Cellular energy metabolism and intracellular pH of intact heart, kidney and liver of small animals were studied before and after burn with an effective technique of phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy. Advantages of this technique are that the measurement can be performed constantly without any complicated and destructive process, that it is possible to use this technique in clinical examination in the near future and that a lot of information can be obtained from only one spectrum.It was demonstrated that there were a short cellular energy source and a cellular acidosis in' heart, kidney and liver during burn shock. The high-energy phosphate compounds-lowered wavily but not straight, which could be divided into stress stage, compensation stage and decompensation stage. Intracellular pH decreased first in the heart, and then in the kidney and liver. In contrast to the results obtained by way of direct blood flow determination, the early and significant intracellular acidosis indicated the insufficiency of blood and oxygen supply in myocardium.

Although the research work on the permanent skin substitutes was started in our country later than in developed countries, the development has been promising in recent years. The results of this field of research would completely change the fact that deep burn wounds could only be repaired with autologous skin grafting. It is our expectation that with the advent of these permanent skin substitutes, the survival rate of severe trauma could be raised and the quality of wound repair could be improved. In a series of papers published in this issue, the development and the status quo of permanent skin substitutes will briefly be introduced.

Insulin resistance(IR) after severe burn may aggravate metabolic disturbance, delay wound healing and increase the probability of infection and MODS. The mechanism of IR may be the combined defects of insulin biological effects at pre-receptor, receptor and post-receptor levels. This article discusses the mechanism and intervention of postburn IR, aiming to improve treatment for extensive burn.

This study was aimed to improve the therapeutic results of composite skin substitutes. Human vascular endothelial cell growth factor (hVEGF 165 ) gene was constructed to an eukaryotic expression vector pcDNA3. Transfection of recombinant vector pcDNA3 hVEGF 165 into human dermal fibroblast cells was performed. VEGF protein level in the supernatant of transfected fibroblasts culture was determined. Its biological activities were tested by observing the growth rate of the human umbilical vein endothelial cells/HUVEC after being stimulated with the said supernatant, and by performing the Miles assay in guinea pigs. The results showed that these transgenic cells were able to secrete VEGF to certain extent, with biological activities to enhance the growth of HUVEC in vitro and improve vascular permeability. It indicated that transgenic fibroblasts could resurface the dermal substitute of a composite skin.

Objective: To prepare a collagen rnembrane and to investigate its feasibi1ity as a derma1 scaffold. Metbods:Pig hide collagen was dispersed in 0. 5 mol/L acetic acid, co-precipitated with chordroitin-6-sulfate and lyophilized for 48 h toproduce a highly porous membrane. Cross-linking was in 0. 25% glutaraldehyde for 24 h. The membrane was implanted be-neath the flaps on the dorsal sites of SD rats. After implantation, biopsies were obtained periodically to study the tissue com-patibility, vascularity and stability. Results: The average pore size of the spongiform collagen membrane was 100 pm. Sub-dermal implantation of it showed that the inflammation was minimal, with no evidence of an acute rejective reaction. Conclu-siou: The spongiform collagen membrane prepared is easy to secure and produce, it has satisfied tissue compatibility, abilityof vascularity and optimum dermal scaffold structure.

To investigate the possibility of constructing composite skin, keratinocytes were cultivated in vitro on the epidermal surface of cell free dermis prepared from pig skin.Keratinocytes grown on the dermal matrix were released at selected time points, followed by determining the proliferative capacity with cell number quantity and cell proliferation test. Cells attaching to the dermal matrix after it were seeded for 1 and 2 weeks were observed with histological section HE staining and electron microscopy scanning. Results showed that the number of keratinocytes was markedly increased with culture time. They maintained their proliferative potential after they were seeded on acellular xeno dermal matrix and reached a confluent monolayer or 3 to 6 layers at the 1st and 2nd week after seeding. The data showed that a living composite skin combined with keratinocytes and acellular dermal matrix could be successfully prepared in vitro.

To investigate the fate of composite skin comprising mixed keratinocytes seeded on acellular dermal matrix (ADM) after its transplantation to the wound. Newborn BALB/c and human keratinocytes were mixed in various ratios, seeded on the surface of ADM, and cocultured. The composite skin substitute were then grafted onto the full thickness skin wounds in BALB/c mice. The fate of human keratinocytes was observed. The results showed that the composite skin substitutes could close the full thickness wounds in BALB/c mice. Human keratinocytes were mainly located in the upper layer of the epidermis, and were gradually replaced by BALB/c keratinocytes. This indicated that the mixed culture of keratinocytes of two different species on ADM could close full thickness wounds, having the advantages such as saving the donor skin and shortening the culture time in vitro .

To reconstruct a composite skin substitute composed of keratinocytes and collagen sponge as dermal scaffold. Collagen was extracted from fresh porcine skin. After being mixed with chondroitin sulfate, it was frozen and dried under vacuum to form a spongy membrane. Keratinocytes were separated from the foreskin with the routine method, and they seeded on the collagen membrane. The keratocytes which were carried by the collagen sponge were cultured, and the growth and proliferation were observed. The result showed that keratinocytes could grow and proliferate to form a confluent layer after 2～3 weeks. It indicated the the collagen sponge membrane showed no toxicity to human epidermal cells, and it could be used as a dermal scaffold in the construction of a composite skin substitute.

To investigate the expression of epidermal growth factor (EGF) of EGF gene transfected keratinocytes in vivo and in vitro after grafting. EGF levels in the supernatant of the culture media of EGF gene transfected keratinocytes cultured for different lengths of time and different passages were determined with ELISA method. Then, the gene transfected keratinocytes were seeded on the surface of acellular dermal matrix, After culture, the composite skin substitutes were grafted onto the full thickness wounds in nude mice. Specimens were harvested at intervals after grafting and stained for EGF with immunohistochemistry. The results showed that keratinocytes transfected with EGF gene secreted EGF, which was detected in the supernatant of the culture, for 5 passages. Immunohistochemical staining method showed that EGF was expressed in the newly generated epidermis 1～3 weeks after grafting of the composite skin substitute. The data showed that gene transfected keratinocytes could express EGF stably in vivo and in vitro , which would be of benefit to the construction of the tissue engineering skin.

Epidermal stem cells (ESCs) play a critical role in homeostasis and wound repair of skin tissue. Since ESCs are rare (fewer than 10% in total basal cells population) and lack specific markers,it is difficult to isolate and identify them from keratinocytes. Currently ,isolation of ESCs was achieved mainly by fast adhesion of ESCs to extracelluar matrix or flow cytometry. Several specific markers have been found in recent years for the identification of ESCs.