Application of Normal Human Fibroblasts and Keratinocytes Cultured In Vitro in the Complex Treatment of Patients with Trophic Ulcers of Venous Origin

The dissertation by Lapin, Andrey Yurievich is dedicated to the application of normal human fibroblasts and keratinocytes cultured in vitro in the complex treatment of patients with trophic ulcers of venous origin. The research encompasses an analysis of pathogenic mechanisms of chronic venous insufficiency, diagnostic and classification methods for trophic ulcers of the lower extremities, and the development of a methodology for transplanting various compositions of cellular material onto the ulcerative defect. The study demonstrates substantial advantages of the proposed cell-based approach over traditional treatment methods and substantiates indications for the use of different types of cellular material in the therapeutic process.

Relevance of the problem.

Trophic ulcers (TUs) of the lower extremities are a problematic and most frequently encountered complication of varicose disease (VD) and chronic venous insufficiency (CVI). (Sarkisov D.S., Alekseev A.A. et al., 1996; Savelyev V.S. et al., 2000; Ruckley C.V., 1997; Burton C.S., 2001).

Existing contemporary methods of conservative treatment of TUs, combining systemic pharmacotherapy and local action on the ulcerative defect, do not allow achieving a positive result — epithelization of the trophic ulcer — in 76% of cases. (Kirienko A.I. 2000). Despite the introduction into clinical practice of modern pharmacological preparations, wound dressings of various functional purposes, the frequency of detection of trophic ulcers remains constant, and in the last decade a clear trend toward rejuvenation of this patient contingent has been observed (Savelyev V.S. 2001, Ruckley C.V., 1997). An effective way to eliminate regional hemodynamic and trophic disturbances in the extremity caused by CVI is surgical intervention (Khokhov A.M. 2002, Byrikhin N.I., Chermakova A.A., 2005), however, the presence of an active trophic ulcer, in the opinion of most specialists, is a contraindication to surgery due to the high risk of developing purulent-septic complications (Khokhov A.M., 1993; Yablokov E.G. et al., 1999; Sedov V.M., 2006). Moreover, in some cases, performing operative treatment may be associated with increased risk or even be impossible due to the presence of severe comorbid systemic pathology in patients. (Kotlukova T.V., 2000; Magomedov M.M., 2001; Prioleau P., Franceschi C., et al., 1994; Raffin V., Bellamy V., et al., 1998). Therefore, to this day the problem of searching for more effective methods of healing TUs remains relevant.

The above has compelled phlebologists to turn to methods of replacement cell therapy. In addition to the use of cell products for restoration of the skin of burns, their application in the treatment of decubitus ulcers, long-healing wounds, trophic ulcers of diabetic, neurotrophic, and venous etiology is described. In the treatment of venous trophic ulcers, cultured in vitro fibroblasts (FB), multilayer keratinocyte sheet (MKPS), and skin equivalent (SE) were used. (Vause S., et al., 2001; Phillips T. et al., 2002; Seidman C.E et al., 2003; Hjerppe A., 2004). The use of a living skin equivalent — a skin analog — for healing TUs is advocated. This method is considered a modern alternative to autodermoplasty, and its application is recognized as quite promising. (Trent J., et al., 1998; Eaglstein W., et al., 1998; Ballard K., et al., 2000; Brem H. et al, 2000; Veves A. et al., 2001; Vowden K. et al., 2002).

Without questioning the results of scientific research, in the available domestic and foreign literature we have not found argued information reflecting the advantages and disadvantages of replacement cell therapy compared to traditional approaches. There is no information on the methodology and technique of transplantation of this or that type of cultured cells and their compositions to patients with trophic ulcers depending on the initial clinical and morphological situation. The place of cell technologies in the overall multi-component structure of the treatment process and the economic feasibility of this method have not been determined. The above-mentioned points determined the goal and objectives of our research.

Objective of the research

Improvement of results and reduction of treatment duration for patients with trophic ulcers of venous origin through the application of various compositions of human skin cells cultured in vitro.

Tasks of the research

1. To assess the state of the vascular bed, microbial landscape, character of inflammatory and reparative changes in the zone of the trophic ulcer in various phases of the wound process (exudation, proliferation).

2. To determine clinical criteria and time frames for readiness of the ulcerative defect for transplantation of cell products.

3. To develop a methodology and technique for transplantation of various compositions of cultured human skin cells onto trophic ulcers, ensuring optimal therapeutic effect in the shortest possible time and rational use of cellular material.

4. To evaluate clinical effectiveness, advantages, and disadvantages of the proposed treatment method compared to traditional approaches.

5. To determine indications, contraindications, and formulate recommendations for the application of various compositions of cultured human skin cells in the treatment of patients with trophic ulcers of venous origin.

Scientific novelty

For the first time in clinical practice, multilayer keratinocyte sheets, human fibroblasts, and their combinations, cultured in vitro, have been comprehensively and differentially applied to patients with trophic ulcers of the lower extremities resulting from varicose disease in combination with post-thrombotic disease and chronic venous insufficiency.

A methodology and technique for the treatment of trophic ulcers with the application of various cell compositions have been developed for the first time.

A substantial advantage of the proposed method over traditional approaches in the treatment of trophic ulcers of venous origin has been demonstrated. Indications for the application of this or that type of cellular material in the treatment process have been determined.

A point-based assessment of the prognosis of treatment results has been proposed.

Practical significance and implementation of research results.

The proposed treatment method allows significantly accelerating the process of healing trophic ulcers, reducing the cost and duration of hospital stay and the period of outpatient treatment, as well as effectively preparing patients for surgical intervention in the shortest possible time.

The method of treatment of trophic ulcers of the lower extremities with the application of cell products has been introduced into the practice of surgical clinics of SPbGMA named after I.I. Mechnikov and the surgical department of GUZ MSC No. 18 of Saint Petersburg. On the basis of GUZ MSC No. 18, together with the department of cell cultures of the Institute of Cytology of the Russian Academy of Sciences in Saint Petersburg, a scientific-practical center has been established for providing consultative and therapeutic assistance to patients with trophic ulcers of the lower extremities of venous origin. The results of scientific research are used in the lecture course and practical sessions at the Department of Surgical Diseases with a Course of Pediatric Surgery of SPbGMA named after I.I. Mechnikov and in the course of training surgeon-residents at the Russian Medical Academy of Postgraduate Education named after S.M. Kirov.

Based on the results of the dissertation research, methodological recommendations for surgeons have been developed, approved at the Scientific Coordination Council of SPbGMA named after I.I. Mechnikov.

Basic provisions submitted for defense

1. Transplantation of cell products does not exert a direct effect on the links of the pathogenesis of chronic venous insufficiency; however, absolute cellular compatibility of in vitro cultured material, absence of local inflammatory tissue reaction to transplantation and rejection reaction after transplantation, as well as active stimulation of reparative processes in the zone of the trophic ulcer allow considering the application of fibroblasts, multilayer keratinocyte sheets, and their combinations a rational method for closing trophic ulcers of the lower extremities of venous origin.

2. The application of cell products substantially reduces the healing time of trophic ulcers compared to traditional treatment methods and allows preparing patients for surgical intervention in optimal time frames.

3. In case of refusal of surgery, a sustained positive treatment effect and absence of ulcer recurrence can be achieved through comprehensive therapy including systemic pharmacotherapy, physiotherapy procedures, elastic compression in combination with local action on the trophic ulcer in the form of transplantation of fibroblasts, multilayer keratinocyte sheets of human skin, and their compositions.

Author's contribution

The literature analysis on the researched problem was conducted independently, and the clinical material was collected and analyzed.

The author personally participated in the development and implementation of the technique for transplantation of cell products and in the subsequent treatment of patients.

The author performed the analysis and statistical processing of the obtained data.

Presentation of the work

The basic provisions of the dissertation research were reported:

- at the city scientific-practical conference "Treatment of Wounds and Trophic Ulcers Using Dermal Equivalent and Keratinocyte Sheets" (Russia, Saint Petersburg, 2003).

- at the international scientific-practical conference "Problems of Cell and Tissue Transplantology" (Ukraine, Ivano-Frankivsk, 2003).

- at the X Surgical Congress "International Surgical Congress. New Technologies in Surgery" (Russia, Rostov-on-Don, 2005).

Publications

Based on the dissertation materials, 7 printed works have been published.

Structure and volume of the dissertation

The dissertation is presented on 177 pages of typed text and consists of an introduction, six chapters, conclusion, findings, practical recommendations, and a list of literature containing 283 works: 169 domestic and 114 foreign authors. The dissertation materials contain 26 tables and 42 figures.