Adhesion strength of the cartilage gels were significantly higher compared to alginate gel, with the 2-wk group showing a near 20-fold higher strength . The in vivo remodeling process analysis of the 2 wk cultured gels showed increased cartilage repair characteristics and stiffness over time, with higher integration-failure stress compared to osteochondral autograft controls at 4 weeks (p < 0.01). In the nonhuman primate investigation, cartilage repair scores were significantly better in the gel group compared to defects alone after 24 weeks (p < 0.001). Cell distribution analysis at 24 weeks showed that human cells remained within the transplanted defects only. A self-assembled, FCPC-based cartilage gel showed chondrogenic repair potential as well as adhesive properties, beneficial for cartilage repair.The aim of this study was to develop a fetal cartilage-derived progenitor cell (FCPC) based cartilage gel through self-assembly for cartilage repair surgery, with clinically useful properties including adhesiveness, plasticity, and continued chondrogenic remodeling after transplantation. Characterization of the gels according to  Despite its sound concept, few TEC products have reached clinical trials1. Significant hurdles towards translation include limit in size, poor integration, and suboptimal microstructural biomimicry of native cartilage2. Scaffolds which are commonly used in TECs often make the construct relatively rigid and fixed in geometry. Such constructs often necessitate additional fixation and substantial debridement of the defect, often resulting in gap formation, damage to normal host tissue, and overall, increasing the difficulty of the surgical procedure3.Tissue-engineered cartilage (TEC) constructs aim to improve current cell therapy for articular cartilage defects such as autologous chondrocyte implantation by direct transplantation of engineered cartilage tissue. Expected benefits include direct cartilage formation by prefabricated hyaline cartilage-like tissue containing viable cells, theoretically providing better resistance towards the knee’s highly loaded environment5. Despite reports of its feasibility and effectiveness, scaffold-free TECs must overcome certain limitations for successful clinical translation. First, a highly proliferative allogenic cell source is preferred, as scaffold-free engineering requires a relatively large number of cells, often difficult to obtain with autologous cells7. Secondly, scaffold-free TECs undergo considerable remodeling in vivo. Transplanted tissue should therefore predictably differentiate and remodel into hyaline cartilage with minimal dedifferentiation or cell loss2. Thirdly, TECs should adhere immediately to the defect site to obviate the need for additional fixation. Overall, current scaffold-free TECs utilizing chondrocytes and mesenchymal stem cells (MSC) have yet to provide a comprehensive answer to these limitations5.Scaffold-free engineering is an alternative approach that improves some previous limitations of TECs. Its relative plasticity enables them to be applied directly over the defects, minimizing gap formation and improving integration9. Furthermore, we have previously reported a scaffold-free, self-assembly method using passaged chondrocytes10. Secondly, we aimed to optimize the cartilage gel’s plastic and adhesive features that would lead to a fixation-free, seamless fit of the cartilage gel into the defect. Finally, we sought to analyze cartilage repair efficacy, remodeling process, and cell distribution of the cartilage gel after transplanation in a nude mouse model and a non-human primate cartilage defect model in vivo.In efforts to overcome these limitations, we first aimed to develop an engineered cartilage construct utilizing fetal cartilage progenitor cells (FCPC) via scaffold-free, self-assembly engineering. According to our previous work, FCPCs boast a high proliferative capacity through late passages and higher chondrogenic capacity compared to adult chondrocytes and bone marrow derived-mesenchymal stem cells (bmMSC)9.Characterization of FCPCs used in this study revealed surface marker expression similar to MSCs  were analyzed, with 8 week osteochondral autologous transplantation (OATS) blocks and NC serving as controls. Overall, cartilage gels showed continued differentiation after in vivo transplantation, expressing increased hyaline-like cartilage qualities over the 12 week time period, with 8-week samples showing lacuna formation  model (n = 10 mice/group) to evaluate ion Fig. . Cellulaion Fig. . Total cion Fig. . Stiffneion Fig. . Cartilaion Fig. . O’Driscion Fig. 11.Figure11.Cartilage gels were transplanted in full-thickness cartilage defects of non-human primate knee joints . Cartilage gels were transplanted on the left knee joint (transplanted knee), within three defects made in the trochlea, medial, and lateral condyle cartilages of the femur. Empty, full thickness defects made in the right knee joint (control knee) served as controls. MRIs were taken at POD 8, 16, and 24 wks to follow the healing process, and animals were euthanized at POD 24 wks for tissue harvest. All defects in the transplanted knees remained filled through the 24 wk follow-up period Fig. . At POD Cell distribution of the gels after 24 weeks showed transplanted cells remaining within the defect. RT-PCR of human specific ALU sequences showed human ALU expression in the repaired cartilage of the transplanted knee Fig. . Human A12. All defect sites were absent from CD45 expression as shown in the IHC figures of the defects  count, blood C-reactive protein (CRP) levels, synovial histology, and transplantation site IHC. On gross examination, no wound problems were noted in all animals on all observation time points. Postoperative knee circumference of the transplanted knees returned to baseline levels (Δ < 3 mm) in all animals by 4 weeks Fig. . WBC anacts Fig. . Overall1. Chondrosphere®  a cartilage tissue aggregate product using autologous chondrocytes, have shown some improvement in clinical scores13. Revaflex™ , a neocartilage product based on allogeneic juvenile chondrocytes, is relatively large (2.2–2.5 cm diameter) and stiff compared to other scaffold-free TECs requiring tailoring and fixation during implantation. A Phase I/II trial showed 67% of patients to have ‘normal to nearly normal’ cartilage repair one year postoperatively, without any adverse immune response potentially associated with allogeneic cells14. Despite early clinical success, we still have little scientific evidence regarding the optimal cell source, therapeutic mode of action, and optimal biomechanical properties for clinical translation. Our study challenged these issues by first utilizing a highly proliferative and chondrogenic cell source and secondly by optimizing clinically relevant biomechanical properties in vitro. Finally, the therapeutic mode of action was investigated through remodeling process and cell distribution analyses, showing that the construct remains within the transplanted defect in the long run, successfully remodeling into hyaline-like cartilage in vivo.Several scaffold-free TECs have reached clinical trial stages with promising early results, with at least two products available on the market7. With intention to treat by a single procedure, a highly proliferative allogeneic stem cells with chondrogenic capacity is desirable. FCPCs are easy to isolate and expand, resulting in high yields (6.5 ± 0.95 × 107 cells/ 1 g tissue)9. Secondly, they possess stem cell characteristics of self-renewal, multilineage differentiation, and immune-modulation, with the potential to be a safe allogeneic cell source18. Thirdly, cartilage tissue formation ability of FCPCs are better than chondrocytes or MSCs, even without the use of exogenous growth factors. Previous studies of FCPCs have shown larger cartilage tissue formation with more GAG production compared to chondrocytes or bmMSCs20. Most importantly, tissue engineering with FCPCs may translate numerous regenerative benefits of fetal tissues. Fetal tissue responds to injury in a fundamentally different manner than adult tissue, resulting in a rapid, scar-less healing in many dense connective tissues22. This enhanced healing potential is intrinsic to the fetal tissue itself rather than the fetal environment, as the healing potential carries over into an adult host after transplantation21. Our findings support previously published FCPC characteristics, where FCPCs dependably differentiated towards cartilage without the use of growth factors and continued ECM production  in high-glucose DMEM containing 1% FBS at 37 °C under 5% CO2. After 12 h, the released cells were centrifuged at 1700 rpm for 10 min, washed twice, and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin at a density of 8 × 103 cells/cm2. When cells reached 80% confluence, the 0.5% Trypsin-EDTA of 10X to 1 ×  were used to detach the cells, which replated by the same way as above. Cells were expanded for two passages with culture medium changed every 3 days. All donor cells showed no significant difference in terms of morphology, proliferation, surface marker, senescence, and chondrogenesis as previously published9. Passage 2 cells were used for this study from all groups during in vitro experiments, and M GA11 wks cells were used for in vivo investigations.All protocols involving human tissue were performed under the approval of Institutional Review Board of Ajou University School of Medicine (AJIRB-MED-SMP-10-268). Human fetal cartilage tissues were harvested from four abortus within 24 hours following elective termination  after informed consent from respective guardians. Cells were isolated from the femoral head cartilage and expanded using a previously published protocolCells at passage 2 were analyzed for expression of stem cell related surface markers by flow cytometry. Briefly, cells in the suspension were incubated with anti-CD29-PE .555443), anti-CD34-FITC , anti-CD45-PE , anti-CD73-FITC , anti-CD90-FITC , anti-CD105-FITC , anti-SOX2-PE   and anti-OCT3/4-APC   for 40 min at 4 °C. Stained cells were determined by BD FACSCanto II flow cytometer  and analyzed using Flowing software . The adipogenic medium consisted of α-MEM supplemented 10% FBS, 1 µM dexamethasone, 10 μg/ml insulin, 0.5 mM isobutyl-methylxanthine, and 0.1 mM indomethacin . After 3 weeks of differentiation, cells were stained with Alizarin red S and Oil red O  to observe the degree of mineralization and the lipid droplets, respectively. For chondrogenic differentiation, 3 × 105 cells at passage 2 were centrifuged at 500 × g for 5 min, and the cell pellet was cultured in a chondrogenic medium. The chondrogenic medium consisted of DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement, 40 μg/ml proline, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate . After 3 weeks of induction, the samples were fixed with 4% formaldehyde and embedded in paraffin wax. Sections with a thickness of 4 μm were prepared and stained with Safranin O  to observe the sulfated glycosaminoglycans.For osteogenic and adipogenic differentiation, cells at passage 2 were plated in 6-well plates at densities of 2 × 10We also examined the proliferation ability and phenotype of the FCPCs according to passage time. The doubling time of FCPCs was determined from passage 1 to passage 15 (n = 4). Cells were subcultured at 80% confluence. The doubling time was calculted using the following formula: DT = (T1 − T0)log2/(logN1 − logN0), where T1 − T0 = the culture period in days, N0 = the plating cell number, and N1 = the harvesting cell number. Accumulated cells numbers were calculated with passages or days.10. Briefly, FCPCs were cultured in a high-density monolayer (at 2 × 105 cells/cm2) with DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement, 40 μg/ml proline, 1.25 mg/ml bovine serum albumin, 100 μg/ml sodium pyruvate. They were cultured until full confluency two dimensionally when cells spontaneously formed a thin membrane. Subsequently, the medium was removed and 1X Trypsin-EDTA was added and incubated for less than 5 min at 37 °C. When the membrane was peeled off from the plate, the enzyme was immediately removed and the membrane was carefully isolated using a wide-bore pipette and moved individually to a 50 ml tube filled with 10 ml of defined medium without exogenous growth factors . Each tube was centrifuged at 100 × g for 20 min to consolidate the membrane into a pellet-type construct. The constructs were incubated for 16 h at 37 °C and then transferred to a 6-well culture plate for extended culture for 1, 2, or 3 weeks in a 37 °C humidified atmosphere of 95% air and 5% CO2. The culture medium (5 ml) was changed every 3 days.Cartilage gels were fabricated using a previously published protocol43.After macroscopic examination, volume of the cartilage gels was measured using micro-CT . Scanning protocols are outlined in the Supporting Information TextFor histological analysis, cartilage gels were fixed in 4% formalin for 3 days and processed for tissue sectioning. Tissue sections of 4 µm in thickness were made and stained with hematoxylin-eosin (H&E), Safranin-O, and immuno-histochemical (IHC) analysis for type I collagen , type II collagen , type X collagen  using the DAB method.44. Briefly, the assay was prepared via two steps: (i) The colour reagent was prepared by dissolving 16 mg dimethylmethylene blue in 1 l water containing 3.04 g glycine, 2.37 g NaCl and 95 ml 0.1 M HCl, to give solution at pH 3.0 . (ii) The papain-digested GAG sample prepared as above was taken, and measured by reference to a standard calibration curve. The result was analyzed by Magellan v.6.4. software and Tecan microplate reader . For total collagen content analysis, samples were measured by chloramine-T hydroxyproline assay45. Souluble peptides and proteins in each standard and tissue sample were hydrolyzed to individual amino acids by adding 500 μl of 4 N sodium hydroxide (NaOH) and incubating at 121 °C and 15 psi above atmospheric pressure for 20 min (using an autoclave). Samples were allowed to cool to room temperature and then neutralized with 500 μl of 4 N HCl. Hydroxyproline amino acids were converted to pyrolle-2-carboxylate by oxidation via addition of 0.625 mL of 0.05 M chloramine-T in 74% v/v H2O, 26% v/v 2-propanol, 0.629 M NaOH, 0.140 M citric acid (monohydrate), 0.453 M sodium acetate (anhydrous), and 0.112 M acetic acid , followed by incubation at room temperature for 20 min. Finally, 0.625 mL of 15% w/v DMAB (1 M) in 2-propanol plus concentrated acid (a.k.a. Ehrlich’s solution) was added to each sample and vortexed immediately to facilitate mixing. Samples were incubated at 65 °C for 20 min and then rapidly cooled by immersion in room temperature water to stop chromophore development.For water content analysis, tissue samples were weighed (wet weight), freeze dried, and weighed again (dry weight). For glycosaminoglycans (GAG) content and DNA content analysis, samples were freeze-dried and digested with papain-digestion solution . For papain digestion, the sample in 1 ml papain-digestion solution was incubated at 60 °C for overnight. The total GAG content was spectrophotometrically measured by using the 1, 9-dimethylmethylene blue colorimetric assay. Chondroitin sulfate sodium salt from shark cartilage  was also used as a standardGenetic markers were analyzed with real-time polymerase chain reaction for 1, 2, and 3 wk cultured cartilage gels, with FCPCs and mature cartilage tissue used as controls. All adult human cartilage and osteochondral tissue were harvested from morphologically normal (ICRS grade 0) lateral condyle osteochondral tissue obtained during joint replacement surgery. Protocols and primer information are outlined in Supporting Information and Supplemental Table 47. The spreadability of cartilage gel was determined by pressing the tissue construct between top and bottom plates covered by paper, then 500 g standardized weigth was put on the upper plate and left for about 5 minutes, as previously described49. Diameters of spread circles were measured in mm and were taken as comparative value for spreadability. Adhesive strength of cartilage gels after transplantation into the human cartilage defect osteochondral blocks was measured using a push-out test as in previously described52. Briefly, The adhesive strength was evaluated as the forces at ultimate failure per unit of the interfacial area and determined using 5 mm in diameter of cylindrical-shaped indenter in an Universal Testing Machine fitted with a 5N maximum load cell. Alginate gels (2%) were fabricated using previously published methods and used for controls during adhesion strength testing47. All in vitro characterization analyses were done 5 times each.Biomechanical analysis of cartilage gels was done by measuring the aggregate modulus, spreadability, and adhesive strength. For aggregate modulus, tissues were subjected to an unconfined compression test using Universal Testing Machine . Each sample was placed on a bottom plate of the machine, and compressed at a speed of 1 mm/min. The machine was stopped automatically after moving a programmed length between the top and bottom plate53. Osteochondral blocks were sampled at 2, 4, 8, and 12 weeks after transplantation and analyzed as described above. Histological images were scored for cartilage repair using O’Driscoll cartilage repair scoring system by two separate pathologists not involved in this study54. For cartilage-to-cartilage integration analysis, a human cartilage cylinder construct model was used as previously described55. Briefly, cartilage gels were implanted in the cylinder constructs and OATS was used as controls. Cylinder constructs were then subcutaneously transplanted into the back of nude mice (n = 7 for each group). The cartilage-to-cartilage integration was evaluated by safranin-O stain and mechanical test for integration strength using using a push-out test as in previously described55. A custom 2 mm in diameter of cylindrical-shaped indenter affixed to a Universal Testing Machine  pushed the cartilage repair out of the cartilage annulus (1 mm/min) while recording load. Failure stress (integration strength) was calculated as the quotient of the load at failure and the interface area.Full thickness cartilage defects of 3 mm diameter were made on 5 mm diameter human osteochondral blocks obtained from joint replacement surgery. Lateral femoral condyles blocks without gross cartilage wear were used. NCs were used as normal controls, obtained from lateral femoral condyles of fresh frozen cadavers without knee joint pathology. The 2-WCG gels were transplanted on the defects using a syringe without additional fixation. OATS was performed on osteochondral blocks as controls. Briefly, 3 mm diameter osteochondral defects were made with a matching size biopsy punch. The osteochondral block was gently reinserted into the defect similar to the OATS procedure using a plastic impactor . Osteochondral blocks were then transplanted subcutaneously into the back of nude mice , after institutional approval of Institutional Animal Care and Use Committee of Ajou University (IACUC No. 2014-0072)Macaca fascicularis) aged 54 to 69 months weighting 4–5.5 kg were used for this study. Procedures for anesthesia and surgery for femoral chondral defects on both knees followed a previously published protocol and outlined in the Supporting Information Text56. Postoperative knee circumference was measured preoperatively, and at postoperative wk 1, 2, 4, 8, 16, and 24. Intravenous blood was drawn for WBC count and C-reactive protein quantification preoperatively, and during postoperative wk 1, 4, 16, and 24. MRI was performed on each knee at postoperative wk 8, 16, and 24. MRI protocols are outlined in the Supporting Information Text. At 24 wks, all animals were euthanized for further analysis. Vital organs  and relevant knee tissue  were harvested and prepared for cell distribution RT-PCR. Chondral defects were prepared for further histological analysis and resultant histological results were further analyzed using O-Driscoll cartilage repair scores by 2 separate pathologists twice that did not participate in this study11. Synovial tissue of each knee harvested from anterior fat pads were also prepared for histological analysis and subsequent chronic synovitis grading in the same fashion12. For cell distribution analysis, PCR for human alu sequences was performed to confirm the presence of transplanted cartilage gels in recipient NHP organs. Total DNA of organ samples was extracted with a QIAamp DNA Mini Kit , according to the manufacturer’s instructions. The primers used were Human Alu F: 5′-GTAAGAGTTCCGTAACAGAGCT-3′, Human Alu R: 5′-CCCCACCCTAGGAGAACTTCTCTTT-3′, NHP gapdh F: 5′-CGGATTTGGTCGTATTGGG-3′ and NHP gapdh R: 5′-GGGATCTCGCTCCTGGAAG-3′. Samples were incubated at 94 °C for 2 min and then amplified for 25 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and extension for 59 s at 72 °C. The PCR products were analyzed by resolution on a 1.5% (w/v)57. Cell tracking within the chondral defects were also performed using human anti-nuclei antibody MAB1281, clone 235-1  immunohistochemistry. Human anti-nuclei antibody (+) cells were counted and compared in 5 histological sections 20 μm apart for each defect. The transplantation site (cartilage defects) was also evaluated for CD45 expression, with IHC using anti-CD 45 antibody .All protocols involving nonhuman primates (NHP) use were approved by Institutional Animal Care and Use Committee of Seoul National University (SNU-13-0251). All experiments were performed in accordance with relevant guidelines and regulations. A total of five skeletally mature male NHPs . Non-parametrical tests were used throughout this study. The test is used as a non-parametric alternative of the independent two-sample t-test (Mann-Whitney) or multiple comparison . Data are expressed as a mean ± standard deviation. P-values less than 0.05 were considered statistically significantSupporting Information.Supplemental online video 1.Supplemental online video 2.


SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either +glo1 or +glo2. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human  SNCAIP) gene, which is expressed in a variety of tissues and enriched in the brain.Lewy body inclusions and Lewy neurites are important hallmarks of neurodegenerative diseases such as Parkinson’s disease (PD) and Lewy body dementia. They consist primarily of α-Synuclein (αSyn) aggregates and occur in different neuronal cell populations including dopaminergic neurons . In addiSNCAIP were studied in a variety of model organisms, including mice models [SNCAIP in wild-type (wt) S. cerevisiae strain resulted in the formation of SY1-containing inclusions, starting with the formation of foci at endomembranes and lipid droplets. Büttner et al. speculated that the interaction with lipid rafts could facilitate dimerisation and self-assembly of SY1 [SNCAIP expression reduced survival and triggered apoptosis and necrotic cell death in a Sir2 dependent manner, most likely due to excessive aggresome build-up during growth and consequential deregulation of autophagic processes. In a co-expression model, SY1 aggravated αSyn aggregation, which was correlated with enhanced αSyn phosphorylation on Ser129 [SY1 is predominantly expressed in neurons and present at presynaptic nerve terminals , and it e models ,9,10,11,e models , neuroble models , and yeae models . Expressy of SY1 . In expoy of SY1  of said n Ser129 .glo1+) from the fission yeast Schizosaccharomyces pombe shares 50% amino acid sequence identity with the Saccharomyces cerevisiae Glo1 [S. cerevisiae has two isoforms of the structural genes for glyoxalase II, GLO2 and GLO4. Expression of GLO2 occurs both in glucose and glycerol media, while GLO4 is expressed only in glycerol medium [S. pombe glo2+ gene has not yet been experimentally validated [S. cerevisiae, S. pombe does not possess a Gre3 ortholog. Aldose reductases such as Yak3/YakC have been identified in S. pombe, but none of these have reported MGO-detoxifying activity [Glycation has primarily been studied in the context of diabetes mellitus, in which dysregulation of glucose metabolism results in the eventual formation of advanced glycation end-products (AGEs) via the non-enzymatic Maillard reaction ,17. Thesiae Glo1 . S. cerel medium . The idealidated . Contraractivity ,31.The observation of AGEs in Lewy bodies in cases of incidental Lewy body disease suggested that AGEs might trigger Lewy body formation in individuals that are considered pre-PD patients . In addiS. cerevisiae deficient in the GLO1, GLO2, and GRE3 genes and S. pombe strain deficient in glo1+ and glo2+.In this research article, we add to the findings described above by assessing the repercussions of a disrupted glyoxalase system and aldose reductase Gre3 activity on SY1 inclusion formation and SY1-mediated growth effects. To this end, we employed a genetic approach by analysing glo1∆, glo2∆, and gre3∆ S. cerevisiae strains transformed with either an empty vector (EV) or a plasmid expressing native SNCAIP were subjected to growth analysis. These results revealed that both wt and glo1∆ cells containing the EV grow equally well  ±0.8, glo2∆ and gre3∆ strains only reached an ODmax of ±0.6  could e W303 in , SNCAIP analysis C left. TWhile Glo1 converts the spontaneously formed MGO-glutathione complex into S-D-lactoylglutathione, Glo2 is responsible for the conversion of S-D-lactoylglutathione into glutathione and D-lactate. As such, Glo2 recycles the cytoplasmic pool of glutathione. Cytoplasmically generated glutathione can either remain in the cytosol or be relocated into the mitochondria via active transport. The mitochondrial pool of glutathione is considered the major redox system in numerous processes, such as the preservation of sulfhydryl groups of mitochondrial proteins in the appropriate redox state and protecting mitochondrial DNA and membrane integrity against ROS-induced oxidative stress. Disruption of cytoplasmic Glo2 activity results in S-D-lactoylglutathione accumulation and consecutively keep glutathione trapped in the cytosol. This can then lead to decreased glutathione availability in the mitochondria, disrupting the glutathione-dependent antioxidant enzyme system ,37.SNCAIP-expressing glo2∆ cells is associated with increased SY1 oxidation. The impact of SNCAIP expression on growth was also assessed in wt, glo1∆, and glo2∆ fission yeast strains. S. pombe does not possess a GRE3 homolog and glyoxalase 2 activity of the proposed S. pombe GLO2 homolog, glo2+, has yet to be established [S. cerevisiae, no growth differences between EV-expressing strains, nor a SNCAIP expression-dependent inhibitory effect on growth was observed in any strain examined  in daughter cells is minimal  ,40.glo1∆ cells on the one hand, and glo2∆ and gre3∆ cells, on the other hand, prompted verification of the SY1 protein level via Western blot analysis. To this end, we subjected whole-cell extracts of wt, glo1∆, glo2∆, and gre3∆ S. cerevisiae, and wt, glo1∆, and glo2∆ S. pombe strains to Western blot analysis using an anti-SY1 antibody. Our analysis confirmed that glo2∆ and gre3∆ S. cerevisiae strains displayed a reduced SY1 protein content in comparison to wt and glo1∆ strains .S. cerevisiae, we did not detect differences in SY1 protein level between the S. pombe strains , juxta/intranuclear quality control compartment (JUNQ/INQ), and insoluble protein deposit (IPOD) were identified in proteins ,45. Whilradation .glo1∆, glo2∆, and gre3∆ cells, dsRed-SY1 inclusions were distantly located. In contrast, inclusions in the majority of wt cells displayed perinuclear localisation , which can be used as a measure of the extent of superoxide and hydrogen peroxide formation in cells. Surprisingly, both the base level, i.e., upon expressing an empty plasmid, and the SY1-associated oxidative stress level detected in glo2∆ and gre3∆ cells were lower than the corresponding values detected in wt and glo1∆ cells. Contrary to wt and glo1∆ cells in which SNCAIP expression reduced or did not affect the percentage of positive cells, respectively, glo2∆ and gre3∆ strains showed a strong increase in the percentage of DHE-positive cells upon expression of SNCAIP when compared with the control  and reduced (glo2∆ and gre3∆) growth upon SNCAIP expression. Moreover, co-localisation of dsRed-SY1 inclusions and actin cables was visible in actively dividing wt and gre3∆ cells, pointing towards retrograde transport of inclusions via actin cables to the mother cell. In addition, in both wt and gre3∆ cells, inclusions were flanked by actin patches, but not to the extent as described previously [During exponential growth, localisation of dsRed-SY1 inclusions adjacent to actin cables was visible in cells which displayed both normal  displayed Glo3 activity and conferred MGO resistance in a glo1∆ strain [S. cerevisiae, we did not observe growth defects in an SY1-expressing glo1∆ strain. This points towards a dominant role of Hsp31-mediated Glo3 activity in detoxifying MGO. As mentioned in the introduction, the glyoxalase 2 activity of the proposed S. pombe Glo2 homolog has yet to be shown [We conducted a comparative study to assess any potential repercussions of a disrupted methylglyoxalase system on the growth of the fission yeast n of MGO . Neverth∆ strain . Similarbe shown .glo2∆ cells in comparison to wt cells indicates a lack of glyoxalase 2 functionality of Glo2. Alternatively, disruption of Glo2 activity might be compensated for by the mitochondrial thioredoxin system, which was shown to act as a back-up system for glutathione-mediated antioxidant activity in fission yeast [SNCAIP.The absence of an SY1-associated growth phenotype and similar levels and intracellular distribution of SY1 inclusions in on yeast . InteresS. cerevisiae, the observed inclusions in S. pombe did not show signs of fusion or retrograde transport via actin cables to facilitate asymmetric distribution into one of the two daughter cells. These findings were in line with the lack of observed SY1-associated growth defect.This points towards the involvement of a transport mechanism actively regulating the intracellular localisation of these inclusions. In fission yeast, polarity factors, deposited at the plasma membrane by microtubule-mediated transport, recruit protein complexes  involved in actin cable assembly . These aS. cerevisiae strains were grown in 3 mL minimal medium, containing glucose  and yeast nitrogen base without amino acids and ammonium sulphate  at 30 °C. Supplements were ammonium sulphate  and complete supplement mixture drop-out depleted for uracil . Cells were grown for 48 h after the time point of inoculation and subsequently subjected to the appropriate analysis. The wt and deletion strains glo1::kanMx, glo2::kanMx, and gre3::kanMx were BY4741 MATa, his3∆1 leu2∆0 met15∆0 ura3∆0. All strains were haploid. SNCAIPWT and dsRed-SNCAIPWT were expressed from a multicopy episomal plasmid (pYX212 backbone with URA3 marker) using the triosephosphate isomerase (TPI) promoter. The cDNA encoding synphilin-1 was isolated from a hippocampal cDNA library via PCR amplification using the primers CATGCCATGGAAGCCCCTGAATACC and CCGCTCGAGTTATGCTGCCTTATTCTTTCC that included, respectively, a NcoI and XhoI restriction site for cloning into the pYX212 plasmid, which allows expression from the constitutive TPI1 promoter [promoter .S. pombe cell cultures were grown in Edinburgh Minimal Medium [S. pombe strain used in the study was h90ura4.D18. The deletion strains glo1::kanMX6 and glo2::kanMX6 were h+ade6-M210/M216 ura4-D18 leu1-32 (Bioneer S. pombe deletion mutant library [SNCAIPWT was cloned in the pINT plasmid backbone [ura4 marker) [leu1 locus, strains simultaneously acquire uracil prototrophy and leucine auxotrophy.l Medium  (Formedi library ). All stbackbone  (ura4 ma marker)  and expr marker) . ExpressS. cerevisiae and S. pombe strains were first grown as described in 2.1, after which the cells were inoculated at an optical density of 600 nm of 0.1 in either a 96-well (S. cerevisiae) or 24-well (S. pombe) plate  for subsequent growth in a Multiskan GO microplate spectrophotometer (Thermo Scientific). Plates were shaken at 400 rpm and optical density was measured at 600 nm every 30 min. For spot assays, serial dilutions of S. cerevisiae and S. pombe precultures  were spotted onto the appropriate solid minimal medium (see 2.1). Two to four days after spotting, colonies appeared and plate scans were made. Growth profiles are based on three clones per strain.S. cerevisiae strains were grown in a 96-well plate as described in 2.1. Accumulation of ROS was analysed based on the superoxide-driven conversion of non-fluorescent dihydroethidium (DHE) to fluorescent ethidium. The DHE assay was carried out as described in the guidelines (ThermoFisher) and quantified using flow cytometry . A two-way ANOVA test  was carried out using GraphPad Prism 8.3.0 .S. cerevisiae were analysed by standard fluorescence microscopy using a Leica DM4000B . To assess differences between strains in inclusion characteristics, a comparative analysis based on at least 800 cells originating from three clones per strain was performed. Only fluorescence positive cells were considered for the comparative analysis of the number and size of inclusions between strains. Statistical analysis  was carried out using GraphPad Prism 8.3.0.SY1 inclusion formation and effects on cytoskeletal architecture in TM Blue CMAC dye (Thermo Fisher) according to the manufacturer’s guidelines.Cell nuclei were stained using DAPI (Thermo Fisher). To this end, cells were fixed in 37% formaldehyde for 1 h at room temperature, after which the cells were washed twice in PBS. Nuclei were subsequently stained in 1:3 DAPI/PBS for 1 h in the dark. Cell vacuoles were stained using CellTrackerTM 488 phalloidin (Thermo Fisher) and conducted as recommended in the kit’s guidelines.Finally, cells were washed trice in PBS and subjected to microscopic analysis. Quantifications are based on the localisation of 100 inclusions in around 50 cells of one clone per strain, grown until the mid-late exponential phase. Due to limitations in the number of independent cell countings, no statistics were performed for the inclusion localisation experiment. The actin staining was performed using Alexa FluorS. cerevisiae whole-cell extracts were prepared according to Vandebroek et al [S. pombe whole-cell extracts were prepared according to Hagan et al. [ek et al , while Sn et al. . Proteinp-value of 0.05) was conducted to assess differences in anti-SY1 antibody immunoreactivity between strains. Standard deviations are based on densitometric data, as determined by Fiji Software (v1.52i), of three clones per strain.A one-way ANOVA test  inverted microscope. Cell fixation, permeabilisation, and antibody application steps were performed according to the protocol described in , omittinS. cerevisiae and S. pombe. We found that lack of Glo2 and Gre3 activity in S. cerevisiae affected both SY1 inclusion number and growth ability. Increased SY1 inclusion formation correlated with enhanced oxidative stress levels and an inhibitory effect on growth, most likely caused by deregulated autophagic degradation due to excess aggresome build-up. These findings illustrate the severe repercussions of enhanced oxidation and glycation on SY1 inclusion formation.In this study, we investigated the impact of a functionally disrupted glyoxalase system and aldose reductase activity on SY1 inclusion characteristics and cell growth in the yeasts glo1∆ gre3∆ could help unravel the connection between both MGO detoxification pathways.Future endeavours could focus on co-expressing SY1 and αSyn to shed more light on their interplay in conditions characterised by increased protein glycation and oxidation. Moreover, the use of double mutants such as SNCAIP expression did not seem to reach a sufficiently high level to impair the growth of any of the tested strains. In addition, our data do not provide any indications that the fission yeast homolog of GLO2, glo2+, carries out glyoxalase 2 activity. Combined, our findings illustrate the relevance of yeasts, especially S. cerevisiae, as complementary model organisms to unravel mechanisms contributing to SY1 protein pathology in the context of neurodegenerative diseases.Finally, while we clearly detected inclusion formation in our fission yeast model, the level of incited stress upon


Severe hypercholesterolemia  affects over 30 million people worldwide. In this study, we validated a new polygenic risk score (PRS) for LDL‐C.Summary statistics from the Global Lipid Genome Consortium and genotype data from two large populations were used.p = 10–41). After risk categorization, the risk of having HC was higher in the high‐ versus low‐risk group . Compared to a 12‐SNP LDL‐C raising score (currently used in the United Kingdom), the PRS explained more LDL‐C variability (8% vs. 6%). Among Finns with severe HC, 53% (66/124) versus 44% (55/124) were classified as high risk by the PRS and LDL‐C raising score, respectively. Moreover, 54% of individuals with severe HC defined as low risk by the LDL‐C raising score were reclassified to intermediate or high risk by the new PRS.A 36‐SNP PRS was generated using data for 2,197 white Americans. In a replication cohort of 4,787 Finns, the PRS was strongly associated with the LDL‐C trait and explained 8% of its variability  affects over 30 million people worldwide. In this study, we generated a new polygenic risk score (PRS) for LDL‐C in 2,197 white Americans and validated our results in a replication cohort of 4,787 Finns. This new PRS explains 30% more LDL‐C variability (8% vs. 6%) and has a better predictive role in identifying HC of polygenic origin compared to existing methods. PRSs have been used to identify patients at risk of several conditions, including cardiovascular , followed by validation on a second cohort of European Finnish individuals. We also compared the performance of the new PRS against the 12‐SNP LDL‐C raising score by Talmud et al.   approved the study. Protocols for the eMERGE network were approved by the Institutional Review Boards (IRBs) at the institutions where participants were recruited; all included participants provided written informed consent prior to inclusion in the study.2.2A study cohort of 2,764 white American individuals was obtained from The Electronic Medical Records and Genomics network   and the allele dosage carried by the individual  according to the formula:The PRS of an individual p < 1 × 10−3) were retrieved from the Global Lipid Genetics Consortium (GLGC). This initial set of SNPs was reduced by performing linkage disequilibrium (LD) pruning, thus retaining only the most significant SNPs in each LD block. Different LD thresholds (r2 between 0.1 and 0.8) were tested  and evaluated by PRSice to identify the best PRS, that is, the one that maximizes the explained phenotypic variance in the white American cohort. At each p‐value threshold, the PRS was incorporated in a linear regression model to explain the LDL‐C continuous trait, while adjusting for the following covariates: age, gender, BMI, and ancestry differences captured by the first two components from multidimensional scaling. From each regression model, an incremental R2 was computed by PRSice and plotted against the p‐value threshold. This R2 is reported as the difference between the R2 of the full regression model (LDL‐C∼PRS + covariates) and the R2 of the null model (LDL‐C∼covariates). The best PRS was the one achieving the highest R2.Sets of SNPs were defined over a range of 2.42 of the models compared. These R2 values were calculated following the same approach described for PRSice : The phenotype was categorized in severe HC (LDL‐C > 4.9 mmol/L), intermediate HC (3.0 ≤ LDL‐C ≤ 4.9 mmol/L), and normal LDL‐C levels (LDL‐C < 3.0 mmol/L), and the classification accuracy of the scores was assessed by receiver operating characteristic (ROC) curves. We used the DeLong test to compare AUCs from different PRSs.The PRS was categorized using the deciles of the distribution: low‐risk (decile third and below), intermediate risk , high‐risk category (deciles seventh and above). This mirrors the score categorization used in the SNP LDL‐C raising score by Talmud et al. , thus alThe distribution of subjects with severe HC was analyzed across PRS categories. We compared the percentage of patients with severe HC in the low‐risk PRS category.The PRS was assessed using the following statistical approaches:The list of genes , and GenBank (RefSeq) identifiers) included in the PRS is presented in Table 3A cohort of 2,197 white American individuals was used to construct the PRS for the LDL‐C trait. The clinical and biochemical characteristics of the individuals included in the study are presented in Table p < 1 × 10−3) were analyzed by the PRSice algorithm. In a regression model  in a multiple regression analysis, which adjusts for covariates.A total of 8,224 SNPs . Individuals in the high category had a significantly higher risk of severe HC relative to individuals in the low category .After score categorization Table , median 3.1Northern Finland Birth Cohort 1966 (NFBC), whose clinical and biochemical data are presented in Table p = 10–63; RR = 4.8 (CI: 2.6–8.9), p = 10–7).The new PRS was applied to a cohort of 4,787 Finnish individuals from the 3.2p = .12 DeLong test) and Finnish populations  and was able to explain 30% more of trait variance (8% vs. 6% in the American population and 8% vs. 6% in the Finnish population).We compared the results obtained with our new PRS to those obtained using the SNP LDL‐C raising score of Talmud et al.  estimateAfterwards, the categorized PRS and SNP LDL‐C raising score were compared. In the white American cohort, 45% (230/506) versus 42% (213/506) of individuals with normal level LDL‐C (<3 mmo/L) were classified in the low‐risk category, and 50% (53/107) versus. 46% (49/107) individuals with severe HC were classified in the high‐risk category using the PRS and SNP raising score, respectively.SD 0.2 versus. 60 yrs, SD 11.5 in the Americans), with a healthier lipid profile , thus confirming a trend toward a better performance of PRS versus SNP raising score in the replication cohort.In the Finnish cohort, which is a younger cohort compared to the Americans  and 42% of severe HC cases in the Finnish cohort .3.3LDLR, APOB, or PCSK9 (approximately 40% of patients who undergo genetic testing for FH)  are also present in the SNP LDL‐C raising score (see Table ‐4) and lipoprotein processes (p = 7 × 10−4). No enrichment was found in KEGG metabolic pathways annotation, which suggests that multiple metabolic pathways may be implicated in the development of severe HC of polygenic origin.The 36 SNPs in the new PRS map to 23 genes Table , six of ε2, ε3, and ε4 haplotype. However, these two SNP are in LD (LD > 0.35 between rs7412 and rs7254892 in TOMM40 and between rs429358 and rs4420638 in APOC4) in both American and Finnish populations , which define the APOE p < 1 × 10−8. Eight genes  were associated with coronary artery disease, five with diabetes mellitus type 2  and three with Alzheimer's disease  (see Table APOB and ABO) were reportedly associated with cancer  and three genes  with Stroke  with variants reported for these traits). The list of pleiotropic SNPs is as follows: association with coronary artery disease , stroke , diabetes mellitus type 2 , and Alzheimer's disease .As severe HC is often associated with other cardiovascular risk factors and the degree of aggressive lipid lowering treatment is dictated by the patient overall cardiovascular risk, we assessed the pleiotropic nature of the 23 genes in the new PRS by examining their association with other traits in the GWAS catalog, using a cutoff for association 4p‐value threshold < 10–3 and demonstrated that it is robustly associated with the LDL‐C trait in two independent populations of white European ancestry from the United States of America and Finland. Compared to the existing 12‐SNP LDL‐C raising score , the new PRS was able to explain 30% more of LDL‐C trait variability and to identify a polygenic risk component, therefore, reclassifying several patients otherwise deemed as low risk for hypercholesterolemia of polygenic origin. The PRS for LDL‐C can have several applications in clinical practice including i) identifying patients with a clinical diagnosis of FH who are at high likelihood of HC of polygenic origin, and ii) inclusion into algorithms for the early stratification of patients at risk of HC and other comorbidities, both cardiovascular and Alzheimer's disease, for early life style modifications.We constructed a new PRS for LDL‐C from an initial set of thousands of SNPs at GWAS The SNP LDL‐C raising score by Talmud et al. , and thep‐value < 5 × 10−8), the field is now migrating toward the use of genome‐wide polygenic scores consisting of thousands of SNPs with higher p‐values  of SNPs at GWAS significant level (In this study, we used an unbiased method for selecting the most informative SNPs associated with LDL‐C from an initial set of over 8,000 SNPs, to construct a polygenic score for LDL‐C. Although the AUC for PRS was only marginally better compared to that of LDL‐C SNP score, the PRS was better in classifying patients into low or high risk compared to the LDL‐C SNP score method. However, this was just a trend possibly because of the small number of patients with severe HC in our two cohorts.The 23 genes harboring the 36 SNPs selected for the PRS show enrichment in Gene Ontology terms related to lipid metabolism, thus further confirming the validity of the selection process. Pathways analysis did not show any enrichment, which suggests that small defects in multiple metabolic pathways may be involved in hypercholesterolemia of polygenic origin.We are still far from understanding the genetic causes of FH, a condition associated with a 20‐fold increased risk of CHD compared to the general population (Nordestgaard et al., We found that many of the genes included in the PRS have pleiotropic effects. We and others have noted that gene pleiotropy is common in genes implicated in both rare (Ittisoponpisan, Alhuzimi, Sternberg, & David, An important limitation of our study is the small number of patients with severe HC in the two cohorts. Future work will involve applying the PRS in FH patient cohorts and, in particular, to FH/M‐ patients. Moreover, it will be important to evaluate the correlation between polygenic risk for HC, as defined by the PRS, to the risk of cardiovascular events.In conclusion, we developed a polygenic biomarker based on 36 SNPs that is able to identify patients at an increased risk of HC and associated comorbidities as a result of their genetic makeup.The authors declare that there is no conflict of interest.AD and LGL conceptualized the study and contributed to the first draft of the manuscript. LGL performed the molecular analyses. AD contributed to the bioinformatic analyses. AD, CH, and MJS contributed to interpretation of bioinformatic data. All authors critically reviewed the manuscript.SupinfoClick here for additional data file.


The relative lengths of the rearfoot talus and calcaneus normalized to the foot length also correlated significantly with the sprint performance . Furthermore, the relative height of the calcaneus, but not the talus, normalized to body height correlated significantly with sprint performance . Additionally, the relative calcaneus height correlated significantly with the foot arch height index , and the foot arch height index correlated significantly with sprint performance . These findings suggest that the taller calcaneus may be a key morphological factor for achieving superior sprint performance, potentially via modeling the longer forefoot and rearfoot bones and functional foot morphology in sprinters.This study examined the relationships between the foot bone morphologies and sprint performance in sprinters. Foot images in 56 male sprinters obtained using magnetic resonance imaging. The relative lengths of the forefoot bones of the big and second toes, which were calculated as total lengths of the forefoot bones for each toe normalized to the foot length, correlated significantly with personal best 100-m sprint time ( Dowson et al.3 reported that greater plantar flexor isokinetic torque correlated with superior sprint performance in athletes, including sprinters. In general, the magnitude of joint torque is primarily determined by the agonist muscle size5; therefore, greater plantar flexor muscle may be related to superior sprint performance in sprinters. Despite the fact that the relationship between the plantar flexor muscle size and sprint performance in sprinters has been inconsistently remained among the results of previous studies10, we and others reported the absence of this relationship10. Therefore, sprinters may have other morphologies than muscle size related to increase in plantar flexor torque while sprinting.Superior sprint performance is achieved using gross torques of the lower limb joints, including the hip, knee, and ankle. Of these components, greater ankle plantar flexor torque plays an important role in increasing ground reaction forces and shortening contact time during the stance phases while sprinting14. This result suggests that the longer forefoot bones may be an essential characteristic for successful sprinters. Furthermore, using computer simulation, Lee and Piazza12 determined that a longer forefoot contributes to the production of greater plantar flexor torque during the push-off phase while sprinting. Given their findings, we demonstrated that longer forefoot bones correlated with better sprint performance in sprinters14. Therefore, the forefoot bone length is an important morphological factor of sprint performance in sprinters.We and others previously demonstrated that the forefoot bones were longer in sprinters than in non-sprinters11 determined that the plantar flexor moment arm (MA) dimension  was shorter in sprinters than in non-sprinters. Raichlen et al.15 reported a positive correlation between the plantar flexor MA and the calcaneus length. Considering these findings, shorter rearfoot bones, especially the calcaneus, may be a required characteristic for achieving superior sprinter performance in sprinters. Nevertheless, Baxter and Piazza16 determined that greater plantar flexor MA correlated with higher plantar flexor isometric and isokinetic torques. Their result is consistent with the results of the knee joint in previous studies18. Because of the close relationship between plantar flexor torque and sprint performance3, a shorter calcaneus may be a disadvantage in producing greater plantar flexor torque and achieving superior sprint performance. Therefore, the first hypothesis of the present study was that longer, rather than shorter, rearfoot bones, especially the calcaneus, would be related to better sprint performance in sprinters.When having the longer forefoot bones, shorter rearfoot bones may be required for modeling the foot, because of a balance between lengths of the forefoot and rearfoot bones. In fact, Baxter et al.14. Based on the results of our previous studies, the second hypothesis of the present study was that, when having a taller rearfoot bones, especially the calcaneus, it would be required for modeling longer forefoot and rearfoot bones and would be related to better sprint performance in sprinters.We previously reported that, although the lengths of the forefoot bones were longer in sprinters than in non-sprinters, the lengths of the mid-foot bones  did not differ between the two groups; therefore, we concluded that the mid-foot bones do not display specific modeling for sprinters21. Hence, the foot arch height may play an important role in producing higher plantar flexor torque because of increased MTP joint torque using the stored energy during push-off in the stance phase21. Morita et al.22 reported that the foot arch height correlated with 50-m sprint time in children. Despite these findings, to the best of our knowledge, no study has examined the relationship between foot arch height and sprint performance in adults, including sprinters. The foot arch height is often associated with the foot length; however, this relationship is inconsistent among the findings of the previous studies24. Nevertheless, a study by Morita et al.22 did not find a correlation between foot length and sprint performance in children. Furthermore, several previous studies reported that the foot length did not differ between faster and slower groups of adult sprinters25. Based on these previous findings, the foot arch height may relate to sprint performance independently of the foot length; in other words, the foot arch height may be a specific morphology for determining sprint performance among the foot characteristics. If there is a correlation between foot arch height and sprint performance in adult sprinters, it may be due to a taller calcaneus, which could contribute to modeling a higher foot arch height, potentially because of increased navicular height. Therefore, the third hypothesis of the present study was that taller calcaneus would be related to higher foot arch height, and that the higher foot arch height would be related to better sprint performance in sprinters.Previous studies reported that the foot arch height may help utilize the elastic energy stored by the metatarsophalangeal (MTP) joint during the stance phase while sprintingTo test these hypotheses, we first compared the lengths and heights of the forefoot and rearfoot bones and foot arch height between faster and slower sprinters who had been divided based on their personal best times in a 100-m sprint race, which is due to understand the foot morphological characteristics in sprinters having superior sprint performance. Thereafter, we examined the relationship between these foot morphologies and sprint performance in sprinters.13. The personal best 100-m sprint time of faster sprinters ranged from 10.21 to 11.18 s . The personal best 100-m sprint time of slower sprinters ranged from 11.19 to 11.90 s . The participants were informed of the experimental procedures and provided written consent to participate in the study. None of the participants had contraindications to magnetic resonance imaging. All participants were informed of the experimental procedures and provided written consent to participate in the study. The study was approved by the Ethics Committee of Ritsumeikan University (BKC-IRB-2016-047) and conducted according to the Declaration of Helsinki.Fifty-six well-trained male sprinters  participated in this study. All sprinters were involved in regular sprint training and competition. Their best personal times in a 100-m sprint ranged from 10.21 to 11.90 s . In order to understand the foot characteristics in sprinters having superior sprint performance, sprinters were divided into two groups, one consisting of faster sprinters (n = 28) and one consisting of slower groups (n = 28), based on the personal best 100-m sprint time, as in our previous studyLength of the right foot of participants was measured in millimeters as the maximum distance from the heel to the end of the big and second toes. The highest value in both toes was considered the maximum foot length, and this length was used to normalize the lengths of the forefoot and rearfoot bones.The scans of the right foot of participants were performed using a 1.5-T magnetic resonance system . The participants were positioned supine on the scanner bed, with both knees fully extended. The subject’s right ankle was carefully set at a neutral position . The foot scans were acquired using a 4-channel foot ankle coil. Three-dimensional isotopic T1-weighted images were obtained with a repetition time of 11.3 s, echo time of 5.1 ms, slice thickness of 1.2 mm, field of view of 28 cm, and matrix size of 256 × 256 pixels. The analyses of the foot morphological variables were conducted using image analysis software . All foot morphological variables were measured twice by a single examiner, and the two values were averaged.14. The coefficient of variations of the two measurements for the forefoot bone lengths of the big and second toes in all participants were: 1.6 ± 1.2, 1.4 ± 1.2, and 0.7 ± 0.5%, respectively, for the distal phalanx, proximal phalanx, and metatarsal of the big toe; 1.8 ± 1.5, 1.7 ± 1.3, 1.7 ± 1.3, and 0.6 ± 0.5%, respectively, for the distal phalanx, middle phalanx, proximal phalanx, and metatarsal of the second toe. The intraclass correlation coefficient (ICC) of the two measurements for each bone length ranged from 0.937 to 0.992. The reproducibility of these forefoot bone measurements has been reported in our previous studies14. The calculated forefoot bone lengths of the big and second toes were totaled to assess overall forefoot bone length for each toe. The total forefoot bones for each toe were normalized to the maximal foot length and used for analyses of this study.Representative magnetic resonance imaging scans for measuring the foot morphological variables bones are shown in Fig. The lengths and heights of the talus and calcaneus were measured as maximal distances between the cortexes at the distal and proximal ends. The coefficient of variations of the two measurements for the lengths and height of the talus and calcaneus in all participants were: 0.5 ± 0.4 for the talus length, 0.4 ± 0.2% for the calcaneus length, 0.6 ± 0.6 for the talus height, and 0.6 ± 0.4% for the calcaneus height. The intraclass correlation coefficient of the two measurements for the lengths and heights of each bone ranged from 0.989 to 0.996. The lengths and heights of the talus and calcaneus were normalized to the maximal foot length and body height, respectively, and these relative values were also used for analyses in this study.22. The foot arch height index was also used for analyses in this study.MRI-measured foot arch height at non-weight bearing was measured as the minimum vertical distance between the navicular and the line connecting the thenar eminence to the bottom of the heel. The coefficient of variation of the two measurements for the foot arch height in all participants was 0.5 ± 0.4%. The intraclass correlation coefficient of the two measurements for the foot arch height was 0.997. The foot arch height was normalized to the maximal foot length , which represented the foot arch height indext-testing. The Cohen’s d effect size using the pooled SD was calculated to determine the magnitudes of differences in the measured variables between the two groups. This effect size were interpreted as small (0.20–0.49), medium (0.50–0.79) and large (> 0.80)26. Relationships between the foot bone morphological variables and personal best 100-m sprint time in sprinters was examined using the Pearson’s product-moment correlation coefficient. The statistical significance level was defined at P < 0.05. All statistical analyses were conducted using IBM SPSS software .The data are presented as the mean ± SD. Comparisons of measured variables between faster and slower groups of sprinters were performed using an unpaired P = 0.009), with medium effect size (d = 0.73). The maximum foot length was also significantly higher in slower group than in faster group (P = 0.017), with medium effect size (d = 0.66).Physical characteristics and foot morphological variables between faster and slower groups of sprinters are listed in Table P < 0.001), with large effect size (d = 1.20).With regard to absolute lengths and heights of the foot bones, the total lengths of the forefoot bones of the big and second toes did not differ significantly between faster and slower groups of sprinters Table . MoreovePs < 0.05), with medium and large effect size . Moreover, the relative length of the calcaneus but not the talus was significantly higher in faster group than in slower group (P = 0.018), with medium effect size (d = 0.65). Furthermore, the relative calcaneus height was significantly higher in faster group than in slower group (P < 0.001), with large effect size (d = 1.22).Comparisons of the relative lengths and heights of the foot bones between faster and slower groups of sprinters are presented in Fig. r =  − 0.293 and − 0.459, both Ps < 0.05). Similarly, the relative lengths of the talus and calcaneus correlated significantly with personal best 100-m sprint time . There was no significant correlations between the relative talus height and personal best 100-m sprint time . In contrast, the relative calcaneus height correlated significantly with personal best 100-m sprint time .Relationships of the relative lengths and heights of the forefoot and rearfoot bones with sprint performance in sprinters are presented in Fig. r =  − 0.413, P = 0.002). Although there was no significant correlation between the relative calcaneus height and the relative forefoot length of the big toe, the relative calcaneus height correlated significantly with the relative forefoot length of the second toe . Similarly, the relative calcaneus height correlated significantly with the lengths of the talus and calcaneus .Correlation coefficients between the relative rearfoot bone heights and the relative foot bone lengths in sprinters are shown in Table Ps ≤ 0.001), with large effect size . Although there was no significant correlation between the talus height and foot arch height , the calcaneus height correlated significantly with the foot arch height . Such a significant correlation was also observed between the relative calcaneus height and foot arch height index . Additionally, the foot arch height correlated significantly with personal best 100-m sprint time . Similarly, a significant correlation was observed between the foot arch height index and personal best 100-m sprint time .Relationships of the foot arch height index with the calcaneus height and sprint performance in sprinters are presented in Fig. We found that the lengths of the forefoot and rearfoot bones correlated with personal best 100-m sprint time in sprinters. Moreover, the height of the calcaneus, but not that of the talus, correlated with the sprint performance. Furthermore, the calcaneus height correlated with the forefoot and rearfoot bone lengths. Additionally, the calcaneus height correlated with the foot arch height, and the foot arch height correlated with sprint performance. These findings suggest that a taller calcaneus may play an important role in modeling longer forefoot and rearfoot bones and higher foot arch height and archiving better sprint performance in sprinters.14; therefore, with an increase in sample size, the present finding further clarify the result of our previous studies. Using computer simulation, Lee and Piazza12 reported that a longer forefoot contributes to the production of greater plantar flexor torque during the push-off phase while sprinting. Additionally, van Werkhoven and Piazza27 determined that a longer hallux correlated with higher jump height in a single-joint jump task, possibly due to increased push-off strength. Jump performance is a major physical performance factor for predicting sprint performance in sprinters29. The findings of the present and previous studies suggest that the longer forefoot bones are advantageous morphologies for superior performance of sprinters, potentially because of increases in MTP flexor  and plantar flexor torques during sprinting.We previously reported that longer forefoot bones correlated with better sprint performance in sprinters27, they reported that longer heel length, in addition to hallux length, correlated with higher single-joint jump height. Their results suggest that both longer forefoot and rearfoot bones can coexist in the foot and may additively contribute to superior sprint performance. To the best of our knowledge, the present study is the first to determine the relationship between the rearfoot bone lengths and athletic performance, including sprinting.Our first hypothesis was that the longer rearfoot bones would be related to superior sprint performance in sprinters. In this study, we found that the calcaneus, but not the talus, was longer in faster group than in slower group of sprinters. Furthermore, longer calcaneus and talus bones correlated with better sprint performance in sprinters. These findings suggest that the lengths of the rearfoot bones, especially the calcaneus, in addition to the forefoot bones may be related to superior sprint performance in sprinters. In a study by van Werkhoven and PiazzaOur second hypothesis was that the taller calcaneus would be required for modeling the favorable foot bone morphologies  and would be related to superior sprint performance. In this study, we found that the calcaneus height was higher in faster group than in slower group of sprinters. Moreover, a taller calcaneus correlated with better sprint performance in sprinters. Furthermore, the taller calcaneus correlated with the longer forefoot and rearfoot bones. These findings support our hypothesis prior to the present study. To the best of our knowledge, the present study is also the first to determine the relationship between the calcaneus height and athletic performance, including sprinting.21. Morita et al.22 reported a positive correlation between foot arch height and sprint performance in children. Despite these findings, to the best of our knowledge, no study has examined this relationship in adults, including sprinters, prior to the present study. Therefore, this is the first study to determine a positive relationship between foot arch height and sprint performance in adult sprinters. In addition, although the foot arch height is often associated with the foot length in non-sprinters24, the present study determined no correlation between foot arch height and foot length  in sprinters . Furthermore, a trend against negative correlation was observed between maximal foot length and sprint performance . Therefore, a higher foot arch height may be related to better sprint performance independently of the foot length in sprinters. Taken together, we suggest that a taller calcaneus may help achieve superior sprint performance, potentially by modeling the functional foot morphology  in addition to the longer forefoot and rearfoot bones.Our third hypothesis was that a taller calcaneus would be related to a higher foot arch height. We also hypothesized that the higher foot arch height would be related to superior sprint performance. In this study, we found that the foot arch height was higher in faster group than in slower group of sprinters. Furthermore, the higher foot arch height correlated with better sprint performance in sprinters. Previous studies reported that the foot arch height may contribute to increasing the MTP joint and plantar flexor torques during the push-off phase while sprinting31. The results of this study showed that although there were no correlations of absolute and relative heights of the talus with the foot arch height and its index, absolute and relative heights of the calcaneus correlated positively with the foot arch height and its index. Using radiographs Agoada and Kramer30 reported that in the course of weight-bearing, a taller calcaneus may be related to a higher foot arch height by showing a positive correlation of the ratio of the height relative to the length of the calcaneus with the foot arch height. In an additional analysis conducted in the present study, we similarly obtained a positive correlation of the ratio of the height to the length of the calcaneus with the foot arch height when non-weight-bearing . By contrast, in a study by Agoada and Kramer30, all variables, including the ratio of the height to the length, related to the height of the talus did not correlate with the foot arch height. Furthermore, the result of additional analysis in the present study showed a negative correlation of the ratio of the height to the length of the talus with the foot arch height , suggesting that a taller talus may be a negative morphological factor for modeling a higher foot arch height. As an explanation of these findings of the present and previous studies, the high arch of the foot is related to an increase in the calcaneal inclination angle , which may contribute to modeling a taller calcaneus31. Contrary to this, the low arch of the foot is related to an increase in the calcaneal-first metatarsal angle , which may contribute to modeling a taller talus31. Therefore, a taller calcaneus due to increased calcaneal inclination angle may be a positive factor for a higher foot arch height, whereas a taller talus due to increased calcaneal-first metatarsal angle may be a negative factor for a higher foot arch height. In summary, the height of the calcaneus, but not the talus, may play an important role in increasing the foot arch height.The calcaneus and talus are known to be pivotal bones for foot arch formation32, the calcaneus may be more sensitive to acquired factors  than other foot bones such as the forefoot bones33. Based on the findings of the present and previous studies, successful sprinters are characterized by greater calcaneus, whereas successful endurance runners may be characterized by smaller calcaneus35, suggesting that the calcaneus morphology may be favorably modeled throughout long-term specific training. Further longitudinal studies are needed to follow-up the growth and development of the foot bones in junior athletes, including sprinters and endurance runners.This study has several limitations. First, although we recruited only Japanese male sprinters, it remains unclear whether the present findings can be generalized to other racial and ethnic groups or to female sprinters. Further studies are needed to determine the relationship between the foot morphological variables and sprint performance in sprinters of different races and sex. Second, although we measured the foot arch height when non-weight bearing because of a technical limitation of magnetic resonance imaging, the foot arch height at weight-bearing may be a more important parameter for predicting sprint performance than that at non-weight bearing. This is potentially due to a general form during human movements. Further studies are needed to determine the relationship between the foot arch height when weight-bearing and sprint performance. Third, although we determined that the foot bone morphologies were different between faster and slower groups of sprinters based on personal best 100-m sprint time, it is unclear whether these differences are derived from genetic and/or long-term training effects. Nevertheless, although the forefoot bones may be determined mainly by genetic factors31, an increase in the foot arch height may become the important target of regular training for enhancing sprint performance in sprinters. This is because the foot arch height can be increased by short foot exercise training38. Mulligan and Cook37 reported that the short foot exercise training increased the foot arch height when weight bearing in healthy young adults. Therefore, further studies are needed to examine the effect of short foot exercise training on the foot arch height and sprint performance in sprinters.In a perspective, it would be generally difficult to remodel the length and height of the foot bones in adult humans. Nevertheless, an increase in calcaneal inclination angle may contribute to increasing the calcaneus height based on an analysis method of this study; this implies that the calcaneal inclination angle may have potential as an important morphological parameter of sprint performance in sprinters. Based on a positive correlation between calcaneal inclination angle and foot arch heightIn conclusion, we suggest that the taller calcaneus may be a key morphological factor for achieving superior sprint performance, potentially via modeling the longer forefoot and rearfoot bones and higher foot arch height. These findings could enhance our understanding of the importance of the foot morphology on athletic performance in athletes.The datasets used for this study are available from the corresponding author on reasonable request.


Malus doumeri leaf flavonoids (MDLF) were used as the research object to observe their in vitro antioxidant stress ability. Hydrogen peroxide (H2O2) was used to induce oxidative stress in 293 T cells.In this study, MTT, flow cytometry, and qPCR were used to verify the effect of MDLF.2O2. It was also observed that MDLF could significantly increase the levels of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) and reduce the level of malondialdehyde (MDA). The results of quantitative polymerase chain reaction (qPCR) showed that MDLF could significantly up-regulate the mRNA expression levels of CAT, SOD, GSH, GSH-Px, B-cell lymphoma-2 (Bcl-2) and downregulate the expression levels of B-cell lymphoma-2 associated x protein (Bax), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa-B (NF-κB) in oxidative stress-injured cells. The HPLC analysis showed that MDLF contained hyperin, isoquercetin, quercitrin, hesperidin, myricetin, baicalin and quercetin.In vitro cell experiments showed that at a concentration of 0–160 μg/mL, MDLF did not affect the normal proliferation of human embryonic kidney 293 T cells (HEK 293 T cells), and MDLF had no cytotoxic effect in this concentration range. It was found that MDLF could maintain the survival of HEK 293 T cells (82.6%) at a high concentration (160 μg/mL). Morphological observation also found that MDLF can inhibit the cell structure imperfection caused by H2O2 in 293 T cells. Therefore, MDLF is a type of natural substance with good anti-oxidant effect, and it has the potential to interfere with many diseases.From the experimental results, it was observed that MDLF has a strong anti-oxidation ability in vitro, and it can interfere with the oxidative stress damage caused by H It increases the expression of the apoptotic gene, but it decreases the expression of the anti-apoptotic protein, leading to normal cell apoptosis and pathological changes in the body [The production of oxidative stress is due to the excessive production of free radicals and the release of a large number of reactive oxygen species (ROS) and other oxidizing substances after the body is injured by endogenous or exogenous factors. However, the ability of the body to resist oxidation is decreased and the accumulation of ROS cannot be inhibited, which leads to a imbalance between the ability of oxidation and anti-oxidation. The oxidation reaction of the body is far beyond the ability of antioxidant clearance. Excessive ROS caused by oxidative stress can stimulate cells to initiate lipid peroxidation reaction, which can damage cell function and structure , 2. The the body . When inthe body . Oxidatithe body .The body balance is disrupted after oxidative stress, and this causes many diseases. Under the stimulation by external adverse factors, the level of skin internal oxidation rises, resulting in the metabolic imbalance of the oxidation system, which directly or indirectly leads to the occurrence of some skin diseases, such as skin photoaging, skin tumors, pigmented diseases, and wound healing and repair . StudiesMalus doumeri is a Rosaceae family plant, whose fruit is consumed. The leaves are also used to a certain extent. Firstly, they are processed into drinks such as tea; secondly, they are used as folk drugs to prevent diarrhea; thirdly, they have antiseptic and bacteriostatic effects, and they are often mixed with other foods and drugs as natural preservatives [Malus that are beneficial to human health; among them, the vitamin C content reaches 177.75 mg/g [Malus doumeri is about 5%, and the content of flavonoids in general tea is only 1–2% of the total polyphenols, while that in the leaves of Malus doumeri is more than 60%. After rough processing, the yield of total flavonoids in the dry leaves of Malus doumeri can be more than 3%, which shows that the content of flavonoids in the leaves of Malus doumeri is high [At present, many plants have been found to have antioxidant effects, which mainly arise from flavonoids, saponins, polysaccharides, and organic acids , 13. Antrvatives . Studies.75 mg/g ; in addi.75 mg/g . The con is high .Malus doumeri. However, with an increasing number of drinks and health products processed from the leaves of Malus doumeri, its biological activity and mechanism need to be studied further. In this study, the components of flavonoids in the leaves of Malus doumeri leaf (MDLF) were analyzed, and the effect of MDLF on oxidative stress injury induced by hydrogen peroxide (H2O2) in human embryonic kidney 293 T cells was studied for the first time, which is the theoretical basis for further utilization of MDLF accumulation.Because of the limited area of application and cognition, there are few researches on Malus doumeri leaves  were crushed and sieved. A voucher specimen is deposited at the herbarium of the Chongqing University of Education under the number CCICFF-201902. Professor Xin Zhao authenticated the sample. Then 200 g of these leaves were weighed and placed in a beaker precisely and 70% ethanol was added according to the liquid to material ratio of 20:1; then they were placed in a 60 °C water bath for 3 h and the liquid was extracted for standby, passing through FL-3 macroporous resin. The liquid passing through the resin was evaporated to dryness through a rotary evaporator, and the extracted MDLF was obtained [After the dried 4 cells/mL suspension; then 200 μL cell suspension was inoculated into a 96 well cell culture plate and cultured at 37 °C for 24 h until cells were attached to the wall. Then the culture medium was discarded, and 200 μL new des medium containing 0–200 μg/mL MDLF was added for 48 h. Then the medium in the 96 well culture plate was discarded, and the deme medium containing 5 mg/mL MTT  was added for continuous culture for 4 h. The medium was discarded again, dimethyl sulfoxide  of 200 μL was added, and the reaction was carried out for 30 min without light and vibration. Finally, the optical density (OD) value of each hole was measured at 494 nm , and the cell survival rate  after the MDLF treatment was calculated / As) × 100%, where Ar: OD value of MDLF treated cells; As: OD value of normal cells) to observe the toxic effect of MDLF in 293 T cells [After being resuscitated, HEK 293 T cells  were inoculated in the deme medium containing 10% fetal bovine serum and 1% penicillin streptomycin double antibody. Then the cells were cultured in a carbon dioxide incubator  for 48 h and digested with 0.25% trypsin. Further, 293 T cells were counted and made into a 1 × 10 T cells .2O2 was added and cultured for 4 h to induce a cell oxidative damage model. After that, the medium was discarded again, the deme medium with concentration of 0–200 μg/mL MDLF was added to culture for 48 h, and then the OD value of each cell was measured at 494 nm according to the cytotoxicity experiment of MDLF on HEK 293 T cells and the inhibition effect of MDLF on oxidative damage induced by H2O2 in HEK 293 T cells was observed by calculating the survival rate of cells after oxidative damage prevention by each concentration of MDLF [Cells were resuscitated and cultured according to the experimental operation in the cytotoxicity experiment of MDLF on HEK 293 T cells. Cells were made into a cell suspension and cultured in 96 well plates. Cells were attached to the wall and then discarded. Further, 200 μL of deme medium containing 0.3 mmol/L H of MDLF .According to the effect of MDLF on the oxidative damage of HEK 293 T cells induced by hydrogen peroxide, the cells were cultured, induced oxidative damage and treated with MDLF for 48 h. The morphology of cells in each group was observed under the microscope .2O2 in HEK 293 T cells, the cells were cultured and treated with MDLF for 48 h; then the cells were fixed, and apoptosis was detected by flow cytometry after staining  [According to the experimental operation of MDLF on oxidative damage induced by HCA, USA) .2O2 in HEK 293 T cells, the cells were cultured and treated with MDLF for 48 h. Then the MDA, SOD, GSH, GSH-Px, and CAT levels in cells of each group were measured according to the kit instructions  [According to the experimental operation of MDLF on oxidative damage induced by H, China) .2O2 in HEK 293 T cells by MDLF, cells in each group were used to extract total RNA of cells by Trizol reagent (Solarbio Life Sciences). Further, 1 μL of oligo (DT) 18 primer  and 1.0 μL of total RNA (1.0 μg/μL) were added to 10.0 μL of nuclease free water and heated in PCR for 5 min (65 °C). Then, 4.0 μL of 5 × reaction buffer, 1.0 μL of ribolock RNase inhibitor (20 U), 2.0 μL of 10 mM dNTP mix, and 1.0 μL of reversible reverse transcription (200 U/μL) were added to the above reaction system solution for cDNA transcription . Then, 1 μL 10.0 μM upstream primer and 1 μL 10.0 μM downstream primer , gradient elution mobile phase C was acetonitrile; mobile phase B was 0.5% glacial acetic acid aqueous solution; flow rate was 0.5 mL/min, column temperature was 35 °C; detection wavelength was 360 nm; and injection volume was 10 μL. Then the content of MDLF was calculated by the external standard peak area method according to the HPLC method , and the formula was as follows: Where: Mc is the content of flavonoids in the sample, mg/g; C1 is the concentration of the standard, mg/ml; Ar is the peak area of the standard; A1 is the peak area of the sample; C is the concentration of the original solution of the sample, 0.0025 g/mL.P < 0.05. The analysis method was Duncan’s multiple range test single factor analysis of variance (ANOVA).All experiments were carried out three times in parallel. The mean ± standard deviation (SD) was used to express the results. SAS9.1 statistical software was used to analyze the significant differences between groups of data at the level of When HEK 293 T cells were treated with MDLF at concentrations of 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 μg/mL, compared with HEK 293 T cells that were not treated with MDLF, MDLF at concentrations of 20–160 μg/mL did not significantly affect the proliferation of HEK 293 T cells, and the survival rate of HEK 293 T cells in this concentration range was more than 95% , while the SOD, GSH, GSH-Px, and CAT levels were significantly lower  pathway, endoplasmic reticulum stress, NF-κB, and Bcl-2 family . In the Oxidative stress and inflammation promote each other. Research shows that TNF-α can enhance the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inhibit the expression of SOD, which leads to an oxidative stress reaction. However, inhibiting the expression of TNF-α can effectively inhibit the oxidative stress reaction . NF-κB iHypericin, isoquercetin, quercitrin, hesperidin, myricetin, baicalin, and quercetin are commonly found flavonoids in plants, and all of them have strong antioxidant activity –42. HypeIn this study, the results showed that MDLF could protect HEK 293 T cells from oxidative stress by regulating oxidation, apoptosis, and inflammation. MDLF contains active flavonoid substances, which make it play these roles. This study preliminarily verified the effect of MDLF, but its antioxidative effect and mechanism in vivo still need to be studied further. In the future, an in-depth study is needed to assess the role of MDLF in the intervention for various diseases through its antioxidant effect.


Implant-associated infections remain a tremendous challenge, and all efforts should be taken to improve the prevention and treatment of this entity. A critical factor in the establishment of implant-associated infections is the colonization of the implant surface and subsequent biofilm formation by bacteria or fungi in the context of the “race for the surface” Antimicrobial coatings technologies offer the option of local protection of the implant surface against the above-mentioned implant colonization and biofilm formation of microorganisms. Despite tremendous research in this field, there are currently only a few clinically available implants with antimicrobial coatings on the market In the narrow sense, implant coatings include technologies with direct modification of the actual implant surface , which can directly be used by surgeons in the operating room. These technologies mainly consist of systems that release agents with antimicrobial activity such as silver, gentamicin and povidone-iodine. These compounds are attached directly to the implant surface during the manufacturing process by orthopaedic device companies clinicaltrials.gov).Implant coatings are different from surface treatment of implants by surgeons. In the latter, the surgeons apply a compound directly to the metallic surface of the implant immediately prior to implantation. Hydrogels or other antimicrobial drug delivery systems, such as antibiotic-loaded calcium sulfates and calcium phosphates, belong in this category Most experimental coatings involve antibiotics or antimicrobial compounds that have been tested in the laboratory setting. Novel compounds include CZ-01127 that allow elution of high antibiotic concentrations Regarding direct coating technologies, passive modification of the implant surface design with modification of its morphology or hydrophilic/hydrophobic properties is another theoretical approach to prevent bacterial adhesion and improve the implant's robustness against infection, but none of these technologies have reached market approval yet in vitro data for orthopedic devices, there is still a lack of human clinical data Current regulatory requirements are challenging for the market approval of antimicrobial coated implants in the orthopaedic field Clinical safety of all currently available technologies has been demonstrated. There is growing evidence to reduce infection rates by direct coatings, mainly for certain antibiotics, silver and povidone iodine. However, these data have been mainly derived from case series or cohort studies on megaendoprostheses in orthopaedic oncology and complex revision cases with an elevated (re)infection risk, but not from prospective randomized controlled trials. Alternative “indirect” coatings,  have not been officially approved as implant coatings. Future technologies, such as covalently bonded antibiotics or silicone-based coatings, are on the horizon but will have to undergo complex regulatory approval processes before their official clinical use.In conclusion, the given clinical safety of the currently available coatings justifies the use of antimicrobial coated implants in “high risk” cases, but individual risk benefit analysis should be performed for each single patient. The orthopaedic community, industry and regulatory agencies are urged to pave the way for further and sound clinical evaluation regarding clinical efficacy of antimicrobial coated implants in randomized controlled clinical trials.