Mineral and cross-linking in collagen fibrils: The mechanical behavior of bone tissue at the nano-scale.

The mineralized collagen fibril is the main building block of hard tissues and it directly affects the macroscopic mechanics of biological tissues such as bone. The mechanical behavior of the fibril itself is determined by its structure: the content of collagen molecules, minerals, and cross-links, and the mechanical interactions and properties of these components. Advanced glycation end products (AGEs) form cross-links between tropocollagen molecules within the collagen fibril and are one important factor that is believed to have a major influence on the tissue. For instance, it has been shown that brittleness in bone correlates with increased AGEs densities. However, the underlying nano-scale mechanisms within the mineralized collagen fibril remain unknown. Here, we study the effect of mineral and AGEs cross-linking on fibril deformation and fracture behavior by performing destructive tensile tests using coarse-grained molecular dynamics simulations. Our results demonstrate that after exceeding a critical content of mineral, it induces stiffening of the collagen fibril at high strain levels. We show that mineral morphology and location affect collagen fibril mechanics: The mineral content at which this stiffening occurs depends on the mineral's location and morphology. Further, both, increasing AGEs density and mineral content lead to stiffening and increased peak stresses. At low mineral contents, the mechanical response of the fibril is dominated by the AGEs, while at high mineral contents, the mineral itself determines fibril mechanics.

PAI-1 uncouples integrin-ß1 from restrain by membrane-bound ß-catenin to promote collagen fibril remodeling in obesity-related neoplasms.

The paracrine actions of adipokine plasminogen activator inhibitor-1 (PAI-1) are implicated in obesity-associated tumorigenesis. Here, we show that PAI-1 mediates extracellular matrix (ECM) signaling via epigenetic repression of DKK1 in endometrial epithelial cells (EECs). While the loss of DKK1 is known to increase ß-catenin accumulation for WNT signaling activation, this epigenetic repression causes ß-catenin release from transmembrane integrins. Furthermore, PAI-1 elicits the disengagement of TIMP2 and SPARC from integrin-ß1 on the cell surface, lifting an integrin-ß1-ECM signaling constraint. The heightened interaction of integrin-ß1 with type 1 collagen (COL1) remodels extracellular fibrillar structures in the ECM. Consequently, the enhanced nanomechanical stiffness of this microenvironment is conducive to EEC motility and neoplastic transformation. The formation of extensively branched COL1 fibrils is also observed in endometrial tumors of patients with obesity. The findings highlight PAI-1 as a contributor to enhanced integrin-COL1 engagement and extensive ECM remodeling during obesity-associated neoplastic development.

Mineral and cross-linking in collagen fibrils: The mechanical behavior of bone tissue at the nano-scale.

The mineralized collagen fibril is the main building block of hard tissues and it directly affects the macroscopic mechanics of biological tissues such as bone. The mechanical behavior of the fibril itself is determined by its structure: the content of collagen molecules, minerals, and cross-links, and the mechanical interactions and properties of these components. Advanced-Glycation-Endproducts (AGEs) cross-linking between tropocollagen molecules within the collagen fibril is one important factor that is believed to have a major influence on the tissue. For instance, it has been shown that brittleness in bone correlates with increased AGEs densities. However, the underlying nano-scale mechanisms within the mineralized collagen fibril remain unknown. Here, we study the effect of mineral and AGEs cross-linking on fibril deformation and fracture behavior by performing destructive tensile tests using coarse-grained molecular dynamics simulations. Our results demonstrate that after exceeding a critical content of mineral, it induces stiffening of the collagen fibril at high strain levels. We show that mineral morphology and location affect collagen fibril mechanics: The mineral content at which this stiffening occurs depends on the mineral's location and morphology. Further, both, increasing AGEs density and mineral content lead to stiffening and increased peak stresses. At low mineral contents, the mechanical response of the fibril is dominated by the AGEs, while at high mineral contents, the mineral itself determines fibril mechanics.

Central versus peripheral thickness in the human cornea explained.

PURPOSE: The 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. METHODS: In 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. RESULTS: Mean 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). CONCLUSIONS: This 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.

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.

In-silico simulation of nanoindentation on bone using a 2D cohesive finite element model.

This study proposed and validated a 2D finite element (FE) model for conducting in-silico simulations of in-situ nanoindentation tests on mineralized collagen fibrils (MCF) and the extrafibrillar matrix (EFM) within human cortical bone. Initially, a multiscale cohesive FE model was developed by adapting a previous model of bone lamellae, encompassing both MCF and EFM. Subsequently, nanoindentation tests were simulated in-silico using this model, and the resulting predictions were compared to AFM nanoindentation test data to verify the model's accuracy. The FE model accurately predicted nanoindentation results under wet conditions, closely aligning with outcomes obtained from AFM nanoindentation tests. Specifically, it successfully mirrored the traction/separation curve, nanoindentation modulus, plastic energy dissipation, and plastic energy ratio obtained from AFM nanoindentation tests. Additionally, this in-silico model demonstrated its ability to capture alterations in nanoindentation properties caused by the removal of bound water, by considering corresponding changes in mechanical properties of the collagen phase and the interfaces among bone constituents. Notably, significant changes in the elastic modulus and plastic energy dissipation were observed in both MCF and EFM compartments of bone, consistent with observations in AFM nanoindentation tests. These findings indicate that the proposed in-silico model effectively captures the influence of ultrastructural changes on bone's mechanical properties at sub-lamellar levels. Presently, no experimental methods exist to conduct parametric studies elucidating the ultrastructural origins of bone tissue fragility. The introduction of this in-silico model presents an invaluable tool to bridge this knowledge gap in the future.

Evaluating the Efficacy of a Thermoresponsive Hydrogel for Delivering Anti-Collagen Antibodies to Reduce Posttraumatic Scarring in Orthopedic Tissues.

Excessive posttraumatic scarring in orthopedic tissues, such as joint capsules, ligaments, tendons, muscles, and peripheral nerves, presents a significant medical problem, resulting in pain, restricted joint mobility, and impaired musculoskeletal function. Current treatments for excessive scarring are often ineffective and require the surgical removal of fibrotic tissue, which can aggravate the problem. The primary component of orthopedic scars is collagen I-rich fibrils. Our research team has developed a monoclonal anti-collagen antibody (ACA) that alleviates posttraumatic scarring by inhibiting collagen fibril formation. We previously established the safety and efficacy of ACA in a rabbit-based arthrofibrosis model. In this study, we evaluate the utility of a well-characterized thermoresponsive hydrogel (THG) as a delivery vehicle for ACA to injury sites. Crucial components of the hydrogel included N-isopropylacrylamide, poly(ethylene glycol) diacrylate, and hyaluronic acid. Our investigation focused on in vitro ACA release kinetics, stability, and activity. Additionally, we examined the antigen-binding characteristics of ACA post-release from the THG in an in vivo context. Our preliminary findings suggest that the THG construct exhibits promise as a delivery platform for antibody-based therapeutics to reduce excessive scarring in orthopedic tissues.

Hyaluronic acid-mediated collagen intrafibrillar mineralization and enhancement of dentin remineralization.

Non-collagenous proteins (NCPs) in the extracellular matrix (ECM) of bone and dentin are known to play a critical regulatory role in the induction of collagen fibril mineralization and are embedded in hyaluronic acid (HA), which acts as a water-retaining glycosaminoglycan and provides necessary biochemical and biomechanical cues. Our previous study demonstrated that HA could regulate the mineralization degree and mechanical properties of collagen fibrils, yet its kinetics dynamic mechanism on mineralization is under debate. Here, we further investigated the role of HA on collagen fibril mineralization and the possible mechanism. The HA modification can significantly promote intrafibrillar collagen mineralization by reducing the electronegativity of the collagen surface to enhance calcium ions (Ca2+) binding capacity to create a local higher supersaturation. In addition, the HA also provides additional nucleation sites and shortens the induction time of amorphous calcium phosphate (ACP)-mediated hydroxyapatite (HAP) crystallization, which benefits mineralization. The acceleration effect of HA on intrafibrillar collagen mineralization is also confirmed in collagen hydrogel and in vitro dentin remineralization. These findings offer a physicochemical view of the regulation effect of carbohydrate polymers in the body on biomineralization, the fine prospect for an ideal biomaterial to repair collagen-mineralized tissues.

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

Mineralized Collagen Fibrils: An Essential Component in Determining the Mechanical Behavior of Cortical Bone.

Bone comprises mechanically different materials in a specific hierarchical structure. Mineralized collagen fibrils (MCFs), represented by tropocollagen molecules and hydroxyapatite nanocrystals, are the fundamental unit of bone. The mechanical characterization of MCFs provides the unique adaptive mechanical competence to bone to withstand mechanical load. The structural and mechanical role of MCFs is critical in the deformation mechanisms of bone and the marvelous strength and toughness possessed by bone. However, the role of MCFs in the mechanical behavior of bone across multiple length scales is not fully understood. In the present study, we shed light upon the latest progress regarding bone deformation at multiple hierarchical levels and emphasize the role of MCFs during bone deformation. We propose the concept of hierarchical deformation of bone to describe the interconnected deformation process across multiple length scales of bone under mechanical loading. Furthermore, how the deterioration of bone caused by aging and diseases impairs the hierarchical deformation process of the cortical bone is discussed. The present work expects to provide insights on the characterization of MCFs in the mechanical properties of bone and lays the framework for the understanding of the multiscale deformation mechanics of bone.

Collagen Fibril Diameter Distribution of Sheep Anterior Cruciate Ligament.

The anterior cruciate ligament (ACL) tissue is a soft tissue connecting the femur and tibia at the knee joint and demonstrates a limited capacity for self-regeneration due to its low vascularity. The currently available clinical procedures are unable to fully restore damaged ACL tissue, and tissue engineering can offer options with a potential of restoring the torn/ruptured ACL by using biomimetic constructs that are similar to native tissue in terms of structure, composition, and functions. However, a model substrate to understand how the ACL cells regenerate the injured tissue is still not available. In this study, it is hypothesized that the nanofiber-based model substrate with bimodal and unimodal fiber diameter distributions will mimic the diameter distribution of collagen fibrils seen in healthy and injured sheep ACL, respectively. The aims were to (i) create an ACL injury in a sheep ACL by applying extensional force to rupture the healthy ACL tissue, (ii) measure the collagen fibril diameter distributions of healthy and injured ACL, (iii) fabricate polycaprolactone (PCL) nanofiber-based model constructs using electrospinning with diameter distributions similar to healthy and injured ACL tissue, and (iv) measure mechanical properties of ACL tissue and PCL electrospun constructs. The results showed that the fiber diameter distributions of PCL electrospun constructs and those of the healthy and injured ACL tissues were similar. The novelty in this investigation is that the collagen fibril diameter distribution of healthy and injured sheep ACL tissues was reported for the first time. The study is significant because it aims to create a model construct to solve an important orthopedic-related clinical problem affecting millions of people globally. The model construct fabricated in this work is expected to have an important impact on ACL regeneration efforts.

A coarse-grained molecular dynamics investigation of the role of mineral arrangement on the mechanical properties of mineralized collagen fibrils.

Mineralized collagen fibrils (MCFs) comprise collagen molecules and hydroxyapatite (HAp) crystals and are considered universal building blocks of bone tissue, across different bone types and species. In this study, we developed a coarse-grained molecular dynamics (CGMD) framework to investigate the role of mineral arrangement on the load-deformation behaviour of MCFs. Despite the common belief that the collagen molecules are responsible for flexibility and HAp minerals are responsible for stiffness, our results showed that the mineral phase was responsible for limiting collagen sliding in the large deformation regime, which helped the collagen molecules themselves undergo high tensile loading, providing a substantial contribution to the ultimate tensile strength of MCFs. This study also highlights different roles for the mineralized and non-mineralized protofibrils within the MCF, with the mineralized groups being primarily responsible for load carrying due to the presence of the mineral phase, while the non-mineralized groups are responsible for crack deflection. These results provide novel insight into the load-deformation behaviour of MCFs and highlight the intricate role that both collagen and mineral components have in dictating higher scale bone biomechanics.

Understanding the inelastic response of collagen fibrils: A viscoelastic-plastic constitutive model.

Collagen fibrils, which are the lowest level fibrillar unit of organization of collagen, are thus of primary interest towards understanding the mechanical behavior of load-bearing soft tissues. The deformation of collagen fibrils shows unique mechanical features; namely, their high energy dissipation is even superior compared to most engineering materials. Additionally, there are indications that cyclic loading can further improve the toughness of collagen fibrils. Recent experiments from Liu at al. (2018) focused on the response of type I collagen fibrils to uniaxial cyclic loading, revealing some interesting results regarding their rate-dependent and inelastic response. In this work, we aim to develop a model that allows interpreting the complex nonlinear and inelastic response of collagen fibrils under cyclic loading. We propose a constitutive model that accounts for viscoelastic deformations through a decoupled strain-energy density function (into an elastic and a viscous parts), and for plastic deformations through plastic evolution laws. The stress-stretch response results obtained using this constitutive law showed good agreement with experimental data over complex loading paths. Ultimately we use the model to gain more insights on how cyclic loading and rate effects control the interplay between viscoelastic and plastic deformation in collagen fibrils, and to extrapolate the results from experimental data, analyzing how complex cyclic load influences energy dissipation and deformation mechanisms. STATEMENT OF SIGNIFICANCE: In this work, we develop a viscoelastic-plastic constitutive model for collagen fibrils with the aim of analyzing the effects of inelasticity and energy dissipation in this material, and more specifically the competition between viscoelasticity and plasticity in the context of cyclic loading and overload. Experimental and theoretical approaches so far have not fully clarified the interplay between viscous and plastic deformations during cyclic loading of collagen fibrils. Here, we aim to interpret the complex nonlinear response of collagen fibrils and, ultimately, suggest predictive capabilities that can inform tissue-level response and injury. To validate our model, we compare our results against the stress-stretch data obtained from experiments of cyclic loaded single fibrils performed by Liu et al. (2018).

Measurement of the Elastic Modulus of Cornea, Sclera and Limbus: The Importance of the Corneal-Limbus-Scleral Biomechanical Unit.

BACKGROUND: Energy storage, transmission and dissipation are important considerations of normal mechanical homeostasis. In this paper we present a new technique termed vibrational optical coherence tomography (VOCT) to study the anterior anatomic structures of the pig eye to better understand how energy applied to the cornea is dissipated without delamination occurring. METHODS: VOCT uses infrared light and an applied sinusoidal audible sound wave to image and measure the resonant frequency and modulus of individual macromolecular components of tissue non-invasively. We have measured the resonant frequencies and calculated the moduli of tissues in the anterior portion of the pig eye using VOCT. RESULTS: While both pig and human eyes have similar resonant frequencies, they do differ in the peak amplitudes near the frequencies of 80, 120, 150 and 250 Hz. It is known that the stroma of pig cornea is much thicker than that of human corneas and these differences may explain the normalized peak height differences. The similarity of the resonant frequency peaks near 80, 120, 150 and 250 Hz of cornea, sclera and limbus suggest that the anatomically described layers in these tissues are connected into a single biomechanical unit that can store external mechanical energy and then transmit it for dissipation. Since the energy stored and dissipated is proportional to the modulus and the ability of the tissue to deform under stress, energy storage in these tissues is related to the stiffness. CONCLUSIONS: It is concluded that stored energy is transmitted to the posterior segment of the eye for dissipation through the attachment with the sclera. This mechanism of energy dissipation may protect the cornea from changes in shape, curvature, and refractive power. However, ultimately, energy dissipation through thinning of the sclera may cause globe elongation observed in subjects with myopia and glaucoma.

A biomimetic synthetic nanofiber-based model for anterior cruciate ligament regeneration.

Reconstructed ACL cannot completely restore its functions due to absence of physiologically viable environment for optimal biomaterial-cell interaction. Currently available procedures only mechanically attach grafts to bone without any biological integration. How the ACL cells perform this biological attachment is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. Availability of biomimetic models would enable the scientists to better explore the behavior of cells at health and during tissue healing. In this study, it is hypothesized that the collagen fibril diameter distribution in rat ACL changes from a bimodal distribution in the healthy ACL to a unimodal distribution after injury, and that this change can be mimicked in synthetic nanofiber-based constructs. This hypothesis was tested by first creating an injured rat ACL model by applying a mechanical tensile force to the healthy ACL tissue until rupture. Secondly, the collagen fibril diameter distributions of healthy and injured ACL tissue were determined, and polycaprolactone (PCL) constructs were created to mimic the distributions of collagen fibrils in healthy and injured tissues. Findings reveal that the fiber diameter distribution of aligned bimodal PCL constructs were similar to that of the collagen fibrils in native ACL tissue. This study is significant because suggested bimodal and unimodal fibrous model constructs, respectively, represent a healthy and injured tissue environment and the behavior of ACL cells cultured on these constructs may provide significant input on ACL regeneration mechanism.

Aging exacerbates the morphological and mechanical response of mineralized collagen fibrils in murine cortical bone to disuse.

Mineralized collagen fibrils (MCFs) are the fundamental building blocks of bone tissue and contribute significantly to the mechanical behavior of bone. However, it is still largely unknown how the collagen network in bone responds to aging and the disuse normally accompanying it. Utilizing atomic force microscopy, nanoindentation and Raman spectroscopy, age-related alterations in the microstructure and mechanical properties of murine cortical tibia at multiple scales were investigated in this study. The potential difference in the responses of bone to disuse at different ages was studied. The results indicated that the age- and disuse-related alterations in bone initiate from MCFs in the bone matrix. The D-periodic spacing, radial elastic modulus of a single MCF and the mineral-to-matrix ratio on the cortical bone surface were larger in aged mice than in adult mice. Disuse, on the other hand, mainly has a major influence on aged mice, particularly on the morphology and mechanical properties of MCFs, but it only has modest effects on adult bone. These findings revealed insights into the morphological and mechanical adaptation of mineralized collagen fibrils in murine cortical bone to aging and disuse. STATEMENT OF SIGNIFICANCE: Bone is a complex structured composite material consisting of an interwoven framework of collagen fibrils reinforced by mineral particles and embedded in an extrafibrillar mineralized matrix. Utilizing atomic force microscopy, nanoindentation and Raman spectroscopy, this study suggests that the effects of aging, as well as the accompanying disuse, on the morphology and mechanical properties of bone initiate from the mineralized collagen fibril level. More interestingly, the MCF in the bone of aged mice seems to be more sensitive to disuse than that in adult mice. These findings significantly further the current understanding of the adaptation process of bone to aging at the mineralized collagen fibril level and provide direct insights into the physiological response of bone to aging and the abnormal mechanical environment.

Research of connective tissue dysplasia influence on teething.

Purpose: This work aimed to study the rate and quality of maturation of the mineral component of retained teeth 3.8, 4.8 and lower jaw fragment of a human in connective tissue dysplasia in different periods of postpartum ontogenesis. Methods: The study involved 102 men (76 with connective tissue dysplasia and 26 without connective tissue dysplasia) divided into groups by age: 31-40, 41-50, 51-60 years. One tooth 3.8, 4.8 and a fragment of the alveolar part of the lower jaw in the projection of teeth 3.8, 4.8 0.5*0.5 cm in size were extracted from each examinee for medical indications. Results: Low optical density values are observed at the age of 41-50 years, at the age of 51-60 years, indicating decreased mineral density and the presence of total areas of hypomineralization from the age 31-40 years in connective tissue dysplasia. At the age of 41-50, 51-60 years, at the boundary of connective tissue structures and periosteum, a pronounced sclerosis and deformation of delineation elements were observed; at the age of 31-40 years, the indicated changes were less pronounced. At the age of 31-40 years, the level of bone plate dissection has a local character, after 40 years, it has a generalized character. Conclusion: Progressive osteoporosis of the mandible and incomplete amelogenesis are an obstacle to the correct and harmonious teething of the lower wisdom teeth after the age of 30.

Tendon-Specific Dicer Deficient Mice Exhibit Hypoplastic Tendon Through the Downregulation of Tendon-Related Genes and MicroRNAs.

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer (Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing.

Collagen fibril formation in vitro: From origin to opportunities.

Sometimes, to move forward, it is necessary to look back. Collagen type I is one of the most commonly used biomaterials in tissue engineering and regenerative medicine. There are a variety of collagen scaffolds and biomedical products based on collagen have been made, and the development of new ones is still ongoing. Materials, where collagen is in the fibrillar form, have some advantages: they have superior mechanical properties, higher degradation time and, what is most important, mimic the structure of the native extracellular matrix. There are some standard protocols for the formation of collagen fibrils in vitro, but if we look more carefully at those methods, we can see some controversies. For example, why is the formation of collagen gel commonly carried out at 37 â€‹°C, when it was well investigated that the temperature higher than 35 â€‹°C results in a formation of not well-ordered fibrils? Biomimetic collagen materials can be obtained both using culture medium or neutralizing solution, but it requires a deep understanding of all of the crucial points. One of this point is collagen extraction method, since not every method retains the ability of collagen to reconstitute native banded fibrils. Collagen polymorphism is also often overlooked in spite of the appearance of different polymorphic forms during fibril formation is possible, especially when collagen blends are utilized. In this review, we will not only pay attention to these issues, but we will overview the most prominent works related to the formation of collagen fibrils in vitro starting from the first approaches and moving to the up-to-date recipes.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment.

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.