Collagen Research Articles

Inhibition of matrix metalloproteases by a chemical cross-linker to halt the corneal degradation in keratoconus.

The need for better and simpler alternative crosslinking strategies to treat keratoconus (KC) is becoming essential as there is only a single approved way to treat it. Recently, conventional UV-A Riboflavin crosslinking is proven to have some disadvantages such as causing damage to the corneal endothelium and inducing keratocyte apoptosis. A chemical cross-linker (CXL) using carbodiimide chemistry and an octanedioic acid spacer is found effective in stiffening the cornea and has the potential to be developed as an alternative therapy to halt KC progression. In order to investigate the molecular changes induced by the cross-linker, we have analyzed the effect of the cross-linker on the activity of matrix metalloproteases (MMPs) in epithelial and stromal layers of KC corneas and in in vitro cellular systems to determine its role in stiffening the KC cornea. At well-optimized concentration, KC corneal buttons were treated with the CXL and the stiffening of the cornea was measured. The collagen fibril assembly in the stroma was analyzed using transmission electron microscopy and the activity of MMPs 2 and 9 were visualized using gelatin zymography. KC corneal fibroblasts in culture and tumor necrosis factor-α (TNF-α) induced human corneal epithelial (HCE) cell line were treated with CXL and secretion of MMPs 1, 2, 3 and 9 were analyzed by enzyme-linked immunosorbent assay (ELISA). We found that the CXL stiffened the KC corneas comparable to the normal corneas, with very less cytotoxicity. The collagen fiber assembly was reorganized in an orderly fashion and fibril density and diameter increased after CXL treatment. The activity of MMPs and cathepsin G in the epithelial and stromal layers of KC tissues decreased post-treatment. Secretion and activity of MMPs from the corneal epithelial and stromal cells after CXL treatment were significantly reduced while the epithelial lysyl oxidase activity increased. The CXL, intended to stop the KC progression, modified the extracellular matrix collagen assembly in the stroma and decreased the secretion of a group of metalloproteases and their activity. We have demonstrated a set of molecular changes effected by the CXL, which might aid in the stiffening of the KC cornea.

AGEing of collagen: The effects of glycation on collagen's stability, mechanics and assembly.

Advanced Glycation End Products (AGEs) are the end result of the irreversible, non-enzymatic glycation of proteins by reducing sugars. These chemical modifications accumulate with age and have been associated with various age-related and diabetic complications. AGEs predominantly accumulate on proteins with slow turnover rates, of which collagen is a prime example. Glycation has been associated with tissue stiffening and reduced collagen fibril remodelling. In this study, we investigate the effects of glycation on the stability of type I collagen, its molecular-level mechanics and its ability to perform its physiological role of self-assembly. Collagen AGEing is induced in vitro by incubation with ribose. We confirm and assess glycation using fluorescence measurements and changes in collagen's electrophoretic mobility. Susceptibility to trypsin digestion and circular dichroism (CD) spectroscopy are used to probe changes in collagen's triple helical stability, revealing decreased stability due to glycation. Atomic Force Microscopy (AFM) imaging is used to quantify how AGEing affects collagen flexibility, where we find molecular-scale stiffening. Finally we use microscopy to show that glycated collagen molecules are unable to self-assemble into fibrils. These findings shed light on the molecular mechanisms underlying AGE-induced tissue changes, offering insight into how glycation modifies protein structure and stability.

REVISITING THE PHYSICAL AND CHEMICAL NATURE OF THE MINERAL COMPONENT OF BONE.

The physico-chemical characteristics of bone mineral remain heavily debated. On the nanoscale, bone mineral resides both inside and outside the collagen fibril as distinct compartments fused together into a cohesive continuum. On the micrometre level, larger aggregates are arranged in a staggered pattern described as crossfibrillar tessellation. Unlike geological and synthetic hydroxy(l)apatite, bone mineral is a unique form of apatite deficient in calcium and hydroxyl ions with distinctive carbonate and acid phosphate substitutions (CHAp), together with a minor contribution of amorphous calcium phosphate as a surface layer around a crystalline core of CHAp. In mammalian bone, an amorphous solid phase has not been observed, though an age-dependent shift in the amorphous-to-crystalline character is observed. Although octacalcium phosphate has been postulated as a bone mineral precursor, there is inconsistent evidence of calcium phosphate phases other than CHAp in the extracellular matrix. In association with micropetrosis, magnesium whitlockite is occasionally detected, indicating pathological calcification rather than a true extracellular matrix component. Therefore, the terms 'biomimetic' or 'bone-like' should be used cautiously in descriptions of synthetic biomaterials. The practice of reporting the calcium-to-phosphorus ratio (Ca/P) as proxy for bone mineral maturity oversimplifies the chemistry since both Ca2+ and PO43- ions are partially substituted. Moreover, non-mineral sources of phosphorus are ignored. Alternative compositional metrics should be considered. In the context of bone tissue and bone mineral, the term 'mature' must be used carefully, with clear criteria that consider both compositional and structural parameters and the potential impact on mechanical properties. STATEMENT OF SIGNIFICANCE: Bone mineral exhibits a unique hierarchical structure and is classified as intrafibrillar and extrafibrillar mineral compartments with distinct physico-chemical characteristics. The dynamic nature of bone mineral, i.e., evolving chemical composition and physical form, is poorly understood. For instance, bone mineral is frequently described as "hydroxy(l)apatite", even though the OH- content of mature bone mineral is negligible. Moreover, the calcium-to-phosphorus ratio is often taken as an indicator of bone mineral maturity without acknowledging substitutions at calcium and phosphate sites. This review takes a comprehensive look at the structure and composition of bone mineral, highlighting how experimental data are misinterpreted and unresolved concerns that warrant further investigation, which have implications for characterisation of bone material properties and development of bone repair biomaterials.

Strontium-substituted hydroxyapatite nanoparticles associated with type I-collagen enhances the obliteration of dentinal tubules and the mineralization by human dental pulp stem cells

Strontium-substituted hydroxyapatite nanoparticles associated with type I-collagen enhances the obliteration of dentinal tubules and the mineralization by human dental pulp stem cells

Identifying health risk determinants and molecular targets in patients with idiopathic pulmonary fibrosis via combined differential and weighted gene co-expression analysis

IntroductionIdiopathic pulmonary fibrosis (IPF) is a rare but debilitating lung disease characterized by excessive fibrotic tissue accumulation, primarily affecting individuals over 50 years of age. Early diagnosis is challenging, and without intervention, the prognosis remains poor. Understanding the molecular mechanisms underlying IPF pathogenesis is crucial for identifying diagnostic markers and therapeutic targets.MethodsWe analyzed transcriptomic data from lung tissues of IPF patients using two independent datasets. Differentially expressed genes (DEGs) were identified, and their functional roles were assessed through pathway enrichment and tissue-specific expression analysis. Protein-protein interaction (PPI) networks and co-expression modules were constructed to identify hub genes and their associations with disease severity. Machine learning approaches were applied to identify genes capable of differentiating IPF patients from healthy individuals. Regulatory signatures, including transcription factor and microRNA interactions, were also explored, alongside the identification of potential drug targets.ResultsA total of 275 and 167 DEGs were identified across two datasets, with 67 DEGs common to both. These genes exhibited distinct expression patterns across tissues and were associated with pathways such as extracellular matrix organization, collagen fibril formation, and cell adhesion. Co-expression analysis revealed DEG modules correlated with varying IPF severity phenotypes. Machine learning analysis pinpointed a subset of genes with high discriminatory power between IPF and healthy individuals. PPI network analysis identified hub proteins involved in key biological processes, while functional enrichment reinforced their roles in extracellular matrix regulation. Regulatory analysis highlighted interactions with transcription factors and microRNAs, suggesting potential mechanisms driving IPF pathogenesis. Potential drug targets among the DEGs were also identified.DiscussionThis study provides a comprehensive transcriptomic overview of IPF, uncovering DEGs, hub proteins, and regulatory signatures implicated in disease progression. Validation in independent datasets confirmed the relevance of these findings. The insights gained here lay the groundwork for developing diagnostic tools and novel therapeutic strategies for IPF.

BMP9 induces postnatal zonal stratification of immature articular cartilage through reconfiguration of the existing collagen framework

Articular cartilage lines bones in synovial joints, and its main structural element, collagen, has an arcade-like arrangement formed from an initially random network in a process called postnatal maturation. This reshaping of the extracellular matrix is similar across all species and is critical for the lifelong strength and durability of cartilage. Collagen remodelling during maturation is difficult to study because it spans a period of time between birth and puberty, and in larger animals this can be months or years. In this study, we show that growth factor bone morphogenetic protein-9 (BMP9) induces collagen remodelling in intact immature articular cartilage explants within 21 days, generating the characteristic arcade-like structure and zonal anisotropic architecture of adult cartilage. In explants exposed to BMP9, collagen fibrils underwent angular displacement from 19° to 78° with respect to the surface, cell density decreased 1.77-fold, and chondrons were significantly larger. The absence of labelling with anti-COL2¾m, a marker of collagen turnover, showed that the existing fibril network was restructured. We found that stromelysin-1 (metalloproteinase-3, MMP3) gene expression was consistently upregulated, whilst other MMP transcript levels were unchanged or reduced. Remodelling was dependent on proteoglycan turnover and could be inhibited using PD166973. These data suggest a possible mechanism whereby MMP3 induces proteoglycan turnover and depolymerises collagen fibrils enabling them to undergo spatial reorganisation. This process may be driven by tissue swelling, which generates directional strain to align fibrils into an arcade-like pattern. The ability to induce tissue maturation advances the potential for engineering durable and functional cartilage for patients requiring joint repair due to diseases such as osteoarthritis.

Identifying Collagenase (MMP-1, -8, -13) Expression and Correlation with Periodontitis Progression Using the Rat Model

Collagenase (MMP-1, -8, and -13) is one of the groups of the matrix metalloproteinases (MMPs) that is responsible for the breakdown of collagen, particularly type-I collagen, which is found in profusion in the extracellular matrix (ECM). It is essential to understand the role of a group of biomarkers in the progression of periodontal disease. This study aims to evaluate the expression of MMP-1, -8, and -13 combined in the periodontitis progression induced by wire ligation and Enterococcus faecalis inoculation using the rat model. Twelve rats were allocated uniformly between the control group 0-day, experimental group 7- and 14-days. Orthodontic wire (0.2 mm) was placed between the proximal space of the right upper first and second molar tooth area and 0.5 μl of 1.5 ´ 108 cfu/ml. Rats in the experimental groups received an injection of E. faecalis suspension into their gingival sulcus. After the respective induction time, the rats were euthanised. Gingival tissue and maxillary jaw samples were obtained from all rats for quantitative real-time PCR and histological examination. The results showed a significant increase in mRNA expression within the tissue samples from the gingiva of MMP-1 (p < 0.05), -8 (p < 0.01), and -13 (p < 0.01) in 7 days as compared to the control. The MMP-8 expression levels were also significantly reduced (p < 0.05). Histological analysis showed a higher inflammatory cell infiltration and the presence of osteoclast in the 7 days, which was reduced in the 14 days. MMP-1, -8, and -13 levels were positively correlated with the presence of inflammatory cells. Therefore, identifying a group of collagenases might be a useful biomarker to detect the progression of periodontitis.

Zonal Characteristics of Collagen Ultrastructure and Responses to Mechanical Loading in Articular Cartilage.

The biomechanical properties of articular cartilage arise from a complex bioenvironment comprising hierarchically organised collagen networks within the extracellular matrix (ECM) that interact with the proteoglycan-rich interstitial fluid. This network features a depth-dependent fibril organisation across different zones. Understanding how collagen fibrils respond to external loading is key to elucidating the mechanisms behind lesion and managing degenerative conditions like osteoarthritis. This study employs polarisation-resolved second harmonic generation (pSHG) microscopy to quantify the ultrastructural organisation of collagen fibrils and their spatial gradient along the depth of bone-cartilage explants at a close-to-in vivo condition. By combining with in-situ loading, we examined the responses of collagen fibrils by quantifying changes in their principal orientation and degree of alignment. The spatial gradient and heterogeneity of collagen organisation were captured at high resolution (1 µm) along the longitudinal plane of explants (0.5 by 2 mm). Zone-specific ultrastructural characteristics were quantified to aid in defining zonal borders, revealing consistent zonal proportions with varying overall thicknesses. Under compression, the transitional zone exhibited the most significant re-organisation of collagen fibrils. It initially allowed large deformation through re-orientation of fibrils, which then tightened fibril alignment to prevent excessive deformation, indicating a dynamic adaptation mechanism in response to increasing strain levels. Our results provide comprehensive, zone-specific baselines of cartilage ultrastructure and micromechanics, crucial for investigating the onset and progression of degenerative conditions, setting therapeutic intervention targets, and guiding cartilage repair and regeneration efforts. STATEMENT OF SIGNIFICANCE: Achieved unprecedented quantification of the spatial gradient and heterogeneity of collagen ultrastructural organisation at a high resolution (1 µm) along the full depth of the longitudinal plane of osteochondral explants (500 by 2000 µm) in close-to-in vivo condition. Suggested new anatomical landmarks based on ultrastructural features for determining zonal borders and found consistent zonal proportions in explants with different overall thicknesses. Demonstrated that collagen fibrils initially respond by re-orientating themselves at low strain levels, playing a significant role in cartilage deformation, particularly within the transitional zone. At higher strain levels, more collagen fibrils changed their alignment, indicating a dynamic shift in the response mechanism at varying strain levels.

Evaluation method for proteoglycans using near-infrared spectroscopy.

Cartilage is a connective tissue composed of mainly water, collagen (COL) and proteoglycans (PGs) including chondroitin sulfate (CS). Near-infrared (NIR) spectroscopy is adequate for examination of soft and hard tissues with large amount of water non-destructively and non-invasively. We measured tablets containing CS and COL using NIR spectroscopy to develop an evaluation method for PGs in cartilage non-destructively and non-invasively. Calibration curves were constructed using the NIR spectra of the tablets that show the quantitative linear relationship between the concentration and height of the second derivative at 4260cm-1 for COL and at 5800cm-1 for COL and CS. An equation to calculate the CS-to-COL ratio was derived from the calibrated slopes at 5800 and 4260cm-1, and the utility of the equation was demonstrated by the evaluation of tablets. Moreover, we conducted an evaluation of the CS-to-COL ratio in the aqueous nucleus pulposus and annulus fibrosus, and the results were consistent with the glycosaminoglycans (GAGs)-to-COL ratios obtained through Raman spectroscopy of the same specimens. Thus, this method is adequate for evaluating PGs with large amount of water non-destructively, non-invasively and with less damage.

A tough soft–hard interface in the human knee joint driven by multiscale toughening mechanisms

Joining heterogeneous materials in engineered structures remains a significant challenge due to stress concentration at interfaces, which often leads to unexpected failures. Investigating the complex, multiscale-graded structures found in animal tissue provides valuable insights that can help address this challenge. The human meniscus root-bone interface is an exemplary model, renowned for its exceptional fatigue resistance, toughness, and interfacial adhesion properties throughout its lifespan. Here, we investigated the multiscale graded mineralization structure and their strengthening mechanisms within the 30-micron soft-hard interface at the root-bone junction. This graded interface, featuring interdigitated structures and an exponential increase in modulus, undergoes a phase transition from amorphous calcium phosphate (ACP) to gradually matured hydroxyapatite (HAP) crystals, regulated by location-specific distributed biomolecules. In coordination with collagen fibril deformation and reorientation, the in situ tensile mechanical experiments and molecular dynamic simulations revealed that immature ACP particles debond from the collagenous matrix and translocate to dissipate energy, while the progressively matured HAP crystals with high stiffness pins propagating cracks, thereby enhancing both the toughness and fatigue resistance of the interface. To further validate our findings, we built biomimetic soft-hard interfaces with phase-transforming mineralization which exhibited boosted strength, toughness, and interface adhesion. This interface model is generalizable to other material joints and provides a blueprint for developing robust soft-hard composites across various applications.

Preparation of Hydroxyapatite-Aligned Collagen Sheets and Their Evaluation for Fibroblast Adhesion and Collagen Secretion.

The structure of many native tissues consists of aligned collagen (Col) fibrils, some of which are further composited with dispersed hydroxyapatite (HAp) nanocrystals. Accurately mimicking this inherent structure is a promising approach to enhance scaffold biocompatibility in tissue engineering. In this study, biomimetic sheets composed of highly aligned Col fibrils were fabricated using a plastic compression and tension method, followed by the deposition of HAp nanocrystals on the surface via an alternate soaking method. The fabricated Col sheets exhibited high solid density, retained the native periodicity (D-band) of Col fibrils, and displayed plate-like HAp nanocrystals dispersed on their surface. In vitro experiments demonstrated that these sheets could regulate fibroblasts adhesion, inducing more elongated nuclei and oriented actin bundles on the aligned Col sheets. Analysis of focal adhesion assembly revealed greater cell focal adhesions on the aligned composite sheets compared to those with random Col fibril structures. Fibroblasts cultured on aligned Col with partly HAp-mineralized sheets exhibited the highest cell-extracellular matrix (ECM) protein secretion, highlighting that HAp incorporation and fibroblast alignment synergistically promote early ECM formation and wound healing. These results suggest that highly aligned Col fibrils with dispersed HAp nanocrystals, closely mimicking the microarchitecture of natural tissues, have significant potential to control cell adhesion and protein secretion for tissue engineering applications.

Unveiling an innovative sustainable blue resource-ecofriendly extraction technique towards a circular economy: Optimization of natural deep eutectic solvent and ultrasonication synergistic pathways for type-I collagen refined recovery from discarded fish scales.

A vast sum of fish waste is being annually discarded by marine fishing industries imposing serious environmental pollution concerns. However, these aquatic discarded matters are captivating sources of collagen, a fibrous protein with eminent social and economic relevance. Collagen is conventionally recovered using outdated complex processes requiring many reagents, multiple steps, and extended periods. Hereupon, the current project is the first work on the isolation of Seabass fish scales (FSC) type-I collagen, with preserved secondary and triple helical structures of the native collagen, developing a simple, green, cost-effective, and eco-friendly methodology, utilizing sustainable natural deep eutectic solvents (NADES)-assisted ultrasonication (US) technical route. The operational conditions were optimized based on the one-factor-at-a-time modeling to maximize the yield with no alteration of collagen integrity. Recorded data confirmed type-I collagen with preserved triple helix integrity and thermal stability, improved bio-functionalities, in vitro fibril formation, and functional performances. Finally, the in vitro hemolysis and cytotoxicity tests confirmed the extracted collagens biocompatibility, demonstrating the feasibility of Seabass FSC waste and a NADES-coupled US brief process (20 min) to establish a more sustainable eco-friendly pathway to isolate high-quality type I-collagen, as an attempt to rise industries awareness about wastes valorization within the scheme of circular economy.

Tendon Inversion Improves Tendon-to-Bone Healing in a Rat Bicep Tenodesis Model.

Tendon-to-bone repair remains a surgical challenge. Although bone tunnel fixation is a common surgical technique whereby soft tissue is expected to heal against a bone tunnel interface, contemporary methods have yet to recapitulate biomechanical similarity to the native enthesis. In this study, we aimed to understand how inside-out longitudinal tendon inversion affects bone tunnel healing with the hypothesis that inversion removes the gliding epitenon surface to facilitate interface healing. Forty male Sprague-Dawley rats underwent either native tendon tenodesis (control group) or tendon inversion tenodesis (experimental group). Interface tissue was harvested 8 weeks after surgery. Biomechanical testing was performed to assess tensile strength and modes of failure. Histology was performed to assess tissue architecture, and immunohistochemistry confirmed the disruption of epitendinous lubricin from interface tissues. Maximum tensile strength increased after tendon inversion compared with control surgery. The extracellular matrix protein lubricin was reduced with tendon inversion, and specimens with tendon inversion had greater healing scores and collagen fibril alignment at the healing interface. Tendon inversion has the potential to improve bone tunnel healing in rats. Our findings suggest that longitudinal tendon inversion, or inverse tubularization, in a rat biceps model improves tendon-to-bone healing in part because of disruption of the epitendinous surface at the bone healing interface. This work provides molecular insight into future improvements for tendon-to-bone repair surgical techniques.

Bone quality in Pycnodysostosis: micropetrosis, locally distorted osteocyte lacuno-canalicular network and heterogenous mineralization pattern in an adult female patient with multiple fractures

Abstract Pycnodysostosis is a very rare skeletal dysplasia caused by biallelic loss-of-function mutations in cathepsin K, a proteolytic enzyme highly expressed by osteoclasts. Deficiency of cathepsin K impairs bone resorption, and bone remodeling and leads to progressive osteosclerosis and bone fragility. Moreover, cathepsin K is also expressed by mature osteocytes. Whether the density, size and viability of osteocytes and the osteocyte lacuno-canalicular network (OLCN) are also altered and thereby impact bone quality in pycnodysostosis has not been explored. We used light microscopy, quantitative backscattered electron imaging and confocal laser scanning microscopy to examine bone material obtained from a 57-year-old female patient obtained during surgical correction after femoral head fracture. The cortex consisted of a compact shell of multilayered collagen fibrils oriented in parallel to the periosteum reflecting vigorous primary bone apposition, multiple osteons with concentrically ordered lamellae and scattered patches of woven bone. The trabecular area was very dense with BV/TV, varying locally from 30.3% to 67.4%. The bone matrix was overmineralized (average calcium content: +7.5% versus reference values, with a fivefold increase of highly mineralized areas >27 weight % calcium). Numerous multinucleated osteoclasts and fringes of demineralized matrix were viewed on bone surfaces. The density (number/mm2: 193 to 223) and area (20 μm2) of the osteocyte lacunae and their canalicular length (0.05 μm/μm3 bone volume) were within normal range. However, numerous bone packets exhibited (hyper)mineralized osteocyte lacunas (micropetrosis) resulting in a locally disrupted OLCN. In summary, our data indicate that in pycnodysostosis not only osteoclast function is impaired but also osteocyte viability is decreased, leading to micropetrosis, distorted OLCN and heterogenous mineralization pattern. Thus, osteoclasts and osteocytes both contribute to reduce bone quality. However, the presence of a dense osteocyte network in large areas of the sample indicates that cathepsin K is not essential for the formation of the OLCN.

HEMA-free versus HEMA-containing adhesive systems: a systematic review

BackgroundHydrophilic monomer 2-hydroxyethyl methacrylate (HEMA)-free adhesive systems are gaining increasing popularity nowadays. Although the addition of HEMA to dental adhesives improves dentin wettability and resin diffusion into demineralized collagen fibrils, HEMA’s high hydrophilicity can lead to hydrolytic degradation of the adhesive interface. Thus, HEMA-free adhesive systems have been developed. Unfortunately, the lack of HEMA in the adhesive composition may lead to a separation phase between hydrophobic and hydrophilic components. The aim of this systematic review was to evaluate the clinical performance of HEMA-free adhesive systems and compare them with HEMA-containing ones.MethodsAn electronic search of The National Library of Medicine (MEDLINE/PubMed) was conducted. Eligibility criteria were reporting empirical data from clinical studies published between 2013 and 2023 about the clinical performance of HEMA-free adhesive systems for direct resin composite restorations. Studies with at least 2-year clinical follow-up done in permanent dentition in any form of cavities were selected. The included studies were assessed for risk of bias using the modified Cochrane Collaboration tool criteria.ResultsThe database search returned 147 studies; a total of 7 studies were included in this review; the majority of studies reported no significant difference between the two types of adhesives for the parameter of retention.ConclusionsHEMA-free adhesive systems exhibited good clinical performance with regard to retention. There was some concern about their influence on marginal adaptation and marginal discoloration due to the conflicted results reported by the included trials. Thus, the results need to be confirmed with long-term evaluations.Systematic review registrationPROSPERO CRD42023448952.

Aging-Induced Discrepant Response of Fracture Healing is Initiated from the Organization and Mineralization of Collagen Fibrils in Callus.

Fracture healing is a complex process during which the bone restores its structural and mechanical integrity. Collagen networks and minerals are the fundamental components to rebuild the bone matrix in callus. It has been recognized that bone quality could be impaired during aging. However, how the structural and mechanical recovery of fracture healing is influenced by aging, particularly from the perspective of organization and mineralization of the collagen network in callus, remains unclear. A tibial fracture model was established for both the young (5 weeks) and aged mice (68 weeks). On the 21st day postfracture, the characteristics of the collagen network, mineralization, and the nanoscale mechanical properties of the callus were assessed. The results indicated that aging postpones the fracture healing process, leading to incomplete microstructure, less mineral content and mineralization, and weaker mechanical properties of callus. In the aged mice, the internal fixation and mechanical immobilization promoted the mineralization of callus by increasing mineral crystal length and mineral-to-matrix ratio by 48 and 42% compared to the internal fixation and free movement control group, respectively. By contrast, in the young mice, the internal fixation and mechanical immobilization induced disordered collagen fibrils and decreased the crystal length and mineral-to-matrix ratio by 32 and 36%, compared to the internal fixation and free movement control group, respectively. The present findings suggested that the aging-induced structure and mechanical differences of callus during fracture healing initiate from the organization and mineralization of collagen fibrils. Multiscale structural and mechanical analysis suggested mechanical immobilization is beneficial to the structure, composition, and mechanics of callus in the aged mice while impairing the organization and mineralization of collagen fibril in the callus of the young mice. These findings suggested that different mechanical intervention strategies should be adopted for fracture healing at different ages, which provides valuable insights for the clinical treatment of bone fracture.

The GLP-1R agonist semaglutide reshapes pancreatic cancer associated fibroblasts reducing collagen proline hydroxylation and favoring T lymphocyte infiltration

BackgroundMetabolic syndrome represents a pancreatic ductal adenocarcinoma (PDAC) risk factor. Metabolic alterations favor PDAC onset, which occurs early upon dysmetabolism. Pancreatic neoplastic lesions evolve within a dense desmoplastic stroma, consisting in abundant extracellular matrix settled by cancer associated fibroblasts (CAFs). Hereby, dysmetabolism and PDAC association was analyzed focusing on CAF functions.MethodsPDAC development upon dysmetabolic conditions was investigated in: 1) high fat diet fed wild type immunocompetent syngeneic mice by orthotopic transplantation of pancreatic intraepithelial neoplasia (PanIN) organoids; and 2) primary pancreatic CAFs isolated from chemotherapy naïve PDAC patients with/without an history of metabolic syndrome.ResultsThe dysmetabolic-associated higher PDAC aggressiveness was paralleled by collagen fibril enrichment due to prolyl 4-hydroxylase subunit alpha 1 (P4HA1) increased function. Upon dysmetabolism, P4HA1 boosts collagen proline hydroxylation, intensifies collagen contraction strength, precluding PDAC infiltration. Noteworthy, semaglutide, an incretin agonist, prevents the higher dysmetabolism-dependent PDAC stromal deposition and allows T lymphocyte infiltration, reducing tumor development.ConclusionsThese results shed light on novel therapeutic options for PDAC patients with metabolic syndrome aimed at PDAC stroma reshape.

Hyperelastic constitutive modeling of healthy and enzymatically mediated degraded articular cartilage.

This research demonstrates a systematic curve fitting approach for acquiring parametric values of hyperelastic constitutive models for both healthy and enzymatically mediated degenerated cartilage to facilitate finite element modeling of cartilage. Several widely used phenomenological hyperelastic constitutive models were tested to adequately capture the changes in cartilage mechanics that vary with the differential/unequal abundance of matrix metalloproteinases (MMPs). Trauma and physiological conditions result in an increased production of collagenases (MMP-1) and gelatinases (MMP-9), which impacts the load-bearing ability of cartilage by significantly deteriorating its extracellular matrix (ECM). The material parameters in the constitutive equation of each hyperelastic model are significant for developing a comprehensive computational interpretation of MMP mediated degenerated cartilage. Stress-strain responses obtained from indentation test were fitted with selected Ogden, polynomial, reduced polynomial, and van der Waals hyperelastic constitutive models by optimizing their adjustable parameters (material constants). The goodness of fit of the 2nd order reduced polynomial and van der Waals model exhibited the closest data fitting with the experimental stress-strain distributions of healthy and degraded articular cartilage. The coefficient of the shear modulus for the 2nd order reduced polynomial decreased gradually by 21.9% to 80.1% with more enzymatic degradation of collagen fibril due to the relative abundance of MMP-1 (collagenases), and 28.5% to 69.2% for the van der Waals model. Our findings showed that the major materials coefficients of the models were reduced in the degenerated cartilages, and the reduction varied differentially with the relative abundance of MMPs-1 and 9, correlating the severity of degeneration. This work advances the understanding of cartilage mechanics and offers insights into the impact of biochemical (enzymatic) effects on cartilage degradation.

Downregulation of collagen IV deposition and ITGB1-FAK signaling pathway to inhibit adipogenesis: A novel mechanism of swertiamarin in treating type 2 diabetes mellitus.

Extracellular matrix (ECM) and integrins are important biological macromolecules. ECM especially collagen IV (COLIV) deposition modulates the integrin-FAK signaling pathway involved in adipogenesis and is strongly associated with insulin resistance. Type 2 diabetes mellitus (T2DM) mice were given swertiamarin (STM) by intragastric administration. STM reduced body weight, blood glucose, and lipid levels and enhanced insulin sensitivity in diabetic mice. The lipid accumulation in liver, gastrocnemius muscle, and inguinal subcutaneous white adipose tissue (igSWAT) were significantly reduced by STM. Bioinformatics analysis revealed a connection between ECM, ITGB1/FAK, and PI3K/Akt signaling pathways. STM downregulated the adipogenesis, IRβ expression, COLIV deposition, ITGB1/FAK, and PI3K/Akt signaling pathways in igSWAT of diabetic mice. In vitro, STM inhibited the glucose uptake and differentiation of adipocytes, and downregulated adipogenesis-related gene and protein expression. STM is bound to ITGB1 and downregulated COLIV deposition, ITGB1/FAK, and PI3K/Akt signaling pathways. When we overexpressed FAK, the effects of STM on downstream PI3K/Akt signaling pathway and adipogenesis were attenuated. In conclusion, STM reduced COLIV deposition and binding with ITGB1 to downregulate ITGB1/FAK signaling pathway, further the downstream PI3K/Akt signaling pathway was inhibited to reduce adipogenesis and ameliorated T2DM. Thus, these signals may be a novel mechanism of STM in treating T2DM.

Impact of High Glucose on Bone Collagenous Matrix Composition, Structure, and Organization: An Integrative Analysis Using an Ex Vivo Model.

Diabetes mellitus is a widespread metabolic disorder linked to numerous systemic complications, including adverse effects on skeletal health, such as increased bone fragility and fracture risk. Emerging evidence suggests that high glucose may disrupt the extracellular matrix (ECM) of bone, potentially altering its composition and organization. Collagen, the primary organic component of the ECM, is critical for maintaining structural integrity and biomechanical properties. However, definitive evidence and a comprehensive understanding of the molecular mechanisms through which high glucose impacts the ECM and collagen remain elusive. This study employed an ex vivo embryonic chicken femur model to investigate the effects of high glucose on the collagenous matrix. A comprehensive approach integrating histological evaluation, histomorphometry, ATR-FTIR spectroscopy, and proteomics was adopted to unravel structural, biochemical, and molecular changes in the ECM. Histomorphometric analysis revealed disrupted collagen fibril architecture, characterized by altered fibril diameter, alignment, and spatial organization. ATR-FTIR spectroscopy highlighted biochemical modifications, including non-enzymatic glycation that impaired collagen crosslinking and reduced matrix integrity. Proteomic profiling unveiled significant alterations in ECM composition and function, including downregulation of key collagen crosslinking enzymes and upregulation of inflammatory and coagulation pathways. High glucose profoundly disrupts the collagenous matrix of bone, weakening its structural integrity and organization. These findings emphasize the critical impact of high glucose environments on extracellular matrix composition and bone quality, offering insights into the mechanisms behind diabetic bone fragility and guiding future research toward targeted therapeutic strategies.