[Growth and migration of umbilical cord mesenchymal stem cells on polycarbonate membrane with different pore sizes].

OBJECTIVE: To observe the growth and migration of human umbilical cord mesenchymal stem cells (hUCMSCs) on polycarbonate membrane with different pore sizes and explore the criteria of selecting optimal Transwell insert for indirect co-culture to induce the differentiation of hUCMSCs. METHODS: hUCMSCs were isolated in vitro and then expanded in culture medium. After the treatment of mitomycin C, the cells were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes of 0.4, 3.0 and 8.0 µm respectively. After culturing for 7 days, the cells were observed and counted on the bottom of each porous membrane. Then the calculation of migration ratio was performed. The growth and migration of hUCMSCs on porous membranes were also examined under scanning electron microscope (SEM). RESULTS: The migration ratios of hUCMSCs on membranes of 0.4, 3.0 and 8.0 µm pore sizes were 0, 1.8% and 8.0% respectively. The migration ratio of cells on 0.4 µm pore size membrane was statistically different from that of the other two pore size groups (P < 0.01). Under SEM, a small portion of cells were growing on the bottoms of membranes and moving through the pores. But there was no cell movement through 0.4 µm pore size membrane. CONCLUSIONS: hUCMSCs can migrate through the polycarbonate membranes of 3.0 µm and 8.0 µm pore sizes but not through the 0.4 µm one. Thus both sides of polycarbonate membrane of 0.4 µm pore size may be used for close indirect co-culture to induce the differentiation of hUCMSCs.

[Early changes in serum neutrophil elastase in rats with burn, blast injury or combined burn-blast injury and its significance].

OBJECTIVE: To explore the early changes in serum neutrophil elastase (NE) in rats with burn, blast injury or combined burn-blast injury and its significance. METHODS: A total of 176 male Sprague Dawley rats were randomly divided into four groups: control (C), burn (BU), blast injury (BL) and burn-blast combined injury (BB). Rats in C group were not injured. Animals in BU group were subjected to 25% TBSA full-thickness burn on back with 94 degrees C water for 12 seconds; Animals in BL group were inflicted with moderate blast injury with 5 g 8701 compressed dynamite stick as the explosion source 75 cm away while left chest facing the explosive source; Rats in BB group were burned immediately after the blast injury similarly as in BL group. During the first 24 h post-injury, animals in BU and BB groups received intraperitoneal injection of sodium lactate Ringer's solution at a dose of 50 ml x kg(-1) x 12 h(-1). Protein concentration in bronchoalveolar lavage fluid (BALF), water content of lung tissue and NE content in serum were determined at 0 h (C group), 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 7 d post-injury. RESULTS: Protein concentration in BALF, water content of lung tissue and NE content in serum in SD rats of the injured groups were significantly higher than those in C group (P < 0.01 or P < 0.05), peaked within 2 d post-injury, especially at 2 d post-injury (NE content in serum: BU group, 319. 85 +/- 19.50 ng/ml; BL group, 467.43 +/- 31.64 ng/ml; BB group, 626.00 +/- 26.38 ng/ml vs. C group, 78.53 +/- 25.10 ng/ml). Overall, protein concentration in BALF, water content of lung tissue and NE content in serum in BB group were significantly higher than BU and BL groups (P < 0.01 or P < 0.05). Correlation analysis showed that within 3 d postinjury, a significant positive correlation was found between the protein concentration in BALF, water content of lung tissue and NE content in serum (r = 0.7910, 0.8078, P < 0.05) in BU group. NE content in serum and protein concentration in BALF were significantly positively correlated in BB group (r = 0.8672, P < 0.05). CONCLUSION: NE may play an important role in early lung injury of burn or blast injury, especially in combined burn-blast injury.

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16-20 h is an effective and simple modified enzyme digestion method.

A novel model of burn-blast combined injury and its phasic changes of blood coagulation in rats.

Burn-blast combined injury has a complex pathological process that may cause adverse complications and difficulties in treatment. This study aims to establish a standard animal model of severe burn-blast combined injury in rats and also to investigate early phasic changes of blood coagulation. By using 54 Wistar rats, distance from explosion source (Hexogen) and size of burned body surface area were determined to induce severe burn-blast combined injury. Thereafter, 256 rats were randomly divided into four groups (n = 64): blast injury group, burn injury group, burn-blast combined injury group, and sham injury group. Gross anatomy and pathological changes in lungs were investigated at 3, 24, 72, and 168 h, respectively. Blood was also collected for analyzing coagulation parameters as prothrombin time, activated partial thromboplastin time, and plasma levels of fibrinogen, D-dimer, antithrombin III, and α2-antiplasmin from 0 to 168 h after injury. Severe burn-blast combined injury was induced by inflicting rats with a moderate blast injury when placing rats 75 cm away from explosion source and a full-thickness burn injury of 25% total body surface area. The rats with burn-blast combined injury had more severe lung injuries when compared with the other three groups. Pathological examination in the BBL group showed diffused alveolar hemorrhage, fluid filling, alveolar atelectasis, rupture and hyperplasia of partial alveolar septum, emphysema-like change, reduced capillary bed, and infiltration of extensive polymorphonuclear cells after injury. The blood of combined injured rats was in a hypercoagulable state within 24 h, shortly restored from 24 to 48 h, and rehypercoagulated from 48 to 72 h after injury. A secondary excessively fibrinolytic function was also found thereafter. The rat model of burn-blast combined injury was successfully established by simulating real explosion characteristics. Rats with burn-blast combined injuries suffered from more severe lung injuries and abnormal coagulation and fibrinolytic function than those induced by a burn injury or a blast injury component. Hence, a time-dependent treatment strategy on coagulation function should be emphasized in clinical therapy of burn-blast combined injury.

[Effect of different concentration of tamoxifen ointment on the expression of TGF-beta2 of hypertrophic scar at rabbit ears].

OBJECTIVE: To observe the effect of different concentration of Tamoxifen ointment on the fibroblasts and transforming growth factor (TGF-beta2) of hypertrophic scar at rabbit ears, so as to explore the possibility of treatment of hypertrophic scar with Tamoxifen. METHODS: The hypertrophic scar model was established in 96 New Zealand rabbits' ears. The wounds were divided into four groups (A, B, C and D), with 144 wounds in each group. Different concentration of tamoxifen ointment (0.5%, 1%, 2%) was topically administered in groups A, B and C respectively, and blank ointment in group D. On postoperative day 30, 60 and 90, the scar samples were harvested. The scar thickness, scar histological change and the content of TGF-beta2 were detected. RESULTS: (1) On the 30th day after operation, the difference of scar tissue thickness among groups A, D and B, C reached statistical significance (group A, D < group B < group C). However, there was a contrary tendency in fibroblasts density and TGF-beta2 content of the scar tissue simultaneously. (2) On 60th, 90th day after injury, there was statistical difference in scar thickness, fibroblasts density and the content of TGF-beta2 in scar of four groups (P < 0.05). The content of TGF-beta2 in group A, B, C, D was (43.97 +/- 3.63) microg/L, (41.92 +/- 3.91) microg/L, (36.69 +/- 4.15) microg/L, (54.90 +/- 4.71) microg/L, respectively, on 60th day; and (45.69 +/- 2.63) microg/L, (40.43 +/- 3.87) microg/L, (38.76 +/- 3.24) microg/L, (52.59 +/- 4.92) microg/L, respectively, on 90th day. The fibroblasts density of scar in groups A, B, C, D was (4392.07 +/- 327.84) point/mm2, (4208.57 +/- 329.76) point/mm2 (4 033.44 +/- 427.91) point/mm2, (4863.03 +/- 387.98) point/mm2, respectively, on 60th day; and (4418.41 +/- 432.52) point/mm2, (4077.65 +/- 386.70) point/mm2, (3844.53 +/- 354.29) point/mm2, (4838.64 +/- 390.52) point/mm2, respectively, on 90th day. The content of TGF-beta2 and fibroblasts density of scar were lined up as group D > group A > group B > group C (P < 0.05). CONCLUSIONS: Topical Tamoxifen can reduce the content of TGF-beta2 and fibroblast, decrease fibroblasts density and the formation of hypertrophic scar at rabbit ears. It offers a new way for the treatment of the hypertrophic scar.