Collagen Cross-linking Research Articles

Dissipation and recovery in collagen fibrils under cyclic loading: A molecular dynamics study.

The hysteretic behavior exhibited by collagen fibrils, when subjected to cyclic loading, is known to result in both dissipation as well as accumulation of residual strain. On subsequent relaxation, partial recovery has also been reported. Cross-links have been considered to play a key role in overall mechanical properties. Here, we modify an existing coarse-grained molecular dynamics model for collagen fibril with initially cross-linked collagen molecules, which is known to reproduce the response to uniaxial strain, by incorporating reformation of cross-links to allow for possible recovery of the fibril. Using molecular dynamics simulations, we show that our model successfully replicates the key features observed in experimental data, including the movement of hysteresis loops, the time evolution of residual strains and energy dissipation, as well as the recovery observed during relaxation. We also show that the characteristic cycle number, describing the approach toward steady state, has a value similar to that in experiments. We also emphasize the vital role of the degree of cross-linking on the key features of the macroscopic response to cyclic loading.

Genome-wide association study for bone quality of ducks during the laying period

The cage-rearing model of the modern poultry industry makes the bones of birds, especially egg-laying birds, more vulnerable to fracture, which poses serious damage to the health of birds. Research confirms that genetic material plays an important role in regulating bone growth, development, and remodeling. However, the genetic architecture underlying bone traits is not well understood. The objectives of this study are to identify valuable genes and genetic markers through a genome-wide association study (GWAS) for breeding to improve the duck bone quality. First, we quantified the tibia and femur quality traits of 260 laying ducks. Based on GWAS, a total of 75 SNP loci significantly associated with bone quality traits were identified, and 67 potential candidate genes were annotated. According to gene function analysis, genes P4HA2, WNT3A, and BST1 et al may influence bone quality by regulating bone cell activity, calcium and phosphate metabolism, or bone collagen maturation and cross-linking. Meanwhile, combined with the transcriptome results, we found that HOXB cluster genes are also important in bone growth and development. Therefore, our findings were helpful in further understanding the genetic architecture of the duck bone quality and provided a worthy theoretical basis and technological support to improve duck bone quality by breeding.

Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Transglutaminase-induced Cornea Collagen Crosslinking Effect on Central Corneal Thickness and Keratocyte Cell Density

Abstract
 Introduction & Objectives : This research is aimed to evaluate the effect of transglutaminase-induced corneal collagen crosslinking (CXL) on central corneal thickness (CCT) and keratocyte cell density in vivo.
 Methods : Twenty-eight white New Zealand rabbits were divided into four groups: the transglutaminase- induced CXL group, the epithelial-off CXL group, the transepithelial CXL group, and the control group. The ocular surface was treated with a 1 U/mL microbial transglutaminase solution, and both the epithelial-off and transepithelial groups were exposed to clinical ultraviolet A-riboflavin (UVA/RF). The efficacy of each group was evaluated on the 14th day after the procedures. Central corneal thickness was evaluated with Corneal Visualization Scheimpflug Technology (Corvis ST) and keratocyte cell density wad evaluated with histopathology examination.
 Results : Transglutaminase-induced CXL group exhibited the highest mean biomechanical CCT (370.14 ± 38.85) in comparison to the UVA/RF epithelial-off group (368.00 ± 25.48), the UVA/RF transepithelial group (369.86 ± 23.43), and the control group (365.14 ± 28.74). Still, there was no significant differences in both biomechanical CCT (p=0.990). Transglutaminase-induced CXL group had the highest mean of keratocyte cell density (43.26 ± 10.65) compared to UVA/RF epithelial-off (29.99 ± 4.79), UVA/RF transepithelial group (42.03 ± 6.55), and control group (34.36 ± 6.76). There was a significant difference between the group, with a p-value of 0.008.
 Conclusion : Transglutaminase shown that it produces favorable results for CCT and keratocyte density after CXL, which are two factors contributing to corneal rigidity. The outcomes are equivalent to riboflavin used as a conventional element in CXL.

Corneal Collagen Cross-Linking (CXL) in Pediatric Keratoconus: Changes in Visual Acuity and Biomechanical Properties

Abstract
 Introduction & Objectives : Keratoconus is a progressive disease that changes the mechanical shape of the cornea due to thinning of the stromal layer of the cornea. Therefore, early detection and correct management should be able to slow the progression of the disease. This study aimed to compare changes in visual acuity and biomechanic properties of cornea in patients who underwent Corneal Collagen Cross- Linking (CXL) in Dr. Sardjito General Hospital
 Methods : This is a prospective study that included all pediatric keratoconus patients without any underlying disease that underwent CXL between July 2022 to December 2022. Uncorrected Visual Acuity (UCVA), Best Corrected Visual Acuity (BCVA), Central Corneal Thickness (CCT) and Intraocular Pressure (IOP) were evaluated preoperatively, 2 weeks, and 6 weeks following CXL.
 Results : Mean age of patients was 11 (range 6-18 y.o). UCVA were 1.00±0.34 logMAR preoperatively, 0.95±0.35 and 0.88±0.37 logMAR at 2 and 6 weeks after CXL (P=0.27 and P=0.02, respectively). BCVA were 0.37±0.28 logMAR preoperatively, 0.46±0.28 and 0.35±0.21 logMAR at 2 and 6 weeks after CXL (P=0.05 and P=0.46, respectively). Mean IOP were 15.21±3.99 preoperatively, 14.45±2.73 and 15.21±3.68 at 2 weeks and 6 weeks after CXL (P=0.33 and P=1.00, respectively). Mean CCT were 503.09±49.43 preoperatively, 508.18±47.68 and 506.82±48.41 at 2 weeks and 6 weeks after CXL (P=0.01 and P=0.11, respectively). No complications occurred during the observation period.
 Conclusion : UCVA and BCVA were improved after CXL. Analysis showed no significant change for IOP and CCT after CXL procedure. Overall, CXL was safe and significantly improve UCVA and BCVA in pediatric keratoconus.

Rigid Gas Permeable (RGP) as Keratoconus Management After Corneal Collagen Cross-Linking (CXL) Procedure: a Case Report

Introduction : Rigid Gas Permeable (RGP) contact lenses are the mainstay for keratoconus patients and widely used. This case report presents RGP as a management for keratoconus after Corneal Collagen Cross- Linking (CXL) procedure.
 Case Illustration : A 22-year-old woman diagnosed with keratoconus since eight months ago, came for vision correction. She had CXL performed on both eyes. Ophthalmologic examination revealed uncorrected visual acuity of 0.63 in the right eye (RE) and 0.08 in the left eye (LE). Her best-corrected visual acuity (BCVA) was 1.0 in the RE with -0.75 D and 0.125 in the LE with -2.00 D. Anterior segment examination of the RE was unremarkable, but the LE has Vogt’s striae and Munson's sign (Figure 1). Corneal topography for the LE showed K1 64.7D/5.2 mm, K2 68.7D/4.9 mm, and Kmean 66.7D/5.1 mm. She was fitted with trial lens of BC 5.40 and the fit was loose. She then fitted with a BC of 5.50 mm showed a good fit result. RGP contact lenses were prescribed with S -8.50, BC 5.50, and 8.6 mm diameter for her LE and combined with spectacle for her RE. Her over-refraction BCVA of the LE was 0.9.
 Discussion : The management goals are to improve the patient’s visual acuity and to decrease the progression. The visual acuity of this patient improved after RGP fitting.
 Conclusion : The combination of CXL procedure and the use of RGP effective to increase visual acuity, regularity of corneal surface, and patient satisfaction. Keratoconus should be monitored regularly to avoid possible progressivity.

Research hot spots and trends of keratoconus in China: a bibliometric analysis

Objective: To review the studies related to keratoconus in China, investigate research hotspots and development trends in this field, and provide reference for future research. Methods: This is a bibliometrics study. The relevant literature written in Chinese was retrieved from the WanFang DATA and the China National Knowledge Infrastructure, English articles were collected from the Web of Science Core Collection database. Searched for journal articles related to keratoconus VOSviewer software, CiteSpace, and Bibliometrix in the R language were employed to create the knowledge map. The analysis encompassed the distribution of published journals, research collaboration networks of countries/regions, institutions, and authors. Additionally, core authors, high-frequency keyword co-occurrence, keyword topic maps, and keyword emergence time ranking were examined. Results: The study ultimately included 1 100 Chinese articles and 668 English articles. Chinese literature and English literature began to increase in 1997 and 2009, respectively, indicating that the field is currently in a developmental stage. The publications involved 244 Chinese journals and 150 English journals, predominantly in the field of ophthalmology. The United States collaborated the most with China, contributing to 123 articles, followed by other countries such as the United Kingdom and Switzerland. Chinese literature and English literature involved 552 and 883 institutions, respectively. The institution with the highest number of Chinese literature publications was the Eye Institute of Shandong First Medical University (63 papers), while Wenzhou Medical University had the highest number of English literature publications (91 papers). Chinese literature involved 2 435 authors, and English literature involved 2 073 authors. The largest collaboration cluster in Chinese literature was formed by the teams of Xie Lixin and Shi Weiyun, while the Gao Hua team formed the largest cluster in English literature. However, collaboration between authors was primarily limited to within each team. A total of 622 and 1 611 keywords were extracted from Chinese and English literature, respectively. The node centrality of the four Chinese keywords, "keratoconus", "cornea", "corneal transplantation" and "myopia" as well as the three English keywords, "keratoconus", "collagen cross-linking" and "penetrating keratoplasty" was greater than 0.1. "Collagen", "riboflavin", "corneal transplantation" and "ultraviolet A" were identified as common core hotspots and important research topics in Chinese and English literature on keratoconus. Keyword emergence analysis indicated that the keywords with the highest intensity of emergence in Chinese and English literature were "myopia" (13.54) and "penetrating keratoplasty" (9.99), respectively. The longest emergence time was observed for "contact lenses" (1995-2006) and "penetrating keratoplasty" (2003-2014). Conclusions: At present, research on keratoconus in China is on the rise, with research hotspots including pathogenesis, various new surgical methods, and improvement in quality of life. The future research trend mainly focuses on early diagnosis and screening methods, artificial intelligence, biomechanical examination, subclinical keratoconus, and small incision lenticule extraction.

Effects of various cross-linked collagen scaffolds on wound healing in rats model by deep-learning CNN

Scar tissue is connective tissue formed on the wound during the wound-healing process. The most significant distinction between scar tissue and normal tissue is the appearance of covalent cross-linking and the amount of collagen fibers in the tissue. This study investigates the efficacy of four types of collagen scaffolds in promoting wound healing and regeneration in a Sprague-Dawley murine model—the histomorphology analysis of collagen scaffolds and developing a deep learning model for accurate tissue classification. Four female rats (n = 24) groups received collagen scaffolds prepared through physical and chemical crosslinking. Wound healing progress was evaluated by monitoring granulation tissue formation, collagen matrix organization, and collagen fiber deposition, with histological scoring for quantification—the EDC and HA groups demonstrated enhanced tissue regeneration. The EDC and HA groups observed significant differences in wound regeneration outcomes. Deep-learning CNN models with data augmentation techniques were used for image analysis to enhance objectivity. The CNN architecture featured pre-trained VGG16 layers and global average pooling (GAP) layers. Feature visualization using Grad-CAM heatmaps provided insights into the neural network’s focus on specific wound features. The model’s AUC score of 0.982 attests to its precision. In summary, collagen scaffolds can promote wound healing in mice, and the deep learning image analysis method we proposed may be a new method for wound healing assessment.

Therapeutic strategies based on non-ionizing radiation to prevent venous neointimal hyperplasia: the relevance for stenosed arteriovenous fistula, and the role of vascular compliance.

We have reviewed the development and current status of therapies based on exposure to non-ionizing radiation (with a photon energy less than 10 eV) aimed at suppressing the venous neointimal hyperplasia, and consequentially at avoiding stenosis in arteriovenous grafts. Due to the drawbacks associated with the medical use of ionizing radiation, prominently the radiation-induced cardiovascular disease, the availability of procedures using non-ionizing radiation is becoming a noteworthy objective for the current research. Further, the focus of the review was the use of such procedures for improving the vascular access function and assuring the clinical success of arteriovenous fistulae in hemodialysis patients. Following a brief discussion of the physical principles underlying radiotherapy, the current methods based on non-ionizing radiation, either in use or under development, were described in detail. There are currently five such techniques, including photodynamic therapy (PDT), far-infrared therapy, photochemical tissue passivation (PTP), Alucent vascular scaffolding, and adventitial photocrosslinking. The last three are contingent on the mechanical stiffening achievable by the exogenous photochemical crosslinking of tissular collagen, a process that leads to the decrease of venous compliance. As there are conflicting opinions on the role of compliance mismatch between arterial and venous conduits in a graft, this aspect was also considered in our review.

S100b treatment overcomes RAGE signaling deficits in myoblasts on advanced glycation end-product cross-linked collagen and promotes myogenic differentiation.

Advanced glycation end-products (AGEs) stochastically accrue in skeletal muscle and on collagen over an individual's lifespan, stiffening the muscle and modifying the stem cell (MuSC) microenvironment while promoting proinflammatory, antiregenerative signaling via the receptor for advanced glycation end-products (RAGEs). In the present study, a novel in vitro model was developed of this phenomenon by cross linking a 3-D collagen scaffold with AGEs and investigating how myoblasts responded to such an environment. Briefly, collagen scaffolds were incubated with d-ribose (0, 25, 40, 100, or 250 mM) for 5 days at 37°C. C2C12 immortalized mouse myoblasts were grown on the scaffolds for 6 days in growth conditions for proliferation, and 12 days for differentiation and fusion. Human primary myoblasts were also used to confirm the C2C12 data. AGEs aberrantly extended the DNA production stage of C2C12s (but not in human primary myoblasts) which is known to delay differentiation in myogenesis, and this effect was prevented by RAGE inhibition. Furthermore, the differentiation and fusion of myoblasts were disrupted by AGEs, which were associated with reductions in integrins and suppression of RAGE. The addition of S100b (RAGE agonist) recovered the differentiation and fusion of myoblasts, and the addition of RAGE inhibitors (FPS-ZM1 and Azeliragon) inhibited the differentiation and fusion of myoblasts. Our results provide novel insights into the role of the AGE-RAGE axis in skeletal muscle aging, and future work is warranted on the potential application of S100b as a proregenerative factor in aged skeletal muscle.NEW & NOTEWORTHY Collagen cross-linked by advanced glycation end-products (AGEs) induced myoblast proliferation but prevented differentiation, myotube formation, and RAGE upregulation. RAGE inhibition occluded AGE-induced myoblast proliferation, while the delivery of S100b, a RAGE ligand, recovered fusion deficits.

Trends and Sociodemographic Patterns in Keratoconus Management 2015–2020: An American Academy of Ophthalmology IRIS® Registry Analysis

PurposeInvestigate trends in keratoconus (KCN) treatment patterns and diagnosis age between 2015-2020 and evaluate sociodemographic associations with treatment approach. DesignRetrospective cohort study. Subjects, Participants, and/or ControlsPatients with a new KCN diagnosis from 2015 to 2020 were identified in the Academy IRIS® Registry (Intelligent Research in Sight). Methods, Intervention, or TestingAssociations between sociodemographic factors and treatment were evaluated using multivariable logistic regression. Main Outcome MeasuresOutcomes included percentages and rates of each treatment (either collagen crosslinking [CXL], keratoplasty, or no procedure) from 2015 to 2020, age at diagnosis during this period, and sociodemographic factors associated with treatment type. Results66,199 patients with a new diagnosis of KCN were identified. The percentage of patients undergoing CXL increased from 0.05% in 2015 to 29.5% in 2020 (P=0.008). The average age (SD) of KCN patients decreased from 44.1 (± 16.9) years in 2015 to 39.2 (± 16.9) years in 2020 (P<0.001). In multivariable analyses comparing CXL versus no procedure and keratoplasty versus no procedure, patients undergoing CXL tended to be younger with the odds of having CXL decreasing with increasing age; e.g., comparing CXL and no procedure patients, using ages 0-20 years as reference, the odds ratio (OR) (95% CI) decreased from 0.62 (0.57-0.67, P<0.0001) for patients 21-40 years to 0.03 (0.02-0.04, P<0.0001) for patients older than 60. Males were more likely than females to have CXL (OR=1.31, 95% CI 1.23-1.40, P<0.0001) and keratoplasty (OR=1.30, 95% CI 1.19-1.42, P<0.0001). Black patients were less likely than White patients to have CXL (OR=0.70, 95% CI 0.63-0.77, P<0.0001) and more likely to have keratoplasty (OR=2.24, 95% CI 2.01-2.50, P<0.0001). Similarly, Hispanic patients had higher odds of CXL (OR=1.12, 95% CI 1.00-1.24, P<0.05) and keratoplasty (OR=1.29, 95% CI 1.12-1.50, P<0.001) compared to non-Hispanic patients. CXL and keratoplasty also varied by region and insurance status. ConclusionsA significant increase in use of CXL was noted from 2015 to 2020. Sociodemographic differences in treatment among KCN patients may reflect differences in access, utilization, or care patterns, and future studies should aim to identify strategies to improve access for all patients.

Effect of collagen cross-linking agents on the depth of penetration of bioceramic sealer and release of hydroxyproline: An in vitro study.

During endodontic treatment, sealers seal off dentinal tubules and prevent microbial attack. Bioceramic sealers have excellent bioactivity, but its high alkalinity is found to have detrimental effects on radicular collagen. Collagen cross linkers have the ability to chemically modify collagen and can prevent the detrimental effects of the sealer. This research was aimed to assess the effect of collagen cross-linking agents on the integrity of radicular collagen matrix and depth of penetration of sealer. Mandibular premolars (n = 48) were taken. Teeth were decoronated; canals were prepared till ProTaper size F2 and were irrigated with 5 mL of 2.5% NaOCl, followed by 3 mL of 17% ethylenediaminetetraacetic acid between instrumentation and finally rinsed with saline following which teeth were divided into three groups based on the surface treatments: Group 1: 6.5% proanthocyanin (PA), Group 2: chlorhexidine (CHX), and Group 3: saline. Teeth were obturated using gutta-percha and bioceramic sealer and stored in artificial saliva. Hydroxyproline (HYP) release was assessed after 14 and 21 days using spectrophotometer. Sealer penetration was assessed using the scanning electron microscope. Wilcoxon signed-rank test and Kruskal-Wallis test for release of HYP and paired t-test and ANOVA for sealer penetration were performed. Significantly lower release of HYP was seen in proanthocyanin-treated group. Sealer penetration was better for both the proanthocyanin- and CHX-treated groups when compared to saline. Surface treatment with collagen cross-linkers caused a decrease in the amount of HYP released, indicating lesser degradation of collagen. Sealer penetration was better due to the removal of smear layer following the surface treatments.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic β-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-β1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

A Novel approach for Corneal Remodeling of Laser Asymmetric Keratectomy with Collagen Cross Linking in Patients with Keratoconus Suspect

Background True Keratoconus Suspect (KCS) is an absolute contraindication to LASIK or Photorefractive Keratectomy (PRK) due to postoperative ectasia. Objective To evaluate the effectiveness of laser asymmetric keratectomy with collagen cross-linking (L-LAK-CXL) in myopic patients with suspected keratoconus (KCS). Methods This study included 40–44-year-old four myopic (-2.50 to -5.50 D) patients (4 eyes), of KCS with focal corneal steepening over +47.0 D and peripheral asymmetric corneal thickness. L-LAK-CXL was performed for both original ablation of refractive errors and crescentic customized ablation of the thicker peripheral cornea selectively and myopic changes due to the ablation of the peripheral thicker cornea simultaneously, followed by CXL without the epithelium. We compared preoperative and postoperative ocular findings, including corneal symmetry (total differences of the corneal thickness in four directions (SUM) and decentration of the thinnest point (DISTANCE)) and tear break-out time (TBUT). Results From preoperative to postoperative, spherical equivalent (D, average) decreased from -3.38 to -0.34, uncorrected distance visual acuity (LogMAR) increased from 0.53 to 0.00, and Kmax (average D) had decreased from +48.3 to +43.95, central pachymetry (CP, µm, average) decreased from 574 to 511. Postoperative corneal symmetry increased markedly owing to decreased SUM and DISTANCE scores. TBUT increased over 15 s postoperatively. No post-operative corneal ectasia was observed. Conclusion L-LAK-CXL improved corneal symmetry in myopic patients with KCS by reducing SUM, decreasing DISTANCE and Kmax, increasing TBUT, and demonstrating good postoperative visual outcomes.

Keratometry Changes Between Year One to Seven After Corneal Cross-Linking in Patients With Keratoconus.

We evaluated the timing at and extent to which midterm to long-term keratometric changes can occur in year 1 to 7 after corneal collagen cross-linking (CXL) in patients with keratoconus. We conducted a subgroup analysis of a retrospective cohort study of all consecutive patients who underwent CXL at our cornea center between 2007 and 2011. The inclusion criteria comprised CXL according to the Dresden protocol and a full set of keratometry parameters collected by Scheimpflug tomography preoperatively and at year 1, 3, 5, and 7 after CXL. In addition, best-corrected visual acuity was evaluated. Sixty-three eyes of 47 patients were enrolled. Mean age was 25.46 years ±7.39 years (80.9% male patients). All relevant keratometric parameters showed significant improvement at year 1 after CXL (except for posterior astigmatism). According to mixed-effects model analysis, they all showed further significant change at different points in time between year 1, 3, 5, and 7 (except for K1). In addition, best-corrected visual acuity improved statistically significant between year 1, 3, 5, and 7. Suspected disease progression was noted in 22.2% of patients, mostly between year 1 and 3 after CXL. After initial improvement 1 year after CXL, keratometric and functional parameters were stable until year 5 after CXL in most cases; further improvement can take place even after up to 7 years post-CXL. By contrast, in case of disease progression, changes seem to occur already between year 1 and 3 after CXL.

A new approach to characterize cardiac sodium storage by combining fluorescence photometry and magnetic resonance imaging in small animal research

Cardiac myocyte sodium (Na+) homoeostasis is pivotal in cardiac diseases and heart failure. Intracellular Na+ ([Na+]i) is an important regulator of excitation–contraction coupling and mitochondrial energetics. In addition, extracellular Na+ ([Na+]e) and its water-free storage trigger collagen cross-linking, myocardial stiffening and impaired cardiac function. Therefore, understanding the allocation of tissue Na+ to intra- and extracellular compartments is crucial in comprehending the pathophysiological processes in cardiac diseases. We extrapolated [Na+]e using a three-compartment model, with tissue Na+ concentration (TSC) measured by in vivo 23Na-MRI, extracellular volume (ECV) data calculated from T1 maps, and [Na+]i measured by in vitro fluorescence microscopy using Na+ binding benzofuran isophthalate (SBFI). To investigate dynamic changes in Na+ compartments, we induced pressure overload (TAC) or myocardial infarction (MI) via LAD ligation in mice. Compared to SHAM mice, TSC was similar after TAC but increased after MI. Both TAC and MI showed significantly higher [Na+]i compared to SHAM (around 130% compared to SHAM). Calculated [Na+]e increased after MI, but not after TAC. Increased TSC after TAC was primarily driven by increased [Na+]i, but the increase after MI by elevations in both [Na+]i and [Na+]e.

Selenium Compounds Affect Differently the Cytoplasmic Thiol/Disulfide State in Dermic Fibroblasts and Improve Cell Migration by Interacting with the Extracellular Matrix.

Deficient wound healing is frequently observed in patients diagnosed with diabetes, a clinical complication that compromises mobility and leads to limb amputation, decreasing patient autonomy and family lifestyle. Fibroblasts are crucial for secreting the extracellular matrix (ECM) to pave the wound site for endothelial and keratinocyte regeneration. The biosynthetic pathways involved in collagen production and crosslinking are intimately related to fibroblast redox homeostasis. In this study, two sets of human dermic fibroblasts were cultured in normal (5 mM) and high (25 mM)-glucose conditions in the presence of 1 µM selenium, as sodium selenite (inorganic) and the two selenium amino acids (organic), Se-cysteine and Se-methionine, for ten days. We investigated the ultrastructural changes in the secreted ECM induced by these conditions using scanning electron microscopy (SEM). In addition, we evaluated the redox impact of these three compounds by measuring the basal state and real-time responses of the thiol-based HyPer biosensor expressed in the cytoplasm of these fibroblasts. Our results indicate that selenium compound supplementation pushed the redox equilibrium towards a more oxidative tone in both sets of fibroblasts, and this effect was independent of the type of selenium. The kinetic analysis of biosensor responses allowed us to identify Se-cysteine as the only compound that simultaneously improved the sensitivity to oxidative stimuli and augmented the disulfide bond reduction rate in high-glucose-cultured fibroblasts. The redox response profiles showed no clear association with the ultrastructural changes observed in matrix fibers secreted by selenium-treated fibroblasts. However, we found that selenium supplementation improved the ECM secreted by high-glucose-cultured fibroblasts according to endothelial migration assessed with a wound healing assay. Direct application of sodium selenite and Se-cysteine on purified collagen fibers subjected to glycation also improved cellular migration, suggesting that these selenium compounds avoid the undesired effect of glycation.

Impact of Type 2 Diabetes Mellitus on the Occurrence of Vertical Root Fracture: A Case Control Study

IntroductionThe aim of this case–control study was to examine the relationship between type 2 diabetes mellitus (DM) and the occurrence of VRFs. The crack extension, dentin sclerosis, and chemical characteristics of root dentin in teeth with VRF from patients with/without DM were also compared. MethodsOne hundred and thirty-two patients diagnosed with VRF in crowned root filled posterior teeth were selected. The study was conducted in 2 parts. In Part-1: The cases were matched with control teeth (1:1) for age (±5 years), sex, tooth type, apical extent of root filling, time period after root filling to a diagnosis of VRF, presence or absence of intracanal post and abutment status. The presence or absence of type 2 DM (HbA1c > 6.5) was recorded. In Part-2: The extracted teeth with VRF from the case control study were used to evaluate the extension of VRF, presence of sclerotic dentin and isthmus using a microscopic analysis; while the levels of pentosidine, collagen cross-linking ratio and mineral-collagen ratio were determined by Fourier-transform infrared spectroscopy. The distribution of DM between cases and controls was analyzed using Pearson Chi-Square test and Odds Ratio estimated. Chemical composition data was analyzed using Mann–Whitney test. The extent of sclerotic dentin was analyzed using Pearson Chi-Square test. ResultsWhen compared to patients without DM, patients with DM had 2.67 (95% CI: 1.6–4.45) folds higher odds for occurrence of VRF. Pentosidine (P = .014), collagen cross-linking ratio(P = .047), mineral-collagen ratio (P = .009) and sclerotic dentin extent (P = .0009) were significantly higher in patients with DM and VRF. ConclusionsType 2 DM was more often associated with VRFs in root canal treated teeth with crowns. Root dentin from patients with type 2 DM and VRF had higher levels of pentosidine, collagen cross-linking ratio, mineral to collagen ratio and sclerotic dentin.

Patterns in refractive error and treatment delay in keratoconus-An Australian study.

Keratoconus is the most common primary corneal ectasia and is associated with significant morbidity. In its early stages, keratoconus is often asymptomatic, making the identification of subclinical disease challenging. Refractive error is a parameter that is documented at most routine optometry visits, yet interestingly, changes in refraction of keratoconic patients over time have not yet been studied and compared with the general population. Early diagnosis of keratoconus facilitates timely referral for treatments such as corneal collagen cross-linking, which has been shown to slow disease progression. In this context, documenting delays between initial presentation to the optometrist and referral for collagen-cross-linking as well as comparing the trends in visual acuity and refractive error between keratoconic and non-keratoconic patients over time are particularly relevant.

Efficacy of enzyme‑induced collagen crosslinking on porcine cornea.

The purpose of the present study was to investigate the effect of a new crosslinking (CXL) method, induced by enzymes, on porcine corneas. Corneal strip (10x3 mm) pairs obtained from 60 fresh porcine eyes were harvested and divided into four groups, Groups A-D. Each pair of corneal strips was incised from the central part of the same cornea; one was incubated in transglutaminase (Tgase) solution (microbial Tgase 2 produced by tissue engineering) and the other remained untreated as a control. CXL strips of Groups A-D were incubated with 2, 1, 0.5 and 0.25 U/ml Tgase solution, respectively at 37˚C for 30 min. After that, tensile strain measurements were performed for all strips. One cornea from each group was chosen randomly for hematoxylin and eosin, and Masson staining to identify histological morphology changes. The elastic modulus of treated corneas of Groups A-D were 6.56±2.93, 4.72±1.29, 5.24±2.13 and 3.48±1.60 MPa (mean ± SD), respectively at a strain of 20%, and had a 66, 43, 36 and -6% increase compared with those of their control strips. Compared with the control strips, the elastic modulus of the treated strips significantly increased in Groups A-C. The central corneal thickness of the treated corneas in Groups A-D were 1.54±0.14, 1.41±0.15, 1.47±0.11 and 1.43±0.13 µm, respectively; however, there was not a statistically significant difference compared with the control group. No reduction in corneal transparency was observed, and no obvious abnormalities were found in corneal morphology. CXL mediated by enzymes can lead to a notable enhancement in the biomechanical characteristics of the cornea while maintaining its structural integrity. Enzyme-induced CXL could be a new generation CXL method for strengthening the cornea.