Abstracts of References
1. Analysis of morphological and immunohistochemical changes during treatment with DerMax - F.P. Lassance

Introduction
There are important differences between healing and chronic wounds. In chronic wounds fibrocytes called quiescents are observed, in healing wounds extremely active can be easily found. One of the latest most important findings is the different expression of endo-peptidases called matrix metalloproteineases (MMPs). The MMPs are capable to degrade all the extracellular matrix (ECM) components. During a normal wound healing there is a balance between “construction” and "destruction" of ECM. In chronic wounds an imbalance is observed, with a high level of MMP-2. DerMax® is a new device in the market that claims to decrease the production of MMP 2. Biopsies from chronic wounds were taken in day 0, 2 weeks and 6 weeks. We made an analysis of the morphological changes in comparison with fibrocytes MMP-2 expression during the healing process.

Materials and Methods
Biopsies from chronic wounds were taken during day 0, after 2 weeks and 6 weeks. We made an analysis of the morphological changes in comparison with fibrocytes MMP-2 expression during the healing process. Fibroblasts imbalance is observed, with a high level of MMP-2. Biopsies were performed in 4 chronic wounds during treatment with DerMax®. The samples were evaluated morphologically and submitted to immunocytochemical techniques using monoclonal MMP-2antibodies (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. Fibrocytes MMP-2 producers were substituted by fibroblasts ECM components producers, leading to the wound healing.

F.P. Lassance, biologist, PhD. , R.B.Karim, MD PhD. , B.L.R.Brito, MD, J.Raaijmakers, MD , J.Joris Hage, MD Ph.D., Department of Plastic and Reconstructive Surgery OLVG Hospital, Amsterdam, The Netherlands
2. Poly Hydrated Ionogens regulate Matrix Metalloproteinases expression and reactive oxygen species production in recalcitrant wounds – M.J. Hoekstra

Objective
Impaired healing in recalcitrant wounds is related to MMP/TIMP imbalance and protracted inflammation. Protracted inflammation is associated with release of hydroxyl radicals, hypochloric acid and peroxynitrate (ROS). 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 metal ions).
  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 again. 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 PI-U caB influence MMP metabolism and induce reepithelialization in recalcitrant wounds. Topical application of PHI caB control MMP/TIMP imbalance, down-regulate ROS production and stimulate re-epithelialization.

M J Hoekstra l, A Pirayesh2, C D Richters3, A J J van den Berg\ R P Dutrieux5
1 Dutch Burns Foundation, Research Department, Beverwijk, The Netherlands,
2 Univ. Hospital Ghent, Plastic Surgery, Ghent, The Netherlands,
3 Dutch Burns Foundation, Euro Skin Bank, Beverwijk, The Netherlands, CD.Richters.Cell@med.vu.nl;
4 PhytogeniX, Fac. Farmacy Univ. Utrecht, Utrecht, The Netherlands, a.j.j.vandenberg@phytogenix.uu.nl;
5 Dutch Burns Foundation, Research Department, Beverwijk, The Netherlands, +31206346614, +31206346730, dutrieux@xs4all.nl
3. Summary of a comparing study on the healing effects of DerMax OBE and DerMax PHI-5; a Pig study – M.J. Hoekstra

In previous animal experiments with daily application of OBE ointment (botanical equivalent of PHI-5) clearly induced re-epithelialization in non-healing wounds in contrast to contra-lateral placebo treated wounds.

Also in human patients induction of epithelialization could be observed in therapy-resistant recalcitrant wounds. In acute wounds in humans as well as pigs an acceleration of epithelialization could be demonstrated also.

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 - A.J.J. van den Berg

Objective
Reactive oxygen species, 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.

Method
Samples of materials were tested in assays for the following: inhibition of reactive oxygen species 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 of reactive oxygen species).

Results
Metal ions were shown to inhibit both PMN production of reactive oxygen species and the activation of complement via the classical pathway,where as 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 may also be important, such as MMPs, these results show that metal ions inhibited human complement activation via the classical pathway and inhibited production of reactive oxygen species by activated PMNs, and that citric acid scavenged superoxide anions. In addition, incubation of PMNs in up to 100µl/ml MI-CA5 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 in part explained by a reduction of reactive oxygen species in these wounds.

A.J.J. van den Berg,1PhD, Associate Professor; S.B.A. Halkes,1 PhD, Senior Researcher; H.C. Quarles van Ufford,1 Senior Technician; M.J. Hoekstra,2 MD, Chief of Research; C.J. Beukelman,1 PhD, Associate Professor;
1 Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Utrecht University,The Netherlands;
2 Burns Research Institute, Red Cross Hospital, Beverwijk,The Netherlands. Email:A.J.J.vandenBerg@ pharm.uu.nl
5. G.S.Schultz PhD, E.Sampson, M.Popp, R.Lobmann, S. Monroe, Polyhydrated Inogens (PHI) alters patterns of gene expression in normal and diabetic fibroblast cultures, Institute for Wound Research, University of Florida, Gainesville, USA.

Introduction
Treatment of chronic wounds with DerMax® dressing was reported to improve healing. DerMax® dressing formulation contains citric acid and a mixture of metal ions (polyhydrated ionogen, or PHI) and was reported to reduce reactive oxygen species in cultures of polymorphonuclear leukocytes (J Wound Care 12:10, 2003). 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.

Materials and 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%), BSA 1%, with 10 mg/ml LPS and 0.25% PHI, and RNA 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, medium was removed and replace with medium containing 10% serum with/out PHI at 0.125% PHI. 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).

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

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

Results
Experiment one
The Principal Component Analysis (PCA) for this data confirms that the gene expression changes are due to treatment of the diabetic dermal fibroblasts 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 two
Effect of PHI on Fibroblast Viability in 10% Serum
Fibroblasts from a diabetic patient or normal volunteer were seeded into 96 well plate 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