Accumulation of fluorescent advanced glycation end products and carboxymethyl-lysine in human cortical and trabecular bone.

Chemical crosslinks known as advanced glycation end-products (AGEs) are associated with increased bone fracture risk and deteriorated bone mechanical properties. However, measurement of bone AGEs via ex vivo and in vitro methods has been limited to quantification of bulk fluorescent AGEs (fAGEs) and pentosidine only, which is a crosslinking fluorescent AGE. However, a non-crosslinking and non-fluorescent AGE such as carboxymethyl-lysine (CML) is found to be 40-100 times higher in quantity than pentosidine, but only one previous study has reported it in cortical bone, and one study reported it in trabecular bone. In our study, we wanted to investigate if accumulation of CML differs in cortical and trabecular compartments and if they are more strongly associated with bone mechanical properties than with fAGEs. We hypothesized that CML and fAGEs level would be higher in the trabecular compartment and show negative correlations to mechanical properties in cortical and trabecular bone. We obtained human cadaveric cortical and trabecular bone specimens, induced the formation of AGEs via the established in vitro ribosylation method, imaged specimens by microcomputed tomography to assess specimen geometry and microarchitecture, and mechanically tested cortical specimens by cyclic reference point indentation and fracture toughness tests and trabecular specimens by compression tests, followed by measurement of fAGEs and CML. fAGEs were 22 % higher in cortical bone (687 ± 44.8 ng Q/mg collagen) compared to trabecular bone (859 ± 317.1 ng Q/mg collagen), whereas CML levels were found to be 148 % higher in trabecular bone (6189.9 ± 866 ng/mg of protein) compared to cortical bone (924.6 ± 576.3 ng/mg of protein). Pooling the specimens from both the control and ribose groups, Spearman correlation analysis indicated that CML levels, but not fAGEs, are moderately associated with cortical porosity (r = +0.505, p ≤ 0.05) and mechanical properties such indentation depth (r = +0.460, p ≤ 0.05), total indentation depth (r = +0.440, p ≤ 0.05), and average energy dissipated (r = +0.465, p ≤ 0.05) in cortical bone. fAGEs showed a trend towards negative association with crack propagation toughness in cortical bone (r = -0.365, p = 0.055). No significant correlations were observed between CML and microarchitecture or mechanical properties in trabecular bone. CML levels were also associated with fAGEs in cortical bone (r = +0.596, p ≤ 0.05) but not in trabecular bone. Our preliminary findings indicate that CML, a non-crosslinking AGE, may affect bone material and mechanical properties differently than bulk fluorescent AGEs, given the higher accumulation of CML in each bone compartment. This study provides direction to future studies to quantify crosslinking and non-crosslinking AGEs separately as their effect on material and mechanical properties may be different and it would help identify better biomarkers for bone strength prediction.

Correction: Chalivendra et al. Effect of Shear Angle and Printing Orientation on Shear Constitutive Response of Additively Manufactured Acrylonitrile Butadiene Styrene. Polymers 2022, 14, 2484.

In the original publication [...].

Effect of Shear Angle and Printing Orientation on Shear Constitutive Response of Additively Manufactured Acrylonitrile Butadiene Styrene.

An experimental investigation was performed to understand the quasi-static shear response of additively manufactured (AM) acrylonitrile butadiene styrene (ABS) via fusion deposition modeling (FDM). A modified flat hat-shaped (FHS) specimen configuration was used for shear testing. The main aim of this study was to investigate the effect of four different shear angles (0°, 5.44°, 13.39°, and 20.83°) and three printing orientations (vertical build, 0°/90°, and 45°/-45°) on the shear constitutive response and shear performance of FDM-printed ABS. Scanning electron microscopy images of the failure surface were used to explain the shear response of the material. The flow shear stress of the shear stress-strain response for vertically printed specimens demonstrated a monotonic increase up to a peak shear stress and then decrease at the end of the shear zone, while for 0°/90° specimens, an increasing trend until the peak value at the end of the shear zone was observed. With increasing shear angles, all specimens printed with three printing orientations exhibited increasing shear zone size and shear strength, and the 0°/90° specimens exhibited the highest shear strength for all four shear angles. However, the specimens of the 45°/-45° orientation demonstrated the highest increase in shear strength by about 60% and in the shear strain at the end of shear zone by about 175% as the shear angle was increased from 0° to 20.83°.

Electrospinning of tyrosine-based oligopeptides: Self-assembly or forced assembly?

Short oligomeric peptides typically do not exhibit the entanglements required for the formation of nanofibers via electrospinning. In this study, the synthesis of nanofibers composed of tyrosine-based dipeptides via electrospinning, has been demonstrated. The morphology, mechanical stiffness, biocompatibility, and stability under physiological conditions of such biodegradable nanofibers were characterized. The electrospun peptide nanofibers have diameters less than 100 nm and high mechanical stiffness. Raman and infrared signatures of the peptide nanofibers indicate that the electrostatic forces and solvents used in the electrospinning process lead to secondary structures different from self-assembled nanostructures composed of similar peptides. Crosslinking of the dipeptide nanofibers using 1,6-diisohexanecyanate (HMDI) improved the physiological stability, and initial biocompatibility testing with human and rat neural cell lines indicate no cytotoxicity. Such electrospun peptides open up a realm of biomaterials design with specific biochemical compositions for potential biomedical applications such as tissue repair, drug delivery, and coatings for implants.

Mechanical and biological properties of chitin/polylactide (PLA)/hydroxyapatite (HAP) composites cast using ionic liquid solutions.

This research investigates the potential development of lobster shell waste-derived chitin reinforced with poly(lactic acid) (PLA) and nano-hydroxyapatite (nHAP) into new materials with potentially superior mechanical and thermal properties for biomedical applications. The ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was used as a solvent to prepare chitin/PLA/nHAP composites. The effect of variation of the polymer concentrations on the conduct of the resulting composite was explored. The detailed physico-mechanical, thermal and surface morphology properties were evaluated with different thermal and optical characterization techniques. When the concentration of PLA in the composite was increased from 20 to 80 wt%, the tensile strength improved by ~77% while the elongation at break and the toughness of the material decreased significantly. The addition of hydroxyapatite was observed to improve strength of the composites up to 140% with an increase in elongation at break up to 465%. Cell growth study show that the composite materials support the growth and proliferation of Ocy 454 osteocyte cells. The materials were shown to have no effect on osteocyte gene expression, as well as minimal cytotoxicity and biodegradability. These results reveal that the biocomposites would be suitable candidates for use in bone regeneration that are not exposed to excessive forces.

In Vitro-Induced High Sugar Environments Deteriorate Human Cortical Bone Elastic Modulus and Fracture Toughness.

Advanced glycation end-products (AGEs) have been suggested to contribute to bone fragility in type 2 diabetes (T2D). AGEs can be induced through in vitro sugar incubations but there is limited data on the effect of total fluorescent AGEs on mechanical properties of human cortical bone, which may have altered characteristics in T2D. Thus, to examine the effect of AGEs on bone directly in T2D patients with uncontrolled sugar levels, it is essential to first understand the fundamental mechanisms by studying the effects of controlled in vitro-induced AGEs on cortical bone mechanical behavior. Here, human cortical bone specimens from female cadaveric tibias (ages 57-87) were incubated in an in vitro 0.6 M ribose or vehicle solution (n = 20/group) for 10 days at 37°C, their mechanical properties were assessed by microindentation and fracture toughness tests, and induced AGE levels were quantified through a fluorometric assay. Results indicated that ribose-incubated bone had significantly more AGEs (+81%, p ≤ 0.005), lower elastic modulus assessed by traditional microindentation, and lower fracture toughness compared with vehicle controls. Furthermore, based on pooled data, increased AGEs were significantly correlated with deteriorated mechanical properties. The findings presented here show that the accumulation of AGEs allows for lower stiffness and increased ability to initiate a crack in human cortical bone. Statement of clinical significance: High sugar levels as in T2D results in deteriorated bone quality via AGE accumulation with a consequent weakening in bone's mechanical integrity. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:972-983, 2020.

Harnessing cellular-derived forces in self-assembled microtissues to control the synthesis and alignment of ECM.

The alignment and blend of extracellular matrix (ECM) proteins give a tissue its specific mechanical properties as well as its physiological function. Various tissue engineering methods have taken purified ECM proteins and aligned them into gels, sponges and threads. Although, each of these methods has created aligned ECM, they have had many limitations including loss of hierarchal collagen structure and poor mechanical performance. Here, we have developed a new method to control ECM synthesis using self-assembled cells. Cells were seeded into custom designed, scaffold-free, micro-molds with fixed obstacles that harnessed and directed cell-mediated stresses. Cells within the microtissue reacted to self-generated tension by aligning, elongating, and synthesizing an ECM whose organization was dictated by the strain field that was set by our micro-mold design. We have shown that through cell selection, we can create tissues with aligned collagen II or aligned elastin. We have also demonstrated that these self-assembled microtissues have mechanical properties in the range of natural tissues and that mold design can be used to further tailor these mechanical properties.

Nano-Mechanical Studies on Polyglactin Sutures Subjected to In Vitro Hydrolytic and Enzymatic Degradation.

An experimental investigation on the effects of in vitro hydrolytic and enzymatic degradation on mechanical properties of polyglactin 910 monofilament sutures was performed by conducting nanoindentation studies using an atomic force microscope (AFM). For hydrolytic degradation, the sutures were incubated in phosphate buffered saline (PBS) solution at three different pH conditions, 5, 7.4, and 10. For enzymatic degradation, esterase was employed at pH condition of 7.4. The property of the sutures changed with time at different conditions were investigated by nanoindentation, tensile test experiments, image analysis using both of scanning electron microscopy (SEM) and AFM, and also Fourier transform infrared spectroscopy (FTIR). The effects of degradation on gradation of Young's modulus values across the cross section of the sutures were studied by doing progressive nanoindentation from center to surface. FTIR studies revealed the formation of new hydroxyl bonds due to both hydrolytic and enzymatic degradations. Nanoindentation results indicated that the degradation does not cause a gradient of Young's modulus of the polyglactin 910 monofilament sutures across the cross section from center to surface at different degradation times for both hydrolytic and enzymatic degradations. However, in general, the Young's modulus of all samples was decreased over 4 weeks of degradation. The microscopic evaluation of the samples also showed both qualitative changes in surface morphology and quantitative changes in surface roughness on the surface of degraded sutures. This study provided a deep understanding of the polyglactin sutures subjected to in vitro hydrolytic and enzymatic degradation, and also opened a new avenue to study the biomaterials at nano-scale.

Effect of stiffness of micron/sub-micron electrospun fibers in cell seeding.

The objective of this study was to develop a predictive model for cell seeding depth in electrospun scaffold as a function of fiber stiffness. Electrospun scaffolds (micron and submicron) and 3T3 fibroblasts are used as scaffold-cell systems under vacuum seeding conditions. Atomic force microscopy is used to determine the Young's modulus (E) as a function of fiber diameter. A higher E value led to a lower depth of cell seeding (closer to the surface) indicating that nanofibrous scaffolds offer higher resistance to cell movement compared to microfibrous scaffold. An energy balance model was developed to predict cell seeding depth as a function of E for various vacuum pressures. Experimental data was used in the model to extract unknown parameters to predict cell seeding depth as a function of vacuum pressure for different stiffness scaffolds.