Contrast-enhanced Micro-CT 3D visualization of cell distribution in hydrated human cornea.

Background: The cornea, a vital component of the human eye, plays a crucial role in maintaining visual clarity. Understanding its ultrastructural organization and cell distribution is fundamental for elucidating corneal physiology and pathology. This study comprehensively examines the microarchitecture of the hydrated human cornea using contrast-enhanced micro-computed tomography (micro-CT). Method: Fresh human corneal specimens were carefully prepared and hydrated to mimic their in vivo state. Contrast enhancement with Lugol's iodine-enabled high-resolution Micro-CT imaging. The cells' three-dimensional (3D) distribution within the cornea was reconstructed and analyzed. Results: The micro-CT imaging revealed exquisite details of the corneal ultrastructure, including the spatial arrangement of cells throughout its depth. This novel approach allowed for the visualization of cells' density and distribution in different corneal layers. Notably, our findings highlighted variations in cell distribution between non-hydrated and hydrated corneas. Conclusions: This study demonstrates the potential of contrast-enhanced micro-CT as a valuable tool for non-destructive, 3D visualization and quantitative analysis of cell distribution in hydrated human corneas. These insights contribute to a better understanding of corneal physiology and may have implications for research in corneal diseases and tissue engineering.

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells.

PURPOSE: To investigate the effect of benzalkonium chloride (BAK)-preserved latanoprost and bimatoprost, polyquad (PQ)-preserved travoprost, and preservative-free (PF) latanoprost and tafluprost, all prostaglandin analogues (PGAs), on human conjunctival goblet cell (GC) survival. Furthermore, to investigate the effect of BAK-preserved and PF latanoprost on the cytokine secretion from GC. METHODS: Primary human conjunctival GCs were cultivated from donor tissue. Lactate dehydrogenase (LDH) and tetrazolium dye colorimetric (MTT) assays were used for the assessment of GC survival. A cytometric bead array was employed for measuring secretion of interleukin (IL)-6 and IL-8 from GC. RESULTS: BAK-preserved latanoprost and bimatoprost reduced cell survival by 28% (p = 0.0133) and 20% (p = 0.0208), respectively, in the LDH assay compared to a negative control. BAK-preserved latanoprost reduced cell proliferation by 54% (p = 0.003), BAK-preserved bimatoprost by 45% (p = 0.006), PQ-preserved travoprost by 16% (p = 0.0041), and PF latanoprost by 19% (p = 0.0001), in the MTT assay compared to a negative control. Only PF tafluprost did not affect the GCs in either assay. BAK-preserved latanoprost caused an increase in the secretion of pro-inflammatory IL-6 and IL-8 (p = 0.0001 and p = 0.0019, respectively) compared to a negative control, which PF latanoprost did not. CONCLUSION: BAK-preserved PGA eye drops were more cytotoxic to GCs than PQ-preserved and PF PGA eye drops. BAK-preserved latanoprost induced an inflammatory response in GC. Treatment with PF and PQ-preserved PGA eye drops could mean better tolerability and adherence in glaucoma patients compared to treatment with BAK-preserved PGA eye drops.

The human lens is capable of trilineage differentiation towards osteo-, chondro-, and adipogenesis-a model for studying cataract pathogenesis.

The potential for trilineage differentiation of cells in tissues represents a model for studying disease pathogenesis and regeneration pathways. Human lens trilineage differentiation has not yet been demonstrated, and so has calcification and osteogenic differentiation of human lens epithelial cells in the whole human lens. Such changes can pose a risk for complications during cataract surgery. Human lens capsules (n = 9) from cataract patients undergoing uneventful surgery were trilineage-differentiated toward osteogenesis, chondrogenesis, and adipogenesis. Furthermore, whole human healthy lenses (n = 3) collected from cadaveric eyes were differentiated into bone and characterized by immunohistochemistry. The cells in the human lens capsules were capable of undergoing trilineage differentiation, while the whole human healthy lenses could undergo osteogenesis differentiation, expressing osteocalcin, collagen I, and pigment epithelium-derived factor. We, hereby, show an ex vivo model for cataract formation through different stages of opacification, as well as provide in vivo evidence from patients undergoing calcified lens extraction with bone-like consistency.

Vitreous Substitutes from Bench to the Operating Room in a Translational Approach: Review and Future Endeavors in Vitreoretinal Surgery.

Vitreous substitutes are indispensable tools in vitreoretinal surgery. The two crucial functions of these substitutes are their ability to displace intravitreal fluid from the retinal surface and to allow the retina to adhere to the retinal pigment epithelium. Today, vitreoretinal surgeons can choose among a plethora of vitreous tamponades, and the tamponade of choice might be difficult to determine in the ever-expanding range of possibilities for a favorable outcome. The currently available vitreous substitutes have disadvantages that need to be addressed to improve the surgical outcome achievable today. Herein, the fundamental physical and chemical proprieties of all vitreous substitutes are reported, and their use and clinical applications are described alongside some surgical techniques of intra-operative manipulation. The major upcoming developments in vitreous substitutes are extensively discussed, keeping a translational perspective throughout. Conclusions on future perspectives are derived through an in-depth analysis of what is lacking today in terms of desired outcomes and biomaterials technology.

Intracorneal Implantation of 3D Bioprinted Scaffolds Containing Mesenchymal Stromal Cells Using Femtosecond-Laser-Assisted Intrastromal Keratoplasty.

Injury of the cornea is a complex biological process. Regeneration of the corneal stroma can be facilitated by the presence of mesenchymal stromal cells (MSCs) and application of tissue equivalents. A new tissue-engineering strategy for corneal stroma regeneration is presented using cellularized 3D bioprinted hydrogel constructs implanted into organ cultured porcine corneas using femtosecond laser-assisted intrastromal keratoplasty. The ex vivo cultured, MSC-loaded 3D bioprinted structures remain intact, support cell survival, and contain de novo synthesized extracellular matrix components and migrating cells throughout the observation period. At day 14 postimplantation, the cellularized tissue equivalents contain few or no cells, as demonstrated by optical coherence tomography imaging and immunofluorescent staining. This study successfully combines a laboratory-based method with modern, patient-care practice to produce a cell-laden tissue equivalent for corneal implantation. Optimal bioink composition and cellularization of tissue equivalents are essential in fine-tuning a method to promote the current technique as a future treatment modality.

The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopaedic Medical Devices: A Review.

Due to the well-known phenomenon of antibiotic resistance, there is a constant need for antibiotics with novel mechanisms and different targets respect to those currently in use. In this regard, the antimicrobial peptides (AMPs) seem very promising by virtue of their bactericidal action, based on membrane permeabilization of susceptible microbes. Thanks to this feature, AMPs have a broad activity spectrum, including antibiotic-resistant strains, and microbial biofilms. Additionally, several AMPs display properties that can help tissue regeneration. A possible interesting field of application for AMPs is the development of antimicrobial coatings for implantable medical devices (e.g., orthopaedic prostheses) to prevent device-related infection. In this review, we will take note of the state of the art of AMP-based coatings for orthopaedic prostheses. We will review the most recent studies by focusing on covalently linked AMPs to titanium, their antimicrobial efficacy and plausible mode of action, and cytocompatibility. We will try to extrapolate some general rules for structure-activity (orientation, density) relationships, in order to identify the most suitable physical and chemical features of peptide candidates, and to optimize the coupling strategies to obtain antimicrobial surfaces with improved biological performance.

Generic benzalkonium chloride-preserved travoprost eye drops are not identical to the branded polyquarternium-1-preserved travoprost eye drop: Effect on cultured human conjunctival goblet cells and their physicochemical properties.

PURPOSE: To investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics. METHODS: The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops. RESULTS: In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28-30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated. CONCLUSION: BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical.

Colorimetric and Electrochemical Screening for Early Detection of Diabetes Mellitus and Diabetic Retinopathy-Application of Sensor Arrays and Machine Learning.

In this review, a selection of works on the sensing of biomarkers related to diabetes mellitus (DM) and diabetic retinopathy (DR) are presented, with the scope of helping and encouraging researchers to design sensor-array machine-learning (ML)-supported devices for robust, fast, and cost-effective early detection of these devastating diseases. First, we highlight the social relevance of developing systematic screening programs for such diseases and how sensor-arrays and ML approaches could ease their early diagnosis. Then, we present diverse works related to the colorimetric and electrochemical sensing of biomarkers related to DM and DR with non-invasive sampling (e.g., urine, saliva, breath, tears, and sweat samples), with a special mention to some already-existing sensor arrays and ML approaches. We finally highlight the great potential of the latter approaches for the fast and reliable early diagnosis of DM and DR.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface.

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells.

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

BACKGROUND: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. METHODS: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. RESULTS: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. CONCLUSIONS: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.

Covalent grafting of titanium with a cathelicidin peptide produces an osteoblast compatible surface with antistaphylococcal activity.

Bacterial infection of orthopaedic implants, often caused by Staphylococcus species, may ultimately lead to implant failure. The development of infection-resistant, osteoblast-compatible biomaterials could represent an effective strategy to prevent bacterial colonization of implants, reducing the need for antibiotics. In this study, the widely used biomaterial titanium was functionalized with BMAP27(1-18), an α-helical cathelicidin antimicrobial peptide that retains potent staphylocidal activity when immobilized on agarose beads. A derivative bearing a short spacer with a free thiol at the N-terminus was coupled to silanized titanium disks via thiol-maleimide chemistry. Tethering was successful, as assessed by Contact angle, Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoelectron Spectroscopy (XPS), with an average surface mass density of 456 ng/cm2 and a layer thickness of 3 nm. The functionalized titanium displayed antimicrobial properties against a reference strain of Staphylococcus epidermidis with well-known biofilm forming capability. Reduction of bacterial counts and morphological alterations of adhering bacteria, upon 2 h incubation, indicate a rapid contact-killing effect. The immobilized peptide was not toxic to osteoblasts, which adhered and spread better on functionalized titanium when co-cultured with bacteria, compared to non-coated surfaces. Results suggest that functionalization of titanium with BMAP27(1-18) could be promising for prevention of bacterial colonization in bone graft applications.

Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface.

AIM: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). MATERIALS & METHODS: Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. RESULTS: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. CONCLUSION: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.

Evaluation of free or anchored antimicrobial peptides as candidates for the prevention of orthopaedic device-related infections.

The prevention of implant-associated infection, one the most feared complications in orthopaedic surgery, remains a major clinical challenge and urges development of effective methods to prevent bacterial colonization of implanted devices. Alpha-helical antimicrobial peptides (AMPs) may be promising candidates in this respect due to their potent and broad-spectrum antimicrobial activity, their low tendency to elicit resistance and possible retention of efficacy in the immobilized state. The aim of this study was to evaluate the potential of five different helical AMPs, the cathelicidins BMAP-27 and BMAP-28, their (1-18) fragments and the rationally designed, artificial P19(9/G7) peptide, for the prevention of orthopaedic implant infections. Peptides were effective at micromolar concentrations against 22 Staphylococcus and Streptococcus isolates from orthopaedic infections, while only BMAP-28 and to a lesser extent BMAP-27 were active against Enterococcus faecalis. Peptides in solution showed activities comparable to those of cefazolin and linezolid, on a molar basis, and also a variable capacity to neutralize bacterial lipopolysaccharide, while devoid of adverse effects on MG-63 osteoblast cells at concentrations corresponding to the MIC. The (1-18) BMAP fragments and P19(9/G7) were selected for further examination, based on better selectivity indices, and showed effectiveness in the presence of hyaluronic acid and in synovial fluid, while human serum affected their activity to variable extents, with BMAP-27(1-18) best retaining activity. This peptide was immobilized on streptavidin-resin beads and retained activity against reference Staphylococcus epidermidis and Staphylococcus aureus strains, with negligible toxicity towards osteoblasts, underlining its potential for the development of infection-resistant biomaterials for orthopaedic application. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.