Many biological systems, however, are not only complex in shape and composition, but also perform unique functions via changes in tissue conformation. The heart, for example, pumps in response to the body's intrinsic electrical system, while many other tissues undergo structural changes following intrinsic or external stimuli. As such, static structures obtained from conventional 3D bioprinting may be unable to elicit realistic biological responses. What's needed is a fourth dimension: time.

"Four-dimensional (4D) bioprinting has been proposed in recent years, by combining smart, stimuli-responsive biomaterials with 3D printing techniques, to introduce this fourth dimension into the fabricated system and emulate the dynamic processes seen in biological tissues," explained Ali Khademhosseini from Harvard Medical School's Biomaterials Innovation Research Center. "This is an exciting field with huge potential for development."

Writing in the journal Biofabrication, Khademhosseini and colleagues examine the potential of various stimuli-responsive materials as bioinks for 4D printing. They also discuss the state-of-the-art and limitations of 3D printing, and look at future directions for 4D bioprinting platforms (Biofabrication 9 012001).

"This is a key point in time where researchers are starting to move their attention to 4D bioprinting," Khademhosseini told medicalphysicsweb. "We wanted to provide a thorough overview on the potential materials that can be possibly used in 4D bioprinting, to spur collective efforts to push this field forward."

Adding motion

Three main techniques have emerged for 3D bioprinting. Inkjet printing uses liquid bioink ejected in droplets to build a 3D volume, and typically offers high printing speed at low cost. Microextrusion printing, where bioinks are physically extruded by a dispensing system, benefits from the continuity of the extruded bioinks, can use bioinks with a wide viscosity range and achieve high cell densities. Finally, laser-induced forward transfer printing works by focusing laser pulses on an absorbing layer, forcing a bioink droplet to deposit and form the desired pattern.

To add shape transformation into printed structures, the next step is to combine such 3D bioprinting platforms with stimuli-responsive biomaterials. One simple way to print structures that can actively transform is to deposit materials with differential properties (such as swelling) to achieve folding and unfolding. By using materials that expand to different degrees as joints between rigid segments, for example, the direction and angle of the joints can be controlled under suitable stimulus.

Khademhosseini and colleagues describe the variety of transformation mechanisms that could potentially be integrated into 4D bioprinting. One approach is to use cells themselves as the active element. In this "cell origami" technique, cell traction force is harnessed to transform 3D microstructures into pre-defined shapes. By combining multiple cell types, complex cell-laden structures may be spontaneously formed in a post-printing procedure.

The other option is to use stimuli-responsive materials as a programmable bioink. Materials that respond to changes in humidity, for example, are widely present in nature. And researchers have developed multi-layered materials whose motion is actuated upon humidity change, for example a bilayer film that starts to bend as the relative humidity increases.

Then there are thermo-responsive materials, which change shape in response to temperature alterations. Hydrogels that transform reversibly from helical into straight structures and hydrogel disks that fold into tubes upon heating have already been demonstrated. Such materials, which have been employed already in tissue engineering, drug delivery and sensing, offer great potential for 4D bioprinting. Work is needed though to lower actuation temperatures to ensure sufficient cell viability and function.

Materials can also be engineered to change size or shape in response to electric or magnetic stimulation. Examples here include a biomorphic robot, created by connecting two folded accordion-like constructs, that moves when a voltage is applied, and magnetic-responsive ferrogels that control the release of drugs. Constructs bioprinted from composite inks based on hydrogels mixed with (highly conductive) carbon nanotubes or magnetic particles have been shown to support adhesion and growth of cells. Such bioinks could also prove suitable for 4D bioprinting.

Finally, there are light-sensitive materials that convert light into mechanical responses, and benefit from far higher precision than abovementioned stimuli. Such materials have been widely applied in applications such as controlled drug delivery or cancer therapy, and it is envisioned that they can be combined with existing bioinks to enable 4D bioprinting.

A bright future

Despite this large range of potential stimuli-responsive biomaterials, their translation into bioinks may not be straightforward. Existing materials must be engineered to meet the requirements for a bioink: strong biocompatibility to ensure accurate functionality in the printed objects; appropriate rheological parameters to ease printability; and suitable stabilization mechanisms. It's also important that embedded cells can survive the stimuli, and that the shape-changing capacity is not diminished by inclusion of cells.

Another challenge lies in ensuring that 4D bioprinted constructs have robust shape-changing capabilities. Previous studies have shown the mechanical properties of printed objects to degrade severely after repeated transformations.

As for the use of 4D bioprinting in clinical applications, this may still be decades away. "Right now the field is still in its infancy and concept stage," Khademhosseini explained. "First, it requires a lengthy time to develop the variety of 4D bioprinting strategies, then we need to assess them in a thorough multi-phase clinical trial. This whole process involves not only the refinement and analysis of the biomaterials and encapsulated cells, but ensuring these smart structures actually behave and transform in the body in the ways that we design them to."

Khademhosseini and colleagues are optimistic about the future. "Although it needs some efforts to overcome the current bottleneck, we believe that with collective efforts, the field of 4D bioprinting will flourish in the next decade," they said.

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