Author: Dr Patrick Thayer, Bioink Officer at CELLINK

The development of a functional tissue from a bioprinted construct requires the incorporation or application of complex cues and stimuli that guide cell organization and differentiation. For certain tissues, such as ligament, tendon, and muscle whose in vivo microstructure is highly organized, it is paramount that cell organization is controlled to ensure development of a physiological extracellular matrix and recapitulation of native mechanical characteristics. Unlike 2D cell culture where the cell growing surface can be modified to possess topographical guidance cues such as grooves, ridges, microfibers, and roughness, incorporating of these cues within a 3D hydrogel-based construct can be difficult. Traditional hydrogels possess low viscosity and can be difficult to pattern topography within. Techniques for doing this include gelatin under flow, electrical stimulation, or mechanical stretching. However, these techniques are difficult to scale with thick constructs and constructs with complex shapes. Bioprinting technology can permit the patterning of different bioinks and with the generation of bioinks that provide topographical cues overcome the shortfalls of traditional 3D hydrogel constructs.

A recent paper published in the Journal Biomaterials titled “Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo” from the labs of Dr. Alberto Rainer (Tissue Engineering Lab, Università Campus Bio-Medico di Roma, Rome, Italy) and Dr. Cesare Gargioli (Department of Biology, Tor Vergata Rome University, Rome, Italy) addressed this challenge. The researchers sought to utilize bioprinting to incorporate topographical cues within a bioprinted construct to guide the organization of cells for skeletal muscle applications. To briefly summarize the manuscript, C2C12 myoblasts were blended with a photocurable PEG-fibrinogen/alginate bioink and bioprinted to form a multilayered construct that consisted of parallel filaments. The myoblasts within the individual filaments began to fuse together and form multinucleated myotubes that were aligned within and in the direction of the filaments. These constructs could be implanted within mice and when analyzed after 28 days, they contained an organized muscle tissue as demonstrated by the presence of striated myofibers.

The project discussed in this manuscript took advantage of a unique characteristic of bioprinted constructs, which is the ability to generate and deposit filaments in any desired pattern. Through the modulation of the chemistry of the bioink one can fabricate filaments that can temporarily isolate cell populations while allowing nutrient and oxygen diffusion. These filaments can restrict cell migration but also guide the orientation of the population as a whole. As with cells seeded in grooves of sufficient size, cell populations will align parallel to the edge, in this case, the surface of the filament cylinder. Control over bioink filament diameter, length, direction, and chemistries using bioprinting technology could allow for the incorporation of topographical guidance cues within a bioprinted construct. This technique could be suitable in the fabrication of any tissue construct whose microstructure is hierarchically organized. The dimensions of bioprinted filaments are in the range of collagen fascicles found in ligament and tendon tissue, osteons found in bone, nerves, and small arteries and arterioles that enrich tissues.  This approach could be valuable in the fabrication of complex tissue constructs that incorporate many of these microstructures.

We are excited to see what research comes out of the Rainer and Gargioli lab in the future. But like any good project, it opens up more questions and opportunities for research. Here are several that comes to mind,

Can infill patterns of bioprinted constructs evolve rather from fulfilling a ‘space filling’ and ‘structural support’ role to mimicking the native tissue microarchitecture?

Can bioinks be developed that encapsulate one cell type but allow its remodeling by another cell type? For example, can a filament be deposited to contain smooth muscle cells and endothelial cells but allow the infiltration of fibroblasts and such to generate connective tissue that incorporates the ‘filament’ into the broader tissue construct as a neo-blood vessel?

In a similar vein, can bioinks be developed that prevent the ingrowth of muscular tissue but allow the outgrowth of nerve axons to interface with the skeletal muscle at neuromuscular junctions?

Could precise control over the filament deposition allow the generation of complex shapes such as nephron units that can be maintained and not overgrown by the bulk tissue around it?

Click here for original manuscript.