Preparation and evaluation of extracellular matrix scaffold of human adipose tissue

To explore the possibility of building tissue-engineered adipose tissue and find a new approach for repairing soft tissue defects. Using enzymatic digestion, adipose tissue-derived stem cells (ASCs) were extracted from the lipid part of human liposuction aspirate, cultured, and underwent adipogenic induction or not. The adipogenic-induced and non-adipogenic-induced ASCs were labeled with 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate (DiI), a fluorescent marker, in vitro to be used as seed cells. Then, they were combined with injectable fibrin glue scaffold at 1 x 10(7)/ml cell density. Six athymic BALB/C mice underwent subcutaneous injection of adipogenic-induced ASCs with fibrin glue scaffold at the density of 1 x 10(7) cells/ml into the left side of the low back (induced group), subcutaneous injection of non-adipogenic-induced ASCs into the right side of the low back (non-induced group), and subcutaneous injection of injectable fibrin glue scaffold into the middle part of the neck (blank control group), with 0.2 ml per injection. Twelve weeks later the mice were killed and the implants were taken out. The wet weight was measured. HE and oil red O staining and light and fluorescence microscopy were used for morphological observation. Adipose tissue-like new-born tissues were found in the injection sites of the induced and un-induced groups. The average wet-weight of the induced group new-born tissue was (28 +/- 15) mg, significantly heavier than that of the un-induced group [(22 +/- 16) mg, P < 0.01]. HE staining and oil red O staining confirmed that the new-born tissue of the induced group was mature adipose tissue and DiI fluorescent staining approved its exogenousness. Most part of the new-born tissues of the un-induced group was fibroid tissue with only a few mature adipose tissues. ASCs extracted from the lipid part after liposuction can be used as seed cells, mixed, after adipose-induction, with injectable scaffold of fibrin glue, and injected into the body to build mature adipose tissue.

Objective To explore the possibility of building tissue-engineered adipose tissue with human adipose-derived stem cells (ADSCs) and adipose tissue extracellular matrix (ECM) scaffold,and provide experimental basis for clinical application of tissue-engineered adipose tissue for the repair of soft tissue defects.Methods ADSCs were isolated from adipose tissue by liposuction with the method of enzymatic digestion.The cells were induced for three-line differentiation and marked with DiI.Human ECM was extracted from adipose tissue obtained by liposuction.The ECM was freeze-dried,sterilized and crushed to powders.The surface characteristics of ECM were observed under the electron microscopy in vitro.The ADSCs were adhered to ECM powers,and the feasibility of ECM powers as scaffold was explorted.Human ADSCs in a density of 2 × 109/L were collected,and mixed them with ECM powers.They were transplanted subcutaneously into nude mice,and the opposite side of the same mice transplanted with scaffold only served as control group.There were 6 mice in total,and each side was transplanted by 0.5 ml.At 8th week post-transplantation,the mice were sacrificed and the implanted tissue were taken to record the wet weight,The hematoxylin and eosin (HE) sections and red oil O staining were detected.Two-sample test of SPSS 13.0 was used for statistical comparison between wet-determination.Results The ADSCs and human ECM powder were obtained successfully from adipose tissue.The ADSCs could differentiate into adipose cells,bone cells and chondrocytes.Scanning election microscopy (SEM) images showed that the powers had porous structure to which ADSCs could adhere easily,with the adhesion rate being (89.87 ±2.59)％.Eight weeks after transplantation,both experimental and control groups formed new tissues.There were significant differences between two groups ( P ＜ 0.05 ) in the wet weight.HE and red oil O staining confirmed that the experimental group could form mature adipose tissue.Conclusion ADSCs and human ECM scaffold could form tissue-engineered adipose tissuein vivo.It can be used as an ideal method to construct tissue-engineered adipose tissue. Key words: Adipose-derived stem cells; Extracellular matrix; Scaffold; Tissue engineering

To develop a primary culture method of human omental preadipocytes and to study their biological properties, such as hyperplasia, hypertrophy and endocrine secretion of human visceral adipose tissue. Using enzyme-digesting method, fibroblast-like cells from the human omental adipose tissues were cultured. The morphological changes of the cultured cells were observed and the growth curve was drawn by MTT method. The intracytoplasmic lipid of the cultured cells was determined by oil red O staining. The leptin and adiponectin levels in the culture supernatants were measured by ELISA. The cultured fibroblast-like cells were homogeneous. Proliferation of cells began at the 3 rd day and the cell numbers increased in indicial way from the 3 rd day to the 9 th day. The doubling time of cells was about 60 hours. During the process of induction by conditional medium, the cells became round and larger, and more adipose droplets were aggregated. On the 21 st day, more than 90% of the cells became adipocytes. Leptin secretion was detected at low level in the preadipocytes and continuously increased during differentiation, with a peak on day 17. It remained constant from day 17 onward. Unlike leptin, adiponectin secretion was not detected until day 7 after induction, when differentiated adipocytes had already been observed. Its secretion increased dramatically between days 7 and 17, and reached a maximum level on day 17, but had a significant reduction on day 21. Extraction of intracytoplasmic lipid stained with oil red O and detection of leptin and adiponectin both verified the isolated cells were preadipocytes functioning actively. A human omental preadipocytes model has been established and different secretion patterns of leptin and adiponectin secretion related to preadipocyte differentiation has been characterized. Adiponectin may be proposed as a specific marker for preadipocyte differentiation.

Objective To explore the feasibility of building the tissue-engineered adipose tissue of breast with adipose- derived stem cells (ASCs) and collagen sponge scaffolds.Methods Enzymatic digestion and centrifugalization were used to isolate ASCs from the inguinal fat pad in adult female Sprague Dawley rats (n=6).After expansion in culture medium,ASCs underwent adipogenic and osteogenic induction and were identified with oil red O staining and yon Kossa staining.For in vitro study,ASCs were seeded onto collagen scaffolds and cultured for 14 days.For in vivo study,ASCs were combined with collagen type Ⅰ sponge scaffolds before implantation.The 6 SD rats underwent implantation of autologous ASCs with collagen sponge into the right side of the upper breast (experimental group),and implantation of collagen sponge into the left side of the upper breast (control group).Twelve weeks later the rats were euthanatized to harvest the implants for wet weight measurement.HE and oil red O staining were used for morphological observation.Results Adipose tissue-like new-born tissues were found in the implantation sites in the experimental group,while collagen sponge-like new-born tissues were found in the implantation sites in the control group.The average wet-weight of the new-born tissue in the experimental group was (121 ±9) mg,significantly heavier than that in the control group [ (77±6) mg,P＜0.05].HE staining and oil red O staining confirmed that most of the new-born tissues in the experimental group were mature adipose tissues,while most of the new-born tissues in the control group were collagen fibers.Conclusion Autologous ASCs combined with collagen type Ⅰ scaffolds can be implanted into the breast to build mature adipose tissue in rats. Key words: Adipose-derived stem cells; Tissue engineering; Type Ⅰ collagen; Rat

Objective To assess the osteogenic ability after co-culture BMSC and ADSC in vivo and in vitro. Methods ADSC and BMSC were obtained by adherent screening method and enzymatic digestion method. Flow cytometry was used to confirm the phenotypes of ADSC and BMSC. Oil red O was used to induce MSC to fat. Alkaline phosphatase (ALP) and alizarin red staining were used in osteogenic group. This sample was divided into four groups, no-induced stem cells group; BMSC osteogenic induction group; ADSC osteogenic induction group; co-culture of BMSC and ADSC osteogenic induction group. ALP activities and Calcium absorbance were determined during different periods of osteogenic introduction. OCN and Runx2 expression level were tested via RT-PCR and western blot methods after osteogenic induction for 2 weeks. Furthermore, cells in each group were seeded on HA/CS/PLLA composite scaffolds, and the scaffolds with cells were planted into bone defects in rat models. The rats were sacrificed by overdose anesthesia at 8 weeks after surgery and the scaffolds were removed for further analysis. Results Oil red O staining demonstrated red after adipogenic induction. Alkaline phosphatase and Alizarin red staining showed flaky red under condition of osteogenic induction. There had no statistical change among each group after osteogenic induction for 3 days, and ALP activity significantly increased after osteogenic induction for 5 days. Meanwhile, the ALP activity in co-culture of BMSC and ADSC group was markedly higher than the other three groups. However, there had no significant change in A value of calcium absorbance among each group after osteogenic induction for 7 days, while it increased at 14th day and ALP activity in co-culture of BMSC and ADSC group was significantly higher than the other three groups. After osteogenic induction for 2 weeks, the mRNA expression of OCN and Runx2 in co-culture of BMSC and ADSC group was 78.24±8.11 and 1 180.13±121.16 respectively, and the protein expression of OCN and Runx2 was 6.54±0.59 and 4.43±0.51. These mRNA and protein expression level in co-culture of BMSC and ADSC group enhanced significant compared with the other 3 groups. Histological assay demonstrated that the new bone tissues formed in co-culture of BMSC and ADSC group were 497.75±7.44 μm2, which was larger than that in the other 3 groups at 8 weeks after implantation. Conclusion Co-culture BMSC and ADSC may up-regulated the osteogenic ability in vivo and in vitro. Key words: Mesenchymal stem cells; Bone marrow; Adipose tissue; Coculture techniques; Tissue engineering

Objective To study the effects of three different granular fats on proliferation, differentiation and migration of adipose-derived stem cells. Methods Ten patients were selected for lumbar liposuction. The adipose tissue was obtained with different sized side-hole fat aspiration devices: 10 ml of Macrofat (n=10), 10 ml of Microfat (n=10) and 10 ml of Nanofat (n=10). Enzyme digestion method was used to separate and extract adipose-derived stem cells(ADSCs). The differences of vascular matrix components in these 3 kinds of fat sources were analyzed. XTT assay was used to detect proliferation and growth ability. The migration ability of the cell injury model was observed in vitro, and the multi-directional differentiation ability was compared by the analysis of adipogenic and osteogenic induction. The experimental data were analyzed using SPSS 13.0 software. One-Way ANOVA was used to compare the difference of multiple groups. P<0.05 was considered as statistically significant. Results The detection of adipose-derived stem cells was by flow cytometry and it showed Macrofat, Microfat and Nanofat was(4.23±0.37)×105, (2.29±0.33)×105 and(1.56±0.16)×105. The content of fat-derived stem cells in Macrofat was the highest, followed by Microfat, and the content of Nanofat was the least (F=209.533, P<0.001). XTT assay showed that the proliferative ability of adipose-derived stem cells in the first two generations of Macrofat was the highest, followed by Microfat, while the proliferation of Nanofat was the lowest (the absorbance in 3 groups increased in a time-dependent manner). There was no significant difference in the proliferative capacity of the third generation of 3 groups (the absorbance of these 3 groups were compared at each time point). The cellular trauma model showed that the first generation of Macrofat-derived stem cells had the best migration ability, followed by Microfat, while the Nanofat had the weakest migration ability(Compared with the remaining area of wounds at 0 h, 12 h, 24 h point between the three groups, F=306.370, 1409.907, P<0.001). From the second generation, the migration ability of each group of ADSCs gradually self-repaired, and the migration ability at 12 h was similar to that of the first generation (F=11.410, P<0.001), but there was no significant difference in 24 h (F=0.070, P=0.933). Oil red O and alizarin red staining showed that the first generation of Macrofat and Microfat had better fat-forming and osteogenic differentiation ability than Nanofat(F=523.532, 620.022, P<0.001). However, there was no significant change after the second generation (F=2.144, 0.866, P=0.137, 0.432). Conclusions In the process of adipose tissue extraction of Nanofat, the production and activity of adipose-derived stem cells was impacted. However, in the process of culture and passage, the cell activity, proliferation ability, migration ability and differentiation ability can be achieved through self-repair, evenclose to the level of Macrofat or Microfat. Key words: Granular fat; Nanofat; Adipose-derived stem cells; Proliferation; Differentiation; Migration

Objective We observed the potential of the rat penile corpus cavernosum muscle-derived stem cells (MDSCs) differentiating into adipose cells in the presence of adipose differentiation culture medium in vitro.Methods MDSCs were purified through density gradient centrifugation and differential attachment.PP6 cells were obtained and detected expression of stem cell markers Sca-1 by immunofluorescence.The third passages PP6 cells were induced to differentiate to adipose cells within 21 days of adipose differentiation culture medium stimulation,while the control group used stem cells culture medium.Induced cells was detected by oil red O staining at 0,7,14 and 21 d respectively in order to observed whether there was a lipid droplets.Results PP6 cells expressed stem cell markers Sca-1 and PP1 cells hardly expressed Sca-1.On the first three days,we observed that the shape of cells had changed from long spindle-shape to circular or triangular,closely arranging as imbricate,meanwhile nucleus clarity and proliferation ability of cells had significantly slowed down.Both of groups had not changed by oil red O staining.On the seventh day,we observed that a small amount of high refractive small lipid droplets was in cytoplasm under phase contrast microscope,uniform size,which mainly concentrated around the cells nucleus,and was dyed red by oil red O staining.On the fourteenth day,the number of lipid droplets in cells was increased,lipid droplets blended into large,and the cells nucleus were crowded to one side or disappeared.On the twentieth day,cell differentiation,lipid droplets volume and number in cytoplasm reached to peak,and even continued to induce,but lipid droplets did not increased.In control group,however,cell morphology did not change and oil red O staining was negative.Conclusion Rat corpus cavernosum MDSCs have potential to differentiate into adipose cells,and MDSCs multi-directional differentiation potential is further confirmed. Key words: Muscle-derived stem cells; Differentiation; Adipose cells; Oil red O

Extracellular matrix proteins in organ transplantation.

Introduction: Mesenchymal stem cells (MSCs) exhibit multiple beneficial properties in treatment of various diseases through their differentiation capacity and secretion of paracrine signals. Therefore, using MSCs as a tool for cell therapy rely on the ability of harvesting a sufficient number of cells in a short time. Methods: In this study, MSCs derived from bone marrow (BM) and adipose tissue (AD) were investigated in terms of success rate of culture and cell yields. To ensure that the isolated cells are MSCs, adipogenic and osteogenic differentiation study were performed by Oil Red O, Alizarin Red S and Alkaline Phosphatase assay, and specific surface markers expression examined by flow cytometry. Results: The results showed that successful culture's rate of AD-MSCs were superior to BM-MSCs. AD-MSCs reached to confluency and passage in a shorter time (faster) and produced further amount of cells at a defined time. It was also demonstrated that the derivation of MSCs from adipose tissue were completely reproducible and not donor dependent rather than BM-MSCs. Conclusion: In conclusion, with our protocol, adipose tissues are proposed the more appropriate source for derivation of MSCs in terms of percentage of cells successfully isolated and subsequent purification as compared to bone marrow.

Objective To investigate the effect of peroxisome hyperplasia activated receptor γ(PPAR-γ) agonist(PGZ) on the differentiation and function of nonadrenergic primary brown adipose tissue cells in mice with high fat diet-induced obesity, and try to provide new treatment for obesity and type 2 diabetes. Methods Primary BAT cells were separated from high fat diet-induced C57BL/6J obesity(HFD) mice and cultured in DMEM culture medium.The primary BAT cells were divided into 10 culture dishes equally, and assigned as PGZ treatment group, and control group(treated with normal saline), 5 dishes each group. The effects of PGZ on BAT cells were assessed by quantitative real-time polymerase chain reaction(RT-PCR), Gene of BAT: uncoupling protein-1(UCP-1); elongase of very long chain fatty acids(ELOVL3); PPAR-γ co-activator-1α(PGC1-α); PPAR-γ co-activator-1β(PGC1-β); PRD1-BF-1-RIZ1 homologous domain containing protein-16(PRDM16); CCAAT/enhancer binding protein β(CEBP/β); adiponectin: adipocyte fatty acid-binding protein2(AP2); cytochrome c oxidase1(CYC1); mitochondrial transcription factor A(TFAM). Western blotting was used to assess the expression of UCP-1 protein; and Oil red O staining was applied to measure the adipogenesis function of BAT. The t test is used between two groups, ANOVA and LSD test is used between multiple groups. Results The expression of mRNA associated with BAT chararcteristic genes(UCP-1、ELOVL3、PGC1-α、PGC1-β), lipogeneic genes(AP2), mitochondrial genes(CYC1、TFAM), and differential genes(PRDM16、CEBP/β) increased in PGZ treated group when compared with those in control group, the relative expression of the genes in the two groups were listed as following: UCP-1: 1100.0±612.0, 2.0±0.4; ELOVL3: 1461.0±617.0, 2.0±1.2; PGC1-α: 8.1±2.8, 2.0±1.1; PGC1-β: 8.3±2.8, 2.0±1.3; adiponectin: 2.6±0.8, 1.0±0.7; AP2: 5.1±2.2, 1.00±0.24; CYC1: 3.1±0.8, 1.0±0.4; TFAM: 1.2±0.4, 1.00±0.25; PRDM16: 4.8±2.6, 2.0±0.3; CEBP/β: 6×108±5×108, 2.0±0.6; t=2.45-5.22, all P<0.05; the expression of BAT functional protein UCP-1 assessed by Western blotting increased in PGZ treated group than that in control(1.24±0.25 vs 1.00±0.14, t=2.63, P<0.05); the function of BAT adipogenesis measured by oil red O test increased in PGZ treated group than that in control (1.2±0.2 vs 1.0±0.1, t=2.45, P<0.05). Conclusion PPAR-γ agonist-pioglitazone(PGZ) may promote the differentiation and function of brown adipocytes. This effect may be one of the reasons for PGZ improving metabolism. Key words: Pioglitazone; Brown adipose tissue; Nonadrenergic; Uncoupling protein-1

To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes (DA) as seed cells. Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic, osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining, alizarin bordeaux staining and alcian blue staining, respectively. Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45, CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.

Background Inflammation and adipogenesis are two parallel processes with increasing activity in severe thyroid-associated ophthalmopathy(TAO),and mevastatin was proved to have the inhibiting effect on the differentiation of adipose.Objective The aim of this work was to investigate the effects of mevastatin on the expression of cyclooxygenase-2(COX-2)and peroxisome proliferator activated receptor-γ(PPAR-γ)and differentiation of TAO-derived orbital preadipocytes,and explore its modulation effects on lipopolysaccharide(LPS)induced inflammation and the differentiation of TAO-derived orbital preadipocytes in vitro.Methods The retroorbital adipose tissue was obtained from 4 TAO patients during the surgery.The orbital fibroblasts were cultured from orbital adipose tissues using explant culture method.To study the suppressing effect of mevastatin on inflammatory response,cultured cells were divided into 5 groups.The 1000 μg/L LPS orbital fibroblasts were stimulated for 8 hours in group A,and 1000 μg/L LPS combined with 5 μmol/L,10 μmoL/L or 20 μmoL/L mevastatin were used respectively for the substitute in the group B,group C and group D.The orbital fibroblasts in group E were cultured routinely without any intervention as control.To observe the inhibiting effect of mevastatin on the differentiation of adipose,the group A were then subdivided into group A1-A6.After 1000 μg/L LPS was used to treat the cells for 8 hours,the ceils were induced to differentiate into adipocytes.All orbital preadipocytes from A1 to A6 were stimulated to differentiate into mature adipocytes with cocktail differentiation medium for a 10-day duration.During the procedure,group A2,A3 and A4 were interfered with 5,10 or 20 μmol/L mevastatin,and in the group A5 and A6,10 μmol/L mevastatin were added at the fourth day or eighth day.Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining.The absorption(A492 nm)was measured in the ceils by enzyme-linked immunosorbent assay(ELISA).Expression of COX-2 and PPAR-γ mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR),and the expression of COX-2 and PPAR-γ protein was detected by Westernblot.The level of PGE2 in the supernatant was detected by ELISA.Results The expression of COX-2 protein and mRNA as well as the PGE2 levels in B,C,D group decreased markedly in comparison with those in A group(P＜0.05).With the increase of mevastatin concentration,the expression of COX-2 protein and mRNA as well as the PGE2 levels in B,C,D groups decreased successively(F =228.380,101.745,1586.881,P＜0.05).The expression of COX-2 protein and mRNA and PGE2 levels in E group were lower significantly than those in A,B and C groups(P＜0.05),but no significant differences were found between E group and D group(P＞0.05).The A492 value and the expressions of PPAR-γ protein and mRNA in differentiated cells showed the successively decrease in A1-A4 group with the elevation of mevastatin concentration(P＜0.05),and the evidently decreased A492 value and the expressions of PPAR-γ protein and mRNA also were seen in A1 and A5 groups compared with A3 group(P ＜ 0.05).Conclusions Mevastatin inhibits LPS-induced COX-2 expression,PPAR-γ expression,PGE2 secretion and differentiation of TAO-derived orbital fibroblasts in vitro in dose-dependent manner.Mevastatin plays these effect more prominently in early stage of adipocytes differentiation. Key words: Thyroid-associated ophthalmopathy; Preadipocytes; Fibroblast; Lipopolysaccharide; Peroxisome proliferator activated receptor-γ; Cyclooxygenase-2

Objective To explore the effect of Wntless (Wls)-mediated Wnt signaling on the development and energy metabolism of brown adipose tissue (BAT). Methods BAT-specific Wls knockout (WlsMyf5Δ/Δ) mice were generated by Cre-loxP system. The differentiations of BAT in WlsMyf5Δ/Δ knockout mice and Wlsfl/fl control mice were analyzed by histological morphology, immunohistochemistry, real-time PCR, and Western blot. After stromal vascular fraction (SVF) cells in BAT were induced to differentiate, oil red O staining, real-time PCR, and cell respiration experiments were performed for analyzing in-vitro cell differentiation and oxygen consumption. The energy metabolism of mice was monitored by rectal temperature, oxygen consumption rate in BAT, and energy expenditure. The adiposity of mice was evaluated by NMR while the glucose metabolism was analyzed by the glucose and insulin tolerance tests. Results The WlsMyf5Δ/Δ knockout mice appeared smaller body size, lower weight, higher percentage of lean fat, lower size of BAT, with higher body temperature on the back as compared to Wlsfl/fl control mice. The differentiation and thermogenesis of BAT in Wls-deficient mice were relatively augmented, along with an increase in Ucp1 mRNA and protein expressions. SVF cells from BAT in WlsMyf5Δ/Δ knockout mice revealed enhanced brown differentiation. Adiposity was decreased and glucose metabolic capacity was enhanced in the WlsMyf5Δ/Δ knockout mice, without significant change in oxygen consumption of the whole body. Conclusion Wls-mediated Wnt signaling decreases the thermogenesis and glucose metabolism of BAT by suppressing its differentiation. Key words: Wnt signaling pathway; Wntless gene; Brown adipose tissue

A cell-derived extracellular matrix (ECM) scaffold was constructed using cultured porcine chondrocytes via a freeze-drying method, and its ability to promote cartilage formation was evaluated in vitro. Scanning electron microscope (SEM) revealed that the scaffold had highly uniform porous microstructure. Then, rabbit chondrocytes were seeded dynamically on ECM scaffold and cultured for 2 days, 1, 2, and 4 weeks in vitro for analysis. Polyglycolic acid (PGA) scaffold was used as a control. On gross observation of neocartilage tissue, a silvery white cartilage-like tissue was observed after 1 week of culture in ECM scaffold, while similar morphology was seen only after 4 weeks in PGA scaffold. The volume of neocartilage-like tissue was significantly increased in both ECM and PGA groups. The compressive strength was gradually increased with time in ECM group, while gradually decreased in PGA group. DNA, glycosaminoglycan (GAG) and collagen contents also increased gradually with time in both groups, but showed more significant increase in ECM group. Histological staining for GAG (Safranin O staining) and type II collagen (immunohistochemistry) showed sustained accumulation of ECM molecules with time, which gradually and uniformly filled porous space in ECM scaffold. On the contrary, they accumulated only at the peripheral area of PGA scaffold. These results suggest that a novel cell-derived ECM scaffold can provide a promising environment for generating a high quality cartilage in vitro.

To investigate the effects of hypoxia chemically induced by CoCl2 on the expression of monocyte chemoattractant protein-1 ( MCP-1 ) in mouse 3T3-L1 adipocytes.3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes.Cell differentiation and lipid accumulation was determined by Oil Red O staining.CoCl2 was used as a chemical hypoxia-inducible reagent to mimic hypoxic microenvironment.The effect of CoCl2 on cell viability was estimated by MTT assay.Hypoxia-inducible factor-1α(HIF-1α) expression under hypoxia was detected by realtime fluorescent PCR and Western blot,while MCP-1 expression was detected by real-time fluorescent PCR and ELISA.CoCl2 induced hypoxia led to a marked recruitment of HIF-1α in mouse 3T3-L1adipocytes.Similarly,both mRNA and protein levels of MCP-1 were up-regulated.Exposure of 3T3-L1 adipocytes to CoCl2 induced hypoxic microenvironment in vitro,and hypoxic induction of MCP-1 expression and secretion may be mediated by HIF-1 α and may contribute to chronic low grade inflammation in adipose tissue. Key words: 3T3-L1 adipocytes; CoCl2; Hypoxia; Hypoxia-inducible factor-1α; Monocyte chemoattractant protein-1