MSCs that were primed briefly into the endochondral and intramembranous pathways on polycaprolactone scaffolds exhibited comparable new bone formation in murine calvarial defects, whereas blood vessel infiltration was higher in the endochondral group . In femoDue to the discrepancies among these studies, we were interested in directly comparing the in vitro and in vivo behavior of bone marrow MSCs differentiated into the two ossification pathways to better understand their capacity for bone formation in vitro and regeneration in vivo. We hypothesized that the cells’ properties, deposition of the matrix and the resulting potential of the engineered constructs for bone formation and regeneration would strongly depend on the cells’ differentiation regime. To test this hypothesis, we induced bone marrow MSCs into osteoblasts and hypertrophic chondrocytes using biologically inert silk scaffolds, which exhibited high potential for bone tissue engineering in our previous studies and demonstrated a muted inflammatory tissue reaction upon implantation ,21,22. ASigma Aldrich  was the source for all purchased materials. The purchased materials were of analytical grade unless otherwise specified.2O for 2 days. Cylinders 2 mm in height and 4 mm in diameter were cut from the material and the scaffolds were disinfected with 70% ethanol.Silk solutions for scaffold generation were prepared at a concentration of 16 wt% bombyx mori silk fibroin in hexafluoroisopropanol (HFIP), following published methods . To gene7 cells/mL. Scaffolds were seeded with 750,000 cells per scaffold using the drip method [Human bone marrow MSCs  were acquired, characterized, and expanded as in previous studies . The expConstructs mimicking intramembranous ossification (termed osteo constructs) were generated by facilitating MSC differentiation over five continuous weeks in an osteogenic medium. The osteogenic medium was composed of low-glucose DMEM , supplemented with 10% fetal bovine serum, a 10 mM HEPES buffer, 1% penicillin/streptomycin (P/S), 50 µg/mL ascorbic acid, 100 nM dexamethasone and 5 mM β-glycerophosphate. Generated in parallel to the constructs mimicking intramembranous ossification, constructs mimicking endochondral ossification (termed hyper constructs) were created by first differentiating the MSCs in chondrogenic medium for two weeks to generate chondrocytes. The chondrogenic medium consisted of high-glucose DMEM (ThermoFisher) supplemented with 50 µg/mL ascorbic acid, 100 nM dexamethasone, 50 µg/mL proline, 1% ITS+, 100 µg/mL sodium pyruvate, 1% P/S, 10 ng/mL TGF-β3 and 10 ng/mL BMP6. After two weeks of chondrogenic differentiation, the MSC-derived chondrocytes were matured over three weeks into hypertrophic chondrocytes. The hypertrophic maturation medium was composed of high-glucose DMEM supplemented with 50 µg/mL ascorbic acid, 1 nM dexamethasone, 50 µg/mL proline, 1% ITS+, 100 µg/mL sodium pyruvate, 1% P/S, 5 mM β-glycerophosphate and 50 ng/mL L-thyroxine.n = 4) from each experimental group were evaluated in duplicate. The ΔCt, defined by the Ct of GAPDH subtracted from the Ct of the gene of interest, was utilized to normalize the quantitations. After five weeks of in vitro cultivation, the RNA was extracted by utilizing TRIzol (ThermoFisher) according to the manufacturer’s instructions. NanoDrop spectrophotometric quantitation (ThermoFisher) was utilized to determine the total RNA content in each scaffold. DNase treatment was utilized to remove contaminating DNA, and then a high-capacity cDNA reverse transcription kit (ThermoFisher) was used to transcribe the RNA into cDNA, according to the manufacturer’s instructions. Quantitative RT-PCR was performed with the transcribed cDNA using the Fast Sybr Green mix (ThermoFisher). All samples (Constructs harvested at the culmination of five weeks were first weighed and then digested with papain (40 units/mg) in a digestion buffer at 60 °C overnight. The digestion buffer consisted of 10 mM cysteine HCl, 0.1 M sodium acetate and 50 mM EDTA at pH 6.0. Quantitation of the DNA in each scaffold digest was analyzed using a Quant-iT PicoGreen assay kit (ThermoFisher) following the manufacturer’s instructions. The manufacturer-supplied lambda DNA standard was used in the quantitation, with each sample’s amount of DNA normalized to the sample’s wet weight.Dimethylmethylene blue assay was utilized to determine the amount of sulfated glycosaminoglycan (GAG) deposited within each scaffold, consistent with prior publications . ChondroA modified protocol  was leveTo assess the calcium content, the harvested constructs were snap-frozen in liquid nitrogen and stored at −20 °C. A 5% trichloroacetic acid solution was utilized to extract the calcium from the constructs, and the sample was quantified using the Calcium (CPC) Liquicolor kit  following the manufacturer’s instructions.−1. The specific regions of interest (900–1200 cm−1 and 1590–1720 cm−1) were isolated, baselined and normalized using Omnic software (ThermoFisher). A genetic algorithm that fit up to 12 Gaussian distributions was created with Matlab software  to de-convolute the normalized data. The peak intensity of the sub-peaks of interest was recorded for each measurement. The presence of mineral within the matrix was measured by analyzing both the phosphate band in the curve in the 900–1200 nm range and the Amide I band in the curve present in the 1600–1700 nm range. The maturity of the mineral, determined by the extent of the mineral’s crystallinity, was assessed by calculating the ratio of the sub-band area at 1030 nm to the sub-band area at 1020 nm.A Nicolet iS5 (ThermoFisher) with a diamond window containing an iD5 ATR accessory was utilized to record the FTIR spectra. Samples were embedded in paraffin, and sections  were sliced for analysis. The FTIR spectra of the samples were measured using scans with a resolution of 400–4000 cmn = 6). To summarize, an initial compressive load of 0.2 N was applied to the sample of interest, followed by a stress relaxation step. The compression during the step occurred at a ramp velocity of 1% per second until a total strain of 10% was reached, with the compressive load then held at 10% strain for 1800 s. The Young’s and equilibrium moduli were calculated from the measured forces.Previously published methods  were utiAfter five weeks of in vitro cultivation, the osteo and hyper constructs  were inserted into subcutaneous pouches in nude mice, following previously approved animal experimentation procedures. The constructs were harvested after 3, 6 and 12 weeks after implantation and analyzed as described below.n = 4) from each experimental group. The scans followed a protocol modified from previously published methods [3. Scanco Medical software was utilized to calculate the bone volume, the bone mineral density (BMD) and the bone surface to volume ratio (BS/BV).Upon completion of in vitro differentiation and prior to implantation, a vivaCT 40 system  was utilized to scan the constructs ( methods . The vivn = 4), 6 weeks (n = 6) and 12 weeks (n = 4) were scanned on a µCT 50 system  utilizing the following scanner settings: current, 0.200 mA; voltage, 70 kV; slice thickness, 10 µm; isotropic resolution, 10 µm. A global thresholding technique set at 282.9 mg HA/cm3 was utilized for three-dimensional reconstructions and quantitation. Software provided by Scanco Medical was used to calculate the bone volume, BMD and BS/BV.Constructs harvested at 3 weeks , osteopontin (OPN) and bone sialoprotein (BSP) were assessed through immunohistochemistry. Separately, the hyper constructs were stained with Alcian Blue for the presence of glycosaminoglycans (GAG), and collagen type 10 (COLX) was evaluated by utilizing immunohistochemistry for hypertrophic chondrocyte matrix deposition to confirm mimicking of the endochondral ossification process. Antigen retrieval was carried out by submerging the samples in a citrate buffer in a container which was placed into a boiling water bath for 20 min. Incubation in 0.3% hydrogen peroxide for 30 min was utilized to block the samples, which were then washed and incubated overnight at 4 °C with the primary antibodies against BSP (1/500 dilution) , OPN (1/500 dilution) (EMD Millipore), ALP (1/250 dilution) and COLX (1/2000 dilution) . The Vectastain Elite Universal staining kit  was utilized to detect primary antibodies. Counterstaining was completed with Hematoxylin QS (Vector Laboratories).n = 6) through semi-quantitation analysis by two independent blinded researchers using NIH ImageJ ).After harvest and scanning with the μCT, subcutaneously implanted constructs were cut into halves. One half of each construct was used for a decalcified histological evaluation. These samples were preserved in 4% formaldehyde for 24 h, rinsed with PBS and decalcified using a formic-acid-based solution . After the samples had been decalcified, they were rinsed with PBS, dehydrated in ethanol solutions, embedded in paraffin and sectioned at 6 µm. Antigen retrieval was conducted on the samples, and the Vectastain ABC Kit for Rabbit IgG (Vector Laboratories) was used for secondary antibody staining. Primary antibodies for M1  (AbCam) and M2  (AbCam) macrophages were incubated overnight at 4 °C, as well as the primary antibody for CD31 (1/50 dilution) (AbCam). Samples were counterstained with Hematoxylin QS (Vector Laboratories) after visualization of the primary antibody. The number of blood vessels, the distance of the blood vessels from the scaffold edge and the areas of the constructs and the blood vessels were determined for the constructs harvested after 6 weeks . Undecalcified and thinly ground sections were prepared along the longitudinal axis of the constructs according to Donath  and stait-test, α = 0.05, with the significance determined by p < 0.05. Analysis was conducted with Prism Software . Post-implantation µCT quantitation was analyzed through one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test, α = 0.05, with the significance determined by p < 0.05 for each construct type. Differences in the distribution of the vessels’ distance from the edge were assessed between the two ossification pathways by ANOVA, α = 0.05, with the significance determined by p < 0.05.All data are presented as the mean ± standard deviation. Significance between the pre-implantation and post-implantation constructs was determined using Student’s Human bone marrow MSCs were seeded on porous silk scaffolds, differentiated by inducing the endochondral and intramembranous ossification pathways for 5 weeks and implanted subcutaneously into nude mice for up to 12 weeks . All theAt the end of cultivation, the engineered tissue constructs from both groups had comparable DNA and collagen contents, but differed in their calcium content, glycosaminoglycan (GAG) content and matrix deposition patterns . The expDifferences in the mineralization patterns were observed between the two groups of constructs, as shown by von Kossa staining B. In theFinally, differences in Young’s elastic and equilibrium moduli were measured through compression tests for the two groups . While tAs cellular signaling mediates the complex process of bone regeneration, the cytokines released in the conditioned media of both construct groups were analyzed A. The hyThe hyper and osteo constructs were implanted subcutaneously in nude mice to evaluate the influence of each differentiation pathway on ectopic bone formation. To quantify the amount of bone deposition and visualize the development of mineralized tissue, samples were scanned before implantation and after 3, 6 and 12 weeks using µCT. Before implantation, the three-dimensional representation of mineral deposition  correspoUpon implantation, these constructs underwent remodeling that differed between the two groups. The hyper constructs did not deposit significantly more mineral  during the 12 weeks in vivo, but instead displayed a significant increase in BMD B. VisualBone tissue formation and remodeling were evaluated in constructs explanted after 3, 6 and 12 weeks using the undecalcified bone histology and Levai–Laczko staining . After 3To develop a better understanding of the regenerative environments of both construct groups after implantation, constructs harvested after 3 weeks were stained for the presence of M1 and M2 macrophages. M1 macrophages, staining positively for iNOS, could not be detected in either group of constructs , even thAngiogenesis of tissue-engineered constructs is essential for successful integration and bone defect regeneration. Vascular endothelial growth factor A (VEGF-A), which stimulates and directs angiogenesis, was found at a significantly higher concentration in the conditioned medium of the osteo constructs compared with that of the hyper constructs prior to implantation A. In conBones form through two main pathways, endochondral and intramembranous ossification. Endochondral ossification occurs in situations that require substantial tissue deposition or the establishment of mechanical stability, and is characterized by the initial deposition of the cartilage matrix, followed by hypertrophic chondrocyte-mediated bone deposition . In contThe in vitro differentiation of the bone marrow MSCs into the two pathways within silk fibroin scaffolds resulted in significantly different construct properties due to the differentiation pathways, including differences in the gene expression levels of osteogenic markers, the deposition patterns of bone matrix proteins, mineralization and growth factor/cytokine secretion. Regarding the gene expression levels, previous in vitro studies have demonstrated the dynamic changes that occur across various timepoints during differentiation. After 5 weeks of differentiation, the hyper constructs had significantly higher expression levels of RUNX2, the master regulator of osteogenic differentiation ,33 and oThe increased gene expression of bone matrix proteins was reflected in the protein level, with the hyper constructs exhibiting dense protein deposits within the bone scaffolds’ pores, which exceeded the deposits observed in the osteo constructs on the scaffolds’ surfaces. These differences in the gene expression and matrix deposition patterns between the two groups could potentially be attributed to the different characteristics of the differentiation pathways. Intramembranous ossification occurs on an established matrix, with the attachment and subsequent polarization of the osteoblasts dictating the direction and location of bone matrix deposition ,35. The Large pores in the silk scaffolds allowed for the recapitulation of endochondral ossification, as demonstrated by the dense matrix containing GAG and COLX within the pore spaces. The bone matrix proteins and mineral deposits within these large pores did not present any structural patterns, except that the matrix surrounded enlarged cell lacunae, which is characteristic of hypertrophic chondrocytes and is similar to native endochondral ossification deposition patterns .The µCT analysis of the cultured constructs revealed that the mineralized matrix was organized into small spindles in the osteo constructs and into aggregated clumps throughout the hyper constructs. These characteristics are consistent with previous studies ,18,20,21The observed differences in the matrix morphology were accompanied by differences in mineral deposition, with the hyper constructs acquiring significantly more calcium/DNA than the osteo constructs during the 5-week culture period. The increased mineral deposition correlated with lower crystallinity, i.e., mineral maturity, in the hyper constructs. The presence of GAG within the matrix deposition environment is known to disrupt mineral formation, leading to smaller crystallinity values ,43. InteUpon implantation, the hyper constructs underwent extensive remodeling, and the immature mineralized matrix deposits were transformed into cortical-like bone containing cement lines and embedded cells, similar to a previous report . The minIn an attempt to better understand the remodeling of the hyper constructs in vivo, we also explored the presence of macrophages. It has been shown that macrophages, and M2 macrophages in particular, are essential for endochondral ossification, as they play unique roles in bone regeneration ,54. We fFinally, it has been shown that vascularization is critical for bone formation and regeneration in vivo , and theOne limitation of this study involved the limited characterization of the undifferentiated MSCs within the silk scaffolds as a comparator. While the focus of this study was on the deposition and behavior caused by the mimicked ossification pathways, future studies should attempt to understand the matrix deposition and gene expression of the undifferentiated MSCs compared with the differentiated pathways. A more comprehensive knowledge of undifferentiated vs. differentiated MSCs’ behavior will permit the tailored development of more complex bone tissue engineered constructs.The implementation of two differentiation pathways—endochondral and intramembranous ossification—resulted in significantly different deposition patterns of the mineralized bone matrix in tissue constructs engineered from bone marrow MSCs on porous silk scaffolds. The hyper constructs quickly deposited immature mineral and instigated extensive remodeling in vivo, accompanied by enhanced vascularization and the elevated presence of M2 macrophages. The osteo constructs deposited mature mineral along the scaffolds’ surfaces and instigated vascularization at the constructs’ periphery, accompanied by initial bone resorption and followed by bone formation. These responses were reflective of the differences in the signaling components for the two construct types. As the goal of our study was to compare the performance and properties of the two different differentiation regimes to aid future translation, we propose that the differentiation of hypertrophic chondrocytes could be utilized when extensive bone deposition, mechanical strength and vascularization are needed. Direct differentiation into osteoblasts could be utilized in more complex settings, in which slow and mature tissue remodeling can take place, and where greater control of the new tissue’s formation patterns and bone shape can be guided through the scaffolds’ architecture.


According to the European Society of Gastrointestinal Endoscopy guidelines, self-expandable metal stents (SEMSs) are preferable to plastic stents (PSs) in the management of pancreatic cancer, regardless of cancer stage. The aim of this study was to compare the therapeutic efficacy and treatment costs of SEMS and PS in the management of malignant biliary obstruction.One hundred and thirty-five patients who underwent endoscopic stent placement were retrospectively enrolled and divided into PS (41 patients), primary SEMS (39 patients) and secondary SEMS (55 patients) groups. We determined the technical and functional success rate, stent patency, and cumulative treatment cost.p < 0.001). Multiple PS implantations and larger stent diameter increased the length of stent patency compared to 7-Fr PSs . The cumulative treatment cost of patients with different survival times did not differ significantly between groups, however, among patients surviving 2–4 months it was higher in PS group than primary SEMS and secondary SEMS groups  due to increased number of biliary reintervention  and longer hospital stay .A total of 111 SEMSs and 153 PSs were placed with similar technical (100% vs. 98.69%) and functional success rate (90.10% vs. 86.27%) but with different stent patency (10.28 vs. 22.16 weeks; In view of treatment costs, the consequences of illness, and the processes of the health care system, SEMS implantation is recommended regardless of patients’ life expectancy.The online version contains supplementary material available at 10.1186/s12876-023-02668-9. Pancreatobiliary malignancies are often diagnosed at a locally advanced or metastatic stage, when curative resection is no longer feasible. These tumors' prognosis is especially dismal, and their 5-years mortality rate reaches 94% . In apprAlthough several international gastroenterological societies recommend the placement of SEMS with ERCP for the management of malignant biliary obstruction, the use of PS has not yet substantially decreased , 7.ThereWe gathered retrospective data about consecutive patients with unresectable primary pancreatobiliary malignancy who underwent endoscopic stent placement for distal biliary obstruction in one of the Hungarian tertiary-level referral gastroenterology centers between 2011 and 2018. Biliary obstruction had been treated before the ESGE guideline updated in 2017 was published and integrated into daily routine. The interventions were performed by one of the three ERCP specialists in our institution. The exclusion criteria were (1) benign lesion such as chronic or autoimmune pancreatitis, identified by clinical or histological examination against the background of suspected malignant biliary obstruction; (2) surgical resection or biliary bypass performed less than 4 weeks after the first biliary stent implantation; (3) concurrent gastric outlet obstruction and malignant biliary obstruction at the time of stent placement; (4) hilar or intrahepatic malignant biliary obstruction; (5) secondary malignant biliary obstruction caused by disseminated extra-pancreatobiliary malignancy with direct tumor spread or lymph node metastasis; (6) moribund condition of patients with end-stage pancreatobiliary malignancy, which could be expressible numerically (5 or 6 points) as the score on the physical status classification of the American Society of Anesthesiologists . This scheduled stent replacement was carried out 3 to 4 months after the PS implantation or earlier if stent complications  occurred. Patients were accordingly divided into groups  during the assessment of cost effectiveness of different stent types. We used 7-Fr and 10-Fr polyethylene straight PSs provided by Blue Neem Medical Devices Private Limited  and CONMED Corporation  and 10-mm covered, partially covered, or uncovered biliary SEMSs with a diameter of 10 mm and a length of 40–80 mm provided by Changzhou Health Microport Medical Device Co., Ltd. , Boston Scientific Corporation ; S&G BioTech Inc. ; ENDO-FLEX GmbH. ; Endo-Technik ; and Taewoong Medical Co., Ltd., .The primary endpoint of the study was to determine and compare the efficacy and treatment costs of PS and SEMS placement in the management of primary distal malignant biliary obstruction. The efficacy of stent implantation was characterized by technical and functional success rates and duration of stent patency. The intervention was considered technically successful if the stent was placed across the stricture in the proper position, as confirmed by radiography and endoscopy. Functional success of the stent was defined as restoration of bile outflow, detected by endoscopy immediately after drainage, and as more than 20% decrease in serum bilirubin level from baseline within a week after stenting. Duration of stent patency was defined as the period between the stent placement and either functional failure or the patient's death. The following complications were investigated: pancreatitis, cholangitis, stent occlusion, cholecystitis, bleeding, perforation. Complication rate was given as the proportion of patients with one or more adverse event. In addition, we determined also the rate of immediate procedure related and disease progression related complications, as well as the rate of early complications (occurred ≤ 4 weeks after stenting) causing stent insufficiency. Reintervention rate was defined as the proportion of patients who required endoscopic, interventional radiological or surgical intervention to ensure bile flow.Length of patient survival time was highly variable in the three groups; therefore, the average treatment costs per month of survival were compared in the cost analysis. In the PS and pSEMS groups, the cost analysis evaluated the cost of the entire follow-up period, while in the sSEMS group we assessed both the entire follow-up period of patients (pre-SEMS and post-SEMS together) and the post-SEMS costs alone in order to determine the cost-effectiveness of switch from a PS to SEMS in comparison to definitive PS or SEMS placement. In addition, we compared the commutative treatment costs of patients with different survival times. In the sSEMS group, only the costs of the post-SEMS period were evaluated to determine whether the cost of switching to SEMS was recovered. During the economic analysis, we assessed only the costs of medical treatment directly associated with the management of biliary obstruction: the cost of stents , interventions , and hospital stay ; p < 0.001; Table Of the 135 patients with primary malignant biliary obstruction, 41 were in the PS group, 39 were in the pSEMS group, and 55 were in the sSEMS group. The clinical characteristics of patients  and neoplasms  did not differ significantly. Mean survival was substantially longer among patients in the sSEMS group (47.07 ± 32.79 weeks) than among those in the pSEMS (21.46 ± 20.87 weeks) and PS groups . Stent failure developed significantly later in cases of younger patients who had only mild comorbidities , and a weak correlation was observed between stent patency and location of obstruction in the SEMS group . These factors did not influence the therapeutic effect of PSs; however, duration of stent patency was increased by implantation of multiple stents (10.88 weeks) and larger stent diameter (10.55 weeks) in comparison with the use of single 7-Fr stents (7.63 weeks), although the difference was not significant (p = 0.4420).The rates of technical success (100% vs. 98.69%) and functional success (90.10% vs. 86.27%) of SEMSs and PSs were similarly high and independent of stent type. The mean duration of patency of SEMSs was significantly longer compared with PS , but the immediate procedure related complications did not differ between groups (2.6% vs. 2.7%). Stent occlusion necessitated the replacement of PSs in 68.05% of cases, and concurrent cholangitis was observed in 64.05% of cases. Total 29 patients with PS (18.95%) had early complications  causing stent insufficiency and requiring biliary reintervention, which was substantially more frequent than for SEMS . No substantial difference was detectable among covered, partially covered and uncovered SEMSs in terms of overall stent complications , tumor ingrowth , cholangitis , cholecystitis , or stent migration .The rate of overall complications of PSs was significantly higher than that of SEMS  was compared with the other two groups. We compared the cumulative treatment costs for patients with different survival times. In the PS group, 31.71% of patients required repeated ERCP and PS implantation due to complications before the planned stent replacement date. Among patients with short survival (≤ 2 months), the cumulative treatment costs did not differ significantly by stent groups: 1681 ± 734€ for PSs, 2302 ± 735€ for pSEMSs, and 207 ± 823€ for sSEMSs (p = 0.1568). Among patients who survived 2 to 4 months, repeated biliary intervention was performed in substantially more PS recipients than pSEMS and sSEMS recipients , and this trend was also observed among patients who survived more than 4 months . The PS implantation was also associated with longer hospitalization among patients surviving longer than 2 months. (Table p = 0.5502), but the differences were not statistically significant , pSEMS 939.11€), and sSEMS groups assessed the whole follow-up period  was superior to that of PS , and that SEMSs had lower rates of occlusion  and reintervention  than did PSs  . Pooled  20.81]) .p = 0.069), median subsequent intervention rate , or median case costs  [The early clinical trials and meta-analyses suggested that SEMS placement is the right choice for cost-effectiveness considerations only if a patient’s life expectancy is more than 4 months –17. Acco= 0.803) .In view of new evidence, the ESGE (in the guideline published in 2017) now recommends SEMS insertion for palliative drainage of malignant extrahepatic biliary obstruction, regardless of the patient's life expectancy . The resThe most appropriate SEMS type in the management of malignant distal biliary obstruction is still debated. Meta-analyses have revealed no significant difference between covered and uncovered metal stents with regard to the survival benefit, overall rate of adverse events, rate of stent dysfunction, and duration of primary stent patency during the period from primary stent insertion to primary stent dysfunction or patient death –23. SomeThe main limitation of this research is its retrospective, single-center design. Thus, some differences were observed in terms of stent choice and timing of stent replacement. The designs of SEMS purchased from different manufacturers varied slightly, and the diameter and the number of PS inserted in the same time were different, but their design was uniform. We considered these differences during the statistical analysis, but the substantial difference in the sizes of group populations limited the detection of statistically significant variance. Because of the retrospective nature of data collection, the only detailed information available concerned the gastroenterological treatment of pancreatobiliary malignancies performed in our tertiary-level clinical center; however, the patients frequently underwent follow-up in primary- or secondary-level medical institutions. Therefore, in the cost-effectiveness analysis, we assessed the direct cost of interventions and hospitalization in relation to malignant biliary obstruction. The concomitant oncologic treatments or coexisting diseases with potential influence on the total cost of patients' medical care would not be included in the analysis. The ASA score represented the clinical condition of patients.Our retrospective cohort study confirmed that SEMS is a better choice than PS in the management of unresectable primary malignant biliary obstruction not only in terms of effectiveness and longer stent patency but also in terms of cost-effectiveness. Because we found no difference in the cumulative treatment costs of patients with different survival times, we recommend SEMS implantation regardless of patients’ life expectancy. Our results also confirmed that multiple stent implantation and larger stent diameter increased the duration of stent patency and decreased the reintervention rate, in comparison with the use of single 7-Fr stents. Therefore, if SEMS is not available, implantation of multiple PSs is recommended.Additional file 1: Table S1. American Society of Anesthesiologists (ASA) Physical Status Classification; Table S2. Costs used in cost-effectiveness analysis of stent implantation in the management of primary malignant biliary obstruction.


The prevalence of metabolic syndrome (MetS) has been reported to be higher in selected populations of people with COPD. The impact of MetS on mortality in COPD is unknown. We used routinely collected healthcare data to estimate the prevalence of MetS in people with COPD managed in primary care and determine its impact on 5-year mortality.Records from 103 955 patients with COPD from the Clinical Practice Research Datalink (CPRD-GOLD) between 2009 to 2017 were scrutinised. MetS was defined as the presence of three or more of: obesity, hypertension, lowered high-density lipoprotein cholesterol, elevated triglycerides or type 2 diabetes mellitus (T2DM). Univariate and multivariable Cox regression models were constructed to determine the prognostic impact of MetS on 5-year mortality. Similar univariate models were constructed for individual components of the definition of MetS.=0.085). The presence of hypertension  and T2DM  increased and obesity  reduced mortality risk.The prevalence of MetS in the COPD cohort was 10.1%. Univariate analyses showed the presence of MetS increased mortality (hazard ratio (HR) 1.19, 95% CI: 1.12–1.27, p<0.001), but this risk was substantially attenuated in the multivariable analysis (HR 1.06, 95% CI: 0.99–1.13, pMetS in patients with COPD is associated with higher 5-year mortality, but this impact was minimal when adjusted for indices of COPD disease severity and other comorbidities. Individual components of the MetS definition exerted differential impacts on mortality suggesting limitation to the use of MetS as a multicomponent condition in predicting outcome in COPD. The presence of MetS in COPD is not significantly associated with increased mortality over 5 years. The individual components of MetS exert differential effects on mortality, limiting the utility of the syndrome in predicting outcome.https://bit.ly/3PooGQd COPD is recognised to have a number of systemic consequences beyond the primary pulmonary pathophysiology and is frequently associated with a high prevalence of multiple comorbid long-term conditions. Metabolic syndrome (MetS) describes the coexistence of linked metabolic pathophysiologies known to be associated with adverse health outcomes, principally resulting from the development of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) . A systeAccepted definitions of MetS comprise several component indices, typically abdominal obesity, dyslipidaemia, hypertension and hyperglycaemia . Whilst The presence of MetS in people with COPD is of potential prognostic and functional importance because COPD itself confers an increased risk of cardiovascular mortality, and indices incorporated in the syndrome (such as obesity) may have a significant impact on symptom burden, activity limitation and longer-term outcomes . HoweverIn this study, we sought firstly to estimate the prevalence of MetS in a general population recorded as having COPD in primary care, using a modified definition of MetS developed and tested for the purpose of estimating prevalence from routinely recorded primary care clinical records (Aim 1) . SecondlWe conducted a retrospective longitudinal cohort study using routinely collected healthcare data provided by the Clinical Practice Research Datalink , the largest source of electronic primary care data representing 5% of people in the general UK population , 9. Linksupplementary appendix S1) [The study population were patients over the age of 35 years with COPD, and alive and registered between the study-interval dates from 1 January 2009 to 31 July 2017 with a minimum of 12 months data prior to study entry. All included patients had a coded COPD diagnosis determined using pre-specified medcodes and validated definitions (see ndix S1) .−2)Obesity (defined as BMI at least 30 kg·mHypertension−1 (men); <50 mg·dL−1 (women))Lowered high-density lipoprotein (HDL) cholesterol (<40 mg·dL−1)Elevated triglycerides (TG) (>150 mg·dLPresence of type 2 diabetes mellitus (T2DM)Other comorbidities were categorised into groups based on their relevance to clinical outcome and health status from previous studies [Using a previously reported definition with information available from primary care records , MetS wa studies . CategorAll-cause 5-year mortality was derived from ONS data. ONS derives the underlying cause of death from death certificates and is coded using ICD-10 codes. Mortality rates were compared between patients with and without MetS, and survival curves over 5-year follow-up were plotted.Supplementary table S1k lists and describes candidate variables which were assigned to demographic, COPD-specific (list) or comorbidity groups together with predicted outcomes of all-cause mortality. COPD-specific covariates were continuous, whereas comorbidities were binary and related to the presence or absence of conditions.supplementary table S1c). Based on GP records, we therefore defined AECOPD as any of the following:antibiotic (ABX) and steroid (OCS) prescriptions prescribed for 5–14 days,two out of three symptoms from cough, sputum, breathlessness and ABX or OCS for 5–14 days,a lower respiratory tract infection,or from hospital data COPD as primary diagnosis.Acute exacerbation of COPD (AECOPD) was expected to be a key predictor for mortality, and we therefore recorded these events in the 12 months prior to case ascertainment, defined using a combination of diagnostic and therapy codes that have been shown to be valid [i.e. mean±sd. For categorical variables, the number of patients (n) belonging to each category and percentage (%) were given. Patient characteristics were compared between those defined as having MetS and those not having MetS.The prevalence of MetS was estimated using descriptive statistics. For baseline characteristics, we assessed demographics of the population using measures of central tendency and variances appropriate to their respective distributions, 1 % predicted), exacerbation frequency, Medical Research Council (MRC) score and comorbidity classification (see supplementary table S1j). Receiver operating characteristic (ROC) area under the curve (AUC) statistics were used to determine the best cut-off value for predicting whether a new patient was likely to present with or without MetS.Multiple logistic regression (LR) models were developed to determine the association between clinical and demographic factors and the presence of MetS. Variables included were: sex, age, ethnicity, smoking status, IMD quintile, forced expiratory volume in 1 s . Univariate and multivariable Cox proportional hazard ratios were calculated for 5-year survival for patients with and without MetS and the individual component conditions of MetS. Covariates chosen for the multivariable analysis were those deemed statistically significant (p<0.001) in the univariate multiple LR models outlined for Aim 2.Demographics: sex, age, ethnicityIMD quintile: (1 being least deprived and 5 being most deprived)1 % predicted, smoking status, number of exacerbationsCOPD severity: MRC dyspnoea grade, FEVComorbidities: liver disease, kidney disease, psychological disease, frailty, asthma, CVD, musculoskeletal disease, GERDMultivariable associations were also explored as part of a sensitivity analysis between MetS and all-cause 5-year mortality when stratified by:sd age of 69±11 years and mean±sd BMI 27.6±6 kg·m−2. Summary statistics are shown in 1 % predicted was 63.1 but with wide variation (sd=23), confirming a broad range of disease severity.We included 40 806 patients with COPD who met eligibility criteria , of whomversus 23.9%). Full demographics and disease characteristics for those with and without MetS are shown in The prevalence of MetS in the COPD cohort was 10.1%. Age and sex were comparable for patients presenting with and without MetS. Over 90% of patients belonged to a White background irrespective of MetS status. Neighbourhood deprivation was analogous for quintiles 1–4, however, there appear to be more adults in the highest deprivation quintile (IMD=5) with MetS compared to without .A multiple LR model identified clinical and demographic predictors most associated with MetS to be sex , age, current smokers, missing smoking status, IMD5 (most severe), FEVThe association of the presence of MetS with all-cause mortality is shown in supplementary table S2a. The univariate associations between individual components of the MetS variables (see Aim 2) and 5-year survival can be found in In this study we estimated the prevalence and prognostic impact of MetS in patients with COPD managed in primary care in the UK. Using a definition of MetS derived from indices routinely recorded in primary care , we estiOur prevalence estimates for MetS are lower than previous studies, which report rates of between 20 and 50% in people with COPD, and was reported as being 34% in a systematic review summarising such studies . Such di−2 as a marker of truncal obesity and the presence only of a coded diagnosis of hypertension or diabetes, whereas international definitions of the syndrome incorporate prospective records of blood pressure, fasting glucose, lipid profiles and waist:hip ratios [We used surrogates for internationally accepted definitions for the components of MetS, for example a BMI >30 kg·mp ratios , 3. Somep ratios  but onlyp ratios . These fp ratios .Whilst the presence of MetS was adversely associated with mortality in the unadjusted analysis, this association disappeared when adjusted for other disease severity variables and comorbid conditions. In our sensitivity analysis, stratification for comorbidities but not disease severity, demographics or the index of socioeconomic deprivation was critical in mediating mortality. We have previously shown that indices of COPD disease severity had a greater impact than multimorbidity on 12-month survival , whereasA strength of the current study is the investigation of a large, unselected population of people with COPD, but we acknowledge a number of limitations from our data. Primary care data are provided from routine patient contacts conducted for the purpose of delivering healthcare rather than acquiring health data. Miscoding and missing data are therefore frequent and require bearing in mind when interpreting the data, particularly with respect to individual (rather than population) health. Examples may be miscoding of diagnoses of asthma and COPD or miscoding of “never” and “ever” smoking status. However, CPRD has been used extensively to study COPD, and the identification of our study population was undertaken using validated medcodes. Likewise, as outlined above, drawing comparisons to other population studies of MetS will be limited by the definition of the syndrome used, and we necessarily used indices that would be available in the primary care record. We note that this definition has been used in CPRD studies previously and does allow the impact of the syndrome (as defined by these indices) on health outcomes.per se. Understanding the impact of managing these comorbid conditions in COPD (for example through weight/diet management programmes for patients with obesity and/or diabetes) will be an important area for future research.In conclusion, we have shown that in patients coded as having COPD in primary care, the presence of MetS is associated with lower 5-year survival but that the impact on mortality was no longer significant when the impact of COPD disease severity and other comorbidities were accounted for. The individual components of our modified definition of MetS exerted differential effects on mortality suggesting there may be limited advantage in recording the presence of MetS as an entity as the individual component conditions of the syndrome exert different effects on future health risk, and we suggest that identifying and treating these components should be the focus of clinical care rather than seeking to diagnose MetS 10.1183/23120541.00211-2022.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.00211-2022.SUPPLEMENTSupplementary material