Julia Buján

Fibroblasts from the transversalis fascia of young patients with direct inguinal hernias show constitutive MMP-2 overexpression.

ObjectiveTo determine the expression pattern of certain metalloproteinases (MMPs) known to be involved in the degradation of the extracellular matrix in cultured fibroblasts from the transversalis fascia (TF) of patients with inguinal hernia. Summary Background DataInguinal hernia is a common pathology, the cause of which remains unknown. It is, however, clear that the TF is one of the anatomical structures that may impede the formation of hernias, and particularly the direct type of hernia. In previous studies the authors found enhanced MMP-2 expression in TF specimens in vivo. The persistence of increased expression in cultured fibroblasts might support the idea of a genetic defect as the cause for this pathology. MethodsFibroblasts from the TF of patients with direct and indirect inguinal hernia were cultured and compared with those obtained from control TF in terms of MMP (MMP-2 and MMP-9) expression. ResultsSignificant active MMP-2 expression was shown by TF fibroblasts from young patients with direct hernias. These findings were confirmed by immunosorbent assay, immunoblotting, and zymography of the fibroblast culture media. No MMP-9 expression was detected. ConclusionThese results indicate that MMP-2 may be involved in the TF matrix degradative process in patients with direct hernia. The persistence of changes in MMP-2 levels in the cell cultures appears to suggest a genetic defect or irreversible change as the origin of this pathology rather than environmental factors, which may later participate in the development of the hernial process.

Integration of biomaterials implanted into abdominal wall: process of scar formation and macrophage response

The behaviour of two biomaterials polytetrafluoroethylene (PTFE) and polypropylene (PL) has been studied, focussing especially on the macrophage response to the implant, as well as on certain aspects of the process of scar formation. A total of 24 animals (white New Zealand rabbits) received prostheses implanted into the anterior abdominal wall in such a way as to involve every layer over an area of 7 cm x 5 cm. The interfaces formed with the visceral peritoneum, subcutaneous tissue layer and the recipient muscle-aponeurotic tissue were assessed. The techniques employed were light microscopy, scanning electron microscopy and immunohistochemical methods, the latter using a monoclonal antibody specific for rabbit macrophages (RAM-11). From the results obtained, it can be seen that: (a) the structure of PL allows its total integration with the reparative tissue, while PTFE becomes encapsulated, on both the subcutaneous and the peritoneal aspects, by orderly connective tissue; (b) the macrophage response, determined on the basis of the presence of labelled macrophages, shows a similar pattern in both biomaterials; and (c) angiogenesis is very intense in the PL mesh, whereas the PTFE prosthesis undergoes almost no vascular colonization.

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.

Tissue response to polypropylene meshes used in the repair of abdominal wall defects.

The degree of integration of biomaterials used in the repair of abdominal wall defects seems to depend upon the structure of the prosthesis. Several polypropylene (PP) prostheses are currently available which differ in the number of PP filaments, the type of weave and the porosity. The aim of this study was to evaluate the integration, adhesion formation and resistance to traction of three types of PP prostheses (Marlex, Trelex and Prolene) used in the partial or total repair of abdominal wall defects. Abdominal wall defects (7 x 5 cm) were created in 54 New Zealand rabbits involving all the tissue layers (total substitutions (TS); n = 27) or all layers excluding the parietal peritoneum (partial substitutions (PS); n = 27). The defects were repaired with PP monofilament prostheses of different weave (1 mm porosity) (Marlex, n = 18; Trelex, n = 18) or bifilament (2 mm porosity) (Prolene; n = 18). They were placed in contact on one side with subcutaneous tissue and on the other with abdominal viscera or parietal peritoneum. Animals were killed at 30, 60 and 90 days and samples of prosthesis and scar tissue processed for light and scanning microscopy. The adhesion formation with viscera was evaluated. Resistance to traction was measured with a tensiometer using strips including the prosthesis and anchorage tissue. Adhesions were detected in all the TS and in four PS. Microscopic analysis revealed total integration of the TS samples by fibrous and disorganized tissue. Prostheses used for PS were integrated by white adipose tissue with the exception of the areas around the mesh nodes and anchorage zones. The foreign body reaction could be seen as a moderate accumulation of white blood cells. Tensiometric analysis showed an increase in resistance to traction with time (P < 0.001) in each type of prosthesis, but no differences were detected (P > 0.001) between them. We concluded that: (a) the formation of adhesions was almost inhibited when the parietal peritoneum was left intact; (b) in both TS and PS, polypropylene prostheses integrated completely although the composition of the scar tissue was seem to differ; and (c) resistance to traction was similar in both TS and PS.

The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107-111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using μCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107-111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material.

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.

Early tissue incorporation and collagen deposition in lightweight polypropylene meshes: bioassay in an experimental model of ventral hernia.

BACKGROUND This study was designed to assess the early host tissue incorporation of several polypropylene lightweight (PP-LW) meshes used to repair abdominal wall defects and to correlate collagen deposition with the biomechanical response shown by PP-LW versus polypropylene heavyweight (PP-HW) meshes. METHODS Ventral hernial defects (7 x 5 cm) were created in the anterior abdominal wall of New Zealand rabbits and repaired by fixing PP-LW mesh of different pore sizes or a low porosity HW mesh to the edges of the defect. Rabbits were killed 14 days after implant, and specimens were taken from the central mesh area to examine collagen deposition by light microscopy, real time reverse transcription polymerase chain reaction, immunohistochemistry, and Western blotting. The biomechanical resistance of the biomaterials was also assessed. RESULTS All the materials showed excellent incorporation in host tissue. Relative amounts of collagen III mRNA were considerably higher than collagen I mRNA. Higher collagen I and III mRNA levels were noted for pore sizes equal to or greater than 3.45 +/- 0.19 mm(2) (Ultrapro/Optilene Elastic. These two meshes showed significantly higher levels of collagen III than Parietene and Surgipro with smaller pores. Biomechanical resistance values for Optilene were significantly higher than those recorded for Surgipro and Parietene. CONCLUSIONS (a) LW meshes of pore size larger than 3 mm(2) induced the genetic overexpression of collagen types I and III; (b) the larger pore-sized LW meshes induced more collagen type III deposition and its faster conversion to collagen I; (c) Optilene, the most porous LW mesh examined, showed the greatest tensile strength 14 days after implant.

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.

Age-Related Changes in the Elastic Tissue of the Human Aorta

Background: Age-related arterial alterations affecting cells, matrix and biomolecules are the main culprit for initiation and progression of cardiovascular disease. The objective of this study is to gain further insights into the complex mechanism of elastic tissue ageing in human aortic blood vessels. Methods: One hundred and nineteen human aortic tissue samples were collected from adult patients (101 males, 18 females; age 40–86 years) undergoing coronary artery bypass grafting. Overall extracellular matrix architecture was examined by multiphoton laser scanning microscopy and histology. Matrix metalloproteinases 2 and 9, corresponding tissue inhibitors 1 and 2 as well as desmosine were determined. mRNA levels of tropoelastin were assessed by quantitative RT-PCR. Results: Age-related destruction of the vascular elastic laminas as well as a loss of interlamina cross-links were observed by laser scanning microscopy. These results were confirmed by histology indicating increasing interlamina gaps. There were no significant differences in matrix turnover or desmosine content. A steady decrease in tropoelastin mRNA by about 50% per 10 years of age increase was observed. Conclusions: Our findings indicate that ageing is accompanied by a destruction of the elastic vascular structure. However, tropoelastin expression analysis suggests that elastogenesis occurs throughout life with constantly decreasing levels.