Negative pressure wound therapy reduces the motility of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and enhances wound healing in a rabbit ear biofilm infection model

Pseudomonas aeruginosa motility, virulence factors and biofilms are known to be detrimental to wound healing. The efficacy of negative pressure wound therapy (NPWT) against P. aeruginosa has been little studied, either in vitro or in vivo. The present study evaluated the effect of negative pressure (NP) on P. aeruginosa motility in vitro, and the effect of NPWT on virulence factors and biofilms in vivo. P. aeruginosa motility was quantified under different levels of NP (atmospheric pressure, ? 75, ? 125, ? 200 mmHg) using an in vitro model. Swimming, swarming and twitching motility were significantly inhibited by NP (? 125 and ? 200 mmHg) compared with atmospheric pressure (p = 0.05). Virulence factors and biofilm components were quantified in NPWT and gauze treated groups using a rabbit ear biofilm model. Biofilm structure was studied with fluorescence microscopy and scanning electron microscopy. Additionally, viable bacterial counts and histological wound healing parameters were measured. Compared with the control, NPWT treatment resulted in a significant reduction in expression of all virulence factors assayed including exotoxin A, rhamnolipid and elastase (p = 0.01). A significant reduction of biofilm components (eDNA) (p = 0.01) was also observed in the NPWT group. The reduction of biofilm matrix was verified by fluorescence- and scanning electron-microscopy. NPWT lead to better histologic parameters (p = 0.01) and decreased bacterial counts (p = 0.05) compared with the control. NPWT treatment was demonstrated to be an effective strategy to reduce virulence factors and biofilm components, which may explain the increased wound healing observed.     

Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l^?1 sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0–53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.     

A porcine model of skin wound infected with a polybacterial biofilm

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

Knockdown of heme oxygenase-2 impairs corneal epithelial cell wound healing

Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti‐oxidative and anti‐inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO‐2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO‐1 and HO‐2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO‐1 and HO‐2 proteins. Injury elicited a rapid and transient increase in HO‐1 and HO activity; HO‐2 expression was unchanged. Treatment with biliverdin or CORM‐A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM‐A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO‐2 expression, but not HO‐1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO‐2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin. J. Cell. Physiol. 226: 1732–1740, 2011. © 2010 Wiley‐Liss, Inc.     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.     

The Migratory Capacity of Human Trophoblastic BeWo Cells: Effects of Aldosterone and the Epithelial Sodium Channel

Aldosterone is a key regulator of the epithelial sodium channel (ENaC) and stimulates protein methylation on the β-subunit of the ENaC. We found that aldosterone (100 nM) promotes cellular migration in a wound-healing model in trophoblastic BeWo cells. Here, we tested if the positive influence of aldosterone on wound healing is related to methylation reactions. Cell migration and proliferation were measured in BeWo cells at 6 h, when mitosis is still scarce. Cell migration covered 12.4, 25.3, 19.6 and 45.1 % of the wound when cultivated under control, aldosterone (12 h), 8Br-cAMP and aldosterone plus 8Br-cAMP, respectively. Amiloride blocked the effects of aldosterone alone or in the presence of 8Br-cAMP on wound healing. Wound healing decreased in aldosterone (plus 8Br-cAMP) coexposed with the methylation inhibitor 3-deaza-adenosine (3-DZA, 12.9 % reinvasion of the wound). There was an increase in wound healing in aldosterone-, 8Br-cAMP- and 3-DZA-treated cells in the presence of AdoMet, a methyl donor, compared to cells in the absence of AdoMet (27.3 and 12.9 % reinvasion of the wound, respectively). Cell proliferation assessed with the reagent MTT was not changed in any of these treatments, suggesting that cellular migration is the main factor for reinvasion of wound healing. Electrophysiological studies showed an increase in ENaC current in the presence of aldosterone. This effect was higher with 8Br-cAMP, and there was a decrease when 3-DZA was present. AdoMet treatment partially reversed this phenomenon. We suggest that aldosterone positively influences wound healing in BeWo cells, at least in part through methylation of the ENaC.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Antimicrobial and antibiofilm potential of injectable platelet rich fibrin—a second-generation platelet concentrate—against biofilm producing oral staphylococcus isolates

Injectable Platelet rich fibrin (i-PRF) is a platelet concentrate that has been extensively used for multiple medical purposes and is a valuable adjunct for the regeneration of damaged tissues in surgical procedures. The enriched bioactive substances in i-PRF are responsible for speeding the wound healing process. Infection of biofilm producing bacteria in surgical wounds is becoming a serious threat. Research in this field is focused on new strategies to fight infections and to reduce the healing time. The present study was aimed to evaluate the in vitro antimicrobial and antibiofilm effects of i-PRF against oral pathogenic biofilm producing staphylococcus bacteria isolated from patient with dental and oral abscess. The antibacterial activity of i-PRF, was determined through broth microdilution as minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). i-PRF exhibited bactericidal activity against both non biofilm and biofilm producing bacteria. i-PRF could be potential antimicrobial peptide used to combat postoperative infections caused by biofilm producing staphylococcus.     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

Soft tissue fibroblasts from well healing and chronic human wounds show different rates of myofibroblasts in vitro

Due to an increasing life expectancy in western countries, chronic wound treatment will be an emerging challenge in the next decades. Because therapies are improving slowly appropriate diagnostic tools enabling the early prediction of the healing success remain to be developed. We used a well-established in vitro assay in combination with the analysis of 27 cytokines to discriminate between fibroblasts from chronic (n = 6) and well healing (n = 8) human wounds. Proliferation and migration of the cells as well as their response to hypoxia and their behaviour in co-culture with microvascular endothelial cells were analyzed. Myofibroblast differentiation, a time-limited essential process of regular wound healing, was also quantified. Besides weaker proliferation and migration significantly higher rates of myofibroblasts were detected in chronic wounds. With respect to the cytokine release, there was a clear trend within the group of chronic wound fibroblasts, which were releasing interferon-γ, monocyte chemotactic protein-1, granulocyte–macrophage colony stimulating factor and basic fibroblast growth factor in higher amounts than fibroblasts from healing wounds. Although the overall response of both groups of fibroblasts to hypoxia and to the contact with endothelial cells was similar, especially chronic wound fibroblasts seemed to benefit from the endothelial interaction during hypoxia and displayed better migration characteristics. The study shows (1) that the assay can identify specific features of fibroblasts derived from different human wounds and (2) that wound fibroblasts are varying in their response to the chosen parameters. Thus, current therapeutic approaches and individual healing prediction might benefit from this assay.     

Wound healing effects of methanol extract of Laurocerasus officinalis roem

Laurocerasus officinalis Roem. (syn: Prunus laurocerasus L.) is a member of Rosaceae family. We investigated the antimicrobial and antioxidant activity of L. officinalis Roem in wound healing both in vivo and in vitro using an excisional wound model model in mice. We used four groups of eight mice as follows: untreated (control), empty gel, extract + gel (L. officinalis + gel), and Madecassol® groups. All treatments were applied topically once daily. The scar area, percentage wound closure and epithelization time were measured. L. officinalis promoted wound healing and increased granulation tissue, epidermal regeneration and angiogenesis. L. officinalis extract, which is known for its antioxidant and antimicrobial activities, may be useful for promoting wound healing.     

An in vivo polymicrobial biofilm wound infection model to study interspecies interactions

Chronic wound infections are typically polymicrobial; however, most in vivo studies have focused on monospecies infections. This project was designed to develop an in vivo, polymicrobial, biofilm-related, infected wound model in order to study multispecies biofilm dynamics and in relation to wound chronicity. Multispecies biofilms consisting of both Gram negative and Gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multi-species biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with four bacterial species throughout the experiment. We observed that wounded mice given multispecies biofilm infections displayed a wound healing impairment over mice infected with a single-species of bacteria. In addition, the bacteria in the polymicrobial wound infections displayed increased antimicrobial tolerance in comparison to those in single species infections. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.     

A Novel Microbial Biofilm for Bioremoval of Nickel from Aqueous Media

This study evaluated the efficacy of a microbial biofilm in removing Ni ions in aqueous media. The biofilm was developed incorporating a garden soil fungus with a bacterium isolated from Ni-rich serpentinite soil. The biofilm was characterized using microscopy, scanning electron microscopy, Fourier transform infrared (FTIR) investigations, and Boehm and potentiometric titrations. Ni removal was determined using batch experiments as a function of pH, Ni concentration, and time. The adsorption isotherm assay was conducted with varying Ni concentrations from 25 to 500 mg/L for 4 days. Isotherm and kinetic modeling were applied to the experimental data to understand the mechanisms of Ni removal. The zero point charge at pH 4.5 indicated the pH values greater than 4.5 is favorable for Ni adsorption. Acidic nature of the biofilm was reflected from Boehm titration data showing higher number of acidic groups than basic groups. With the increase in initial Ni concentration, the uptake increased from 3.43 to 38.16 mg/g. Hill, the best-fitted isotherm model, indicated a maximum adsorption capacity of 165.37 mg/g. After 4 days, the adsorption rate reached an equilibrium with a maximum sorption of ～30 mg/g for an initial concentration of 100 mg/L. Kinetic model fitting with Power function further demonstrated the chemisorptive interaction of Ni with the biofilm surface. A clear involvement of functional groups of the biofilm in Ni bonding was observed from the attenuated total reflection (ATR)-FTIR spectrum. The microbial biofilm showed an efficient but slow removal of Ni from aqueous media.     

Genome analysis and clinical implications of the bacterial communities in early biofilm formation on dental implants restored with titanium or zirconia abutments

This cross-sectional study aimed to identify and quantify up to 42 target species colonizing the early biofilm of dental implants restored with titanium or zirconia abutments. A total of 720 samples from 20 healthy individuals were investigated. Biofilm samples were collected from the peri-implant sulci, inner parts of implants, abutment surfaces and prosthetic crowns over a functioning period of 30 days. Checkerboard DNA–DNA hybridization was used for microbial detection and quantitation. Clinical characteristics (probing depth, bleeding on probing, clinical attachment level and marginal bone loss) were also investigated during the monitoring period. Genome counts were low at the implant loading time point for both the abutment materials, and increased over time. Both the titanium and the zirconia groups presented similar microbial counts and diversity over time, and the microbiota was very similar to that colonizing the remaining teeth. Clinical findings were consistent with a healthy condition with no significant difference regarding marginal bone loss between the two materials.     

Terminal RFLP analysis to determine the oral microbiota with hyposalivation

Previous studies of oral microbiota by culture-dependent or targeted DNA approaches demonstrated that hyposalivation, a reduction in salivary secretions, might increase the amount of certain oral pathogens. However, the relationship between hyposalivation and the balance of oral microbiota, especially uncultivable bacteria, remains still unclear. The aim of this study was to elucidate the relationship between hyposalivation and oral microbiota by analyzing terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNA. The 61 subjects were divided into two groups, hyposalivation group and normo-salivation group. The microbiota of tongue-coating samples was analyzed by T-RFLP. The amount of saliva, the number of Candida albicans, and also the dental status including plaque index, gingival index, bleeding on probing, probing pocket depth and decayed, missing, and filled teeth (DMFT) were assessed. Regarding the dental status, none of the evaluated factors were significantly different between the groups except the number of DMFT. According to the T-RFLP profiles, the patterns of microbiota in the tongue coating were classified into two groups, Clusters I and II. Cluster I is made up 76 % of subjects with hyposalivation, while Cluster II is made up 61 % of subjects with normo-salivation (p < 0.001). Compared with the microbiota found in Cluster II, that in Cluster I had higher proportions of T-RFs corresponding to genera Veillonella, Dialister, Prevotella, Fusobacterium, and Streptococcus. T-RFLP analysis showed a significant role of salivary volume in determining the composition of the microbial community, regardless of the cultivability of the bacteria.     

Secretory leukocyte protease inhibitor is a novel inhibitor of fibroblast-mediated collagen gel contraction

Cultured epithelial cells, including those from the oral epithelium, have been successfully applied in the promotion of scarless wound healing. Factors released from the epithelial cells are thought to contribute significantly to the beneficial effects. In the conditioned medium of human oral epithelial cells, we found a factor that inhibited fibroblast-mediated collagen gel contraction, an in vitro model of wound healing and scar formation. Biochemical analysis identified the factor to be human secretory leukocyte protease inhibitor (SLPI). Fibroblasts transfected with SLPI cDNA showed reduced gel-contracting activity. SLPI purified from the conditioned medium inhibited gel contraction in a dose-dependent manner, and anti-SLPI antibody counteracted this activity. Upon SLPI treatment, human skin fibroblasts in collagen gel became shorter in length and were inhibited in pseudopodia extension. Furthermore, after SLPI treatment, alpha(1)-integrin immunoreactivity decreased, and cyclic AMP levels increased. Excessive gel contraction was observed when fibroblasts treated with TGF-beta1 and fibroblasts from hypertrophic and from keloid scar tissue were cultured in collagen gel. SLPI was also effective in inhibiting gel contraction in the above three models of scar formation. These results suggest that SLPI may be useful in promoting scarless wound healing.     

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.