Nano-Scale Stiffness and Collagen Fibril Deterioration: Probing the Cornea Following Enzymatic Degradation Using Peakforce-QNM AFM

Under physiological conditions, the cornea is exposed to various enzymes, some of them have digestive actions, such as amylase and collagenase that may change the ultrastructure (collagen morphology) and sequentially change the mechanical response of the cornea and distort vision, such as in keratoconus. This study investigates the ultrastructure and nanomechanical properties of porcine cornea following incubation with α-amylase and collagenase. Atomic force microscopy (AFM) was used to capture nanoscale topographical details of stromal collagen fibrils (diameter and D-periodicity) and calculate their elastic modulus. Samples were incubated with varying concentrations of α-amylase and collagenase (crude and purified). Dimethylmethylene blue (DMMB) assay was utilised to detect depleted glycosaminoglycans (GAGs) following incubation with amylase. Collagen fibril diameters were decreased following incubation with amylase, but not D-periodicity. Elastic modulus was gradually decreased with enzyme concentration in amylase-treated samples. Elastic modulus, diameter, and D-periodicity were greatly reduced in collagenase-treated samples. The effect of crude collagenase on corneal samples was more pronounced than purified collagenase. Amylase was found to deplete GAGs from the samples. This enzymatic treatment may help in answering some questions related to keratoconus, and possibly be used to build an empirical animal model of keratoconic corneas with different progression levels.

Technology for obtaining an ultrathin posterior lamellar corneal graft at the Eye Tissue Bank

Objective: to develop technologies for preoperative preparation of the posterior lamellar corneal graft based on our own formulation of the preservation medium for optimal dehydration of the donor cornea and a technique for cutting out an ultrathin flap using an optimized method at the Eye Tissue Bank. Materials methods. In a series of experimental studies, we obtained data on the hydration level of cadaveric donor corneas that were preserved in various solutions at different observation periods. Using 16 corneas, analytical weighing and pachymetry were performed via optical coherence tomography in the experimental (n = 8) and control (n = 8) groups. Morphological and functional characteristics of the corneal endothelium were then assessed. At the next stage of work, ultrathin grafts were formed from 16 corneas after hypothermic preservation in the experimental (n = 8) and control (n = 8) solutions by single-pass microkeratome, followed by microscopy of the samples using a scanning electron microscope. Results. After the first days of preservation in the proposed solution, there was dehydration of 9% cornea in the experimental group in comparison with the samples of the control group. After 4 days of preservation, there was no reliable difference found between the groups (p > 0.05) in the study of the endothelial cell viability of ultra-thin corneal grafts by immunofluorescent microscopy using the «Live and dead» marker. Scanning electron microscopy revealed that corneal stromal collagen fibers, preserved in the proposed medium, retained their integrity. Conclusion. The proposed technology can be recommended for use at eye banks for formation of an ultra-thin corneal graft at the preoperative stage.

Non-disruptive collagen characterization in clinical histopathology using cross-modality image synthesis

The importance of fibrillar collagen topology and organization in disease progression and prognostication in different types of cancer has been characterized extensively in many research studies. These explorations have either used specialized imaging approaches, such as specific stains (e.g., picrosirius red), or advanced and costly imaging modalities (e.g., second harmonic generation imaging (SHG)) that are not currently in the clinical workflow. To facilitate the analysis of stromal biomarkers in clinical workflows, it would be ideal to have technical approaches that can characterize fibrillar collagen on standard H&E stained slides produced during routine diagnostic work. Here, we present a machine learning-based stromal collagen image synthesis algorithm that can be incorporated into existing H&E-based histopathology workflow. Specifically, this solution applies a convolutional neural network (CNN) directly onto clinically standard H&E bright field images to extract information about collagen fiber arrangement and alignment, without requiring additional specialized imaging stains, systems or equipment.

Palatal Wound Healing with Primary Intention in a Rat Model—Histology and Immunohistomorphometry

Background and objectives: Subepithelial connective tissue graft (SCTG) from the palate has been considered as the “gold standard” for the treatment of deep gingival recessions. A single-incision technique was reported to allow primary wound healing. A palatal single incision was performed in a rat model. The present study assessed the histology and histomorphometry of palatal wound healing following surgical closure with primary intention. Materials and Methods: Twenty-six 6-month-old male Wistar rats weighing 427–650 g. An incision was made on the maxillary palate. A full thickness flap was raised palatally, and then repositioned and sutured. Two experimental groups: S—Study group, I—Intact control group. Half of the animals were sacrificed 7 days and the remaining 14 days postoperatively. Outcome parameters included—epithelial gap; inflammatory infiltration; vascular fraction, expression of myofibroblasts and stem cell markers within the oral epithelium and stromal cells and physical properties of stromal collagen fibers. Investigations were performed at two time-points (7 and 14 days) during the wound healing process. Results: The epithelial gap closed completely after 14 days. The inflammatory reaction and vascular fraction were relatively low. Surgical trauma downregulated the expression of cytokeratin (CK) 14 and CK 15, which returned to normal after 14 days. Epithelial differentiation was mediated through upregulation of connective tissue sex- determining-region-Y-box2 (SOX2). Epithelial SOX2, CD34, alpha smooth muscle actin (αSMA) and physical properties of stromal collagen fibers were not influenced by the surgical trauma. Conclusions: Surgical trauma followed by palatal wound healing with primary intention in a rat model heals within 14 days. It induces minimal inflammatory infiltration and vascular proliferation. Epithelization is exerted through promotion of epithelial differentiation from stem cells by connective tissue SOX2.