“Only when the last tree has died and the last river has been poisoned and the last fish has been caught will we realize we cannot eat money.” Cree Indian Proverb

The table I have under my laptop while writing this article, the materials used for my laptop, the cover case for my phone, the pen I have by my phone, the package for the mail I have received, the dividers I have in my notebook, the hair dryer I have for drying my samples before performing FT-IR on my samples, the FT-IR machine itself … They are all made up of plastics. I could go on and on, giving examples of what I observe in my immediate environment made of plastics. It would not be exaggerated to say that after the Stone Age, Bronze Age, and Iron Age, we are now living in the “Plastic Age” given the fact that the production of plastics has increased from 1.5 million tons per year in the 1950’s to 260 million tons per year in 2007.1 The majority of plastics we use in our daily life are petroleum-based plastics. What that means is, the starting materials of these plastics are chemicals derived from crude oil. There are some major concerns related with these petroleum based plastics – the Earth may run out of oil one day, or the questionable durability of how these plastics biologically degrade. Further environmental concerns exist, such as the toxic additives these plastics contain, including plasticizers like adipates and phthalate. Burning these plastics can release billions of tons of toxic pollutants every year; moreover, most plastic production reactions are done in toxic solvents, so the disposal of these solvents becomes a problem.2 Reflecting on it, it’s an incredible mercy that we have been able to get away with all the waste we have produced up to this point. But the question is: how much longer can we get away with such wasteful behavior?

One of Paulo Coelho’s passages from his book The Winner Stands Alone exactly describes my attitude and desire to “go green.” My heart pounds as I read the sentences that so touched me:

It seems now that-despite wars, famine in Africa, terrorism, the violation of human rights, and the arrogant attitude of certain developed countries-our main preoccupation is saving poor planet Earth from the many threats created by human society. “Ecology. Save the planet. How ridiculous.”

Hamid knows, however, that there’s no point in fighting the collective unconscious. The colors, the accessories, the fabrics, the so-called charity events attended by the Superclass, the books being published, the music being played on the radio, the documentaries made by ex-politicians, the new films, the material used to make shoes, the new bio-fuels, the petitions handed in to members of parliament and congressmen, the bonds being sold by the largest of the world banks, everything appears to focus on one thing: saving the planet. Fortunes are made overnight; large multinationals are given space in the press because of some completely irrelevant action they are taking; unscrupulous NGOs place advertisements on the major TV channels and receive hundreds of millions of dollars in donations because everyone seems obsessed with the fate of the Earth. Whenever he reads articles in newspapers or magazines written by politicians using global warming or the destruction of the environment as a platform for their electoral campaigns, he thinks:

“How can we be so arrogant? The planet is, was, and always will be stronger than us. We can’t destroy it; if we overstep the mark, the planet will simply erase us from its surface and carry on existing. Why don’t they start talking about not letting the planet destroy us? Because ‘saving the planet’ gives a sense of power, action, and nobility. Whereas ‘not letting the planet destroy us’ might lead to feelings of despair and impotence, and to a realization of just how very limited our capabilities are.” 3

On that note I would like to share some amazing facts I found while searching articles written on bacterial biopolymers, but first of all I would like to introduce some definitions on the concepts I will be writing about.

Plastics have many definitions, but usually, in a daily conversation, plastics mean “anything that can be molded or shaped.” Scientifically, a plastic is a sub category of a polymer. Poly- meaning “more than one” and -mer meaning “member of a particular group.”[4] Basically, a polymer is a naturally occurring or synthetic compound made of many relatively simple repeating units that are linked together in the same fashion, forming a carbon rich backbone in most cases. For example, PVC is a well known synthetic polymer, in which the monomer (the repeating unit) as seen in Figure 1 is repeated several times. A well known natural polymer is cellulose, in which the monomer as seen in Figure 2 is repeated several times.

Here it is important to note the difference between a polymer and a plastic. All plastics are polymers, as in the example of PVC, whereas not all polymers are plastics, as in the example of cellulose. The combination of the chemicals, and the type of bonds these chemicals are linked to each other by, determines the properties and applications of the polymers. The molecular weight of the polymer depends on how many times the monomer repeats itself. The molecular weight of polymers can be controlled during production with chemical techniques. One significant difference between natural vs. synthetic polymers is the molecular weight distribution. When the polymer is synthesized in the lab, the polymer product is a combination of different molecular weight chains. In other words, when a polymerization reaction takes place, lots of polymer chains are produced and one chain is never the same length or weight as another. Instead, there is a molecular weight distribution as seen in Figure 3, where most of the polymer chains in the solution have a molecular weight close to the value of Mw. So in the solution, we will have polymer chains that have molecular weights close to each other, and some extreme short or long polymer chains. It is impossible to synthesize a polymeric solution where all the polymer chains are of identical length and weight; therefore, we speak about the average molecular weight when the case is synthetic polymers. However, when we look at any polymer produced in nature, we see that the polymer chain length and molecular weight are the same every time the polymer is produced. So instead of a molecular weight distribution, natural polymers have a molecular weight value. This is important because the narrower the molecular weight distribution is, the better.

When talking about bio plastics, it is important to make the differentiation between bio-derived plastics and bio-based plastics. As Dr. R. Narayan explained in his talk at Johnson County Community College[5] , bio-derived plastics means that the plastic is isolated from a living organism, meaning that the living organism performs the polymerization reaction and then you extract the polymer from the organism.

On the other hand, bio-based plastics mean that the starting material of the plastic is derived from a living organism instead of a petroleum-based material, but it is polymerized into a plastic by humans. Therefore, not all bio-based plastics are biodegradable; however, the fact that the starting material is from a plant that can be replaced in a couple of years rather than a petroleum-based product which can only be replaced after a couple million years, drives motivation for their usage. There is the ethical concern that bio-based plastics are usually made from food sources, such as corn, however Dr. R. Narayan, who is one of the leaders in the field, argues that if the situation is handled appropriately, this should not be a problem. He argues that one up-side of the situation would be to increase values of crops and the prevention of mass migration to big cities. It’s your call to decide which side you favor more.

What is more interesting to me is the polymers being created in nature. A chemistry doctorate, Dr. Lon J. Mathias, writes that “We humans make nylons in tons per day in huge chemical plants where simple molecules are joined together in large quantities to give products that we need or want. Nature is much more careful and concise in how she does things. For a living organism to make an enzyme, another enzyme or active species must be involved. The synthesis always involves a template, or recording, of how the individual amino acids are to be joined together to give the final polymer. The enzyme adds a single amino acid, one at a time, as indicated by the mRNA. This is a slow and tedious process and takes a long time. Sometimes the enzyme gets frustrated, waiting for the right amino acid to come along, and slaps a wrong one on instead. To compensate for this, the enzyme is made to back up occasionally to check its work. If it has made a mistake, it has a process for clipping out the wrong amino acid and inserting the right one. We humans never do this. If we make a mistake, we simply grind it up and throw it away.”6

Dr. Mathias goes on, comparing the manufacturing conditions between nature’s form of polymerization and humanity’s. He says polypeptides in nature are synthesized in water, whereas we synthesize our polypeptides in toxic organic solvents. “This leads us to a problem: what do we do with the organic solvents when we’re through? Sometimes we burn them, but more commonly we try to recycle these materials, which not only are getting more expensive to buy in the first place (compared to cheap water, which is everywhere, or almost everywhere) but are also a responsibility for their recycling, purification, and final disposal. An example of how nature uses water in this way, and one which we still haven’t figured out, is the production of spider silk. Spiders spin their webs from solutions of polypeptides in water. These solutions are squeezed through the spider’s tiny spinneret and elongated quickly to form the spider webs which we’ve all seen and sometimes become tangled in. What’s really weird is that, once these spider webs form, they are no longer soluble in water. If we could just figure out how spiders first make spider silk in water and then spin their webs from it, we could make nylon the same way. This might save us a lot of waste disposal problems, and money.”6

Another spectacular creation in nature is polymers produced in bacteria which can be used as plastics once isolated from the bacteria. A wide range of biopolymers that are synthesized in bacteria serve diverse biological functions and have material properties suitable for numerous industrial and medical applications.7 Different carbon sources are efficiently converted into a diverse range of polymers with varying chemical and material properties.7 To be a little more specific, four major classes of polymers are produced by bacteria: polysaccharides, polyesters, polyamides and inorganic polyanhydrides (such as polyphosphates).7 These polymers serve various biological functions, for example, as reserve material or as part of a protective structure, and can provide a substantial advantage for bacteria under certain environmental conditions.7 Some of these biopolymers can be isolated from bacteria and can be used as plastic. Biopolymers are, by definition, biodegradable, and so their application as commodity products becomes increasingly attractive in view of the desire to avoid the use of recalcitrant oil based polymers that will accumulate in the environment.7 Biodegradable means that when exposed to the microbial flora present in a given environment (for example, in soil or water), biopolymers are fully degraded and mineralized to CO2 and H2O.5 The reason biopolymers are 100% degradable is, as they are produced in bacteria as storage material, they have sites where bacterial enzymes could attack to break them down when they search for nutrients. Whereas other polymers – even bio based polymers – will not have these enzymatic sites, so they are not always biodegradable.

One popular class of polymers produced by bacteria which can be used as plastics is called polyhydroxyalkanoates (PHA’s). PHA’s are a class of polymers produced in nature by the bacterial fermentation of sugar or lipids. They are produced by bacteria to store carbon and energy when there is a nutrient lacking from the environment. Many kinds of bacteria are able to produce PHA’s, such as soil inhabiting bacteria, and many bacteria in activated sludge, high seas, or extreme environments. 8 As we store fats in our bodies, the bacterium store PHA’s. In an environment that contains all of the necessary nutrients, bacteria grow and reproduce – in other words they produce biomass. However, when subjected to specific nutrient depletion (nutrients such as nitrogen or phosphorus) and excess amount of carbon resources, the bacterium starts storing PHA granules (Picture 3). The moment the missing nutrient is introduced back into the environment, the bacterium starts degrading the PHA granules and continues to produce biomass. Therefore, by manipulating the nutrient resources in the environment and providing optimum conditions, bacterium can be pushed to produce PHA’s.[9]

There are metabolic pathways involving various enzymes for the conversion of carbon sources to polymers. Scientists have been trying to genetically engineer bacteria for the increased production of these polymers. In some cases it is possible to over-express the key enzymes in the pathways to achieve increased production of PHA. However, this kind of research takes a lot of time and effort because altering biological activity is a very complicated process and in most cases, cells give unpredictable responses to alterations. By feeding the bacterium with different carbon sources at different conditions, it is also possible to alter the composition of the polymers. Moreover, different strains of bacterium produce different types of polymers; therefore, the range of biopolymer research is very wide. With over 150 different PHA monomers (the repeating unit of polymers) being reported, PHA with flexible thermal and mechanical properties have been developed. 7 Such diversity has allowed the development of various applications.

During his speech at the “2nd International PLASTiCE Conference Trends in Bioplastics” in Slovenia, 9 Dr. Martin Koller explained that there are two types of PHA’s that a microorganism produces. The first type are short length PHA’s (3-5 carbons in the backbone) and the second type are medium chain length PA’s (6-12 carbons in the backbone). While the medium chain length PHA’s can be used for biodiesel production, the short chain length PHA’s can be used as thermoplastics (plastics that can melt with heat, and can therefore be processed with the help of heat). These thermoplastics can be isolated from the organisms they are produced in by solvent extraction, mechanical disruption, or by using hypotonic media (having the lower osmotic pressure of two fluids) for cells that have high intracellular osmotic pressure.9 In the last case, the cells will explode due to the pressure difference and release the PHA’s; deionized water can be used as the hypotonic media. However, only specific strains can be treated with this method. At the moment, the most common technique used for extraction is solvent extraction. These solvents – such as chloroform or dichloromethane – are generally toxic, therefore creating a contradiction with the point of producing biopolymers.

Although not mainstream, some of these bacterial plastics are produced in the industrial world.8 The simplest and widest application for bacterial plastics is for packaging purposes. They can also be used in therapeutic applications, as they are generally biocompatible. Drugs can be incorporated into them, therefore as they biodegrade, they release the drug in a controlled time frame.9 For example, Dr. Martin Koller and his group have just finalized a project called “BRIC – BioResorbable Implants for Children,” funded by the Austrian Research Promotion Agency (FFG).10 Their purpose was to isolate a biocompatible polymer produced from bacterium which could be degraded and removed from the body within a certain time. The point of this project is based on the fact that in contrast to the traditional implants that need to be removed from the body after a certain amount of time, such as plates, screws or pins, the newly developed implants could be degraded and removed from the body naturally, preventing the need for a second surgery. This is a great advantage, especially for children, who would suffer greatly from additional surgeries.

Bacterial bioplastics have many other applications; however the biggest obstacle for their usage is the cost of production. During his speech, Dr. Keller stated the production of bacterial bioplastics is around five times more costly than petroleum based plastics. Most of the cost is related with the bioreactors needed to grow the bacterium and the solvents used to extract the polymers. The scientists are hoping to develop new techniques to reduce the cost of the polymers.

It is breathtaking that these creatures we cannot even see with the naked eye have been synthesizing polymers as well as we do, if not even better, and for a lot longer than us. The polymers they synthesize are completely biodegradable, have a constant molecular weight, and do not require toxic chemicals for their production, unlike the synthetic polymers we produce in the lab. They don’t harm nature as we do. And THAT is powerful.

References

1- Simon, Tristan (2007). “Experience Curves in the World Polymer Industry” Utrecht University, Netherlands.

2- Lei Pei, Markus Schmidt and Wei Wei (2011). “Conversion of Biomass into Bioplastics and Their Potential Environmental Impacts, Biotechnology of Biopolymers.” InTech.

3- Coelho Paulo(2008), “The Winner Stands Alone.” pg: 139.

4- http://dictionary.reference.com/

5- Narayan, Ramani (2013)”Bioplastics and Reducing Carbon Footprint.” JCCC Video. Johnson County Community College, USA.

6- Mathias, Lon J. (2005).”Natural Polymers.” Polymer Science Learning Center. The University of Southern Mississippi, USA.

7- Rehm, Bernd H.A.(2010). “Bacterial polymers: biosynthesis, modifications and applications” Nature Reviews Microbiology. Massey University, New Zealand.

8- Chen, Guo-Qiang (2010). “Plastics Completely Synthesized by Bacteria: Polyhydroxyalkanoates”. Plastics from Bacteria: Natural Functions and Applications, Microbiology Monographs, Springer. Tsinghua University, China.

9- Koller, Martin (2012). “Polyhydroxyalkanoates: Biodegradable polymeric materials from renewable resources” Plastice Project Video. 2nd International PLASTiCE Conference Trends in Bioplastics, Slovenia.

10- No name (2013).”Plastics from Renewable Raw Materials:Body automatically breaks down implants” Graz University of Technology, Austria.

11- Nishiyama, Yoshiharu; Langan, Paul; Chanzy, Henri (2002). “Crystal Structure and Hydrogen-Bonding System in Cellulose Iβ from Synchrotron X-ray and Neutron Fiber Diffraction”. J. Am. Chem.The University of Tokyo, Japan.

12- Ritter, Stephen(2005). “Green Success.” Science and Technology. pg: 40-43.

13- Waters Co. (2013). “GPC-Gel Permeation Chromatography”. Web.

In general, the most common approach in tissue engineering is to develop tools as needed. Physicians treat patients and define the requirements for a better cure. Then, biologists study the targeted problem and learn what the mechanism is that caused the failure. Later, chemists and materials scientists manufacture the tools needed to treat the problem. Finally, the tools are delivered to doctors to treat the patients. Thus, tissue engineering requires a good understanding of how body parts work and come into existence, and this involves precise and sensitive application. Precise, aware and regular study of the interactions involved in tissues and organs must be practiced by the researchers who are interested in developing techniques for the manufacture of potential body parts. One of the first scientific approaches used for tissue engineering is to simply inject the body with molecules, such as growth factors, which are known to promote organ formation.

The growth factors are naturally occurring proteins which are assigned for cell proliferation and differentiation. Different parts of the body require different types of growth factors to signal to the cells to multiply or to replace the cells which have died or have been damaged. For example, it has been discovered that bone morphogenic proteins are responsible for the beginning of bone cell reproduction. For someone with a fractured bone that can not heal on its own within a reasonable period of time, the injection of bone growth factors to the site can direct the body to where bone cells are needed to be produced to repair the fracture.

In more severe conditions, the body may not receive the signal only with a simple injection of the growth factors. In this case, there is a need for more intricate treatment. Another way to treat organ malfunction starts with the harvesting of cells from the patient. The harvested cells can be multiplied in an artificial scaffold to eventually be implanted into the wound site. Because cells inhabit a different world than we do, we need a way to speak their language. The artificial scaffold should provide everything a cell needs and be able to direct the targeted cells toward the desired purpose. Basic knowledge gained from biology can help us to design potential artificial environments for cells.

A critical challenge in tissue engineering is how to design and make the artificial scaffolds. The cells must be fed through the blood vessel and are grown in the scaffold by the body; the scaffold should be able to communicate with the cells and finally the scaffold should disappear when its mission has been completed. The best example of a perfect scaffold is the natural environment of the cells, the extracellular matrix (ECM). The ECM provides support and anchorage for the cells and regulates communication between cells. There are various biological signals found in the ECM that help cell survival. For example, proteins called collagens provide mechanical support for cells through adhesive proteins in the ECM and the handles on the cell surface, known as integrins. Cell adhesion is crucial for cell survival and proliferation. Growth factors are also found in the ECM for cell organization. Some growth factors promote blood vessel formation, which can provide nutrients for cells. Therefore, a simple artificial environment should include various biological signals found in the ECM.

Currently, there are natural and synthetic scaffolds that are being used to generate the optimal environment for cells. Natural polymers such as collagen, chitosan or glycosaminoglycans, and synthetic polymers, including polylactic acid, polyglycolic acid, polycaprolactone or self-assembled nanofibers, are some of the materials used or considered for scaffold production. Natural polymers can be obtained easily, however biological contamination is a concern since they are produced using components from animals or microorganisms. Synthetic polymers can usually avoid the problem of contamination. Sometimes the ability to process the polymers can be problematic. Researchers have developed self-assembled nanofibers to overcome the problems that arise with synthetic and natural polymers. These nanofibers are composed of small molecules which are programmed to come together under control and to form larger structures. The nanofibers in the solution can form a three-dimensional network and convert into a self-supporting gel which can encapsulate cells as an artificial scaffold. In general, small bioactive molecules can be conjugated to the self-assembled molecules or can be encapsulated in situ in the 3-D network of fibers.

One of the recent uses of tissue engineering is to replace tissue that has been damaged by cancer. Cancer surgery is one of the most challenging types of surgery in that the defective tissue must be reconstructed afterwards. Improvement in surgical technology gives the chance of transferring a tissue from different sites of the body but unfortunately most of the time it is not the same tissue, and does not have the same texture or function. Reconstructing a resected tongue or the feeding tube is possible with the use of skin from the leg or forearm. But this skin does not provide the normal mucosal function, so it does not enable taste or sense to be perceived in the same way nor does it produce mucus in the same way. Together with advances in tissue engineering surgeons have started using the tissue-engineered mucosa of patients to reconstruct the mouth and feeding passage defects, instead of using the skin from chest, leg or forearm skin. These clinical applications of tissue engineering are in their very early stages, but it would not be surprising if we were able to reconstruct a lost organ from a similar one in the future. It would be exciting to be able to replace the tongue of a tongue cancer patient with a brand new tongue grown from his/her own tissues produced in a laboratory. Tasting the same…sensing the same…moving and even articulating the same…instead of having a piece of meat from another part of the body…

Innovative and imaginative work which has been inspired by natural materials demonstrates how the treatment of organ malfunctions is feasible. Efforts in biotechnology to develop tissue-engineered products will benefit many people who are searching for a healthier life. Potentially, in the near future, tissue-engineered products will be more widely used to treat bone fractures, serious skin burns, spinal cord injuries, diabetes, and heart diseases. Before implanting the tissue-engineered products, it is vital that there be extensive testing of the materials to be used. Toxicology and efficacy studies should be performed on the materials to prevent damage to the original healthy cells, and the new cells and regenerated tissue must be compared to original healthy cells and tissue.

Mustafa Guler has a PhD in chemistry. He is currently a research associate at Northwestern University, Chicago, IL. Joseph Coreman is a medical doctor at the Ohio State University Medical College, Columbus, OH.

References

• Khariwala SS, Vivek PP, Lorenz RR, Esclamado RM, Wood B, Strome M, Alam DS. Swallowing outcomes after microvascular head and neck reconstruction: a prospective review of 191 cases. Laryngoscope. 2007 Aug; 117(8):1359-63.
• Sauerbier S, Gutwald R, Wiedmann-Al-Ahmad M, Lauer G, Schmelzeisen R. Clinical application of tissue-engineered transplants. Part I: mucosa. Clin Oral Implants Res. 2006 Dec; 17(6):625-32.
• Hotta T, Yokoo S, Terashi H, Komori T. Clinical and histopathological analysis of healing process of intraoral reconstruction with ex vivo produced oral mucosa equivalent. Kobe J Med Sci. 2007;53(1-2):1-14.
• Ratner, Buddy D. “Biomaterials Science – An Introduction to Materials in Medicine” Elsevier, 2004.
• Lanza, Robert P., Robert S. Langer, William L. Chick, “Principles of Tissue Engineering”, Academic Press, 1997.
• Alberts, Bruce, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, “Molecular Biology of the Cell” Garland Science, 2002.