Hydrogel, Blueberries, and a Pinch of Turmeric: Artificial Vascular System

Which organ is the most important in the human body? Romantics will say the heart, pragmatists will say the brain, and realists will say everything. And this is so, because the human body is a harmonious mechanism consisting of many parts, large and small, working in unison. If we talk about the most important fuel for such a mechanism, then one of the first, of course, comes to mind oxygen. And the delivery of oxygen is the cardiovascular system. Today we will meet with you a study in which scientists managed to create an artificial vascular labyrinth from a photopolymerizable hydrogel. How to create artificial vessels, how effective are they, are they inferior in some ways to real vessels, and what does turmeric have to do with it? This and not only we learn from the report of the research group. Go.

Study basis

At the heart of artificial vessels, the main task of which is the transfer of fluid, lies a material that works with liquids just fine. This material is called hydrogel.

A hydrogel is a combination of hydrophilic * polymer chains, sometimes found in the form of a colloidal gel, in which water is a dispersion medium * .

Hydrophilicity * - the ability to absorb water well, the antipode of hydrophobicity (the ability of a molecule to repel water).

Dispersed system * - a compound of several phases that do not mix and do not react chemically with each other. A striking example of a dispersed system is air, cloud, composite materials, etc.

A three-dimensional solid from a hydrogel is formed due to cross-bonds holding hydrophilic polymer chains. Because of this, the structural integrity of the hydrogel network does not dissolve even at high water concentrations. At the same time, hydrogel is an excellent absorbent.

Another important feature of the hydrogel for this study is its flexibility, comparable to the flexibility of natural tissues, which is associated with a high water content.

Not only was the material unusual, but also the method of its application. Since the morphology of the vascular and pulmonary systems is very complicated and confusing, using conventional 3D printing methods would be wrong. Scientists used stereolithography to create soft hydrogels containing the necessary vascular “labyrinths” inside.

Unlike standard extrusion printing, when voxels * are applied sequentially, photo stitching * allows you to use image projection and create millions of voxels simultaneously.

Voxel * - an element of a three-dimensional image, like a pixel in a two-dimensional image.

Photocrosslinking * (photocrosslinking) - the photoinduced formation of a covalent bond between two macromolecules or between two different parts of one macromolecule.

In stereolithography, the xy resolution is determined by the passage of light, while the z resolution is determined by light-absorbing additives that absorb excess light and limit the polymerization to the desired layer thickness, thereby improving the accuracy of the pattern of the created object.

It is worth clarifying that the term "resolution" in three-dimensional printing has several definitions at once, due to the presence of three-dimensionality, that is, the x , y and z axes .

Resolution xy is the least motion that occurs laser or extruder during the three-dimensional printing a single layer. The lower this indicator, the more accurate the result. Resolution z is already the thickness of the layer itself.

If photoabsorbing additives are not used, then the hydrogel model will be extremely limited in terms of shape and complexity of the structure. And one problem arises: it is impossible to use conventional light-blocking chemicals that are used to structure photoresist or to manufacture plastic parts (for example, Sudan I - C ₁₆ H ₁₂ N ₂ O) due to the toxicity and carcinogenicity of such substances. But scientists are not so easily discouraged. They proposed the use of synthetic and natural food colors, which do an excellent job with photoabsorption and are safe for human health.

The researchers initially tried to create a monolithic hydrogel, consisting mainly of water and polyethylene glycol diacrylate with a cylindrical channel with a diameter of 1 mm inside, oriented perpendicular to the axis of light projection. But even such a simple model is very difficult to create due to the fact that the low mass fraction of elements to be combined and the need for a longer polymerization lead to solidification in narrow channels, which should naturally be hollow.

In order to solve this problem, it was necessary to select certain constituent elements of the future model, including food coloring. Scientists have found that using tartrazine (a yellow food coloring, E102), curcumin (from turmeric) or anthocyanin (from blueberries) makes it possible to obtain a hydrogel with a vascular labyrinth without hardening, blocking the flow of fluid through the channel.

Among inorganic compounds, excellent results were shown by gold nanoparticles (50 nm), which are characterized by a high degree of light absorption and good biocompatibility.

Research results

Combining all the above discoveries and previous developments, the researchers started the practical implementation of a hydrogel containing a vascular network.

The first step was to test chaotic mixers (mixers), that is, intravascular topologies that homogenize * fluids as a result of interactions between fluid flows and vessel geometry.

Homogenization * is the process of reducing the heterogeneity of the distribution of chemicals and phases over the volume of a common system for them.

A monolithic hydrogel was created with a built-in static (fixed) mixer consisting of three-dimensional swirling blades (150 mm in thickness) with alternating chirality inside a 1-mm cylindrical channel.


Image No. 1

To test the operability of such a mixer, laminar fluid flows were applied to a static mixer with a low Reynolds number (0.002). As a result, rapid mixing per unit length ( 1A ) was observed and depending on the number of blades.

Next, scientists created a three-dimensional bicuspid venous valve ( 1B) The valves of this valve were dynamic (mobile) and quickly responded to pulsating anterograde (forward motion) and retrograde (reverse motion) fluid flows. It is also worth noting the formation of stable vortices in the sinuses of the valve, which is fully consistent with the behavior of this valve.


Demonstration of the work of artificial three-dimensional hydrogel bicuspid venous valve.

The next step is more complex and intricate vascular systems, which may consist of several labyrinths. The most important thing is that they should not intersect, otherwise the result will be one large maze when two or more separate, independent from each other flows are needed. The mathematical algorithms for filling space and fractal topology used by scientists have shown good results in the design of two vascular labyrinths that do not intersect.


Image No. 2

Researchers tested several architecture options with two disjoint channels: a spiral around a direct (axial) channel ( 2A ); Hilbert curves 1 ° and 2 ° ( 2B); bicontinuous cubic lattice ( 2C ); toric knot around the torus ( 2D ).


Demonstration of all variants of vascular architecture, consisting of two independent channels.

Next, scientists checked how effectively their artificial vascular system fulfills its main responsibilities - oxygen transportation. A fluid with deoxygenated red blood cells (oxygen saturation ≤ 45%) passed through a spiral channel ( 2E ) enriched with moistened gaseous oxygen (7 kPa). At the output, you can see the color change from dark red to bright red, which indicates the saturation of red blood cells with oxygen during the passage of fluid through the channel ( 2F and 2G ). The red blood cell analysis after this test confirmed an increase in oxygen saturation.

Such a spiral vascular system is quite simple, as the scientists themselves say. And despite the excellent oxygenation results, it is necessary to test the model under more stringent conditions. The model of our lung is perfect for this, since in this case it is necessary to take into account not only the possibility of building a complex network of blood vessels, but also their elasticity - an important indicator due to the dynamics of the lungs. Scientists, based on their previous achievements and the works of their colleagues, created an alveolar model with an enveloping vascular network, which is based on the principle of a complex three-dimensional structure of “Weir-Felan foam”.


Image No. 3

Weir-Phelan foam is based on convex polyhedra, but this does not stop creating concave ones that will resemble alveolar air sacs with a common atrium of the airways ( 3A ). The resulting model consisted of 185 vascular segments and 113 intersection points.

Next, the model was applied for printing. The size of the bills was 5 pl, and the printing time was 1 hour ( 3B) The cyclic ventilation of the combined airways with humidified gaseous oxygen led to a noticeable stretching and curvature of the concave airways. Perfusion of deoxygenated red blood cells at the entrance to the vascular system (from 10 to 100 mm / min) during cyclic ventilation led to a noticeable compression and clearance of red blood cells from vessels adjacent to the concave regions of the respiratory tract ( 3C ).


Demonstration of the alveolar model with an enveloping vascular network.

Analysis data of the computational model confirmed anisotropic stretching of the concave airways during inflation, i.e. expansion ( 3D ).

While the hydrogel volume (0.8 ml) in the alveolar model is about 25% of the volume of the spiral model, the oxygenation efficiency of both models is almost identical ( 3E ).

Scientists believe that the branched (mesh) topology of the hydrogel and its extension, as well as the redirection of flows during ventilation, can increase the absorption of oxygen by red blood cells, that is, their oxygenation.


Comparison of deoxygenated (left) red blood cells and oxygenated (right) red blood cells inside the fabricated vascular system.

One of the most important points is scalability. In other words, it is necessary to take into account the location of the entry / exit of the vascular system and the duct so that this architecture is as close as possible to the real lungs. The initial test volume of the hydrogel resulted in a highly branched system ( 3F ). Entrance and exit vascular systems should be located at an angle of 180 degrees relative to each other and be topologically displaced from the respiratory tract. The vessels themselves should reach the farthest branches, that is, to the alveolar vesicles, consisting of 354 vascular segments and 233 vascular intersection points ( 3G ).

Testing of the obtained alveolar model showed that it is able to withstand more than 10,000 ventilation cycles at a pressure of 24 kPa and a frequency of 0.5 Hz for 6 hours. At the same time, humidified oxygen and humidified nitrogen ( 3H , 3J ) were used during the test .

On image 3I it is clearly seen that the developed system provides the mixing of red blood cells and bidirectionality of flows within individual segments of blood vessels.


Demonstration of a pulmonary model consisting of several alveolar.

The developed system shows excellent results during the tests, as we already understood, but another important question remains - is the hydrogel model compatible with living cells.

To verify this, scientists used stereolithography to make the same models as described above, but already containing living mammalian cells. Human mesenchymal stem cells acted as such cells. Analysis of the resulting system showed that cells within the hydrogel structure remain viable and can undergo osteogenic differentiation.

Such positive results could not be left without verification, because the scientists decided to conduct a series of tests to establish the degree of usefulness of this method of manufacturing biocompatible artificial systems.

The liver was taken as the basis, for this organ performs a number of the most important functions in the body, the success of which depends heavily on the structural topology of this organ.


Image No. 4

Researchers have created a complex hydrogel structure consisting of many unicellular tissues and hydrogel carriers containing hepatocyte aggregates ( 4A - 4C ).

The promoter activity of albumin of tissue carriers containing aggregates was increased by more than 60 times compared with the activity of implanted tissues containing single cells ( 4B , 4C ). In addition, with a thorough examination of the tissues after resection, the hydrogel carrier tissues were more integrated with the tissue and blood of the test mouse ( 4D ).

Hepatic aggregates are better than single cells, but they add complexity to the process of creating hydrogel models, because their size exceeds the lowest resolution of voxels (50 mm).

In order to solve this problem, scientists created their own aggregate carrier architecture ( 4E) The microchannel network was seeded with human umbilical vein endothelial cells, as this improves tissue survival. Further, this artificial system was transplanted into the liver with chronic damage to the rodent. 14 days after implantation, the activity of the albumin promoter was observed, which indicates the survival of functional hepatocytes, i.e., the viability of the transplanted liver cells ( 4F ). Immunohistological analysis showed the presence of hepatic aggregates on the surface of the printed hydrogel components ( 4F and 4G ). In addition, a conventional analysis of the images showed the presence of blood of the carrier individual inside the implanted hydrogel system, which once again confirms the absence of any rejection.

For a more detailed acquaintance with the nuances and details of the study, I recommend that you look into the report of scientists and additional materials to it.

Epilogue

The result of this study is a vascular system based on hydrogel and natural / artificial food colors, which perfectly copes with its main tasks, in particular with oxygen transfer. In addition, scientists used a not quite standard printing method (stereo lithography), which allows you to create complex architectures in a fairly short time. In the future, scientists intend to improve their brainchild, because the vascular system of each organ or body part has its own characteristics, which must be considered, studied and taken into account in the development of a more advanced hydrogel artificial analogue.

The creation of artificial tissues, their aggregates and subsequently organs is a painstaking and very complex process. But good deeds are very often fraught with difficulties. And this study can not be called anything other than a good deed. The first problem that a sick person in need of transplantation of any organ faces is the expectation. For example, according to some sources, 20 people die in a queue for a liver transplant in the United States every day. The second problem is the donor. You can’t just take the organ of one person and transplant it to another. Compatibility of a number of parameters is required. And the second problem smoothly feeds the first, lengthening the waiting time for the rescue operation.

Of course, the mass cultivation of organs and systems, like tomatoes on a farm, with further transplantation is only the future, but how far it depends on such studies and their success. Speaking specifically about today's work, we can say that such a future has become a little closer.

Thank you for your attention, remain curious and have a good working week, guys!

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