Abstracts of Key Studies

  1. Analysis of morphological and immunohistochemical changes during treatment with DerMax.

    Karim RB, Brito BL, Dutrieux PR, Lassance FP, Hage JJ.
    Adv. Skin Care, 2006, 19(6): 324-327.

    Introduction:
    There are important differences between healing wounds and chronic wounds. In chronic wounds quiescent fibrocytes are observed, whereas in healing wounds extremely active fibrocytes can be easily found. Fibrocytes synthesize components of the extracellular matrix (ECM). One of the latest most important findings is the different expression in these two types of wounds of endo-peptidases called matrix metalloproteinases (MMPs). The MMPs are capable of degrading all the ECM components. During normal wound healing there is a balance between “construction” and "destruction" of ECM. In chronic wounds an imbalance is observed, with an elevated level of MMP-2, thereby favoring "destruction". DerMax® is a new device in the market that claims to decrease the production of MMP 2.

    Materials and Methods:

    Biopsies from chronic wounds were taken on day 0, and then after 2 weeks and 6 weeks of Dermax® treatment. We analyzed morphological changes in relation to fibrocytic MMP-2 expression during the healing process. Before treatment with Dermax®, fibroblast imbalance was observed, with a high level of MMP-2. Biopsies were performed in four chronic wounds during treatment with DerMax®. The samples were evaluated morphologically and submitted to immunocytochemical techniques using monoclonal MMP-2 antibodies (NeoMarkers).

    Results:

    During treatment with DerMax® the most striking (immuno-) histological changes were the loss of the fibro-necrotic cap covering the wound bed, the reactivation of the fibroblasts in the granulation tissue and the sharp decline in the expression of fibroblast MMP-2.

    Conclusion:

    In this study the application of DerMax® induced an improvement of wound healing both clinically and histologically. MMP-2-producing fibrocytes were substituted by fibroblasts producing ECM components, leading to wound healing.

  2. Poly Hydrated Ionogens Regulate Matrix Metalloproteinase Expression and the Production of Reactive Oxygen Species in Recalcitrant Wounds.

    Hoekstra MJ1, Pirayesh A2, Richters CD1, van den Berg AJJ3, Dutrieux RP.1
    1Burns Research Institute, Red Cross Hospital, Beverwijk, The Netherlands;
    2University Hospital Ghent, Plastic Surgery, Ghent, The Netherlands;
    3PhytogeniX, Faculty of Pharmacy, University of Utrecht, Utrecht, The Netherlands.

    Objective:

    Impaired healing in recalcitrant wounds is related to Matrix Metalloproteinase/Tissue Inhibitor of Metalloproteinases (MMP/TIMP) imbalance and protracted inflammation. Protracted inflammation is associated with release of Reactive Oxygen Species (ROS) including hydroxyl radicals, hypochloric acid and peroxynitrate. Poly Hydrated Ionogens (PHI) regulate protease imbalance, down-regulate ROS production and stimulate re-epithelialization.

    Methods:

    1. Experimental burns treated with carbodiimide (cross-linking agent) showed strong MMP-2 expression in three pigs. A topical ointment with a low pH in an inert synthetic carrier containing PHI (DerMax®) was applied daily. Contra-lateral "mirror image" wounds were treated with a placebo (ointment without PHI).

    2. MMP-2 was estimated qualitatively by immunohistochemical staining tissue biopsies of recalcitrant wounds.

    3. The influence of PHI on ROS production by granulocytes was estimated in vitro.

    Results:

    1. Non-healing burn wounds treated with PHI showed MMP-2 down-regulation in fibroblasts and re-epithelialization. There was no healing in placebo treated wounds.

    2. Recalcitrant wounds had a high expression of MMP-2 in fibroblasts and endothelium. Within two weeks of treatment MMP-2 expression was down-regulated and re-epithelialization initiated.

    3. In vitro ROS production of granulocytes was down-regulated.

    Conclusions:

    1. PHI influences MMP metabolism and induces re-epithelialization in recalcitrant wounds.

    2. Topical application of PHI controls MMP/TIMP imbalance, down-regulates ROS production and stimulates re-epithelialization.

  3. A Comparative Study of the Healing Effects of DerMax OBE and DerMax PHI-5 in Pigs.

    Hoekstra MJ.
    Burns Research Institute, Red Cross Hospital, Beverwijk, The Netherlands.

    Summary:
    In previous animal experiments, daily application of Dermax OBE ointment (botanical equivalent of Dermax®) clearly induced re-epithelialization in non-healing wounds, in contrast to contra-lateral placebo treated wounds.

    Also in human patients Dermax OBE induced epithelialization in therapy-resistant recalcitrant wounds. Additionally, in acute wounds in humans, as well as in pigs, an acceleration of epithelialization could be demonstrated.

    The burn wound model in the Yorkshire pig can be used for the macroscopic evaluation of differences in wound healing in a mirror image way. Differences in epithelialization, contraction and scar tissue formation can be observed clearly.

  4. A Novel Formulation of Metal Ions and Citric Acid Reduces Reactive Oxygen Species in vitro.

    van den Berg AJ, Halkes SB, van Ufford HC, Hoekstra MJ, Beukelman CJ. J. Wound Care, 2003, 12(10): 413-418.

    Objective:

    Reactive oxygen species (ROS), including superoxide anions, are thought to play an important role in impairing wound healing. Additionally, superoxide anions react with nitric oxide produced by macrophages to form peroxynitrite, another strong oxidant with detrimental effects on surrounding tissue. This in vitro study investigated whether samples of metal ions and citric acid are able to reduce levels of reactive oxygen species.

    Methods:

    Samples of materials were tested in assays for the following: inhibition of ROS production by human polymorphonuclear neutrophils (PMNs); antioxidant activity (scavenging of superoxide anions in a cell-free system); inhibition of human complement (limiting the generation of complement factors that attract and stimulate PMNs, thereby reducing levels ROS).

    Results:

    Metal ions were shown to inhibit both PMN production of reactive oxygen species and the activation of complement via the classical pathway,whereas citric acid was found to be a scavenger of superoxide anions.

    Conclusion:

    The beneficial effects of using formulations containing metal ions and citric acid on chronic wounds may be explained in part by a reduction of reactive oxygen species in these wounds.

    Summary:

    Although other factors governing wound healing (such as MMP activity) may also be important, these results show:

    1. that metal ions inhibited human complement activation via the classical pathway

    2. that metal ions inhibited the production of reactive oxygen species by activated PMNs

    3. that citric acid scavenged superoxide anions

    4. that incubation of PMNs in up to 100µl/ml PHI-5 did not have any cytotoxic effects. Unpublished data suggesting the beneficial effects of formulations containing metal ions and citric acid (DerMax®) on chronic ulcers may be explained in part by a reduction of ROS in these wounds.

    5. Polyhydrated Ionogens (PHI) Alters Patterns of Gene Expression in Normal and Diabetic Fibroblast Cultures.

      Schultz GS1, Sampson E1, Popp M1, Lobmann R1, Monroe S2.
      1Institute for Wound Research, University of Florida, Gainesville, Florida;
      2Greystone Pharmaceuticals, Inc.

      Introduction:

      Treatment of chronic wounds with DerMax® dressing was reported to improve healing. DerMax® dressing formulation contains a mixture of metal ions (polyhydrated ionogen, or PHI) and citric acid and was reported to reduce reactive oxygen species in cultures of polymorphonuclear leukocytes (van den Berg et al., 2003, J. Wound Care 12(10): 413-418). To further investigate the effect of PHI on gene expression in fibroblasts, cultures of dermal fibroblasts from normal and diabetic patients were incubated with concentrations of PHI that do not alter cell viability, both in the presence and absence of serum.

      Methods:

      Cultures of dermal fibroblasts were established from punch biopsies taken from the thigh of normal volunteers and from the base of chronic foot ulcers of diabetic patients. Cells were grown in defined culture medium (DMEM) supplemented with 10% calf serum.

      Microarray Analysis of Gene Expression in Fibroblasts Incubated with PHI:

      • In Experiment 1, cultures of diabetic fibroblasts were grown to initial confluency in complete medium containing 10% serum in T-75 culture flasks, the medium was removed and replaced by medium with low serum (0.125%), 1% BSA, 10 mg/ml LPS and 0.25% PHI, and the RNA was harvested 24 hours later.

      • In Experiment 2, cultures of normal and diabetic fibroblasts were grown to initial confluency in complete medium containing 10% serum in T-75 culture flasks. This initial medium was then removed and replaced with new medium containing 10% serum with or without PHI (0.125%). After 30 hours of incubation, total RNA was isolated using the RNeasy protocol (Qiagen, Inc., Valencia, CA). Aliquots of RNA were used as templates for cDNA synthesis with the Superscript Choice System kit (Invitrogen Life Technologies, Gaithersburg, MD).

      6. After hybridization, each array was stained with a streptavidin-phycoerythrin conjugate (Molecular Probes, Eugene, Oregon) and then washed and scanned with a Genearray Scanner (Agilent Technologies, Palo Alto, CA).

      Genes that were not expressed under any of the four experimental conditions (absent on all four chips) were removed from further analysis. Signal intensities for the remaining genes were variance normalized, and genes with the greatest variation (± 3 SD) in signal values were selected and normalized. Signal values were analyzed by both 2-way hierarchical clustering and K-Means cluster analysis.

      Results:
      Experiment 1:

      The Principal Component Analysis (PCA) for this data confirms that the gene expression changes are due to treatment of the diabetic dermal fibroblast cell cultures with PHI. PCA is a powerful, well-established technique for data reduction and visualization. Objects with similar patterns are plotted closest to each other.

      Experiment 2 - Effect of PHI on Fibroblast Viability in 10% Serum:

      Fibroblasts from a diabetic patient or normal volunteer were seeded into 96 well plates and cultured with PHI at the indicated concentrations for 48 hours. Then MTT was added and the numbers of cells were measured by absorbance at 490 nm. PHI showed no toxicity at 0.5% or lower concentrations.

      Conclusions:
      • PHI induced substantial changes in patterns of gene expression in fibroblast cultures from normal and diabetic patients at concentrations that do not alter cell viability
      • Microarray analysis of biopsies from chronic wounds treated with PHI will further identify changes in gene expression produced by PHI