Diabetes alters inflammation, angiogenesis, and fibrogenesis in intraperitoneal implants in rats

Microvascular Research 93 (2014) 23–29 Contents lists available at ScienceDirect Microvascular Research journal homepage: www.elsevier.com/locate/ymvre Diabetes alters inflammation, angiogenesis, and fibrogenesis in intraperitoneal implants in rats Teresa Oviedo-Socarrás a,b, Anilton C. Vasconcelos a, Irma X. Barbosa a,c, Nubia B. Pereira a, Paula P. Campos a, Silvia P. Andrade d,⁎ a Department of General Pathology - Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil University of Córdoba, Montería, Córdoba, Colombia University of Tolima, Ibagué, Tolima, Colombia d Department of Physiology and Biophysics - Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Av. Antônio Carlos, 6627 — Pampulha, CEP 31270-901, Brazil b c a r t i c l e i n f o Article history: Accepted 25 February 2014 Available online 1 March 2014 a b s t r a c t The increased prevalence of diabetes worldwide is associated with increasing numbers of diabetic individuals receiving synthetic matrices and biomedical implants to repair and/or replace biological tissues. This therapeutic procedure invariably leads to adverse tissue healing (foreign body reaction), thus impairing the biomedical device function of subcutaneous implants. However, the influence of diabetes on abnormal tissue healing in intraperitoneal implants is unclear. We investigated key components of foreign body reactions in diabetic rats. Polyether-polyurethane sponge discs were placed intraperitoneally in rats previously injected with streptozotocin for induction of diabetes and in non-diabetic rats. Implants removed 10 days after implantation were assessed by determining the components of the fibrovascular tissue (angiogenesis, inflammation, and fibrogenesis). In implants from diabetic rats, fibrous capsule thickness and fibrovascular tissue infiltration (hematoxylin & eosin and picrosirius staining) were reduced in comparison with implants from non-diabetic rats. Hemoglobin (Hb) content (vascular index) and VEGF levels (pro-angiogenic cytokine) were increased after diabetes. However, the number of vessels (H&E and CD31-immunostaining) in the fibrovascular tissue from diabetic rats was decreased when compared with vessel numbers in implants from non-diabetic animals. Overall, all inflammatory parameters (macrophage accumulation-NAG activity; TNF-α and MCP-1 levels) increased in intraperitoneal implants after diabetes induction. The pro-fibrogenic cytokine (TGFβ-1) increased after diabetes, but collagen deposition remained unaltered in the implants from diabetic rats. These important diabetes-related changes (increased levels of pro-inflammatory and angiogenic and fibrogenic cytokines) in peritoneal implant healing provide an insight into the mechanisms of the foreign body response in the diabetic environment in rats. © 2014 Elsevier Inc. All rights reserved. Introduction Wound healing in mammalian tissue is an essential biological response to tissue in\jury, responsible for maintaining of body integrity and function. Despite having obvious etiological and clinical distinctions, cutaneous or serosal injuries share common overlapping events (hemostasis, inflammation, proliferation, angiogenesis, and remodeling of the extracellular matrix). Thus, when disruption of blood vessels occurs after injury, a leakage of blood components into the wound space triggers a well-known pattern of events: platelet activation, clot formation, leukocyte recruitment, endothelial/mesothelial cell and fibroblast recruitment/activation. If the insult ceases, the cutaneous wound or peritoneal damage is repaired (Chegini, 2002; Singer and ⁎ Corresponding author. Fax: +55 31 34092924. E-mail address: [email protected] (S.P. Andrade). http://dx.doi.org/10.1016/j.mvr.2014.02.011 0026-2862/© 2014 Elsevier Inc. All rights reserved. Clark, 1999). While many phases of wound repair are common to various types of injury, a number of factors (systemic diseases, chronic inflammatory processes) can lead to healing deficiencies/alterations. In diabetes, for example, the integrity of many tissues is affected and healing of cutaneous tissue is impaired. The diabetes-associated healing impairment is characterized by a decreased inflammatory response, amount of fibrin, collagen synthesis, tensile strength, angiogenesis, and altered production of cytokines (Altavilla et al., 2001; Lerman et al., 2003; Marchant et al., 2009). Local inflammatory processes induced by implantation of biomaterials also lead to defective healing. Prolonged inflammatory signals at the site of the injury elicit fibrosis, formation of foreign body giant cells, and encapsulation of the device (Kyriakides and MacLauchlan, 2009). This adverse healing response has been shown to be present at distinct anatomical sites, including subcutaneous and intraperitoneal sites (Le et al., 2011; Mooney et al., 2010). In diabetic animals bearing subcutaneous implants, 24 T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 granulation tissue and connective tissue ingrowth were reduced (Gerritsen et al., 2000; Thomson et al., 2010). However, we found no study that has investigated the influence of diabetes on key components (inflammation, angiogenesis and fibrosis) of this adverse tissue healing (peritoneal fibrosis and adhesion) after peritoneal damage or implantation of a medical device. Here, we proposed to apply a model of peritoneal fibroproliferation induced by implantation of a synthetic matrix to characterize the effects of diabetes in healing implants (intraperitoneal foreign body reaction) in rats. In this model, the implants have been shown to adhere firmly to visceral organs (liver and/or intestines), forming an adhesion-like structure in which the assessment of relevant components of the fibrovascular tissue (angiogenesis, inflammatory cell recruitment/activation, extracellular matrix deposition) could be determined (Araújo et al., 2011; Mendes et al., 2007, 2009). Our findings have demonstrated for the first time important diabetes-related differences in the formation of fibrovascular tissue induced by a synthetic matrix located in the peritoneal cavity. Understanding the influence of diabetes on host reaction to a foreign body is critical in the development of implantable biomaterials and in the management of this reaction in healing processes within the abdominal cavity. Materials and methods Animals The use of animals and procedures in the present study were approved by the Ethics Committee in Animal Experimentation (CETEA/UFMG). We used male Wistar laboratory rats, weighing 300–350 g provided by Centro de Bioterismo (CEBIO) of the Universidade Federal de Minas Gerais (UFMG). The animals were housed in polypropylene cages inside a well-ventilated room, provided with chow pellets and water ad libitum and maintained under a 12-hour light/dark cycle. All animal procedures were in accordance with the standards set forth in the guidelines for the care and use of experimental animals by our local Institutional Animal Welfare Committee. Induction of diabetes mellitus Streptozotocin (STZ) was obtained from Sigma-Aldrich, St. Louis, MO, USA. STZ was dissolved in a 10 mM citrate buffer (pH 4.5) and always prepared for immediate use within 5 to 10 min. STZ doses were determined according to the body weight of animals and were administered intravenously in a single injection of 60 mg/kg. The blood glucose level was measured before injecting STZ and subsequently every 4 days with On Call® Plus Blood Glucose Meter (ACON Laboratories, Inc.). Animals whose blood glucose level exceeded 200 mg/dl after treatment were considered diabetic. killed by cervical dislocation. The sponge discs were carefully dissected from adherent tissue, removed and weighed. They were then processed as described below for the various assays. Histological staining, immunohistochemistry, and morphometric analysis The sponge implants from both groups (non-diabetic and diabetic) were fixed in 10% buffered formalin (pH 7.4) and processed for paraffin embedding. Sections with 5 mm thickness were stained with hematoxylin/eosin (H&E) and processed for light microscopic studies. Picrosirius-red staining followed by polarized-light microscopy was used to visualize and determine collagen fibers. Immunohistochemistry (IHC) reactions for the detection of endothelial cells/blood vessels were performed using the monoclonal antibody clone CD 31 (Fitzgerald MA, USA). Tissue sections (5 μm) were dewaxed and antigen retrieval was performed in citrate buffer (pH 6). The slides were boiled in citrate buffer for 25 min at 95 °C and then cooled for 1 h in the same buffer. Sections were incubated for 5 min in 3% hydrogen peroxide to quench the endogenous tissue peroxidase. Nonspecific binding was blocked by using normal goat serum for 10 min (1:10 in phosphate-buffered saline) with 1% bovine serum albumin (in phosphate-buffered saline). The sections were then immunostained with monoclonal antibody to CD31 (1:40 dilution, Dako Corporation, Carpinteria, CA, USA) for 60 min at room temperature. After washing in Tris–HCl buffer, sections were incubated for 30 min at room temperature with biotinylated Link Universal Streptavidin-HRP (Dako; Carpinteria, CA, USA). The reactions were revealed by applying 3,3′-diaminobenzidine in chromogen solution (DAB) (Dako; Carpinteria, CA, USA). The sections were counterstained with hematoxylin and mounted in Permount (Fisher Scientific; NJ, USA). Immunostaining was performed manually, and the spleen was used as a positive control. Negative controls were carried out with omission of the primary antibody, resulting in no detectable staining. The expression of these proteins was evaluated on the basis of extent of cytoplasmic immunolabeling in endothelial cells forming lumen in six high-power fields, regardless of staining intensity (×400). The area of total collagen, capsule thickness and blood vessels were measured morphometrically. To perform morphometric analysis of the number of blood vessels, images of cross sections obtained from 35 fields (132.043 μm2/field) were captured with a planapochromatic objective (40 ×) in light microscopy (final magnification = 400 ×). For collagen analysis and wall thickness, images were obtained from three representative fields at 20× (Final magnification = 200×). The images were digitized through a JVC TK-1270/JCB microcamera and transferred to an analyzer (software Image-Pro Plus 4.5 (Media Cybernetics, Inc.). A countable vessel was defined as a structure with a lumen that may or may not contain red blood cells. Tissue extraction and measurement of hemoglobin (Hb) Preparation of sponge discs and implantation Polyether–polyurethane sponge discs, 5 mm thick × 12 mm diameter (Vitafoam Ltd, Manchester, U.K.) were used as the matrix for fibrovascular tissue growth. The sponge discs were soaked overnight in 70% ethanol and sterilized by boiling in distilled water for 30 min before the implantation surgery. Thirteen days after STZ injection in the diabetic groups, all animals were anesthetized with a mixture of ketamine and xylazine (60 mg/kg and 10 mg/kg, respectively). The abdominal hair was shaved and the skin wiped with 70% ethanol. The sponge discs were aseptically implanted inside the abdominal cavity through a 1 cm long ventral midline incision in the line alba of the abdomen, respectively. The incisions were closed with silk braided nonabsorbable suture. At 10 days postimplantation (23 days after induction of diabetes), the animals were anesthetized with ketamine and xylazine and later The vascularization in the sponge implants was evaluated indirectly by the amount of Hb in the tissue detected by the Drabkin method. This method has been modified and used to determine angiogenesis in various experimental models and tissues, including the sponge implant model (Araújo et al., 2011; Campos et al., 2008; Mendes et al., 2009). All implants were individually homogenized (Tekmar TR-10, Cincinnati, OH) in 5 mL of Drabkin reagent (Labtest, São Paulo, Brazil) and centrifuged at 12,000 rpm for 20 min. The supernatants were filtered through 0.22 mm filter (Millipore, São Paulo, Brazil). Hb concentration in the samples was determined spectrophotometrically by measuring absorbance at 540 nm using an enzyme linked immunosorbent assay (ELISA) plate reader and comparing against a standard curve of Hb. The content of Hb in the implant sponge was expressed as μg Hb/mg of wet tissue. T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 25 Fig. 1. Glycemic levels and body weight of non-diabetic and diabetic animals. A significant increase in blood glucose level (A) and decrease in body weight (B) was observed after streptozotocin (STZ) injection in rats compared with non-diabetic rats during the entire experiment. Data are expressed as means ± SEM, from 11 animals in each group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. Fig. 2. Representative photograph of intraperitoneal implant in situ in rat (A) and wet weight values of implants from diabetic and non-diabetic animals removed 10 days postimplantation (B). Data are expressed as means ± SEM from groups of 11 animals. *Significant difference between non-diabetic and diabetic; P b 0.05; Student's t-test. NIP, non-diabetic implant; DIP, diabetic implant. Determination of myeloperoxidase (MPO) activity Neutrophil infiltration in the implants was measured indirectly by assaying MPO activity as previously described (Campos et al., 2008; Ferreira et al., 2004; Mendes et al., 2007). After using the sponge implant for measurement of Hb, as indicated above, a part of the pellet was weighed and homogenized in 2 mL of pH 4.7 phosphate buffer (0.1 M NaCl, 0.02 M Na3PO4, 0.015 M NaEDTA, pH 4.7), and centrifuged at 12,000 ×g for 15 min. The pellet was re-suspended in 0.05 M sodium phosphate buffer (pH 5.4) containing 0.5% hexa-1,6bisdecyltrimethylammonium bromide (HTAB, Sigma). Later, the suspensions were freeze–thawed three times using liquid nitrogen 'and centrifuged at 10,000 ×g for 10 min. MPO activity in the supernatant samples was evaluated by quantifying the change in absorbance (optical density, OD) at 450 nm using tetramethylbenzidine (1.6 mM) and H2O2 (0.3 mM). The reaction was finished by adding of 50 μl of H2SO4 (4 M). Results were expressed as a change in OD/mg of wet tissue. Fig. 3. Representative histological sections (5 μm, stained with H&E) of fibrovascular tissue in 10-day old implants from non-diabetic (A) and diabetic rats (B). The pores of the sponge matrix, seen as triangular shapes, are composed of spindle-shaped fibroblasts, microvessels and inflammatory infiltrate. However, cellularity was less in implants of diabetic rats and microvessels were more dilated; bar 50 μm. 26 T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 Fig. 4. Representative histological sections and analysis of wall thickness of 10-day old implants from non-diabetic (A) and diabetic (B) rats (H&E staining). Data are expressed as means ± SEM from 27 measurements/group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. (C) NIP, non-diabetic implant; DIP, diabetic implant. Inset—foreign body giant cell; bar 50 μm. Determination of N-acetyl-β-D-glucosaminidase (NAG) activity The extent of mononuclear cells in the sponge implants was quantitated by measuring the levels of the lysosomal enzyme, N-acetyl-glucosaminidase (NAG) present in high levels in activated macrophages (Campos et al., 2008; Ferreira et al., 2004; Mendes et al., 2007). The implants were homogenized in 2 mL NaCl solution (0.9% w/v) containing 0.1% v/v Triton X-100 (Promega, Madison, WI) and centrifuged at 3000 rpm; 10 min at 4 °C. One hundred microliters of the supernatant was incubated for 10 min with 100 mL of pnitrophenyl-N-acetyl-b-D-glucosaminide (Sigma, Saint Louis, MO) prepared in citrate/phosphate buffer (0.1 M citric acid, 0.1 M Na2HPO4; pH 4.5) with a final concentration of 2.24 mM. The reaction was terminated by adding of 100 μl of 0.2 M glycine buffer (pH 10.6). Hydrolysis of the substrate was determined by the color absorption at 400 nm. A standard curve was constructed with p-nitrophenol (0–500 nmol/ml) and NAG activity was expressed as a change in nmol/mg of wet tissue. Measurement of VEGF, TNF-α, MCP-1 and TGF-β1 content of the sponge implants Implants were homogenized in PBS pH 7.4 containing 0.05% Tween and centrifuged at 10,000 ×g for 30 min. The cytokines VEGF, TNF-α, MCP-1 and TGF-β1 were measured in 100 μl of the supernatant using Immunoassay Kits (R and D Systems, USA) and following the manufacturer's protocol. Dilutions of cell-free supernatants were added to ELISA plates coated with a specific murine monoclonal antibody against the cytokine, followed by the addition of a second horseradish peroxidase-conjugated polyclonal antibody, also against the cytokine. After washing to remove any unbound antibody–enzyme Fig. 5. Hemoglobin content (A) and VEGF levels (B) in 10-day old implants from non-diabetic and diabetic rats. Both angiogenic markers were increased after diabetes induction. Values shown are expressed as mean ± SEM from 11 animals in each group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. NIP, non-diabetic implant; DIP, diabetic implant. T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 27 Fig. 6. Vascularization in 10-day old implants of non-diabetic and diabetic rats. CD31-immunostained sections (A). Morphometric analysis showed decreased number of vessels in implants from diabetic compared with non-diabetic rats (B). Values shown are expressed as mean ± SEM from 105 fields/group; 3 animals in each group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. NIP, non-diabetic implant; DIP, diabetic implant. reagent, a substrate solution (50 μL of a 1:1 solution of hydrogen peroxide and tetramethylbenzidine 10 mg/ml in DMSO) was added to the wells. The color development was stopped after 20 min incubation, with 2 N sulfuric acid (50 μL) and the intensity of the color was measured at 540 nm on a spectrophotometer (E max — Molecular Devices). Standards were 0.5-log10 dilutions of recombinant murine cytokines from 7.5 pg/ml to 1000 pg/ml (100 μl). The results were expressed as a picogram of cytokine/mg of wet tissue. Statistical analysis Results are presented as mean ± SEM. The assumptions of normality and homoscedasticity were determined for subsequent statistical analysis. Comparisons between two groups (non-diabetic and diabetic groups) were performed using Student’s t-test for unpaired groups. P b 0.05 was considered significant. Results A single intravenous injection of streptozotocin (60 mg/kg) rendered the rats diabetic with blood glucose levels at 425.8 ± 12 mg/dl 5 days after the treatment, which remained unaltered for the entire experimental period (23 days). The body weight of diabetic animals was affected by the diabetogenic treatment. At the beginning of the experiment, the mean weight of the animals was 229 ± 4.4 g. Twenty-three days after the diabetogenic treatment, the animals Fig. 7. Markers of inflammation in 10-day old implants from non-diabetic (A) and diabetic (B) rats. An overall increase in the inflammatory reaction was observed after diabetes induction. (MPO — myeoloperoxidase (A); NAG — n-acetyl-β-D-glucosaminidase (B); MCP-1 (C) and TNF-α (D). Values shown are expressed as mean ± SEM from 11 animals in each group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. NIP, non-diabetic implant; DIP, diabetic implant. 28 T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 Fig. 8. Markers of fibrogenesis in 10-day old implants from non-diabetic and diabetic rats. The levels of TGFβ1 were increased in implants of diabetic rats compared with that of nondiabetic (A). The amount of collagen, B) was similar in both groups, but after diabetes green collagen (type 3) was predominant in the implants. Representative histological sections (5 μm; picrosiurus-red staining) of implants from non-diabetic and diabetic rats showing distinct types of collagen in both implants (C and D). Values shown are expressed as mean ± SEM from 6 animals in each group. *Significant difference between non-diabetic and diabetic; P b 0.05. Student's t-test. NIP, non-diabetic implant; DIP, diabetic implant. weighed 267 ± 8.3 g and the control animals gained 96.3 g (325 ± 7) (Figs. 1A and B). Gross appearance and histological examination of sponge implants Macroscopically, no signs of rejection were observed at the site of implantation in diabetic and non-diabetic rats. Representative images of intraperitoneal implants revealed that the synthetic matrix was integrated to the surrounding tissue forming an adhesion-like tissue between the intestine and the implant (Fig. 2A). The wet weight of the implants decreased after diabetes (Fig. 2B). Histological sections of intraperitoneal implants of diabetic and non-diabetic rats evidenced the induction of a fibrovascular response, causing the synthetic sponge matrix to be filled with fibrovascular tissue (H&E staining). The granulation tissue was composed of spindle-shaped fibroblasts, microvessels, and dense inflammatory infiltrate (Figs. 3A and B). In implants from diabetic rats, decreased cellularity was seen (Fig. 3B). Because fibrous capsule formation is a hallmark of foreign body reaction, we measured this parameter in implants from diabetic and non-diabetic animals. The thickness of the capsule in implants of non- diabetic rats was 175.8 ± 20 μm, after diabetes it was reduced to 130.8 ± 8 μm (Figs. 4A–C). Measurement of angiogenesis Quantitative measurement of angiogenesis was performed by hemoglobin content, VEGF levels and by the number of vessels (H&E stained and CD31-immunostained sections). Diabetogenic treatment was able to increase the amount of hemoglobin and VEGF levels intra-implant in comparison with the values of implants from nondiabetic animals (Figs. 5A and B). Conversely, the number of vessels was decreased in implants from diabetic rats when compared with that of non-diabetic animals (Figs. 6A and B). Inflammation in sponge implants Several measurements of the inflammatory component of the implants (inflammatory enzyme activities and pro-inflammatory cytokines) were performed. As shown in Figs. 7A–D, there were diabetes-related differences in leukocyte recruitment/activation in these parameters in 10-day old implants. In implants from diabetic rats, NAG activity, TNF-α and MCP-1 levels were increased when compared with the implants from non-diabetic rats. Measurement of TGFβ1 levels and total collagen deposition Fibrogenesis is an important mechanism of wound healing and this was measured by the cytokine TGF-β1 levels and total area of collagen (μm2) in the sponge implants (picrosirius staining). A significant increase in TGF-β1 levels intra-implant was observed after diabetes in comparison with the levels in implants from non-diabetic rats. Although collagen deposition decreased in implants from diabetic rats, the drop did not reach statistical significance (Figs. 8A–D). Discussion We have studied the influence of diabetes on intraperitoneal injury induced by implantation of synthetic matrix in rats. Implantation of T. Oviedo-Socarrás et al. / Microvascular Research 93 (2014) 23–29 medical devices has been widely used to repair and/or replace biological tissues but, invariably, a collagenous fibrous capsule confines the implanted device, preventing it from interacting with surrounding tissues. This unwanted reaction compromises the efficiency of the implanted material leading to device failure (Kyriakides and Bornstein, 2003; Le et al., 2011; Morais et al., 2010). Much has been reported on the skin healing deficiencies in diabetes, but information is scarce on internal wound healing parameters in individuals with this condition (Le et al., 2011). By studying the inflammatory, angiogenic, and fibrogenic components of the tissue induced by polyether–polyurethane implants located intraperitoneally, we have been able to disclose that the diabetic environment greatly altered the healing response (foreign body reaction) in most of the parameters that were analyzed. Fibroproliferative tissue infiltration (wet weight, fibrous capsule formation, and cellularity) was decreased by diabetes. In one study that investigated subcutaneous implants in diabetic baboons, reduced granulation tissue formation was observed (Gerritsen et al., 2000; Thomson et al., 2010). However, we are not aware of any systematic study that evaluated intraperitoneal implant healing in diabetes. It has been pointed out that studies that focus on the diabetic response to biomedical devices are essential to improving implant performance in these individuals since, in this medical condition, tissue replacement/implantation is likely to occur (Le et al., 2011). Decreased numbers of vessels were observed in implants from diabetic rats (H&E stained sections and in CD31-immunostained sections) when compared with implants from non-diabetic rats, contrasted with an increased amount of Hb and VEGF levels in the former implants. Decreased angiogenesis has been demonstrated in cutaneous wound healing in diabetes and in subcutaneous implants of diabetic rats and was associated with decreased levels of VEGF (Altavilla et al., 2001; Teixeira et al., 1999). However, rather than decreasing, the VEGF levels in our implants increased. Thus, it is likely that the high amount of Hb resulted from the VEGF's well-known vasodilator effect. It may be relevant to point out that the significant activation of macrophages (NAG activity) of the peritoneal implants may have contributed to increased production of this growth factor, since these activated cells produce a number of molecules, including VEGF (Miao et al., 2012; Raggi et al., 2013). We measured four markers of inflammation (MPO, NAG, and two cytokines, TNF-α and MCP-1), which were generally augmented by diabetes. Interestingly, deficient inflammation is a normal response in dermal tissue in diabetes (Gimeno et al., 2003; Gu et al., 2013). Thus, increased inflammation observed in intraperitoneal implants from diabetic animals is a novel finding. However, an increase in inflammatory cells (40%) when compared with subcutaneous devices was seen around the percutaneous implants from diabetic animals (Gerritsen et al., 2000). Therefore, the inflammatory process in the diabetic environment differs among tissues at distinct anatomical locations. Stromal fibrovascular tissue is regulated by complex interactions of pro- and anti-fibrogenic proteins within the inflammatory tissue. TGF-β1 is a key pro-fibrogenic cytokine that induces differentiation of fibroblasts into myofibroblasts which, in turn, synthetize collagen (Bonniaud et al., 2005; Leask and Abraham, 2004). We found increased levels of TGF-β1 and altered maturation of collagen in intraperitoneal implants after diabetes. It is possible that an increased pro-fibrogenic cytokine level was an attempt to stimulate intraperitoneal implants to improve collagen deposition/type in the implant compartment. Our findings show for the first time that intraperitoneal implantation of synthetic matrix in diabetic rats modifies the adverse intraperitoneal healing response (foreign body reaction) observed in non-diabetic 29 animals. 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