3D print organs 50 times faster than industry standards

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It looks like science fiction: A machine dips into a shallow vat of translucent yellow goo and pulls out what becomes a life-sized hand.

But the seven-second video, which is sped-up from 19 minutes, is real.

The hand, which would take six hours to create using conventional 3-D printing methods, demonstrates what University at Buffalo engineers say is progress toward 3-D-printed human tissue and organs—biotechnology that could eventually save countless lives lost due to the shortage of donor organs.

“The technology we’ve developed is 10-50 times faster than the industry standard, and it works with large sample sizes that have been very difficult to achieve previously,” says the study’s co-lead author Ruogang Zhao, Ph.D., associate professor of biomedical engineering.

The work is described in a study published Feb. 15 in the journal Advanced Healthcare Materials.

It centers on a 3-D printing method called stereolithography and jelly-like materials known as hydrogels, which are used to create, among things, diapers, contact lenses and scaffolds in tissue engineering.

The latter application is particularly useful in 3-D printing, and it’s something the research team spent a major part of its effort optimizing to achieve its incredibly fast and accurate 3-D printing technique.

A machine dips into a shallow vat of translucent yellow goo and pulls out what becomes a life-sized hand. But the seven-second video, which is sped-up from 19 minutes, is real. Credit: University at Buffalo

“Our method allows for the rapid printing of centimeter-sized hydrogel models. It significantly reduces part deformation and cellular injuries caused by the prolonged exposure to the environmental stresses you commonly see in conventional 3-D printing methods,” says the study’s other co-lead author, Chi Zhou, Ph.D., associate professor of industrial and systems engineering.

Researchers say the method is particularly suitable for printing cells with embedded blood vessel networks, a nascent technology expected to be a central part of the production of 3-D-printed human tissue and organs.


Large scale cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication is faced with challenges in achieving clinically-relevant size and hierarchical structures (1). 3D bioprinting is an emerging technology for hydrogel fabrication and has been successfully used to create hydrogel models with biomimetic structures and functions (2, 3); however, its application in large, solid hydrogel fabrication has been limited by the slow printing speed that can affect the part quality and the biological activity of the encapsulated cells (4, 5).

Due to the point-by-point deposition process used in nozzle-based bioprinting techniques, extended printing time is required to fabricate a large-sized model with fine structures (6, 7). Prolonged exposure of the encapsulated cells to a variety of printing-induced environmental factors, such as the shear stress, the low oxygen level and the temperature shock, has been shown to cause serious cellular injury and cell death (8, 9).

The effort to improve the printing resolution by using small diameter nozzles can cause further damage to the cells (9, 10). Additionally, due to the low mechanical strength of the hydrogel scaffold materials, it is very challenging for point-by-point deposition methods to create overhanging or hollow structures such as vascular channels inside solid parts.

To address this limitation, Atala group utilized rigid polymeric scaffolds to support the printing of cell-laden hydrogel materials (11), and Feinberg group extruded hydrogel material in a secondary supporting hydrogel to print biomimetic structures such as a heart chamber (12); however, these approaches suffer from either the high rigidity of the supporting material or the complexity of the post-processing steps.

Although extrusion printing of dissolvable templates composed of sacrificial materials such as fugitive inks and carbohydrate glass has enabled the creation of perfusable vascular channels in casted hydrogel constructs (13–15), this approach has very limited capacity to create fine tissue structures other than vascular channels due to the simple casting method used.

Digital mask projection-stereolithography (MP-SLA) is a photopolymerization-based, layer-by-layer 3D printing technology that features multi-scale fabrication capacity with high spatial resolution, allowing the bulk geometry and fine structure of a complex 3D model to be built through one single process (16, 17).

The liquid resin provides natural self-support for the fabrication of hollow structures. This approach has been used to fabricate hydrogel models such as nerve conduits and muscle-powered biobots. Recently, multivascular networks have been created in hydrogels by controlling the spatial resolution of hydrogel photopolymerization using selected food dye photoabsorbers (18–20); however, the layer-by-layer process used in these studies limited the printing speed, which can potentially cause dehydration-induced part deformation and reduced cell viability during the fabrication of large-sized hydrogel parts.

Recently, the development of continuous liquid interface production (CLIP) technology drastically increased the fabrication speed of MP-SLA through continuously building the layers of a 3D part immediately above a “dead zone” formed by oxygen inhibition of photopolymerization (21).

In the dead zone, the flow of liquid water-insoluble-resin (WI-resin) enables continuous material replenishment at the polymerization interface. However, due to the low fluidity of the WI-resin material and the corresponding large suction force at the curing interface, the fabrication ability of the CLIP technology is limited to thin-walled parts (21–23). The fabrication of a centimeter-sized solid hydrogel part has not yet been achieved using CLIP.

In this work, we established low suction force-driven, high-velocity flow of the hydrogel prepolymer for continuous MP-SLA printing through precisely controlling the photopolymerization condition. The high-velocity flow supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth.

This method, the Fast hydrogeL prOjection stereolithogrAphy Technology (FLOAT), allows the creation of a centimeter-sized, multiscale solid hydrogel model within several minutes. We showed that this process is unique to the hydrogel prepolymer solution and cannot be achieved using resin without externally supplemented oxygen.

The rapid printing of centimeter-sized hydrogel models using FLOAT was shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in layer-by-layer based printing methods. Media perfusion in the printed vessel network was shown to promote cell survival and metabolic function in the deep core of the large-sized hydrogel model over long term.

The FLOAT is compatible with multiple photocurable hydrogel materials and the printed scaffold supports the endothelialization of prefabricated vascular channels.Together, these studies demonstrate a rapid hydrogel 3D printing method and highlight the potential of this method in the fabrication of large-sized engineered tissue models.

The effects of rapid printing on part quality and cellular function in large-sized models.(A-E) Demonstration of FLOAT printing process of a centimeter-sized human hand model. Sequential images of the hand model that was continuously formed in 10% PEGDA 400 Da preploymer pool at 5 min, 10 min and 15 min and completed at 20 min. It would take 6.5 hours to print the same model using traditional layer-by-layer printing method. In panel D, the length of the completed hand model is 5.6 cm. (E) Fingers were easily bent under compression, showing the compliance of the hydrogel hand model. (F) Hand model printed in 2 hours using the traditional layer-by-layer SLA process. Severe layer detachment and finger distortion occurred due to dehydration. (G) 3D reconstructed image of FLOAT-printed hand model from MRI scanning. The channels were visualized with the aid of a contrast agent. Metabolic activity and cytotoxicity measured by XTT assay (H) and LDH assay (I) shortly after the printing of centimeter-sized, cell-laden samples. Cells in FLOAT-printed samples experienced much less printing-induced injury and have much higher metabolic activity than those in layer-by-layer printed samples. The above cell-laden samples were printed using 7% GelMA plus 2% PEGDA 8k Da. n = 9. All box plots with whiskers represent the data distribution based on five number summary (maximum, third quartile, median, first quartile, minimum). **, p < 0.001; *, p < 0.05; #, p = 0.1 determined by two-tailed t-test.

Discussion
Hydrogel fabrication has been one of the main focused areas in bioprinting, but much of the efforts have been made on improving the printing resolution (28, 29). Although the spatial resolution limited by nozzle size and material morphology control has been improved recently for extrusion-printing, which allows the creation of tissue models with biomimetic features such as vascular channels (11, 14), such point-by-point material deposition method is still limited by its inability to print at multiple length scales and its slow printing speed that exposes cells to prolonged environmental stresses.

The addition of colored dyes as photoabsorbers has allowed the fabrication of multivascular network using the layer-by-layer SLA method; however, this method still suffers from the slow printing speed, which requires anti-settling agents such as Xanthan gum and glycerol to prevent cell settlement during the fabrication of large-sized cell-laden tissue models (20).

To address these challenges, FLOAT method combines high printing speed with multiscale printing capacity to allow the fabrication of a centimeter-sized hydrogel model in several minutes, as compared to several hours needed in extrusion-based printing and layer-by-layer SLA printing for a similarsized part (11, 31).

The development of the FLOAT method is achieved through studying the interactive effect of the process parameters and precisely controlling the prepolymer formula to enable high-velocity flow. Our studies demonstrated the different photopolymerization conditions that permit and inhibit the high-velocity flow, which allowed the formulation of the optimal prepolymer composition. The low suction force occurred in the optimal curing condition was shown to be critical to drive the prepolymer flow and to maintain good printing quality, thus unveiling the fundamental mechanism of the process.

In the current study, we showed that the continuous SLA process is unique to the hydrogel prepolymer solutions and cannot be achieved in resins without externally supplemented oxygen. It is possible that continuous hydrogel printing can also be achieved with the O2 permeable window and external oxygen supply as described in the CLIP setup (21), though the experimental setup will be more complicated.

The current studies on the prepolymer flow and suction forces under controlled photopolymerization conditions should provide guidelines for such studies. Suction force is one of the limiting factors for the continuous printing of large-size models. We showed that the suction force in FLOAT hydrogel printing is several hundred times less than that in continuous resin printing. This can potentially be explained by the fluid mechanics theory of Stefan’s adhesion where separation force is inversely proportional to the cube of the separation distance and linearly proportional to the fluid viscosity (23, 32).

Although we did not measure the dead zone thickness in our continuous resin printing, it should be smaller than that in the CLIP method (120 μm). Since the liquid flow layer thickness is more than 650 μm in the FLOAT method, the more than 5 times difference in the separation distance can lead to several hundred times difference in the separation force between the two methods. The difference in the viscosity (~10 cP for PEGDA MW 4000 prepolymer and 100 – 1000 cP for WI-resin) further contributes to the difference in the separation force.

The low suction force in FLOAT method permits the fabrication of large, solid parts with good quality, thus improving over continuous resin printing where only small-size, thin-walled parts can be fabricated due to the large suction force of the WI-resin (21–23).

In the current study, we performed material optimization to seek hydrogel formula with both good printability and good biocompatibility. One of the major factors affecting the printability is the mechanical strength of the material. Sufficient mechanical strength is needed to avoid layer delamination or part breakage during continuous printing. Owing to the very low suction force in FLOAT, we show that a low, soft tissue-like mechanical strength (1 – 8 kPa) of the printed part is sufficient to support the continuous printing.

In the current work, we added PEGDA to the GelMA mixture (total polymer less than 13% v/v) to enhance its mechanical strength. Future strategies to enhance the mechanical strength of polymeric materials may include improving photo-crosslinking chemistry (33) or developing nanomaterial doped polymer composites (34).

However, it should be noted that high mechanical strength out of the stiffness range of the soft tissues (1 – 100 kPa) should be avoided for tissue engineering applications, since it will not support the growth and function of encapsulated cells. In studies that do not involve cell encapsulation, such as the nerve conduits fabricated by Zhu et al. (35), high stiffness hydrogel parts (300 kPa – 3000 kPa) have been produced by using high concentration precursor solutions (total polymer 32.5% v/v). Relatively high concentration precursor solutions (total polymer 20% v/v) was used to build cell-laden constructs using layer-by-layer SLA, but they only supported short term cell viability and function within 24 hours (20).

reference link:https://www.biorxiv.org/content/10.1101/2020.10.22.345660v1.full


More information: Nanditha Anandakrishnan et al, Fast Stereolithography Printing of Large‐Scale Biocompatible Hydrogel Models, Advanced Healthcare Materials (2021). DOI: 10.1002/adhm.202002103

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