Aging and injury affect nuclear shape heterogeneity in tendon.

Tissue level properties are commonly studied using histological stains assessed with qualitative scoring methods. As qualitative evaluation is typically insensitive, quantitative analysis provides additional information about pathological mechanisms, but cannot capture structural heterogeneity across cell subpopulations. However, molecular analyses of cell and nuclear behavior have identified that cell and more recently also nuclear shape are highly associated with cell function and malfunction. This study combined a Visually Aided Morpho-Phenotyping Image Recognition analysis that automatically segments cells based on their shape with an added capacity to further discriminate between cells in certain protein-rich extracellular matrix regions. We used tendon as a model system given the enormous changes in organization and cell and nuclear shape they undergo during aging and injury. Our results uncover that multiple shape modes of nuclei exist during maturity and aging in rat tendon and that distinct subgroups of cell nuclei shapes exist in proteoglycan-rich regions during aging. With injury, several immunomarkers (αSMA, CD31, CD146) were associated with more rounded shape modes. In human tendons, the cell nuclei at sites of injury were found to be more rounded relative to uninjured tissues. To conclude, the tendon tissue changes occurring during aging and injury could be associated with a variation in cell nuclear morphology and the appearance of various region-specific subpopulations. Thus, the methodologies developed allow for a deeper understanding of cell heterogeneity during tendon aging and injury and may be extended to study further clinical applications.

Aging and matrix viscoelasticity affect multiscale tendon properties and tendon derived cell behavior.

Aging is the largest risk factor for Achilles tendon associated disorders and rupture. Although Achilles tendon macroscale elastic properties are suggested to decline with aging, less is known about the effect of maturity and aging on multiscale viscoelastic properties and their effect on tendon cell behavior. Here, we show dose dependent changes in native multiscale tendon mechanical and structural properties and uncover several nanoindentation properties predicted by tensile mechanics and echogenicity. Alginate hydrogel systems designed to mimic juvenile tendon microscale mechanics revealed that stiffness and viscoelasticity affected Achilles tendon cell aspect ratio and proliferation during aging. This knowledge provides further evidence for the negative impact of maturity and aging on tendon and begins to elucidate how viscoelasticity can control tendon derived cell morphology and expansion. STATEMENT OF SIGNIFICANCE: Aging is the largest risk factor for Achilles tendon associated disorders and rupture. Although Achilles tendon macroscale elastic properties are suggested to decline with aging, less is known about the effect of maturity and aging on multiscale viscoelastic properties and their effect on tendon cell behavior. Here, we show dose dependent changes in native multiscale tendon mechanical and structural properties and uncover several nanoindentation properties predicted by tensile mechanics and echogenicity. Alginate hydrogel systems designed to mimic juvenile tendon microscale mechanics revealed that stiffness and viscoelasticity affected Achilles tendon cell spreading and proliferation during aging. This knowledge provides further evidence for the negative impact of maturity and aging on tendon and begins to elucidate how viscoelasticity can control tendon derived cell morphology and expansion.

Enhanced tendon healing by a tough hydrogel with an adhesive side and high drug-loading capacity.

Hydrogels that provide mechanical support and sustainably release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J m-2) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainably released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with 'Janus' surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications.

3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands.

Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical-microbead-based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high-aspect-ratio hydrogel microstrands are introduced to overcome these limitations. Pre-crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40-100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co-printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.