Cleft palate cells can regenerate a palatal mucosa in vitro.

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.

Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds.

In diabetic patients, wound healing is impaired. We studied the pathogenesis behind this clinical observation by characterizing the pattern of deposition of extracellular matrix (ECM) molecules and the cellular infiltrate in chronic (>8 wk) diabetic wounds, compared with chronic venous ulcers and an acute wound healing model. Punch biopsies were obtained from the chronic ulcer margins and control samples were collected from upper leg skin 5, 19, 28 d and 12 and 18 mo postwounding (p.w.). T cells, B cells, plasma cells, granulocytes and macrophages, and the ECM molecules fibronectin (FN), chondroitin sulfate (CS), and tenascin (TN) were visualized using immunohistochemical techniques. Expression of FN, CS, and TN was detected in dermal tissue early in normal wound healing (5-19 d p.w.). Abundant staining was seen 3 mo p.w., returning to prewounding levels after 12-18 mo p.w. In the dermis of chronic diabetic and venous ulcers with a duration of 12 mo or more, a prolonged presence of these ECM molecules was noted. Compared with normal wound healing: (i) the CD4/CD8 ratio in chronic wounds was significantly lower (p < 0.0027) due to a relatively lower number of CD4+ T cells; (ii) a significantly higher number of macrophages was present in the edge of both type of chronic ulcers (p < 0.001 versus day 29 p.w.); and (iii) more B cells and plasma cells were detected in both type of chronic wounds compared with any day in the acute wound healing model (p < 0.04 for CD20+ and p < 0.01 for CD79a+ cells). These data indicate that important differences exist in the cellular infiltrate and ECM expression patterns of acute, healing versus chronic wounds, which may be related to the nonhealing status of chronic wounds.

Living skin substitutes: survival and function of fibroblasts seeded in a dermal substitute in experimental wounds.

The healing of full-thickness skin defects requires extensive synthesis and remodeling of dermal and epidermal components. Fibroblasts play an important role in this process and are being incorporated in the latest generation of artificial dermal substitutes. We studied the fate of fibroblasts seeded in our artificial elastin/collagen dermal substitute and the influence of the seeded fibroblasts on cell migration and dermal substitute degradation after transplantation to experimental full-thickness wounds in pigs. Wounds were treated with either dermal substitutes seeded with autologous fibroblasts or acellular substitutes. Seeded fibroblasts, labeled with a PKH-26 fluorescent cell marker, were detected in the wounds with fluorescence microscopy and quantitated with flow cytofluorometric analysis of single-cell suspensions of wound tissue. The cellular infiltrate was characterized for the presence of mesenchymal cells (vimentin), monocytes/macrophages, and vascular cells. Dermal substitute degradation was quantitated by image analysis of wound sections stained with Herovici's staining. In the wounds treated with the seeded dermal substitute, fluorescent PKH-26-labeled cells were detectable up to 6 d and were positive for vimentin but not for the macrophage antibody. After 5 d, flow cytofluorometry showed the presence of 3.1 (+/-0.9) x 10(6) (mean +/- SD, n = 7) PKH-26-positive cells in these wounds, whereas initially only 1 x 10(6) fluorescent fibroblasts had been seeded. In total, the percentage of mesenchymal cells minus the macrophages was similar after 5 d between wounds treated with the seeded and the acellular substitutes. In the wounds treated with the seeded substitute, however, 19.5% of the mesenchymal cells were of seeded origin. Furthermore, the rate of substitute degradation in the seeded wounds was significantly lower at 2-4 wk after wounding than in wounds treated with the acellular substitute. Vascular in-growth and the number of infiltrated macrophages were not different. In conclusion, cultured dermal fibroblasts seeded in an artificial dermal substitute and transplanted onto full-thickness wounds in pigs survived and proliferated. The observed effects of seeded fibroblasts on dermal regeneration appeared to be mediated by reducing subcutaneous fibroblastic cell migration and/or proliferation into the wounds without impairing migration of monocytes/macrophages and endothelial cells. Moreover, the degradation of the implanted dermal substitute was retarded, indicating a protective activity of the seeded fibroblasts.

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.

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.

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

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 [3H]thymidine incorporation. Fibroblast morphology was studied by light and transmission electron microscopy. Diabetic ulcer fibroblasts, measured by [3H]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 mM) and low-glucose (5.5 mM, 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.

The suitability of cells from different tissues for use in tissue-engineered skin substitutes.

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.

Effectiveness and Safety of the PEGT/PBT Copolymer Scaffold as Dermal Substitute in Scar Reconstruction Wounds (Feasibility Trial).

Skin defects left after excision of hypertrophic scars were treated with a dermal substitute and split-thickness skin grafts transplanted after vascularisation of the substitute. The used substitute was a synthetic porous scaffold made from the biodegradable copolymer polyethyleneglycol-terephtalate and polybuthylene-terephtalate. The study was designed to assess the rate of granulation tissue formation, graft take, and after 3 and 12 months the quality of life (pain, comfort of treatment, cosmetic or functional nuisance), scar formation and wound contraction. In addition, scaffold biodegradation and scar tissue formation were evaluated histologically.Seven patients with different causes of burn injury were enrolled, of which 5 completed the study. In the first 4 patients the time between scaffold application and split-thickness skin overgrafting was in between 17 and 24 days. The time point of overgrafting was significantly reduced to 10-12 days by meshing of the dermal scaffold as evidenced in the last 3 patients. Histological evaluation at 3 months revealed normal generation of dermal tissue, however, the collagen bundles were parallel organized like in scar tissue. In the deeper layers of the neodermis, fragments of the dermal substitute were present, causing a mild inflammatory response. One year post-treatment, some fragments of the copolymer were still observed. The extent of wound contraction after successful overgrafting ranged from 30% to 57% after 1 year. All 5 patients showed an improvement in the total Vancouver Scar Score compared to the value before scar removal being similar to what can be expected when treated with split-thickness skin grafts alone. No unanticipated adverse effects due to application of the substitute were observed.We conclude that although this synthetic dermal substitute can be safely used in humans, the presence of 3D dermal template in a full-thickness skin defect will not automatically improve the skin tissue regeneration process or inhibit wound contraction.

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

Contraction 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.

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, 1x10(5) (0-DS10) and 5x10(5) cells/cm(2) (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 (alpha-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.