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Biomedical subjects

S T Boyce

Publications and source records attributed to S T Boyce.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Administration, Oral↗
PubMed1995

Noncytotoxic combinations of topical antimicrobial agents for use with cultured skin substitutes.

Cultured skin grafts are destroyed more easily than split-thickness skin grafts by common burn wound organisms, including gram-negative and gram-positive bacteria and fungi. To increase the survival and engraftment of cultured skin grafts, formulations of antimicrobial agents were tested for cytotoxicity to cultured human keratinocytes and fibroblasts and for activity against common organisms from burn wounds. On the basis of previous studies, a base formulation containing neomycin (40 micrograms/ml), polymyxin B (700 U/ml), and mupirocin (40 micrograms/ml) was prepared, to which ciprofloxacin (20 micrograms/ml) or norfloxacin (20 micrograms/ml) and amphotericin B (0.25 microgram/ml) or nystatin (100 U/ml) were added. Toxicity to cultured human cells was determined by the growth response of cell cultures (n = 6) to each drug combination over 4 days. Activity against clinical isolates (n = 40) of Staphylococcus aureus, Pseudomonas aeruginosa, other gram-negative bacteria, and Candida spp. was determined by the wet disc assay. Analysis of variance testing showed no significant differences in the growth of keratinocytes or fibroblasts under control or experimental conditions. Medium without antimicrobial agents was not effective against any of the 40 microbial strains tested. The base formulation was effective against all bacterial strains tested but against none of the fungi, while all experimental formulations were effective against all microbial strains tested. These findings suggest that neomycin, mupirocin, and polymyxin B may be combined with a quinolone and an antimycotic agent to provide broad antimicrobial activity for a formulation for topical use with cultured skin on burns. However, the formulations described here are strictly experimental and are not recommended for clinical use without further evaluation.

Administration, Topical↗
PubMed1995

Case studies: treatment of chronic wounds with cultured skin substitutes.

This report of four patients (2 male, 2 female) presents preliminary results of treatment of chronic skin wounds with cultured skin substitutes prepared from allogeneic human skin cells and collagen substrates. The patients ranged in age from 64 to 73 years (mean: 67.25). The etiologies of their wounds were saphenous veinectomy, venous stasis, peripheral vascular disease, and venous stasis, with wound sizes 10cm2, 20cm2, 36cm2 and 184cm2. After the wounds were debrided, the cultured grafts, overlaid with a porous, non-adherent dressing, were stapled to the wounds. The procedure included frequent irrigations with essential nutrients containing broad spectrum antimicrobials, followed by dressing with antimicrobial ointments, and periodic examination of the wounds. Complete healing was obtained in two patients, partial healing in one with gradual recurrence due to poor patient compliance, and no healing in one. The two healed wounds did not recur after more than one year of observation and achieved closure in shorter periods of time than their duration before treatment with the skin substitutes. These results suggest that grafts of cultured allogeneic skin cells and collagen may accelerate healing of qualified chronic wounds. Qualifications include, but are not limited to, sufficient perfusion of the affected site and adequate debridement to viable tissue.

Aged↗
PubMed1994

Agar well diffusion assay testing of bacterial susceptibility to various antimicrobials in concentrations non-toxic for human cells in culture.

Previously, we showed that microbial susceptibility to antimicrobials in concentrations non-toxic for human cells in culture could be tested using the wet disc topical antimicrobial assay. In this report, wet disc assay and agar well diffusion assay results were compared testing the susceptibility of Ps. aeruginosa isolates from burn patients to concentrations of Polymyxin B non-toxic for cultured cells. Both assays were performed on the same agar plates. No differences in results were observed. Further agar well diffusion assay testing showed that susceptibility/resistance could be demonstrated when testing several antimicrobials in concentrations non-toxic for cultured cells against a variety of bacteria isolated from burn patients. Therefore, the more familiar agar well diffusion as well as the wet disc assay can be used to test microbial susceptibility to these concentrations of antimicrobials.

Anti-Infective Agents↗
PubMed1994

Genetically modified skin to treat disease: potential and limitations.

Molecular definition of disease at the level of the gene and advances in recombinant DNA technology suggest that many diseases are amenable to correction by genes not bearing the defective elements that result in disease. Many questions must be answered before this therapy can be used to correct chronic diseases. These questions fall into safety and efficacy categories. Experience with transplanting cellular elements of skin or skin substitutes (defined as skin that possess the cell types and a dermal structure to develop into a functioning skin) to athymic rodents is considerable and is seen as a system where these questions can be answered. This paper reviews these questions and presents our early analysis of genetically modified cells in skin substitutes in vivo and in vitro. Experimental data demonstrate that both a matrix of woven nylon, housing a fibroblast generated collage, and dead dermis can be utilized to shuttle genetically modified human fibroblasts from the laboratory to an in vivo setting. Genetically modified fibroblasts do not migrate from the shuttle to the surrounding tissue. The survival of significant numbers, approximately 70%, of genetically modified fibroblasts for at least 6 weeks in these shuttles, supports this general approach as having clinical utility. It is also concluded that skin substitute systems can be used to generate a genetically modified skin in vitro that has the capacity to develop into functional skin in vivo. Further, as genetically modified keratinocytes differentiate there is increased production by the transgene, supporting the concept that keratinocytes have true potential as shuttles for therapeutic genes. This work demonstrates that transplantation of systems containing genetically modified cells of the skin can be used to experimentally define many aspects of gene therapy using skin before this technology is taken to the clinic. Examples include determining the effect of gene transduction and expression on structure and function of the genetically modified skin as well as on distant skin and an assessment of the translational capacity of the transgene as function of time and cell number.

3T3 Cells↗
PubMed1993

Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers.

Closure of large skin wounds (i.e., burns, congenital giant nevus, reconstruction of traumatic injury) with split-thickness skin grafts requires extensive harvesting of autologous skin. Composite grafts consisting of collagen-glycosaminoglycan (GAG) substrates populated with cultured dermal fibroblasts and epidermal keratinocytes were tested in a pilot study on full-thickness burn wounds of three patients as an alternative to split-thickness skin. Light microscopy and transmission electron microscopy showed regeneration of epidermal and dermal tissue by 2 weeks, with degradation of the collagen-GAG implant associated with low numbers of leukocytes, and deposition of new collagen by fibroblasts. Complete basement membrane, including anchoring fibrils and anchoring plaques, is formed by 2 weeks, is mature by 3 months, and accounts for the absence of blistering of healed epidermis. All skin antigens tested (involucrin, filaggrin, laminin, collagens IV and VII, fibronectin, and chondroitin-sulfate) were expressed by 16 days after grafting. This cultured skin analogue provides an experimental alternative to split-thickness skin graft that develops histiotypic markers of skin anatomy and antigen expression after wound closure.

Adolescent↗
PubMed1993

Selection of topical antimicrobial agents for cultured skin for burns by combined assessment of cellular cytotoxicity and antimicrobial activity.

Cultured epidermal skin has become an adjunctive therapy for treatment of major burn injuries, but its effectiveness is greatly limited due to destruction by microbial contamination. To evaluate candidate drugs for use with cultured skin, a combined cytotoxicity-antimicrobial assay system was developed for determination of toxicity to cultured human keratinocytes and fibroblasts, and to common burn wound organisms (20 bacterial and 4 fungal strains). Candidate agents including Hibiclens (n = 3), amikacin, piperacillin, norfloxacin, and nystatin were tested separately and in combination (n = 6 each) for inhibition of growth of human cells and lytic activity on microorganisms in the wet disc assay. The data showed that: (1) Hibiclens was uniformly toxic to both cultured human cells and microorganisms; (2) norfloxacin had dose-dependent toxicity to human cells and broad effectiveness against microorganisms; and (3) norfloxacin (25 micrograms/mL) plus nystatin (100 U/mL) had low toxicity to human cells and high toxicity to both Gram-positive and Gram-negative bacteria (20 of 20) and fungi (4 of 4). Selection of topical antimicrobial drugs by these assays may improve effectiveness of cultured skin for burns and may be extended to the control of other surgical wound infections.

Administration, Topical↗
PubMed1993

Lipid supplemented medium induces lamellar bodies and precursors of barrier lipids in cultured analogues of human skin.

Barrier function of cultured skin substitutes (CSS) is required for their effective use in clinical treatment of skin wounds, and for percutaneous absorption in vitro. Arachidonic, palmitic, oleic, and linoleic free fatty acids, in conjunction with the antioxidant alpha-tocopherol acetate (lipid supplements, "LS"), were added to nutrient media of CSS to provide precursors of epidermal barrier lipids. CSS were composed of human keratinocytes (HK), fibroblasts (HF), and collagen-glycosaminoglycan substrates, and were incubated for 14 d submerged or lifted to the air-liquid interface in media based on MCDB 153 +/- LS. Duplicate samples (30 cm2) were harvested and the epidermal analogue was analyzed for total protein, total DNA, total lipid, lipid fractions including acylglucosylceramide (AGC), and presence of lamellar bodies. Significant increases (p < 0.05) were detected between CSS incubated in +LS medium for total lipid, total DNA, ceramide, glucosylceramide, triglycerides, and diglycerides. AGC and lamellar bodies were detected only in epithelia of CSS incubated in +LS medium. These data show that free fatty acids, vitamin E, and lifting of CSS promote increased epithelial morphogenesis compared to CSS cultured submerged without lipid supplements. Presence of lamellar bodies and AGC suggests enhanced production in vitro of barrier-associated epidermal lipids.

Cell Cycle↗
PubMed1993

Pigmentation and inhibition of wound contraction by cultured skin substitutes with adult melanocytes after transplantation to athymic mice.

Wound closure with cultured skin substitutes results in epithelium that is consistently hypopigmented. Hypothetically, addition of human melanocytes to cultured skin grafts may result in normal pigmentation of healed skin. Skin substitutes were composed of human epidermal keratinocytes and melanocytes, dermal fibroblasts, and collagen-glycosaminoglycan substrates, and were incubated for 12 d in media for keratinocyte growth (KG, n = 4), for keratinocyte differentiation containing four fatty acids and vitamin E with basic fibroblast growth factor (KDF, n = 6) or epidermal growth factor (KDE, n = 6), or for melanocyte growth (MG, n = 6) with phorbol ester and 5% fetal bovine serum. Skin substitutes were grafted orthotopically to full-thickness skin wounds (2 x 2 cm) on athymic mice, and scored for percent original wound size (+/- SEM), visible pigmentation (number pigmented/n), and positive staining for human leukocyte antigens (HLA)-ABC after 6 weeks on the mice. The data show that cultured skin grafts containing human melanocytes that are incubated in KDE or MG media have statistically significant reduction in wound contraction, 1:1 correlation of expression of pigment and HLA-ABC, and increased frequency of pigmentation after healing compared to incubation in KG or KDF media. Transmission electron microscopy confirmed the presence of melanocytes, melanosomes, and pigment transfer to keratinocytes in pigmented skin. These results suggest that survival and differentiated function of cultured epithelium can support melanization of skin, and that skin analogues exposed to phorbol ester in vitro can support skin pigmentation after wound healing.

Adult↗
PubMed1993

Rate of stratum corneum formation in the perinatal rat.

During late gestation, the fetal rat exhibits marked hyperplasia of the interfollicular epidermis and accelerated cornification in preparation for birth. In this study, we utilized simple morphometric techniques to provide quantitative estimates of the rate of stratum corneum (SC) formation during the perinatal period. Cryostat sections of dorsal epidermis from rat pups between -48 and +72 h of age were expanded under alkaline conditions and the number of corneocyte interfaces counted from photomicrographs. This method yielded the following regression lines: prenatal, y = 0.19x + 13.07, r = 0.93; postnatal, y = 0.13x + 13.00, r = 0.93, where y = number of SC layers and x = age in hours. Lack of desquamation was assured by the postnatal persistence of the granular periderm. Adhesive stripping of the epidermis followed by phase-contrast microscopy revealed intact monolayer sheets of SC. Quantitation using a computerized image analysis system gave an average corneocyte surface area in the newborn rat of 1908 +/- 36 mu 2 (mean +/- SEM). Treatment of neonatal rats with epidermal growth factor (500 ng/g BW) increased the number of SC layers over the first 24 h of life (p < 0.05) and resulted in marked hyperkeratosis by one week of age. These results allow the following conclusions: 1) in the prenatal rat, SC forms at a rate of one layer every 5 h; 2) postnatally, SC formation slows to one layer every 8 h; 3) to support normal corneum synthesis at birth, dorsal keratinocytes must enter transition at a rate of approximately 3 cells/second/cm2 of body surface; and 4) treatment with exogenous EGF augments the rate of terminal differentiation of perinatal rat epidermis.

Animals↗
PubMed1993

Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds.

Cultured skin substitutes consisting of implantable collagen (COL) and cultured human skin cells often fail clinically from destruction by microbial contamination. Hypothetically, addition of selected antimicrobial drugs to the implant may control microbial contamination and increase healing of skin wounds with these materials. As a model for drug delivery, bovine skin COL (1 mg/ml) and amikacin (AM; 46 micrograms/ml) were modified by covalent addition of biotin (B-COL and B-AM, respectively) from B-N-hydroxysuccinimide and bound together noncovalently with avidin (A). B-COL was incubated with A and then with B-peroxidase (B-P) or by serial incubation with B-AM and B-P, before P-dependent chromogen formation. Colorimetric data (n = 12 per condition) from spot tests on nitrocellulose paper were collected by transmission spectrophotometry. Specificity of drug binding in spot tests was determined by (i) serial dilution of B-COL; (ii) reactions with COL, AM, or P that had no B; (iii) removal of A; or (iv) preincubation of B-COL-A with B before incubation with B-P. Binding of B-AM was (i) dependent on the concentration of B-COL; (ii) specific to B-COL, A, and B-P (P < 0.05); and (iii) not eluted by incubation in 0.15 or 1.0 M NaCl. B-AM was found to block binding of B-P to the B-COL-A complex and to retain bacteriocidal activity against 10 clinical isolates of wound bacteria in the wet disc assay. Antimicrobial activity of B-AM was removed from solution by treatment with magnetic A and a permanent magnet. These results suggest that selected antimicrobial drugs can be biotinylated for attachments to COL-cultured cell implants without loss of pharmacologic activity. Because this chemistry utilizes a common ligand, any molar ratio of agents may be administered simultaneously and localized to the site of implantation.

Amikacin↗
PubMed1992

Absence of tumorigenicity in athymic mice by normal human epidermal keratinocytes after culture in serum-free medium.

The very rapid growth rate (1 population doubling/day) of normal human epidermal keratinocytes (HK) cultured in serum-free medium can be utilized for wound closure in burn treatment. However, rapid growth in vitro may present the possibility of neoplastic transformation. To investigate this possibility, HK were cultured from primary isolation to large populations in MCDB 153 medium supplemented with epidermal growth factor (EGF, 10 ng/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), and Bovine Pituitary Extract (BPE, 70 micrograms/ml). HK were studied for their ability to form tumors in athymic mice after subcutaneous inoculation. Sixteen separate HK strains were inoculated from primary cultures, or from secondary cultures either before or after storage in liquid nitrogen. Transformed cell lines, SCC 13 and FL, derived from human epithelial carcinomata were used as controls for tumor formation. HK formed no tumors (0/79) after 26 weeks incubation, SCC 13 formed nodular tumors (3/5) after 20 weeks incubation, and FL formed tumors (5/5) after 4 weeks incubation. HK cells were not found by histological examination of inoculation sites of keratinocyte cultures derived from primary culture from skin. In contrast, palpable tumors from both SCC 13 and FL were returned to tissue culture and continued to proliferate. These results support the conclusion that the rapid growth rate of human epidermal keratinocytes in vitro can be attributed to permissive culture conditions, and not to neoplastic transformation.

Animals↗
PubMed1991

Reconstitution of the histologic characteristics of a giant congenital nevomelanocytic nevus employing the athymic mouse and a cultured skin substitute.

This study addresses the development of an animal model for human giant congenital nevomelanocytic nevi (GCNN). Skin grafts were made from 1) non-involved split-thickness skin from a 12-month-old GCNN patient, 2) nevus split-thickness skin from the same GCNN patient, 3) nevus full-thickness skin, and 4) cadaveric human split-thickness skin. For groups 1) and 2), human epidermal and dermal cells were enzymatically isolated and expanded in tissue culture. Composite grafts were made by placing the cultured dermal cells into a collagen-glycosaminoglycan (GAG) matrix, followed by placement of the epidermal cells onto the opposite, laminated side of the matrix. All grafts were placed onto full-thickness wounds of athymic mice and biopsies were obtained from 6 to 38 weeks later for light microscopy including S-100 immunoperoxidase staining, and electron microscopy. The GCNN cultured skin mice (group 2) developed black, raised skin in the healed wounds. None of the group 1 mice developed lesions, grossly or histologically. All of the nevus full-thickness mice retained the nevus grossly. Histopathologic examination at 38 weeks of the black, raised plaques of group 2 demonstrated a reconstituted dermis similar to group 3. Nevus cells were larger and more epithelioid in the upper dermis, as seen with true GCNN. These nevomelanocytes were not seen in the dermis at 24 weeks, suggesting that the nevus cells migrated from the epidermal component of the cultured graft to the dermis during this time frame (24-38 weeks). The melanocyte identity of these cells was confirmed with S-100 immunoperoxidase staining and electron microscopy. These findings are unique to this composite cultured graft system. The ability to culture specific types of melanocytes and place them int skin substitutes on athymic mice provides a basis for the study of GCNN and melanocyte biology in vivo.

Animals↗
PubMed1991

Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors.

Skin wound closure remains a major problem in acute and reconstructive skin grafting after large burns because of limited availability of donor skin. This report evaluates six protocols for preparation in vitro of skin substitutes composed of cultured human cells, biopolymers, and growth factors for wound closure. Full-thickness wounds in athymic mice treated in a single procedure with cultured skin substitutes were compared directly to treatments with murine skin autograft, human skin xenograft, or no graft. Rectilinear planimetry of healed wounds 6 weeks after surgery showed that skin substitutes cultured in serum-free medium, and for 24 hours before surgery in defined medium with basic fibroblast growth factor (100 ng/ml), were not statistically different (p less than 0.05) in size from treatment with human skin xenograft. Acceptance and persistence of skin substitutes cultured in serum-free media were 70% at 6 weeks after surgery, as determined by staining of healed skin with a fluorescein-labeled monoclonal antibody against human HLA-ABC antigens. Ultrastructural examination of wounds with cultured human skin 6 weeks after treatment showed complete basement membrane, including anchoring fibrils, presence of melanocytes and pigment transfer to keratinocytes, and innervation of healed skin adjacent to basement membrane. These findings demonstrate effectiveness of cultured skin substitutes for closure of skin wounds and illustrate important capabilities to modulate the natural processes of wound repair, to increase supply of materials used for wound repair, and to enhance quality of wound healing.

Animals↗
PubMed1990

Cytotoxicity to cultured human keratinocytes of topical antimicrobial agents.

Cultured skin grafts administered clinically for closure of burn wounds may be contacted by topically applied antimicrobial agents. A study was performed to assess whether commonly used topical antimicrobial agents are toxic to cultured human keratinocytes (HK) in vitro. Serum-free MCDB 153 culture medium containing Neosporin G.U. irrigant (Neomycin, 40 micrograms/ml-polymyxin B sulfate, 200 units/ml) and a standard tissue culture antimicrobial agent of penicillin (10,000 units/ml)-streptomycin (10,000 micrograms/ml)-amphotericin B (25 micrograms/ml) had no effect on the keratinocyte growth rates when compared to standard MCDB 153 medium without antibiotics. Medium containing Sulfamylon (mafenide acetate, 0.85%), Polysporin (polymyxin B sulfate, 1 x 10(4) units/ml-bacitracin, 500 units/ml), gentamicin sulfate (0.1%), modified Dakins solution (25%), and acetic acid (0.25%) all showed statistically significant (P less than 0.01) decreases in keratinocyte growth rates. This data suggests that commonly applied antimicrobials may not be appropriate for cultured grafts in the concentrations that are used clinically.

Acetates↗
PubMed1990

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.

Animals↗