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The skin plays a crucial role in protecting the integrity of the body's internal milieu. The loss of this largest organ is incompatible with sustained life. In reconstructive surgery or burn management, substitution of the skin is often necessary. In addition to traditional approaches such as split-or full-thickness skin grafts, tissue flaps and free-tissue transfers, skin bioengineering in vitro or in vivo has been developing over the past decades. It applies the principles and methods of both engineering and life sciences toward the development of substitutes to restore and maintain skin structure and function. Currently, these methods are valuable alternatives or complements to other techniques in reconstructive surgery. This review article deals with the evolution and current approaches to the development of in vitro and in vivo epidermis and dermis.

At 60-801% confluence keratinocyte cultures were assigned as controls or for gene transfer by infection with one or the other of two recombinant retrovirus vectors: (i) an amphotropic helper-free murine leukemia virus vector, MFG-lacZ, containing the (-galactosidase gene, lacZ (R.C.M., unpublished), or (ii) a vector, a-SGC-hGH, containing the gene sequence from hGH (L. Cohen and R.C.M., unpublished). Subconfluent keratinocyte cultures were incubated on 3 consecutive days with the retrovirus in Polybrene (8 pg/ml) and 10% fetal bovine serum containing Dulbecco's modified Eagle's medium (DMEM; GIBCO).During transfection the modified Waymouth medium was removed and replaced daily by an equal amount of DMEM containing the retrovirus at a concentration of 1-2 x 107 plaque-forming units/ml. After 6 hr the virus-containing medium was removed and replaced by Waymouth medium. This procedure was repeated on 3 consecutive days. At confluence, keratinocytes were released from the flasks with 0.01% trypsin and resuspended in normal unbuffered saline (0.9%O) containing 100 international units ofpenicillin and 100 pg of streptomycin per ml, at a concentration of 3 x 106 cells per ml. Keratinocytes resuspended in saline were found to be >90% viable, as determined by trypan blue staining.The procedure for making 170 reproducible standardized f-thickness skin wounds on the backs of 13 anesthetized pigs for these experiments was as follows. Under aseptic conditions using a 2.25-cm2 square template the skin was excised with a scalpel to the level ofthe panniculus carnosus muscle with careful hemostasis. Wound margins were tattooed with India ink, allowing for photoplanimetric evaluation of the surface area on sequential standardized photographs. The chambers, serving as an in vivo cell culture device, were applied to each wound (1, 4). The chamber (P. A. Medical, Columbia, TN) consists of a flexible transparent vinyl top bonded to an adhesive base. The base has a central opening fitting the wound margins. Chambers were filled with 1.2 ml of isotonic saline containing 100 pg of streptomycin and 100 international units penicillin per ml (1, 4). Twenty-two wounds were treated with lacZ transfected Abbreviations: hGH, human growth hormone; H&E, hematoxylin/ eosin; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside. t~o whom reprint requests should be addressed. 9307The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to b-cell exhaustion; this study examines the effect of chronic GK activation on b-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key b-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk del/wt ). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPb compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk del/wt islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key b-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.

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