F. Jurado

Study of biochemical substrate and role of metalloproteinases in fascia transversalis from hernial processes

The aim of this study was to examine the fascia transversalis (FT) from patients with direct and indirect hernia in an attempt to identify possible differences between each type of hernia. FT samples were obtained from 36 patients presenting inguinal hernia (23 indirect hernia and 13 direct hernia) who underwent surgery. We have analysed the ultrastructure of the fascia surrounding the hernial lesions, the proline and lysine hydroxylation in the tissue, the type I–type III collagen ratio and the presence of metalloproteinases. We have not detected ultrastructural differences in the collagen fibrils from FT in direct and indirect hernias. However, the interfibrillar matrix was more abundant in direct hernias, showing abundant electron‐dense particles. No differences in proline hydroxylation were observed between each type of hernia. A small decrease in lysine hydroxylation was detected in patients with direct hernia. Enzyme‐linked immunosorbent assays (ELISAs) showed no statistically significant differences in the type I–type III collagen absorbance ratios. Immunohistochemistry revealed no differences in the expression of matrix metalloproteinase‐1. FT from patients presenting direct hernia showed a very strong staining vs. metalloproteinase‐2 when compared with that observed in indirect hernia.

Peritoneal Regeneration after Implant of a Composite Prosthesis in the Abdominal Wall

Abstract. Prosthetic materials currently used to repair abdominal wall defects occasionally must be placed in direct contact with the visceral peritoneum. The prosthesis–peritoneum interface is the site of several possible problems, including the formation of adhesions and erosion of the intestinal loops, which may lead to the formation of fistulas. This investigation was designed to compare the behavior of two prosthetic biomaterials in composite form at the level of the peritoneum. Defects (7 × 5 cm) were created in the abdominal wall of 18 white New Zealand rabbits weighing approximately 2500 g. The defects (involving aponeurotic and muscular planes and the parietal peritoneum) were repaired with polypropylene (PL) + ePTFE (Preclude dura substitute) or Parietex composite (PC) prostheses. The prostheses were secured to the edges of the defect by continuous PL sutures interrupted at the corners of the implant. Three study groups were established according to the type of implant: group I (n= 6) (controls)—PL; group II (n= 6)—PL + ePTFE; and group III (n= 6)—PC. The animals were sacrificed 14 days after implant, and the prostheses were examined by light microscopy and scanning electron microscopy (SEM). The formation of adhesions at the prosthesis–visceral peritoneum interface were quantified according to a protocol previously described by us. The biomechanical resistance of the implant was evaluated using strips comprising prosthetic material and anchorage tissue. The Mann-Whitney U-test was used to compare data corresponding to each group. There was no postimplant mortality. No infection or rejection of the prosthesis was observed in any of the animals. Firm adhesions were detected in the PL implants, whereas in the PL + ePTFE and PC implants the adhesions were loose. The mean prosthetic surface areas covered by adhesions were 7.67, 0.10 and 0.19 cm2 for groups I, II, and III, respectively, showing a significant difference between values corresponding to groups I and II and to groups I and III (p < 0.05). Comparison of values recorded for groups II and III yielded no significant difference (p > 0.05). In groups II and III, the neoperitoneum was homogeneous and composed of organized and vascularized connective tissue covered by a mesoendothelium that was interrupted by accumulations of fibroblasts and white blood cells. In contrast, a disorganized neoperitoneum of rough texture was observed in the group I specimens. At times, areas of hemorrhage and necrosis corresponding to the sites of adhesion formation could be observed. Resistance to traction of composite implants (mean ± SD: 15.72 ± 1.32 and 15.89 ± 2.73) was similar to that of the PL implants (15.03 ± 2.92) (Mann-Whitney U-test, p < 0.05). It may be concluded that (1) composite prostheses show optimum behavior in terms of adhesion formation at the prosthesis–visceral peritoneum interface; (2) the neoperitoneum formed after the implant of a composite prosthesis almost physically and functionally replaces the normal peritoneum; (3) a significantly greater degree of peritoneal regeneration is achieved after implant of a PC prosthesis; and (4) there was no significant difference regarding biomechanical resistance between PL prostheses and PL + ePTFE and Parietex composites.

Macrophage response to experimental implantation of polypropylene prostheses

We have assessed the macrophage response to polypropylene mesh (Marlex) implanted into the abdominal wall of New Zealand white rabbits, using RAM-11, a monoclonal antibody specific for rabbit macrophages. The response diminishes during the course of the first 90 days after implantation, although the presence of other cell types typical of foreign-body reactions increases. We have also confirmed the high degree of integration of the biomaterial into the wall achieved after 9 weeks. This has been determined using light and scanning electron microscopy. Likewise, we have observed the formation of numerous adhesions between the polypropylene mesh and the viscera of the abdominal cavity.

In vitro interaction of bacteria with polypropylene/ePTFE prostheses

The infection of an implanted prosthetic material can have serious consequences on the tissue integration of the implant and the scarring process in the host, and may even necessitate replacement of the prosthesis. This study was designed to explore the in vitro effects of Staphylococcus aureus (Sa) and Staphylococcus epidermidis (Se) on polypropylene (PL) and expanded polytetrafluoroethylene (ePTFE) prostheses. Fragments of PL and ePTFE were placed in a medium previously inoculated with Sa, Se or Sa + Se (1 x 10(8) cfu Sa or/and Se). Bacterial effects on the biomaterials were evaluated for 30 days through scanning electron microscopy (SEM). In order to confirm the presence of bacteria on the prostheses, specimens were Gram stained and challenged with an antibody against protein-A (a specific Sa bacterial wall component). In both methods, specimens were examined by light microscopy. The presence of bacteria as microcolonies or biofilms was detected at PL filament cross-over regions after 30 days. Bacterial colonization of the ePTFE fragments was observed in internodal areas, which led to the deformation of prosthetic filaments. The present findings indicate that Sa and Se colonize the cross-over regions of the PL filaments, whereas in ePTFE prostheses, it is the internodal areas which are mostly affected. The latter areas are of difficult access to defence agents.

Similarity in behavior of polytetrafluoroethylene (ePTFE) prostheses implanted into different interfaces

The biomaterial ePTFE is widely used in the clinical environment for vascular replacement or bypass, as well as in the repair of tissue defects, especially those involving the abdominal wall. The objective of this study was to evaluate the healing response to ePTFE prostheses implanted into a circulatory interface and a tissue interface, each in a different animal species. For vascular implants, the Sprague-Dawley rat (n = 60) was used, while the New Zealand white rabbit (n = 20) was used in the tissue replacement model. In the former, a vascular microprosthesis measuring 5 mm in length and 1 mm in internal diameter, having a porosity of 30 microns, was implanted into the common iliac artery. In the rabbit, a 7 x 5-cm fragment of ePTFE (Soft-Tissue Patch) was implanted into a defect in anterior abdominal wall that involved all the tissue layers. In this case, the prosthesis was left touching the intestinal loops. The implants were studied between 14 and 90 days of postimplantation by means of light microscopy, scanning electron microscopy, and immunohistochemistry. The latter involved the use of anti-rat (MAC-341) and anti-rabbit (RAM-11) macrophage-specific monoclonal antibodies. The behavior of the ePTFE in the different interfaces (vascular and abdominal wall) was similar with respect to the following aspects: the prosthesis presented a process of encapsulation which was more intense on the outer surface; colonization of the implant was limited to the outermost two thirds, with minimal invasion of the middle portion; colonization was absent on the edges of the prosthesis; collagenization of the interstice of the mesh occurred late; the foreign body reaction taking place on the outer surface was similar in both interfaces, with formation of a barrier consisting of macrophages and giant cells that did not penetrate the prosthesis; and, finally, in neither of the two models was vascular colonization of the PTFE prosthesis observed; rather, the angiogenic process was limited to the periprosthetic zones. The integration of the implant made of ePTFE is similar despite the differences in interfaces and the use of different animal species. The macrophage response does not determine the success or failure of the implant.

Use of nonporous polytetrafluoroethylene prosthesis in combination with polypropylene prosthetic abdominal wall implants in prevention of peritoneal adhesions

One of the drawbacks of using macroporous polypropylene prostheses in the repair of the abdominal wall is the formation of adhesions with the viscera. However, polytetrafluoroethylene (PTFE) has low adhesion formation, although it provides less resistance to traction in the repaired zone. The aim of the present study was to reduce the formation of adhesions to a polypropylene implant Prolene (PL) by introducing a nonporous expanded PTFE layer (Preclude Dura-Substitute) (PR) between the polypropylene prosthesis and the abdominal viscera. The scarring process and resistance to traction in the repaired zone were also evaluated. Thirty-six rabbits were divided into three groups and were treated as follows: group I, a PR patch (7 x 5 cm) was secured to the parietal peritoneum; group II, an abdominal wall defect (7 x 5 cm) was created and repaired using a PL patch; group III, an abdominal wall defect was as in group II and was repaired using a combined PR/PL prosthesis. Specimens were evaluated at 14, 30, 60, and 90 days postimplant for extent of adhesion formation and morphological analysis was performed using light and scanning electron microscopy. Biomechanical resistance of the implant was evaluated using strips comprising prosthetic material and anchorage tissue. Group I and group III prostheses showed loose adhesions only, but group II adhesions were firmly attached. The mean surface areas covered by adhesions were 0.08 cm2 (group I), 7.67 cm2 (group II), and 0.1 cm2 (group III). PR implants (group I) were encapsulated by organized tissue. In group II the formation of disorganized tissue invading the prosthesis was observed. In group III the PR impeded the growth of disorganized scar tissue and the lower surface of the implant was covered by an orderly neoperitoneum. Resistance to traction of the double implants (group III) (mean +/- SD, 33.32 +/- 0.9 N) was similar to that of the Prolene implants (group II) (33.76 +/- 0.46 N) (Mann-Whitney U test, p < 0.05). We concluded the presence of a PR layer between the PL implant and viscera greatly reduced the incidence of adhesion formation without affecting the high resistance to traction provided by the PL implant or the evolution of the scarring process.

Effect of phosphatidylcholine on the process of peritoneal adhesion following implantation of a polypropylene mesh prosthesis

The postimplantation peritoneal adhesions formed with some biomaterials continue to represent a serious problem when these biomaterials are placed in contact with the organs of the abdominal cavity. The objective was to test the effects of phosphatidylcholine (PC) in the attempt to modulate the adhesive process, thus improving the biomaterial/visceral peritoneum interface. We have carried out an experimental study using the New Zealand white rabbit, implanting a 7 x 5 cm2 polypropylene prosthesis into the anterior abdominal wall in such a way that it replaced all the tissue layers and was in contact with the intestinal loops. Three study groups were established: group I (control), in which only implantation of the biomaterial was performed; group II, in which the prosthesis was pretreated with a solution of PC; and group III, in which the implants were treated as in group II and the animals underwent intraperitoneal administration of 10 ml of the same solution. The animals were killed on postimplantation day 14. The total surface of the prosthesis occupied by adhesions was measured and light and scanning electron microscopy were performed to analyse the healing process. In comparison with the controls, groups II and III did not present significant differences with respect to the resistance or extension of the adhesions. The microscopic results showed a rapid formation of disorderly and well-vascularised scar tissue enveloping the entire prosthesis. Thus, PC was unable to modulate the process of adhesion formation between the prosthesis and the organs of the abdominal cavity; nor did it induce changes in the cells of sufficient importance to alter the results of the healing process in the presence of this biomaterial.

In vitro mesothelialization of prosthetic materials designed for the repair of abdominal wall defects

The aim of this study was to evaluate the in vitro response of mesothelial cells (MC) in terms of their ability to cover different biomaterials. MC were harvested from human omentum. The MC from the first passage were seeded onto different biomaterials from 10 min to 24 h: PL-PU99 (polypropylene-polyurethane); DM (ePTFE); PL (polypropylene); and PL + Col (polypropylene-collagen). The prosthetic surface covered was examined by microscopy and quantified. PL-PU99: The MC were adhered to the biomaterial 10 min post-incubation. At 4 h, the 53.12±7.86% of the prosthesis were coated with polygonal cells. At 12 h, 96.32±11.32% of the biomaterial was coated. DM: between 30 min to 8 h, the MC cells form small, round colonies. At 12 h, polygonal and fusiform secretory cells were observed (68.94±5.78%). 93.54±11.49% of surface was coated after 24 h. PL: only isolated cells were observed on the prosthesis. PL + Col: MC form a monolayer over prosthetic surface after 18 h (90.21±9.76). We conclude: (a) MC formed a stable monolayer over all the biomaterials tested with the exception of the PL due to its porosity. (b) The PL-PU99 showed the greatest potential for in vitro mesothelialization compared to the PL-Col and DM prostheses.

Improvement of the tissue integration of a new modified polytetrafluoroethylene prosthesis: Mycro Mesh®

We studied the behaviour of the different tissue interfaces formed on a new type of prosthesis used for the repair of abdominal wall defects, Mycro Mesh (W. L. Gore and Ass., Flagstaff, AZ, USA), which consists of perforated layers of polytetrafluoroethylene (PTFE). In 20 New Zealand white rabbits, a full-thickness (except skin) 7 cm x 5 cm defect was created in the anterior abdominal wall. The defects were repaired with a prosthetic implant (Mycro Mesh) that was placed in direct contact with abdominal viscera and subcutaneous tissue. At 14, 30, 60 and 90 d post-implantation, samples were obtained from the tissue interfaces formed between the prosthesis and subcutaneous tissue, visceral peritoneum and receptor tissue, respectively. Samples were studied by optical microscopy and scanning electron microscopy. The immunohistological study was made with RAM-11, a monoclonal antibody specific for rabbit macrophages. Tensile strength was measured with an Instron tensiometer using 2 cm wide strips obtained parallel to the shorter axis of the implant. Strips included the prosthesis and two anchor zones on the receptor tissue. Macroscopically, the prosthesis induced little adhesion formation on the visceral peritoneum interface. Microscopically, an organized neoperitoneum and abundant tissue formed on the subcutaneous interface. In the prosthesis perforations, bridges of tissue linked the peritoneal and subcutaneous sides. The macrophage response decreased significantly in intensity between day 14 and day 90 (Student-Newman-Keuls test, P = 0.01). Tensile strength increased significantly (Wilcoxon test, P < 0.05) at every study period. To conclude: the Mycro Mesh prosthesis proved suitable for implantation in sites where it comes in contact with abdominal viscera and it provided good support for the formation of an organized neoperitoneum; the perforations in the prosthetic material improved implant integration; the macrophage response was not altered by the biomaterial and the tensile strength of the prosthesis increased as scar tissue formation and tissular integration of the prosthesis progressed.

Evaluation of a new composite prosthesis (PL-PU99) for the repair of abdominal wall defects in terms of behavior at the peritoneal interface

This study was designed to evaluate the behavior of a new composite polypropylene-polyurethane (PL-PU99) when placed in direct contact with the visceral peritoneum during the repair of an abdominal wall defect. Full-thickness abdominal wall defects (7×5 cm) were created in 36 anaesthetized white New Zealand rabbits. The defects were repaired with polypropylene prostheses or PL-PU99 prostheses (comprised of PL and a polyurethane sheet glued to the PL with acrylic adhesive) to establish two study groups (n=18 each). Animals were sacrified 14, 30, or 90 days after implantation and prosthesis/surrounding tissue specimens were subjected to light and electron microscopy and morphometric analysis of the newly formed peritoneum. Immunohistochemical analysis was performed using the rabbit specific monoclonal antibody RAM-11. The biomechanical strength of the implants was also assessed. Firm adhesions were detected in the PL implants, whereas adhesions were practically non-existent in the PL-PU99 implants. The surface area covered by adhesions was greater (p<0.01) in the PL group (7.36 vs. 0.11 cm2). The neoperitoneum formed after the implantation of a PL prosthesis was disorganized in structure, whereas that formed at the interface with the PL-PU99 prosthesis was structurally similar to the host peritoneum. The excellent performance of the PL-PU99 prosthesis shown in this study warrants further investigation into its use for the repair of abdominal wall defects when the prosthetic patch needs to be placed in contact with the intestinal loops.RésuméLe but de cette étude a été d’évaluer le comportement biologique d’une nouvelle prothèse composite (PL-PU99) placée directement en contact avec les viscères lors de la réparation pariétale. Sous anesthésie, on a créé des pertes de substance complètes de la paroi abdominale (7×5 cm) chez 36 lapins blancs New Zealand. Les defects ont été réparés avec soit du polypropylène (PL) (n=18), soit la prothèse PL-PU99 (composée de PL et une feuille de polyuréthane collée sur la feuille de PL par une adhesive acrylique) (n=18). Les animaux ont été sacrifiés 14, 30 et 90 jours après l’insertion de la prothèse et des prélèvements tissulaires comprenant la prothèse et les tissus environnants ont été examinés en microscopie classique et électronique; le néopéritoine a été analysé morphométriquement. L’analyse immunohistochimique a été réalisée par des anticorps monoclonaux spécifiques du lapin RAM-11. La solidité bio-mécanique des implants a été évaluée. De fortes adhérences ont été détectées en ce qui concerne les plaques PL, alors qu’il n’y en avait pratiquement pas en cas de prothèses PL-PU99. La surface couverte par des adhérences était plus grande (p<0.01) dans le groupe PL (7.36 vs. 0.11 cm2). La structure du néopéritoine était désorganisée au niveau de la prothèse PL alors que celle à l’interface avec la prothèse PL-PU99 ainsi que l’étude de la fonction étaient similaires au péritoine natif. L’excellent comportement de la prothèse PL-PU99 démontré dans cette étude mérite d’autres investigations en ce qui concerne son utilisation pour la réparation des defects de la paroi abdominale lorsqu’il y a besoin de poser une prothèse au contact des anses intestinales.ResumenSe realiza este estudio para evaluar el comportamiento de una nueva prótesis mixta (PL-PU99) al colocarla en contacto directo con el peritoneo visceral durante la reparación de un defecto de la pared abdominal. Se realizaron, bajo anestesia, defectos de la totalidad de la pared abdominal de 7×5 cm en 36 conejos blancos de Nueva Zelanda. Los defectos se repararon con prótesis de polipropileno (PL) o de PL-PU99 (se trata de una prótesis mixta constituida por PL y una hoja de poliuretano pegada al PL por un adhesivo acrílico). Los animales se distribuyeron en dos grupos, cada uno de 18 conejos. Se sacrificaron a los 14, 30 ó 90 días tras la implantación; la prótesis y tejidos circundantes fueron estudiados mediante microscopía de luz y electrónica. Se analizó la morfometría del neoperitoneo formado. También se efectuaron análisis inmunohistoquímicos empleando el anticuerpo monoclonal RAM-11, específico del conejo. Así mismo se evaluó la resistencia biomecánica de los implantes. En los implantes de PL se detectaron firmes adherencia, mientras que éstas fueron prácticamente inexistentes en el grupo con prótesis PL-PU99. La superficie cubierta con adherencias fue mucho mayor (p<0.01) en el grupo PL (7.36 vs 0.11 cm2). El neoperitoneo formado tras el implante de PL aparecía estructuralmente desorganizado mientras que, el formado en contacto con la superficie de la prótesis PL-PU99 era estructural y funcionalmente semejante al peritoneo del huésped. El excelente comportamiento demostrado en este estudio de la prótesis PL-PU99 justifica nuevas investigaciones por lo que a su empleo en el tratamiento de los defectos de la pared abdominal se refiere, sobre todo cuando el implante debe colocarse en contacto con las asas intestinales.