Melanocyte repopulation in full-thickness wounds using a cell spray apparatus.

Melanocyte restoration is critical in reconstituting skin color. We developed a spotted (piebald) pig wound model to study methods of restoring melanocytes to the epidermis. Paired, full-thickness, porcine wounds were covered with nonpigmented, fully expanded, 3:1 meshed, split-thickness skin grafts and were sprayed with an epidermal cell suspension. The suspensions were highly pigmented skin (HPS) cell isolates for half of the wounds (n = 16) and nonpigmented skin (NPS) cell isolates for the remaining wounds (n = 16). Histologic sections showed 6.0 +/- 3.0 and 15 +/- 4.0 pigmented melanocytes per high-power field on days 8 and 20 in HPS-treated wounds and no pigmented melanocytes in NPS-treated wounds. Melanin pigment was dispersed in all layers of the epithelium for the HPS group on day 20 compared with a lack of melanin pigment observed in the NPS group. Cell spraying may provide a clinical method to restore color to skin; further work is needed to control the expression of melanin.

Assessment of adhesion assays for use with keratinocytes.

Cell attachment, as a biological process, is an important aspect with respect to graft survival and "take". With the ever-increasing use of cultured epithelial autografts for coverage and re-epithelialization of wounds, the assessment of keratinocyte adhesion in vitro has become a more common requirement in studies involving extracellular matrix proteins and their receptor molecules. Cell adhesion has been well-documented in immunological research, however keratinocyte adhesion has been investigated by manual counting (using methylene blue) or other less sensitive colorimetric methods. With the increase in number of fluorogenic probes available, their use as a sensitive alternative to radioactive labelling has been promoted in the literature. This study was carried out to investigate the possibility of using fluorescent probe 5,6-carboxyfluorescein diacetate succidimyl ester to achieve a more standardized assay in the assessment of keratinocyte adhesion. Adhesion was assessed on extracellular matrix proteins such as fibronectin, collagen types I & IV and laminin. We concluded that the fluorescent probe might provide greater sensitivity in measuring adhesion, however it may be cytotoxic to keratinocytes. Pre-labelling of keratinocytes may affect cellular functions such as adhesion and even proliferation and consequently the probe must be chosen with care.

The treatment of hypopigmented lesions with cultured epithelial autograft.

Hypopigmentation may be a significant problem after burn injury. It is often difficult to predictably repair with conventional surgical techniques. It has been our experience that epidermal cells cultured from patients with dark skin produce pigment within the epidermal cell sheets, which indicates the presence of melanocytes. The presence of melanocytes and melanin in these cell sheets was demonstrated with the use of histochemical techniques. The results indicate that repigmentation with cultured epithelial autograft is possible. We describe a novel technique of dermabrasion and a co-culture of epidermal cells and melanocytes.

Sprayed keratinocyte suspensions accelerate epidermal coverage in a porcine microwound model.

Keratinocyte suspensions can potentially treat a variety of epidermal defects, but the mechanism of action has not been fully determined. We developed a porcine model to study the effect of sprayed cell suspensions delivered on small wounds within a meshed autograft. Paired full-thickness surgically excised wounds were covered with a fully expanded 3:1 meshed split-thickness autograft. A keratinocyte cell suspension was sprayed onto half of the wounds at a seeding density of 2.8 x 10(3) cells/cm2; the remaining wounds were sprayed with cell culture medium alone. Histologic analysis at days 5 and 8 showed an increase in average epidermal thickness, confluence, keratin cysts, and blood vessels in the keratinocyte cell suspension group compared with the cell culture medium control group. Wounds sprayed with the cell suspension showed faster and better quality of epithelialization than wounds sprayed with cell culture medium alone.

Systemic factors influencing the growth of cultured epithelial autograft.

This is a retrospective study of 23 consecutive patients who had biopsies taken for cultured epidermal autograft between February 1993 and September 1994. Keratinocyte cultures were initiated on all of these biopsies. Of these 23 biopsies, it was noted that the cells obtained from three patients grew particularly slowly or failed to grow at all. Taking into account that the biopsy and culture conditions were standard for all patients, we investigated whether the patient's previous medical history may have had any effect on the ability of the cells to be cultured in vitro. Our results indicated that the keratinocyte cultures of patients with a significant past medical history, and particularly those with underlying pathology affecting their general physical condition, have a decreased growth rate. This raises the question that general patient condition can possibly influence the clinical use of the technique.

Streptavidin-biotin immunotoxins: a new approach to purging bone marrow.

Immunotoxins have been used both experimentally and clinically to purge bone marrow of tumor cells or T cells before transplantation. We describe the synthesis of a streptavidin-biotin-toxin conjugate using whole ricin. Streptavidin-biotin-ricin (SA-BR) conjugates were synthesized by biotinylation of whole ricin, which was then complexed with streptavidin. Hybrid molecules consisting of a single biotinylated ricin moiety linked to a streptavidin molecule were separated by gel filtration. This SA-BR conjugate was used in an indirect cytotoxicity assay. The assay involved sensitizing of target cells with biotinylated monoclonal antibody (B-MCAB) followed by treatment with dilutions of SA-BR conjugate. The assay demonstrated a specific antibody-directed cytotoxicity. The strength of this SA-BR system is that a single conjugate was able to be used in conjunction with a library of B-MCABs to selectively target phenotypically different cell types. The application of the SA-BR conjugate is thus only restricted by the availability of B-MCABs specific for the desired target cells. The high affinity of avidin for biotin (Kd approximately 10(-15)) and the ability of a single conjugate to target phenotypically different cells through utilization of a library of B-MCABs gives SA-BR conjugates great potential in the selective targeting of individual cell types.