Musculoskeletal Tissue Regeneration: Biological Materials and Methods

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Fiber diameter also modulates stem cell differentiation into different neural cell types. Additionally, the cellular response to fiber diameter could depend on differentiation state. Fiber diameter also maintains stemness. Evaluation of morphology includes measurements such as cells size and shape; cell, cytoskeleton, and nuclear alignment; or focal adhesion number and size. However, there are exceptions where hMSCs 80 and endothelial cells 91 had larger surface areas on smaller fibers and cases where the fiber diameter did not affect rat MSC rMSC area 85 or human U glioblastoma multiform cell elongation or aspect ratio.

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While there are multiple types of cell migration, migration by protrusion of the actin cytoskeleton was the most common type identified in this review. Cell migration is not restricted to a single fiber when the opportunity exists to interact with other fibers. Fiber alignment provides an inductive environment for certain lineages. Fiber alignment has no effect on cell proliferation in human ligament fibroblasts, HUVECs, human aortic smooth muscle cells, human induced pluripotent stem cells, or U astrocytoma cells.

Neurite formation increased, with greater neurite extension , on aligned fibers. In contrast, on randomly oriented fibers, cells typically form a round 85 , 92 , , or polygonal 91 , , , morphology, with the cells exhibiting random orientation.

Musculoskeletal Tissue Regeneration: Biological Materials and Methods

However, on randomly oriented fibers, cells spread in all directions, resulting in a rounded morphology with random orientation. Fiber alignment drives cell migration more than chemotactic gradients.

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Further, cells migrate in the direction of aligned fibers, regardless of fiber diameter. Whereas, the migration velocity of dermal fibroblasts on aligned fibers was low initially, but doubled over 4 days. Mouse cardiosphere-derived cells increase cardiomyogenesis on high fiber density scaffolds. In a 3D-printed, scaffold with constant fiber diameter, square pores supported greater chondrogenic differentiation, while rhomboidal pores supported greater osteogenic differentiation, with osteochondral media.

The effect of fiber density and scaffold porosity on tenogenic, adipogenic differentiation and a range of other differentiation pathways are not well understood.

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On pore sizes of similar dimensions to cell size, HEKT cells exhibited minor pseudopodia but had obvious pseudopodia extension on pores larger than cell size. Larger pore size increased migration speed of HT human fibrosarcoma on 3D fibers and also increased osteoblast motility on aligned 2D fiber systems. As cells attached to single fibers in scaffolds with large pores, the large pores leave the cells with one option — follow the only fiber available — leading to increased migration speeds. Smaller pores 6. In addition to the fiber parameters, the mechanical properties of the fibers can guide cell response.

Type I collagen expression was upregulated on the stiffest PGA substrate. While the PGA degradation did not affect hMSC response via decreased scaffold alignment, no further effects of degradation on cell response were investigated. Smooth muscle cells SMCs were observed to exhibit greater proliferation on a poly urethane :collagen blend that had a greater elastic modulus Cells spread more on fibers with greater stiffness stiffness range of 1.

Surface wettability hydrophobicity and hydrophilicity affects differentiation and proliferation. Octadiene-allylamine polymers modified with a gradient of increasing allylamine to increase hydrophilicity increased mouse ESC proliferation and differentiation toward mesodermal and ectodermal lineages, while the same polymer surface modified with methyl groups to increase hydrophobicity did not promote mouse ESC differentiation or proliferation.

Surface wettability did not have an evident effect on mouse ESC differentiation toward endodermal lineages. Surface roughness affects cell morphology. Hydrophilic surfaces modified with amine groups led to increased branching and osteocyte-like morphology in HDPSCs, while HDPSCs maintained an MSC morphology on the more hydrophobic surfaces modified with methyl groups and hydrophilic surfaces modified with hydroxyl or carboxyl groups. Rougher surfaces promote cell migration over smoother surfaces. The increased roughness led to a two-fold increase in migration area in vascular cells, as well as a three-fold increase in migration area in corneal cells.

Additionally, hydrophobic surfaces promote cell migration in vascular endothelial cells and corneal cells. Conversely, hydrophilic surfaces promote cell adhesion.

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Peak macrophage elongation occurred on smaller fibers. Recent studies have investigated cell behavior in response to dynamic scaffolds that mimic the ever-changing, in vivo environment. While fiber parameters can drive cell responses, a limitation of synthetic polymer scaffolds is the lack of cell signaling cues provided by the native ECM.

Therefore, tissue engineering commonly incorporates proteins and other biomimetic factors from the ECM into synthetic polymer scaffolds to provide additional cell signaling cues to the enhanced structural and architectural cues provided by synthetic scaffolds. These biomimetic factors are incorporated through various means: coating a scaffold, covalently linking to the scaffold, or by adding nanoparticles to the system that release the biomimetic factors over time. In all cases, the addition of biomimetic factors seeks to further enhance and guide cell response to the desired end.

Incorporating growth factors and other biomimetic molecules into the fiber system is commonly used to further induce differentiation using various strategies. Coating poly L-lactic acid PLLA electrospun fibers with polydopamine induced osteogenic differentiation in hMSCs, with greater expression of ALP , RUNX2 , bone sialoprotein, and interleukin 8 on the polydopamine-coated fibers than on the uncoated fibers — both scaffolds with comparable fiber parameters. The addition of graphene oxide to the fibers increased their tensile strength two-fold.

The increased mechanical properties could be the mechanism leading to the increased osteogenic expression, as high substrate elasticity and stiffness guide cells toward osteogenic lineages. Bioprinting is yet another method to include growth factors to influence differentiation. Aligned nanofibers promote osteogenesis, 46 , 80 , 81 tenogenesis, 45 , 98 , 99 and myogenesis depending on cell type.

Incorporation of proteins into the fiber system also enhances differentiation. Human aortic SMCs did not proliferate on randomly oriented or aligned polyurethane nanofibers, but the addition of collagen into the fibers increased SMC proliferation. Many biological tissues exhibit endogenous electric fields, which have been characterized during development and regeneration.

Alternatively, piezoelectric fibers 6. Coating aligned, electrospun nanofibers with fibronectin improved neurite extension in NG neuroblastoma and glioma cells compared to the uncoated fibers. Human skeletal muscle myoblasts and fibroblasts elongated on nanofibers coated with laminin or collagen while they exhibited polygonal morphology on the uncoated fibers. Topographical cues from fiber alignment dominated HUVEC motility over a chemical gradient of vascular endothelial growth factor orthogonal to fiber alignment but had an additive effect when the two were parallel.

The literature demonstrates that fibers drive many cellular responses. In native ECM, the fibrous proteins provide signaling cues to drive cell differentiation, proliferation, adhesion, and migration. Tissue engineering controls the fiber parameters of scaffolds to regulate cell response during engineered development Table 3. Fiber diameter regulates differentiation in a lineage-dependent manner: nanofibers drive osteogenesis, fiber diameter has a biphasic effect on chondrogenesis, the effect of fiber diameter on tenogenesis changes over time, and differing fiber diameters can drive cells toward specific neural lineages.

Larger fiber diameters lead to greater cell elongation and alignment. Cells migrate at higher speeds on smaller fibers, while they migrate farther distances on larger fibers.

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Increased fiber alignment can drive cells into tenogenic, cardiomyogenic, and neuronal lineages, while non-aligned fibers guide cells toward osteogenic and glial differentiation. Cells elongate and align with underlying fibers, forming a spindle shape morphology on aligned fibers, while they form a rounded morphology on randomly oriented fibers. Cells will follow aligned fibers preferentially across chemotactic gradients.

Low scaffold porosity, or high fiber density, leads to greater cell proliferation. Porosity also guides differentiation into multiple cell lineages as a function of pore size and shape. When the pores are small, cells can extend across multiple fibers, leading to a more rounded morphology and lower migration speed. Conversely, when the pores are large, cells will attach and align with single fibers, which also results in increased migration speeds. While synthetic fibers can drive these responses, incorporating biomimetic factors into the scaffolds can further improve the desired response and modulate the response via interactions with the scaffold structure and architecture.

Cells tend to align and elongate along microfibers and aligned fibers through common intracellular mechanisms. Commonly on both microfibers and aligned fibers, cells formed larger or greater numbers of focal adhesions along the increased cell-contact area.

This also occurred in cells along large pores i. The increased focal adhesions lead to the actin cytoskeleton aligning along the fibers, which generated the elongated, spindle shaped morphology. However, cells on microfibers had lower migration speeds than on nanofibers. Microfibers increase cell migration directionality along the fibers but saw lower cell velocities than nanofibers. Cells form larger focal adhesions on the microfibers than on the nanofibers, which generally predicts higher migration speeds.

The major limitation facing many current studies is the failure to consider all fiber parameters in toto, instead focusing only on one.

Changing scaffold production to affect one parameter often changes others simultaneously, and if these other parameters are not adequately controlled, it raises questions about the effects seen. Many studies characterize fiber parameters and then look for a desired outcome, however, the mechanisms driving the outcomes are not investigated as often.

Similarly, including biomimetic factors into fibrous scaffolds can change the structure and architecture of fibers and needs to be controlled. Additionally, material properties such as stiffness or wettability cannot be ignored when investigating the effects of fiber parameters on cell response. While there have been substantial advances in our understanding of cell—fiber interactions, some remaining gaps include: 1 investigating the effect of a larger range of fiber diameters: many studies investigate the effects of nanofibers for cell differentiation and migration.

However, there is growing evidence that microfibers guide cells toward specific lineages.

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Screening a wider range of fiber diameters from nanofibers to microfibers for a wide range of cell types could inform tissue engineering design to achieve the most desired outcome for a variety of tissues. While doing this, the fiber parameters and material properties need to be considered, as they modulate cellular response to the biomimetic cues. While much work is still needed to achieve this feat of tissue engineering, understanding how fiber parameters guide cell response helps to pave the way.

Further information on research design is available in the Nature Research Reporting Summary linked to this article. Danielson, K. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. Cell Biol. Schonherr, E.

Musculoskeletal Tissue Regeneration: Biological Materials and Methods Musculoskeletal Tissue Regeneration: Biological Materials and Methods
Musculoskeletal Tissue Regeneration: Biological Materials and Methods Musculoskeletal Tissue Regeneration: Biological Materials and Methods
Musculoskeletal Tissue Regeneration: Biological Materials and Methods Musculoskeletal Tissue Regeneration: Biological Materials and Methods
Musculoskeletal Tissue Regeneration: Biological Materials and Methods Musculoskeletal Tissue Regeneration: Biological Materials and Methods
Musculoskeletal Tissue Regeneration: Biological Materials and Methods Musculoskeletal Tissue Regeneration: Biological Materials and Methods

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