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A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

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Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) are encapsulated in hyaluronic acid (HA)-based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion-controlled interfacial bioorthogonal crosslinking is carried out on the soft cellular construct using HA modified with a ferocious dienophile, trans-cyclooctene (TCO). Cultures are maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restricts cell spreading. Compared to the nLP controls, sLP7 conditions upregulate the expression of fibrous matrix proteins (Col I, DCN, and FN EDA), classic fibroblastic markers (TNC, FAP, and FSP1), and matrix remodeling enzymes (MMP2, TIMP1, and HAS3). Day 7 stiffening also upregulates the catabolic activities, enhances ECM turnover, and promotes YAP expression. Overall, in situ delayed matrix stiffening promotes a fibroblast transition from MSCs and enhances YAP-regulated mechanosensing.

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Crosslinked, degradable, and cell-adhesive hydrogel microfibers were synthesized via interfacial polymerization employing tetrazine ligation, an exceptionally fast bioorthogonal reaction between strained trans-cyclooctene (TCO) and s-tetrazine (Tz). A hydrophobic trisTCO crosslinker and homo-difunctional poly(ethylene glycol) (PEG)-based macromers with the tetrazine group conjugated to PEG via a stable carbamate (PEG-bisTz1) bond or a labile hydrazone (PEG-bisTz2) linkage were synthesized. After laying an ethyl acetate solution of trisTCO over an aqueous solution of bisTz macromers, mechanically robust microfibers were continuously pulled from the oil-water interface. The resultant microfibers exhibited comparable mechanical and thermal properties but different aqueous stability. Combining PEG-bisTz2 and PEG-bisTz3 with a dangling arginine-glycine-aspartic acid (RGD) peptide in the aqueous phase yielded degradable fibers that supported the attachment and growth of primary vocal fold fibroblasts. The degradable and cell-adhesive hydrogel microfibers are expected to find utility in a wide array of tissue engineering applications.

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Healthy articular cartilage supports load bearing and frictional properties unmatched among biological tissues and man-made bearing materials. Balancing fluid exudation and recovery under loaded and articulated conditions is essential to the tissue's biological and mechanical longevity. Our prior tribological investigations, which leveraged the convergent stationary contact area (cSCA) configuration, revealed that sliding alone can modulate cartilage interstitial fluid pressurization and the recovery and maintenance of lubrication under load through a mechanism termed 'tribological rehydration.' Our recent comparative assessment of tribological rehydration revealed remarkably consistent sliding speed-dependent fluid recovery and lubrication behaviors across femoral condyle cartilage from five mammalian species (equine/horse, bovine/cow, porcine/pig, ovine/sheep, and caprine/goat). In the present study, we identified and characterized key predictive relationships among tissue properties, sliding-induced tribological rehydration, and the modulation/recovery of lubrication within healthy articular cartilage. Using correlational analysis, we linked observed speed-dependent tribological rehydration behaviors to cartilage's geometry and biphasic properties (tensile and compressive moduli, and permeability). Together, these findings demonstrate that easily measurable biphasic tissue characteristics (e.g., bulk tissue material properties, compressive strain magnitude, and strain rates) can be used to predict cartilage's rehydration and lubricating abilities, and ultimately its function in vivo. STATEMENT OF SIGNIFICANCE: In healthy cartilage, articulation recovers fluid lost to static loading thereby sustaining tissue lubricity. Osteoarthritis causes changes to cartilage composition, stiffness, and permeability associated with faster fluid exudation and presumably poorer frictional outcomes. Yet, the relationship between mechanical properties and fluid recovery during articulation/sliding remains unclear. Through innovative, high-speed benchtop sliding and indentation experiments, we found that cartilage's tissue properties regulate its exudation/hydration under slow sliding speeds but have minimal effect at high sliding speeds. In fact, cartilage rehydration appears insensitive to permeability and stiffness under high fluid load support conditions. This new understanding of the balance of cartilage exudation and rehydration during activity, based upon comparative tribology studies, may improve prevention and rehabilitation strategies for joint injuries and osteoarthritis.

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OBJECTIVE: Many in vivo procedures to repair chondral defects use ultraviolet (UV)-photoinitiated in situ polymerization within the cartilage matrix. Chemical species that absorb UV light might reduce the effectiveness of these procedures by acting as light absorption barriers. This study evaluated whether any of the individual native biochemical components in cartilage and synovial fluid interfered with the absorption of light by common scaffolding photosensitizers. MATERIALS: UV-visible spectroscopy was performed on each major component of cartilage in solution, on bovine synovial fluid, and on four photosensitizers, riboflavin, Irgacure 2959, quinine, and riboflavin-5'-phosphate. Molar extinction and absorption coefficients were calculated at wavelengths of maximum absorbance and 365 nm. Intact articular cartilage was also examined. RESULTS: The individual major biochemical components of cartilage, Irgacure 2959, and quinine did not exhibit a significant absorption at 365 nm. Riboflavin and riboflavin-5'-phosphate were more effectual light absorbers at 365 nm, compared with the individual native species. Intact cartilage absorbed a significantly greater amount of UV light in comparison with the native species. CONCLUSION: Our results indicate that none of the individual native species in cartilage will interfere with the absorption of UV light at 365 nm by these commonly used photoinitiators. Intact cartilage slices exhibited significant light absorption at 365 nm, while also having distinct absorbance peaks at wavelengths less than 300 nm. Determining the UV absorptive properties of the biomolecules native to articular cartilage and synovial fluid will aid in optimizing scaffolding procedures to ensure sufficient scaffold polymerization at a minimum UV intensity.

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There remains no routine treatment for congenital tracheal abnormalities affecting more than 1/3 of the length. Natural and artificial prostheses are plagued by mechanical failure and inconsistent outcomes. Mimicking native tissue mechanics in an engineered replacement may improve functional and patient outcomes. We synthesized tubular constructs comprising photo-cross-linked methyl acrylate-co-methyl methacrylate, p(MA-co-MMA), with patterned r- and z-axes in order to achieve mechanical properties similar to lamb tracheae. Hard and soft alternating bands, and a soft vertical section, mimic tracheal architecture. Patterned constructs were capable of 46% elastic longitudinal extension. The construct longitudinal composite modulus, 0.34 ± 0.09 MPa, was not significantly different from ovine tracheae. The superior of two geometries evaluated supports up to a 46% reduction of internal volume within the physiological range of transmural pressures. Thus, these patterned hydrogels yielded longitudinal elasticity and radial rigidity while allowing for radial deformation required for effective coughing.