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Biofabrication

Live cells survive in bioprinted bone

24 Aug 2018 Belle Dumé
A bioprinted construct
A ight micrograph of a bioprinted construct

Researchers in Germany have shown that a material based on calcium phosphate could offer a viable support material for bioprinting replacement bone tissue. The team, led by Michael Gelinksy at the Technical University Dresden, say that the technology opens up new possibilities for plastic and reconstructive surgeries, since it could be used to fabricate patient-specific bone tissue constructs, as well as more complex structures consisting of, for example, bone and cartilage or bone and soft tissue.

According to Gelinsky, calcium phosphate is the ideal scaffold material for these applications because it offers the same mineral structure and mechanical properties as natural bone. His team has been experimenting with scaffolds made from calcium phosphate cement (CPC), a pasty material that is easy to process into various shapes using a low-temperature extrusion-based technique called 3D plotting.

Recent work has shown that sensitive bio-components, like growth factors, can be integrated into printed CPC scaffolds without their biological activity being affected. The problem is that live cells cannot be suspended in the same scaffold because they can’t survive in such a solid and stiff support material.

Gelinsky and colleagues have now overcome this barrier by combining 3D plotting of CPCs with cell printing using a specially developed bioink. “Using a mechanically stable, self-setting CPC as a printable support material that nicely mimics the mineral component of bone, as in our work, is a big step forward to when it comes to bioprinting bone tissue constructs,” he says.

Towards stronger scaffolds

Until now, explains Gelinsky, the only scaffold materials that have been used successfully in bioprinting applications have been thermoplastic polymers (such as PCL/polycaprolactone) or highly concentrated biopolymer hydrogels. “The soft hydrogels typically used for cell printing are mechanically too weak for printing constructs for tissues like bone,” he says. “And since bone is a mineralized tissue (more than half its weight by volume comprises the calcium phosphate mineral phase hydroxyapatite), a polymer like PCL is not really a good substitute here either.”

Gelinsky’s team has already optimized a process for fabricating CPC scaffolds using 3D plotting. They have studied the way that the CPC paste solidifies after extrusion, and have found that pre-setting in a humid environment for three days prevents the formation of micro-cracks that compromise the strength of printed scaffolds. “In our previous work, we already showed that we could co-print CPC with cell-free alginate-based hydrogels,” Gelinsky continues. “So it was relatively easy for us to go a step further and co-print CPC with an alginate-based bioink that is laden with live human cells.”

The challenge for Gelinsky and his team was to find a fabrication regime that would enable the live cells to survive the setting process. Their first task was to co-print the CPC with a bioink laden with human mesenchymal stroma cells, which they did with three-channel extrusion printer that alternates printing between the CPC and the bioink. This creates a biphasic scaffold with an open pore structure, which is vital to ensure that oxygen and nutrients can reach the cells and allow them to grow.

However, setting the CPC in a humid environment for three days would kill the cells, so the researchers tested the impact of reducing the setting time on both micro-crack formation and cell viability. They found that a setting period of 20 minutes in a high-humidity environment was sufficiently long to create mechanically strong scaffolds, while also allowing almost all the live cells to survive (Biofabrication 10 045002).

One remaining issue, say the researchers, is that the fresh CPC paste is slightly cytotoxic for cells that are in direct contact within the bioink strands – which is probably caused by a slight pH shift during the cement setting reaction. “We have already come some way in overcoming this problem by using a novel type of bioink in which we haven’t seen dead cells at the crossing points of CPC and bioink strands,” says Gelinsky.

The team also plans to print bi- or tri-layered constructs with different types of human cells. “Until now, we have simply used fluorescent microbeads to demonstrate proof of principle for such complex implants,” notes Gelinsky.

  • Read our special collection “Frontiers in biofabrication” to learn more about the latest advances in tissue engineering. This article is one of a series of reports highlighting high-impact research published in Biofabrication.

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