Multiscale spatiotemporal variability of fecal indicator bacteria and associated particle size distributions in the sandy bottom sediments of a Pennsylvania creek.

Concentrations of the fecal indicator bacteria (FIB) Escherichia coli and enterococci are used to assess microbial impairment in irrigation and recreation water sources. Although the FIB concentrations' variability at large temporal scales, such as seasons, and large spatial scales encompassing different land use has been studied, the knowledge about smaller scale variability remains sparse. This work aimed to research the small-scale variability of E. coli and enterococci in a montane creek with sandy bottom sediments. Sediment samples were collected weekly for a year in triplicate at sampling sites in a forested headwater, an agricultural area, and a mixed urban-agricultural area. The average weekly change in concentrations was from two times at the forested site to five times at the urban-agricultural site. Mean relative deviations from averages across sampling locations increased from -25% at the forested site to 45% at the urban-agricultural site. This trend was also observed separately over the cold and warm seasons. Over a week without precipitation, E. coli concentrations decreased on average by 20% in warm period and by 45% in cold period; the enterococci concentration declined by 12% in both cold and warm periods. The sediment particle size distributions were significantly different among the three sites and between the cold and warm seasons. Rankings of sediment fine mass fractions and FIB concentrations were positively correlated at two of three sampling sites in more than 70% of observation dates. The results of this work indicate the need to evaluate the uncertainty of sediment FIB concentrations before designing sediment FIB monitoring quality.

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice.

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.

Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice.

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4-7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin.

Reduced engraftment and wound closure of cryopreserved cultured skin substitutes grafted to athymic mice.

Cryopreservation of cultured skin substitutes is a requirement for establishment of banks of alternative materials for treatment of acute and chronic skin wounds. To determine whether cryopreservation of skin substitutes that contain cultured cells reduces their efficacy for wound closure, cell-biopolymer grafts were frozen, recovered into culture, and grafted to wounds on athymic mice. Grafts consisted of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates that were frozen in cell culture medium with 20% serum and 10% DMSO at a controlled rate and stored overnight in liquid nitrogen. After recovery into culture for 24 h, frozen or unfrozen (control) skin substitutes were grafted to full-thickness wounds on athymic mice. Wound area and surface electrical capacitance were measured at 2, 3, and 4 weeks after grafting at which time animals were sacrificed. Wounds were scored for presence of human cells by direct immunofluorescence staining with a monoclonal antibody to HLA-ABC. The data demonstrate that cell-biopolymer grafts are less efficacious after controlled-rate cryopreservation using 10% DMSO as a cryoprotectant. Frozen grafts at 4 weeks after surgery have significantly smaller wound areas, higher capacitance (wetter surface), and fewer healed wounds that contain human cells. The results suggest that these conditions for cryopreservation of cultured grafts reduce graft viability. Improved conditions for cryopreservation are required to maintain viability and efficacy of cultured skin substitutes after frozen storage.

Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice.

Collagen-based implants have been described as vehicles for transplantation of cultured skin cells for treatment of burn wounds. To optimize vascularization and repair of connective tissue, collagen solubility and glutaraldehyde crosslinking were evaluated. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates that were prepared from acid-insoluble, or partially soluble collagen. Substrates were crosslinked with 0% or 0.25% glutaraldehyde, populated with cells, and grafted to full-thickness wounds on athymic mice (n = 6/condition). After 6 weeks, the wound area was measured by planimetry, and healed wounds were scored by histochemistry for immunoreactivity to HLA-ABC and bovine collagen. Data analysis shows that crosslinking of collagen implants with glutaraldehyde is associated (p < 0.001) with detection of the implant. No association was found between solubility of bovine collagen and immunodetection. Epidermis of all wounds was positive for HLA-ABC, and no differences in wound areas were found. These results suggest that glutaraldehyde crosslinking of collagen implants decreases the rate of biodegradation. Delayed degradation of crosslinked collagen may result clinically in reduced engraftment of skin substitutes.

Effective management of microbial contamination in cultured skin substitutes after grafting to athymic mice.

Cultured skin substitutes have become therapeutic alternatives for treatment of acute and chronic skin wounds, but all models of these substitutes are avascular and susceptible to microbial destruction during vascularization. To develop a practical management protocol for increased survival of skin substitutes, experimental wounds were contaminated with Pseudomonas aeruginosa and treated with a formulation of noncytotoxic antimicrobial agents (polymyxin B, neomycin, ciprofloxacin, mupirocin, amphotericin B) in a nutrient medium (vehicle). Cultured skin substitutes consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan sponges were grafted to 2 x 2 cm full-thickness wounds on athymic mice that were contaminated with the strain SBI-N of P. aeruginosa at 1 x 10(4), 1 x 10(5), and 1 x 10(6) organisms/wound. Experimental wounds were irrigated with 1 ml/day topical antimicrobial solution for 10 days, and controls received vehicle only. Two, three, and four weeks after grafting, wounds were traced and swabbed for microbial culture and areas were measured with planimetry. At 4 weeks, biopsy samples were scored histochemically for immunoreactivity to HLA antigens. Data analysis by chi-square, analysis of variance, and Tukey's test shows that treatment of contaminated wounds with noncytotoxic topical antimicrobials is associated with an increased area of healed wounds, positive detection of HLA antigens, and negative cultures for P. aeruginosa. These results show that microbial contamination of cultured skin substitutes on full-thickness wounds may be managed effectively during graft vascularization. However, this formulation of antimicrobial agents is not currently approved for human use and is investigational only. Effective management of microbial contamination suggests that clinical efficacy of avascular tissue analogs may be increased by local application of noncytotoxic antimicrobial agents.

Surface electrical capacitance as a noninvasive index of epidermal barrier in cultured skin substitutes in athymic mice.

Restoration of an epidermal barrier is a definitive requirement for wound closure. To determine formation of an epidermal barrier as a function of hydration of the stratum corneum, we measured surface electrical capacitance (SEC) of the epidermis in cultured skin substitutes (CSS) in vitro and after grafting to athymic mice. CSS were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. On culture days 3, 7, 14, 17, and 21, SEC was measured in situ. CSS (n = 18; mean +/- SEM) showed a time-dependent decrease of SEC (picoFarads, "pF") from 4721 +/- 28 pF on day 3 to 394 +/- 117 pF on day 14, and subsequent increase to 1677 +/- 325 pF on day 21. After 14-d incubation, parallel CSS samples (n = 5) or murine autografts (n = 5) were grafted orthotopically to athymic mice. After grafting, CSS showed decreases in SEC from 910 +/- 315 pF at 2 wk to 40 +/- 10 pF at 4 wk with no significant decreases thereafter. Control values for murine autograft were 870 +/- 245 pF at 2 wk, and 87 +/- 30 pF at 4 wk. SEC values for native murine skin (n = 10) were 91 +/- 18 pF, and for native human skin (n = 10) were 32 +/- 5 pF. The data demonstrate that SEC decreases with time in culture and that healed or intact skin has approximately 10- to 100-fold lower SEC than CSS in vitro. This noninvasive technique provides a quantitative index of epidermal barrier in CSS in vitro and demonstrates the development of functional epidermal barrier during healing of wounds treated with cultured skin substitutes.

Expression of interleukin-1alpha, interleukin-6, and basic fibroblast growth factor by cultured skin substitutes before and after grafting to full-thickness wounds in athymic mice.

OBJECTIVES: Cultured skin substitutes (CSSs), consisting of human keratinocytes and human fibroblasts attached to collagen-glycosaminoglycan substrates, have been demonstrated to cover wounds, and may release detectable quantities of growth factors that promote wound healing. MATERIALS AND METHODS: Basic fibroblast growth factor (bFGF), interleukin-1alpha (IL-1alpha), and interleukin-6 (IL-6) were assayed by enzyme linked immunosorbent assay and immunohistochemistry in CSSs in vitro and at days 1, 3, 7, 14, and 21 after grafting to full-thickness wounds in athymic mice. MEASUREMENTS AND MAIN RESULTS: When isolated cells were tested, IL-1alpha was found to come primarily from the keratinocytes, whereas bFGF was from the fibroblasts. Combinations of both cell types in the CSSs resulted in a synergistic enhancement of IL-6 expression. Quantities of all three cytokines from CSSs were greater in vitro compared with in vivo levels at all time points after grafting. bFGF increased from day 1 to day 7, and then remained relatively constant until day 21. At day 3 maximal levels of IL-1alpha were observed. By day 7, IL-1alpha decreased to approximately 40% of maximal levels, and subsequently increased until day 21. IL-6 levels were highest at day 7 after grafting. All cytokines had reached elevated levels during the time of wound revascularization (days 3-7). CONCLUSIONS: The sequence of cytokine synthesis in the wounds (i.e., rapid IL-1alpha increase followed by IL-6 expression) parallels serum levels reported after a septic challenge. These findings support the hypothesis that the wound is a source of systemic cytokines.

Capillary morphogenesis during healing of full-thickness skin grafts: an ultrastructural study.

Biologic mechanisms by which skin grafts become revascularized after transplantation are poorly understood. To investigate graft revascularization, we examined the pattern of capillary growth in full-thickness skin grafts at serial time points. Full-thickness skin (2 x 2 cm) was excised to muscle fascia from the bilateral hind limbs of adult male Lewis rats. The graft/wound base boundary was identified by placement of a polypropylene mesh on the wound beneath the graft. Excised skin was replaced in its original orientation and secured with silk sutures tied over a gauze bolster dressing. After 3, 5, 7, and 10 days, animals were killed, and their aortas were cannulated and infused with an acrylic polymer to generate vascular casts. Grafts were excised, tissues were digested, and casts were examined with the use of scanning electron microscopy. Transmission electron microscopy was performed on tissues infused with the acrylic polymer that were not digested. At day 3, an immature lobular pattern was observed extending from the neovascular plexi on the graft side of the polypropylene mesh. At day 5, defined vessels with lobular ends occurred with high frequency. At day 7, the number of observed lobular structures was greatly reduced, and high frequencies of depressions in acrylic casts suggested protrusion of endothelial cell nuclei. By day 10, lobular structures were rare, well-defined microvascular plexi were contiguous with larger vessels, and depressions from endothelial cell nuclei appeared more shallow and less frequent. These findings suggest that (1) an immature lobular pattern representing either capillary outgrowth or extracapillary leakage occurs at day 3; (2) these immature lobules decrease, and more discrete capillaries increase by day 5; (3) vascular integrity is reestablished by day 7; (4) vascular plexi has regained full continuity, and there are suggestions that endothelial cell proliferation has subsided by day 10.

Topical nutrients promote engraftment and inhibit wound contraction of cultured skin substitutes in athymic mice.

Routine treatment of burns with cultured skin substitutes (CSS) has been limited by poor engraftment and by scarring. Hypothetically, topical application of essential nutrients and/or growth factors may support epithelial survival temporarily during graft vascularization. CSS, composed of human epidermal keratinocytes and dermal fibroblasts attached to collagen-glycosaminoglycan substrates, were incubated for 19 d in media optimized for keratinocytes. CSS, human xenografts, murine autografts, or no grafts were applied orthotopically to full-thickness skin wounds (2 x 2 cm) in athymic mice. Wounds were irrigated for 14 d with 1 ml/d modified cell culture medium or with saline containing epidermal growth factor, or were treated with dry dressings. After 6 weeks, treated sites were scored for percentage original wound area (mean +/- SEM) and percentage HLA-ABC-positive healed wounds [(number positive/n) x 100], and tested for significance (analysis of variance, p < 0.0001; Tukey test, p < 0.05). The data showed that CSS irrigated with nutrient medium were not statistically different in wound area (67.8 +/- 5.1%) from murine autografts (63.3 +/- 2.9%) but were statistically larger than human xenograft, no graft, or CSS treated with saline irrigation or dry dressings. HLA-ABC expression was 100% in CSS with nutrient irrigation, 86% in CSS with saline irrigation, 83% in CSS without irrigation, and 75% in xenografts with nutrient irrigation. These findings suggest that availability of essential nutrients supports keratinocyte viability during graft vascularization of CSS.

Pigmentation and microanatomy of skin regenerated from composite grafts of cultured cells and biopolymers applied to full-thickness burn wounds.

Rapid coverage and epithelial closure of extensive burns remains a major requirement for patient recovery. Although many skin substitutes have been described, permanent regeneration of both epithelial and connective tissues after a single surgical application of a skin substitute has not become routine. To replace both dermal and epidermal skin, cultured skin substitutes (CSS) were prepared from autologous keratinocytes and fibroblasts seeded onto collagen-glycosaminoglycan (C-GAG) substrates. CSS were applied to excised, full-thickness burns on 5 patients. Histologic analysis showed a fully stratified, hyperkeratotic epidermis within 12 days of grafting with little to no evidence of an inflammatory reaction. Epidermal and connective tissues are interdigitated in analogy to rete pegs and dermal papillae, and the neovascular plexus approximates the dermal-epidermal junction. Transmission electron microscopy identified a continuous basement membrane with hemidesmosomes and anchoring fibrils that connected the epidermis with the underlying connective tissue. Within 14-28 days, the C-GAG had been degraded and replaced by newly synthesized collagen in regenerated connective tissue. Spontaneous repigmentation of healing CSS from passenger melanocytes in keratinocytes culture was observed within 2 months after grafting. Electron microscopy revealed the presence of numerous melanosomes within the keratinocytes, illustrating pigment transfer between melanocytes and keratinocytes after wound closure. These results demonstrate that the CSS develop into functional permanent skin tissue capable of spontaneous repigmentation after grafting onto burn wounds.

Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides.

The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes. Here we present conclusive evidence that alpha-melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM alpha-MSH for 48 hr. The mitogenic effect gradually increased to 50-270% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM alpha-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days of treatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that alpha-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM alpha-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of alpha-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.

Characterization of ultraviolet radiation-induced damage to keratinocytes in a skin equivalent in vitro.

The human skin equivalent (HSE) provides a convenient model for studying the dermatological effects of exposure to ultraviolet (UV) radiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were maintained submerged for 1 week after the addition of epidermal cells and then raised to the air-liquid interface for an additional 3 weeks. HSEs were exposed to sublethal doses of UV radiation ranging from 0 to 500 J/m2, incubated up to 48 h in medium containing 3H-thymidine and fixed for ultrastructural and autoradiographic analysis. Exposure to radiation doses greater than 50 J/m2 led to vacuolation of the cornified envelopes and enlargement of intercellular spaces. These doses also led to the formation of dense cytoplasmic bodies, and separation and vesiculation of the nuclear envelope in the basal cells. DNA synthesis in the basal cells was analyzed autoradiographically. Maximal numbers of labeled basal cells were observed 24 h after exposure to UV radiation at 50 J/m2. Although the proportions of labeled cells varied among different epidermal donors, the maximal responses and time-course of 3H-thymidine incorporation remained consistent, supporting the usefulness of the HSE in studying the effects of UV irradiation on human skin.

Cornification and basement membrane formation in a bilayered human skin equivalent maintained at an air-liquid interface.

Human keratinocytes that were grown in a skin equivalent at an air-liquid interface were analyzed morphologically and biochemically to demonstrate differentiation approaching that of human skin. Within 3 weeks of growth at the interface, cuboidal basal cells, distinct spinous and granular zones, and a fully developed cornified layer of enucleated cells formed the multilayered epidermis. Ultrastructurally, the keratinocytes in the upper granular layer contain tonofilament bundles and membrane-coating granules. These cells form cornified squames that are resistant to degradation by sodium dodecyl sulfate/dithiothreitol. Basal cells are attached to a developing basement membrane with hemidesmosomes. Immunogold silver staining analysis with monoclonal antibodies demonstrated the expression of basement membrane collagens IV and VII. This level of differentiation might improve "take" of human grafts and provides a useful system with which to study topical carcinogens and tumor promoters in vitro.