Collagen Fibril Density Research Articles

A unique and biocompatible corneal collagen crosslinking in vivo

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases through increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated parameters important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anaesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm2 for 5 min. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration on days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity on day 6. The ruthenium group also exhibited significantly reduced corneal (p < 0.01) and limbal neovascularization (p < 0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images confirmed high density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study is unique that demonstrates in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This approach can prevent toxicity caused by UV-A light and can be an immediate alternative compared to the existing crosslinking procedures that have side effects and clinical risks for the patients.

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Effect of Quadriceps Tendon Autograft Preparation and Fixation on Graft Laxity During Suspensory Anterior Cruciate Ligament Reconstruction: A Biomechanical Analysis.

Favorable collagen fibril density and thickness combined with advances in graft preparation and fixation have significantly increased interest in the quadriceps tendon (QT) autograft for anterior cruciate ligament (ACL) reconstruction. While various suspensory techniques have been described, the biomechanical profile of these constructs is largely undefined. To compare the biomechanics of suspensory techniques for soft tissue QT autograft fixation in an in vitro model of ACL reconstruction. Controlled laboratory study. Full-thickness QT grafts were harvested using a 9-mm graft blade. Adjustable-loop devices (ALDs) were secured to the graft (n = 6 per group) with a combination implant containing the ALD and suture tape-reinforced whipstitching (tape-reinforced [TR] group), tethered superficially to the graft with a whipstitch (onlay [OL] group), luggage-tagged through and around the graft (luggage tag [LT] group), or staggered behind superficial suturing (staggered [SG] group). Grafts were tested on an electromechanical testing machine following a validated in vitro reconstruction model of intraoperative workflow and postoperative ACL kinematics, cyclic loading, and load to failure. The TR group had significantly less postcyclic tension loss (mean, 24%) compared with the OL (56%; P = .002), LT (69%; P < .001), and SG (90%; P < .001) constructs. Cyclic elongation was below the 3.0-mm threshold defined as clinical failure for TR (1.6 mm), but not for OL (3.3 mm), LT (7.9 mm), and SG (11.3 mm). All constructs were within native ACL stiffness limits (220 ± 72 N/mm) without significant differences. Ultimate loads significantly exceeded a normal ACL loading limit of 454 N for TR (739 N; P = .023), OL (547 N; P = .020), and LT (769 N; P = .001), but not for SG (346 N; P = .236). The TR ALD construct demonstrated the most favorable time-zero biomechanical properties of modern soft tissue QT suspensory constructs, with 32% less tension loss and 52% less cyclic elongation versus the closest construct. Failure loading of all constructs was acceptable with respect to the native ACL except for the SG group, which had suboptimal ultimate load. TR ALD implants may protect soft tissue QT autografts before graft-bone healing in ACL reconstruction by minimizing time-zero laxity and fixation failure.

A potential therapeutic strategy of an innovative probiotic formulation toward topical treatment of diabetic ulcer: an in vivo study

BackgroundThe probiotic potential of Lacticacid bacteria has been studied in various medical complications, from gastrointestinal diseases to antibiotic resistance infections recently. Moreover, diabetic ulcer (DU) is known as one of the most significant global healthcare concerns, which comprehensively impacts the quality of life for these patients. Given that the conventional treatments of DUs have failed to prevent later complications completely, developing alternative therapies seems to be crucial.MethodsWe designed the stable oleogel-based formulation of viable probiotic cells, including Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus casei (L. casei), Lactobacillus fermentum (L. fermentum), and Lactobacillus acidophilus (L. acidophilus) individually to investigate their effect on wound healing process as an in vivo study. The wound repair process was closely monitored regarding morphology, biochemical, and histopathological changes over two weeks and compared it with the effects of topical tetracycline as an antibiotic approach. Furthermore, the antibiofilm activity of probiotic bacteria was assessed against some common pathogens.ResultsThe findings indicated that all tested lactobacillus groups (excluded L. casei) included in the oleogel-based formulation revealed a high potential for repairing damaged skin due to the considerably more levels of hydroxyproline content of tissue samples along with the higher numerical density of mature fibroblasts cell and volume density of hair follicles, collagen fibrils, and neovascularization in comparison with antibiotic and control groups. L. acidophilus and L. rhamnosus showed the best potential of wound healing among all lactobacillus species, groups treated by tetracycline and control groups. Besides, L. rhamnosus showed a significant biofilm inhibition activity against tested pathogens.ConclusionsThis experiment demonstrated that the designed formulations containing probiotics, particularly L. acidophilus and L. rhamnosus, play a central role in manipulating diabetic wound healing. It could be suggested as an encouraging nominee for diabetic wound-healing alternative approaches, though further studies in detailed clinical trials are needed.

Central versus peripheral thickness in the human cornea explained

PurposeThe human cornea is thicker in the periphery than the center and it has been suggested that this must be due to greater numbers of lamellae in the peripheral corneal stroma. The purpose of this study was to use high-resolution ultrastructural imaging to determine if the greater thickness of the peripheral cornea is due to the presence of more lamellae or if there is some other anatomical explanation. MethodsIn this study, full thickness corneas from three human donors were processed for light microscopy (LM) and transmission electron microscopy (TEM). Images were taken in three distinct stromal regions (anterior, middle, and posterior) from the central and peripheral cornea. Stromal thickness was evaluated by LM while TEM was used to evaluate numbers and thicknesses of lamellae, mean collagen fibril diameter, and mean collagen fibril density. ResultsMean stromal thickness was significantly thinner in the central (415 ± 34 µm) compared to the peripheral (536 ± 29 µm) cornea (P = 0.009). Numbers of lamellae were not significantly different between central (246 ± 14) and peripheral (251 ± 14) cornea. Average lamellar thickness was not different across all regions of the cornea, except for the peripheral posterior where the lamellae were approximately 50 % thicker (P < 0.05). Collagen fibril diameters were larger in the peripheral cornea by approximately 30 % when compared to the central cornea, in all regions (P < 0.01). ConclusionsThis study shows that it is an increase peripheral posterior lamellar thickness, rather than an increase in the number of lamellae, that accounts for the increase in corneal stromal thickness in the periphery of the human cornea. While collagen fibril diameters are greater throughout the peripheral stroma, the lamellae in the mid and anterior peripheral stroma are not thicker than centrally.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia

PurposeWe hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). MethodsCorneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. ResultsSlit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased ∼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. ConclusionTgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.

Evaluation of desmoplasia in pancreatic and colon tumors by morphometric analysis

Abstract: Objectives: This study aims to quantify collagen deposition in the desmoplasia of pancreatic and colonial tumors. Methods: Samples of colon and pancreas tumors were selected from the Hospital das Clínicas de Pernambuco and submitted to trichomy with Gomori's Trichrome. Photomicrographic analysis was performed using the integrated Panthera imaging analysis system L. Results: It was identified that 31 colon tumors and 22 pancreatic tumors presented desmoplasia with the visualization of fibrillar collagen distributed irregularly around the tumor. There was a statistically significant difference (p = 0.0031) between collagen fiber deposition in desmoplastic and non-desmoplastic pancreatic tumors, as well as for colonic tumors (p &lt; 0.001). Conclusion: It was observed that more than half of the tumors analyzed presented desmoplasia with a difference of density of collagen fibrils statistically significant.

Polyphenol extracts from Ascophyllum nodosum protected sea cucumber (Apostichopus japonicas) body wall against thermal degradation during tenderization

To retard the protein degradation during sea cucumber processing, polyphenol extracts from Ascophyllum nodosum (PhE) was used as a potential antioxidant to maintain the structural integrity of sea cucumber body wall. Accordingly, the protection effects of PhE (0, 0.5, 1.0 and 1.5 mg PhE/g SFBW) against thermal degradation of the solid fragments of body wall (SFBW) have been investigated in order to evaluate their impact on the oxidation level and structural changes. Electronic Spin Resonance results showed that PhE could significantly inhibit the occurrence of oxidation by scavenging the free radicals. The effect of PhE on chemical analysis of soluble matters in SFBW was characterized by SDS-PAGE and HPLC. Compared with thermally treated SFBW, samples with PhE presented a decrease in protein dissolution. Thermal treatment resulted in the disintegration of collagen fibrils and fibril bundles in SFBW samples, while the density of collagen fibrils was increased, and the porosity decreased in samples with PhE. The results of FTIR and intrinsic tryptophan fluorescence confirmed that the structures of SFBW were modified by PhE. Besides, the denaturing temperature and decomposition temperature were both improved with the addition of PhE. These results suggested that PhE appeared to have a positive effect on lowering oxidation and improving thermostability and structural stability of SFBW, which could provide a theoretical basis for protecting sea cucumber body wall against degradation during thermal tenderization.

Correlation of collagen fibril properties and inner limiting membrane thickness with vitreoretinal adhesion in human eyes

Vitreoretinal adhesive strength is thought to play a mechanical role in various retinal diseases; however, collagen fibril properties and inner limiting membrane (ILM) thickness have not been quantitatively correlated to adhesive strength. In this work, we quantified the relationship between collagen fibril density, angle, length, and ILM thickness with vitreoretinal adhesive strength to advance our understanding of structure-function relationships in vitreoretinal adhesion. Following mechanical peel tests, human retinal sections from the equator and posterior pole of donors 42-89 years of age were extracted and processed for transmission electron microscopy. Collagen fibrils at the vitreoretinal interface were segmented and fibril density, angle, length, and ILM thickness quantified. Morphological measurements were correlated with vitreoretinal adhesion measured in the same location. We found that collagen fibril density was 1.6 times greater in the equator compared to the posterior pole across all ages (p=0.0305). Steady-state peel force showed a slight positive correlation with increasing density in both the equator and posterior pole, but was only statistically significant in the equator (p<0.05). Collagen fibril angle and length did not significantly vary with age or region. ILM thickness was 3.8 times thicker in the posterior pole compared to the equator (p<0.0001). ILM thickness was 1.8 times greater in eyes ≤60 years of age compared to eyes >60 of age (p=0.0136). Maximum peel force was significantly correlated with increasing ILM thickness in the equator (p=0.015). A similar trend was seen in the posterior pole, but this was not significant. These data suggest that collagen contributes to adhesion at the vitreoretinal interface, but the structure of the ILM is more influential on the initiation of separation between the vitreous and retina.

1464-P: Computational Modeling for Design of Next Generation Microphysiological Systems for Evaluation of ß-Cell Health and Function

Dynamic insulin secretion is a key focus of β cell research, with microfluidic devices emerging as a potential means to effectively evaluate this performance parameter in vitro. However, most devices maintain isolated islets in suspension despite evidence that collagen-islet interactions are critical to β cell viability and function. When designing a microphysiological system with encapsulated islets, important considerations include 1) sufficient oxygenation, 2) rapid exchange of glucose and insulin between cells and perfusate, and 3) detectable levels of secreted insulin. Computational models provide a means to simulate different device design parameters prior to fabrication, reducing the time and cost of testing multiple physical devices and microenvironmental conditions. For this study, COMSOL was used to model a microfluidic device containing islets macroencapsulated in a fibrillar collagen scaffold. A range of flow rates, collagen fibril densities, and islet-collagen construct dimensions were tested, with outcome measures including spatiotemporal changes in oxygen, glucose, and insulin. Simulations for all designs tested showed maintenance of islet viability as indicated by less than 1% of the construct area falling below the hypoxia-induced dysfunction threshold. Additionally, decreasing the construct thickness and increasing the medium glucose concentration yielded more rapid delivery of the target glucose stimulus to the islets, since glucose transport is primarily driven by diffusion within the construct. By contrast, transport of secreted insulin was found to be flow-limited and the presence of an encapsulation material altered the shape of the insulin secretion curve. Collectively, these results support the integrated use of computational models, together with experimental validation, as an efficient strategy for the creation of next generation microphysiological systems for evaluation of β cell health and function. Disclosure E.Vanderlaan: None. A.Buganza tepole: None. S.L.Harbin: Other Relationship; GeniPhys, Inc.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils.

BACKGROUNDLiver transplantation is a therapy for irreversible liver failure; however, at present, donor organs are in short supply. Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes.AIMTo investigate the possibility that hepatic progenitor cells (HPCs) prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine, we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells.METHODS In vitro liver organoid tissue has been generated by accumulating collagen fibrils, fibroblasts, and HPCs on a mesh of polylactic acid fabric using a bioreactor; this was subsequently implanted into syngeneic wild-type mice.RESULTSThe in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy.CONCLUSIONLiver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system. This tissue was comparable to liver lobules, and with fibroblasts embedded in the network collagen fibrils of this artificial tissue, it is useful for reconstructing the hepatic interstitial structure.

Recurrence in Reis Bücklers corneal dystrophy

We report a case series highlighting clinical correlation of recurrence of Reis–Bucklers corneal dystrophy (RBCD) with histopathology (HPE), genetic analysis, and electron microscopy (EM). This was an interventional case series of three cases in three generations of one family. The index case was a 23-year-old male with bilateral recurrent RBCD following OD PTK and OS ALTK done 6 years earlier. His father had undergone OU PK. His 35-year-old sister and 9-year-old niece were also affected who underwent OU ALTK. Clinical examination, histopathological examination (HPE), and electron microscopy (EM) evaluation were done. The recurrence of RBCD was noted in one of the three members of the same family. Corneal examination showed a dense reticulate pattern of epithelial-stromal involvement. On HPE, disrupted Bowman's membrane and underlying abnormal subepithelial collagen staining positive with PAS and Masson's trichrome were noted. On EM, degenerated stroma with dense collagen fibrils in aggregates, dispersed among regular collagen fibrils, were noted. All four members showed TGFß1 mutation (chromosome 5q31) with Arg124Leu mutation. A trend toward more advanced presentation with increasing age of cases was noted. The clinical and pathological correlation was performed. Clinical correlation with HPE, EM with genetic analysis of RBCD is interesting with presentation severity varying in different generations.

Increased Fibrosis in a Mouse Model of Anti-Laminin 332 Mucous Membrane Pemphigoid Remains Unaltered by Inhibition of Aldehyde Dehydrogenase.

Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the basal membrane zone of skin and surface-close epithelia and predominant mucosal lesions. The oral cavity and conjunctivae are most frequently affected, albeit clinical manifestations can also occur on the skin. MMP-associated lesions outside the oral cavity typically lead to scarring. Mechanisms underlying scarring are largely unknown in MMP and effective treatment options are limited. Herein, we assessed the collagen architecture in tissue samples of an antibody-transfer mouse model of anti-laminin-332 MMP. In MMP mice, increased collagen fibril density was observed in skin and conjunctival lesions compared to mice injected with normal rabbit IgG. The extracellular matrix of MMP skin samples also showed altered post-translational collagen cross-linking with increased levels of both lysine- and hydroxylysine-derived collagen crosslinks supporting the fibrotic phenotype in experimental MMP compared to control animals. In addition, we evaluated a potential anti-fibrotic therapy in experimental anti-laminin-332 MMP using disulfiram, an inhibitor of the aldehyde dehydrogenase (ALDH), which has been implicated in immune-mediated mucosal scarring. In addition, disulfiram also acts as a copper chelator that was shown to block lysyl oxidase activity, an enzyme involved in formation of collagen crosslinks. Topical use of disulfiram (300 μM in 2% [w/v] methocel) did not improve ocular lesions in experimental MMP over the 12-day treatment period in disulfiram-treated mice compared to vehicle-treated mice (n=8/group). Furthermore, C57BL6/J mice (n=8/group) were treated prophylactically with 200 mg/kg p.o. disulfiram or the solvent once daily over a period of 12 days. Systemic treatment did not show any reduction in the severity of oral and ocular lesions in MMP mice, albeit some improvement in skin lesions was observed in disulfiram- vs. vehicle-treated mice (p=0.052). No reduction in fibrosis was seen, as assessed by immunohistochemistry. Whilst blocking of ALDH failed to significantly ameliorate disease activity, our data provide new insight into fibrotic processes highlighting changes in the collagenous matrix and cross-linking patterns in IgG-mediated MMP.

Effect of travoprost, latanoprost and bimatoprost PGF2α treatments on the biomechanical properties of in-vivo rabbit cornea

Prostaglandin F2α analogues (PGF2α), one of the most commonly prescribed classes of hypotensive agents, could decrease collagen fibril density and remodel the extracellular matrix in cornea. We hypothesized that PGF2α′s would induce corneal softening, reduce the accuracy of intraocular pressure (IOP) measurement and lead to uncertainty in the effectiveness of the therapy. We determined the stress-strain behavior of rabbit cornea after PGF2α usage and evaluated the effect of biomechanical changes associated with PGF2α treatment on IOP measurements by Goldmann Applanation Tonometry (GAT). The tangent modulus decreased after PGF2α treatment, while the stromal interfibrillar spacing increased. PGF2α was shown to also affect the lateral eye with lower effect, which did not undergo direct eyedrop treatment. Significant decreases in the numerical predictions of GAT-IOP were predicted in all treated groups relative to control groups. Different PGF2α′s (travoprost, latanoprost and bimatoprost) were associated with different extents of reduction in tissue stiffness and changes in corneal microstructure. PGF2α-induced changes in corneal mechanical properties could reduce the accuracy of IOP measurement and may cause an overestimation of the effect of PGF2α in lowering IOP, possibly leading to uncertainties in glaucoma management.

Ultrastructural Characteristics of Chronically Failed Reconstructed Anterior Cruciate Ligament

Background In the present study, we have examined the ultrastructure of chronically failed reconstructed anterior cruciate ligament (ACL). We aimed to investigate a faulty ligamentization process of a failed reconstructed anterior cruciate ligament. In other words, we want to study ultrastructural alterations a failed ACL graft undergoes. Methods Two patients who underwent revision ACL reconstruction for nontraumatic failure without discontinuity of the graft were included in the study. The first patient was a 40-year-old male who had undergone ACL reconstruction of his right knee 21 years back using the bone-patellar tendon-bone autograft. The second patient was a 23-year-old male who had sustained an ACL tear with a medial collateral ligament injury treated by isolated ACL reconstruction 3 years back using hamstring tendon autograft. We collected punch biopsy specimens from the failed ligaments of both the patients during revision ACL reconstruction. These specimens were examined for the density of collagen fibrils within a fascicle (per 1.5 mm2), cellular metabolism, and fibril diameter (nm) by transmission electron microscopy. Results Fibroblasts of both the ligaments showed features of increased metabolism, more so in the first patient. Compared to the second patient, the fascicles of the first specimen were more loosely arranged. Both ligaments had a unimodal distribution of collagen fibrils. The first patient had a mean fibril diameter of 45.2 (+/−8.5) nm and an average fibril density of 376.8 fibrils per 1.5 mm2. The second patient had an average fibril diameter of 64.1 nm (+/−7) and a mean fibril density of 152.9 fibrils/1.5 mm2. The difference in these parameters of the two patients was statistically significant (P &lt; 0.001). Conclusion Our study suggests that the absence of thicker collagen fibrils with unimodal distribution, the altered density of the collagen fibrils within a fascicle, and ovoid fibroblasts with increased metabolism may symbolize bad ligamentization changes.

An in silico Framework of Cartilage Degeneration That Integrates Fibril Reorientation and Degradation Along With Altered Hydration and Fixed Charge Density Loss.

Injurious mechanical loading of articular cartilage and associated lesions compromise the mechanical and structural integrity of joints and contribute to the onset and progression of cartilage degeneration leading to osteoarthritis (OA). Despite extensive in vitro and in vivo research, it remains unclear how the changes in cartilage composition and structure that occur during cartilage degeneration after injury, interact. Recently, in silico techniques provide a unique integrated platform to investigate the causal mechanisms by which the local mechanical environment of injured cartilage drives cartilage degeneration. Here, we introduce a novel integrated Cartilage Adaptive REorientation Degeneration (CARED) algorithm to predict the interaction between degenerative variations in main cartilage constituents, namely collagen fibril disorganization and degradation, proteoglycan (PG) loss, and change in water content. The algorithm iteratively interacts with a finite element (FE) model of a cartilage explant, with and without variable depth to full-thickness defects. In these FE models, intact and injured explants were subjected to normal (2 MPa unconfined compression in 0.1 s) and injurious mechanical loading (4 MPa unconfined compression in 0.1 s). Depending on the mechanical response of the FE model, the collagen fibril orientation and density, PG and water content were iteratively updated. In the CARED model, fixed charge density (FCD) loss and increased water content were related to decrease in PG content. Our model predictions were consistent with earlier experimental studies. In the intact explant model, minimal degenerative changes were observed under normal loading, while the injurious loading caused a reorientation of collagen fibrils toward the direction perpendicular to the surface, intense collagen degradation at the surface, and intense PG loss in the superficial and middle zones. In the injured explant models, normal loading induced intense collagen degradation, collagen reorientation, and PG depletion both on the surface and around the lesion. Our results confirm that the cartilage lesion depth is a crucial parameter affecting tissue degeneration, even under physiological loading conditions. The results suggest that potential fibril reorientation might prevent or slow down fibril degradation under conditions in which the tissue mechanical homeostasis is perturbed like the presence of defects or injurious loading.

Stress–Strain Index Map: A New Way to Represent Corneal Material Stiffness

PurposeTo introduce a new method to map the mechanical stiffness of healthy and keratoconic corneas.MethodsNumerical modeling based on the finite element method was used to carry out inverse analysis of simulated healthy and keratoconic corneas to determine the regional variation of mechanical stiffness across the corneal surface based on established trends in collagen fibril distribution. The Stress–Strain Index (SSI), developed and validated in an earlier study and presented as a parameter that can estimate the overall stress–strain behavior of corneal tissue, was adopted in this research as a measure of corneal stiffness. The regional variation of SSI across the corneal surface was estimated using inverse analysis while referring to the common features of collagen fibrils’ distribution obtained from earlier x-ray scattering studies. Additionally, for keratoconic corneas, a method relating keratoconic cone features and cornea’s refractive power to the reduction in collagen fibril density inside the cone was implemented in the development of SSI maps. In addition to the simulated cases, the study also included two keratoconus cases, for which SSI maps were developed.ResultsSSI values varied slightly across corneal surface in the simulated healthy eyes. In contrast, both simulated and clinical keratoconic corneas demonstrated substantial reductions in SSI values inside the cone. These SSI reductions depended on the extent of the disease and increased with more considerable simulated losses in fibril density in the cone area. SSI values and their regional variation showed little change with changes in IOP, corneal thickness, and curvature.ConclusionSSI maps provide an estimation of the regional variation of biomechanical stiffness across the corneal surface. The maps could be particularly useful in keratoconic corneas, demonstrating the dependence of corneal biomechanical behavior on the tissue’s microstructure and offering a tool to fundamentally understand the mechanics of keratoconus progression in individual patients.

ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL STUDIES OF REACTIVE AND ADAPTIVE TRANSFORMATIONS OF THE GREATER OMENTUM AT TUMOR LESIONS OF THE OVARIES

The greater omentum is an organ in which metastases of malignant neoplasms of the female reproductive system are often formed. The issues of adaptive and reactive transformations of the greater omentum under conditions of a tumor process in the organs of the female reproductive system have been insufficiently studied. The aim of the study was to elucidate morphological and functional ultrastructural and immune-histochemical changes in the greater omentum in women with ovarian tumor lesions. Using the methods of light and electron microscopy, immunohistochemistry and morphometry, the ovaries of 48 women were examined, who were diagnosed with poorly differentiated serous-papillary adenocarcinoma stage II (20 patients, group 1), stage III (28 patients, group 2). In stage III patients, numerous tumor metastases were detected in the greater omentum. The results of the study showed that in the patients of the 2nd group, in the peri-tumor areas of the greater omentum, the vessels of the microvasculature were dilated, in the connective tissue, moderate edema and leukocyte infiltration were observed. Immuno-histochemical markers showed an increase in the number of immunocompetent cells, mainly T-lymphocytes in the tumor and in the peri-tumor areas. In the tumor and in the surrounding areas of the greater omentum, the process of neoplasm of blood capillaries was noted, differences in the density of collagen fibrils in different parts of the serous membrane were revealed, and the presence of numerous holes was shown, the sizes of which were reduced in areas with metastases. The results obtained show the adaptive and reparative capabilities of the structures of the greater omentum during the tumor process in the organs of the female reproductive system, indicate the important role of the greater omentum in providing protective reactions in the abdominal cavity. In response to the invasion of tumor cells, the processes of angiogenesis are activated in the greater omentum, and the number of immunocompetent cells increases in neo-lymphogenesis. The materials of the study indicate a significant plasticity and reactivity of the greater omentum under conditions of a tumor process in the body. However, the presence of a pronounced proliferative activity of tumor cells in tumor metastases indicates that these adaptive capacities are insufficient to resist tumor invasion into the greater omentum.

Fibril density reduction in keratoconic corneas.

This study aims to estimate the reduction in collagen fibril density within the central 6 mm radius of keratoconic corneas through the processing of microstructure and videokeratography data. Collagen fibril distribution maps and topography maps were obtained for seven keratoconic and six healthy corneas, and topographic features were assessed to detect and calculate the area of the cone in each keratoconic eye. The reduction in collagen fibril density within the cone area was estimated with reference to the same region in the characteristic collagen fibril maps of healthy corneas. Together with minimum thickness and mean central corneal refractive power, the cone area was correlated with the reduction in the cone collagen fibrils. For the corneas considered, the mean area of keratoconic cones was 3.30 ± 1.90 mm2. Compared with healthy corneas, fibril density in the cones of keratoconic corneas was lower by as much as 35%, and the mean reduction was 17 ± 10%. A linear approximation was developed to relate the magnitude of reduction to the refractive power, minimum corneal thickness and cone area (R2 = 0.95, p < 0.001). Outside the cone area, there was no significant difference between fibril arrangement in healthy and keratoconic corneas. The presented method can predict the mean fibril density in the keratoconic eye's cone area. The technique can be applied in microstructure-based finite-element models of the eye to regulate its stiffness level and the stiffness distribution within the areas affected by keratoconus.

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.