ObjectiveTo review the research progress of constructing injectable tissue engineered adipose tissue by adipose-derived stem cells (ADSCs). MethodsRecent literature about ADSCs composite three-dimensional scaffold to construct injectable tissue engineered adipose tissue is summarized, mainly on the characteristics of ADSCs, innovation of injectable scaffold, and methods to promote blood supply. ResultsADSCs have a sufficient amount and powerful ability such as secretion, excellent compatibility with injectable scaffold, plus with methods of promoting blood supply, which can build forms of injectable tissue engineered adipose tissue. ConclusionIn despite of many problems to be dealt with, ADSCs constructing injectable tissue engineered adipose tissue may provide a promising source for soft-tissue defect repair and plastic surgery.
The application of stem cell therapy for ischemic heart disease has aroused widespread interest. There have been many experimental studies concerning a variety of tissue stem cells such as bone marrow，blood，skin and skeletalmuscle stem cells，and their origins, differentiation and protein expressions are compared. In recent years，it is found that adipose-derived stromal cells （ADSCs） have potential advantages over other types of stem cells in that they are widely available and easily harvested through a simple liposuction procedure，and have a high regenerative capacity and therapeuticpotential for myocardial infarction. This review describes molecular and biological properties of ADSCs，their differentiationpotential，and regenerative and therapeutic potential for myocardial repair.
Objective To review the latest progress in the major biological properties of adipose-derived stem cells (ADSCs) and ADSCs assisted autologous lipotransfer in breast repair and reconstruction. Methods Recent literature about ADSCs assisted autologous lipotransfer in breast repair and reconstruction was reviewed. Results ADSCs have multipotential differentiation capacity, and they could promote angiogenesis and regulate immune reactions. ADSCs assisted autologous lipotransfer can obtain satisfactory effectiveness in breast repair and reconstruction with few complications, but more studies are needed to confirm the long-term safety. Conclusion ADSCs assisted autologous lipotransfer has good effectiveness in breast repaired and reconstruction. But further clinical trials are needed to confirm the long-term safety.
Objective To investigate the effects of the misshapen auricular chondrocytes from microtia in inducing chondrogenesis of human adipose derived stem cells (ADSCs) in vitro. Methods Human ADSCs at passage 3 and misshapen auricular chondrocytes at passage 2 were harvested and mixed at a ratio of 7 ∶ 3 as experimental group (group A, 1.0 × 106 mixed cells). Misshapen auricular chondrocytes or ADSCs at the same cell number served as control groups (groups B and C, respectively). All samples were incubated in the centrifuge tubes. At 28 days after incubation, the morphological examination was done and the wet weight was measured; the content of glycosaminoglycan (GAG) was detected by Alcian blue colorimetry; the expressions of collagen type II and Aggrecan were determined with RT-PCR; and HE staining, toluidine blue staining, Safranin O staining of GAG, and collagen type II immunohistochemical staining were used for histological and immunohistochemical observations. Results At 28 days after incubation, all specimens formed disc tissue that was translucent and white with smooth surface and good elasticity in groups A and B; the specimens shrank into yellow spherical tissue without elasticity in group C. The wet weight and GAG content of specimens in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in the wet weight (t=1.820 3, P=0.068 7) and in GAG content (t=1.861 4, P=0.062 7). In groups A and B, obvious expressions of collagen type II and Aggrecan mRNA could be detected by RT-PCR, but no obvious expressions were observed in group C; the expressions in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in collagen type II mRNA expression (t=1.457 6, P=0.144 9) and Aggrecan mRNA expression (t=1.519 5, P=0.128 6). Mature cartilage lacunas and different degrees of dyeing for the extracellular matrix could be observed in groups A and B; no mature cartilage lacunas or collagen type II could be observed in group C. The expression of collagen type II around cartilage lacuna was observed in groups A and B, but no expression in group C; the gray values of groups A and B were significantly lower than that of group C (P lt; 0.01), but no significant difference was found between groups A and B (t=1.661 5, P=0.09 7 0). Conclusion Misshapen auricular chondrocytes from microtia can induce chondrogenic differentiation of human ADSCs in vitro.
Objective To evaluate the synergistic effect of bone morphogenetic protein 14 (BMP-14) and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells (ADSCs) so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits (weighing, 1.5 kg and 2.0 kg). The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction (alizarin red staining), chondrogenic induction (alcian blue staining), and adipogenic induction (oil red O staining). The optimum multiplicity of infection (MOI) of transfection of adenovirus-cytomegalovirus (CMV)-BMP-14-internal ribosome entry site (IRES)-human renilla reniformis green fluorescent protein 1 (hrGFP-1) was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups: group A, co-culture of ADSCs transfected by BMP-14 and chondrocytes (1 ∶ 1 in Transwell chambers); group B, co-culture of ADSCs and chondrocytes (1 ∶ 1 in Transwell chambers); group C, culture of ADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan (GAG) content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C (P lt; 0.05), and groups B and D were significantly higher than group E (P lt; 0.05), but no significant difference was found between groups B and D (P gt; 0.05). Conclusion It can promote differentiation of ADSCs into chondrocytes by BMP-14 co-culture with chondrocytes, and they have a synergistic effect.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Objective To study biological rule of recombinant human bone morphogenetic protein 2 (rhBMP-2) in regulating the expression of vascular endothelial growth factor (VEGF) of adipose-derived stem cells (ADSCs) at different induced concentrations and time points at gene level and protein level. Methods ADSCs were separated from adult human adipose tissues and cultured until passage 3. After ADSCs were induced by rhBMP-2 in concentrations of 0, 50, 100, and 200 ng/ mL respectively for 24 hours, and by 100 ng/mL rhBMP-2 for 3, 6, 12, 18, 24, 36, and 48 hours (ADSCs were not induced at corresponding time point as controls) respectively, the VEGF mRNA and protein expressions were detected by RT-PCR and ELISA. Results The VEGF mRNA and protein expressions induced by rhBMP-2 were concentration-dependent; the expressions were highest in a concentration of 100 ng/mL. The VEGF mRNA expression in concentrations of 50, 100, and 200 ng/mL were significantly higher than that in a concentration of 0 ng/mL (P lt; 0.05); and the expression in concentration of 100 ng/ mL was significantly higher than that in concentrations of 50 and 200 ng/mL (P lt; 0.05). The VEGF protein expression in a concentration of 100 ng/mL was significantly higher than that in the other concentrations (P lt; 0.05). The VEGF mRNA and protein expressions induced by rhBMP-2 were time-dependent. The VEGF mRNA and protein expressions at 3 and 6 hours after induction were significantly lower than those of non-induced ADSCs (P lt; 0.05); the expressions were lower at 12 hours after induction, showing no significant difference when compared with those of non-induced ADSCs (P gt; 0.05); the expressions reached peak at 18 and 24 hours after induction, and were significantly higher than those of non-induced ADSCs (P lt; 0.05); the expressions decreased in induced and non-induced ADSCs at 36 and 48 hours, showing no significant difference between induced and non-induced ADSCs (P gt; 0.05). Conclusion rhBMP-2 adjusts VEGF expression of ADSCs in a concentration- and time-dependent manner. The optimum inductive concentration of rhBMP-2 is 100 ng/mL, induced to 18-24 hours is a key period when rhBMP-2 is used to promote tissue engineering bone vascularization.
Objective To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury. Methods ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells. Results The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05). Conclusion The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
Objective To introduce the related issues in the clinical translational application of adipose-derived stem cells (ASCs). Methods The latest papers were extensively reviewed, concerning the issues of ASCs production, management, transportation, use, and safety during clinical application. Results ASCs, as a new member of adult stem cells family, bring to wide application prospect in the field of regenerative medicine. Over 40 clinical trials using ASCs conducted in 15 countries have been registered on the website (http://www.clinicaltrials.gov) of the National Institutes of Health (NIH), suggesting that ASCs represents a promising approach to future cell-based therapies. In the clinical translational application, the related issues included the quality control standard that management and production should follow, the prevention measures of pathogenic microorganism pollution, the requirements of enzymes and related reagent in separation process, possible effect of donor site, age, and sex in sampling, low temperature storage, product transportation, and safety. Conclusion ASCs have the advantage of clinical translational application, much attention should be paid to these issues in clinical application to accelerate the clinical translation process.
【Abstract】 Objective To discuss the impact of adi pose-derived stem cells (ADSCs) combined with vascular bundle implantation on vascularized tissue engineering scaffolds in vivo so as to provide a theoretical basis for the repair ofavascular necrosis of the femoral head. Methods ADSCs were isolated from 4-month-old Sprague Dawley (SD) rats andcultured, then were induced to osteogenesis and identified. ADSCs at the 3rd passage were seeded on the nano-hydroxyapatide/ polyamide-66 (nHA/PA66) to prepare the composite scaffolds. The compound condition of cells and scaffold materials were observed under scanning electronic microscope (SEM). Twenty-four 4-month-old SD rats (weighing 350-400 g) were randomly divided into 3 groups (n=8). In group A and group B, the inferior epigastric artery and vein of rats were implanted into composite scaffold cultured for 10 days or simple nHA/PA66 scaffold, respectively. In group C, two composite scaffolds cultured for 10 days were embedded into quadriceps femoris muscle of both thighs, respectively. After 2 and 4 weeks of operation, angiogenesis was observed by HE staining and CD34 immunohistochemical staining. Results Cells isolated from adi pose were identified as ADSCs. SEM showed that the number of cells increased after being cultured for 10 days, cell morphology stretched fully with a shape of long spindle. HE staining and immunohistochemical staining showed that a large number of angiogenesis was observed around the implanted artery and vein in group A, which was superior to groups B and C in the number of blood vessels and the maturity of blood vessel wall. After 2 and 4 weeks of operation, the blood vessel density and blood vessel diameter were significantly higher in group A than in group B and group C, and in group B than in group C (P lt; 0.05). Conclusion Combined application of ADSCs and vascular bundle implantation can promote the degree of vascularization, which could make the scaffold vascularization rel iable.