The importance of the problem of postoperativeventral hernias (POVHs) of the anterior abdominalwall treatment continues to attract the attention ofsurgeons all over the world due to the high incidence ofthis pathology and the unsatisfactory results of surgicaltreatment (Barkov and Movchan, 1995; Grubniket al., 2001; Timoshin et al., 2003; Zhebrovskii, 2005;Sukovatykh et al., 2006). Nowadays, POVHs are con�sidered to be a complex polyetiological disease respon�sible for multiple disorders of the functioning of vis�ceral organs. The quantity of patients with postsurgeryhernias is continuing to increase (Zarivchatskii andYakovkin, 1996; Egiev, 2002; Timoshin et al., 2003).
The major principle of hernias’ surgical treatmentis performance of plastic surgery without tissue inten�tion. The use of various endoprostheses makes it pos�sible to solve this problem reliably. Since the momentof the appearance of plastic materials their usage hasbecome an attracting perspective of radical POVHtreatment. Introduction of synthetic implants in surgi�cal practice depends on relative simplicity of themethod. Of course, the use of synthetic endoprosthe�ses cannot solve every problem. The main cause ofunsatisfactory outcomes of endoprosthesing is thedevelopment of atrophic, degenerative processes, andfibrous tissue alterations in the zone of interventionand decreased firmness reparation properties and thepresence of foreign material in tissues, often leading tovarious complications (seromas, ligature fistulas, sup�puration of wounds) and to impairment of healing
The way to solve these problems is to study ofendoprostheses’ biocompatibility and possibilities ofinfluencing reparation of a patient’s tissues. Introduc�tion of cultivated fibroblasts shortens the period ofconnective tissue maturation and improves the scarstructure and its firmness (Smith et al., 1997; Hebdaand Dohar, 1999).
Fibroblasts are the most valuable cells participatingin healing. Fibroblasts produce carbohydrate–proteincomplexes of the basic substance and form collagen,elastic, and reticular fibers. All these functions con�tribute to formation of connective tissue and stimulateproliferation of other cells. Cultivated embryonicfibroblasts are used in thoracic surgery and cambusti�ology for acceleration of the process of scarring puru�lent cavities and wounds. However, death of fibroblastsin the case of infected wounds is a serious restrainingfactor for wide application of cultivated fibroblasts inthese fields of surgery. Treatment of POVHs under thecondition of “clean” surgery can solve this problem.
The activating action of exogenous fibroblasts ontissue reparative processes, clinical stable positiveeffect, and low immunogenesis are of particular valuein application of synthetic implants. Therefore, use ofallogenic embryonic fibroblasts is of unquestionableinterest in anterior abdominal wall POVH treatment.
The goal of this work was a comparative experi�mental study of the morphological picture and cellcompositions of the anterior abdominal wall of miceusing two different endoprostheses—polypropylene
Comparative Tissue Morphology in Using Prosthesesfrom Polypropylene and Polytetrafluoretilen
S. V. Ivanov, I. S. Ivanov*, G. N. Goryainova, A. V. Tsukanov, and T. P. KatuninaKursk State Medical University, Kursk, Russia
Abstract⎯To treat hernias, polymer net implants are widely used. We carried out a comparative study of themorphological picture of inflammation, cellular composition of tissues, and dynamics of scar formation inmice in using the implants Esfil and Uniflex under conditions of single and double introduction of cultivatedfibroblasts and without fibroblasts in the area of disposition of the endoprostheses. A more expressed inflam�matory reaction throughout the entire period of time was observed in implantation of the material Esfil. Inusing the material Uniflex, there was revealed a higher percent of fibroblasts than in using the Esfil prosthesis.Introduction of cultivated fibroblasts in using both materials modified the curve of dynamics of the inflam�matory process by making it smoother. Use of the Uniflex endoprosthesis is preferable.
Keywords: postoperative hernia, biopolymers, morphology, net prosthesis, fibroblasts.
DOI: 10.1134/S1990519X12030042
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Esfil and polyvinylidenfluoride PVDF�Uniflex (Lin�tex, St. Petersburg) with and without introduction ofallogenic cultivated fibroblasts.
MATERIALS AND METHODS
This work was prosthesis (1 × 0.5 cm) was placedonly above the muscular–aponeurotic layer imitatingthe plastics of POVHs of the onlay type. The standardprostheses Esfil and Uniflex were used. To comparethe tissue cell composition, the animals were dividedinto two groups: I—control, in which the Esfil net wasstitched into the animals; II—experimental, in whichthe Uniflex net was stitched into the animals.
Each group was divided into three subgroups: IAand IIA—animals into which fibroblasts were notintroduced; IB and IIB—animals into which fibro�blasts were introduced above the implant 7 days afterthe moment of endoprosthesing the abdominal wall;IC and IIC—animals into which fibroblasts wereintroduced twice: 7 and 10 days after endoprosthesing.The time of introduction of cultivated fibroblasts, inour opinion, is optimal taking into consideration thestages of the inflammatory process.
Allogenic fibroblasts were isolated from culture ofembryonic fibroblasts. As the initial material, whitemouse embryos from weeks 2 or 4 of pregnancy wereused. The embryos were placed into 199 medium withantibiotics (1000 U/mL penicillin, 1000 µg/mL strep�tomycin, 4 µg/mL amphotericin B) and delivered tothe laboratory of the Department of Cell Transplanta�tion and Immunotherapy of Kursk Regional ClinicalHospital 2 h after collection. After exposure for 4 h at4°C, the cell culture was treated with sterile Hanks’solution. Fragments of the embryo extremities andtrunk were minced with scissors until obtaining piecesof 1–2 mm3 in size and then placed into 0.25% trypsin(Serva, United States) heated to 37°C. Trypsinizationwas performed in a thermostat at 37°C for 20 min withconstant stirring.
To block the enzyme, at the end of trypsinization,cattle serum was added to the trypsin solution. Then,the cell suspension was collected into sterile test tubesand centrifuged at 1000 rpm for 10 min. The cell sedi�ment was resuspended in 199 medium with 10% cattleserum and seeded into cultural mattresses. After 48 h,the medium was changed.
After 7–10 days, the cell monolayer was formed.The confluent monolayer was removed from the sub�strate with Hanks’ solution containing 0.05% trypsinand 0.02% Na4EDTA heated to 37°C. The cellsdetached from the substrate were collected into centri�fuge test tubes and sedimented by centrifugation at1000 rpm for 10 min at room temperature. The sedi�mented cells were washed out three times with199 medium. Then, the cells were counted and seededinto cultural flasks. Culturing was performed in com�plete RPM 1640 medium with addition of 10% embry�
onic calf serum until formation of the fibroblastmonolayer.
The animals were sacrificed after 10, 30, and60 days regardless of introduction of the cultivatedfibroblasts. The studies were carried out with compli�ance to the principles espoused in the Convention ofProtection of Vertebrate Animals Used for Experi�mental and Other Purposes. From the obtained site ofthe anterior abdominal wall with an implant measur�ing 1.5 × 1 cm, there was prepared a series of sectionsthat were stained with hematoxylin with eosin and byvan Gieson. Then, the sections were examined toreveal organism�specific local reactions to implanta�tion of foreign material. The study in the experimentof the biocompatibility of endoprostheses was basedon determination of qualitative and quantitative char�acteristics of the population of fibroblasts, macroph�ages, lymphocytes, and segmentonuclear leukocytes.The data were processed with the aid of the StatPlus 2006and Microsoft Excel software.
RESULTS AND DISCUSSION
Comparative analysis was performed of the cellularcomposition of the tissues directly adjacent to theimplanted Esfil and Uniflex prostheses, without intro�duction of cultivated fibroblasts, with single introduc�tion at the seventh day and twofold introduction at theseventh and tenth days. Peculiarities of cellular reac�tions and the of dynamics of fibrous structures forma�tion around the implants are revealed. According tohistological parameters, these peculiarities depend onthe implant chemical structure and on introduction ofcultivated fibroblasts.
Comparative analysis of cytograms and histologicalpreparations of tissues in the bed of implanted Esfiland Uniflex nets was performed on the 10th, 30th, and60th days, corresponding to the time of acute inflam�matory change reduction after surgical trauma, as wellas to the time of granulation tissue development andmaturation and formation of the fibrous tissue.
In the subgroups of animals that were not given cul�tivated (allogenic) fibroblasts, there were revealed dif�ferences in the ratio of fibroblasts and time of granula�tion tissue formation and its maturation to connectivetissue, as well as in the characteristics of the cytogramreflecting the acuity of the oxidative reaction and ratioof exudation and proliferation phases of the inflam�mation process.
On the tenth day, the inflammatory reaction wasmore pronounced in use of the Esfil endoprosthesis. Inhistological preparations, inflammatory infiltrateswere revealed in capsule walls around the net threadsinvolving neutrophils and lymphocytes. Giant cellsreflecting the organism’s reaction to foreign materialwere also present (Fig. 1). The cytogram demonstrateda large number of inflammatory cells of the inflamma�tion acute phase (neutrophils and macrophages) inusing the Esfil material (Table 1).
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The number of fibroblasts had no significant differ�ences depending on the endoprosthesis type. The cap�sule walls around the net threads were thin and repre�sented by the granulation tissue, in which cell ele�ments and amorphous matrix were predominant. Theformed capsules were weakly connected with the sur�rounding tissues.
On the 30th day, the cytogram showed that thenumber of neutrophils and macrophages—the cellelements reflecting acute inflammatory reaction—became equal. At the same time, the number of lym�phocytes using the Esfil net was significantly elevated,which might be due to sensitization to the material.The amount of fibroblasts was of no significance.
In the microscopic picture of tissues aroundendoprosthesis, in both cases, formation of loose con�nective tissue of the myxoid character was revealed.The endoprosthesis capsule walls were thin with iso�morphous connective tissue fibers in them. In usingthe Esfil net, lymphocytic infiltrates appeared in thecapsule walls and around them. The connective tissuecapsules surrounding the endoprosthesis threads wereloosely connected with the surrounding tissues.
At the 60th day, in using the Uniflex net, in themicroscopic picture of the endoprosthesis bed, fea�tures of inflammatory processes were almost absent(Fig. 2). This is correlated with the parameters of cyto�grams: the number of neutrophils and lymphocyteswas significantly less (2.4 and 3.1%) than the 13 and16%, respectively, in using Esfil (Table 1).
The amount of fibroblasts in the area of the Uniflexendoprosthesis in this period of time exceeded by1.5 times the same parameter for Esfil (Fig. 3).
The capsule walls around thread of the Uniflex netin 60 days were significantly thickened due to accumu�lation of isomorphous connective tissue fibers, amongwhich mature and maturing fibroblasts were revealed.Around the capsules, connective tissue fibrous bandswere formed, which were closely connected with tis�sues around the endoprosthesis bed. At the same time,the capsule walls around the Esfil endoprosthesis werethinner and found to contain foci of inflammatory cell
Fig. 1. Histological structure of the Esfil transplant bed onthe tenth day (without introduction of fibroblast culture).A thin capsule is seen around the net fibers (arrow). Stain�ing with hematoxylin and eosin. Obj.: 40×, eyepiece 10×.
Table 1. Characteristics of cellular composition in the area of net location (without cultured fibroblasts)
Time, days Material Fibroblasts Macrophages Lymphocytes Segmentonuclear leukocytes
* p ≤ 0.01 (in comparison between materials in the same period of time).
Fig. 2. Histological structure of the Uniflex net bed on the60th day (without introduction of fibroblast culture).Longitudinal section of the net thread (arrow). Staining byvan Gieson. Obj.: 20×, eyepiece 10×.
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infiltrates. The connection with the surrounding tis�sues was looser.
Thus, the peculiarities of the reaction to the Esfilmaterial consisted in a longer course and greaterexpression of acute exudative and proliferative phasesof the inflammatory reaction and a decrease in theamount of fibroblasts and in formation of connectivetissue fibers as compared with use of the Uniflex net.This indicates that, in the case of use of a Uniflexendoprosthesis without introduction of allogenicfibroblasts, there was achieved a more favorable ratioof inflammation and organization with formation ofconnective tissue in the area of the endoprosthesis bed.
Large cavities were present around the net threads;the capsule walls were represented by the loose con�nective tissue enriched by eosinophilic matrix andfibroblasts. The granulation tissue around capsulescontained foci of fibroblast degeneration, neutrophilinfiltrates, as well as conglomerates of newly formedvessels of the sinusoidal type surrounded by immaturefibroblasts.
The cytogram demonstrated some decrease in theamount of fibroblasts as compared with cases without
introduction of cultivated fibroblasts, this being espe�cially pronounced in using the Esfil material (Table 2).Such a paradoxical result may be due to the death ofsome of the introduced fibroblasts, as well as to ele�vated activity of macrophages and neutrophils inintroduction of allogenic material; their representa�tion in the cytogram on the tenth day is moreexpressed than in cases without introduction of fibro�blasts. Thus, during use of the Esfil endoprosthesis,the number of macrophages was more than three timesas large.
After 30 days, in the case of single introduction ofcultivated cells, there was microscopically revealedformation of fibrous connective tissue in the capsulewalls; cavities around the net threads were narrowedsignificantly and did not contain exudate. Alongsidewith young fibroblasts, the mature cells appeared.Inflammatory infiltrates were small, focal; in usingEsfil, they contained many infiltrates, which corre�lated with the data of the cytogram.
In using the Uniflex endoprosthesis, the prolifera�tive inflammatory reaction phase was more pro�
100
90
80
70
60
50603010
Time, days
%Uniflex
Esfil
Fig. 3. Dynamics of the number of fibroblasts (withoutintroduction of allogenic embryonic fibroblasts).Curves 1 and 2—Uniflex and Esfil, respectively.
Table 2. Characteristics of cellular composition in the area of net location (introduction of cultured fibroblasts on the sev�enth day)
Time, days Material Fibroblasts Macrophages Lymphocytes Segmentonuclear leukocytes
* p ≤ 0.01 (at comparison between materials at the same time).
10
8
6
4
2
0603010
Time, days
%
Uniflex
Esfil
16
14
12
Fig. 4. Dynamics of the number of lymphocytes (singleintroduction of cultural fibroblasts on the seventh day).Curves 1 and 2—Uniflex and Esfil, respectively.
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COMPARATIVE TISSUE MORPHOLOGY IN USING PROSTHESES 313
nounced; in the cytogram, lymphocytes predomi�nated (Fig. 4).
The number of fibroblasts increased after 30 daysand was compared in cases with Esfil and Uniflex. Bythe 60th day after the single introduction of fibroblasts,cavities around net threads were narrow and the cap�sule walls were denser. In the walls, there were revealedisomorphous connective tissue fibers, accumulationsof fibroblasts of different degree of maturity (Fig. 5).
When using the Esfil net, numerous giant cells offoreign bodies were present, reflecting the develop�ment of chronic granulomatous inflammation as areaction to foreign material. In capsule walls sur�rounding the endoprosthesis threads, there werepresent foci of vascular neoformation, rare focal lym�phocytic infiltrates. The connection with the tissuesurrounding the endoprosthesis was loose.
In the cytogram, the amount of fibroblasts wasessentially elevated in both groups (Table 2). Thenumber of neutrophils and lymphocytes decreasedsignificantly as compared with that at the 10th and30th days. However, in using the Esfil net, the numberof macrophages exceeded by about three times that inusing Uniflex; this, alongside the presence of giant for�eign body cells, indicated an intense phagocytic reac�tion to endoprosthesis.
Thus, the number of fibroblasts in single introduc�tion of cultivated cells and without their administra�tion did not differ essentially in cytogram. Someincrease of their amount was noted 60 days after usingthe net “Esfil”; however, maturation of the connectivetissue and accumulation of fibrous fibers in the capsule
walls around the endoprosthesis threads took placemore intensely in the case of using Uniflex. In usingUniflex, the inflammatory reaction subsided faster.
In double introduction of allogenic fibroblasts on theseventh and tenth days, there was noted an earlier (aswell as on the 30th day) formation of isoformous con�nective tissue fibers in the capsule walls; in using Uni�flex, fibroblasts accumulated inside the cavitiesaround the endoprosthesis threads and the capsulewalls were represented by the dense connective tissue.A significant amount of the newly formed vessels ofthe sinusoid type was detected in using both nets. In60 days. the capsule walls around the Uniflexendoprosthesis threads were fundamentally thickerand denser. In the capsule walls, numerous isomor�phous mature connective tissue fibers were formed(Fig. 6).
In the cytogram, the number of fibroblasts in60 days did not essentially differ from the results of sin�gle introduction of fibroblasts (Table 3). However, thehistological picture exhibited a significant increase inthe number of connective tissue fibers, which indi�cated its earlier maturation into fibrous tissue.According to the cytogram, the inflammatory reactionto Uniflex practically ceased: the number of segmen�tonuclear leukocytes was reduced approximately byseven times, and of macrophages by more than twice.
In using the Esfil material, the macrophage phago�cytic activity was less pronounced than on the tenthday; however, the amount of macrophages in cytogramwas twice as high as when using the material Uniflex
Fig. 5. Histological structure of the Esfil net bed on the60th day (single introduction of cultivated fibroblasts onthe seventh day).Fibroplastic elements of different maturity with the pres�ence of single proliferating cells (arrow). Formed capsulearound net. Staining with hematoxylin and eosin. Obj.:40×, eyepiece 10×.
Fig. 6. Histological structure of the Uniflex net bed on the60th day (double introduction of cultivated fibroblasts onthe seventh and tenth days).Numerous fibroblastic elements in the area of the net bed(arrow). Staining with hematoxylin and eosin. Obj.: 40×,eyepiece 10×.
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material, which indicated greater “uniformity” of theEsfil net (Table 2, Fig. 7).
In using the net Uniflex net, a significantly higherlevel of fibroblasts was noted after 60 days in the serieswithout administration of allogenic fibroblasts ascompared both with the previous period of time(30 days) and with the Esfil net. In the series of intro�duction of allogenic fibroblasts, a gradual increase innumber of fibroblastic elements was noted, despite the“initial” percentage of fibroblasts being lower than inthe control series. The data of morphometry indicateda “smoother,” gradual increase in the number of fibro�blasts with simultaneous increase in the number offibroblasts in single and double introduction of fibro�blasts; this is the basis for the conclusion that the moreoptimal course of reparative processes in the zone ofsurgical intervention is provided with administrationof allogenic embryonic fibroblasts.
In using the Esfil net, after 60 days there wasrevealed a positive effect of the repeated introductionas a significant increase in number of fibroblasts from
65% in the control group to 79.8% in the experimentalone and optimization of the ratio of their young andmature forms. In using the Uniflex material, nothinglike this was observed.
Implantation of embryonic allogenic fibroblasts incertain periods of time changed the course of theinflammatory process. A certain percentage of allo�genic fibroblasts died, but release of various cytokinesactively affected the functional activity of the granula�tion tissue cells by accelerating the maturation pro�cess.
Thus, the common tendency of cellular composi�tion changes in the preparations in administration ofcultivated fibroblasts in all periods of time was a grad�ual increase in number of fibroblasts and connectivetissue fibers and decrease in the amount of segmento�nuclear leukocytes, macrophages, and lymphocytes,especially in using the Uniflex net. Such a tendencyreflects the predominance of processes of organizationover inflammation and alteration. In all periods oftime, the number of fibroblasts prevails in using Uni�flex, especially in the series without administration ofallogenic fibroblasts, this is important for practicalherniology. The inflammatory reaction is more pro�nounced in animals with the Esfil endoprosthesis.
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Table 3. Characteristics of cellular composition in the area of net location (double introduction of cultured fibroblasts onthe seventh and tenth days)
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* p ≤ 0.01 (at comparison between materials at the same term).
25
20
15
10
5
0603010
Time, days
%
Uniflex
Esfil
Fig. 7. Dynamics of the number of macrophages (doubleintroduction of cultivated fibroblasts on the seventh andtenth days).Curves 1 and 2—Uniflex and Esfil, respectively. * p < 0.01(in comparison between materials in the same period oftime).
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