Rendering dynamic 3D printed scaffolds

Ultrasound and phase segregation as tools for remote nanovibration-driven tissue regeneration
Rendering dynamic 3D printed scaffolds

Tissues are formed while subjected to constant mechanical stimuli. Cells are directed, organised and specialised following patterns of intrinsic and extrinsic forces that are present as early as in embryonic stages. These forces directly interact with cell surface receptors which in turn can regulate multiple cellular processes such as migration, proliferation, survival and differentiation. Many of these tissue are further developed and remodelled during skeletal maturation as it is the case for skeletal tissues.

When one of these tissues suffers from a traumatic event, it is damaged or degenerated, our body tries to heal it but, it some cases with little success. Scientist have been trying to tackle this issue since already a couple of decades with the emergence of the tissue engineering and regeneration fields. Based on the production of scaffolds that can recapitulate certain aspects of the damaged tissue, tissue regeneration has already brought some products to the market and is certainly making its way through. However, there is still a lot of work to be done.

The team of Prof. Lorenzo Moroni at the Complex Tissue Regeneration Department of the MERLN Institute for Technology-inspired Regenerative Medicine has been dedicated at the regeneration of musculoskeletal tissues with success, launching some products to the market. Their approach is based on the exploitation of biofabrication techniques, including 3D printing, that allow fabrication of patient specific scaffolds matching multiple characteristics of the tissue, such as porosity, mechanical properties or surface chemistry. Yet, one of the main disadvantages of current scaffolds is that they are intrinsically static, while the development, regeneration and remodelling of tissues are a dynamic process.

The idea behind this approach came up by accident, as many scientific discoveries, just that in this case was a long-term one. When I joined forces at the MERLN institute, Prof. Moroni suggested to apply the physicochemical concepts that we demonstrated earlier in electrospun scaffolds to 3D printed robust structures. The concept here was to create self-assembled or phase-segregated structures during the printing process, allowing thus the control of not only the structure of the fabricated structure but also the spatial chemical distribution of the building materials.

Figure 1| Biofabrication of phase-segregated 3D printed structures, including Janus structures [1]. The phase segregation process followed the traditional nucleation and growth and spinoidal decomposition. However, we were very surprised to observed the formation of Janus structures along each of the fibers of 3D printed scaffolds which was, moreover, oriented on the same direction along the entire structure.

The idea behind this approach came up by accident, as many scientific discoveries, just that in this case was a long-term one. With a broken leg and plenty of time to read and think, we started to wonder how this process could be accelerated or improved. After bone fracture and hematoma formation osteoprogenitor cells differentiate forming fibrocartilage tissue, known as the soft callus. The soft callus starts to form woven or trabecular bone. At this point, the callus attains the maximum size that is the initial template for bone formation. Further, the formed woven bone is remodelled to cortical bone on a process governed by applied mechanical loads. Indeed, after a bone fracture patients are advised to have a minimum of 2 weeks resting time and later initiate a process of partial loading to promote bone formation. We came then to the realisation that the use of dynamic scaffolds, capable of providing a mechanical stimulation to cells while implanted in the body would better mimic natural regeneration and remodelling processes.

Figure 2| Concept behind remote mechanical stimulation of cells. Image courtesy of Dr. Camarero-Espinosa. 

But, how to render a 3D printed scaffold dynamic? This was perhaps the key question to be answered here. While material scientist can play many tricks to create shape-memory or shape-morphing materials, finding a stimulus that could remotely activate a mechanical signal on a robust scaffolds and compatible with physiological environments seemed challenging at first. However, the answer was already out there. Ultrasounds have been used for over decades on clinical environments to, for example, warm up the tissue been treated by a physiotherapist. Ultrasounds, as pressure waves, produce a vibration while crossing different environments. We modelled the vibration (or nanodeflection) that an ultrasounds wave would generate on our 3D printed scaffolds and realised that could easily control the amplitude of the deflections with the applied ultrasound frequency.

Figure 3|Ultrasoun-driven scaffold nanovibration [1]

Janus structures come into play. Vibrations exerted to a mechanical structure are generally “uncontrolled” and thus the industry of ultrasounds transducers makes use of an active material, capable of great vibration, and a backing material with a damping character. Ultrasound transducers, formed as a sandwich composite of the active and the damping materials have, thus, the capability to vibrate with a controlled amplitude and a controlled pulse length. Similarly, our Janus scaffolds were composed of a vibrating polylactide phase and a damping polycaprolactone phase that regulated the amplitude and the pulse length of the naovibration induced by remote ultrasound stimulation.

We explored the effects of the ultrasound-generated nanovibration of 3D printed Janus scaffolds during bone formation with human bone marrow derived stem cells. We were very excited to verify that the mechanical stimulation of cells with our approach to dynamic scaffolds resulted on an enhanced cell proliferation, osteogenic differentiation, mineralization and, thus, bone formation. Moved by the curiosity, we further investigated the mechanisms underlying this process, that we could further demonstrate to be a consequence of the activation of voltage-gated calcium ion channels.

Figure 4| Dynamic culture activates voltage-gated calcium ion channels. Human bone marrow-derived stem cells (right, F-actin in red) cultured under daily ultrasound stimulation present activated voltage-gated calcium ion channels (right, DHPR channels in blue and RyR receptor in green)

This study, although still preliminary, describes what we believe could a new concept on tissue regeneration were scaffolds are not a static component but a dynamic one, guiding the formation of new tissues. We have demonstrated that sound waves can be used to render scaffolds dynamic and that we can control the amplitude and pulse of these nanovibrations, affecting the cell response. We further believe that different tissues would require the adjustment of these parameters. But, deciphering which music would each tissue like to dance, weather low pitches of Bach or the high ones of Metallica, is still to be investigated.

[1] S. Camarero-Espinosa, L. Moroni, Janus 3D printed dynamic scaffolds for nanovibration-driven bone regeneration, Nature Communications 12(1) (2021) 1031.