[Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells].

OBJECTIVE: To explore the most appropriate method for the isolation of human umbilical cord mesenchymal stem cells (MSCs) through a comparison of different methods. METHODS: Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline (PBS) and sliced into 1 mm(3) tissue blocks after removal of umbilical vessels and external membrane. These tissue blocks were averagely divided into 4 groups after washing and centrifuge. Then four methods for the isolation of human umbilical cord MSCs were compared: an explant culture and three enzymatic methods of collagenaseII, collagenaseII/trypsin and collagenaseII/hyaluronidase. The count of living cells was evaluated by trypan blue dye exclusion test. Cell morphology was observed under inverted microscope. The expressions of cell surface markers CD105, CD90, CD73, CD31, CD44, CD45, human leukocyte antigen-I (HLA-I) and human leukocyte antigen class IImolecules (HLA-DR) were detected by immunofluorescent staining. Cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). RESULTS: The human umbilical cord MSCs were successfully isolated by four isolated methods. However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells. Isolated cells using four methods were counted at (5.44 ± 0.21)×10(5), (4.03 ± 0.24)×10(5), (4.91 ± 0.33)×10(5) and (5.94 ± 0.40)×10(5) respectively. More cells were obtained with collagenaseII/hyaluronidase than other three methods (all P < 0.05). Cells out of tissue blocks were observed at Day 9-11 and cells were observed at Day 2 with three types of enzyme digestion. The fusion time of cells were (18.5 ± 3.5), (8.0 ± 1.0), (7.5 ± 1.5) and (3.5 ± 0.5) days respectively. The fusion time of cells obtained with collagenaseII/hyaluronidase was lower than other methods (all P < 0.05). Cell morphology: polygonal, irregular and of large volume for explant culture; relatively short and small for collagenaseII and collagenaseII/trypsin methods; thin spindle for collagenaseII/hyaluronidase method. Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were expressed in all groups while there was no expression of CD31, CD45 or HLA-DR. And the cells obtained with collagenaseII/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods. CONCLUSION: The collagenaseII/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.

Basic fibroblast growth factor/vascular endothelial growth factor in the serum from severe burn patients stimulates the proliferation of cultured human umbilical cord mesenchymal stem cells via activation of Notch signaling pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) are the leading cellular constituents used in regenerative medicine. MSCs repair and reconstruct wounds of acute traumata and radiation-induced burns through proliferation, differentiation, and trophic activity. However, repair effect of MSCs on severe burn wounds remain to be clarified because severe burns are much more complex traumata than radiation-induced burns. Survival and proliferation of MSCs in microenvironments affected by severe burns are very important for improving wound repair/regeneration. This study aimed to elucidate the survival and proliferation effects and the potential proliferation mechanism of serum from severe burn patients (BPS) on human umbilical cord MSCs (hUCMSCs) in vitro. METHODS: The hUCMSCs were isolated, cultured, and identified. Next, we evaluated the effects of BPS on cell numbers, cell cycle progression, cyclin D expression, and key proteins and genes of the Notch signaling pathway. Putative mechanisms underlying the proliferation of hUCMSCs were investigated. RESULTS: BPS markedly increased the number of hUCMSCs, and the results of the cell cycle studies indicated that BPS induced cell cycle progression into the M phase. Cyclin D expression was higher with BPS than in the control group. Moreover, Notch-1, a key determinant of hUCMSC activation and proliferation, and its target gene Hes-1 were overexpressed after BPS treatment. Proliferation numbers of hUCMSC, rate of proliferation period (G2/M+S), and the expression of cyclin D, Notch-1, and Hes-1 were markedly decreased by Notch signaling inhibitors (DAPT/GSI). In the case of BPS, basic fibroblast growth factor and vascular endothelial growth factor were the key factors that promoted hUCMSC proliferation. CONCLUSION: This study provides novel evidence for the role of BPS in the survival and rapid proliferation of hUCMSCs and suggests that these cells could be used for cell therapy-based clinical applications for treating severe burns. Furthermore, hUCMSC proliferation was induced by basic fibroblast growth factor/vascular endothelial growth factor in BPS through activation of Notch signal.

Treatment strategies for mass burn casualties.

BACKGROUND: Mass burn casualties are always a great challenge to a medical team because a large number of seriously injured patients were sent in within a short time. Usually a high mortality is impending. Experiences gained from successful treatment of the victims may be useful in guiding the care of mass casualties in an armed conflict. METHODS: Thirty-five burn victims in a single batch, being transferred nonstop by air and highway from a distant province, were admitted 48 hours post-injury. All patients were male with a mean age of (22.4 +/- 8.7) years. The burn extent ranged from 4% to 75% ((13.6 +/- 12.9)%) total body surface area. Among them, thirty-two patients were complicated by moderate and severe inhalation injury, and tracheostomy had been performed in 15 patients. Decompression incisions of burn eschar on extremities were done in 17 cases before transportation. All the thirty-five patients arrived at the destination smoothly via 4-hour airlift and road transportation. Among them, twenty-five patients were in critical condition. RESULTS: These thirty-five patients were evacuated 6 hours from the scene of the injury, and they were transferred to a local hospital for primary emergency care. The patients were in very poor condition when admitted to our hospital because of the severe injury with delayed and inadequate treatment. Examination of these patients at admission showed that one patient was suffering from sepsis and multiple organ dysfunction syndrome. Dysfunction of the heart, lung, liver, kidney, and coagulation were all found in the patients. Forty-eight operations were performed in the 23 patients during one month together with comprehensive treatment, and the function of various organs was ameliorated after appropriate treatment. All the 35 patients survived. CONCLUSIONS: A well-organized team consisting of several cooperative groups with specified duties is very important. As a whole, the treatment protocol should be individualized, basing on the extent of the injury and the care that the patient had received at the spot. During airlift, the stretchers should be arranged perpendicular to the longitudinal axis of the cabin. The treatment protocol in our hospital consisted mainly of prompt effective relief of all life-threatening complications, followed by early closure of burn wounds, appropriate use of anti-infection therapy, emphasis on nutritional support, correction of metabolic disorders, alleviation of immunosuppression, correction of coagulopathy, and effective support and protection of organ function.

Characteristics of and strategies for patients with severe burn-blast combined injury.

BACKGROUND: Severe burn-blast combined injury is a great challenge to medical teams for its high mortality. The aim of this study was to elucidate the clinical characteristics of the injury and to present our clinical experiences on the treatment of such cases. METHODS: Five patients with severe burn-blast combined injuries were admitted to our hospital 77 hours post-injury on June 7, 2005. The burn extent ranged from 80% to 97% (89.6% +/- 7.2%) of TBSA (full-thickness burns 75% - 92% (83.4% +/- 7.3%)). All the patients were diagnosed as having blast injury and moderate or severe inhalation injury. Functions of the heart, liver, kidney, lung, pancreas and coagulation were observed. Autopsy samples of the heart, liver, and lungs were taken from the deceased. Comprehensive measures were taken during the treatment, including protection of organ dys function, use of antibiotics, early anticoagulant treatment, early closure of burn wounds, etc. All the data were analyzed statistically with t test. RESULTS: One patient died of septic shock 23 hours after admission (four days after injury), the others survived. Dysfunction of the heart, liver, lungs, pancreas, and coagulation were found in all the patients on admission, and the functions were ameliorated after appropriate treatments. CONCLUSIONS: Burn-blast combined injury may cause multiple organ dysfunctions, especially coagulopathy. Proper judgment of patients' condition, energetic anticoagulant treatment, early closure of burn wounds, rational use of antibiotics, nutritional support, intensive insulin treatment, timely and effective support and protection of organ function are the most important contributory factors in successful treatment of burn-blast combined injuries.

[Changes in the expression of rat hepatic lipid metabolism related genes in response to burn wound sepsis as assessed by DNA arrays].

OBJECTIVE: To examine the expression profile of lipid metabolism-related genes in liver of scalded rats with wound sepsis, and to analyze its significance. METHODS: Sixty male Wistar rats with 30% TBSA full-thickness scald wound on the back were enrolled in the study and randomly divided into wound sepsis group (n = 30) and control group (n = 30, with scald). Pseudomonas aeruginosa was inoculated to the wounds in sepsis group. Corresponding indices were determined to verify the diagnosis of wound sepsis. The rats were sacrificed and fresh liver tissues were obtained at 96 post-scald hours (PSH). Total RNA of liver was isolated with Trizol and the different expression of lipid metabolism related genes in response to burn wound sepsis was assessed by DNA microarray. RESULTS: By comparing expression profile of the two groups, totally 47 genes were observed to be differentially expressed in rat hepatic tissues, among them 9 genes were related to lipid metabolism. Among them, those which were upregulated were genes in relation with transportation and activation of fatty acid, and those downregulated were genes related in providing energy for fatty acid oxidation in mitochondria. CONCLUSION: The occurrence of wound sepsis in scalded rats can induce changes in the expression of hepatic lipid metabolism related genes in hepatic tissues, and aggravate

[The influence of lipopolysaccharide on adipose metabolism in liver during shock stage of scalded rats].

OBJECTIVE: To investigate the influence of lipopolysaccharide (LPS) on adipose metabolism in liver during shock stage of scalded rats. METHODS: Sixty adult Wistar rats were inflicted with 30% TBSA full thickness scald and were randomly divided into 3 groups: i. e. sham group (control, n = 20), simple scald group [(n = 20) and LPS group (n = 20, with intra-peritoneal injection of 3.0 mg/kg LPS at 2 postscald hour (PSH)]. The contents of LPS, tumor necrosis factor alpha (TNF-alpha), free fatty acids (FFA) in plasma and adenosine triphosphate (ATP), triglyceride (TG), malonaldehyde (MDA) in liver in each group were determined at 24 and 48 PSH. The histological changes in hepatic tissue in each group were also observed. RESULTS: The plasma contents of FFA in LPS group at 24 and 48 PSH were 2.3 +/- 0.3 mmol/L and 2.5 +/- 0.4 mmol/L, respectively, which were obviously higher than those in control (0.4 +/- 0.3 mmol/L, 0.5 +/- 0.3 mmol/L) and scald (0.9 +/- 0.3, 1.2 +/- 0.5 mmol/L, P <0.01) groups. Meanwhile, there was obvious difference in the contents of TG and ATP in liver between LPS group (TG: 530 +/- 30 mmol/g, ATP: 1.7 +/- 0.5 micromol/g) and scald group (TG: 242 +/- 27 mmol/g, ATP: 6.0 +/- 2.4 micromol/g, P < 0.01). Pathological examination revealed that adipose denaturalization and injury to mitochondria in hepatocytes in scald group were significantly milder than those in LPS group. The morphology of hepatocyte in control group appeared normal. CONCLUSION: LPS challenge to burn subjects could induce impairment in utilizing fat derived energy, and it would aggravate adipose denaturalization in the liver.

[Effect of growth hormone on non-protein respiratory quotient and oxidation rate of protein after burn injury].

OBJECTIVE: To investigate the changes in non-protein respiratory quotient (NPRQ) and oxidation rate of protein after burn injury, and the effect of growth hormone (GH) administration on metabolism. METHODS: Sixteen male minipigs were subjected to 35% total body surface area (TBSA) full thickness burn, and the burned minipigs were randomly divided into two groups: GH group and control group. NPRQ and oxidation rate of protein were monitored by means of the metabolic cart. RESULTS: After burns the oxygenation rate of protein increased slowly, with NPRQ reduced significantly. The extent of reduction was higher in GH group compared to the control group, but there was no significant difference in oxidation rate between the two groups. CONCLUSION: The results suggest that administration of GH could enhance lipid catabolism, with no obvious effect on protein catabolism. However, hyperglycemia is apt to occur.

[The effects of early escharectomy on resting energy expenditure in the severely burned patients].

OBJECTIVE: To investigate the effect of early escharectomy on resting energy expenditure (REE) in severely burned patients dynamically with the metabolic monitoring and diagnostic system. METHODS: Fifty-six adult male patients with severe burns were divided into early escharectomy (group A, n = 39, escharectomy within 5 PBDs) and non-early escharectomy (group B, n = 17, escharectomy after 5 PBDs) groups. The wounds of full thickness and deep partial thickness burn in the two groups were all excised and covered with allogeneic skin and autologous micro-skin in the first operation. The changes in REE were observed dynamically at the bedside of the patients with the metabolic monitoring and diagnostic system. The plasma contents of IL-6, IL-8, TNF-alpha and LPS from 9 patients in group A and 7 in group B were also determined dynamically. RESULTS: All patients survived. The REE in both groups was elevated markedly, but REE in group A was lower compared with group B before and after escharectomy within 14 days. (P < 0.05). The plasma level of IL-6, IL-8, TNF-alpha and LPS in group A were obviously lower than those in group B (P < 0.05). CONCLUSION: The hypermetabolic response of burn patients with severe burns could be lowered by early escharectomy, and it seemed to be related to the decrease of the release of proinflammatory mediators.