Filippo Renò

In vitro mechanical compression induces apoptosis and regulates cytokines release in hypertrophic scars.

Hypertrophic scars resulting from severe burns are usually treated by continuous elastic compression. Although pressure therapy reaches success rates of 60–85% its mechanisms of action are still poorly understood. In this study, apoptosis induction and release of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were evaluated in normal (n = 3) and hypertrophic (=7) scars from burns after in vitro mechanical compression. In the absence of compression (basal condition) apoptotic cells, scored using terminal deoxyribonucleotidyl transferase assay, were present after 24 hours in the derma of both normal scar (23 ± 0.4% of total cell) and hypertrophic scar (11.3 ± 1.4%). Mechanical compression (constant pressure of 35 mmHg for 24 hours) increased apoptotic cell percentage both in normal scar (29.5 ± 0.4%) and hypertrophic scar (29 ± 1.7%). IL‐1β released in the medium was undetectable in normal scar under basal conditions while in hypertrophic scar the IL‐1β concentration was 3.48 ± 0.2 ng/g. Compression in hypertrophic scar‐induced secretion of IL‐1β twofold higher compared to basal condition. (7.72 ± 0.2 ng/g). TNF‐α basal concentration measured in normal scar medium was 8.52 ± 4.01 ng/g and compression did not altered TNF‐α release (12.86 ± 7.84 ng/g). TNF‐α basal release was significantly higher in hypertrophic scar (14.74 ± 1.42 ng/g) compared to normal scar samples and TNF‐α secretion was diminished (3.52 ± 0.97 ng/g) after compression. In conclusion, in our in vitro model, mechanical compression resembling the clinical use of elastocompression was able to strongly increase apoptosis in the hypertrophic scar derma as observed during granulation tissue regression in normal wound healing. Moreover, the observed modulation of IL‐1β and TNF‐α release by mechanical loading could play a key role in hypertrophy regression induced by elastocompression. (WOUND REP REG 2003;11:331–336)

Effect of in vitro mechanical compression on Epilysin (matrix metalloproteinase-28) expression in hypertrophic scars

Epilysin, designated matrix metalloproteinase (MMP)‐28, is the newest member of this family of proteases expressed by keratinocytes in response to an injury. MMP‐28′s physiological role and specific substrates are unknown, but its expression pattern suggests that it may serve a role in both tissue homeostasis and wound healing. The aim of this preliminary study was to observe the presence of MMP‐28 protein in normotrophic and hypertrophic scars and to evaluate the effect of in vitro mechanical compression on its expression. Biopsies from normotrophic and hypertrophic scars resulting from burns were divided into two samples, one to be used as control (uncompressed) and the other to be compressed in an oxygenated organ chamber for 24 hours in the presence of a serum‐free medium, using an electromechanical load transducer (stable pressure = 35 mmHg). Analysis of MMP‐28 protein secretion, assessed by Western blot and β‐casein zymography in scar conditioned media, revealed that normotrophic scar did not release MMP‐28 in any condition while hypertrophic scar released active MMP‐28 both in control conditions and after compression. MMP‐28 immunohistochemistry revealed a light protein presence in normotrophic scar keratinocytes and a strong MMP‐28 positivity in hypertrophic scar keratinocytes in control conditions, while compression increased MMP‐28 staining in normotrophic scar and induced a significant reduction of the protein presence in hypertrophic scar keratinocytes. As it has been suggested that MMP‐28 may restructure the skin basal membrane (Saarialho‐Kere et al., 2002), our data indicate that mechanical compression directly acts to modulate the remodeling phase of wound healing, altering release and activity of MMP‐28 in hypertrophic scars.

The induction of MMP-9 release from granulocytes by Vitamin E in UHMWPE

Ultra-high molecular weight polyethylene (UHMWPE) is a biopolymer widely used in orthopaedic implants and its oxidation is considered as major responsible for inflammation and the prosthesis failure. We have studied the effect on the activation of resting human granulocytes of the addition of Vitamin E (Vit.E, alpha-tocopherol), a natural biological antioxidant and antiinflammatory agent, to UHMWPE. We have measured changes in granulocytes morphology and respiratory burst by flow cytometry using Dihydrorhodamine 123 and matrix metalloproteinase 9 (MMP-9, gelatinase B) release and activity in the growth medium using substrate zymography following contact (60 min at 37 degrees C) with cell grade polystyrene (PS), normal UHMWPE (PE) and Vit.E added UHMWPE (PE Vit.E). FTIR analyses showed that the surfaces of PE and PE-Vit.E were not significantly different. PS, PE and PE Vit.E did not alter granulocytes morphology and respiratory burst as showed by the mean fluorescence emitted (PS=12.0+/-0.1, PE=13.0+/-0.4, PE Vit.E=14.5+/-0.1). PE Vit.E was able to increase MMP-9 release compared to PS and normal PE (215+/-16% of the control, p<0.001). The PE Vit.E-induced MMP-9 release was abolished by okadaic acid (0.5 nM), suggesting a direct role of Vit.E in the phenomenon.

UHMWPE oxidation increases granulocytes activation: a role in tissue response after prosthesis implant

Ultra-high molecular weight polyethylene (UHMWPE), a biopolymer widely used in orthopaedic implants, is oxidized during gamma-ray sterilization; such surface oxidation is considered as major responsible for inflammation and prosthesis failure. As granulocytes are involved in first contact inflammation, we have measured their oxidative burst by flow cytometry using dihydrorhodamine 123 (DRH) to evaluate their activation following contact with normal and oxidized UHMWPE. Peripheral blood cells (obtained by lysed blood) were loaded with DRH, seeded onto polystyrene, normal and heat-oxidized UHMWPE disks for 30min and then collected for analysis. Granulocytes were individuated using FSC and SSC signals and their cell associated green fluorescence was analyzed. Both normal and oxidized UHMWPE stimulated granulocytes activation as showed by the mean fluorescence emitted (109.3+/-3.8 and 150.1+/-9.2, respectively) compared to control samples (81.6+/-0.3). Moreover oxidized UHMWPE activated a significantly higher percentage of granulocytes (73.35+/-5.2%) compared to not-oxidized UHMWPE (21.5+/-3.8%). UHMWPE surface oxidation responsible for increased granulocyte activation seems to play a role in tissue response to implants.

An Efficient Rose Bengal Based Nanoplatform for Photodynamic Therapy

Organically modified mesoporous silica nanoparticles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen ((1)O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.

Chronic exposure to cigarette smoke increases matrix metalloproteinases and Filaggrin mRNA expression in oral keratinocytes: Role of nicotine stimulation

The vegetal alkaloid nicotine has been proved to modify the expression of many keratinocyte markers. In this study, the basal expression of MMP-2, MMP-9, MMP-28, and Filaggrin has been evaluated in oral keratinocytes, in order to collect information about the ability of cigarette smoke to modify the basal expression pattern of these key enzymes in the absence of evident clinical signs in the oral epithelium. MMP-2, MMP-9, MMP-28, and Filaggrin basal expression was investigated by RT-PCR in oral keratinocytes derived from smokers (n=11), non-smokers (n=11), and ex-smokers (n=6) healthy volunteers. Moreover keratinocytes from non-smokers volunteers were stimulated in vitro by a single dose administration of nicotine (10 μM) in order to estimate the effect of nicotinic receptors activation on the basal expression of the studied markers. RT-PCR analysis showed that all the markers studied were overexpressed in keratinocytes from smoker donors compared to control keratinocytes, while a single dose of nicotine was able to induce only Filaggrin expression in keratinocytes from non-smoking donors. Markers expression in ex-smoker donors was similar to that observed in normal non-smoker donors. These data indicate for the first time that cigarette smoking affects basal expression of some important markers in oral mucosa keratinocytes in vivo in the absence of clinical signs and that smoke quitting restores basal expression levels of these markers.

Bacterial adhesion to poly-(D,L)lactic acid blended with vitamin E: toward gentle anti-infective biomaterials.

Anti-infective properties of biomedical materials are often achieved by loading or coating them with powerful bactericides. Undesirably, these bioactive molecules can damage the host cells at the biomaterial-tissues interface and, sometimes, even determine systemic toxic effects. The search for biomaterials able to actively resist infection while displaying a safe cytocompatibility profile toward eukaryotic cells is being progressively developed. Poly-(D,L)lactic acid (PLA) is a broadly used resorbable material with established biocompatibility properties. The dissolving surfaces of a biodegradable material tend to be per se elusive for bacteria. Here, films of pristine PLA, of PLA blended with vitamin E (VitE) and PLA blended with vitamin E acetate (VitE ac) were challenged in vitro with the biofilm-producers Staphylococcus epidermidis RP62A and Staphylococcus aureus ATCC25923. The bacterial adhesion properties of the different materials were investigated on small film disc specimens by a method based on microtiter plates. Adherent bacteria were quantified by both CFU plating and bioluminescence. Significant decrease in bacterial adhesion and biofilm accumulation was found on the surface of both the enriched polymers. These findings, together with the favorable intrinsic properties of PLA and the desirable bioactivities conferred by VitE, point up the VitE-blended PLA polymers as gentle anti-infective biomaterials.

1α,25-Dihydroxycholecalciferol (Vitamin D3) induces NO-dependent endothelial cell proliferation and migration in a three-dimensional matrix.

Background/Aims: The 1α,25-dihydroxycholecalciferol (Vit. D) induces eNOS dependent nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). To our knowledge, there are no reports directly relating Vit. D induced NO production to proliferation and/or migration in endothelial cells (EC). The aim of this study was to evaluate whether Vit. D addition to porcine EC could affect their proliferation and/or migration in a three-dimensional matrix via NO production. Materials and Methods: Porcine aortic endothelial cells (PAE) were used to evaluate Vit. D effects on cell proliferation and migration in a three-dimensional matrix. Results: Vit. D induced NO production in PAE cells. Moreover, it induced a significant increase in cellular proliferation and migration in a three-dimensional matrix. These effects were NO dependent, as inhibiting eNOS activity by L-NAME PAE migration was abrogated. This effect was strictly related to MMP-2 expression and apparently dependent on Vit. D and NO production. Conclusions: Vit. D can promote both endothelial cells proliferation and migration in a three-dimensional matrix via NO-dependent mechanisms. These findings cast new light on the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in such fields as tissue repair and wound healing.

Mechanical stretching modulates growth direction and MMP-9 release in human keratinocyte monolayer

Cells within human skin are exposed to mechanical stretching that is considered a trigger stimulus for keratinocyte proliferation, while its effect on keratinocyte migration has been poorly investigate. In order to explore the effect of stretching on keratinocyte migration spontaneously immortalized human keratinocyte (HaCaT) monolayers seeded onto collagen I-coated silicon sheets were stimulated 3 times for 1 hour every 24 hours (total time = 72 hours) by mechanical stretching increasing substrate deformations (10%) applied both as static (0 Hz) and cyclic (0.17 Hz) uniaxial stretching. At the end of stimulations monolayer areas measured in both static and cyclic samples appeared reduced and strongly oriented in a direction perpendicular to the stress direction compared to unstimulated ones. Moreover during the mechanical stimulation period HaCaT monolayers strongly increased the release in the medium of matrix metalloproteinase 9 (MMP-9), a proteolytic enzyme necessary for keratinocyte migration.

Adsorption of matrix metalloproteinases onto biomedical polymers: a new aspect in biological acceptance

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in the remodelling of connective tissues during the development and wound healing. Moreover, two MMPs, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are also present in body fluids such as blood and urine and, therefore, they can be in contact with implanted biomaterials and can be adsorbed onto their surface. In order to test this hypothesis disks of different polymers (polystyrene (PS), polyvinyl chloride (PVC), poly(D,L-lactide) (PLA), polymethyl methacrylate (PMMA) and poly(2-hydroxyethyl methacrylate) (PHEMA)) have been exposed to human plasma and adsorbed proteins have been eluted and analyzed. Using Western blot and substrate zymography analysis, we observed that both MMP-2 and MMP-9 adsorbed onto the surfaces of all the polymers, especially hydrophilic ones (PMMA and PHEMA) and PLA, in both the active and inactive forms. Furthermore, we observed that adhesion of human granulocyte neutophils to PMMA, the polymer that adsorbed the higher quantity of MMP-2 and MMP-9 compared to the others, was reduced by more that 50% by the presence of a gelatinase inhibitor. This data suggest a surprising role of these absorbed enzymes in the adhesion of neutrophil onto some polymeric biomaterials surface and, therefore, in the setting of inflammation.