Enzymatic Cross-links Research Articles

The role of the interfaces and cross-links on the mechanical behavior of mineralized collagen fibrils. A numerical approach

Mechanical properties of bone tissue are highly dependent on its hierarchical structure. The presence of microcracks and diffuse damage in lamellar bone is correlated with the failure of the collagen-mineral interface in mineralized collagen fibrils (MCF). The main goal of this work is to evaluate the mechanical behavior of the interfaces and quantify the stiffness loss of the MCF associated with different failure mechanisms, under controlled in-plane displacement. Additionally, we aim to study the role of the cross-links on the fibril mechanical response, beyond the interface failure. Inter- and intra-microfibrilar cross-links are analyzed. In order to address the first issue, a detailed representative volume of the MCF is analyzed by means of the finite element method, under the assumption of plane strain and periodic boundary conditions. In this model the interfaces between constituents are modeled with an exponential cohesive law. Enzymatic cross-links, located at the molecular terminals connecting each 4D (D=67nm) staggered molecules, are represented by non-linear springs. Three in-plane controlled deformations are applied. The results of this work provide the anisotropic stiffness loss of the tissue involved in the different failure mechanisms at the nano-scale length. The initiation of microcracks and the presence of damage zones are compatible with the failure mechanisms observed at interfaces. Interface failure entails a progressive stiffness loss, bringing a non-linear behavior of bone. The strength obtained for the longitudinal maximum deformation is more than 20 times the transverse strength and 3.5 times the shear strength. The quantification of the reduction percentage in the elastic moduli and the shear stiffness when the fibril is damaged, has a potential application in improving failure criteria based on degradation of elastic constants. When longitudinal elongation is applied, the mechanical contribution of the cross-links in delaying the failure initiation of the interface is shown. Likewise, results of this work confirm the scarce influence of the cross-links in the strain range analyzed. Additionally, a three-dimensional numerical model of several microfibrils is defined with the aim of analyzing the mechanical relevance of inter- and intra-microfibrilar cross-links, beyond the interface failure. Results confirm that cross-links transfer the load when strain increases, being highlighted the mechanical competence of the trivalent cross-links.

Enzymatic and Non-enzymatic Collagen Cross-Links and Fracture Occurrence in Type 1 Diabetes Patients.

Increased fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD) by DXA. Advanced glycation end-products (AGEs) have been implicated in the increased fracture risk in T1D, yet recent publications question this. To test the hypothesis that enzymatic collagen cross-links rather than AGEs correlate with fracture incidence in T1D, we analyzed iliac crest biopsies from sex-matched, fracturing T1D patients (N = 5; T1DFx), 6 non-fracturing T1D patients (T1DNoFx), and 6 healthy subjects, by Raman microspectroscopy as a function of tissue age (based on double fluorescent labels), in intracortical and trabecular bone, to determine pyridinoline (Pyd), ε-N-Carboxymethyl-L-lysine, and pentosidine (PEN)). There were no differences in the clinical characteristics between the T1DFx and T1DNoFx groups. At trabecular forming surfaces, T1DFx patients had higher PEN and Pyd content compared to T1DNoFx ones. Previous studies have shown that elevated PEN does not necessarily correlate with fracture incidence in postmenopausal, long-term T1D patients. On the other hand, the elevated Pyd content in the T1DFx patients would be consistent with published studies showing a significant correlation between elevated trivalent enzymatic collagen cross-links and fracture occurrence independent of BMD. Collagen fibers with high Pyd content are more brittle. Thus, a plausible suggestion is that it is the enzymatic collagen cross-links that either by themselves or in combination with the adverse effects of increased AGE accumulation that result in fragility fracture in T1D.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Validation of Fourier Transform Infrared Microspectroscopy for the Evaluation of Enzymatic Cross-Linking of Bone Collagen.

Enzymatic cross-linking of the bone collagen is important to resist to crack growth and to increased flexural strength. In the present study, we proposed a new method for assessment of enzymatic cross-link based on Fourier transform infrared (FTIR) microspectroscopy that takes into account secondary structure of type I collagen. Briefly, femurs were collected from sham or ovariectomized mice and subjected either to high-performance liquid chromatography-mass spectrometry or embedded in polymethylmethacrylate, cut and analyzed by FTIR microspectroscopy. FTIR acquisition was recorded before and after ultraviolet (UV) exposure or acid treatment. In addition, femurs from a second animal study were used to compare gene expression of Plod2 and Lox enzymes and enzymatic cross-links determined by FTIR microspectroscopy. We evidenced here that intensities and areas of subbands located at ~1660, ~1680, and ~1690cm-1 were positively and significantly associated with the concentration of pyridinoline (PYD), deoxypyridinoline, or immature dihydroxylysinonorleucine/hydroxylysinonorleucine cross-links. Seventy-two hours exposure to UV light significantly reduced by ~86% and ~89% the intensity and area of the ~1660cm-1 subband. Similarly, 24h of acid treatment significantly reduced by 78% and 76% the intensity and area of the ~1690cm-1 subband. Plod2 and Lox expression were also positively associated to the signal of the ~1660 and ~1690cm-1 subbands. In conclusion, our study provided a new method for decomposing the amide I envelope of bone section that positively correlates with PYD and immature collagen cross-links. This method allows for investigation of tissue distribution of enzymatic cross-links in bone section.

The influence of AGEs and enzymatic cross-links on the mechanical properties of collagen fibrils

Collagen, one of the main building blocks for various tissues, derives its mechanical properties directly from its structure of cross-linked tropocollagen molecules. The cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. For instance, enzymatic cross-links (ECLs), one particular type of cross-links, are known for stabilizing the structure of the fibril and improving material properties, while cross-linking AGEs (Advanced-Glycation Endproducts) have been shown to accumulate and impair the mechanical properties of collageneous tissues. However, the reasons for whether and how a given type of cross-link improves or impairs the material properties remain unknown, and the exact relationship between the cross-link properties and density, and the fibrillar behavior is still not well understood. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules, which leads to failure by breaking of bonds within the tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behavior due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.

Correlation analysis of cartilage wear with biochemical composition, viscoelastic properties and friction

Healthy articular cartilage exhibits remarkable resistance to wear, sustaining mechanical loads and relative motion for decades. However, tissues that replace or repair cartilage defects are much less long lasting. Better information on the compositional and material characteristics that contribute to the wear resistance of healthy cartilage could help guide strategies to replace and repair degenerated tissue. The main objective of this study was to assess the relationship between wear of healthy articular cartilage, its biochemical composition, and its viscoelastic material properties. The correlation of these factors with the coefficient of friction during the wear test was also evaluated. Viscoelastic properties of healthy bovine cartilage were determined via stress relaxation indentation. The same specimens underwent an accelerated, in vitro wear test, and the amount of glycosaminoglycans (GAGs) and collagen released during the wear test were considered measures of wear. The frictional response during the wear test was also recorded. The GAG, collagen and water content and the concentration of the enzymatic collagen crosslink pyridinoline were quantified in tissue that was adjacent to each wear test specimen. Finally, correlation analysis was performed to identify potential relationships between wear characteristics of healthy articular cartilage with its composition, viscoelastic material properties and friction. The findings suggest that stiffer cartilage with higher GAG, collagen and water content has a higher wear resistance. Enzymatic collagen crosslinks also enhance the wear resistance of the collagen network. The parameters of wear, composition, and mechanical stiffness of cartilage were all correlated with one another, suggesting that they are interrelated. However, friction was largely independent of these in this study. The results identify characteristics of healthy articular cartilage that contribute to its remarkable wear resistance. These data may be useful for guiding techniques to restore, regenerate, and stabilize cartilage tissue.

Application of steam explosion treatment on the collagen peptides extraction from cattle bone

In this study, steam explosion (SE) treatment was applied to extract collagen peptides from cattle bone. The SE treatment conditions included steam pressure: 1.5 MPa (200.43 °C), 2.0 MPa (213.85 °C), and 2.5 MPa (224.99 °C), and reaction time: 10 min, 20 min, and 30 min. With the increase of pressure and reaction time, the protein recovery rate improved, and reached to 60.5% at 2.0 MPa–30 min. SE significantly decreased the molecular weight of collagen peptides (P < 0.05) with increasing pressure and reaction time. However, at 2.5 MPa, SE darkened the sample color resulting in dark yellow. Particle size analysis and SEM images indicated that SE decreased the particle size and destroyed microstructure of cattle bone powder. FT-IR analysis showed that SE induced distinct characteristic peaks of hydroxyapatite in cattle bone powder. SE facilitated peptides release from collagen by destroying peptide bonds and enzymatic cross-links. Amino acid analysis indicated SE could not change the amino acid composition of collagen peptides. The calcium-binding ability and osteoblast proliferative activity of extracted collagen peptides were 44.7 μg/mg and 126.7% (200 μg/mL), respectively. The findings put forward data support and a scientific basis for the application and development of SE technology to collagen peptides extraction and high-value utilization of cattle bone.

Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay.

DNA-protein crosslinks (DPCs) are steric hindrances to DNA metabolic processes and the removal and repair of DPCs is a rapidly evolving area of research. A critical component of deciphering this repair pathway is developing techniques that detect and quantify specific types of DPCs in cells. Here we describe a protocol for direct detection of enzymatic DPCs from mammalian cells-the RADAR assay. The method involves isolating genomic DNA and DPCs from cells and binding them to nitrocellulose membrane with a vacuum slot blot manifold. DPCs are detected using antibodies raised against the protein of interest and quantified by normalizing to a DNA loading control. The RADAR assay allows for the detection of specific types of DPCs and the sensitive analysis of the DNA-protein crosslinking activity of various drugs, is adaptable across different cell types and conditions, and requires little specialized equipment.

Collagen cross-link profiles and mineral are different between the mandible and femur with site specific response to perturbed collagen

Compromises to collagen and mineral lead to a decrease in whole bone quantity and quality in a variety of systemic diseases, yet, clinically, disease manifestations differ between craniofacial and long bones. Collagen alterations can occur through post-translational modification via lysyl oxidase (LOX), which catalyzes enzymatic collagen cross-link formation, as well as through non-enzymatic advanced glycation end products (AGEs) such as pentosidine and carboxymethyl-lysine (CML). Characterization of the cross-links and AGEs, and comparison of the mineral and collagen modifications in craniofacial and long bones represent a critical gap in knowledge. However, alterations to either the mineral or collagen in bone may contribute to disease progression and, subsequently, the anatomical site dependence of a variety of diseases. Therefore, we hypothesized that collagen cross-links and AGEs differ between craniofacial and long bones and that altered collagen cross-linking reduces mineral quality in an anatomic location dependent. To study the effects of cross-link inhibition on mineralization between anatomical sites, beta-aminoproprionitrile (BAPN) was administered to rapidly growing, 5–8 week-old male mice. BAPN is a dose-dependent inhibitor of LOX that pharmacologically alters enzymatic cross-link formation. Long bones (femora) and craniofacial bones (mandibles) were compared for mineral quantity and quality, collagen cross-link and AGE profiles, and tissue level mechanics, as well as the response to altered cross-links via BAPN. A highly sensitive liquid chromatography/mass spectrometry (LC-MS) method was developed which allowed for quantification of site-dependent accumulation of the advanced glycation end-product, carboxymethyl-lysine (CML). CML was ∼8.3× higher in the mandible than the femur. The mandible had significantly higher collagen maturation, mineral crystallinity, and Young's modulus, but lower carbonation, than the femur. BAPN also had anatomic specific effects, leading to significant decreases in mature cross-links in the mandible, and an increase in mineral carbonation in the femur. This differential response of both the mineral and collagen composition to BAPN between the mandible and femur highlights the need to further understand how inherent compositional differences in collagen and mineral contribute to anatomic-site specific manifestations of disease in both craniofacial and long bones.

Divergent mechanical properties of older human male femora reveal unique combinations of morphological and compositional traits contributing to low strength.

Bone strength is generally thought to decline with aging and prior work has compared traits between younger and older cohorts to identify the structural and compositional changes that contribute to fracture risk with age. However, for men, the majority of individuals do not fracture a bone in their lifetime. While fracture occurrence is multifactorial, the absence of fracture in the majority of males suggests that some individuals maintain bone strength or do not lose enough strength to fracture, whereas others do lose strength with aging. Consequently, not all structural and material changes observed with age may lead to strength-decline. We propose that consideration of different subgroups of older individuals will provide a more precise understanding of which structural and material changes directly contribute to strength-decline. We identified subgroups using latent profile analysis (LPA), which is a clustering-based algorithm that takes multiple continuous variables into account. Human cadaveric male femoral diaphyses (n=33, 26-89years) were subjected to whole bone and tissue-level mechanical tests. Morphological traits, porosity, and cortical tissue mineral density (Ct.TMD) were obtained, as were measures of enzymatic cross-links and the advanced glycation end product, pentosidine (PEN). A univariate analysis first identified a younger cohort (YNG, n=11) and older cohort (n=22). LPA was then conducted using three mechanical traits (whole bone strength, tissue-level strength, and tissue-level post-yield strain), resulting in a further stratification of the older group into two similarly aged groups (p=0.558), but one with higher (OHM, n=16) and another with lower mechanical properties (OLM, n=6). The OLM group exhibited lower whole bone strength (p=0.016), tissue-level strength (p<0.001), and tissue-level post-yield strain (p<0.001) compared to the YNG group. Meanwhile, the OHM only exhibited significantly lower tissue-level post-yield strain (p<0.001), compared to the YNG group. Between the two older groups, the OHM group exhibited higher whole bone strength (p=0.037), tissue-level strength (p=0.006), and tissue-level post-yield strain (p=0.012) than the OLM group. Probing the morphological and compositional relationships among the three groups, both OHM and OLM exhibited increased PEN content (p<0.001, p=0.008 respectively) and increased Log(cortical pore score) relative to YNG (p=0.003, p<0.001 respectively). Compared to the OHM group, the OLM also exhibited increased marrow area (p=0.049), water content (p=0.048), and decreased Ct.TMD (p=0.005). The key traits that were unique to the OLM group compared to YNG were decreased Ct.TMD (p<0.001) and increased Log(porosity) (p=0.002). There were many properties that differed between the younger and older groups, but not all were associated with reduced mechanical properties, highlighting the relative importance of certain age-related traits such as porosity, Ct.TMD, water content, and marrow area that were unique to the OLM group. Overall, this work supports the hypothesis that there are subgroups of men showing different strength-decline trajectories with aging and establishes a basis for refining our understanding of which age-related changes are directly contributing to decreased strength.

Cytoprotection of Human Progenitor and Stem Cells through Encapsulation in Alginate Templated, Dual Crosslinked Silk and Silk-Gelatin Composite Hydrogel Microbeads.

Susceptibility of mammalian cells against harsh processing conditions limit their use in cell transplantation and tissue engineering applications. Besides modulation of the cell microenvironment, encapsulation of mammalian cells within hydrogel microbeads attract attention for cytoprotection through physical isolation of the encapsulated cells. The hydrogel formulations used for cell microencapsulation are largely dominated by ionically crosslinked alginate (Alg), which suffer from low structural stability under physiological culture conditions and poor cell-matrix interactions. Here the fabrication of Alg templated silk and silk/gelatin composite hydrogel microspheres with permanent or on-demand cleavable enzymatic crosslinks using simple and cost-effective centrifugation-based droplet processing are demonstrated. The composite microbeads display structural stability under ion exchange conditions with improved mechanical properties compared to ionically crosslinked Alg microspheres. Human mesenchymal stem and neural progenitor cells are successfully encapsulated in the composite beads and protected against environmental factors, including exposure to polycations, extracellular acidosis, apoptotic cytokines, ultraviolet (UV) irradiation, anoikis, immune recognition, and particularly mechanical stress. The microbeads preserve viability, growth, and differentiation of encapsulated stem and progenitor cells after extrusion in viscous polyethylene oxide solution through a 27-gauge fine needle, suggesting potential applications in injection-based delivery and three-dimensional bioprinting of mammalian cells with higher success rates.

Multiscale Characterization of Type I Collagen Fibril Stress-Strain Behavior under Tensile Load: Analytical vs. MD Approaches.

Type I collagen is one of the most important proteins in the human body because of its role in providing structural support to the extracellular matrix of the connective tissues. Understanding its mechanical properties was widely investigated using experimental testing as well as molecular and finite element simulations. In this work, we present a new approach for defining the properties of the type I collagen fibrils by analytically formulating its response when subjected to a tensile load and investigating the effects of enzymatic crosslinks on the behavioral response. We reveal some of the shortcomings of the molecular dynamics (MD) method and how they affect the obtained stress–strain behavior of the fibril, and we prove that not only does MD underestimate the Young’s modulus and the ultimate tensile strength of the collagen fibrils, but also fails to detect the mechanics of some stretching phases of the fibril. We prove that non-crosslinked fibrils have three tension phases: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime related to the stretching of the backbone of the tropocollagen molecules, and (iii) a plastic regime dominated by molecular sliding. We also show that for crosslinked fibrils, the second regime can be subdivided into three sub-regimes, and we define the properties of each regime. We also prove, analytically, the alleged MD quadratic relation between the ultimate tensile strength of the fibril and the concentration of enzymatic crosslinks (β).

Regulators of collagen crosslinking in developing and adult tendons.

Tendons are collagen-rich musculoskeletal tissues that possess the mechanical strength needed to transfer forces between muscles and bones. The mechanical development and function of tendons are impacted by collagen crosslinks. However, there is a limited understanding of how collagen crosslinking is regulated in tendon during development and aging. Therefore, the objective of the present review was to highlight potential regulators of enzymatic and non-enzymatic collagen crosslinking and how they impact tendon function. The main collagen crosslinking enzymes include lysyl oxidase (LOX) and the lysyl oxidase-like isoforms (LOXL), whereas non-enzymatic crosslinking is mainly mediated by the formation of advanced glycation end products (AGEs). Regulators of the LOX and LOXL enzymes may include mechanical stimuli, mechanotransducive cell signaling pathways, sex hormones, transforming growth factor (TGF)β family, hypoxia, and interactions with intracellular or extracellular proteins. AGE accumulation in tendon is due to diabetic conditions and aging, and can be mediated by diet and mechanical stimuli. The formation of these enzymatic and non-enzymatic collagen crosslinks plays a major role in tendon biomechanics and in the mechanisms of force transfer. A more complete understanding of how enzymatic and non-enzymatic collagen crosslinking is regulated in tendon will better inform tissue engineering and regenerative therapies aimed at restoring the mechanical function of damaged tendons.

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA–protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

The Effect of Enzymatic Crosslink Degradation on the Mechanics of the Anterior Cruciate Ligament: A Hybrid Multi-Domain Model

The anterior cruciate ligament’s (ACL) mechanics is an important factor governing the ligament’s integrity and, hence, the knee joint’s response. Despite many investigations in this area, the cause and effect of injuries remain unclear or unknown. This may be due to the complexity of the direct link between macro- and micro-scale damage mechanisms. In the first part of this investigation, a three-dimensional coarse-grained model of collagen fibril (type I) was developed using a bottom-up approach to investigate deformation mechanisms under tensile testing. The output of this molecular level was used later to calibrate the parameters of a hierarchical multi-scale fibril-reinforced hyper-elastoplastic model of the ACL. Our model enabled us to determine the mechanical behavior of the ACL as a function of the basic response of the collagen molecules. Modeled elastic response and damage distribution were in good agreement with the reported measurements and computational investigations. Our results suggest that degradation of crosslink content dictates the loss of the stiffness of the fibrils and, hence, damage to the ACL. Therefore, the proposed computational frame is a promising tool that will allow new insights into the biomechanics of the ACL.

Habitual side-specific loading leads to structural, mechanical, and compositional changes in the patellar tendon of young and senior lifelong male athletes.

Effects of lifelong physical activity on tendon function have been investigated in cross-sectional studies, but these are at risk of "survivorship" bias. Here, we investigate whether lifelong side-specific loading is associated with greater cross-sectional area (CSA), mechanical properties, cell density (DNA content), and collagen cross-link composition of the male human patellar tendon (PT), in vivo. Nine seniors and six young male lifelong elite badminton players and fencers were included. CSA of the PT obtained by 3-tesla MRI and ultrasonography-based bilateral PT mechanics were assessed. Collagen fibril characteristics, enzymatic cross links, nonenzymatic glycation (autofluorescence), collagen, and DNA content were measured biochemically in PT biopsies. The elite athletes had a ≥15% side-to-side difference in maximal knee extensor strength, reflecting chronic unilateral sport-specific loading patterns. The PT CSA was greater on the lead extremity compared with the nonlead extremity (17%, P = 0.0001). Furthermore, greater tendon stiffness (18%, P = 0.0404) together with lower tendon stress (22%, P = 0.0005) and tendon strain (18%, P = 0.0433) were observed on the lead extremity. No effects were demonstrated from side-to-side for glycation, enzymatic cross link, collagen, and DNA content (50%, P = 0.1160). Moreover, tendon fibril density was 87 ± 28 fibrils/μm2 on the lead extremity and 68 ± 26 fibrils/μm2 on the nonlead extremity (28%, P = 0.0544). Tendon fibril diameter was 86 ± 14 nm on the lead extremity and 94 ± 14 nm on the nonlead extremity (-9%, P = 0.1076). These novel data suggest that lifelong side-specific loading in males yields greater patellar tendon size and stiffness possibly with concomitant greater fibril density but without changes of collagen cross-link composition.NEW & NOTEWORTHY The present data demonstrate that lifelong side-specific loading yields greater patellar tendon structure on the lead extremity without affecting glycation that is associated with aging. These novel data suggest that lifelong side-specific habitual loading induce structural alterations that may serve to improve the mechanical properties of the tendon.

Molecular insights into the repair mechanism of enzymatic DNA-protein cross-link damage induced by a DNMT inhibitor.

DNA-protein cross-links (DPCs) are a special type of DNA damage that is formed when a protein that participates in DNA transactions is irreversibly and covalently linked to DNA bases. DPCs are formed after exposure to chemicals, anticancer drugs, and ionizing radiation resulting in enzymatic and non-enzymatic DPCs. Up to now, the exact repair mechanism of DPCs has not been fully identified. For this reason, the outputs of the current study provide molecular insights about the repair mechanism of a specific type of enzymatic DPCs formed by DNA-trapped DNMT. In the current study, cells treated with 5-azadC show a clear cytotoxic effect with LD20 ranging from 0.4 to 5 µM. The analysis of DPCs by fluorescence labeling reveals that 5-azadC induces DPCs in a dose-dependent manner. Moreover, cells that are deficient in homologous recombination (HR) pathway (RAD51D and XRCC3) were 2-4 folds sensitive to 5-azadC compared to wild type. In contrast, cells deficient in nucleotide excision repair (NER) pathway (XPD and XPF) and FANC pathway (Fanc A, B, and C) didn’t show any sensitivity to 5-azadC doses. Unexpectedly, mutation in nonhomologous end-joining (NHEJ) gene (DNA-PKcs) gives cells a great survival. Also, double-strand breaks (DSBs) were significantly detected in HR mutant (RAD51D) compared with a wild type indicating that the trapped DNMT by 5-azadC hamper the progression of stalled replication fork generating DSBs. Further investigations are required to understand the accurate mechanism by which cells can repair DPCs that will provide good knowledge regarding the targeting of DPCs in cancer treatment.

Mineral and organic matrix composition at bone forming surfaces in postmenopausal women with osteoporosis treated with either teriparatide or zoledronic acid.

The ability of bone to resist fracture is dependent on the composite nature of its mineral and organic matrix content. Teriparatide (TPTD) and zoledronic acid (ZOL) are approved anabolic and antiresorptive therapies, respectively, to reduce fracture risk in women with postmenopausal osteoporosis. In the SHOTZ study, postmenopausal women with osteoporosis were treated with TPTD (20 μg daily, subcutaneous) or ZOL (5 mg/year, intravenous infusion) for 24 months. Iliac crest biopsies were obtained at 6 months and again at 24 months from approximately one third of the original study cohort. To investigate the early effects of these two drugs on the quality of newly formed bone, we used vibrational spectroscopic techniques to analyze tetracycline-labelled transiliac biopsies obtained from participants at the 6-month time point. Raman spectra were acquired at forming trabecular and intra-cortical surfaces (identified by fluorescent double labels), to determine mineral, organic matrix, glycosaminoglycan, and tissue water content, as well as mineral maturity/crystallinity at three specific tissue ages (1-5, 15, and ≥25 days). Fourier transformed infrared microspectroscopy was used to determine pyridinoline/divalent collagen cross-link ratios. At 6 months, treatment with TPTD versus ZOL resulted in lower mineral and higher organic matrix content, increased tissue water content, and lower mineral/matrix, mineral maturity/crystallinity, glycosaminoglycan content, and pyridinoline/divalent enzymatic collagen cross-link ratio. Our results suggest that TPTD and ZOL have differential effects on material properties of newly formed bone at individual remodeling sites, highlighting their different mechanisms of action.

Mass spectrometric quantitation of AGEs and enzymatic crosslinks in human cancellous bone

Advanced glycation end-products (AGEs) deteriorate bone strength. Among over 40 species identified in vivo, AGEs other than pentosidine were roughly estimated as total fluorescent AGEs (tfAGEs) due to technical difficulties. Using LC-QqTOF-MS, we established a system that enabled the quantitation of five AGEs (CML, CEL, MG-H1, CMA and pentosidine) as well as two mature and three immature enzymatic crosslinks. Human bone samples were collected from 149 patients who underwent total knee arthroplasty. Their clinical parameters were collected to investigate parameters that may be predictive of AGE accumulation. All the analytes were quantitated and showed significant linearity with high sensitivity and precision. The results showed that MG-H1 was the most abundant AGE, whereas pentosidine was 1/200–1/20-fold less abundant than the other four AGEs. The AGEs were significantly and strongly correlated with pentosidine, while showing moderate correlation with tfAGEs. Interestingly, multiple linear regression analysis revealed that gender contributed most to the accumulation of all the AGEs, followed by age, tartrate-resistant acid phosphatase-5b and HbA1c. Furthermore, the AGEs were negatively correlated with immature crosslinks. Mass spectrometric quantitation of AGEs and enzymatic crosslinks is crucial to a better understanding of ageing- and disease-related deterioration of bone strength.

External bone size identifies different strength-decline trajectories for the male human femora

Understanding skeletal aging and predicting fracture risk is increasingly important with a growing elderly population. We hypothesized that when categorized by external bone size, the male femoral diaphysis would show different strength-age trajectories which can be explained by changes in morphology, composition and collagen cross-linking. Cadaveric male femora were sorted into narrow (n = 15, 26–89 years) and wide (n = 15, 29–82 years) groups based upon total cross-sectional area of the mid-shaft normalized to bone length (Tt.Ar/Le) and tested for whole bone strength, tissue-level strength, and tissue-level post-yield strain. Morphology, cortical TMD (Ct.TMD), porosity, direct measurements of enzymatic collagen cross-links, and pentosidine were obtained. The wide group alone showed significant negative correlations with age for tissue-level strength (R2 = 0.50, p = 0.002), tissue-level post-yield strain (R2 = 0.75, p < 0.001) and borderline significance for whole bone strength (R2 = 0.14, p = 0.108). Ct.TMD correlated with whole bone and tissue-level strength for both groups, but pentosidine normalized to enzymatic cross-links correlated negatively with all mechanical properties for the wide group only. The multivariate analysis showed that just three traits for each mechanical property explained the majority of the variance for whole bone strength (Ct.Area, Ct.TMD, Log(PEN/Mature; R2 = 0.75), tissue-level strength (Age, Ct.TMD, Log(DHLNL/HLNL); R2 = 0.56), and post-yield strain (Age, Log(Pyrrole), Ct.Area; R2 = 0.51). Overall, this highlights how inter-individual differences in bone structure, composition, and strength change with aging and that a one-size fits all understanding of skeletal aging is insufficient.