Collagen Lattice Research Articles

The effects of replicative senescence and telomerase on contraction and motility of fibroblast cells

Fibroblast cells are important in wound healing as they differentiate into myofibroblasts that can migrate into a wound and create tension and aging skin seems to take longer to repair a wound. This study was designed to look at the migration and contraction ability of early (EP) and late (LP) passage cells as compared to cells with telomerase (hTERT), an enzyme that lengthens telomeres delaying senescence. To assess contraction, all cells were grown on coverslips with and without transforming growth factor beta, a wound healing agent, and stained for alpha smooth muscle actin (α‐sma) a myofibroblast marker. Collagen lattice assays were used to assess contraction by measuring lattice diameter as it contracted over time then stained for the presence of α‐sma. Finally, a scratch assay was used to measure the ability for each type of cell to migrate into a "wounded" area. Contraction by EP cells was greater than LP cells in a five day collagen lattice. Results on migration assays and α‐sma in coverslips and collagen lattice are still being analyzed; however expected results would show that EP and hTERT cells should migrate faster and have more α‐sma than LP cells. If these results are shown, they would suggest that the formation of α‐sma in EP and hTERT cells allows faster migration and better contraction than in LP cells. Funding provided by the University of Central Oklahoma.Grant Funding SourceUCOIRB

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

Emerging clinical evidence suggests that intense pulsed light (IPL) treatment may exert some beneficial effects on photoaged skin. The molecular mechanisms underlying this IPL effect have not been fully elucidated. To examine the effects of IPL irradiation on normal human dermal fibroblasts grown in contracted collagen lattices. Human skin fibroblasts cultured in contracted collagen lattices were irradiated with IPL with triple pulses of 7 ms with a pulse interval of 70 ms and fluences of 20, 50, and 75 J/cm(2). Twenty-four hours after the irradiation, cell viability, messenger RNA (mRNA), and protein levels of extracellular matrix proteins (e.g., collagen I, collagen III, and fibronectin) and transforming growth factor beta-1 (TGF-beta1) were evaluated using dye exclusion, real-time reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. A dose-dependent increase in viable cells was demonstrated after the IPL irradiation. There was no significant change in mRNA levels of collagen I and fibronectin. Upregulated expression of collagen III and TGF-beta1 in dermal fibroblasts was verified. The analytical results presented here provide a potential mechanistic explanation for the mechanism of clinical photorejuvenation effects of IPL that involves the increase of extracellular matrix construction by upregulating the gene expressions of collagen III and TGF-beta1.

Overexpression of manganese superoxide dismutase in human dermal fibroblasts enhances the contraction of free floating collagen lattice: implications for ageing and hyperplastic scar formation

Cell-matrix interactions are of significant importance for tissue homeostasis of the skin and, if disturbed, may lead to ageing and hyperplastic scar formation. We have studied fibroblasts stably overexpressing manganese superoxide dismutase (MnSOD) with a defined capacity for the removal of superoxide anions and concomitant accumulation of hydrogen peroxide to evaluate the role of enhanced MnSOD activity on the dynamics of cell-matrix interactions in the three-dimensional collagen lattice contraction assay. MnSOD overexpressing fibroblast populated collagen lattices revealed a significantly enhanced contraction compared to collagen lattices populated with vector control cells. The enhanced collagen lattice contraction was in part due to an increase in active TGF-beta1 and the accumulation of H2O2 in MnSOD overexpressing fibroblasts populated collagen lattices. Inhibition of TGF-beta1 signalling by the ALK4,5,7 kinases' inhibitor SB431542 at least partly inhibited the enhanced collagen lattice contraction of MnSOD overexpressing fibroblasts populated lattices. In addition, supplementation of vector control fibroblast populated collagen lattices with recombinant TGF-beta1 concentration dependently enhanced the collagen lattice contraction. In the presence of the antioxidant Ebselen, a mimic of H2O2 and other hydroperoxides/peroxynitrite-detoxifying glutathione peroxidase, collagen lattice contraction and the activation of TGF-beta1 were significantly reduced in collagen lattices populated with MnSOD overexpressing fibroblasts. Collectively, these data suggest that H2O2 or other hydroperoxides or peroxynitrite or a combination thereof may function as important second messengers in collagen lattice contraction and act at least in part via TGF-beta1 activation.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF – 1 mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL's and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p < 0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL.

Functional consequences of mitochondrial DNA deletions in human skin fibroblasts: increased contractile strength in collagen lattices is due to oxidative stress-induced lysyl oxidase activity

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor -aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl--phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both -aminopropionitrile and N-tert-butyl--phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin. (Am J Pathol 2009, 175:1019–1029; DOI: 10.2353/ajpath.2009.080832)

Increased levels of large scale deletions of mtDNA of skin fibroblasts result in increased collagen degradation in dermal skin equivalents

Aged tissues contain increased levels of large-scale deletions of the mitochondrial (mt) DNA. Especially extrinsically aged skin has been shown to carry a significant burden of mtDNA deletions along with several other structural and functional impairments. Until now the functional role of mtDNA mutations for functional and structural changes of aged skin is not understood. Therefore we studied how human skin cells harbouring mtDNA mutations affect their microenvironment by comparing normal human fibroblasts (NHF) to dermal fibroblasts derived from patients suffering from Kearns-Sayre syndrome (KSS) in 3D collagen gels resembling human skin (dermal equivalents, DE). KSS fibroblasts carry a 10,000 fold higher amount of mtDNA deletions exemplified by quantitative measurement of the 4977 bp Common Deletion. We here examined the degradation of components of the extracellular matrix in DE during cultivation. During six weeks we detected more fragmented collagen measured by the marker hydroxyproline (HYP) via GC-MS. Moreover we observed a less robust collagen lattice structure in dermal equivalents with KSS fibroblasts in comparison to NHF by picrosirius red staining of histological sections. In line with that, we could measure higher mRNA expression levels of the dominant collagen degrading enzyme matrixmetalloproteinase-1 (MMP-1) in KSS DE using realtime PCR and increased MMP-1 protein amounts in histological sections over the whole cultivation time. We conclude that increased levels of photo-inducible mtDNA deletions are functionally relevant for intensified matrix degradation and are therefore responsible for detrimental changes in photoaged skin.

Modèles de cultures cellulaires kératinocytaires de prépuce. Possibilité d’application chez les enfants brûlés

We tested in vitro the keratinocytes capacity for division and differentiation. The donor site was the human foreskin. For 12 months, we harvested 18 foreskins after circumcision. The middle age of the operated children was four years. The keratinocytes were isolated after double enzymatic digestion (thermolysin and trypsin, respectively). After filtration and centrifugation we put the keratinocytes in culture. In parallel, the keratinocytes were cultivated on the surface of collagen lattices. The keratinocytes were cultured in submerged condition for two days and then in an air-liquid interface condition for further differentiation. After nine days of culture, a histological examination and immunostain were used. An immunohistologic analysis made it possible to highlight the markers characteristic of epidermal skin differentiation. We obtained an average of 8.8 10(6) cells per foreskin. After seven days of culture, we obtained on average 23.7 10(6) cells by culture. In contact with the collagen lattices, we obtained an epidermal skin and we highlighted the markers of keratinocytes differentiation as well as the markers of the dermoepidermic junction. The keratinocytes resulting from foreskin have a high capacity of division. These cells can divide a long time before differentiation. The observations enable us to propose with our patients the keratinocytes from foreskin for wound healing especially for burns in children.

Recombinant human decorin inhibits TGF-β 1-induced contraction of collagen lattice by hypertrophic scar fibroblasts

Decorin was reported to bind transforming growth factor-beta (TGF-β 1) and neutralise some of its activity as a key regulator of wound contraction and hypertrophic scar formation. In this study, we investigated whether recombinant human decorin affected TGF-β 1-induced fibroblast contractile activity, by using fibroblast-populated collagen lattice with decorin added to the collagen gel. Hypertrophic scar fibroblasts showed greater basal contraction of collagen gels than normal fibroblasts, and the addition of TGF-β 1 significantly enhanced this. Decorin inhibited both the basal and TGF-β 1-enhanced contraction of collagen gel by both normal and hypertrophic scar fibroblasts. Decorin also inhibited TGF-β 1-induced α-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1) protein and mRNA expressions in normal and hypertrophic scar fibroblasts. These results suggest that decorin may have therapeutic potential for excessive skin contraction as observed in hypertrophic scarring.

QS208. Recombinant Human Decorin Inhibits TGF-β 1-Induced Contraction of Collagen Lattice by Hypertrophic Scar Fibroblasts

QS208. Recombinant Human Decorin Inhibits TGF-β 1-Induced Contraction of Collagen Lattice by Hypertrophic Scar Fibroblasts

Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

BackgroundPreterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha) and the non-steroidal anti-inflammatory indomethacin.MethodsCell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs) and immortalised myometrial smooth muscle cells (hTERT-HM): a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests.ResultsTNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up-regulated in hUtSMCs isolated from collagen gel lattices, following thrombin-stimulated contractility.ConclusionTNF alpha and thrombin increased uterine smooth muscle cell collagen contractility while indomethacin had the opposite effect. Thrombin-induced collagen contractility resulted in F2R activation which may in part be mediated by the ROCK-1 pathway. This study established the in vitro human myometrial model as a viable method to assess the effects of a range of uterotonic or uterorelaxant agents on contractility, and also permits investigation of the complex regulatory pathways involved in mediating myometrial contractility at labour.

Effect of low-level laser treatment of tissue-engineered skin substitutes: contraction of collagen lattices

Fibroblast-populated collagen lattices (FPCL) are widely used in tissue-engineered artificial skin substitutes, but their main drawback is that interaction of fibroblasts and matrix causes contraction of the lattice, reducing it to about 20% of its original area. The effect of low-level laser treatment (LLLT) on the behavior of 3T3 fibroblasts seeded in collagen lattices containing 20% chondroitin-6-sulphate was investigated to determine whether LLLT could control the contraction of FPCL. A He-Ne laser was used at 632.8 nm to deliver a 5-mW continuous wave with fluences from 1 to 4 J/cm(2). Laser treatment at 3 J/cm(2) increased contraction of collagen lattices in the absence of cells but decreased contraction of cell seeded lattices over a 7-day period. The effect was energy dependent and was not observed at 1, 2, or 4 J/cm(2). There was no alteration in fibroblast viability, morphology, or mitochondrial membrane potential after any laser treatments, but the distribution of actin fibers within the cells and collagen fibers in the matrices was disturbed at 3 J/cm(2). These effects contribute to the decrease in contraction observed. LLLT may offer a means to control contraction of FPCL used as artificial skin substitutes.

Collagen Fragmentation Promotes Oxidative Stress and Elevates Matrix Metalloproteinase-1 in Fibroblasts in Aged Human Skin

Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and alpha2beta1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and alpha2beta1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ(10) significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging.

Stem cell marker expression, proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.

MMP dependence of fibroblast contraction and collagen production induced by human mast cell activation in a three-dimensional collagen lattice

Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

An In Vitro Tier Evaluation for the Identification of Cosmetic Ingredients Which are Not Ocular Irritants

A tier evaluation system was assessed as an alternative method for the identification of cosmetic ingredients which are not ocular irritants. The system employed monolayer cultures of SIRC cells, an established cell line originally derived from the rabbit cornea, and a three-dimensional living dermal model (LDM), MATREXtrade mark, which consists of human dermal fibroblasts in a contracted collagen lattice. Effects on the cell monolayer cultures were determined by using SIRC cell-Crystal Violet staining (SIRC-CVS), and effects on the LDM were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A non-irritating ingredient was defined as a compound having a maximal average total score (MAS) of 5 or less in the Draize eye test, as this is the criterion used in the Japanese draft guidance for evaluating cosmetic ingredients. Among 34 test substances with known characteristics, 30 were classified accurately. Based on these encouraging results, the possibility of simplifying the MTT assay on the LDM for more-practical use, by selecting only three concentration levels to discriminate non-irritants from irritants, was assessed. The simplified method, involving a three-dose set (the three-dose method), was confirmed as being suitable for the identification of non-irritating ingredients, with triethanolamine used as a negative reference standard. Finally, the LDM was used to evaluate compounds at similar concentrations to those tested in vivo, aiming to predict the concentration at which an ingredient can be formulated into products without causing eye irritation. On the basis of previous validation data and our additional results, it was found that test samples that resulted in a cell viability of 50% or more in this model, could be classified as non-irritating ingredients. In all, these results indicate that the tier evaluation system may be suitable for the evaluation of ingredients intended to be used in cosmetics and medicated cosmetics in Japan.

Matrix Metalloproteinase Inhibition Reduces Contraction by Dupuytren Fibroblasts

Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

A reappraisal of the biological effects of hyaluronan on human dermal fibroblast

Hyaluronan (HA) is a fundamental component of many biomaterials. Reports on the biological effects of HA are, however, both inconsistent and contradictory. This particularly refers to fibroblast contraction in the presence of high concentrations of HA. In this study we used a broad range of molecular weight (MW) and concentrations of HA to assay its influence on human dermal fibroblasts in both two and three-dimensional culture models. High-MW HA in high concentrations demonstrated inhibitory effects on both cell attachment and proliferation. HA-coated surfaces appeared less adhesive to fibroblasts than collagen-coated surfaces. In the fibroblast-populated collagen lattice, HA supplementation dose-dependently retarded gel contraction, which was associated with reduced cell division and spreading. Although gene transcripts of collagen type I and III were not significantly upregulated in the monolayer culture as determined by real-time PCR analysis, more type III collagen was present in HA-incorporated collagen gels by Sirius red staining. Type III collagen was homogeneously distributed and well organized in the presence of HA. Our data indicates that HA has both a qualitative and quantitative effect on cell-matrix interactions. Optimum parameters in terms of MW and concentration need to be determined when HA-based biomaterials are being prepared for tissue-repair purposes.

A review of fibroblast‐populated collagen lattices

Bell's introduction of the fibroblast-populated collagen lattice (FPCL) has facilitated the study of collagen-cell interactions. As a result of the numerous modifications of the casting of FPCLs, the in vivo applications of these in vitro findings have been confusing. Here experimental FPCL contraction findings are viewed in regard to three proposed mechanisms responsible for lattice contraction. The cellular mechanisms responsible for generating FPCL contraction are cell contraction, cell tractional forces related to cell locomotion, and initial cell elongation and spreading.

Transplantation of Autologous Keratinocyte Suspension in Fibrin Matrix to Chronic Venous Leg Ulcers

BACKGROUND The transplantation of keratinocytes suspended in fibrin carrier represents a candidate regimen for chronic ulcer treatment in an outpatient setting. We evaluated the integration and survival of autologous individualized keratinocytes applied within fibrin matrix onto chronic venous leg ulcers in vivo. Parallel in vitro culture was used to validate keratinocyte survival and apoptosis in fibrin compared to collagen matrix carrier. METHODS Seven patients with chronic venous leg ulcers were transplanted with autologous keratinocytes suspended in fibrin sealant after isolation and expansion from full-skin biopsy. The fibrin carrier was removed in three patients after 7 days, whereas four patients served as control with fibrin remaining. In parallel in vitro cultures, primary keratinocyte movement in fibrin as well as viability in three-dimensional (3D) fibrin versus collagen lattices was examined. RESULTS Complete ulcer healing was observed in four of seven ulcers after a mean duration of 14.5 weeks. If the fibrin layer was removed, complete wound healing occurred in three of three patients, compared to one of four in the control group. In vitro, keratinocytes formed a monolayer underneath but remained isolated and nonmobile within the fibrin matrix, suggesting reepithelialization along the lower fibrin interphase. Keratinocyte culture in 3D fibrin at clinically used concentration (90 mg/mL) caused high levels of apoptosis, similar to 3D collagen, which was prevented by diluting fibrin concentration to 3 mg/mL. CONCLUSIONS Transplantation of autologous keratinocytes suspended in fibrin is efficient in the treatment of chronic venous leg ulcers. Due to an antimigratory and survival-compromising effect, the presently used fibrin carrier should be removed after a few days of transplantation.

Wound healing and antioxidant activities of extracts from Musa paradisiaca L. peel

The process of wound healing involves a variety of complex processes such as inflammation, cell proliferation and contraction of the collagen lattice formed. In addition, the healing process may be hampered by the presence of oxygen free radicals or microbial infection [1]. The process of wound healing is promoted by several plants natural products, which are composed of active principles such as triterpenes, alkaloids and biomolecules. These agents usually influence one or more phases of the healing process [2].