Evert N. Lamme

Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation.

Abstract Patients with diabetes mellitus experience impaired wound healing often resulting in chronic foot ulcers. Hospital discharge data indicate that 6–20% of all diabetic individuals hospitalized (mostly with type 2 diabetes) have a lower extremity ulcer. Maintaining glucose levels at acceptable levels (below 10 mmol/l) is considered to be an important part of the clinical treatment, but the exact mechanism by which diabetes delays wound repair is not yet known. We studied this phenomenon by determining the potential of fibroblasts isolated from the ulcer sites of four patients with non-insulin-dependent diabetes mellitus to proliferate in vitro. Controls were fibroblasts isolated from normal skin of the upper leg of five healthy age-matched volunteers and of six non-insulin-dependent diabetes patients. Proliferative capacity was analysed by evaluation of plates after trypsinization and [ 3 H]thymidine incorporation. Fibroblast morphology was studied by light and transmission electron microscopy. Diabetic ulcer fibroblasts, measured by [ 3 H]thymidine incorporation, proliferated significantly more slowly than the nonlesional control fibroblasts ( P < 0.00047) and age-matched control fibroblasts ( P < 0.00003). After culturing the fibroblasts for a prolonged period in high-glucose (27.5 m M ) and low-glucose (5.5 m M , i.e. physiological) medium, this difference in proliferation rate between diabetic ulcer fibroblasts and nonlesional diabetic fibroblasts remained ( P < 0.0001 for high-glucose and P < 0.0009 for low-glucose on day 7). Fibroblast proliferation in all three groups was slightly lower in high-glucose than in low-glucose medium, although not significantly at any time-point. Light microscopy showed diabetic ulcer fibroblasts to be large and widely spread. Transmission electron microscopy of cultured diabetic ulcer fibroblasts and nonlesional diabetic skin fibroblasts revealed a large dilated endoplasmic reticulum, a lack of microtubular structures and multiple lamellar and vesicular bodies. These results show a diminished proliferative capacity and abnormal morphology of fibroblasts derived from diabetic ulcers of non-insulin-dependent diabetes patients.

Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation

Cultured skin substitutes are increasingly important for the treatment of burns and chronic wounds. The role of fibroblast numbers present in a living‐skin equivalent is at present unknown. The quality of dermal tissue regeneration was therefore investigated in relation to the number of autologous fibroblasts seeded in dermal substitutes, transplanted instantaneously or precultured for 10 days in the substitute. A full‐thickness porcine wound model was used to compare acellular dermal substitutes (ADS) with dermal substitutes seeded with fibroblasts at two densities, 1×105 (0‐DS10) and 5×105 cells/cm2 (0‐DS50), and with dermal substitutes seeded 10 days before operation at the same densities (10‐DS10 and 10‐DS50) (n=7 for each group, five pigs). After transplantation of the dermal substitutes, split‐skin mesh grafts were applied on top. Wound healing was evaluated blind for 6 weeks. Cosmetic appearance was evaluated and wound contraction was measured by planimetry. The wound biopsies taken after 3 weeks were stained for myofibroblasts (α‐smooth muscle actin), and after 6 weeks for scar tissue formation (collagen bundles organized in parallel and the absence of elastin staining). Collagen maturation was investigated with polarized light. For wound cosmetic parameters, the 10‐DS50 and 0‐DS50 treatments scored significantly better than the ADS treatment, as did the 10‐DS50 treatment for wound contraction (p<0.05, paired t‐test). Three weeks after wounding, the area with myofibroblasts in the granulation tissue, determined by image analysis, was significantly smaller for 0‐DS50, 10‐DS10, and 10‐DS50 than for the ADS treatment (p<0.04, paired t‐test). After 6 weeks, the wounds treated with 0‐DS50, 0‐DS10, and 10‐DS50 had significantly less scar tissue and significantly more mature collagen bundles in the regenerated dermis. This improvement of wound healing was correlated with the higher numbers of fibroblasts present in the dermal substitute at the moment of transplantation. In conclusion, dermal regeneration of experimental full‐skin defects was significantly improved by treatment with dermal substitutes containing high numbers of (precultured) autologous fibroblasts. Copyright

Extracellular matrix characterization during healing of full-thickness wounds treated with a collagen/elastin dermal substitute shows improved skin regeneration in pigs.

We investigated the architecture of the extracellular matrix (ECM) during healing of full-thickness wounds in the pig. Two different treatments, one based on epidermal transplantation (split skin mesh grafts, SP wounds) and one consisting of a combination of epidermal transplantation and a dermal matrix substitute (MA wounds) were compared. The dermal matrix consisted of native bovine collagen coated with elastin hydrolysate. The latter treatment reduced wound contraction and improved tissue regeneration. The expression patterns of fibronectin, von Willebrand factor, laminin, chondroitin sulfate, and elastin, detected by immunohistochemistry, were examined in time and indicated different stages of healing. During the early phase of healing the dermal matrix induced more granulation tissue, a different fibronectin expression pattern, and rapid vascular cell ingrowth (von Willebrand factor). Furthermore, in the MA wounds chondroitin sulfate was detected earlier in the basement membrane and fibronectin staining disappeared more rapidly. During later stages of healing, chondroitin sulfate expression was selective for areas in which ECM remodeling was active; in these specific areas elastin staining reappeared. ECM remodeling and elastin regeneration occurred both in the upper and lower dermis for the MA wounds but only in the upper dermis for the SP wounds. Electron microscopic evaluation of the wounds after 2 weeks showed many myofibroblasts in the SP wounds, whereas in the MA wounds cells associated with the dermal matrix had characteristics of normal fibroblasts. The results suggest that the biodegradable dermal matrix served as a template for dermal tissue regeneration, allowed faster regeneration, and improved the quality of healing in large full-thickness skin defects.

Porcine wound models for skin substitution and burn treatment.

Skin regeneration is an important field of tissue engineering. Especially in larger burns and chronic wounds, present treatments are insufficient in preventing scar formation and promoting healing. Initial screening of potentially interesting products for skin substitution is usually done by in vitro tests. Before entering the clinic, however, in vivo studies in immunocompetent animals are necessary to prove efficacy and provide information on safety aspects. We have obtained extensive experience using the domestic pig as test animal for studies on skin replacement materials, including tissue engineered skin substitutes, and burn wound treatment. Two models are described: an excisional wound model for testing of dermal and epidermal substitutes and a burn wound model for contact and scald burns, which allows testing of modern wound dressings in comparison to the present gold standards in burn treatment. The results of these experiments show that in vivo testing was able to reveal (dis)advantages of the treatments which were not detected during in vitro studies.

Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides.

BackgroundThe antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism.ResultsHuman LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1.ConclusionAlthough Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.

Morphometry of dermal collagen orientation by Fourier analysis is superior to multi‐observer assessment

In human dermis, collagen bundle architecture appears randomly organized, whereas in pathological conditions, such as scar tissue and connective tissue disorders, collagen bundle architecture is arranged in a more parallel fashion. Histological examination by one or two observers using polarized light is the most common method to determine collagen orientation. The hypothesis on which this study is based is that an objective image analysis technique, Fourier analysis, would improve the reliability (are the measurements reproducible?) and the accuracy (does the method measure what it is supposed to measure?) of collagen orientation assessment, compared with observer ratings. Fourier analysis was applied to 271 images of scar tissue and normal skin that were acquired by confocal laser‐scanning microscopy. Observers rated the same areas using polarized light as well as the confocal microscopy images. Computer images consisting of different types of ellipses were generated with a fixed orientation. Observers and Fourier analysis evaluated the images to evaluate accuracy. The inter‐observer reliability was acceptable when at least three observers rated polarized light images (r > 0.69), whereas two observers were sufficient for rating confocal microscopy images (r > 0.71). Fourier analysis correlated better with observer ratings of confocal microscopy images (r = 0.69) than with polarized light microscopy images (r = 0.42). Fourier analysis was more accurate than four observers for the evaluation of the ‘true’ orientation for almost all types of computer‐generated images. For the first time it is shown that Fourier image analysis is suitable for the morphometry of dermal collagen orientation and leads to a superior measurement of collagen orientation compared with subjective histological evaluation by several experts. If an evaluation is performed by conventional light microscopy, at least three observers are required to attain an acceptable inter‐observer reliability. Copyright

Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation.

In the present study, we compared the use of autologous versus allogeneic fibroblasts in dermal skin substitutes in a porcine wound model. The allogeneic fibroblast populations were isolated from female and a male pig (allo‐1, ‐ 2 and ‐ 3) and the controls, autologous fibroblasts, from female graft‐recipient pigs (control). The histocompatibility of the three donor pigs with the recipient pigs was determined with a mixed lymphocyte reaction. In two pigs, full‐thickness wounds were treated with the fibroblast‐seeded dermal substitutes (n=5 per animal) and immediately overgrafted with meshed split‐skin autografts. After 6 weeks, wound contraction was measured by planimetry and scar formation was scored. At 2, 4, and 6 weeks biopsies were taken and evaluated for the presence of inflammatory reactions, myofibroblasts, and scar formation. The mixed lymphocyte reaction of both recipient pigs showed the highest responses on peripheral blood mononuclear cells of the allo‐3 donor pig, and was low or negative for allo‐1 and allo‐2. In all “allogeneic” wounds, more inflammatory cells were observed over time along with inflammatory foci consisting of a mix of lymphocytes and granulomatous cells. After 4 weeks, myofibroblasts were absent in the control wounds, whereas in “allogeneic” wounds, myofibroblasts colocalized with inflammation foci. The final scar tissue of the “allogeneic” wounds showed granulating areas with thin, immature collagen bundles. In contrast, the control wounds showed a dermal tissue with mature collagen bundles organized randomly like in normal skin. The wounds treated with allo‐3 fibroblasts showed in both pigs a significant increase in scar formation and wound contraction when compared with control wounds. In conclusion, for optimal restoration of dermal skin function with minimal scar formation, skin substitutes containing autologous fibroblasts are preferred over skin substitutes with allogeneic fibroblasts. (WOUND REP REG 2002;10:)

Modulation of scar tissue formation using different dermal regeneration templates in the treatment of experimental full-thickness wounds.

The recovery of skin function is the goal of each burn surgeon. Split‐skin graft treatment of full‐thickness skin defects leads to scar formation, which is often vulnerable and instable. Therefore, the aim of this study was to analyze wound healing and scar tissue formation in acute full‐thickness wounds treated with clinically available biopolymer dermal regeneration templates. Full‐thickness wounds (3 × 3 cm) on both flanks of Gottingen mini pigs (n= 3) were treated with split‐thickness skin graft alone or in combination with a 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC) cross‐linked‐collagen scaffold, Integra, or a polyethyleneglycol terephthalate‐polybutylene terephthalate (PEGT/PBT) scaffold. The wounds (n= 12 per group) were examined weekly for six weeks to evaluate graft take, contraction (planimetry), and cosmetic appearance. Histologic samples taken after one and six weeks were used to assess scaffold angiogenesis, biocompatibility, and scar tissue quality. In all wounds, one week postwounding graft take was between 93 and 100 percent. The control wound, treated with split‐skin graft, showed little granulation tissue formation, whereas the EDC‐collagen treated wounds showed two to three times more granulation tissue formation. The collagen scaffold was completely degraded within one week. The Integra and PEGT/PBT scaffolds showed angiogenesis only through two‐thirds of the scaffold, which resulted in loss of integrity of the epidermis. Only basal cells survived, proliferated, and regenerated a fully differentiated epidermis within three weeks. Granulation thickness was comparable to collagen scaffold‐treated wounds. After six weeks, control wounds showed a wound contraction of 27.2 ± 6.1 percent, Integra‐treated wounds 34.6 ± 6.4 percent, collagen scaffold‐treated wounds 38.1 ± 5.0 percent, and PEGT/PBT scaffold‐treated wounds 54.5 ± 3.9 percent. The latter wounds had significantly more contraction than wounds of other treatment groups. Microscopically, the control and collagen scaffold‐treated wounds showed an immature scar tissue that was two times thicker in the EDC‐collagen treated wounds. The Integra‐treated wounds showed nondegraded collagen scaffold fibers with partly de novo dermal tissue formation and partly areas with giant cells and other inflammatory cells. The PEGT/PBT scaffold was almost completely degraded. Scaffold particles were phagocytosized and degraded intracellularly by clusters of macrophages. The scar tissue was in the early phase of ECM remodeling. In conclusion, this study showed that the rate of dermal tissue formation and scarring is influenced by the rate of scaffold angiogenesis, degradation, and host response induced by the scaffold materials.

Long-term results of a clinical trial on dermal substitution.: A light microscopy and Fourier analysis based evaluation

Although dermal substitution is a main topic of current wound healing research, there is a paucity of clinical trials with a long-term clinical and histopathological evaluation. A clinical trial was conducted to perform an intra-individual comparison of conventional treatment (split-thickness autograft) to a collagen/elastin dermal substitute in combination with an autograft. Promising results with this substitute were obtained with respect to dermal organisation and scar elasticity in animal studies and clinical trials with a short-term follow-up. Twenty-nine of the 42 pairs of the burn wounds and 28 of the 44 pairs of the scar reconstructions enrolled in the study were biopsied after 1 year. Promising but not statistically significant differences were found between substituted groups and control groups for epidermal thickness, basement membrane maturation, rete ridges (P=0.055), fibroblasts, myofibroblasts, inflammatory cells, vessels and extracellular matrix maturation. An objective and accurate technique, Fourier analysis, was used to evaluate collagen bundle orientation and packing. However, no statistically significant differences were obtained for these parameters. This microscopic evaluation provided no convincing evidence for a long-term effectiveness of a dermal substitute despite promising data over a short-term in in vitro and in vivo studies with the same material.

Cadexomer-iodine ointment shows stimulation of epidermal regeneration in experimental full-thickness wounds

The use of iodine in wound healing is still controversial. Both wound healing-stimulating effects and toxic effects leading to impaired wound healing have been reported. In order to study the direct effects of iodine on wound healing without interference of infectious pathogens, we investigated wound-healing parameters in noninfected experimental full-thickness wounds in the pig. Topical iodine treatment with an ointment consisting of a combination of iodine and cadexomer (modified starch), was compared with cadexomer ointment, the vehicle without iodine, and with treatment with saline. Treatment lasted for 30 days, followed by 30 days of wound assessment. The rate of epithelialization, wound contraction, systemic iodine absorption and several immunohistochemical markers were evaluated. All 36 wounds healed without macroscopic signs of wound infection and reepithelialized within 21 days. During the first 9 days of treatment, wounds treated with cadexomer-iodine ointment showed significantly more epithelialization than the wounds treated with either cadexomer or saline. In addition, the epidermis of wounds treated with cadexomer-iodine ointment had significantly more epithelial cell layers from day 12 to day 30, and these wounds stained for chondroitin sulphate proteoglycans in the newly formed basement membrane zone, which was not observed with the other treatments. No negative effects of cadexomer- iodine ointment on the formation of granulation tissue, neovascularization or wound contraction were observed. During the treatment systemic iodine absorption was physiologically acceptable. These results showed that treatment with cadexomer-iodine-containing ointment had positive effects on epidermal regeneration during the healing of full-thickness wounds in the pig compared with ointment alone or saline treatment.