Esther Middelkoop

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 5×105u2009cells/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.

Reduced wound contraction and scar formation in punch biopsy wounds. Native collagen dermal substitutes. A clinical study

In full‐thickness skin wounds dermal regeneration usually fails, resulting in scar formation and wound contraction. We studied dermal regeneration by implantation of collagenous matrices in a human punch biopsy wound model.

Dermal regeneration in native non-cross-linked collagen sponges with different extracellular matrix molecules.

Collagenous dermal templates can prevent scarring and wound contraction in the healing of full‐thickness defects. In a porcine wound model, full‐thickness wounds were substituted by reconstituted and native collagen sponges in combination with autologous split‐skin mesh grafts and covered with a semipermeable wound membrane. Native collagen sponges were also linked with either hyaluronic acid, elastin, or fibronectin. Reconstituted collagen matrixes, composed of cross‐linked small collagen fibrils, disintegrated within a week and did not contribute to dermal regeneration, whereas native collagen matrixes, composed of intact collagen fibers, disintegrated within 2 weeks and did contribute to dermal regeneration. Addition of extracellular matrix proteins retarded the disintegration to 4 weeks. However, fibronectin‐treated matrixes caused aberrant epithelization. When hyaluronic acid was added, matrixes were invaded by more fibroblasts and myofibroblasts. This process correlated with fibrosis and wound contraction. In contrast, the native collagen/elastin matrix reduced the amount of fibroblasts and myofibroblasts. This latter matrix resulted in optimal dermal regeneration and little wound contraction.

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, ‐u20032 and ‐u20033) 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 6u2003weeks, wound contraction was measured by planimetry and scar formation was scored. At 2, 4, and 6u2003weeks 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 4u2003weeks, 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:)

Dermal substitutes for full-thickness wounds in a one-stage grafting model

We tested different biodegradable matrix materials as dermal substitutes in a porcine wound model. Matrixes were covered with a split‐skin mesh graft and protected with a microporous, semipermeable membrane, which prevents blister formation, wound infection and provides ultimate healing conditions. Evaluation parameters were as follows: epithelization, dermal reconstitution, wound contraction, and cosmetic and functional aspect. A microfibrillar matrix of nondenatured collagen gave the best result, with immediate fibroblast ingrowth and epidermal outgrowth. Slight inflammatory reaction and minimal wound contraction were observed. Application of a split‐skin mesh graft, in combination with this collagen matrix, generated a thicker dermal layer than did a split‐skin mesh graft directly applied on a wound bed. However, the histologic dermal architecture was less optimal than one obtained with a full‐thickness punch graft method. Other matrixes caused inflammatory reactions, interfering with epithelization and dermal reconstitution. We conclude that a nondenatured collagen matrix, in combination with a split‐skin mesh graft, can provide a substitute dermis in a full‐thickness wound. This combination is preferable to a split‐skin mesh graft directly applied on the wound bed. With our microporous semipermeable membrane, the combined use of a dermal substitute and a split‐skin mesh graft can be applied in a single‐stage operation.

Reliability, validity and clinical utility of three types of pain behavioural observation scales for young children with burns aged 0-5 years

&NA; Pain measurement is a prerequisite for individualized pain management and research into pain interventions. There is a need for reliable and valid pain measures for young children with burns. The aim of this study was to investigate whether the pain observation scale for young children (POCIS), the COMFORT behaviour scale (COMFORT‐B) and the nurse observational visual analogue scale (VAS obs) are reliable, valid and clinically useful instruments to measure pain in children with burns aged 0–5 years. Participating trained nurses (N = 102) rated pain of 154 children during hospitalization. Two trained nurses simultaneously assessed pain at fixed intervals by using the previous mentioned measures. Cronbachs alpha for POCIS was .87 for background and .89 for procedural pain. Intraclass Correlation Coefficients (ICCs) were .75 for background and .81 for procedural pain. COMFORT‐B observations yielded Cronbachs alpha of .77 for background and .86 for procedural pain and ICCs of .83 for background and .82 for procedural pain. The VAS obs resulted in ICCs of .55 for background and .60 for procedural pain. Correlation coefficient between POCIS and COMFORT‐B was .79 (p < .01), Standardized response mean was 1.04 for both POCIS and COMFORT‐B. Background pain measured with POCIS and COMFORT‐B was lower than procedural pain (p < .001). Nurses found POCIS easier and quicker to use, but COMFORT‐B was found to indicate pain more accurately. Both POCIS and COMFORT‐B are reliable, valid and practical scales for pain measurement in young children with burns and can be used in practice and research. The VAS obs was found to be unreliable.

Production and characterisation of monoclonal antibodies against native and disassembled human catalase

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetrameric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoclonal antibody (17E10) was shown to interact with both native tetramer catalase and--to a lesser extent--with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase.

Topology of catalase assembly in human skin fibroblasts.

The biogenesis, assembly and import of the peroxisomal enzyme catalase was studied in human skin fibroblasts from control persons and from patients with the Zellweger syndrome. For this purpose, two monoclonal antibodies were generated which are able to discriminate between the monomeric or dimeric form and the tetrameric, enzymically active conformation of the enzyme. Metabolic labelling studies showed that catalase is assembled to the tetrameric conformation within one hour after its synthesis, while it is still in the cytosol of the cell. Subsequently, the enzyme becomes particle-bound in the control cells, a process that is retarded by addition of the catalase inhibitor 3-amino-1,2,4-triazole. However, the tetramer remains in the cytosol in cells from Zellweger patients. It is concluded that newly synthesized catalase can be assembled to a tetramer in the cytosol in human skin fibroblasts. Unfolding of this tetramer prior to import into peroxisomes is indicated.

Does aminotriazole inhibit import of catalase into peroxisomes by retarding unfolding

Fusion of complementary cell lines from patients with diseases of peroxisome biogenesis leads to peroxisome assembly in the heterokaryons and to uptake of cytosolic catalase by the newly peroxisomes. Here we show that catalase import is inhibited by prior binding to catalase of the inhibitor 3‐amino‐1,2,4‐triazole, which appears to retard unfolding of the protein.