Tracheae of Сontention
Andrey Panteleyev: When, after telling the doctor about bioengineering, you hear from him that there is a patient on which he is immediately ready to try, you realize that you wasted your time
Photo; Colored scanning electron micrograph of a section through the wall of a trachea. Credit: Steve Gschmeissner/Science Photo Library/Getty Images.
It could be said that Paolo Macchiarini’s case is coming now to an end. Some of his papers have been retracted from prestigious scientific journals, others have to be retracted in the near future. Criminal cases on the results of investigations into the deaths of his patients will sooner or later reach the court. Comprehending devastating consequences of this story, I would like to clarify two questions. Could these widely advertised experiments lead to a success in principle? Does this mean that the bioengineering of the human respiratory tract is now closed to research? I talked to Andrey Panteleev, the head of the tissue engineering laboratory at the Kurchatov Institute in Moscow.
— Andrei Aleksandrovich, since when are you engaged in the problem of bioengineering of tracheae?
— I have been researching the epithelial tissues almost the whole of my life — for 30 years, at least. Bioengineering — for 10 years. And the respiratory epithelium directly — for about 4 years.
— Why did you start this work? Is this somehow connected with the sensational stories of tracheal transplantation?
— To some extent this situation stimulated our choice. A few years ago I attended one of the conferences held by Paolo Macchiarini in Krasnodar. At that time, everyone was interested in his work, his story became famous in the West. He had a lot of publications in leading scientific journals. (Now two Macchiarini’s articles in The Lancet and one article in Nature Communications are retracted — A.A.). It became obvious to me that the problem of tracheal bioengineering is much more complicated than bioengineering of the skin, and therefore it is more interesting.
— What was your impression of the conference?
— Several leading Western experts made reports at the conference. Macchiarini himself delivered only an introductory lecture. Directly about his work told the students in small reports on fragments of the overall project. On the one hand, when so many young people participate in the work, it always makes a positive impression. On the other hand, this practically makes it impossible to conduct a scientific discussion — students have little knowledge of the material, and it is difficult to ask questions to an enthusiastic young man who is convinced that he is involved in the sacraments of great science.
— In other words, you still had the questions.
— Naturally. And firstly the question about the long-term consequences of using bioengineered equivalents of the trachea. A patient with tracheal dysfunction can get a plastic tube instead of a trachea, and he will live relatively well for several weeks. For some time, the main problems will be removed, respiration will be restored.
— He will have time, like Julia Tuulik, to appear in front of the cameras, to give an interview…
— Yes. These weeks will be enough. However, due to the poor survivability of the artificial graft and its inability to perform normal functions of the trachea, it will be rejected and necrotic. This process ends most often sadly.
— Those things did not turn you off from the bioengineering trachea?
— On the contrary, bioengineering, including that of tracheae, is a promising direction. This is most likely the future of medicine. Although Macchiarini was much talked about as an example of success in the field of bioengineering, it was obvious that his work has an indirect relationship to it. For this reason it is rather difficult for me to evaluate Maccharini’s professionalism, we work in different areas. He is a thoracic surgeon (as the professionals told me, an outstanding one) and maybe a manager. And I all my life have been studying the mechanisms of development and functioning of epithelial tissues. My work has an indirect relationship to medicine, like Macchiarini’s activity — to biology and bioengineering. Probably, his mistakes were connected with this. In any case, if someone makes mistakes, it allows others to learn from these mistakes. And to avoid them in the future.
— Macchiarini’s method is known. The trachea was used as a carcass — synthetic or donor (deprived of cells to avoid immune reactions). The cells of the patient’s bone marrow were applied to it. All this was put in a box …
— In an incubator. This is a fairly simple device that helps to cultivate isolated tissues or artificial tissue equivalents of a rather large volume. The incubator not only maintains a certain temperature, but also ensures that the culture medium is pumped through the tissue sample. This makes it possible to maintain the viability of the tissue for a long time. Without an incubator, the natural diffusion of the culture medium supports only the surface layer of cells. The inner part of the sample dies.
— Macchiarini claimed that when the trachea was placed in the patient’s body, the body itself became a natural incubator in which epithelium grew out of these cells. Is it possible?
— This is only possible for very thin or very small tissue samples. Trachea, including its sub-mucous membrane and cartilage matrix, is a rather massive organ. In addition, the conditions in which there is a trachea in the body are far from incubation. It is located inside the body, but its lumen communicates with the environment and is subject to the most powerful effects of the environment. There is a constant flow of air through the trachea — this air is not sterile. In addition, the temperature of the inhaled and exhaled air varies greatly. This is combined with significant pressure swings during inspiration and exhalation and huge mechanical stresses when coughing and sneezing. Under these conditions, the epithelium which is not formed and not protected by mucus, will dry, become infected, or simply detach from the surface of the trachea. Usually all this things happen together. Under these conditions, the maximum that the cells planted can do is to begin to divide and somehow «condition» the surface of the trachea with certain factors that they can synthesize before they die. But this is ineffective. In the patient’s body, on the surface of the trachea, there are no conditions for the formation of a normal epithelium.
— Besides we know that the cartilage of decellularised donor trachea was quickly collapsed after the transplantation…
— This is one of the main problems of using not a synthetic tracheal carcass, but a natural, donor one. Decellularised donor trachea very quickly loses its mechanical properties and begins to collapse during intense breathing or coughing. But the main problem is to get the patient’s own cells to populate the artificial carcass. And not just populate, but form a certain structure. The trachea has a special epithelium — we call it pseudostratified. All of its cells have contact with the basal membrane, but they are different. Each has its own functions. There are basal cells that divide and maintain the viability of the epithelium. Goblet cells produce mucus, which helps to trap dust and microbes entering the trachea from the inhaled air. There are long cells with cilia on the end that cover the entire inner surface of the trachea. Cilia rhythmically swing, pushing the mucus up. At the level of the larynx, the mucus is reflexively swallowed. This active mechanism of evacuation of mucus (with dust and bacteria) is very important for humans. Our trachea is located vertically, unlike the majority of other mammals. Complications in patients who have received «bioengineered» trachea, are largely related to the lack of evacuation of mucus. Mucus accumulates in the lower part of the trachea and in the bronchi and causes putrefactive processes.
— If you do not follow Macchiarini, then which way you go?
— I am convinced that if we talk about the implantation of an artificial, and not a donor organ, then we can only talk about the absolutely functional equivalent of a trachea. Bioengineered trachea should not be transplanted with any «stem» cells with undetermined potencies, but with a fully formed epithelium capable of performing evacuation and barrier functions immediately after transplantation. The creation of such an equivalent of a trachea is a very difficult task. It can be solved only in stages. Therefore, we decided to start with a simpler task, with the creation of the surface layer of the trachea, its epithelium, in vitro. This epithelium should contain all the cellular elements of the normal epithelium (basal, goblet and ciliate cells), produce mucus and perform an evacuation function — the cilia must beat synchronously. Considering the pressure that a trachea must withstand, we needed a matrix with good mechanical properties for such an epithelium. But at the same time such a matrix would provide growth and, most importantly, the correct differentiation of cells. The point is that we can not use the donor epithelium to create the equivalent of tracheal epithelium — it will not be immunologically compatible with the patient’s tissues. Therefore, the epithelium must be grown from the patient’s own cells which we get from his nasal sinuses. When the problem of the matrix for the respiratory epithelium is solved, we can proceed to the next stage, co-cultivation of the resulting functional epithelium with a cartilage base.
— Did you find such a material?
— We tested a lot of materials, and we chose a copolymer of specific properties, developed by our colleagues from the Academy of Sciences. Probably, this is not an ideal option, but this was the best thing that we had in our hands. Now our work looks like this: the copolymer for the matrix is given to us by colleagues from the Russian Academy of Sciences. In the crystallography department of our institute, a matrix of a specific structure is obtained from this copolymer. Then the surface of the matrix is modified in a certain way and we plant cells on it. Our matrix is two-layered. The upper layer is more dense, it supports the growth of epithelial cells. The lower layer — more friable — supports the growth of mesenchymal cells, fibroblasts. We try to achieve that both types of cells grow successfully and support each other. There are a lot of technical problems here. It is necessary to learn how to support the growth of cells of different types in one culture medium, so that they specifically interact and correctly differentiate. For example, under certain conditions, epithelial cells tend to differentiate into chondrocytes, that is, instead of forming the epithelium form the cartilage. We have to make certain efforts to keep them in the way that we need.
— Did you get a full respiratory epithelium on this matrix?
— Yes. On the samples obtained by us, human progenitor epithelial cells are transformed into goblet cells alternating with ciliate cells. In this case, the goblet cells produce the mucus and the ciliate cells produce specific proteins that support their beat. We do not know yet if the beat of these ciliate cells is organized and effective. In principle, this can be estimated by micro-survey.
— It is known that in the work funded by the Russian Science Foundation, Macchiarini had a co-contractor, the Kurchatov Institute. Monkeys in his experiment received a «patch» of polymer in the esophagus. Did he use your material?
— Our laboratory has never cooperated with Macchiarini. But «patches» — this is what we now put in the tracheae of rabbits along with doctors from the First Moscow State Medical University.
— Do you work with Professor Parshin?
— He participates in this project as a consultant. The clinical part of the work is carried out by students under supervision of Professor Dydykin. We take the cells from the nose of a rabbit, grow the respiratory epithelium on a rigid polymer matrix. We place the «patch» in the trachea of the rabbit — for this purpose, a special way of fixing the matrix was invented. Preliminarily we make special damage to the trachea — such that it can not be restored by itself. These operations are quite complex, but the students do a great job. They are very responsible, capable and motivated guys. They did the very first operation for seven to eight hours, now they manage to do it for 30 minutes. Approximately in one and a half months all rabbits are taken out of the experiment. At different stages, we make a histology and see what happened to the «patch». Some results of this work were accepted for publication in the journal The Laryngoscope a few days ago. Other publications are being prepared.
— What’s next? Do you plan to create an artificial trachea for a human?
— We do not set such a task in front of us. We work with models of the respiratory epithelium, which can later be used to treat tracheal defects. It is necessary to create a functional analogue of the tracheal epithelium in vitro, to force the cilia of the epithelium to beat in a certain rhythm and direction. Only when all this works, when a certain composition of mucus is secreted, this system can be used to create a more complex structure, including cartilage and possibly elements of the vascular system. But it will be necessary to solve many other problems for this, first of all, the problems of mechanics. There are different approaches here — you can apply bioengineering methods, growing chondrocytes, forming cartilage, and you can use pieces of cartilage taken from the patient’s ribs. But so far, these are just plans. Now we are thinking about a series of experiments with epithelial «patches» on pigs. And here the students can not cope. We need experienced surgeons who can work with large animals.
— Do surgeons want to cooperate?
— Yes, we are in contact with clinicians from different hospitals. They show great interest in what we do. However, sometimes we are hampered by the desire of surgeons to transfer the results of our work to clinic as soon as possible. When after telling the doctor about the difficulties and the most complicated problems of bioengineering, you hear from him that there is a patient on which he is ready to try your developments already at the end of this week, you realize that you wasted your time. Of course, we want to bring our work to the end and achieve results, including clinical. But the desire for rapid high-profile success will only hurt. Unfortunately, the examples are well known.
The Story in Development:
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