To the content 1 . 2015

Organ "bioengineering", stem cells and scientific integrity in surgery

Keywords:bioethics, bioengineered trachea, transplantology

Clin. Experiment. Surg. Petrovsky J. – 2015. – N 1. – Р. 80–85.

The first issue of our journal in 2013 was opened by an exceptional in depth and analysis article by Professors P. Tischenko and B. Yudin on integrity in scientific research [1]. It was fundamental for the Editorial Board to indicate our level of evalution of scientific results offered to the reader.

That is why we could not help but be interested in the discussion that took place in the pages of one of the most prestigious surgical journals: Journal of Thoracic and Cardiovascular Surgery [2]. The core item of the discussion is that Professors P. De- laere and D.Van Raemdonch considered unreliable the medical data on the first bioengineered organ - the trachea. The author of this technique, Professor P. Macchiarini, on the other hand, is convinced that the results of his experimental studies and clinical experience allow him to speak confidently about tissue engineering of such a complex (anatomically, morphologically and functionally) organ as the trachea.

The author of this comment has comprehensive experience in the engineering of enzyme-treated decellularized vascular xeno-bioprostheses, acellular allogeneic and xenogeneic heart valves with reduced immunogenicity, in experimental studies of the processes of morphological adaptation of synthetic and biologic implants [3]. The general conclusion of these works is the following: all bioprostheses with reduced immunogenicity or vascular grafts of increased porosity have the long-term viability, but nonetheless remain lifeless carcass material. Any tissue engineering construct is out of the question here.

This information and point of view were expressed in the debate to Professor P. Macchiarini during his report on the results of tracheal tissue engineering at the Academician B.V. Petrovsky National Research Center of Surgery (NRCS). The keywords of Professor P. Macchiarini’s report were as follows: bioengineered construct, in-situ bioreactor, stem cells, angiogenesis stimulators, nanotechnology.

However, taking into account the reports on several successful transplantations of the tracheal allografts engineered according to P. Macchiarini’s technique in clinical practice of some other hospitals, we decided to conduct such surgery in our Center (Prof. V.D. Parshin). The patient’s consent and the positive decision of our Center’s Committee on Bioethics were received. Professor P. Macchiarini was directly involved in the surgery. The tracheal allograft was engineered and delivered from an Italian labora- tory.

Clinical observation

Patient I., 25 y/o female (medical history # 30,298,333) was admitted to the Academician B.V. Petrovsky NRCS with the diagnosis of tracheal stenosis for transplant program examination on 25 of October, 2010. Complaints on admission: ex-ertional dyspnea, choking when eating, headaches, sleep disorders, memory impairment, past episodes of seizures. From the case history: in April, 2006 she got a severe head injury, broken shins in a car accident. Craniotomy was performed for cerebral coma and progressive brain edema and tracheostomy was placed for prolonged mechanical ventilation. The patient recovered consciousness 8 months after injury. Tracheostomy healed on its own, but a month later stridor appeared. In a hospital of Shanghai (China) a tracheal mesh stent was placed. In the period from 2007 to 2010 granulation tissue removal and endoscopic recanalization of the trachea were repeatedly performed. In September, 2010 the patient was consulted at an Israeli clinic where conservative treatment including hormones was continued. In NRCS X-ray examination showed a mesh tracheal prosthesis 6 cm in length and 1.5 cm in diameter at the level of C7–T4. Echocardiogram results revealed no pathology: heart cavities were not expanded, the valves were not changed. VC was within normal limits. Generalized tracheal obstruction; tracheal stenosis were revealed. Disruptions in the structure and function of the abdominal cavity organs were not found. MRI of the brain showed cystic glial changes in the right temporo-parietal region. Bronchoscopy: the vocal folds were mobile, scar line changed, partially missing. There was a moderate narrowing of the trachea 1 cm from the subglottic larynx. Further a longitudinal scar was visible on the front wall, cartilaginous rings were not visualized. Below that there was a scar deformation and narrowing up to 8 mm and then tapering to 6×7 mm. Then over 2.5 cm further on the tracheal lumen narrowed to 9×8 mm due to granulation tissue, mucus in the area being hyperemic with the fibrous plaque. Both tracheobronchial angles were involved in this process. Because of severe subtotal stenosis of the trachea in the cervical, cervical-thoracic and thoracic parts, the council considered it expedient to offer the patient transplantation of the trachea on the vascular pedicle. There were no contraindications to that procedure.

Subsequently the decision was taken to trans- plant the tracheal allograft prepared according to P. Macchiarini’s technique using the technology of regenerative medicine. On 04 of November, 2010 tra- cheal bougienage and endoscopic recanalization by removing granulation tissue were performed. On 06 of December, 2010 preparing for reconstructive surgery the patient’s bone marrow was harvested and preserved. The total volume of bone marrow obtained was 300 ml. On 07 of December, 2010 transplantation of a tracheal allograft engineered according to the method of Regenerative Medicine (Fig. 1) was performed (Professor Parshin V.D. with the participation of Professor P. Macchiarini). The deformed trachea was removed almost completely. A section of the mucosa was taken from the resected trachea and divided into pieces of 3×3 mm for further placement on the inner surface of the donor trachea. The suspension of mononuclear cells of the patient’s bone marrow was inserted into the wall of the decellularized trachea at the rate of 1 ml per each two tracheal half-rings. Also erythropoietin, Neupogen, TGF were applied to the graft wall. After completion of the anastomoses, a self-expanding mesh stent overlapping the anastomoses was placed into the lumen of the graft. The greater omental pedicled flap was prepared through an upper median laparotomy, transposed to the tra- chea and fully wrapped around the graft.

Fig. 1. General view of allo- trachea bioprosthesis made in the Italian laboratory. Injection of mononuclear cell suspension

The description of pre-transplant morphology of the allo-trachea: necrotic changes of soft tissues without inflammatory reaction and degenerative changes in the hyaline cartilage were revealed in the donor trachea (Fig. 2, 3).

Fig. 2. Fragment of tracheal bioprosthesis before surgery: no epithelial lining, cartilaginous tissue dystrophic changes in the form of "speckled" pattern of ground substance and individual chondrocytes cytoplasm vacuolation are presented. Van Gieson's stain. × 50.

Fig. 3. Fragment of tracheal bioprosthesis before surgery: hyaline cartilage degeneration, chondrocytes vacuolation and karyopyknosis. Autolysis of soft tissue graft. No inflammatory infiltration. Hematoxylin and eosin stain. × 100.

Hospital and remote post-transplant periods

The patient was extubated on the second day after surgery. The therapy protocol included hormones, erythropoietin, Neupogen. Bronchoscopy on the 15th day after surgery: the lumen of the trachea was satisfactory, the anastomoses were airtight, the graft walls were covered with a thin layer of fibrin.

On 21 of January, 2011 endoscopic removal of the endoprosthesis was performed because of its partial destruction. In the lumen of the graft there were small overgrowth of dense granulation tissue on the front semicircle near the anastomoses and grayish masses with the changed endoprosthetic mesh inclusion eas- ily peeling off the graft’s wall on the back semicircle.

24 of January, 2011. The patient was discharged to outpatient treatment.

21 of February, 2011. Emergency hospitalization to remove the remnants of the endoprosthesis because of its dislocation. Tracheoscopy: the lumen at the proximal anastomosis was narrowed down to 3×5 mm by granulation tissue proliferation. In the place of the removed prosthesis the tracheal wall had a mesh-like surface with bleeding granulation on the membranous part; it was not possible to judge about revascularization and epithelialization of the cartilage.

07 of March, 2011. Stenosis of the tracheal graft, stridor at rest, intubation and tracheoscopy: there were overlapping and narrowing of both anastomoses throughout the graft. A day later the patient was extubated.

10 of March, 2011. Due to severe cicatricial and granulation stenosis, emergency endoscopic tracheal stenting was performed.

25 of March, 2011. The next self-expanding tracheal stent insertion.

25 of April, 2011. Due to late cicatricial tracheal stenosis both laryngoplasty and tracheal resection with T-shaped tube placement were performed. Histological examination of the donor tracheal wall revealed the masses of granulation tissue in various stages of its formation, inflammatory infiltration. The epithelial lining was missing. Cartilage and muscle tissue was not found; fibrous tissue with mature vessels.

In subsequent months, the change of T-shaped tube and electrocoagulation of tracheal granulation tissue above the tracheal bifurcation were repeatedly performed.

29 of February, 2012. The next time we changed the T-shaped tube we found lumenal narrowing of the entire trachea; during cough the trachea collapsed almost completely due to the convergence of the front and back tracheal walls, indicating the lack of almost any natural framing of the donor tracheal allograft.

Conclusion

Thus, the data presented in this article confirm the viewpoint of P.R. Delaere and и D. Van Raemdonch that the information about creating a “new” bioengineered trachea is not a proven fact. Transplantation of a donor trachea made and used in clinical practice as described by Professor P. Macchiarini eventually led to the graft’s stenosis due to scarring and granulation tissue formation without signs of preservation of native tracheal structure and function. One clinical observation, which we have, is not scientific basis for the findings and conclusions, of course. At the same time, we cannot help but support the view that the manipulation of modern terms in surgery gives our patients unreasonable expectations and undermines the authority of science and medicine.

P.S. But life goes on. And recently Professor P. Macchiarini has received a grant from the Russian Science Foundation to create a “bioengineered” esophagus.





References

1. Tishchenko P.D., Yudin B.G. Fair dealing for scientific studies. Klinicheskaya i eksperimental’naya khirurgiya. Zhurnal imeni akademika B.V. Petrovskogo [Clin Experiment Surg Petrovsky J]. 2013; Vol. 1: 5–12. (in Russian)

2. Delaere P.R., D. Van Raemdonck. The trachea: The first tissue-engineered organ? J Thoracic Cardiovasc Surg. 2014; Vol. 4: 1128–31.

3. Macchiarini P. Reply to the Editor. J. Thoracic Cardiovasc. Surg. 2014; Vol. 148 (1): 365–6.

4. Dzemeshkevich S.L., Konstantinov B.A., Gromova G.V. et al. The mitral valve replacement by the new type bio prostheses (features of design and long-term results). J Cardiovasc Surg. 1994; Vol. 35 (1): 189–93.