Steven T. Boyce

The process of microbial translocation

The process of microbial translocation was studied using Candida albicans, Escherichia coli, or endotoxin instilled into Thiry-Vella loops of thermally injured guinea pigs and rats. Translocation of C. albicans occurred by direct penetration of enterocytes by a unique process different from classical phagocytosis. Translocation between enterocytes was not observed. Internalization was associated with a disturbance of the plasma membrane and brush border, but most internalized organisms were not surrounded by a plasma membrane. Passage of the candida into the lamina propria appeared to be associated with disruption of the basal membrane with extrusion of cytoplasm of the cell and candida. Organisms in the lamina propria were commonly phagocytized by macrophages but also were found free in lymphatics and blood vessels. Translocation of E. coli and endotoxin also occurred directly through enterocytes rather than between them, but translocated endotoxin diffused through the lamina propria and muscular wall of the bowel wall by passing between rather than through the myocytes. These descriptive phenomena provide new insight into the role of the enterocyte and intestinal immune cells in the translocation process.

Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes.

Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patients sera with anti-double-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific anti-SSA/Ro sera bound to irradiated keratinocytes, since this antibody antigen system is highly associated with photosensitive cutaneous LE.

Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum-free media

Methods are described for serum-free culture of human epidermal keratinocytes derived from neonatal foreskin tissue. Cultures are initiated, stored frozen, and returned to active growth, all with bovine pituitary extract as the only undefined supplement. Clonal growth assays are then performed in a biochemically defined medium. The degree of stratification and differentiation in the defined medium (and also with pituitary extract) is controlled by the extracellular calcium ion concentration.

Comparative assessment of cultured skin substitutes and native skin autograft for treatment of full-thickness burns.

ObjectiveComparison of cultured skin substitutes (CSSs) and split-thickness autograft (STAG) was performed to assess whether the requirement for autologous skin grafts may be reduced in the treatment of massive bums. Summary Background DataCultured skiasubstitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been demonstrated to close full-thickness skin wounds in athymic mice and to express normal skin antigens after closure of excised wounds in burn patients. MethodsData were collected from 17 patients between days 2 and 14 to determine incidence of exudate, incidence of regrafting, coloration, keratinization, and percentage of site covered by graft (n = 17). Outcome was evaluated on an ordinal scale (0 = worst; 10 = best) beginning at day 14, with primary analyses at 28 days (n = 10) and 1 year (n = 4) for erythema, pigmentation, epithelial blistering, surface roughness, skin suppleness, and raised scar. ResultsSites treated with CSSs had increased incidence of exudate (p = 0.06) and decreased percentage of engraftment (p < 0.05) compared with STAG. Outcome parameters during the first year showed no differences in erythema, blistering, or suppleness. Pigmentation was greater, scar was less raised, but regrafting was more frequent in CSS sites than STAG. No differences in qualtative outcomes were found after 1 year, and antibodes to bovine collagen were not detected in patientsera. ConclusionsThese results suggest that outcome of engrafted CSSs is not different from STAG and that increased incidence is related to decreased percentage of initial engraftment. Increased rates of CSSs may lead to improved outcome for closure of burn wounds, allow greater availability of materials for grafting, and reduce requirements for donor skin autogratt.

The 1999 clinical research award. Cultured skin substitutes combined with Integra Artificial Skin to replace native skin autograft and allograft for the closure of excised full-thickness burns.

Prompt and permanent closure of excised full-thickness burns remains a critical factor in a patients recovery from massive burn injuries. Hypothetically, Integra Artificial Skin (Integra) may replace the need for allografts for immediate wound coverage, and cultured skin substitutes (CSS) that contain stratified epithelium may replace the need for autografts for definitive wound closure. To test this hypothesis, 3 patients with full-thickness burns of greater than 60% of their total body surface areas had their eschar excised within 14 days of admission. Integra was applied, and a skin biopsy was collected from each patient for the preparation of CSS. At 3 weeks or more after the application of the Integra and the collection of skin biopsies, the outer silastic cover of the Integra was removed and CSS were grafted. The CSS were irrigated with nutrients and antimicrobials for 6 days and then dressed with antimicrobial ointment and cotton gauze. Treated wounds were traced on days 14 and 28 after the grafting of CSS for determination of engraftment and wound closure, respectively. Cost analysis was not performed. Engraftment on postoperative day (POD) 14 was 98%+/-1% (mean +/- standard error of the mean), the ratio of closed:donor areas on POD 28 was 52.3+/-5.2, and no treated sites required regrafting. The histology of the closed wounds showed stable epithelium that covered a layer of newly formed fibrovascular tissue above the reticulated structure of the degrading Integra. The clinical outcomes of the closed wounds after POD 28 demonstrated smooth, pliable, and hypopigmented skin. Two patients who had received CSS grafts over Integra on their backs were positioned supine on air beds from POD 8 or POD 9 with minimal graft loss because of mechanical loading. One patient with a full-thickness burn of 88% of the total body surface area was covered definitively at 55 days postburn. These results demonstrate that the combination of CSS and Integra can accomplish functionally stable and cosmetically acceptable wound closure in patients with extensive full-thickness burns. This combination of alternatives to the conventional grafting of split-thickness skin permits the substitution of cadaveric allograft with Integra and the substitution of donor autograft with CSS. This approach to the closure of excised full-thickness burns is expected to reduce greatly the time to definitive closure of burn wounds and to reduce the morbidity associated with the harvesting of donor sites for split-thickness skin autografts.

Principles and practices for treatment of cutaneous wounds with cultured skin substitutes

BACKGROUND Skin substitutes prepared from cultured skin cells and biopolymers may reduce requirements for donor skin autograft, and have been shown to be effective in treatment of excised burns, burn scars, and congenital skin lesions. DATA SOURCES Cultured skin substitutes (CSS) generate skin phenotypes (epidermal barrier, basement membrane) in the laboratory, and restore tissue function and systemic homeostasis. Healed skin is smooth, soft and strong, but develops irregular degrees of pigmentation. Quantitative analysis demonstrates that CSS closes 67 times the area of the donor skin, compared to less than 4 times for split-thickness skin autograft. CONCLUSIONS CSS reduce requirements for donor skin autograft for closure of excised, full-thickness cutaneous wounds, and demonstrate qualitative outcome that is not different from meshed, split-thickness autograft. These results offer reductions in morbidity and mortality for the treatment of burns and chronic wounds, and for cutaneous reconstruction.

Engineered Human Skin Fabricated Using Electrospun Collagen–PCL Blends: Morphogenesis and Mechanical Properties

Engineered human skin is commonly fabricated using collagen scaffolds that often have poor mechanical properties. To improve the strength of collagen-based scaffolds, poly(caprolactone) (PCL) was blended with collagen and formed into submicron fibers using electrospinning. At concentrations < 10% PCL (M(PCL)/[M(Collagen) + M(PCL)] x 100), the PCL component was evenly distributed within the collagen matrix. Increasing the PCL component to 30% caused separation of the collagen and PCL phases forming local domains of PCL within the collagen matrix. Tensile testing indicated that 10-100% PCL concentrations significantly improved the strength and stiffness of the acellular scaffolds. Engineered skin (ES) made with blended collagen-PCL at a concentration of up to 10% PCL did not significantly alter the stratification of the cells, cell proliferation, or epidermal differentiation compared to the 100% collagen group. Ultimate tensile strength of ES fabricated with the collagen-PCL blends was not significantly greater than that of ES made with 100% collagen scaffolds (0% PCL). The 30% PCL group had the least amount of mechanical strength likely caused by poor epidermal formation and reduced cell viability. These results indicate that minimal additions of PCL can be blended with collagen to produce scaffolds suitable for tissue engineering of human skin. However, the increase in scaffold strength with higher PCL concentrations did not result in significantly stronger ES, indicating that high cell viability and proper development of the epidermis are important factors for developing ES with high strength.

Design principles for composition and performance of cultured skin substitutes.

Engineering of skin substitutes provides a prospective source of advanced therapies for treatment of acute and chronic skin wounds. Hypothetically, engineering of skin substitutes can allow deliberate fabrication of biologic materials with properties that address specific patho-biologic conditions (e.g., burns, scars, cutaneous ulcers, congenital anomalies). By design and incorporation of specific therapeutic properties in skin substitutes, reduction of morbidity and mortality from full-thickness skin wounds may be facilitated. Morbidity from grafting of autologous, split-thickness skin [1] occurs at both the treatment site and the donor site [2]. Acute wounds that require grafting include excised burns, burn scars, and congenital cutaneous anomalies (i.e. giant nevus). Patients with acute wounds, in general, do not have healing impairment, but may not have sufficient donor sites to cover their wounds if large total body surface areas (TBSA) are involved. Estimates for hospitalizations from burns range from 60 000–80 000 annually, and costs for recovery from acute injuries range from US

Cultured Skin Substitutes Reduce Donor Skin Harvesting for Closure of Excised, Full-Thickness Burns

36 000–117 000 per patient [3–5]. Increased availability of skin grafts would prospectively provide advantages over conventional therapy including, but not limited to: reduction of donor site area required to close wounds permanently; reduction of surgical procedures and hospitalization time; grafting of patients who are poor candidates for donation of skin grafts; reduction of mortality and morbidity from scarring; and delivery of genetically-modified cells [6–10]. The ultimate objective for skin substitutes is restoration of the anatomy and physiology of uninjured skin after treatment and healing of the wound. At present, only autologous full-thickness skin grafts, free flaps or pedicle flaps [11,12] restore all of the structures and functions of uninjured skin but donor sites and treatment sites must be equal in size. Tissue expanders can stretch skin by an approximate factor of 2, but are associated with complications including rupture or infection of the expander, and necrosis of expanded skin before transplantation [13]. Meshed split-thickness skin grafts may be expanded by ratios of 1:4 with characteristic scarring of mesh interstices. Skin substitutes that contain cultured cells can provide large quantities of grafts for wound treatment, but restore only a subset of anatomic structures and physiologic functions of skin. Therefore, the full potential for engineering of skin substitutes has not yet been realized.

Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice

ObjectiveComparison of cultured skin substitutes (CSS) and split-thickness skin autograft (AG) was performed to assess whether donor-site harvesting can be reduced quantitatively and whether functional and cosmetic outcome is similar qualitatively in the treatment of patients with massive cutaneous burns. Summary Background DataCultured skin substitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been shown to close full-thickness skin wounds in preclinical and clinical studies with acceptable functional and cosmetic results. MethodsQualitative outcome was compared between CSS and AG in 45 patients on an ordinal scale (0, worst; 10, best) with primary analyses at postoperative day 28 and after about 1 year for erythema, pigmentation, pliability, raised scar, epithelial blistering, and surface texture. In the latest 12 of the 45 patients, tracings were performed of donor skin biopsies and wounds treated with CSS at postoperative days 14 and 28 to calculate percentage engraftment, the ratio of closed wound:donor skin areas, and the percentage of total body surface area closed with CSS. ResultsMeasures of qualitative outcome of CSS or AG were not different statistically at 1 year after grafting. Engraftment at postoperative day 14 exceeded 75% in the 12 patients evaluated. The ratio of closed wound:donor skin areas for CSS at postoperative day 28 was significantly greater than for conventional 4:1 meshed autografts. The percentage of total body surface area closed with CSS at postoperative day 28 was significantly less than with AG. ConclusionsThe requirement for harvesting of donor skin for CSS was less than for conventional skin autografts. These results suggest that acute-phase recovery of patients with extensive burns is facilitated and that complications are reduced by the use of CSS together with conventional skin grafting.