Down-regulation of collagen XI during late post-natal corneal development is followed by up-regulation after injury

Collagen XI plays a role in nucleating collagen fibrils and in controlling fibril diameter. The aim of this research is to elucidate the role that collagen XI plays in corneal fibrillogenesis during development and following injury. The temporal and spatial expression of collagen XI was evaluated in C57BL/6 wild type (WT) mice. For wound healing studies in adult mice, stromal injuries were created using techniques that avoid caustic chemicals. The temporal expression and spatial localization of collagen XI was studied following injury in a Col11a1 inducible knockout mouse model. We found that collagen XI expression occurs during early maturation and is upregulated after stromal injury in areas of regeneration and remodeling. Abnormal fibrillogenesis with new fibrils of heterogenous size and shape occurs after injury in a decreased collagen XI matrix. In conclusion, we found that collagen XI is expressed in the stroma during development and following injury in adults. Collagen XI is a regulator of collagen fibrillogenesis in regenerating corneal tissue.

Evaluation of the Filum Terminale in Hereditary Equine Regional Dermal Asthenia

The objectives of this study were to describe the anatomy, histology, and ultrastructure of the equine filum terminale (FT) and to describe the FT in hereditary equine regional dermal asthenia (HERDA), a model of human Ehlers-Danlos syndromes (EDS). Those humans suffer from tethered cord syndrome (TCS) caused by an abnormally structured FT wherein its attachment at the base of the vertebral column leads to long-term stretch-induced injury to the spinal cord. The pathophysiology of TCS in EDS is poorly understood, and there is a need for an animal model of the condition. Histopathologic and ultrastructural examinations were performed on FT from HERDA ( n = 4) and control horses ( n = 5) and were compared to FT from human TCS patients with and without EDS. Adipose, fibrous tissue, and neuronal elements were assessed. CD3 and CD20 immunohistochemistry was performed to clarify cell types (HERDA n = 2; control n = 5). Collagen fibrils were assessed in cross-section for fibril diameter and shape, and in longitudinal section for fibril disorganization, swelling, and fragmentation. The equine and human FT were similar, with both containing fibrous tissue, ependyma, neuropil, and nerve twigs. Hypervascularity was observed in both HERDA horses and human EDS-TCS patients and was not observed in equine or human controls. Moderate to severe abnormalities in collagen fibril orientation and architecture were observed in all HERDA horses and were similar to those observed in human EDS-TCS patients.

Pilot study on the effects of preservatives on corneal collagen parameters measured by small angle X-ray scattering analysis

Objective Small angle X-ray scattering (SAXS) analysis is a sensitive way of determining the ultrastructure of collagen in tissues. Little is known about how parameters measured by SAXS are affected by preservatives commonly used to prevent autolysis. We determined the effects of formalin, glutaraldehyde, Triton X and saline on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen using SAXS analysis. Results Compared to sections of sheep and cats’ corneas stored frozen as controls, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Sections of corneas preserved in Triton X had significantly increased collagen fibril diameters and decreased fibril diameter distribution. Those preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.

Change in Collagen Fibril Diameter Distribution of Bovine Anterior Cruciate Ligament upon Injury Can Be Mimicked in a Nanostructured Scaffold

More than 200,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US. There is an unmet clinical demand for improving biological attachment between grafts and the host tissue in addition to providing mechanical support. For biological graft integration, it is important to provide a physiologically feasible environment for the host cells to enable them to perform their duties. However, behavior of cells during ACL healing and the mechanism of ACL healing is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. This study aims at (i) investigating the change in fibril diameter of bovine ACL tissue upon injury and (ii) fabricating nanofiber-based scaffolds to represent the morphology and structure of healthy and injured ACL tissues. We hypothesized that distribution and mean diameter of ACL fibrils will be altered upon injury. Findings revealed that the collagen fibril diameter distribution of bovine ACL changed from bimodal to unimodal upon injury with subsequent decrease in mean diameter. Polycaprolactone (PCL) scaffold fiber diameter distribution exhibited similar bimodal and unimodal distribution behavior to qualitatively represent the cases of healthy and injured ACL, respectively. The native ACL tissue demonstrated comparable modulus values only with the aligned bimodal PCL scaffolds. There was significant difference between mechanical properties of aligned bimodal and unaligned unimodal PCL scaffolds. We believe that the results obtained from measurements of diameter of collagen fibrils of native bovine ACL tissue can serve as a benchmark for scaffold design.

Scleraxis-lineage cell depletion improves tendon healing and disrupts adult tendon homeostasis

Despite the requirement for Scleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLin cells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTR mice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLin cells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTR mice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLin cell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

Effects of Preservatives on Corneal Collagen Parameters Measured by Small Angle X-Ray Scattering Analysis

Objective: Small angle X-ray scattering (SAXS) analysis is a sensitive method for determining the ultrastructure of collagen in various tissues. Little is known about how parameters measured by SAXS are affected by preservatives, commonly used to prevent autolysis and strengthen sample tissues. We determined the effects of formalin, glutaraldehyde, Triton X and saline on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen using SAXS analysis. Results: Compared to control sheep and cats’ corneas, frozen at -80 °C, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Corneas preserved in Triton X had significantly increased collagen fibril diameters and decreased fibril diameter distribution. Corneas preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.

Swelling of individual cellulose nanofibrils in water, role of crystallinity: an AFM study

Atomic force microscopy (AFM) can be used to quantitatively study nanomaterials in different media, e.g. vacuum, air, or submerged in a liquid. A technique was developed to study swelling of individual cellulose nanofibrils (CNFs) using AFM. As a case study, CNFs with different degrees of crystallinity (DoC) were examined for swellability going from dry to wet (submerged in de-ionized water). Swelling was found to depend on DoC, but no significant correlation between fibril diameter and swellability was seen. Upon introduction of de-ionized water high DoC samples ($$65\pm 2\%$$ 65 ± 2 % ) were found to have a diameter increase of 34% on average, whereas low DoC ($$44\pm 2\%$$ 44 ± 2 % ) were found to have a diameter increase of 44% on average. A tested control, consisting of platinum nanowires on silisium, did not swell. Graphic abstract

Effects of Preservatives on Corneal Collagen Parameters Measured by Small Angle X-ray Scattering Analysis

Background: Collagen is a prominent structural protein in biological tissue, and little is known about the effect of preservatives, commonly used to preserve and study tissue, on collagen structures. Method: The study determined the effects of commonly used tissue preservatives on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen made using small angle X-ray scattering (SAXS) analysis. Results: Compared to control sheep and cats’ corneas that were preserved frozen at -80 °C, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Corneas preserved in Triton X had significantly increased mean collagen fibril diameters and orientation indexes with decreased fibril diameter distribution. Corneas preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Conclusions: Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.