Wound healing capacity of using mesenchymal stem cell-derived exosomes originated from human adipose tissue and cold atmospheric plasma.

Background: Recently, cold atmospheric plasma (CAP), as well as adipose mesenchymal stem cells derived exosomes (ADMSCs-EX), have been applied separately to wound healing treatment. However, no study has investigated the additive effect on the healing mechanism of these two methods in the same skin lesion treatment model. Aim: We conduct this study to describe the results of using CAP and human ADMSCs-EX on in vitro wound healing. Methods: Exosomes were isolated from donor adipose tissue samples by ultracentrifugation method, characterized by transmission electron microscopy (TEM) and Western blot. Assessment in vitro wound healing on proliferation and migration evaluation experiments on human fibroblasts with culture medium supplemented with 10µg total exosomal proteins/1 mL and irradiated with CAP with an intensity of 30 seconds/cm2. Results: Experimental results to evaluate the ability to stimulate fibroblast migration, showed that cell migration speed in the group supplemented with ADMSCs-EX was equivalent to the group with a combination of CAP and ADMSCs-EX and had the highest rate with 87.8 ± 4.2 % and 84.4 ± 5.3 % while in the control group it was the lowest with 61.9 ± 11.4% (p<0.05). The group supplemented with CAP gave fibroblast proliferation and migration results similar to the control group (p>0.05), showing the safety of CAP with the growth of the cells. Conclusions: Therefore, in animal models, we intend to use a combination of these two therapies by using ADMSCs-EX injection therapy into the dermis at the wound edge to avoid the impact of CAP affecting the cell proliferation.

In-vitro cell migration enhancing constituents from Decalepis hamiltonii, a plant used in the preparation of 'Pinda oil', a medicinal oil used in Ayurveda for wound management.

Decalepis hamiltonii, Wight & Arn. (Apocyanaceae) is a one of the raw materials used in the preparation of 'Pinda oil', a medicinal oil which is used for treatment of wounds in Ayurveda. Of the hexanes, dichloromethane, and ethyl acetate extracts derived from the plant raw materials used to prepare 'Pinda oil', the hexanes extract of D. hamiltonii exhibited the highest mean percentage wound closure (75.1 ± 2.9) compared to the negative controls (1% DMSO in DMEM, 4.2 ± 1.2 and 100% DMEM, 4.1 ± 0.9) in the scratch wound assay (SWA). Fractionation of the hexanes extract of stem of D. hamiltonii led to the isolation of 2-hydroxy-4-methoxybenzaldehyde (1) and a mixture of long chain esters of lupeol (2), which showed enhanced cell migration in SWA. It was observed that the esters of lupeol bind to the cell membrane and/or enter the cells during the SWA. It was found that these constituents are also present in 'Pinda oil' which may contribute to the enhancement of wound healing activity of 'Pinda oil'.

Transglutaminase 2 Facilitates Murine Wound Healing in a Strain-Dependent Manner.

Transglutaminase 2 (TG2) plays a role in cellular processes that are relevant to wound healing, but to date no studies of wound healing in TG2 knockout mice have been reported. Here, using 129T2/SvEmsJ (129)- or C57BL/6 (B6)-backcrossed TG2 knockout mice, we show that TG2 facilitates murine wound healing in a strain-dependent manner. Early healing of in vivo cutaneous wounds and closure of in vitro scratch wounds in murine embryonic fibroblast (MEF) monolayers were delayed in 129, but not B6, TG2 knockouts, relative to their wild-type counterparts, with wound closure in 129 being faster than in B6 wild-types. A single dose of exogenous recombinant wild-type TG2 to 129 TG2-/- mice or MEFs immediately post-wounding accelerated wound closure. Neutrophil and monocyte recruitment to 129 cutaneous wounds was not affected by Tgm2 deletion up to 5 days post-wounding. Tgm2 mRNA and TG2 protein abundance were higher in 129 than in B6 wild-types and increased in abundance following cutaneous and scratch wounding. Tgm1 and factor XIIA (F13A) mRNA abundance increased post-wounding, but there was no compensation by TG family members in TG2-/- relative to TG2+/+ mice in either strain before or after wounding. 129 TG2+/+ MEF adhesion was greater and spreading was faster than that of B6 TG2+/+ MEFs, and was dependent on syndecan binding in the presence, but not absence, of RGD inhibition of integrin binding. Adhesion and spreading of 129, but not B6, TG2-/- MEFs was impaired relative to their wild-type counterparts and was accelerated by exogenous addition or transfection of TG2 protein or cDNA, respectively, and was independent of the transamidase or GTP-binding activity of TG2. Rho-family GTPase activation, central to cytoskeletal organization, was altered in 129 TG2-/- MEFs, with delayed RhoA and earlier Rac1 activation than in TG2+/+ MEFs. These findings indicate that the rate of wound healing is different between 129 and B6 mouse strains, correlating with TG2 abundance, and although not essential for wound healing, TG2 facilitates integrin- and syndecan-mediated RhoA- and Rac1-activation in fibroblasts to promote efficient wound contraction.

Effects of heated tobacco products and conventional cigarettes on dental implant wound healing: experimental research.

Smoking affects wound healing and is associated with dental implant failure. Heated tobacco products (HTPs) appear to be less harmful than conventional cigarettes (CCs); however, there is limited analytical data to support this claim. This study aimed to compare HTPs and CCs for their impact on wound healing using L929 mouse fibroblast cells and evaluate whether HTPs also lead to failure in implant therapy. Materials and methods: Cigarette smoke extract (CSE) was obtained from CCs (Marlboro, Philip Morris) and HTPs (Marlboro Heat Sticks Regular for IQOS, Philip Morris) and initiated a wound-healing assay with a cell-free area created in the centre of a titanium plate by sticking a 2-mm-width line tape. The L929 mouse fibroblast cells were exposed with 2.5 and 5% CSE from HTPs and CCs and then seeded in the titanium plate. A scratch wound-healing assay was initiated when all samples were at 80% confluence. The number of cells migrating to the wound site was counted after 12, 24, and 48 h. Results: Cell migration decreased after CSE exposure from both CCs and HTPs. At each time-point with 2.5% CSE, cell migration in the HTP group was less than that of the CC group. There were significant differences between the 2.5% CC and 2.5% HTP groups and the 5% CC and 5% HTP groups after 24 h. HTPs and CCs had similar effects in the wound-healing assay. Conclusion: Therefore, HTP use may be a risk factor for poor dental implant healing.

Proline conjugated chitosan as wound healing material: In vitro studies on the influence of the scaffold on collagen production and wound healing.

The present study reports the development of L-proline conjugated chitosan scaffold for wound healing application. Proline plays a beneficial role in collagen synthesis, and as a biochemical, it has the potential to modulate wound healing. In this regard, amino acid L-proline was conjugated onto chitosan, and the scaffolds were synthesised. FTIR and NMR analysis confirmed amino acid conjugation. The prepared scaffold was characterized by studies such as swelling, dissolution, tensile strength, porosity, water-vapor transmission rate and in-vitro healing properties. Cell viability assay showed that the scaffold has no cytotoxicity against the L929 and HaCaT cells. The in-vitro wound healing potential of the scaffold by scratch wound assay on the L929 cell line showed 53.35 ± 2.3 %, 72.96 ± 2.2 %, and 50.89 ± 0.3 % wound closure for CS-P 200, CS-P 400 and CS-P 600, respectively when compared to native CS scaffold (38.86 ± 1.6 %). A similar observation was found with HaCaT cells too. The studies showed that the modified scaffold promotes collagen deposition from fibroblast cells. These findings suggest that scaffold cues remodel the wound microenvironment for a better wound-healing state, and the L-proline conjugated scaffold may have considerable potential as a wound dressing to improve wound healing.

Reassignment of NMR spectroscopic data of oleana-9(11),12-diene-3ß-ol isolated from Jeffreycia zeylanica and its wound healing potential in terms of cell migration and proangiogenic activity1.

Jeffreycia zeylanica (Asteraceae), a plant endemic to Sri Lanka, is used for the treatment of wounds. The scratch wound assay (SWA) guided fractionation of hexanes extract of J. zeylanica led to the isolation of oleana-9(11),12-diene-3ß-ol (1) which showed enhanced cell migration in SWA and significant proangiogenic response in chorioallantoic membrane (CAM) assay. Since the reported 1H NMR assignments of 1 were incomplete, and some 13C NMR assignments were inconsistent with our observations, reassignment of NMR spectroscopic data of 1 was carried out. Herein we report unambiguous assignment of NMR data of 1 based on 1D and 2D NMR spectra. This is the first report of 1 in J. zeylanica.

Analysis of mRNA Subcellular Distribution in Collective Cell Migration.

The movement of groups of cells by collective cell migration requires division of labor between group members. Therefore, distinct cell identities, unique cell behaviors, and specific cellular roles are acquired by cells undergoing collective movement. A key driving force behind the acquisition of discrete cell states is the precise control of where, when, and how genes are expressed, both at the subcellular and supracellular level. Unraveling the mechanisms underpinning the spatiotemporal control of gene expression in collective cell migration requires not only suitable experimental models but also high-resolution imaging of messenger RNA and protein localization during this process. In recent times, the highly stereotyped growth of new blood vessels by sprouting angiogenesis has become a paradigm for understanding collective cell migration, and consequently this has led to the development of numerous user-friendly in vitro models of angiogenesis. In parallel, single-molecule fluorescent in situ hybridization (smFISH) has come to the fore as a powerful technique that allows quantification of both RNA number and RNA spatial distribution in cells and tissues. Moreover, smFISH can be combined with immunofluorescence to understand the precise interrelationship between RNA and protein distribution. Here, we describe methods for use of smFISH and immunofluorescence microscopy in in vitro angiogenesis models to enable the investigation of RNA and protein expression and localization during endothelial collective cell migration.

In-vitro cell migration enhancing and pro-angiogenic active secondary metabolites from Jeffreycia zeylanica (L.) H. Rob., S.C. Keeley & Skvarla (Asteraceae).

Jeffreycia zeylanica (L.) H. Rob., S. C. Keeley & Skvarla is used for the treatment of wounds in indigenous medicine practiced in Sri Lanka. The scratch wound assay (SWA) guided fractionation of hexanes extract of J. zeylanica led to the isolation of ethuliacoumarin (1), stigmasterol (2), ß-amyrin (3) and lupeol (4) and a non-resolved triterpene alcohol mixture HF5D1, all of which showed enhanced cell migration. The mixture HF5D1 contained glut-5-en-3ß-ol (5) and friedelin-3ß-ol (6). The identities of compounds 1-6 were established by the analysis of spectroscopic data and comparison of them with those reported. The compounds 1-4 and the non-resolved triterpene alcohol mixture, HF5D1 also exhibited significant proangiogenic response in chorioallantoic membrane (CAM) assay in addition to the enhanced cell migration. This is the first report of the occurrence of the compounds 1, 2, 4 and 5 in this plant.

Tenascin-C fibronectin D domain is involved in the fine-tuning of glial response to CNS injury in vitro.

Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an in vitro injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1ß upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of ß1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.

A review of diabetic wound models-Novel insights into diabetic foot ulcer.

Diabetic foot ulcer (DFU) is a major debilitating complication of diabetes. Many research investigations have been conducted with the aims to uncover the diabetic wound healing mechanisms, develop novel therapeutics, and screen bioactive wound dressings in order to improve the current management of DFU. These would have not been possible without the utilization of an appropriate wound model, especially in a diabetic wound context. This review focuses on the different in vitro research models used in DFU investigations such as the 2D scratch wound assay, 3D skin model, and 3D angiogenesis model as well as their limitations. The current efforts and challenges to apply the 2D and 3D in vitro models in a hyperglycemic context to provide insights into DFU modeling will be reviewed. Perspectives of utilizing 3D bioprinting and skin-on-the-chip model as a diabetic wound model in the future will also be highlighted. By leveraging knowledge from past experiences and current research, an improved experimental model for DFU is anticipated to be established in near future.

Modeling and Predicting the Cell Migration Properties from Scratch Wound Healing Assay on Cisplatin-Resistant Ovarian Cancer Cell Lines Using Artificial Neural Network.

The study of artificial neural networks (ANN) has undergone a tremendous revolution in recent years, boosted by deep learning tools. The presence of a greater number of learning tools and their applications, in particular, favors this revolution. However, there is a significant need to deal with the issue of implementing a systematic method during the development phase of the ANN to increase its performance. A multilayer feedforward neural network (FNN) was proposed in this paper to predict the cell migration assay on cisplatin-sensitive and cisplatin-resistant (CisR) ovarian cancer (OC) cell lines via scratch wound healing assay. An FNN training algorithm model was generated using the MATLAB fitting function in a MATLAB script to accomplish this task. The input parameters were types of cell lines, times, and wound area, and outputs were relative wound area, percentage of wound closure, and wound healing speed. In addition, we tested and compared the initial accuracy of various supervised learning classifier and support vector regression (SVR) algorithms. The proposed ANN model achieved good agreement with the experimental data and minimized error between the estimated and experimental values. The conclusions drawn demonstrate that the developed ANN model is a useful, accurate, fast, and inexpensive method to predict cancerous cell migration characteristics evaluated via scratch wound healing assay.

Cytocompatibility Properties of an Herbal Compound Solution Support I n vitro Wound Healing.

The aim of this study was to evaluate the cytocompatibility of an herbal extract compound oral rinse [StellaLife VEGA (SLife)] against relevant human cellular models of oral surgical wound healing. SL was compared to the gold standard for peri-/post-operative oral surgical use, i.e., Chlorhexidine (CHX) and to a commonly utilized essential-oil (EO) based antiseptic rinse. Fibroblasts and primary oral stem cells of the apical papilla (SCAPs) were employed to assess its comparative cytotoxicity to the active comparator antiseptic rinses and its effects on wound healing in vitro. In cytotoxicity assays, multiple timepoints were tested ranging from clinically relevant of 60-s rinsing to protracted challenge of up to 5 min, to determine dose-dependent toxicity. The SLife group consistently demonstrated minimal cytotoxicity as compared to active comparators across experimental timepoints and different cells lines. At concentrations up to 20% v/v SLife-challenged fibroblasts and SCAPs demonstrated no significant toxicity as compared to unstimulated controls (p > 0.05). When assessing wound healing, a scratch wound assay revealed significantly accelerated cell migration for SLife as compared to CHX (p < 0.05). Notably, all active comparator antiseptic rinses affected wound healing responses by significantly reducing total collagen deposition after intermittent "rinsing" intervals that simulated post-surgical oral rinsing. Nonetheless, intermittent as well as continuous challenge of cells with SLife had a positive effect in functional collagen assays. An herbal extract compound-based oral rinse was found to be cytocompatible to cells critical to oral wound healing and to promote fibroblast migration and differentiation, contrary to existing antiseptic rinses that lack selective cytotoxicity.

Extracellular matrix influences astrocytic extracellular vesicle function in wound repair.

Astrocytic injury responses are known to be influenced by the extracellular matrix (ECM). Astrocytes are also recognized as a source of extracellular vesicles (EVs) that can impact the activity and function of other astrocytes and cell types. Whether the ECM influences the function of astrocytic EVs in the context of wound recovery has not been previously studied. We report EVs from astrocytes cultured on varied ECM substrates are sufficient to elicit distinct injury responses in naive astrocytes that recapitulate the effects of the ECM of origin. When compared with wound recovery on control substrates, EVs from ECM-exposed astrocytes elicited accelerated rates of wound recovery that varied based on each ECM. When EVs were collected from IL-1ß treated and ECM-exposed astrocyte cultures, we found that IL-1ß arrested wound recovery in naive astrocytes treated with EVs from astrocytes cultured on ECM but adding EVs from IL-1ß treated Tenascin-c-cultured astrocytes increased wound recovery. To confirm that ECM was a primary influence on these astrocytic EV functions, we tested the contribution of ß1-integrin, a major integrin receptor for the ECM molecules tested in this study. We found that the ß1-integrin inhibitor Ha2/5, resulted in EVs that significantly attenuated the wound recovery of naive astrocytes. This provides new information on the importance of culture substrates on astrocytic responses, EV functions and injury responses that may impact the understanding of astroglial responses related to ECM compositional differences in diverse physiological states.

Perfusion Flow Enhances Viability and Migratory Phenotype in 3D-Cultured Breast Cancer Cells.

Conventional 2D cell culture, a traditional tool in pre-clinical studies, can hardly be regarded as a representation of a natural cell microenvironment. In this respect, it might result in altered cellular behaviors. To overcome such a limitation, different approaches have been tested to conduct more representative in vitro studies. In particular, the use of 3D cell culture introduces variables, such as cell-cell and cell-extracellular matrix interactions; cell features such as survival, proliferation and migration are consequently influenced. For an example, an enhanced drug resistance and increased invasiveness are shown by cancer cells when cultured in 3D versus 2D conventional culture models. In this setting however, non-uniform cell distribution and biological behaviors appear throughout the scaffold, due to reduced diffusion of oxygen and nutrients. Perfusion in bioreactor systems can be used to improve medium transport. In this line of reasoning, this study proposes a breast cancer cell culture model sustained by an integrated approach that couples a 3D environment and a fluid perfusion. This model improves viability and uniformness of cell distribution, while inducing morphological, functional and molecular cancer cell remodeling.

A novel system for dynamic stretching of cell cultures reveals the mechanobiology for delivering better negative pressure wound therapy.

Serious wounds, both chronic and acute (e.g., surgical), are among the most common, expensive and difficult-to-treat health problems. Negative pressure wound therapy (NPWT) is considered a mainstream procedure for treating both wound types. Soft tissue deformation stimuli are the crux of NPWT, enhancing cell proliferation and migration from peri-wound tissues which contributes to healing. We developed a dynamic stretching device (DSD) contained in a miniature incubator for applying controlled deformations to fibroblast wound assays. Prior to the stretching experiments, fibroblasts were seeded in 6-well culture plates with elastic substrata and let to reach confluency. Squashing damage was then induced at the culture centers, and the DSD was activated to deliver stretching regimes that represented common clinical NPWT protocols at two peak strain levels, 0.5% and 3%. Analyses of the normalized maximal migration rate (MMR) data for the collective cell movement revealed that for the 3% strain level, the normalized MMR of cultures subjected to a 0.1 Hz stretch frequency regime was ~ 1.4 times and statistically significantly greater (p < 0.05) than that of the cultures subjected to no-stretch (control) or to static stretch (2nd control). Correspondingly, analysis of the time to gap closure data indicated that the closure time of the wound assays subjected to the 0.1 Hz regime was ~ 30% shorter than that of the cultures subjected to the control regimes (p < 0.05). Other simulated NPWT protocols did not emerge as superior to the controls. The present method and system are a powerful platform for further revealing the mechanobiology of NPWT and for improving this technology.

Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation.

This chapter focuses on the in vitro biological evaluation of multisensitive nanocontainers as drug delivery systems for cancer treatment. Cancer tissues possess some unique characteristics such as increased temperature due to inflammation, thermal vulnerability (40-45 °C), low cellular pH, and redox instabilities. The employment of polymers bearing pH, thermo, and/or redox sensitivities in the synthesis of hollow polymeric nanostructures has led to the formulation of a variety of drug delivery vehicles that are capable of targeted delivery and trigger specific drug release. The cavity in the structure allows for the encapsulation of anticancer drugs as well as other moieties with anticancer activity, like iron oxide magnetic nanoparticles. The drug loading and release capability of the nanocontainers is evaluated prior to biological studies in order to determine the concentration of the drug in the structure. The in vitro assessment includes cytotoxicity studies, quantitatively through the colorimetric MTT assay as well as qualitatively via the scratch-wound healing assay, on both cancer and healthy cell lines. The cellular localization of the studied drug-loaded and unloaded nanocontainers is determined through confocal fluorescence microscopy.

Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.

The migration of epidermal keratinocytes is the basis for skin reepithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact on results.

Electroporation does not affect human dermal fibroblast proliferation and migration properties directly but indirectly via the secretome.

Aesthetic wound healing is often experienced by patients after electrochemotherapy. We hypothesized that pulsed electric fields applied during electrochemotherapy (ECT) or gene electrotransfer (GET) protocols could stimulate proliferation and migration of human cutaneous cells, as described in protocols for electrostimulation of wound healing. We used videomicroscopy to monitor and quantify in real time primary human dermal fibroblast behavior when exposed in vitro to ECT and GET electric parameters, in terms of survival, proliferation and migration in a calibrated scratch wound assay. Distinct electric field intensities were applied to allow gradient in cell electropermeabilization while maintaining reversible permeabilization conditions, in order to mimic in vivo heterogeneous electric field distribution of complex tissues. Neither galvanotaxis nor statistical modification of fibroblast migration were observed in a calibrated scratch wound assay after application of ECT and GET parameters. The only effect on proliferation was observed under the strongest GET conditions, which drastically reduced the number of fibroblasts through induction of mitochondrial stress and apoptosis. Finally, we found that 24 h-conditioned cell culture medium by electrically stressed fibroblasts tended to increase the migration properties of cells that were not exposed to electric field. RT-qPCR array indicated that several growth factor transcripts were strongly modified after electroporation.

Bioassay-guided fractionation and identification of wound healing active compound from Pistacia vera L. hull extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Pistachio hull has traditionally been used to treat peptic ulcer, hemorrhoids, oral and cutaneous wounds. AIM OF THE STUDY: On the basis of its traditional uses and previous pharmacological reports, a bioassay guided fractionation procedures on pistachio (Pistacia vera L.) hulls was performed to define the fractions and bioactive compound that are responsible for wound healing activity of hulls. MATERIAL AND METHODS: A bioassay-guided fractionation of the total extract (MeOH 80%) of Pistacia vera L. hulls was carried out to evaluate wound healing activity by scratch assay on NIH/3T3 murine fibroblast cells. A combination of solvent-solvent partitioning, column chromatography, preparative thin layer chromatography and crystallization were used to obtain fractions/sub-fractions and pure compound. The wound healing potential of isolated compound was examined by fibroblasts migration and proliferation using scratch assay and CFSC dilution assay, respectively. In addition, we evaluated the gene expression of some inflammatory markers which are involved in healing process using Real Time PCR. Chemical structure of active compound was elucidated by spectrometric methods. RESULTS: Due to the higher wound healing activity of CHCl3 fraction from P. vera hulls, it was fractionated by successive chromatographic techniques to yield the active compound. 3-Epimasticadienolic acid was isolated and crystallized as a white powder. This active compound (200 µg/ml) significantly increased the fibroblast proliferation and migration, resulting in reduction of the scratch area about 45%. It showed a strong inhibitory effect on gene expression of IL-6 and TNF-α, and a stimulation effect on NF-κB gene expression at the same dose. CONCLUSION: The present study supported the traditional uses of P. vera hulls for wound-healing and 3-epimasticadienolic acid showed significantly potent on wound repair.

Scratch Wound Healing Assay.

Cell migration is a crucial step for wound healing. Assays able to evaluate cell migration are very useful to evaluate in vitro wound healing. Scratch wound assay creates a gap in confluent monolayer of keratinocytes to mimic a wound. The protocol of scratch wound is based on few steps: cell culture preparation, scratch wound assay, data acquisition, and data analysis.