Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.
To isolate and culture adi pose-derived stem cells (ADSCs), and to study the effects of the conditioned medium of ADSCs (ADSC-CM) treated with insul in on HaCaT cells. Methods ADSCs were isolated from adipose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5 × 104 cells/mL. The cells were divided into 2 groups: group A in which the cells were incubated in 1 × 10-7 mol/ Linsul in for 3 days, and group B in which the cells were not treated with insul in. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor (HGF). HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups: group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1 × 10-7 mol/ L insul in; group D1, 1 mL 2%FBS. Prol iferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration abil ity was measured by in vitro wound-heal ing assay 0, 12, 24, 36 and 48 hours after culture. Results The level of VEGF in groups A and B was (643.28 ± 63.57) and (286.52 ± 46.68) pg/mL, respectively, and the level of HGF in groups A and B was (929.95 ± 67.52) and (576.61 ± 84.29) pg/mL, respectively, suggesting differences were significant between two groups (Plt; 0.05). Cell prol iferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881 ± 0.039, 0.804 ± 0.041, 0.663 ± 0.027 and 0.652 ± 0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05). The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23% ± 1.98%, 8.82% ± 2.59%, 31.70% ± 8.85% and 29.60% ± 8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.05), group B1 was significantly higher than group A1 (P lt; 0.05). The migration distance of HaCaT cells in groups A1, B1,C1 and D1 at 36 hours was (0.184 6 ± 0.019 2), (0.159 8 ± 0.029 4), (0.059 2 ± 0.017 6) and (0.058 2 ± 0.012 3) mm, respectively, whereas at 48 hours, it was (0.231 8 ± 0.174 0), (0.205 1 ± 0.012 1), (0.079 2 ± 0.008 1) and (0.078 4 ± 0.011 7) mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05) at 36 and 48 hours, no significant difference was evident at other time points(P gt; 0.05). Conclusion ADSCs treated with insul in can significantly promote the prol iferation and the migration of HaCaT cells and inhibit their apoptosis.
Objective To find a kind of simple and effective method for purifying and label ing stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for cl inical application of SVFs. Methods The subcutaneous adi pose tissue were harvested form volunteers. The adi pose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase,respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, 1’-dioctadecyl-3, 3, 3’, 3’-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent label ing and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation. Results The number of SVFs was (138.68 ± 11.64) × 104, (183.80 ± 10.16) × 104, and (293.07 ± 8.31) × 104 in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P lt; 0.01). The rates of viable cells were 91% ± 2%, 90% ± 2%, and 81% ± 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P lt; 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs. Conclusion The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.
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.
Objective To review the mechanism of improved revascularization of free fat grafting with adipose-derived stem cells (ADSCs). Methods The literature related to the basic researches of ADSCs in free fat grafting and angiogenesis was reviewed. Results Angiogenesis is a sequence process in time and space which is regulated by various factors. ADSCs possess the capability of secreting many angiogenic growth factors and differentiating into various lineages.Conclusion ADSCs affect every process of angiogenesis with clear improved angiogenic effects, however, the mechanisms of angiogenic effects need the further researches.
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 introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared with BMSCs which are commonly used as adult stem cells, ADSCs and DFAT cells have extensive sources and can be easily acquired. While compared with ADSCs, DFAT cells have good homogeneity and b proliferation capacity. Conclusion As a potential source of stem cells, adipose tissue will provide a new promising for regenerative medicine.
ObjectiveTo summarize the research progress of adipose-derived stem cells (ADSCs) in promoting the repair of peripheral nerve injury.MethodsThe related literature at home and abroad in recent years was widely reviewed, the mechanism of ADSCs promoting the repair of peripheral nerve injury was introduced, and its basic research progress was analyzed and summarized. Finally, the clinical transformation application of ADSCs in the treatment of peripheral nerve injury was introduced, the existing problems were pointed out, and the new treatment regimen was prospected.ResultsADSCs have the function of differentiation and paracrine, and their secreted neurotrophic factors, antiapoptosis, and antioxidant factors can promote the repair of peripheral nerve injury.ConclusionADSCs are rich in content and easy to obtain, which has a definite effectiveness on the repair of peripheral nerve injury with potential clinical prospect.
ObjectiveTo investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.MethodsThe ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.ResultsADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).ConclusionADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
Objective To summarize the recent advances in the research of adipose-derived stem cells (ADSCs) for the treatment of refractory wounds. Methods The related literature about using ADSCs for treating refractory wounds in recent years was reviewed, and their repair mechanism and treatment progress were summarized in detail. Results Tremendous progress has been achieved in using ADSCs in combination with single stent technology, sheet technology, and other methods to promote the healing of refractory wounds. ADSCs can accelerate wound angiogenesis and promote the healing of refractory wounds through its own mechanisms of paracrine, proangiogenic, anti-oxidative and apoptosis. Conclusion With the advantages of adequate sources, easy to extract and culture, non-immune rejection, multidirectional differentiation potential, and significant angiogenic potential, ADSCs has become the ideal seed cells of tissue regeneration. However, it is necessary to improve stem cell transmission technology and develop biomaterials for clinical application in order to improve the refractory wounds healing.