Alberto Cifuentes

Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents

The article deals with the design, preparation, and evaluation of a new bilayered dressing for application in the healing of compromised wounds. The system is based on the sequential release of two complementary bioactive components to enhance the activation of the regeneration of dermal tissue. The internal layer is a highly hydrophilic and biodegradable film of gelatin and hyaluronic acid (HG), crosslinked with the natural compound genipin, which reacts with the amine groups of gelatin. This film is loaded with the proangiogenic, anti-inflammatory, and antibacterial peptide, proadrenomedullin N-terminal 20 peptide (PAMP), that is released slowly in the wound site. The external layer, more stable and less hydrophilic, is constituted by a biodegradable polyurethane derived from poly(caprolactone) and pluronic L61. This layer is loaded with resorbable nanoparticles of bemiparin (a fractionated low molecular weight heparin), which promotes the activation of growth factors, FGF and VEGF, and provides a good biomechanical stability and controlled permeability of the bilayered dressing. Experiments carried out in mice demonstrate the excellent angiogenic effect of the HG film in the dermal tissue. Application of the bilayered dressing in the wound healing rabbit ear model shows an improved cicatrization of the wound in both ischemic and non-ischemic defects, favoring epithelialization and reducing noticeably the contraction and the inflammation.

Chemical Adhesion Barriers: Do They Affect the Intraperitoneal Behavior of a Composite Mesh?

ABSTRACT Introduction: The intraperitoneal behavior of a prosthetic material used to repair a hernia is key to the success of the postimplant repair process. This study was designed to laparoscopically examine the real-time behavior of three composite meshes incorporating a chemical adhesion barrier when placed in contact with the visceral peritoneum. Material and Methods: The defects of 7 × 5 cm were created in the ventral abdominal wall of 18 New Zealand White rabbits and repaired using Parietex Composite® (n = 6), Sepramesh® (n = 6), or Proceed® (n = 6). At 24 hr, 3, 7, and 14 days postimplant, adhesion formation was quantified by subjecting photographs obtained during laparoscopy to image analysis. At 14 days, specimens of the implants and surrounding host tissue were obtained for histologic, morphometric, and immunohistochemical analyses. Results: There were no cases of infection and/or rejection of the implant. Adhesion formation followed by laparoscopy 3, 7, and 14 days after implant was significantly lower for Parietex® than the other biomaterials. Degradation of the chemical barrier occurred earliest in Sepramesh®, this barrier being most stable at 14 days for the Parietex® implants. Macrophage counts were significantly greater for Sepramesh®. The thickness of the neoformed peritoneum formed on the three implants varied significantly (p < .05): 276.89 ± 38.87 μm, 84.49 ± 19.05 μm, and 161.97 ± 47.05 μm, respectively for Paritex®, Sepramesh®, and Proceed®. Conclusions: (a) The most stable barrier against biodegradation was that of Parietex®; (b) the first postimplant week was the most critical period for adhesion formation; and (c) all three biomaterials showed good intraperitoneal behavior.

The angiogenesis promoter, proadrenomedullin N-terminal 20 peptide (PAMP), improves healing in both normoxic and ischemic wounds either alone or in combination with autologous stem/progenitor cells.

A combination of vascular pathologies and other complicating factors results in chronic wounds which constitute a serious burden for both patients and national health systems, due to prolonged hospital stays, high costs, and prolonged nursing staff dedication. Here we investigate whether proadrenomedullin N-terminal 20 peptide (PAMP), a naturally occurring peptide of the skin with antimicrobial and proangiogenic properties, either alone or in combination with autologous skeletal muscle stem/progenitor cells, acts as a wound healing factor. The rabbit ear was chosen as a test system, since it offers a reliable model for normoxic and ischemic wounds. Topical treatments with PAMP, stem/progenitor cells, or a combination of both, resulted in significant improvements of healing, when compared to untreated wounds. PAMP was very effective in promoting reepithelialization and angiogenesis, whereas treatment with stem/progenitor cells alone resulted in less wound contraction. Interestingly, the combination of PAMP and stem/progenitor cells, while maintaining angiogenic potency, reverted to the contraction levels observed in the untreated controls. Under ischemic conditions, generalized necrosis of the dermis and the underlying cartilage was observed in untreated wounds. Treatments of these wounds with PAMP or stem/progenitor cells allowed a timely recovery. In conclusion, PAMP either alone or in combination with autologous stem/progenitor cells may provide a useful tool for improving wound healing both in normoxic and ischemic conditions.

Role of lysyl oxidases in neointima development in vascular allografts.

Background: Extracellular matrix deposition is the main factor inducing stenotic lesions in arterial grafts. Lysyl oxidases (LOX) play a key role in stabilizing collagen and elastin. Objective: To examine the repair response to arterial allografts in terms of LOX expression and collagen/elastin deposition using LOX inhibitors. Methods: Lewis/Fisher-344 rats were used as donors/recipients. Donor segments were grafted to the right iliac artery of recipients and retrieved 14/30 (short-term) or 90/180 days (long-term) after surgery. One group of animals was injected with a potent irreversible LOX inhibitor daily for 30 days. Results: Intimal hyperplasia increased in thickness until 90/180 days postsurgery. Elastin showed great expression in the neointima at 14/30 days and in the media at 90/180 days. LOX/LOXL1 were similarly expressed in the arterial wall during the first month. In the long term, their overexpression was confined to neointimal layers. At 14 days, collagen types I/III were identified in the grafts. The neointima acquired collagen I over time. In the group of animal treated with the LOX inhibitor, intimal hyperplasia was significantly inhibited. Conclusion: LOX were overexpressed in late stages of intimal hyperplasia in the allografts. LOX inhibitors prevented the development of the neointimal layer, such that their modulation could reduce the excessive extracellular matrix deposition that leads to stenosis.

Effects of a novel NADPH oxidase inhibitor (S42909) on wound healing in an experimental ischemic excisional skin model.

Chronic wounds are a serious healthcare problem. As non‐healing wounds involve continuous pathologic inflammatory stage, research is focused on anti‐inflammatory treatments. Our objective was to analyze the effect of S42909, a potent NADPH oxidase inhibitor activity, with vascular anti‐inflammatory properties. An ischemic rabbit ear ulcer model (24 New Zealand white rabbits) was used to evaluate the reepithelialization/contraction areas, anti‐/pro‐inflammatory cytokines mRNA (TGF‐β1/IL‐10/IFN‐γ/VEGF) by qRT‐PCR, collagen I/III deposition, and neovascularization (TGF‐β1/VEGF) by morphological and immunohistochemical analyses. Three different doses were administered by gavage for 2 weeks: 10 and 30 mg/kg/d in self‐microemulsion drug delivery system (SMEDDS) and 100 mg/kg/d in arabic gum. Each vehicle was used as control. No signs of infection or necrosis were found. Reepithelialization was almost complete whatever the groups reaching 95% at the dose of 100 mg/kg. Wound contraction was significantly reduced in all S42909‐treated groups. A significant increase in anti‐inflammatory cytokines TGF‐β1 mRNA and IL‐10 mRNA was observed at the dose of 100 and 30 mg/kg/d, respectively. No changes were observed in pro‐inflammatory factors INF‐γ and VEGF mRNA. Ischemic skin wound areas had scarce expression of collagen I/III and showed rich glycosaminoglycans content. Treatment increased the collagen deposition and TGF‐β1 protein expression and decreased glycosaminoglycan content dose dependently; however, no effect in VEGF was appreciated. Therefore, our results indicate that S42909 improved healing process by dampening excessive inflammation and facilitating collagen deposition without wound contraction phenomena. S42909 might be a promising therapy to treat chronic wounds as venous leg ulcers.

Elastin Development-Associated Extracellular Matrix Constituents of Subepithelial Connective Tissue in Human Pterygium

PURPOSE We evaluated the expression of several extracellular matrix constituents implicated in the synthesis and reticulation of elastin in human pterygium, according to age and sex of the patients. METHODS Pterygia and normal conjunctiva samples were divided into groups according to age (<50/≥50 years) and sex. Tissue was subjected to immunohistochemical staining with anti-lysyl oxidase (LOX), lyxyl oxidase-like 1 (LOXL-1), fibulin (FBLN)-5 and FBLN4, and fibrillin-1 (FBN1) antibodies. Specific primers for the same constituents were used in a quantitative real-time PCR (qRT-PCR) analysis to determine gene expression. RESULTS The LOXL-1 (P = 0.0002), FBLN5 (P = 0.0035), and FBN1 (P < 0.0001) mRNAs were significantly higher in pterygium than conjunctiva. No differences were found for LOX and FBLN4 mRNA. The expression of LOXL-1 was not affected by age or sex; however, pterygium from men and patients over 50 years old exhibited significantly higher FBLN5/FBN1 expression than did controls. The LOX gene expression was higher in the pathologic samples from the over 50-year-olds compared to the conjunctiva (P = 0.0396) and in mens versus womens pterygium (P = 0.0173). In general, the levels of LOX, LOXL-1, and FBLN5 decreased with age in healthy samples, while the pathology seemed to have increased expression of the three proteins, and even more so in older patients. The FBLN4 and FBN1 immunostaining was slight in all samples, displaying no differences between groups. CONCLUSIONS Several extracellular matrix constituents, LOXs, FBN1, and FBLN5, implicated in the development of elastin, are overexpressed in the subepithelial connective tissue extracellular matrix of human pterygium, supporting our hypothesis that elastic synthesis and reticulation is dysregulated in this type of pathology.

High Sensitivity of Human Adipose Stem Cells to Differentiate into Myofibroblasts in the Presence of C. aspersa Egg Extract

Introduction Regeneration therapy using adipose-derived stem cells (ADSC) has been proposed in the treatment of skin aging. Myofibroblast plays a relevant role in the organization of the extracellular matrix of the damaged skin. A natural extract was derived from the eggs of the mollusk Cryptomphalus aspersa (e-CAF) that seems to play a role on skin repair. We have investigated the potential effects of e-CAF in the differentiation of ADSC. Materials and methods ADSC were cultured in the absence or presence of e-CAF (50 and 200 μg/mL) for 24 hours and 7 days. Real-time cell assay, morphological, immunofluorescence, and RT-PCR techniques were used to evaluate the cell culture and expression of αSMA, collagen I, and tropoelastin. Results e-CAF induced significant reduction in the rate of growth of ADSC from 24 hours to 7 days of culture. e-CAF also induced bigger sizes, higher levels of cytoplasmic refringence and complexity, and a more polyhedral morphological changes in the cultured ADSC. The protein and mRNA expression of αSMA was significantly increased in e-CAF-cultured ADSC. Conclusion e-CAF promotes ADSC differentiation to myofibroblasts and should be considered as a potential agent for the prevention and treatment of skin aging.

Remodeling of Noncrosslinked Acellular Dermal Matrices in a Rabbit Model of Ventral Hernia Repair

Background: Bioprostheses represent a significant advance in the abdominal wall reconstruction since they become degraded until their complete elimination in the recipient organism. This study examines remodeling in the host of three noncrosslinked porcine dermal collagen biomeshes: Strattice™ (St; LifeCell Corp.), XCM Biologic® Tissue Matrix (XCM; Synthes CMF) and Protexa® (Pr; Deco Med S.R.L.). Methods: Partial ventral hernia defects created in New Zealand White rabbits were repaired using the biomeshes that were placed in an inlay, preperitoneal position. At 14 and 90 days after implantation, explants were assessed in terms of their host tissue incorporation by morphological studies, collagen gene/protein expression (quantitative real-time PCR/immunofluorescence), macrophage response (immunohistochemistry) and biomechanical strength. Results: There were no cases of mortality or infection. Among our macroscopic findings, the mesh detachment detected in one third of the Pr implants at 90 days was of note. The host tissue response to all the biomeshes was similar at both time points, with a tendency observed for their encapsulation. There were no appreciable signs of mesh degradation. The extent of host tissue infiltration and collagenization was greater for St and Pr than for XCM. Macrophages were observed in zones of inflammation and tissue infiltration inside the mesh. XCM showed a greater macrophage response at 90 days (p < 0.05). Improved tensile strength was observed for St (p < 0.05) over Pr and unrepaired defects. Conclusions:St showed the best behavior, featuring good collagenization and tensile strength while also inducing a minimal foreign body reaction.