Confocal Microscopy of Filtering Blebs after Trabeculectomy

Introduction: Filtration surgery is the most effective method of lowering intraocular pressure (IOP) in patients with insufficient medical control. It consists in facilitating the drainage of the intraocular fluid (IOF) from the anterior chamber to the subconjunctival space and subsequent lowering of IOP. The formation of filtration blebs (FB) and the processes of scarring occurring in the conjunctiva are of particular importance in glaucoma surgery. In many cases, the appearance of FB does not match the IOP values, and what causes the failure after trabeculectomy often remains unclear. Often, over time, there is a change in the structure of the FB, as fibrous tissue grows, which prevents the IOF drainage. Laser scanning in vivo confocal microscopy is a non-invasive study allowing the production of layered images at the microstructural level with high resolution of both the cornea and other structures of the anterior ocular surface. Aim: To evaluate the morphological structure and function of filtering blebs after trabeculectomy using in vivo confocal microscopy taking into account the type of implant and when the surgery was performed. Materials and methods: The study included 33 patients, 46 eyes with glaucoma. Twenty-six of the eyes had primary open-angle glaucoma (POAG), 18 eyes had pseudoexfoliative glaucoma and 2 eyes had juvenile glaucoma. All patients underwent trabeculectomy with fornix-based flap, and three of the eyes underwent retrabeculectomy. Mitomicyn C (MMC) was administered intraoperatively to all patients. The study of the filtering bleb was performed by in vivo confocal microscopy (CFM) (Heidelberg Retina Tomograph II (HRT II) /Rostock Cornea Module/ (Heidelberg Engineering GmbH, Heidelberg, Germany), the period from trabeculectomy and examination being from 1 year to 22 years. An Express implant was placed in 14 eyes, Ologen implant in 7 eyes, and 25 eyes had no implant placed. In the analysis of the morphological structure of the filtering blebs, three indicators were evaluated: the type of epithelium, the type of stroma, and blood vessels. Results: Statistical significance was established with regard to the function and morphological structure of the filtering bleb (p=0.009). Blebs with fine collagen mesh and dense collagen mesh demonstrate good function. In the case of blebs with insufficient function, those with a dense collagen network and hyper-reflective tissue predominated and there were no blebs with a fine collagen network, and in non-functioning blebs most common were those with a pronounced collagen network and hyper-reflective tissue. With regard to vascularization, we found that the functioning blebs in the shortest postoperative period were dominated by those with one blood vessel (stage 1) and there was no stage 3, with weak tortuosity, while in non-functioning blebs in the late postoperative period, there was moderate to severe vascularization and tortuosity (p=0.037), (p=0.043), (p=0.047), (p=0.021). The type of implant affects the tortuosity of the blood vessels of the filtering bleb (p=0.026). The blebs with Express implants show a slight tortuosity, followed by the blebs with Ologen implants. The highest percentage of highly kinked blood vessels occurred in blebs without an implant. Conclusions: In vivo confocal microscopy is an innovative method which allows visualization of the internal structure of the filtering blebs at a cellular level, giving us a new insight into the ongoing healing processes, premising the function of the filtering blebs after glaucoma surgery.

Efficient Decellularization by Application of Moderate High Hydrostatic Pressure with Supercooling Pretreatment

Decellularized tissues are considered superior scaffolds for cell cultures, preserving the microstructure of native tissues and delivering many kinds of cytokines. High hydrostatic pressure (HHP) treatment could remove cells physically from biological tissues rather than chemical methods. However, there are some risks of inducing destruction or denaturation of extracellular matrices (ECMs) at an ultrahigh level of HHP. Therefore, efficient decellularization using moderate HHP is required to remove almost all cells simultaneously to suppress tissue damage. In this study, we proposed a novel decellularization method using a moderate HHP with supercooling pretreatment. To validate the decellularization method, a supercooling device was developed to incubate human dermal fibroblasts or collagen gels in a supercooled state. The cell suspension and collagen gels were subjected to 100, 150, and 200 MPa of HHP after supercooling pretreatment, respectively. After applying HHP, the viability and morphology of the cells and the collagen network structure of the gels were evaluated. The viability of cells decreased dramatically after HHP application with supercooling pretreatment, whereas the microstructures of collagen gels were preserved and cell adhesivity was retained after HHP application. In conclusion, it was revealed that supercooling pretreatment promoted the denaturation of the cell membrane to improve the efficacy of decellularization using static application of moderate HHP. Furthermore, it was demonstrated that the HHP with supercooling pretreatment did not degenerate and damage the microstructure in collagen gels.

Genipin-Based Crosslinking of Jellyfish Collagen 3D Hydrogels

Collagen-based hydrogels are an attractive option in the field of cartilage regeneration with features of high biocompatibility and low immunogenic response. Crosslinking treatments are often employed to create stable 3D gels that can support and facilitate cell embodiment. In this study, we explored the properties of JellaGel™, a novel jellyfish material extracted from Rhizostoma pulmo. In particular, we analyzed the influence of genipin, a natural crosslinker, on the formation of 3D stable JellaGel™ hydrogels embedding human chondrocytes. Three concentrations of genipin were used for this purpose (1 mM, 2.5 mM, and 5 mM). Morphological, thermal, and mechanical properties were investigated for the crosslinked materials. The metabolic activity of embedded chondrocytes was also evaluated at different time points (3, 7, and 14 days). Non-crosslinked hydrogels resulted in an unstable matrix, while genipin-crosslinked hydrogels resulted in a stable matrix, without significant changes in their properties; their collagen network revealed characteristic dimensions in the order of 20 µm, while their denaturation temperature was 57 °C. After 7 and 14 days of culture, chondrocytes showed a significantly higher metabolic activity within the hydrogels crosslinked with 1 mM genipin, compared to those crosslinked with 5 mM genipin.

Bioprinting of biomimetic self-organised cartilage with a supporting joint fixation device

Despite sustained efforts, engineering truly biomimetic articular cartilage (AC) via traditional top-down approaches remains challenging. Emerging biofabrication strategies, from 3D bioprinting to scaffold-free approaches that leverage principles of cellular self-organisation, are generating significant interest in the field of cartilage tissue engineering as a means of developing biomimetic tissue analogues in vitro. Although such strategies have advanced the quality of engineered cartilage, recapitulation of many key structural features of native AC, in particular a collagen network mimicking the tissue’s ‘Benninghoff arcade’, remains elusive. Additionally, a complete solution to fixating engineered cartilages in situ within damaged synovial joints has yet to be identified. This study sought to address both of these key challenges by engineering biomimetic AC within a device designed to anchor the tissue within a synovial joint defect. We first designed and fabricated a fixation device capable of anchoring engineered cartilage into the subchondral bone. Next, we developed a strategy for inkjet printing porcine mesenchymal stem/stromal cells (MSCs) into this supporting fixation device, which was also designed to provide instructive cues to direct the self-organisation of MSC condensations towards a stratified engineered AC. We found that a higher starting cell-density supported the development of a more zonally defined collagen network within the engineered tissue. Dynamic culture was implemented to further enhance the quality of this engineered tissue, resulting in an approximate 3 fold increase in glycosaminoglycan and collagen accumulation. Ultimately this strategy supported the development of AC that exhibited near-native levels of glycosaminoglycan accumulation (>5% WW), as well as a biomimetic collagen network organisation with a perpendicular to a parallel fibre arrangement (relative to the tissue surface) from the deep to superficial zones via arcading fibres within the middle zone of the engineered tissue. Collectively, this work demonstrates the successful convergence of novel biofabrication methods, bioprinting strategies and culture regimes to engineer a hybrid implant suited to resurfacing AC defects.

Resveratrol against Cardiac Fibrosis: Research Progress in Experimental Animal Models

Cardiac fibrosis is a heterogeneous disease, which is characterized by abundant proliferation of interstitial collagen, disordered arrangement, collagen network reconstruction, increased cardiac stiffness, and decreased systolic and diastolic functions, consequently developing into cardiac insufficiency. With several factors participating in and regulating the occurrence and development of cardiac fibrosis, a complex molecular mechanism underlies the disease. Moreover, cardiac fibrosis is closely related to hypertension, myocardial infarction, viral myocarditis, atherosclerosis, and diabetes, which can lead to serious complications such as heart failure, arrhythmia, and sudden cardiac death, thus seriously threatening human life and health. Resveratrol, with the chemical name 3,5,4′-trihydroxy-trans-stilbene, is a polyphenol abundantly present in grapes and red wine. It is known to prevent the occurrence and development of cardiovascular diseases. In addition, it may resist cardiac fibrosis through a variety of growth factors, cytokines, and several cell signaling pathways, thus exerting a protective effect on the heart.

Tissue scale properties of the extracellular matrix regulates nuclear shape, organisation and fate in the embryonic midline sutures

Balancing self-renewal and differentiation is a key feature of every stem cell niche and one that is tuned by mechanical interactions of cells with their neighbors and surrounding extracellular matrix. The fibrous stem cell niches that develop as sutures between skull bones must balance the complex extracellular environment that emerges to define them with self-renewal and bone production. Here, we address the role for physical stimuli in suture development by probing the relationship between nuclear shape, organization and gene expression in response to a developing collagen network in embryonic midline sutures. This work complements genetic approaches used to study sutures and provides the first quantitative analyses of physical structure in these sutures. By combining multiple imaging modalities with novel shape description, in addition to network analysis methods, we find the early emergence of a complex extracellular collagen network to have an important role in regulating morphogenesis and cell fate. We show that disrupted collagen crosslinking can alter ECM organization of midline sutures as well as stimulate expression of bone differentiation markers. Further, our findings suggest that in vivo, skeletal tissues can uncouple the response of the nuclear lamina from collagen mediated tissue stiffening seen in vitro. Our findings highlight a crucial relationship between the cellular microenvironment, tissue stiffness and geometry with gene expression in normal development and maintenance of progenitor fate in embryonic sutures.

Murine Metatarsus Bone and Joint Collagen-I Fiber Morphologies and Networks Studied With SHG Multiphoton Imaging

Chronic inflammatory disease of bones and joints (e.g., rheumatoid arthritis, gout, etc.), but also acute bone injury and healing, or degenerative resorptive processes inducing osteoporosis, are associated with structural remodeling that ultimately have impact on function. For instance, bone stability is predominantly orchestrated by the structural arrangement of extracellular matrix fibrillar networks, i.e., collagen-I, -IV, elastin, and other proteins. These components may undergo distinct network density and orientation alterations that may be causative for decreased toughness, resilience and load bearing capacity or even increased brittleness. Diagnostic approaches are usually confined to coarse imaging modalities of X-ray or computer tomography that only provide limited optical resolution and lack specificity to visualize the fibrillary collagen network. However, studying collagen structure at the microscopic scale is of considerable interest to understand the mechanisms of tissue pathologies. Multiphoton Second Harmonic Generation (SHG) microscopy, is able to visualize the sterical topology of the collagen-I fibrillar network in 3D, in a minimally invasive and label-free manner. Penetration depths exceed those of conventional visible light imaging and can be further optimized through employing decalcification or optical clearing processing ex vivo. The goal of this proof-of-concept study was to use SHG and two-photon excited fluorescence (2-PEF) imaging to mainly characterize the fibrillary collagen organization within ex vivo decalcified normal mouse metatarsus bone and joint. The results show that the technique resolved the fibrillar collagen network of complete bones and joints with almost no artifacts and enabled to study the complex collagen-I networks with various fiber types (straight, crimped) and network arrangements of mature and woven bone with high degree of detail. Our imaging approach enabled to identify cavities within both cortical and trabecular bone architecture as well as interfaces with sharply changing fiber morphology and network structure both within bone, in tendon and ligament and within joint areas. These possibilities are highly advantageous since the technology can easily be applied to animal models, e.g., of rheumatoid arthritis to study structural effects of chronic joint inflammation, and to many others and to compare to the structure of human bone.

Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids

Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.

Research on functional boards and plastic films based on animal skin and hydroxyapatite

Nano-Hydroxyapatite precursors with calcium and phosphorus salts (Ca/P = 1.67) were introduced into a three-dimensional collagen network matrix (3DCM) based on goat skin pretreated with glutaraldehyde for in situ growth, and a functional HAG-3DCM board was obtained. After plasticity compression, a transparent protein plastic film was formed. Response surface methodology based on plasticity pressure conditions was used, and the strength, hardness and water resistance of the HAG-3DCM plastic film was significantly improved. This study demonstrates a new approach for the preparation of animal skin materials with new application value.