Collagen Network Research Articles

An improved linear systems model of hydrothermal isometric tension testing to aid in assessing bone collagen quality: Effects of ribation and type-2 diabetes.

This study sought to further develop and validate a previously proposed physics-based model that maps denaturation kinetics from differential scanning calorimetry (DSC) to the isometric tension generated during hydrothermal isometric tension (HIT) testing of collagenous tissues. The primary objectives of this study were to verify and validate two physics-based model parameters: α, which indicates the amount of instantaneous isometric tension developed per unit of collagen denaturation, and β, which captures the proportionality between temperature and the generated isometric tension post denaturation initiation. These parameters were used as measures of bone collagen quality, employing data from HIT and DSC testing of human bone collagen from two previous studies. Additionally, given the physical basis of the model, the study aimed to further validate Max.Slope, the rate of change in isometric tensile stress with change in temperature, as an independent measure of collagen network connectivity. Max.Slope has previously been positively correlated with measures of cortical bone fracture resistance. Towards this verification and validation, the hypotheses were a) that α would correlate strongly with HIT denaturation temperature, Td, and the enthalpy of melting (ΔH) from DSC, and b) that β would correlate positively and strongly with Max.Slope.The model was employed in the analysis of HIT-DSC data from the testing of demineralized bone collagen isolated from cadaveric human femurs in two prior studies. In one study, data were collected from HIT-DSC testing of cortical bone collagen from 74 donors. Among them, 38 had a history of type 2 diabetes +/− chronic kidney disease, while the remaining 36 had no history of T2D again with or without CKD. Cortical bone specimens were extracted from the lateral mid-shaft. The second study involved 15 donor femora, with four cortical bone specimens extracted from each. Of these four, two specimens underwent a 4-week incubation in 0.1 M ribose at 37 °C to induce non-enzymatic ribation and advanced glycation endproducts, while the other two served as non-ribated controls.The examination involved investigating correlations between the model parameters α and β and various measures, such as Max.Slope, Td, ΔH, age, and duration of type 2 diabetes. The results revealed positive correlations between the model parameter β and Max.Slope (r = 0.55–0.58). The parameter α was found to be associated with Td, but also sensitive to the shape of the HIT curve around Td resulting in difficulties with variability and interpretation. As a result, while both hypotheses are confirmed, Max.Slope and β are better indicators of bone collagen quality because they are measures of the connectivity or, more generally, the integrity of the bone collagen network.

Bone collagen tensile properties of the aging human proximal femur

Despite the dominant role of bone mass in osteoporotic fractures, aging bone tissue properties must be thoroughly understood to improve osteoporosis management. In this context, collagen content and integrity are considered important factors, although limited research has been conducted on the tensile behavior of demineralized compact bone in relation to its porosity and elastic properties in the native mineralized state. Therefore, this study aims (i) at examining the age-dependency of mineralized bone and collagen micromechanical properties; (ii) to test whether, and if so to which extent, collagen properties contribute to mineralized bone mechanical properties.Two cylindrical cortical bone samples from fresh frozen human anatomic donor material were extracted from 80 proximal diaphyseal sections from a cohort of 24 female and 19 male donors (57 to 96 years at death). One sample per section was tested in uniaxial tension under hydrated conditions. First, the native sample was tested elastically (0.25 % strain), and after demineralization, up to failure. Morphology and composition of the second specimen was assessed using micro-computed tomography, Raman spectroscopy, and gravimetric methods. Simple and multiple linear regression were employed to relate morphological, compositional, and mechanical variables with age and sex.Macro-tensile properties revealed that only elastic modulus of native samples was age dependent whereas apparent elastic modulus was sex dependent (p < 0.01). Compositional and morphological analysis detected a weak but significant age and sex dependency of relative mineral weight (r = −0.24, p < 0.05) and collagen disorder ratio (I∼1670/I∼1640, r = 0.25, p < 0.05) and a strong sex dependency of bone volume fraction while generally showing consistent results in mineral content assessment. Young's modulus of demineralized bone was significantly related to tissue mineral density and Young's modulus of native bone.The results indicate that mechanical properties of the organic phase, that include collagen and non-collagenous proteins, are independent of donor age. The observed reduction in relative mineral weight and corresponding overall stiffer response of the collagen network may be caused by a reduced number of mineral-collagen connections and a lack of extrafibrillar and intrafibrillar mineralization that induces a loss of waviness and a collagen fiber pre-stretch.

HtrA3 paves the way for MSC migration and promotes osteogenesis

Mesenchymal stem cell (MSC) migration determines the healing capacity of bone and is crucial in promoting bone regeneration. Migration of MSCs is highly dependent on degradation of extracellular matrix by proteolytic enzymes. However, the underlying mechanisms of how enzymolysis paves the way for MSCs to migrate from their niche to the defect area is still not fully understood. Here, this study shows that high-temperature requirement A3 (HtrA3) overcomes the physical barrier and provides anchor points through collagen IV degradation, paving the way for MSC migration. HtrA3 is upregulated in MSCs at the leading edge of bone defect during the early stage of healing. HtrA3 degrades the surrounding collagen IV, which increases the collagen network porosity and increases integrin β1 expression. Subsequently, integrin β1 enhances the mechanotransduction of MSCs, thus remodeling the cytoskeleton, increasing cellular stiffness and nuclear translocation of YAP, eventually promoting the migration and subsequent osteogenic differentiation of MSCs. Local administration of recombinant HtrA3 in rat cranial bone defects significantly increases new bone formation and further validates the enhancement of MSC migration. This study helps to reveal the novel roles of HtrA3, explore potential targets for regenerative medicine, and offer new insights for the development of bioactive materials.

Multi-acrylamides improve bond stability through collagen reinforcement under physiological conditions

ObjectivesAcrylamides were shown to significantly improve bonding stability in adhesive restorations, but the reinforcement mechanism has not been fully elucidated. We tested the hypothesis that hydrogen bonding reinforcement of the collagen network (with secondary or tertiary acrylamides), as well as degree of crosslinking of the polymer network (with di- or tri-functional acrylamides), can be two of the factors at play. MethodsTwo-step total etch adhesives comprising UDMA (60 wt%) and 40 wt% of: TAAEA, TMAAEA (secondary, tertiary tri-acrylamides), BAAP, DEBAAP (secondary, tertiary di-acrylamides) or HEMA (mono-methacrylate - control) were formulated. Simulated composite restorations (n = 5) were tested after cyclic mechanical and biological (S. mutans biofilm) challenges. Gap formation before and after aging was assessed with SEM imaging. Micro-tensile bond strength (μTBS, n = 6) was assessed after seven-day incubation in water or S. mutans-containing culture medium. Collagen reinforcement was assessed with hydroxyproline assay (n = 10) and rheology (n = 3). Data were analyzed with one-way/two-way ANOVA/Tukey’s test (alpha=5%). ResultsGap formation increased and bond strength decreased for all monomers after biofilm incubation (p < 0.001). Except for DEBAAP, secondary and tertiary di/tri-acrylamides showed lower occlusal gap width values, but no significant differences overall gap length compared to HEMA. μTBS increased for tri-acrylamides compared with HEMA. Samples treated with multi-acrylamides had lower concentration of hydroxyproline (by-product of collagen degradation) (p < 0.001), except for DEBAAP, which showed values close to HEMA (p > 0.05). Dentin shear modulus increased for all acrylamides after 72 h, especially TMAAEA. SignificanceIn general, multi-acrylamides promote collagen reinforcement, leading to reduced gap formation, and stabilize the bond strength under physiological conditions.

Photochemical Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Evaluation in Ex Vivo Human Tissue

Purpose: Experimental investigation in human eyelids to confirm that exposing excised tarsal plates to ultraviolet-A radiation can induce a stiffening effect through the riboflavin-photosensitized crosslinking of tarsal collagen. Methods: Thirteen tarsal plates excised from nonfrozen human cadavers were irradiated with ultraviolet-A rays (365 nm wavelength) at an irradiance of 75 mW/cm2 for 3 minutes, equivalent to a radiation fluence of 13.5 J/cm2, in the presence of a riboflavin derivative as a photosensitizer. The tensile stress (strength) and Young’s modulus (stiffness) of both nonirradiated and irradiated specimens were measured with the BioTester 5000 in the uniaxial mode at a strain of 10% and analyzed statistically. Individual specimens excised from 2 cadavers were also examined by routine histopathologic protocols to assess the effect of radiation on the Meibomian glands and collagen organization. Results: The irradiation enhanced both stiffness and strength of the human tarsal specimens, the difference between the test samples and controls being statistically significant (p &lt; 0.0002 for n = 13). Histology indicated no damage to tarsal connective tissue or to Meibomian glands, and revealed a more compact packing of the collagen network located around the glands, which may be beneficial. The existence of collagen compaction was also supported by the reduction of samples’ thickness after irradiation (p = 0.0645). Conclusions: The irradiation of tarsal tissue with ultraviolet-A light of tarsus appears to be a safe and effective method for reducing eyelid laxity in human patients.

Cell-adhesive double network self-healing hydrogel capable of cell and drug encapsulation: New platform to construct biomimetic environment with bottom-up approach

This study presents the development and characterization of a novel double-network self-healing hydrogel based on N-carboxyethyl chitosan (CEC) and oxidized dextran (OD) with the incorporation of crosslinked collagen (CEC-OD/COL-GP) to enhance its biological and physicochemical properties. The hydrogel formed via dynamic imine bond formation exhibited efficient self-healing within 30 min, and a compressive modulus recovery of 92 % within 2 h. In addition to its self-healing ability, CEC-OD/COL-GP possesses unique physicochemical characteristics including transparency, injectability, and adhesiveness to various substrates and tissues. Cell encapsulation studies confirmed the biocompatibility and suitability of the hydrogel as a cell-culture scaffold, with the presence of a collagen network that enhances cell adhesion, spreading, long-term cell viability, and proliferation. Leveraging their unique properties, we engineered assemblies of self-healing hydrogel modules for controlled spatiotemporal drug delivery and constructed co-culture models that simulate angiogenesis in tumor microenvironments. Overall, the CEC-OD/COL-GP hydrogel is a versatile and promising material for biomedical applications, offering a bottom-up approach for constructing complex structures with self-healing capabilities, controlled drug release, and support for diverse cell types in 3D environments. This hydrogel platform has considerable potential for advancements in tissue engineering and therapeutic interventions.

Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Chondroitin Sulfate/Hyaluronic Acid-Blended Hydrogels Suppress Chondrocyte Inflammation under Pro-Inflammatory Conditions.

Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

Collagen-polyurethane-dextran hydrogels enhance wound healing by inhibiting inflammation and promoting collagen fibrillogenesis.

Diabetic foot ulcers are a serious complication of uncontrolled diabetes, emphasizing the need to develop wound healing strategies that are not only effective but also biocompatible, biodegradable, and safe. We aimed to create biomatrices composed of semi-interpenetrated polymer networks of collagen, polyurethane, and dextran, to enhance the wound healing process. The hydrogels were extensively characterized by various analytical techniques, including analysis of their structure, crystallinity, thermal properties, gelation process, reticulation, degradation, cell proliferation, and healing properties, among others. Semi-interpenetrated hydrogels containing dextran at levels of 10%, 20%, and 30% exhibited porous interconnections between collagen fibers and entrapped dextran granules, with a remarkable crosslinking index of up to 94% promoted by hydrogen bonds. These hydrogels showed significant improvements in mechanical properties, swelling, and resistance to proteolytic and hydrolytic degradation. After 24 h, there was a significant increase in the viability of several cell types, including RAW 264.7 cells, human peripheral blood mononuclear cells, and dermal fibroblasts. In addition, these hydrogels demonstrated an increased release of interleukin-10 and transforming growth factor-beta1 while inhibiting the release of monocyte chemotactic protein-1 and tumor necrosis factor-alpha after 72 h. Furthermore, these hydrogels accelerated the wound healing process in diabetic rats after topical application. Notably, the biomaterial with 20% dextran (D20) facilitated wound closure in only 21 days. These results highlight the potential of the D20 hydrogel, which exhibits physicochemical and biological properties that enhance wound healing by inhibiting inflammation and fibrillogenesis while remaining safe for application to the skin.

Collagen organization and structure in FBLN5-/- mice using label-free microscopy: implications for pelvic organ prolapse.

Pelvic organ prolapse (POP) is a gynecological disorder described by the descent of superior pelvic organs into or out of the vagina as a consequence of disrupted muscles and tissue. A thorough understanding of the etiology of POP is limited by the availability of clinically relevant samples, restricting longitudinal POP studies on soft-tissue biomechanics and structure to POP-induced models such as fibulin-5 knockout (FBLN5-/- ) mice. Despite being a principal constituent in the extracellular matrix, little is known about structural perturbations to collagen networks in the FBLN5-/- mouse cervix. We identify significantly different collagen network populations in normal and prolapsed cervical cross-sections using two label-free, nonlinear microscopy techniques. Collagen in the prolapsed mouse cervix tends to be more isotropic, and displays reduced alignment persistence via 2-D Fourier transform analysis of images acquired using second harmonic generation microscopy. Furthermore, coherent Raman hyperspectral imaging revealed elevated disorder in the secondary structure of collagen in prolapsed tissues. Our results underscore the need for in situ multimodal monitoring of collagen organization to improve POP predictive capabilities.

Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFβ1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

Effect of hydroxyapatite and pulsed electromagnetic field on osteogenic differentiation of human stem cells seeded onto 3D‐printed polycaprolactone scaffolds for future bone graft transplantation

AbstractBone graft transplantation is a risky procedure mainly due to postoperative complications. Tissue engineering is an alternative to regenerating bone tissue by seeding mesenchymal stem cells into 3D scaffolds by osteoinductive signaling. Poly(ε‐caprolactone) (PCL) is one of the most used biopolymers for fabricating scaffolds for bone tissue engineering. PCL is enriched with hydroxyapatite (HA) to promote the production of the bone‐mineralized matrix. Moreover, the application of pulsed electromagnetic fields (PEMFs) on scaffolds loaded with HA can significantly benefit collagen and HA networks favoring the interaction between organic and mineral phases of bones. In this context, we investigated, for the first time, the effect exerted by a PEMF exposure on the osteogenic differentiation of human adipose stem cells (hASCs) seeded onto 3D printed PCL‐HA scaffolds and cultured in growth and osteogenic medium (OM). After 21 days of culture, the results showed excellent colonization of scaffolds and the deposition of extracellular matrix (ECM) secreted by hASCs. Immunofluorescence staining confirmed the deposition of collagen type I and osteopontin both on scaffolds. Gene expression analysis indicated that both HA crystal and PEMF treatment were able to trigger osteogenic differentiation, as documented by the increase of osteogenic markers. These results suggest that HA, osteogenic factors, and PEMFs converge on the same signaling pathways as there are no additive effects and no change in protein synthesis between scaffolds with HA or treated with PEMFs for cells cultured in OM. The proposed study lays the foundations for exploring new approaches in bone graft transplantation.

High angular resolution diffusion imaging (HARDI) of porcine menisci: a comparison of diffusion tensor imaging and generalized q-sampling imaging.

Diffusion magnetic resonance imaging (MRI) allows for the quantification of water diffusion properties in soft tissues. The goal of this study was to characterize the 3D collagen fiber network in the porcine meniscus using high angular resolution diffusion imaging (HARDI) acquisition with both diffusion tensor imaging (DTI) and generalized q-sampling imaging (GQI). Porcine menisci (n=7) were scanned ex vivo using a three-dimensional (3D) HARDI spin-echo pulse sequence with an isotropic resolution of 500 µm at 7.0 Tesla. Both DTI and GQI reconstruction techniques were used to quantify the collagen fiber alignment and visualize the complex collagen network of the meniscus. The MRI findings were validated with conventional histology. DTI and GQI exhibited distinct fiber orientation maps in the meniscus using the same HARDI acquisition. We found that crossing fibers were only resolved with GQI, demonstrating the advantage of GQI over DTI to visualize the complex collagen fiber orientation in the meniscus. Furthermore, the MRI findings were consistent with conventional histology. HARDI acquisition with GQI reconstruction more accurately resolves the complex 3D collagen architecture of the meniscus compared to DTI reconstruction. In the future, these technologies have the potential to nondestructively assess both normal and abnormal meniscal structure.

Abstract 4281: BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer

Abstract The relationship between colon stem cells (SCs), its surrounding niche microenvironment like the extracellular matrix (ECM) and the mesenchymal niche cells is complex and dynamic. ECM proteins are not only a scaffolding system for SCs, but their biophysical properties also contribute to localizing signals and creating gradients leading to the SCs homeostasis. Foxl1+-Telocytes (TCFoxL1+) are mesenchymal niche cells implicated in cell-cell communication and the production of BMP ligands. Upon chronic inflammation, mice lacking BMP signaling in TCFoxL1+ (BmpR1aΔFoxL1+), developed colonic tumors with microenvironment exhibiting abnormal TCFoxL1+ structure, increased BMP expression and myofibroblastic-like cancer-associated fibroblasts (myCAF) subpopulation. Yet, how the TCFoxL1+ influences SCs niche ECM proteins during tumorigenesis is less understood. We aimed to investigate how the loss of BMP signaling in TCFoxL1+ affects ECM network impacting the niche microenvironment promoting the development of CAC. DSS-based chronic inflammatory challenge in mutant and control mice, quantitative-MS-based strategy was performed to determine the proteome of the transformed regions solely in epithelial-mesenchymal tissue following colon deconstruction. Expression of the proteins of interest were validated by Western Blots or immunostainings. Quantitative analysis of collagen network was assessed using phyton pipeline U-net in Sirius red-stained colon analyzed under polarized light. Differential significant expression of 798 proteins, with 342 upregulated and 456 downregulated, were found in the tumors of mutant mice compared to controls. Mfap5 involved in CAF activation was the highest upregulated protein. We found upregulated, laminin subunits α4 and β2, decorin, lumican as well as transgelin (myCAF marker), which are proteins known for contributing to the stiffening of the ECM during colorectal cancer. Adamdec1 which prevents aberrant ECM accumulation and thrombospodin-1, an ECM glycoprotein whose low levels have been related to tumor growth, were both found to be downregulated. Data extracted from Python's U-Net pipeline demonstrated that tumors, in mutant mice, exhibited higher frequencies of straighter collagen fibers compared to wavier fibers in controls. Collagen fiber distribution was shown to present an even angle distribution in controls whereas BmpR1aΔFoxL1+ mice presented a preferential orientation. These results suggest that defective TCFoxL1+ commits the surrounding niche microenvironment, especially its ECM, toward the development and progression of CAC. Citation Format: Vilcy Reyes Nicolas, Alain B. Alfonso, Jennifer Raisch, François M. Boisvert, Marc-Antoine Lauzon, Nathalie Perreault. BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4281.

Mechanical Models of Collagen Networks for Understanding Changes in the Failure Properties of Aging Skin.

Skin undergoes mechanical alterations due to changes in the composition and structure of the collagenous dermis with aging. Previous studies have conflicting findings, with both increased and decreased stiffness reported for aging skin. The underlying structure-function relationships that drive age-related changes are complex and difficult to study individually. One potential contributor to these variations is the accumulation of nonenzymatic crosslinks within collagen fibers, which affect dermal collagen remodeling and mechanical properties. Specifically, these crosslinks make individual fibers stiffer in their plastic loading region and lead to increased fragmentation of the collagenous network. To better understand the influence of these changes, we investigated the impact of nonenzymatic crosslink changes on the dermal microstructure using discrete fiber networks representative of the dermal microstructure. Our findings suggest that stiffening the plastic region of collagen's mechanical response has minimal effects on network-level stiffness and failure stresses. Conversely, simulating fragmentation through a loss of connectivity substantially reduces network stiffness and failure stress, while increasing stretch ratios at failure.

Characterization of the collagen network of human cheek skin using ultrasonic microscopy

Dermal collagen is the most abundant component of human skin and has a network structure that regulates the mechanical properties of the skin. Therefore, non-invasive characterization of the collagen network would be beneficial for the evaluation of skin conditions. The microscopic substructures of the network, which are individual bundles and fibers, have been optically investigated. However, the macroscopic structure of the collagen network has not been assessed. To evaluate the dermal collagen network, we developed two new indicators, volume filling factor (VFF) and collagen fiber texture (CFT), to analyze three-dimensional echo intensity maps of high-frequency ultrasonic microscopy. By identifying the difference in the elastic modulus components of the dermal layer of facial skin, the density and texture of the collagen network were characterized using VFF and CFT, respectively. These new indicators revealed that the density decreased and the texture became fine with facial age. This study demonstrates that ultrasonic microscopy is useful for investigating skin conditions, paving the way for diagnostic applications in dermatology and aesthetic medicine.

Independent and combined effects of obesity and traumatic joint injury to the structure and composition of rat knee cartilage

ABSTRACT Osteoarthritis (OA) is a multifactorial joint disease characterized by articular cartilage degradation. Risk factors for OA include joint trauma, obesity, and inflammation, each of which can affect joint health independently, but their interaction and the associated consequences of such interaction were largely unexplored. Here, we studied compositional and structural alterations in knee joint cartilages of Sprague-Dawley rats exposed to two OA risk factors: joint injury and diet-induced obesity. Joint injury was imposed by surgical transection of anterior cruciate ligaments (ACLx), and obesity was induced by a high fat/high sucrose diet. Depth-dependent proteoglycan (PG) content and collagen structural network of cartilage were measured from histological sections collected previously in Collins et al.. (2015). We found that ACLx primarily affected the superficial cartilages. Compositionally, ACLx led to reduced PG content in lean animals, but increased PG content in obese rats. Structurally, ACLx caused disorganization of collagenous network in both lean and obese animals through increased collagen orientation in the superficial tissues and a change in the degree of fibrous alignment. However, the cartilage degradation attributed to joint injury and obesity was not necessarily additive when the two risk factors were present simultaneously, particularly for PG content and collagen orientation in the superficial tissues. Interestingly, sham surgeries caused a through-thickness disorganization of collagen network in lean and obese animals. We conclude that the interactions of multiple OA risk factors are complex and their combined effects cannot be understood by superposition principle. Further research is required to elucidate the interactive mechanism between OA subtypes.

Quantitative assessment of glomerular basement membrane collagen IV α chains in paraffin sections from patients with focal segmental glomerulosclerosis and Alport gene variants

Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α 3 α 4 α5(IV) and α 1 α 1 α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV): α1/2(IV) mean fluorescence intensities ( α5: α1/2 intensity ratios) and thicknesses ( α5: α1/2 thickness ratios) were calculated to represent the levels of collagen α 3 α 4 α5(IV) relative to α 1 α 1 α2(IV). The α5: α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.

Fractal analysis of extracellular matrix for observer-independent quantification of intestinal fibrosis in Crohn’s disease

Prevention of intestinal fibrosis remains an unresolved problem in the treatment of Crohn’s disease (CD), as specific antifibrotic therapies are not yet available. Appropriate analysis of fibrosis severity is essential for assessing the therapeutic efficacy of potential antifibrotic drugs. The aim of this study was to develop an observer-independent method to quantify intestinal fibrosis in surgical specimens from patients with CD using structural analysis of the extracellular matrix (ECM). We performed fractal analysis in fibrotic and control histological sections of patients with surgery for CD (n = 28). To specifically assess the structure of the collagen matrix, polarized light microscopy was used. A score to quantify collagen fiber alignment and the color of the polarized light was established. Fractal dimension as a measure for the structural complexity correlated significantly with the histological fibrosis score whereas lacunarity as a measure for the compactness of the ECM showed a negative correlation. Polarized light microscopy to visualize the collagen network underlined the structural changes in the ECM network in advanced fibrosis. In conclusion, observer-independent quantification of the structural complexity of the ECM by fractal analysis is a suitable method to quantify the degree of intestinal fibrosis in histological samples from patients with CD.

Collagen Networks under Indentation and Compression Behave Like Cellular Solids.

Simple synthetic and natural hydrogels can be formulated to have elastic moduli that match biological tissues, leading to their widespread application as model systems for tissue engineering, medical device development, and drug delivery vehicles. However, two different hydrogels having the same elastic modulus but differing in microstructure or nanostructure can exhibit drastically different mechanical responses, including their poroelasticity, lubricity, and load bearing capabilities. Here, we investigate the mechanical response of collagen-1 networks to local and bulk compressive loads. We compare these results to the behavior of polyacrylamide, a fundamentally different class of hydrogel network consisting of flexible polymer chains. We find that the high bending rigidity of collagen fibers, which suppresses entropic bending fluctuations and osmotic pressure, facilitates the bulk compression of collagen networks under infinitesimal applied stress. These results are fundamentally different from the behavior of flexible polymer networks in which the entropic thermal fluctuations of the polymer chains result in an osmotic pressure that must first be overcome before bulk compression can occur. Furthermore, we observe minimal transverse strain during the axial loading of collagen networks, a behavior reminiscent of open-celled cellular solids. Inspired by these results, we applied mechanical models of cellular solids to predict the elastic moduli of the collagen networks and found agreement with the moduli values measured through contact indentation. Collectively, these results suggest that unlike flexible polymer networks that are often considered incompressible, collagen hydrogels behave like rigid porous solids that volumetrically compress and expel water rather than spreading laterally under applied normal loads.