RU2848131C1 - Method for studying the course of the wound healing process in modelling alloplasty in laboratory animals - Google Patents

Abstract

FIELD: medicine; experimental abdominal surgery.

SUBSTANCE: invention can be used in modelling the treatment of postoperative ventral hernias. After performing alloplasty on the 7th and 14th days of the experiment, blood samples are taken, the number of rod-shaped neutrophils, ESR and regeneration index are determined. On the 14th day of the experiment, histological material containing abdominal wall tissue with the transplant is collected, and biomechanical and morphological studies are performed. The biomechanical properties of the material are determined using an INSTRON-5944 tensile testing machine, and the stress (MPa), deformation with the maximum load value (N), and Young's modulus (Pa) are determined at the moment of change in the phases of biomechanical behaviour and at the moment of maximum deformation preceding the rupture of the flap. The average values of stress, load and Young's modulus are determined. The morphological properties of the material are determined by staining the sections using the OKG method according to Zerbin and Lukashevich. The presence or absence of immature collagen, leukocyte infiltration, cystic formations, muscle fibre atrophy is determined, and the integration of the mesh graft with muscle fibres is determined.

EFFECT: study of the wound healing process through comprehensive determination of the biomechanical and morphological properties of the biomaterial and the inflammatory response.

1 cl, 7 dwg, 1 ex

Description

Field of technology

The invention relates to the field of medicine, namely to experimental abdominal surgery, and can be used in scientific research, in modeling the treatment of postoperative ventral hernias.

State of the art

Postoperative ventral hernias develop in 4-15% of patients undergoing laparotomy and are the second most common type after inguinal hernias. Postoperative ventral hernias most frequently occur after emergency procedures, and midline hernias predominate. The issues of surgical treatment of ventral hernias still remain among the most important problems of abdominal surgery (see Belyansky I. et al. A novel approach using the enhanced view totally extraperitoneal (eTEP) technique for laparoscopic retromuscular hernia repair. Surg Endosc, 2018. Vol. 32. No. 3. pp. 1525-1532; see Ermolov A.S. et al. Surgical treatment of patients with large and giant postoperative ventral hernias. Surgery. Journal im. N.I. Pirogov, 2019. No. 9. pp. 38-43; see Ermolov A.S. Choice of surgical tactics depending on the size of the ventral hernia and the deficiency of abdominal wall tissue. Moscow surgical journal, 2018. Vol. 3. No. 61. P. 78).

Over the past three decades, the priority direction has been plastic surgery using synthetic prostheses (see Blagovestnov D.A. et al. The role and place of prosthesis fitting methods of forward belly wall plasticity in primary median hernia surgery. Vestnik novykh meditsinskikh tekhnologiy. 2011; Vol. 1. No. 18. pp. 153-155; Ramakrishna H.K., Lakshman K. Intra peritoneal polypropylene mesh and newer meshes in ventral hernia repair: what EBM says? Indian J Surg 2013. Vol. 75. No. 5. pp. 346-351). By now, it has reached the point where there is a need for an in-depth analysis of the accumulated experience. Of particular relevance in this regard are issues related to the need for an individual approach to the selection of a method of prosthetic repair with the development of new methods, a thorough study of immediate and long-term results and the prognosis of late complications associated with mesh migration and hernia recurrence.

At the current stage of development of alloplasty, a clear position has been formed on the insufficient reliability of autoplastic techniques in the absence of a differentiated approach to their use (see Rodríguez M. et al. Polymer Hernia Repair Materials: Adapting to Patient Needs and Surgical Techniques. Journal Materials (Basel), 2021. Vol. 14. No. 11. pp. 27-90; Sheen A.J. et al. Comparison of Mesh Fixation Techniques in Elective Laparoscopic Repair of Incisional Hernia - ReliaTack™ v ProTack™ (TACKoMesh) - A double-blind randomized controlled trial. BMC Surg, 2018. Vol. 18. No. 1. p. 46).

A method for evaluating the main alloplasty techniques to optimize treatment for patients with postoperative ventral hernias (see https://stm-journal.ru/ru/numbers/2011/2/735/pdf) is known. It compares the results of different alloplasty techniques to develop a system for optimizing surgical treatment for patients with postoperative ventral hernias. The method considers an improved version of the sublay alloplasty technique and uses CT-assisted hernioabdominometry to determine the appropriate type of repair for giant and large hernias, allowing for optimized treatment. An analysis of complications and long-term treatment outcomes in 132 patients with postoperative ventral hernias showed that sublay alloplasty yields the best immediate and long-term results.

A method for predicting and preventing wound complications in the treatment of postoperative ventral hernias is known (see https://dgmu.ru/wp-content/uploads/2023/04/Avtoreferat.-Data-razmeshheniya-25-aprelya-2023g..pdf), according to which, at the first stage of the experiment, a defect in the peritoneal-muscular-aponeurotic layer in the area of the white line of the abdomen was created in experimental animals in order to obtain a ventral hernia model. The second stage of the operation was performed 6 months after the formation of the ventral hernia in 12 experimental animals. The experiments were divided into 2 series: 6 animals in each. In the experimental group, prosthetic hernioplasty with fixation of an implant treated with a 2.5% argovit solution was performed according to the developed technique. In the control group, surgery was performed, but without treatment of the mesh implant. To conduct morphological studies, tissue samples were taken from the surgical site. Fragments measuring 2.5-1.5 cm were taken from the peripheral aponeurosis, muscles, and implant on days 3, 7, and 14 postoperatively to study the tissue response dynamics at designated time points with and without implant treatment with 2.5% argovit solution.

A comparison of the methods of alloplasty of the anterior abdominal wall in the experiment is known (see http://www.nauteh-journal.ru/files/ce860e67-8501-4539-93ab-5482099c8ca2?ysclid=m7a9wcjhf2472913814). Due to the introduction of various methods of ventroplasty of the anterior abdominal wall, as well as the lack of pathogenetic substantiation of indications for each of them, the authors of the article carried out work on studying the dependence of the biomechanical and morphological properties of prosthetic ventroplasty on the technique of suturing the mesh implant. 36 laboratory rats of the Standard breed, weighing 400 ± 50 g, were used as biological models in the experiment. Under acute experimental conditions, alloplasty with a mesh implant (Esfil, Russia) using the sublay technology was performed on laboratory animals under general anesthesia in aseptic conditions. Two observation groups (18 operated rats in each group) were formed depending on the method of alloplasty performed. In Group 1, alloplasty was performed with implant fixation using through-and-through U-shaped sutures, while in Group 2, the implant was not fixed. On the 21st day, specimens were collected for biomechanical and morphological studies. It was found that the difference in biomechanical parameters after simulating prosthetic alloplasty using two different methods (with and without fixation) was statistically insignificant in the area of elastic-plastic deformations. Biomechanical and morphological studies demonstrated that the strength of the abdominal wall during posterior separation alloplasty is independent of the method of implant fixation to the abdominal muscles. Thus, the problem of assessing the quality of ventroplasty performed in the experiment, as described in the specialized literature, exists.

However, the above decisions do not allow for the selection of a technique for performing abdominal wall alloplasty that reduces the risk of late wound complications. Long-term outcomes of various ventroplasty techniques are analyzed by assessing the incidence of hernia recurrence, but this approach is unacceptable in experimental surgery.

Disclosure of the essence of the invention

The technical problem of the proposed solution is the development of a method for evaluating the technique of performing abdominal wall alloplasty surgery in an experiment.

The technical result consists of experimental modeling of a method for treating postoperative hernia, which allows for a reduction in the risk of late wound complications.

The technical result is achieved by the fact that in the method for studying the course of the wound process in modeling alloplasty in laboratory animals, according to the solution, after performing alloplasty on the 7th and 14th days of the experiment, blood is collected, the number of band neutrophils, ESR and regeneration index are determined, on the 14th day of the experiment, histological material containing abdominal wall tissue with a transplant is collected, biomechanical and morphological studies are carried out; wherein the determination of the biomechanical properties of the material is carried out on the tensile testing machine "INSTRON-5944", stress (MPa), deformation with a maximum load value (N) and Young's modulus (Pa) are determined at the moment of change of phases of biomechanical behavior and at the moment of maximum deformation preceding the rupture of the flap, the average values of stress, load and Young's modulus are determined; The morphological properties of the material are determined by staining the sections using the OKG method according to Zerbino and Lukasiewicz; the presence or absence of immature collagen, leukocyte infiltration, cystic formations, muscle fiber atrophy are determined; the integration of the mesh transplant with muscle fibers is determined.

Brief description of the drawings

The invention is explained by drawings, where Fig. 1 is a table showing the average values of the complete blood count (CBC) on the 7th day; Fig. 2 is a table showing the average values of the complete blood count (CBC) on the 14th day; Fig. 3 is a table showing the results of biomechanical tests of the experimental samples; Fig. 4 is a morphological picture of the tissue samples of the abdominal wall fragments of the 1st group; Fig. 5 is a morphological picture of the tissue samples of the abdominal wall fragments of the 2nd group; Fig. 6 is a morphological picture of the tissue samples of the abdominal wall fragments of the 3rd group; Fig. 7 is a morphological picture of the tissue samples of the abdominal wall fragments of the 4th group.

Implementation of the invention

The method includes carrying out alloplasty on groups of laboratory animals, analyzing regenerative processes on the 7th and 14th days of the experiment by taking blood for laboratory testing for the presence of an inflammatory process, taking material for biomechanical and morphological studies on the 14th day, assessing the biomechanical and morphological properties and selecting an alloplasty technique based on the results of the reference values obtained as a result of the assessment.

The method is carried out as follows.

All manipulations and animal care were regulated by the local ethics committee of Reaviz Medical University (protocol No. 9, September 10, 2024). The conditions for keeping laboratory animals in the vivarium are regulated by RD-APK 3.10.07.02-09 "Methodological recommendations for keeping laboratory animals in vivariums of research institutes and educational institutions", order of the Ministry of Health of the Russian Federation dated April 1, 2016 No. 199n "On approval of the rules of good laboratory practice", GOST 33216-2014 "Guidelines for the care and maintenance of laboratory animals. Rules for the care and maintenance of laboratory rodents and rabbits" (updated January 1, 2021).

Forty laboratory animals, Standard rats weighing 450±50 g, were used as biological models in the experiment. In an experimental operating room, the animals underwent alloplasty with fixed and non-fixed antemuscular and retromuscular mesh graft placement (Esfil, Russia) under general anesthesia and aseptic conditions. Four observation groups were formed (10 experimental rats in each group) depending on the alloplasty technique. In the 1st group, retromuscular alloplasty with mesh graft fixation was performed, in the 2nd group, retromuscular alloplasty without mesh graft fixation was performed; in the 3rd group, antemuscular alloplasty with mesh graft fixation was performed, in the 4th group, antemuscular alloplasty without mesh graft fixation was performed.

To simulate alloplasty with retromuscular placement of the mesh graft, after dissecting the anterior wall of the rectus sheath, a retromuscular bed was created and a 2x2 cm graft was placed, simulating posterior separation alloplasty. By reconstructing the linea alba, the graft was covered with the rectus muscles and the anterior plates of their sheaths.

To simulate alloplasty with antemuscular placement of the mesh graft, the anterior abdominal wall was incised down to the fascial layer, an antemuscular bed was created, and a 2x2 cm mesh graft was placed, simulating posterior separation alloplasty. The graft was covered with a subcutaneous fat layer after restoration of the linea alba.

To assess regenerative processes, blood was collected for laboratory testing on days 7 and 14 of the experiment. On day 14, samples were collected for biomechanical and morphological studies. Biomechanical properties were assessed using an INSTRON-5944 tensile testing machine. To analyze the abdominal wall tensile test data, stress and strain graphs were plotted over time. Stress (a parameter used to compare the tension of structural tissue elements, obtained by multiplying the relative tensile load by the tissue density; MPa), strain, with the maximum load (N), and Young's modulus (modulus of elasticity) (Pa) were analyzed at the moment of biomechanical behavior phase transition and at the moment of maximum strain preceding flap rupture.

Tissue samples from abdominal wall fragments containing grafts were subjected to morphological examination. Sections were stained using the OKG (orange-red-blue) method modified by D.D. Zerbino and L.L. Lukasevich (1993), which includes the use of orange, which is used to identify collagen fibers, red (for staining erythrocytes and other cellular structures containing hemoglobin), and blue (designed to contrast cell nuclei and some connective tissue components), as well as their transition shades. This made it possible to identify changes in the vessel walls caused by the presence of fibrin and to evaluate the overall morphological picture of the tissues. The examination and photography of the preparations were conducted under a Leica Scope A1 light microscope with an integrated Leica DC-500 digital camera at 100x magnification.

The digital data obtained from the INSTRON-5944 tensile testing machine were processed using variation analysis. Quantitative data in the study groups were presented as the arithmetic mean (M) and standard deviation (SD; σ). The confidence coefficient was defined as the marginal sampling error ΔXср., calculated using the formula ΔXср.=tμср., i.e., the marginal sampling error is equal to t-fold the number of mean sampling errors. Differences were interpreted as significant at an error probability of less than 5% (p<0.05); i.e., if p>0.05, the null hypothesis was not rejected.

Thus, during the experiment, a laboratory study of the rats' blood on the 7th day (Fig. 1) revealed a slight increase in band neutrophils, a regeneration index of 0.12, and an erythrocyte sedimentation rate (ESR) in the complete blood count, indicating a minor inflammatory process. On the 14th day of the experiment (Fig. 2), in the groups with a retromuscular position of the mesh graft, the band neutrophil and ESR indicators were within normal values. However, in the groups with an antemuscular position of the mesh graft, both on the 7th and 14th days, a persistent increase in band neutrophils and ESR was observed, while the regeneration index was 0.2, indicating a persistent inflammatory process, suggesting possible complications.

In addition, it is important to note that with the antemuscular method of installing the mesh implant, an increase in both lymphocytes and monocytes was maintained, while on the 14th day with the retromuscular method of installing the mesh implant, reference values for these indicators were observed.

Subsequently, by comparing the biomechanical properties of the abdominal wall, the following average values of load (N), stress (MPa), and Young's modulus (Pa) were obtained, presented in Fig. 3. It should be clarified that up to 35 seconds, the studied material behaved as elastic-plastic, while afterward, inelastic fracture processes were detected. Therefore, the characteristics for up to 35 seconds were subject to comparison.

Analyzing digital data from the INSTRON-5944 tensile testing machine, shown in Fig. 3, the best results were found in Groups 1 and 2 (after alloplasty with retromuscular mesh placement). However, it should be noted that no significant changes were observed between Groups 1 and 2, suggesting that fixation of the mesh graft to the abdominal wall tissue does not significantly improve the strength of the operated abdominal wall.

Morphological signs of a favorable wound healing process in modeling alloplasty in laboratory animals include the appearance of immature collagen with noticeable capillarization processes, as well as the integration of the mesh graft with muscle fibers within 14 days after surgery with minimal leukocyte infiltration (Figs. 4, 5); signs of an unfavorable wound healing process include the absence of immature collagen within 14 days after surgery with pronounced leukocyte infiltration, with visualization of multiple cystic formations, the presence of foreign body cells, and marked muscle fiber atrophy (Figs. 6, 7). For additional interpretation of the identified changes and their use for further diagnostics, it was established that a regular change in the tinctorial properties of fibrin molecules in the vessel walls occurs. Fibrin clots form in the microcirculatory bed because the microcirculation acts as a morphological substrate for blood and lymph flow and the removal of metabolic waste products. Thus, microcirculation is a broad concept that encompasses not only the influx of arterial blood, rich in oxygen, biologically active substances (BAS), and nutrients, to tissue structures, but also the exchange and outflow of venous blood and lymph, as well as the removal of catabolic waste products (V.V. Kupriyanov et al., 1975). It is important to note that various damaging catabolites originating from tissues are found in the highest concentrations in the blood vessels of the microcirculatory bed. Under certain conditions, these substances can have pathological effects on blood composition. However, as venous blood flows away from the organs, the concentration of these harmful substances decreases, so such effects are usually not observed in large vessels. Analyzing the results obtained with the retromuscular position of the mesh graft, it should be noted that by day 14, pronounced compensatory processes are noticeable, as well as the absence of diffuse leukocyte infiltration of all layers of the peritoneum. This cannot be observed with the results of the antemuscular position of the mesh graft (Figs. 6, 7), in which the cellular reaction was represented by focal accumulations of neutrophilic leukocytes with a tendency toward diffuse distribution, and the arterial and venous vessels were unevenly filled with blood, with increasing edema in the soft tissue area.

Thus, the study results demonstrated that the OKG method (as modified by D.D. Zerbino and L.L. Lukasevich (1993)) reliably detects fibrin, effectively differentiating it from surrounding tissues and establishing its age and morphological structure. The structure of intra- and extravascular fibrin differed slightly, while fibrin color during staining was independent of mesh graft placement. However, it should be noted that leukocyte infiltration was absent on day 14 with a retromuscular mesh graft, which is a highly valuable criterion.

Morphological examination also confirmed that fixation of the mesh transplant to the abdominal wall tissues does not lead to a significant improvement in the strength characteristics of the operated abdominal wall, but the location of the mesh transplant is important.

Thus, based on the analysis of the experimental data, it can be concluded that morphological signs of collagen immaturity and the cellular pattern of leukocyte infiltration in the postoperative scar are unfavorable indicators that impair the quality of ventroplasty. Conversely, morphological signs of mature collagen in the postoperative scar, minimal leukocyte infiltration, and the presence of fibroblasts in the scar indicate a favorable wound healing process and the satisfactory quality of the ventroplasty performed. The significance of morphological examination of the postoperative scar on the 14th day after surgery is confirmed by subsequent testing of the operated abdominal wall on a tensile testing machine: an unfavorable morphological pattern is associated with a decrease in the strength characteristics of the abdominal wall.

Claims (1)

A method for studying the course of the wound process in modeling alloplasty in laboratory animals, characterized by the fact that after performing alloplasty on the 7th and 14th days of the experiment, blood is collected, the number of band neutrophils, ESR and regeneration index are determined, on the 14th day of the experiment, histological material containing abdominal wall tissue with a graft is collected, biomechanical and morphological studies are carried out; in this case, the biomechanical properties of the material are determined on the tensile testing machine "INSTRON-5944", stress (MPa), deformation with a maximum load value (N) and Young's modulus (Pa) are determined at the moment of change of phases of biomechanical behavior and at the moment of maximum deformation preceding rupture of the flap, the average values of stress, load and Young's modulus are determined; The morphological properties of the material are determined by staining the sections using the OKG method according to Zerbino and Lukasiewicz; the presence or absence of immature collagen, leukocyte infiltration, cystic formations, muscle fiber atrophy are determined; the integration of the mesh transplant with muscle fibers is determined.