John F. Hansbrough

In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh.

The design of a skin-substitute must address the need for a dermal component, as this mesenchymally-derived tissue is important in maintaining the integrity and function of skin. An in vivo study was undertaken to assess the use of two biodegradable meshes, polyglycolic acid and polyglactin-910, as carriers for cultured human fibroblasts in a living dermal replacement. The consistent vascularization and epithelialization of these grafts placed on athymic mice showed that this has potential in re-creating the dermis in a skin-substitute.

The Cytotoxic Effects of Commonly Used Topical Antimicrobial Agents on Human Fibroblasts and Keratinocytes

This study evaluated the cytotoxicity of commonly used topical agents to human dermal fibroblasts and epidermal keratinocytes, which play a prominent role in wound healing. The effects of these topical agents were assessed using two separate assays for the fibroblasts--tritiated thymidine incorporation and the uptake of a vital dye (neutral red). Keratinocytes were evaluated with the neutral red assay. Serial dilutions of each of 10 commonly used topical agents produced decreases in both the uptake of neutral red and the incorporation of thymidine at clinically relevant doses. Only Neosporin G.U. irrigant showed no significant difference compared with controls in the assays for both the fibroblasts and the keratinocytes. Careful attention must be paid to which agent is used in the clinical setting, since many of these can have profound effects on cells that influence wound healing.

Altered helper and suppressor lymphocyte populations in surgical patients. A measure of postoperative immunosuppression.

Although a wealth of evidence has suggested that cell-mediated immunity is suppressed after simple surgical trauma, there have been contradictory results using stimulation assays of lymphocyte function. We quantitated T-lymphocyte subsets in 11 patients undergoing routine cholecystectomy by immunofluorescence microscopy using specific monoclonal antibodies. T-helper to T-suppressor cell ratios were calculated on the preoperative day and the first postoperative day in all patients, and on the third or fourth postoperative day in five patients. Helper to suppressor ratios decreased in all patients on the first postoperative day (p greater than 0.01), but returned to within normal limits on subsequent days. Changes were due more to decreases in helper cells than to increases in suppressor cells, although changes in both populations were statistically significant. The measurement of T-cell subsets by antibody-specific labeling and immunofluorescence microscopy may prove to be a more sensitive, quantifiable, and reproducible assay of immune function in surgical or traumatized patients than use of stimulation assays. Measurements of specific helper and suppressor lymphocyte populations may prove useful in predicting morbidity and mortality, and may also help in studying the effect of immunomodulating agents on the immune response.

Safety and Efficacy of TransCyte* for the Treatment of Partial-Thickness Burns

Standard treatment for extensive partial-thickness burns in the United States and in much of the world involves the application of topical antimicrobial agents and repetitive wound débridements and dressing changes. We evaluated a new biologic wound covering, TransCyte (Advanced Tissue Sciences, La Jolla, Calif, formerly marketed as Dermagraft-Transitional Covering), for the treatment of partial-thickness burns. This material is composed of human newborn fibroblasts which are then cultured on the nylon mesh of Biobrane (Dow B. Hickam, Inc, Sugarland, Tex); the thin silicone membrane bonded to the mesh provides a moisture vapor barrier for the wound. A prospective, randomized, comparison study of silver sulfadiazine and TransCyte was performed with the use of paired wound sites on 14 patients. Wounds treated with TransCyte healed more quickly (mean 11.14 days to 90% epithelialization vs 18.14 days, P = .002). A noncomparison evaluation was then done for an additional 18 patients, and it confirmed excellent wound healing and an absence of infections. There were no infections in the 32 wound sites treated with TransCyte. In the first study group, late wound evaluations (3, 6, and 12 months postburn) were performed with use of the Vancouver Scar Scale. The results indicated that wound sites treated with TransCyte healed with less hypertrophic scarring than sites treated with silver sulfadiazine (P < .001 at 3 and 6 months, P = .006 at 12 months).

Video Recording Trauma Resuscitations: An Effective Teaching Technique

Since the initial hour after injury is the most crucial time for trauma patients, resuscitation technique is of vital importance. Standardized courses for first-hour management (ATLAS) have been widely accepted. A teaching format based upon video recording of every resuscitation has been developed. Tapes are reviewed by the staff and by the individuals involved in a particular resuscitation. In a weekly resuscitation review conference, actual footage is presented to the trauma team members, specific aspects of a resuscitation are critiqued, and supplemental didactic information is presented. Legal problems have been avoided by making the review and conference a part of the quality assurance process. Patient anonymity is preserved by positioning the video camera at the foot of the resuscitation bed. Tapes are erased after each conference. Video recording allows analysis of: 1) priorities during the resuscitation; 2) cognitive integration of the workup by the team leader; 3) physical integration of the workup by the team leader; 4) team member adherence to assigned responsibilities, resuscitation time, errors or breaks in technique; and 5) behavior change over time. In 3 1/2 years, more than 2,500 resuscitations have been recorded. Over a 3-month period, average resuscitation time to definitive care decreased for age- and injury severity-matched patient groups cared for by one team. Resuscitations have become more efficient and adherence to assigned responsibilities better. Weekly review of resuscitation contributes to improved technique and trauma care.

A multicenter clinical trial of a biosynthetic skin replacement, dermagraft-TC, compared with cryopreserved human cadaver skin for temporary coverage of excised burn wounds

This multicenter study compared the use of a biosynthetic human skin substitute with frozen human cadaver allograft for the temporary closure of excised burn wounds. Dermagraft-TC (Advanced Tissue Sciences, Inc.) (DG-TC) consists of a synthetic material onto which human neonatal fibroblasts are cultured. Burn wounds in 66 patients with a mean age of 36 years and a mean burn size of 44% total body surface area (28% total body surface area full-thickness) were surgically excised. Two comparable sites, each approximately 1% total body surface area in size, were randomized to receive either DG-TC or allograft. Both sites were then treated in the same manner. When clinically indicated (> 5 days after application) both skin replacements were removed, and the wound beds were evaluated and prepared for grafting. DG-TC was equivalent or superior to allograft with regard to autograft take at postautograft day 14. DG-TC was also easier to remove, had no epidermal slough, and resulted in less bleeding than did allograft while maintaining an adequate wound bed. Overall satisfaction was better with DG-TC.

Composite grafts of human keratinocytes grown on a polyglactin mesh-cultured fibroblast dermal substitute function as a bilayer skin replacement in full-thickness wounds on athymic mice.

We have developed and tested in athymic mice a new, cultured, dermal-epidermal graft composed of two human cell types coupled with a biodegradable dermal scaffold. Cultured, proliferating human keratinocytes (HK) were applied to the surface of a living dermal tissue replacement that is composed of human fibroblasts cultured on a polyglactin mesh. After 4 to 6 days of coculture, proliferating HKs achieved confluency on the surface of the living dermal tissue replacement. Grafts were then transferred to full-thickness wounds on the dorsum of athymic mice. Sixteen animals were grafted, and the mean percentage of graft take (original wound area covered) on day 20 after grafting was 51.25%. Staining with antibody specific for human involucrin confirmed the presence of HKs on closed wounds, and staining with antibody specific for human laminin revealed a continuous layer of laminin at the dermal-epidermal junction on day 20. Animals closed with living dermal tissue replacement alone markedly contracted, whereas application of living dermal tissue replacement-HK grafts appeared to retard contraction. Because polyglactin mesh fibers are absorbed by hydrolysis rather than by enzymatic degradation, this living composite graft may be more resistant to destruction when placed on excised human wounds than are composite grafts, which are composed of a collagen matrix. The inclusion of the living dermal substitute may ultimately provide better skin quality than is achieved from the use of cultured keratinocytes alone. Fragility of the epidermal layer is probably due to the short-term culture of HKs on the living dermal tissue replacement, and further efforts to develop a thicker epithelial layer may improve graft durability.

Wound healing in partial-thickness burn wounds treated with collagenase ointment versus silver sulfadiazine cream.

During burn care the wounds must be repeatedly debrided of adherent and loose debris until the decision is made to surgically excise and graft the wound or to await epithelialization. Though native proteolytic enzymes in the skin or those produced by colonizing bacteria can speed eschar separation, the use of exogenous enzymes for wound debridement may accelerate wound cleaning and healing. Collagenase digests native and denatured collagen in necrotic tissue. This multicenter trial of 79 patients with partial-thickness wounds compared the efficacy of collagenase ointment applied with polymyxin B sulfate/bacitracin powder with the efficacy of standard topical antimicrobial therapy (control) in which silver sulfadiazine cream (1%) was used to debride paired burn sites. Patients selected for the study had two noncontiguous, partial-thickness, comparably sized, and anatomically similar burn wounds. Ages of patients ranged from 5 to 60 years (mean 33 years). The total body surface area burned ranged from 2% to 30% (mean 13.6%). Mean burn sizes used for study treatment were 366 cm2 (26 to 2310 cm2) for collagenase sites and 355 cm2 (26 to 2394 cm2) for control sites. Sites on each patient were randomly assigned to treatment with either collagenase or control. Endpoints were time to clean wound bed (absence of retained debris) and time to healing (complete epithelialization). The sites treated with collagenase cleaned in less time (mean 9.3 days) than the control sites (mean 11.6 days). Similarly the collagenase sites healed faster than the control sites (mean 19 vs 22.1 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Acellular human dermis promotes cultured keratinocyte engraftment

In full-thickness skin injury, loss of dermis may result in compromised wound repair, including contracture, hypertrophic scarring, and wound breakdown. This report examines the effect of an acellular dermal matrix on in vivo skin repair. Human keratinocytes cultured onto a synthetic hydrophilic dressing were applied with (N = 9) and without (N = 11) an acellular dermal matrix to full-thickness skin defects on athymic mice. Host cells progressively repopulated the acellular dermal component of the grafts. All animals with dermal matrix revealed fully differentiated epidermis by postoperative day 21. Human keratinocytes persisted in all animals grafted with dermal matrix, compared to only 63.6% of those animals without a dermal component. Planimetric analysis revealed significantly reduced wound contraction (p = 0.016) in animals receiving the dermal matrix. Histologic, immunohistochemical, and electron microscopic analyses also were performed. These studies suggest that an acellular dermal matrix can effectively direct regeneration of normal skin morphology.

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.