Depth of the graft bed influences split-skin graft contraction.

Item does not contain fulltextContraction of a split-thickness skin graft used for coverage of large defects remains a great problem in plastic, burn and reconstructive surgery. In this study we evaluated healing of split-thickness skin grafts transplanted in wounds on the subcutaneous fat and muscle fascia in pigs. Four young domestic female pigs were included in the study, and the contraction was measured planimetrically during a 3-months' follow-up. At the end of the study the scar tissue was histologically assessed. From day 42 till the end of the study grafts transplanted on the muscle fascia were significantly more contracted than grafts on the subcutaneous fat without (p < 0.001) and with (p < 0.005, unpaired t-test) correction for the growth of the animal. The histological assessment showed that after 3 months the regenerated dermal tissue in the muscle fascia wounds was thicker, and less remodeled (higher tissue cellularity and thinner collagen bundles). In conclusion, in pigs, split-thickness skin grafts transplanted into deeper wounds contract more, and scar tissue maturation seems to last longer

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.

Item does not contain fulltex

Neovascularization of poly(ether ester) block-copolymer scaffolds in vivo: long-term investigations using intravital fluorescent microscopy.

Item does not contain fulltextPoly(ether ester) block-copolymer scaffolds of different pore size were implanted into the dorsal skinfold chamber of balb/c mice. Using intravital fluorescent microscopy, the temporal course of neovascularization into these scaffolds was quantitatively analyzed. Three scaffold groups (diameter, 5 mm; 220-260 thickness, microm; n = 30) were implanted. Different pore sizes were evaluated: small (20-75 microm), medium (75-212 microm) and large pores (250-300 microm). Measurements were performed on days 8, 12, 16, and 20 in the surrounding normal tissue, in the border zone, and in the center of the scaffold. Standard microcirculatory parameters were assessed (plasma leakage, vessel diameter, red blood cell velocity, and functional vessel density). The large-pored scaffolds showed significantly higher functional vessel density in the border zone and in the center (days 8 and 12) compared with the scaffold with the small and medium-sized pores. These data correlated with a larger vessel diameter and a higher red blood cell velocity in the large-pored scaffold group. Interestingly, during the evaluation period the microcirculatory parameters on the edge of the scaffolds returned to values similar to those found in the surrounding tissue. In the center of the scaffold, however, neovascularization was still active 20 days after implantation. Plasma leakage and vessel diameter were higher in the center of the scaffold. Red blood cell velocity and functional vessel density were 50% lower than in the surrounding tissue. In conclusion, the dorsal skinfold chamber model in mice allows long-term study of blood vessel growth and remodeling in porous biomedical materials. The rate of vessel ingrowth into poly(ether ester) block-copolymer scaffolds is influenced by pore size and was highest in the scaffold with the largest pores. The data generated with this model contribute to knowledge about the development of functional vessels and tissue ingrowth into biomaterials

Porcine wound models for skin substitution and burn treatment.

Item does not contain fulltextSkin 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

Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation.

Contains fulltext : 49512schalkwijk.pdf (publisher's version ) (Closed access)Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression

Upside-down transfer of porcine keratinocytes from a porous, synthetic dressing to experimental full-thickness wounds.

Contains fulltext : 59124.pdf (publisher's version ) (Closed access)Currently, the use of cultured epithelial autografts as an alternative to split-thickness skin autografts for coverage of full-thickness wounds is limited due to fragility of the sheet and variability in the outcome of healing. This could be circumvented by the transfer of proliferating keratinocytes, instead of differentiated sheets, to the wound bed and the "in vivo" regeneration of epidermis. The aim of this study was to achieve re-epithelialization on experimental full-thickness wounds in the pig using a porous, synthetic carrier seeded with proliferating keratinocytes. Porcine keratinocytes were isolated by enzymatic digestion and cultured in Optimem basal medium with mitogens. In a full-thickness wound model, carriers with different seeding densities were transplanted upside down onto the wound bed. Keratinocytes were labeled using a fluorescent red membrane marker, PKH-26 GL. Transfer of keratinocytes and re-epithelialization were recorded macroscopically and histologically. On day 4 after transplantation, transfer of fluorescently labeled keratinocytes was shown by their presence in the granulation tissue. An immature epidermis, as well as epithelial cords and islands, formed as early as day 8. At day 12 a stratified epidermis and wound closure were established and epithelial cysts were formed by differentiation of epithelial islands. Wounds treated with seeding densities as low as 50,000 cells/cm(2) showed wound closure within 12 days, whereas wounds treated with 10,000 cells/cm(2) or the nonseeded (acellular) carriers did not show complete re-epithelialization before day 17 after treatment. This study showed that porcine keratinocytes, transplanted "upside down" in experimental full-thickness wounds using a synthetic carrier, continued to proliferate and started to differentiate, enabling the formation of a new epidermis in a time frame of 12 days

Cleft palate cells can regenerate a palatal mucosa in vitro.

Contains fulltext : 69579.pdf (publisher's version ) (Closed access)Cleft palate repair leaves full-thickness mucosal defects on the palate. Healing might be improved by implantation of a mucosal substitute. However, the genetic and phenotypic deviations of cleft palate cells may hamper tissue engineering. The aim of this study was to construct mucosal substitutes from cleft palate cells, and to compare these with substitutes from normal palatal cells, and with native palatal mucosa. Biopsies from the palatal mucosa of eight children with cleft palate and eight age-matched control individuals were taken. Three biopsies of both groups were processed for (immuno)histochemistry; 5 were used to culture mucosal substitutes. Histology showed that the substitutes from cleft-palate and non-cleft-palate cells were comparable, but the number of cell layers was less than in native palatal mucosa. All epithelial layers in native palatal mucosa and mucosal substitutes expressed the cytokeratins 5, 10, and 16, and the proliferation marker Ki67. Heparan sulphate and decorin were present in the basal membrane and the underlying connective tissue, respectively. We conclude that mucosal cells from children with cleft palate can regenerate an oral mucosa in vitro

Cleft palate cells can regenerate a palatal mucosa in vitro.

Contains fulltext : 69579.pdf (publisher's version ) (Closed access)Cleft palate repair leaves full-thickness mucosal defects on the palate. Healing might be improved by implantation of a mucosal substitute. However, the genetic and phenotypic deviations of cleft palate cells may hamper tissue engineering. The aim of this study was to construct mucosal substitutes from cleft palate cells, and to compare these with substitutes from normal palatal cells, and with native palatal mucosa. Biopsies from the palatal mucosa of eight children with cleft palate and eight age-matched control individuals were taken. Three biopsies of both groups were processed for (immuno)histochemistry; 5 were used to culture mucosal substitutes. Histology showed that the substitutes from cleft-palate and non-cleft-palate cells were comparable, but the number of cell layers was less than in native palatal mucosa. All epithelial layers in native palatal mucosa and mucosal substitutes expressed the cytokeratins 5, 10, and 16, and the proliferation marker Ki67. Heparan sulphate and decorin were present in the basal membrane and the underlying connective tissue, respectively. We conclude that mucosal cells from children with cleft palate can regenerate an oral mucosa in vitro