Objective Extracellular matrix is one of the focus researches of the adi pose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sl iced into2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adi pose-derived stem cells (hADSCs) were isolated from adi pose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation abil ity. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Results Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibil ity. The CA and PI staining showed the seeded hADSCs had the potential of spread and prol iferation on the matrix. Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adi pose tissue engineering.
Objective To review the research status of the neovascularization of adi pose tissue engineering in the past decade so as to provide theoretical references for the development of the rapid revascularization of tissue engineered adi pose. Methods The l iterature about the revascularization of adi pose tissue engineering was extensively reviewed andanalyzed, centering on 5 elements: specificity of histological structures and blood supply, revascularization mechanism, coculture of different seed cells, modification of scaffold, and microenvironment. Results Adi pose tissue engineering offers a new solution for soft tissue defects. However, there is still the unfulfilled need in the size of engineered adipose tissue (less than 1 mL), which was determined by the degree of neovascularization in engineered tissue. Overall, rapid neovascularization in engineering tissue is a key l ink of experimental study changing into cl inical appl ication. Conclusion Providing a sufficient supply with nutrients and oxygen by means of a sufficient and rapid neovascularization will be at the heart of any attempts to obtain bigger tissue engineered adipose to meet the demand of repairing large soft tissue defect.
Objective Seed cells are the hotspot of tissue engineering research. To study the seed cells with high potential of adipogenic differentiation for applying the adipose tissue engineering and increasing the constructing efficiency of adipose tissue engineering. Methods Mature adipocytes (MA) and adipose-derived stromal cells (ADSCs) were harvestedfrom human fat aspirates via l iposuction by collagenase digestion. MA were cultured and induced to dedifferentiated adipocytes (DA) by ceil ing adherent culture method. DA and ADSCs were induced to adipogenic differentiation. The adipogenic abil ities of DA and ADSCs were compared by inverted phase contrast microscope observation, absorption spectrometry assay of oil red O staining, and cell counting of oil red O staining. Results MA could dedifferentiate into fibroblast-shaped DA. After adi pogenic differentiation, the inverted phase contrast microscope observation showed that there were much more l i pid droplet in DA than in ADSCs. Absorption spectrometry assay of oil red O staining showed there were significant l ipid droplet aggregation in DA 4 days of adipogenic induction. However, the same phenomenon could be observed in ADSCs at 10 days after differentiation. After 12 days, the absorption value of DA was higher than that of ADSCs, showing significant difference (P lt; 0.05). The cell counting of oil red O staining demonstrated that the adipogenic rates of DA and ADSCs were 65% ± 6% and 35% ± 5%, respectively, showing significant difference (P lt; 0.05). Conclusion The potential of adipogenic differentiation of DA is ber than that of ADSCs. DA is a promising seed cell of adipose tissue engineering.
【Abstract】 Objective To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibil ity for stem cells labell ing and tracer means. Methods Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro . The 3rd passage of ADSCs was incubated with BrdU at 5, 10, 15 and 20 μg/mL (groups A, B, C and D）for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labell ing. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labell ing index (positive rate) and the cells’ activity for different time after BrdU labell ing. The ADSCs without BrdU labell ing were used as control (Group E）. Results The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblastsand adipocytes under corresponding inductive medium. The ADSCs’ nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labell ing ratio increased in groups A, B, C and D after incubating 12 hours, the mean labell ing ratio were 30.6％ ±2.3％，32.4％ ±1.9％，45.8％ ±1.8％，50.8％ ±3.1％ , respectively, and the labell ing ratio of Group E was 0. There were significant differences between groups C, D and Group A (P lt; 0.01). The labell ing ratio of groups A, B, C and D were 45.9％ ±2.0％，87.9％ ±3.3％，90.6％ ±2.9％，91.7％ ±3.2％，respectively after 24 hours and the labell ing ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P lt; 0.01). The results of all groups after incubating48 hours and 72 h ours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences（P gt; 0.05）. Conclusion The most appropriate time for BrdU labell ing ADSCs is 48 hours, the most appropriate concentration is 10 μg/mL. The labell ing rate is high and cytotoxicity is l ittle.
Objective To investigate the possibility of theadipose tissue-derived stromal cells(ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutylmethylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extendedperiod with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and βⅢ-tubulin, characteristics of the neurons. Isobutyl-methyxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0.1%0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and βⅢ-tubulin, but some neuron-like cells also expressed thesmooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Ourresults support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stemcell source for transplantation related to the central nervous system.
Objective To review research progress of adipose tissuederived stromal cells (ADSCs).Methods The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated.Results A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes,endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate intomultiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells.Conclusion ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.
ObjectiveTo investigate the necessity of subcutaneous suture for gynecologic abdominal incision, and to prove the clinical value of suture without subcutaneous ligature. MethodsWe retrospectively studied the clinical data of 210 cases of gynecologic abdominal incision treated between May 2010 and May 2013. A total of 111 cases had the suture without subcutaneous ligature and 99 received the traditional suture. ResultsOne patient (0.90%) had fat liquefaction in the group of suture without subcutaneous ligature, while 7 (7.07%) of fat liquefaction were found in the traditional suture group, and the difference was statistically significant (χ2=3.883, P=0.049). No hospital infection occurred. The healing period averaged (15.1±4.7) days, and the patients were followed up for 2 months without any complication of abdominal incision in all the patients. ConclusionSuture without subcutaneous ligature is simple and easy to practice, with precise effect, which deserves clinical application.
ObjectiveTo study effect of expression levels of serum inflammatory factors and insulin receptor substrate(IRS)-1/2 in visceral adipose tissue after Roux-en-Y gastric bypass(RYGB) on type 2 diabetes mellitus(T2DM) rats, and explore possible mechanism in treatment of T2DM. MethodsThe T2DM rats models were established, which were divided into 3 groups by intervention: T2MD-RYGB group(n=14), T2MD-sham operation(T2MD-SO) group(n=10), and T2MD group(n=10), and 10 normal rats were selected as control group. The rats of the T2MD-RYGB group were received the RYGB, and of the T2MD-SO group were received transection and reanastomosis of the gastroin-testinal tract. The fasting plasma glucose(FPG), fasting insulin(FINS), C-reaction protein(CRP), tumor necrosis factor-α(TNF-α), free fatty acid(FFA), homestasis model assessment for insulin resistance(HOMA-IR), adipose tissue insulin resistance(Adipo-IR) were tested respectively before operation and on week 1, 4, 8 after operation(synchronous detec-tion of rats with or without surgical intervention). The IRS-1 and IRS-2 protein contents of the rat epididymal adipose tissue were tested on week 8 after operation. ResultsThe FPG, FINS, CRP, TNF-α, FFA levels, and HOMA-IR, Adipo-IR indexes in the T2DM rats were significantly higher than those in the normal rats(P < 0.05) before operation, the above indicators on week 4, 8 after operation were significantly lower than those before operation in the T2MD-RYGB group(P < 0.05). The differences of changes among the other groups were not statistically significant(P > 0.05). The IRS-1 and IRS-2 protein expressions in the adipose tissue of the rats were significantly increased in the T2MD-RYGB group as compared with these indicators in the T2MD group and T2MD-SO group(P < 0.05), but which were significantly lower than those in the control group(P < 0.05). ConclusionsRYGB could increase IRS-1/2 expression levels in adipose tissue, which could enhance insulin sensitivity, decrease serum inflammatory factors levels, and improve insulin resistance ultimately. This might be one of the mechanisms in treatment of T2DM.