Diaphragma frozen homograft for rats' abdominal wall defects repair

Abstracts

OBJECTIVE: To analyse the morphology and biomechanics of diaphragma frozen homografts used for rats' abdominal wall defects repair. METHOD: Thirty wistar rats with abdominal wall resection and reconstruction using frozen diaphragma homografts were compared to twenty other rats submitted to abdominal wall incision and closure (control). Animals were euthanized after 3 and 6 months and abdominal walls were avaliated histologically with H/E and Picrosiriud Red staining and tensiometric evaluation. RESULTS: The tensiometric parameters were greater in the experimental group at 3 months after surgery. The percentage of mature collagen was significantly greater at 6 months after surgery in control and experimental groups. Tensiometric parameters and type I collagen as well maturation collagen index and rupture maximal strength were statistically correlated after 3 and 6 months. CONCLUSION: Frozen diaphragma homograft can be an alternative to repair the defects of abdominal wall.

Wound healing; Homografts; Diaphragm; Abdominal wall; Rats

OBJETIVOS: Análise morfológica e biomecânica do enxerto homólogo congelado de diafragma para correção de defeito da parede abdominal em ratos. MÉTODOS: Os animais foram distribuidos em controle (20 ratos Wistar) e experimento (30 ratos Wistar). Os do grupo controle foram submetidos à laparotomia mediana e sutura da parede abdominal; já os do grupo experimento, à ressecção da parede abdominal e reconstrução com enxerto homólogo congelado de diafragma. Os animais foram submetidos à eutanásia no 3° e 6° mês de pós-operatório e avaliados quanto à presença de complicações pós-operatórias, integridade do enxerto, presença de aderências, avaliação tensiométrica, avaliação histopatológica com H/E e com sirius-red (colágeno tipo I e III). RESULTADOS: Houve integração do enxerto em todos os animais sem complicações. Aderências foram semehantes entre os grupos controle e experimento após três e seis meses. Observaram-se maior força máxima, força de ruptura e tensão nos animais do grupo experimento aos três meses de pós-operatório (p=0,001; p=0,012 e p=0,001, respectivamente). Na correlação entre as diferentes variáveis estudadas houve correlação estatisticamente significante entre força máxima e tensão nos grupos controle e experimento (p=0,001 e p= 0,001), e nos subgrupos três e seis meses (p=0,002 e p=0,001). Correlacionaram-se força máxima e colágeno tipo I (p=0,04) e Índice de Maturação do Colágeno (IMaC) e força máxima (p=0,03) ambos somente no grupo controle, mas nos subgrupos 3 e 6 meses (p=0,045 e p=0,038). O número de monomorfonucleares e força máxima também apresentou significância estatística tanto para o grupo controle quanto para o experimento (p=0,005 e p=0,004, respectivamente). CONCLUSÃO: O enxerto homólogo congelado de diafragma mostrou ser boa alternativa no reparo de grandes defeitos da parede abdominal em ratos.

Cicatrização de feridas; Transplante homólogo; Diafragma; Parede abdominal; Ratos

ORIGINAL ARTICLES

Diaphragma frozen homograft for rats' abdominal wall defects repair

Paola Maria Brolin Santis-Isolan^I; Miguel Angelo Agulham^II; Gustavo Rassier Isolan^III; Elizabeth Milla Tambara^IV; Sérgio Bernardo Tenório^IV; Ana Cristina Lira Sobral^V; Antônio Ernesto da Silveira^II

^IM.Sc. in Clinical Surgery, Universidade Federal do Paraná (UFPR), Curitiba, PR., Brazil

^IIAssociate Professor, Pediatric Surgery, UFPR , Curitiba, PR, Brazil

^IIIAssociate Professor, Neurosurgery, Pontifícia Universidade Católica (PUC), Curitiba, PR, Brazil

^IVAssociate Professor, Anesthesiology, UFPR, Curitiba, PR, Brazil

^VProfessor, Faculdade Evangélica do Paraná, Curitiba, PR, Brazil

Correspondence address

ABSTRACT

OBJETIVE: To analyze the morphology and biomechanics of diaphragm frozen homografts used for the repair of abdominal wall defects in rats.

METHODS: Thirty Wistar rats with abdominal wall incision and reconstruction using frozen diaphragm homografts were compared to twenty other rats submitted to a sham operation (control). Animals were euthanized after 3 and 6 months and abdominal walls were evaluated histologically with H-E and sirius-red staining and tensiometric evaluation.

RESULTS: The tensiometric parameters were greater in the experimental group at 3 months after surgery. The percentage of mature collagen was significantly greater at 6 months after surgery in the control and experimental groups. Tensiometric parameters and type I collagen as well as maturation collagen index and ultimate strength were statistically correlated after 3 and 6 months.

CONCLUSION: Frozen diaphragm homografts can be an option in the repair of abdominal wall defects.

Key words: Wound healing. Homografts. Diaphragm. Abdominal wall. Rats.

INTRODUCTION

The surgical repair of the major congenital or acquired defects of the abdominal wall has always been a great challenge for surgeons, since, in addition to the unfavorable clinical status of the patient, no ideal surgical technique exists. Neither are there comparative studies with a high level of clinico-epidemiological evidence to validate one type of surgical procedure over the others.

The use of prostheses improved the patients' quality of life, yet the material does not meet all the biocompatibility criteria, thus causing complications over the long term and eventually necessitating reoperations. Biological materials have biocompatibility features; however, they pose greater technical difficulties and cause large defects at the donor site, with consequences such as muscle dystrophy and atrophy. The ideal material should be musculotendinous without producing new defect areas. Therefore, the tissues stored in musculoskeletal tissue banks offer all the qualities of an ideal material for the repair of large abdominal wall defects¹. Their widespread use has founded new horizons for research in wound healing concerning abdominal wall defects.

The objective of the present study was to create an experimental model of the repair of abdominal wall defects in rats with the interposition of a frozen diaphragm homograft from a tissue bank and evaluate it both morphologically and biomechanically.

METHODS

The animals were kept and operated on at the Instituto de Pesquisas Médicas (Institute for Medical Research) of the Hospital Universitário Evangélico de Curitiba, in compliance with Federal Law 6638 and the guidelines of the Colégio Brasileiro de Experimentação Animal (COBEA, Brazilian College of Animal Experimentation).

Seventy female Wistar rats (Rattus norvegicus albinus, Rodentia mammalia) were used in the study, 50 at the age of 20 days and mean weight of 110 g (receptors), and 20 at the age of 12 months and mean weight of 840 g (donors), all from the breeding colony of the Instituto de Tecnologia do Paraná (TECPAR) in Curitiba, PR, Brazil.

The Experimental Group (30 rats) and the Control Group (20 rats) were divided into subgroups according to the period of euthanasia and wound healing assessment: subgroup EA (Experimental 1A), composed by 15 animals with euthanasia on the 90th day of life; subgroup EB (Experimental 1B), 15 rats with euthanasia on the 180th day; subgroup CA (Control 2A) 10 rats, euthanasia on the 90th day, and subgroup CB (Control 2B), 10 rats with euthanasia on the 180th day.

The donor animals were submitted to euthanasia with a lethal dose of ethyl ether and resection en bloc of the diaphragm. The diaphragm grafts were kept hydrated in a container with 0.9% saline, at room temperature, until they were placed in individualized plastic bags and tagged. The whole procedure was carried out according to asepsis and antisepsis guidelines for the preparation of sterile material. The grafts were transported, in styrofoam boxes with ice, to the Banco de Ossos e Tissues (Bone and Tissue Bank) of the Hospital de Clínicas of the Universidade Federal do Paraná in Curitiba, where the material was re-packed in sterile conditions under laminar airflow. The following step was freezing the grafts at -85°C, which remained thus preserved for six months.

After inhalation anesthesia with 97% ethyl ether, a 5-cm midline incision was made in the control group animals, from 2 cm below the xiphoid process and encompassing all planes of the anterior abdominal wall. Closure was performed in a single plane with 4-0 polypropylene involving the aponeurosis and musculature with individual stitches 0.5 cm apart. The skin was closed with 5-0 polyglecaprone 25 (Monocryl) by a running suture.

On the day of the experiment, the frozen materials were taken from the Banco de Ossos e Tecidos and transported in ice boxes to the experimental surgery laboratory. The diaphragm allografts were thawed at room temperature and kept in 0.9% saline until used.

After anesthesia, the experimental group animals underwent a 5-cm midline abdominal incision and a traumatic defect of the anterior wall of the abdomen was produced through the resection of a 5 cm x 4 cm segment mimicking a large congenital abdominal wall defect. The diaphragm allograft was subsequently sutured to the abdominal wall using monofilament 4-0 polypropylene individual stitches, closing all the abdominal defect. The skin was sutured with 5-0 polyglecaprone 25 (MonocrylÒ) running suture.

In the preliminary post-mortem evaluation, the rats were weighed and examined for the presence of ventral herniations and the appearance of the surgical wound. The inspection included gross characteristics of the abdominal cavity such as adhesions or other gross intraabdominal alterations, e.g., intraabdominal abscess, volvulus and stenosis. The adhesions were classified according to the semiquantitative adhesion scoring system developed by Jenkins et al.².

The resection of the anterior abdomen was performed en bloc, comprising the musculoaponeurotic segment and graft (experimental group) or surgical scar (control group). The segments were stretched out on filter paper and divided into three parts: two 1 cm x 2 cm peripheral fragments and one 2 cm x 2 cm central fragment. The peripheral fragments were referred for histological examination (sirius-red and hematoxylin-eosin) and the central fragment, for mechanical tensiometric testing.

The test specimens (central fragments) were prepared according to a previously described technique^3,4. The analyses were carried out on a computed tensile tester InstronÒ model 4467 with pneumatic grips and 3-ton capacity at the Mechanical Testing Laboratory of the Instituto de Tecnologia para Desenvolvimento (Lactec) at the Centro Politécnico (Polytechnology Center) of the Universidade Federal do Paraná. The parameters were ultimate strength, burst strength and stress.

In the histological sections stained by hematoxylin-eosin, the quality and the intensity of the inflammatory reaction were evaluated. Three fields per histological section were studied, and 100 cells were counted at 400x magnification. The quality of the inflammatory reaction was evaluated by the occurring cell types. The predominance of polymorphonuclear cells characterized an acute inflammatory reaction, while the predominance of monomorphonuclear cells, a chronic inflammatory reaction. If neither cell type prevailed, an acute chronic inflammatory reaction.

The sirius-red-stained histological sections were analyzed under an OlympusÒ polarized light microscope at 200x in two fields of the wound healing area. The images were captured by a SonyÒ CCD Iris camera and relayed to a monitor (Mídia Cibernética) connected to a PentiumÒ III 733 MHz computer at the Anatomical Pathology Laboratory of the Hospital de Clínicas of the Universidade Federal do Paraná. The percentage of surface area occupied by type I collagen – mature (thick, strongly birefringent reddish fibers) and by type III collagen – immature (thin, dispersed, weakly birefringent greenish fibers, ) was calculated for each animal, considering that the percentage was proportional to the amount of each fiber type found in the histological sections. A Collagen Maturation Index (ImaC, Índice de Maturação do Colágeno) was also used. The index is based on the percentages of type I and type III collagen and is defined as the ratio between those percentages. Values higher than 1 indicate that the percentage of type I collagen is greater than that of type III collagen.

Student's t-test was used for statistical analysis. Statistical significance was established for P = 0.05. The non-parametric tests of Mann-Whitney, Fisher's Exact, Pearson were also applied, as well as the coefficient of logistic regression.

RESULTS

No surgical complications were found in any of the cases, and the graft was intact in all animals in the experiment.

With respect to adhesions, neither in the control group, nor in the experimental group were there adhesions of grades II, III or IV. No statistical differences were found between groups or subgroups (Table 1).

In the analysis of the results for ultimate strength, a statistically significant correlation (p=0.001) was found between the experimental and control group at three months. On the other hand, the value of p was 0.849 for the correlation at six months. Regarding the results for burst strength, it was greater in the experimental group compared with the control group at three months (p=0.012). In the correlation at six months, the difference between the two groups was not statistically significant (p=0.832). Results were similar for the variable stress, in which the value of p at three and six months was 0.001 and 0.605, respectively.

In the correlations between the tensiometric parameters, the results proved statistically significant for the variables stress and ultimate strength (p<0.001) as well as for ultimate strength and burst strength (p<0.001), and stress and burst strength (p<0.001).

Polymorphonuclear cells were present in three cases in the experimental group an none in the control group at three months; in four animals of the experimental group and three in the control group at six months. In the study of monomorphonuclear cells, the values of p between the control and experimental groups were 0.238 and 0.031 at three and six months, respectively. Hence, there was a greater number of monomorphonuclear cells in the experimental group at six months postoperatively.

No statistically significant correlation existed between the control and experimental group in relation to type I and III collagen. However, there was statistically significant predominance of type I collagen in the animals evaluated at six months, both in the control and the experimental group, with P=0.00012 (Figure 1).

Regarding the ImaC, the values of p between the control and experimental group were 0.21 and 0.28, respectively. However, in the correlation between subgroups three and six months, p was lower than 0.001.

In the correlation between the different study variables (tensiometry, histology and collagen), a statistically significant correlation occurred between ultimate strength and number of monomorphonuclear cells in the groups control and experimental (p=0.0014 and p=0.0011, respectively), as well as between those variables in the subgroups three and six postoperative months (p=0.001 and p=0.0012, respectively).

When collagen distribution and the tensiometric variables were analyzed, statistically significant correlations were only found in the control group animals. Correlation values for the percentage of type I collagen were p=0.04 with ultimate strength, p=0.048 with stress, and p=0.047 with burst strength. The values for the correlations between those variables in the experimental group animals were p=0.934 (percentage of type I collagen and ultimate strength), p=0.965 (percentage of type I collagen and stress), and p=0.975 (percentage of type I collagen and burst strength) – therefore, not statistically significant.

The IMaC also showed statistically significant correlations for the control group animals in relation to the tensiometric variables. IMaC and ultimate strength correlated with p=0.003. IMaC and stress showed a correlation with p=0.004. The value of p for the correlation between IMaC and burst strength was 0.0049. In turn, the results of those correlations for the experimental group animals were p=0.801 (IMaC and ultimate strength), p=0.956 (IMaC and stress), and p=0.872 (IMaC and burst strength), i.e., not statistically significant.

Figure 2 demonstrates a statistically significant correlation between the tensiometric variables both in the control and experimental group. Values of p=0.0001 were found for all correlations involving the three variables – between ultimate strength and stress, ultimate strength and burst strength, and stress and burst strength – in the control and experimental group.

Correlations of IMaC and type I collagen percentage with the tensiometric variables ultimate strength, stress and burst strength at three and six months showed no statistical significance. The correlation values for IMaC with ultimate strength were p=0.871 (three months) and p=0.857 (six months); for IMaC with stress, the values of p were 0.864 (three months) and 0.866 (six months), and IMaC with burst strength resulted in p=0.853 (three months) and p=0.841 (six months). Type I collagen percentages were correlated with ultimate strength and the values of p were 0.921 (three months) and 0.988 (six months), while with stress the values were p=0.992 (three months) and p=0.989 (six months). IMaC and percentage of type I collagen also exhibited a correlation with each other at three and six months, with statistically significant values (P=0.05 and P=0.04, respectively).

The correlation between ultimate strength and stress at three and six months resulted in p=0.0001. Ultimate strength and burst strength correlated for the same periods resulted in p=0.00012. Similarly, when stress was correlated with burst strength, a value of p=0.00011 was found.

The absolute values of the correlations between the predictive variables number of monomorphonuclear cells, type I collagen percentage, collagen maturation index (IMaC), ultimate strength, stress and burst strength were analyzed through logistic regression. The variables that showed a statistically significant correlation with each other were number of monomorphonuclear cells and ultimate strength, with p=0.009, and ultimate strength with stress, p=0.041. When testing the correlation of the two histological variables and one tensiometric variable, it was found that all sustained values that were comparable to those of their individual correlations; the associations were considered to be independent.

The other variables, said not predictive with their absolute values, were correlated through logistic regression, and had no statistically significant correlations.

DISCUSSION

It has already been recognized that the use of musculoskeletal tissue grafts is a form of transplant, thus falling under government regulations. As a result, an increase in demand has been observed, and not only in the field of orthopedics.

The choice for the frozen homologous musculotendinous graft in the present study was based on previous studies in the literature, as well as the need to evaluate the diaphragm muscle as a source of material.

Frozen homografts constitute good grafting options, since the low temperature promotes a reduction in the immune response (graft-versus-host response), thus providing enhanced graft incorporation and the maintenance of biomechanical properties. Some authors⁵ reported those data and, additionally, emphasized less morbidity, muscle weakness and muscle fiber relaxation in the homografts compared with autografts. The present study assessed tensiometric strengths comparing a frozen diaphragm homograft with the original abdominal wall (control group) and corroborated those data.

The risk of graft infection has always been a strong concern regarding its utilization. Tomford et al.⁶ reported values of 5% for frozen homograft infection rates. Mae et al.⁷ studied the utilization of gamma irradiation in the sterilization process of tendon homografts implanted in 142 rats. Infection rate was zero, yet there was a tensile strength decrease of up to 50% in comparison with the frozen homografts that were not irradiated. In the present study, no infection-related complications were found.

The large majority of the articles found in the literature state the superiority of frozen homografts with respect to ease of conservation and manipulation as well as their ability to maintain the primary properties of tensile and burst strength, and collagen remodeling. Those arguments influenced the choice of frozen homografts for the present study and were proved true as tensiometric values, collagen and histological variables were equal to or better than the original material (control group).

The most widely used musculoskeletal tissue in humans in the present day is the fascia lata, due to the act that it is fully tendinous. However, in animals, the best tissue depends on particular characteristics. The fascia lata of the rat, for instance, is scant, while the diaphragm is a good option given the large proportion of tendon⁸. However, the diaphragm has been scarcely used as a homograft in experimental studies or in clinical practice. This fact, in conjunction with its anatomical features, decisively influenced the choice of the diaphragm as a frozen homograft in this study. The muscle proved to be a good option for a frozen homograft, since it preserved its biological and mechanical characteristics, as found in the present study.

Daily inspection was performed for the following surgical complications: herniations, dehiscence, evisceration and surgical wound infection. No surgical complications were found. Conversely, Klinge et al.⁹, when testing four different types of meshes for the repair of abdominal wall defects in 225 rats, found varying rates of complications according to the type of mesh they used. Also conflicting with the results found herein, van't Riet et al.¹⁰, when evaluating two groups of 28 rats for 7 and 30 days, sacrificed two animals on the fifth day, as they presented with surgical wound dehiscence from urinary retention and accidental suture of the colon. In addition, the infection rate was significantly higher in the group of polypropylene mesh with collagen when compared with the group that used the polypropylene mesh alone. Levine and Saltzman⁸ introduced freeze-dried diaphragm homografts in the omental pouch of 30 rats. The diaphragm grafts remained intact in 83.6% of cases. In the present study, 100% of the frozen diaphragm homografts were intact.

The present study identified no statistically significant differences in adhesions between groups or subgroups. Significantly better tensiometric values were detected at 90 postoperative days in the experimental group. Some authors¹¹ described the biomechanical alterations sustained by frozen homografts. From baseline to four weeks, they exhibited poorer biomechanical properties than the autograft. Nonetheless, after 24 weeks there were no significant differences between grafts with respect to the strength variables. In fact, homografts could at times show higher stress values¹, due to their hypercellularity and greater type I collagen deposition. In the present study, the experimental group had statistically higher ultimate strength, burst strength and stress values than the control group at three months postoperatively. At six months, on the other hand, those tensiometric values had similar values between groups. Monaco and Lawrence¹² report that, after three months, the injured tissues would have recovered 80% of their tensile strength compared with the intact tissue. According to the same authors, that is due to the predominance of collagen fibers in the extracellular matrix. Those fibers, despite being thin fibers – type III collagen –, reveal restored spatial distribution as well as reduced interfibrillar spaces with a peak of elastin deposition. Those factors provide biomechanical properties to the scar tissue, thereby affording tensiometric values that are equivalent to, or even better than those for the intact tissue.

The existence of correlations between ultimate strength, burst strength and stress are logical physical concepts and well-established in the literature. However, Van Winkle W Jr¹³ reported that burst strength may be affected by external factors such as the age of the donor of the tissue – it may decrease even if ultimate strength and stress are elevated. That fact could answer the questions raised by the findings of the present study.

Inflammatory response starts after the trauma and corresponds to the first phase of wound healing. Such phenomenon is often studied in experimental research on wound healing. There are, however, few studies evaluating the reaction of the abdominal wall to different types of materials used in grafts by the hematoxylin-eosin technique. Furthermore, there are no studies analyzing the inflammatory reaction in a diaphragm graft from a tissue bank, only in freeze-dried homografts⁸. Gamba et al.¹⁴, in a study evaluating the use of an acellular matrix in the repair of abdominal wall defects in rabbits, demonstrated that the homologous acellular diaphragm graft provided a substrate for fibroblast migration, neoformed collagen deposition and neovascularization. The cell infiltrate included polymorphonuclear and monomorphonuclear cells. Their presence was similar in all groups and remained with fewer than 10 cells per high-power field in the animals analyzed after two years. The present study detected the predominance of monomorphonuclear cells in the two studied groups; however, there were significantly more cells in the experimental group at six months. That result differs from another study¹¹ describing biological alterations sustained by frozen homografts and fresh autografts during the different phases of wound healing. It was found that from the fourth to the sixth week more pronounced hypercellularity occurs in frozen homografts compared with fresh autografts. After six months, however, no differences existed in the number of cells between the graft types. One aspect that is worth stressing in order to explain the hypercellularity that persisted at six months is the loss of cell apoptosis in scar tissues.

The authors of the present study found that the repair of the abdominal wall with acellular matrix diaphragm homologous grafts showed fibroblast migration, neoformed collagen deposition and neovascularization. In this study, type I collagen predominated in the animals evaluated at six months, both in the control and the experimental group, with statistically significant differences. The IMaC supported those results with higher values at six months, i.e., showing a larger amount of mature or type I collagen . The present study diverges from that by Forrest¹⁵, who reported the normalization of the distribution and morphometry of the collagen fibers of the tissues after 18 months of wound healing. However, Shino and Horibe¹⁶ noted that, at six months, the frozen homografts exhibited hypercellularity, probably fibroblasts, and the predominance of large-caliber fibers, i.e., type I collagen fibers – data that corroborate the findings of the present study.

In the present study, a statistically significant correlation was found between the number of monomorphonuclear cells and the tensiometric variable ultimate strength both at three and six months in the control and experimental groups. Such correlation is likely due to the fact that monomorphonuclear cells – including fibroblasts – are the collagen producers. That protein provides tissues with biomechanical properties. Therefore, the hypercellularity of frozen homografts would promote greater collagen synthesis and higher ultimate strength. The correlations between the tensiometric variables and type I collagen or IMaC, which occurred in the present study only for control group animals at three months, were not found in the literature. The late phases of wound healing, despite the presence of thick and more abundant collagen fibers, may not have a correlation with the tensiometric variables, as found in present study.

In summary, frozen diaphragm homografts proved to be a good option for the repair of large abdominal wall defects in rats, since it showed no postoperative complications, good integrity, low adhesion index, good wound healing and the ability to maintain stress and collagen percentage parameters.

The next step in the evolution of abdominal wall defect repairs, according to Zhang and Chang¹⁷, is tissue engineering. The production of musculotendinous tissues involves using cell culture and growth factors for the formation of grafts, and the association of tissue engineering techniques with the new research studies on stem cells. They will be the great contributions of the 21^st century.

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Publication Dates

• Publication in this collection
  09 Nov 2009
• Date of issue
  Aug 2009

History

• Received
  30 Oct 2008
• Accepted
  07 Jan 2009