• The second quantum revolution is building the technology of the futureRead More
  • EditorialRead More
  • Green silver nanoparticles: Synthesis, characterization and usesRead More
  • Arsenite removal with Fe3O4, MnFe2O4 and CoFe2O4 NanoparticlesRead More
  • SENTINELRead More

Modern trends in regenerative medicine


Engineering and life sciences toward the development of biological substitutes

Krzysztof Tadyszak  Ph.D.

NanoBioMedical Centre, Adam Mickiewicz University, ul. Umultowska 85, 61614 Poznań, Poland.

Institute of Molecular Physics Polish Academy of Sciences ul. M. Smoluchowskiego 17. 60-179 Poznań.

E-mail: krztad@amu.edu.pl

One of the biggest goals in medicine is to regenerate/replace any tissue when it starts to malfunction or when it is heavily damaged. One of the possible ways to do it is to help the body heal itself faster by promoting cellular grow and replace the damaged or missing tissues (regenerative medicine). This can be achieved, for example, through preparing conditions and organizing space around cells, by using specific scaffolds. Typically, porous structures, which can mimic different tissues like skin, bones, heart valves etc. made of organic polymers/composites are used. This possibility is also the main reason why they found their way into the bio-medical sciences.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue or organ function [1]. A broader term generally used is regenerative medicine, which aside of tissue engineering incorporates research on self-healing of living organisms (Fig. 1). It is well known that cells are the building blocks of tissues, the basic units of organs, which in turn are designed to perform more sophisticated functions than a single tissue could burden. Groups of cells make and secrete their own support structure, called extra-cellular matrix (scaffold). These structures have to do more than just support the cells, they are also responsible for enabling proliferation, multiplication, transporting nutrition, and also act as relay station for various signaling molecules send by other cells in the local environment. That is the reason why most of the requirements set for scaffolds demand that the structures allow the migration of various chemical substances and/or cells, which typically means pore sizes of 5-500 µm [2, 3]). In addition to the conditions mentioned above, artificial structures should exhibit low toxicity to the cells, which will populate the structure, additionally, should not cause inflammation, be biodegradable and exhibit specific structural robustness (enough mechanical strength to retain shape, and provide support for growing tissues). Potential materials with these characteristics include natural/synthetic polymers, ceramics, and metals.


Figure 1.  Principles of tissue engineering in regenerative medicine.

There is a large group of biologically important polymers derived from animals or plants which can be converted into porous polymers, functionalized and then adapted to fulfill a new bio-role. They can be divided into biological (natural) polymers (e.g. carbohydrates, lipids, proteins, nucleic acids, gelatine, glycosaminoglycan, pectin, chitin, chitosan, starch and agar) and synthetic polymers: poly(alpha-hydroxy esters), polyanhydrides, polyorthoesters, polyphosphazens, polyglycolic acid (PGA), polylactic acid (PLA) and their copolymers. Many of the biological tissues are polymeric structures, like parts of cardiovascular system, skin, and bone like structures.

Porous solid systems, with predominance of open pores in the structure, are used in biology/medicine. They form a diversified class of synthetic materials which exhibit a lot of remarkable properties dependent on the structure type, pore size/shape, surface functionalization, etc. For example, the porosity of graphene aerogel can be so high that the density riches 0.16 mg/cm3. Aerogels are gels in which the liquid was replaced by air, while maintaining the solid structure intact. The porosity in such materials is sometimes so large that the term “science of empty space” is used [4]. When designing the pore structure in a controllable fashion aerogels can gain additional robustness and resilience against applied force or mechanical deformation (10000 times higher stiffness than their starting material [5]). Most of these properties became extraordinary, special when using mass as a normalization factor, which of course, in some cases is justified [6].

Bioprinting

The current need for organ/tissue replacement, repair, and regeneration, with the current low supply of donors, has forced scientists to develop scaffolds as an alternative to transplantation. Preparing personalized tissue replacements demands a technique which is reliable, versatile and fast, which is currently the main obstacle. Nevertheless, a technique which might fulfill all the requirements is called bioprinting, and it is the next step in 3D printing technology used today. It uses bioinks – suspensions of cells, growth factors and polymers which can be used in living organisms (as scaffold), which are able to withstand the forced strain of printing and bio-application. To accelerate the process printing is done simultaneously by multiple nozzles, creating in real time the entire structure saddled by cells. A proposed bioprinter design is showed in Fig. 2.

Figure 2.  Bio Printer design “BioFAB 4500 Aimed At Printing Complete Human Organs” [7, 8].

This technology allows the manufacturing of structures with optimal pore size, spatial distribution, geometry, and provides an adequate mechanical support for tissue regeneration. Currently, there are two trends in tissue preparation: i) creating general multipurpose/patient tissues or ii) customized, after extracting patients own cells. The second case suggests better results in terms of biocompatibility, unfortunately it is harder to perform. This is due to the time and cost needed when creating a tissue from scratch in emergency room conditions. These disadvantages hopefully may be overcome by 3D bioprinters. Basically, to print a tissue replacement, patients own cells are extracted, separated from unwanted cells, proliferated in a Petri dish with specific growth factors and applied during scaffold printing (Fig. 1). The time necessary for gelation and cell growth in between each step is dependent on the scaffold and cells used. As expected, this factor is one of the most time consuming steps of the whole process. The time demands can be minimized by using fewer scaffold layers, faster scaffold aging procedures, and enhanced cells which will settle the new environment faster (effectively reducing culture period). Although this methodology remains under development. It is not hard to visualize how a “mature” technology, will have the potential to print not only tissues but entire organs [9].

Skin

Over the ages people have had to deal with different types of skin damage, such as cuts, chafes, or, more commonly, burns. As a result, a lot of protective covers were invented, from primitive dressings to cotton (cellulose)-based sterile bandages as we know them today.

The concept of wound covering textiles with pharmaceutics agents, started evolving when the building fabrics gained the ability to transport drugs. This has been attributed to the development of pore structure and surface modifications, which has allowed different kinds of substances to be absorbed/adsorbed and released in wounds, as complementary treatment. Some of the most promising wound covers are liquid bandages, applied in spray from a can, creating a solid, porous and breathable structure on the wound. Their biggest advantages are: very short time of application, sterility and easily modification for drug transport. This technology is developing so fast, right now it is even possible to print/spray on the body a layer which becomes an item of clothing.

A further step in the evolution of bandages is an artificial skin – a cover that simulates the natural skin. It is clear that between bandages and artificial skin there is a huge difference. First of all, bandages should stop the blood loss, allow oxygen transport, protect the wound from biological contamination and not necessarily interact any further with the wound. However, a skin replacement should simulate at best the natural skin, additionally; it is not removable and should, in optimal conditions, become part of the body. Nevertheless, natural skin does much more than these. It is a complicated structure made of multiple layers of specialized tissues, functioning as protection (e.g. biological, physical, chemical) and transducing information about the surrounding. To be able to simulate skin functions, at least partially, we need to use a material that has the necessary mechanical, chemical and biological properties, ensuring that it can be left on wounds for a long time. Additionally, it has to be biodegradable in a time scale of days (25 days), protect from bacteria, and work as a template for the synthesis of neodermal tissue, which means it has to maintain the necessary moisture level, allow transport of new skin cells and nourish them. Cell transport in the new skin needs to start immediately, and not after the structure degrades. To be able to do that the structure needs to be porous (pore size ≥ 5 um). Many attempts have been made in order to obtain such materials, starting with polymers like collagen (collagen crosslinked with forrmaldehyde, crosslinked collagen-glycosaminoglycan precipitate) [10-13], curcumin cross-linked collagen aerogels with anti-proteolytic, pro-angiogenic properties with enhanced physical and mechanical properties [14].

One of the methods of covering wounds while simultaneously creating artificial skin is so called cell spray-grafting. It is based on donor skin cells which are separated mechanically and chemically with a modified two enzyme isolation technique involving dispase and trypsin, together with cell washing by centrifugation, and then sprayed on the wound directly [15]. This method is fast, efficient and can be regarded as the equivalent of 3D printing, which can also be done directly on the body/wound but with higher precision, repetition and accuracy if necessary.

The artificial skin can be fabricated from different substances forming two specified layers: first, a porous layer (dermis) which is covered with the second layer, a collagen rich layer (epidermis).

The 3D bioprinting attempts started with a stack of collagen layers with inclusions of fibroblasts and keratinocytes on poly(dimethylsiloxane) [16], but unfortunately this procedure was to time consuming. Further attempts decreased the number of layers to two.  The lower layer was a plasma-derived fibrin matrix populated with human fibroblasts and the upper layer was formed by human keratinocytes, seeded on the top of the fibrin scaffold forming a human plasma-based bilayered skin.  It is very important to remark, that the skin made of donor’s own cells has two advantages: it is less often rejected and has identical pigmentation with the rest of the patient’s skin. Time of printing of 100 cm2 is around 35 min which is more than enough for commercial application [17]. Further improvements will rely on decreasing the cell flocculation time and polymerization time of fibrin based gels.

The further evolution of artificial skin tissue will more likely go into the direction that connects new gels with flexible electronics, like stretchable and self-healing conductors, semiconductors, and substrates. An example was made by epidermal electronic systems, which are electronic devices glued on the skin, to measure electrophysiological signals: electrocardiograms, electromyograms, as well as temperature and mechanical strain [18]. That is also the first step for binding two apparently different technologies like artificial skin described above and “e-skin”. The second model is an artificial skin dedicated for robotics or prosthetics. It is an artificial material covered by a vast amount of sensors which, at least in theory, should transmit signals from e.g. robotic limbs to brain through artificial interface [19, 20]. The developments in this field could be applied for handicapped people with missing limbs. Due to electrical connection between limbs and brain, people could be using the new parts as they own.

In this aspect, a much more futuristic vision relays on combining an exoskeleton with e-skin and scaffolds, where the porous part would serve as the inside protective interface between the skeleton and the human body. The gel interface could actively absorb excessive impact due to its porous (sponge) structure and, if necessary, release drugs or hinder blood loss by changing locally aerogel parameters (pore structure – swelling), making human body less fragile and maybe even stronger due to the robotic exoskeleton. For sure this kind of technology will find its main application in the army, police, for motorbike suits or anywhere where human life is at risk.

Bones

Bones are organs made of two types of bone tissue – cancellous (inner, 50 – 90 vol% porosity) and cortical (outer, 10 vol% porosity) bone, which differ in constituents and functions [21]. Porous bone scaffolds can be made by a variety of chemical methods, however all of them exhibit disadvantages regarding their pore sizes or structural properties [22]. These disadvantages can be overcomed by using bottom up approach, e.g. 3D bioprinting, which allows to carefully selecting the shape of pores, and thus, improving mechanical parameters with pores large enough to maintain cell migration. Additionally, 3D bioprinting technology has already allowed the creation of three dimensional structures designed on demand, dependent on a specific injury, which was previously known from CT or MRI scans. Therefore, implants for prosthetics like ears or noses can be made much faster and in the desired shape (Fig. 3). One disadvantage that occurs and repeats in artificially produced scaffolds is the insufficient elastic stiffness and compressive strength compared to that of human bone, which is the main disqualifying factor when using them under heavy and/or dynamic load. One of the possibilities to overcome this problem is to enhance/reinforce the structures by making composites e.g. with iron-doped nanoparticles [23].

Figure 3.  Ear scaffold implanted in mice [24, 25].

Additionally to this, it is possible to recreate the porous structures which cover the outer surface of bones: cartilage at joints of long bones and periosteum everywhere else. The first is a smooth elastic tissue, rubber-like padding that covers and protects the ends of long bones at the joints. Thanks to 3D printing technology it was possible after taking stem cells from under the knee cap to recreate this protective tissue. This could help to treat cancer, osteoarthritis and traumatic injury [26, 27].

The second structure (periosteum), which is a porous structure of 500 µm thickness acting as interface between bone and muscles and providing an attachment for muscles and tendons, can be replicated using natural protein fibers [28]. Dependent on the material used throughout the entire regeneration process, the scaffold could be completely replaced by newly formed bone [29].

Cardiovascular system

The cardiovascular system is made out of three parts: heart, blood vessels and blood. Scientists are struggling to replicate each of them due to the fact that cardiovascular diseases are the most common cause of death in the Western world. The attempts to recreate parts of this system, like valves or veins, are important in general but also are crucial in other related studies – advanced organ reconstruction. Large cell structures have to be supplied with different types of molecules in an amount that is impossible to provide by a simple porous structure. In those cases a network of tubes (veins, arteries) of different sizes needs to be created. Early attempts to develop blood vessel substitutes from purely synthetic polymers, like expanded polytetrafluoroethylene or polyethylene terephthalate, have led to failure, especially in small diameter vessels (<6 mm). The reason for that can lay in the fact that natural veins are more complicated – made of three layers of cells which differ in structure and functions. One of the strategies is to “borrow” an existing vascular network instead of creating one – for example by taking a plant (spinach leave) vascular network, removing the plant cells and populating the system with human heart cells [30]. Being able to make blood vessels in the lab from a patient’s own cells could mean better treatments for cardiovascular disease. Most common practice is to use vascular grafts – artificial constructs in the shape of tubes – substitutes of blood vessels – made of biocompatible materials. There is always the struggle between biocompatibility and mechanical and dynamical properties of engineered materials [31].

An interesting strategy is to take cells out of the patient and prepare vessels invitro and graft them without being rejected. Scientist forced the donor cells to release proteins (collagen, elastin, fibronectin) that covered the surface of artificially prepared tubes. These proteins structure mimics the shape of tubes and can be further grafted to the body. Animal (sheep) test showed that this scaffold after flushing the floating cells out did not cause any immune reaction and was able to replace a part of animal artery [32].

Artificial heart devices have been surgically implanted since the 1980s, but no device has been able to replace the human heart as effectively as a healthy biological one. Common problem with it lies in the valve, which normally allows blood to flow in only one direction. If damaged they are operatively replaced with synthetic structures (Fig. 4). Due to the constant movement they perform, it is necessary to made them from a material resistant to these conditions. They are usually made of polyurethane and in the shape of a native heart valve (with soft leaflets). The most common are surfactant-templated polyurea-nanoencapsulated macroporous silica aerogels, tetramethylorthosilicate (TMOS) or copolymer structures.

Fig. 4 Artificial polymeric vein (left [33]) and heart valve (right [34]).

Organs

After the preliminary stage, where only tissues are being made in the lab, the next step is to combine the different obtained structures into more complex “factories” – organs. Much effort has been put into building new organs up to now and with some positive results. It was possible to obtain artificial thymus – an organ crucial to the human immune system, that could produce special cancer-fighting T-cells in the body [35], ears [36], bionic ear [37], limb (rat leg) [38], bladder, liver [39], trachea, back discs, kidney and human skeletal muscle [40].

The applicability of basically porous materials is not limited to tissue engineering. They can be used among others for biosensors [41-46], neural cell scaffolds [47], as membranes [48, 49] and filters [50, 51]. Fast development of the 3D printing technology, previously in the industry and now also in biology/medicine, clearly shows a picture of a new type of “printing quality”, with a great applicability potential, standing at the doorstep of commercialization. It is in fact a tantalizing possibility that such printed structures could someday enhance human capabilities and many healthcare treatments.

ACKNOWLEDGEMENTS

Author is financed from the Polish National Science Centre (UMO-2016/21/D/ST3/00975 and UMO-2014/15/B/ST4/04946).

References

[1] Chapekar, M. S. J Biomed Mater Res 53, 6, (2000).

[2] Yang, S., Leong, K. F., Du, Z., Chua, C. K. Tissue Eng 7, 6, (2001).

[3] Yang, S., Leong, K. F., Du, Z., Chua, C. K. Tissue Eng 8, 1, (2002).

[4] Gash, A., Reese, D. Aerogels: The Materials Science of Empty Space.  Available from: https://step.llnl.gov/programs/science-on-saturday/lecture/425 [Access Date Jun. 24, 2017].

[5]Zheng, X. R. MIT researchers develop new ultralight, ultrastiff 3D printed materials. MIT News  Available from: https://www.llnl.gov/news/lawrence-livermore-mit-researchers-develop-new-ultralight-ultrastiff-3d-printed-materials#.U6gIr42Szee. [Access Date Jun. 24, 2017].

[6] Chandler, D. L. Porous, 3-D forms of graphene developed at MIT can be 10 times as strong as steel but much lighter. MIT News  Available from: https://news.mit.edu/2017/3-d-graphene-strongest-lightest-materials-0106. [Access Date Jun. 24, 2017].

[7] Barnatt, C. Bioprinting.  Available from: http://explainingthefuture.com/bioprinting.html [Access Date Jun. 24, 2017].

[8]  Barnatt, C., 3D Printing: Third Edition. CreateSpace Independent Publishing Platform,   2017).

[9] Lavars, N. Seven life-changing surgeries made possible by 3D printing.  Available from: http://newatlas.com/seven-life-changing-surgeries-3d-printing/35186/ [Access Date Jun. 24, 2017].

[10] Yannas, I. V., Orgill, D. P., Skrabut, E. M., Burke, J. F., Skin Regeneration with a Bioreplaceable Polymeric Template, in Polymeric Materials and Artificial Organs. (American Chemical Society,   1984)

[11] Yannas, I. V., Burke, J. F. Journal of Biomedical Materials Research 14, 1, (1980).

[12] Yannas, I. V., Burke, J. F., Gordon, P. L., Huang, C., Rubenstein, R. H. Journal of Biomedical Materials Research 14, 2, (1980).

[13] Dagalakis, N., Flink, J., Stasikelis, P., Burke, J. F., Yannas, I. V. Journal of Biomedical Materials Research 14, 4, (1980).

[14] Dharunya, G., Duraipandy, N., Lakra, R., Korapatti, P. S., Jayavel, R. et al. Biomed Mater 11, 4, (2016).

[15] Gerlach, J. C., Johnen, C., McCoy, E., Bräutigam, K., Plettig, J. et al. Burns 37, 4,

[16] Lee, W., Debasitis, J. C., Lee, V. K., Lee, J.-H., Fischer, K. et al. Biomaterials 30, 8, (2009).

[17] Mandrycky, C., Wang, Z., Kim, K., Kim, D.-H. Biotechnology Advances 34, 4, (2016).

[18] Yeo, W.-H., Kim, Y.-S., Lee, J., Ameen, A., Shi, L. et al. Advanced Materials 25, 20, (2013).

[19] Chortos, A., Liu, J., Bao, Z. Nat Mater 15, 9, (2016).

[20] Hammock, M. L., Chortos, A., Tee, B. C. K., Tok, J. B. H., Bao, Z. Advanced Materials 25, 42, (2013).

21. Salgado, A. J., Coutinho, O. P., Reis, R. L. Macromol Biosci 4, 8, (2004).

22. Bose, S., Vahabzadeh, S., Bandyopadhyay, A. Materials Today 16, 12, (2013).

[23] De Santis, R., Russo, A., Gloria, A., D’Amora, U., Russo, T. et al. Journal of Biomedical Nanotechnology 11, 7, (2015).

[24] Cao, Y., Vacanti, J. P., Paige, K. T., Upton, J., Vacanti, C. A. Plast Reconstr Surg 100, 2, (1997).

[25] Vacanti mouse. 2017.

[26] Cottingham, K. 3-D printing could one day help fix damaged cartilage in knees, noses and ears (video).  Available from: https://www.acs.org/content/acs/en/pressroom/newsreleases/2016/march/3d-printed-cartilage.html [Access Date Jun. 24, 2017].

[27] Enriquez, J. (2016).

[28] Ng, J. L., Knothe, L. E., Whan, R. M., Knothe, U., Tate, M. L. K. Sci. Rep. 7, (2017).

[29] Rezwan, K., Chen, Q. Z., Blaker, J. J., Boccaccini, A. R. Biomaterials 27, 18, (2006).

[30] Gershlak, J. R., Hernandez, S., Fontana, G., Perreault, L. R., Hansen, K. J. et al. Biomaterials 125, (2017).

[31] Zhang, W. J., Liu, W., Cui, L., Cao, Y. J Cell Mol Med 11, 5, (2007).

[32] Syedain, Z., Reimer, J., Lahti, M., Berry, J., Johnson, S. et al. Nature Communications 7, (2016).

[33] Gefäßprothese.  Available from: https://commons.wikimedia.org/wiki/

You may also like

Green silver nanoparticles: Synthesis, characterization and uses

Green silver nanoparticles: Sy..

Environmentally sustainable production of silver nanoparticles

read more

Leave a Reply

Your email address will not be published. Required fields are marked *