Objective To investigate the possibility of enhancing the inducing rate of adipose-derived stem cells (ASCs) into epidermal cells in the medium containing all-trans retinoic acid (ATRA) by supplementing with HaCaT condition medium. Methods ASCs were isolated and identified by detecting the expression of CD34, CD45, CD73, CD90, and CD105 with flow cytometry and differentiating into adipose and osteoblast lineage in the induction medium. The air-liquid interface cell culture model was established with the Transwell Room. The induction medium A contained ATRA, epidermal growth factor (EGF), and keratinocyte growth factor (KGF), while the induction medium B contained ATRA, EGF, KGF, and HaCaT condition medium. Experiment was divided into three groups cultured for 12 days: induction medium A (group A), induction medium B (group B), basic medium (group C). The epidermal cell surface markers: cytokeratin (CK) 14, 15, 16, 19 (Pan-CK) were detected by flow cytometry and CK14 were identified by immunofluorescence stain. Results After induction for 12 days, flow cytometry showed that the positive rate of Pan-CK in group B [(22.0±3.5)%] was higher than that in group A [(11.9±2.7)%], which were both higher than that in group C [(1.1±0.3)%], and the differences were statistical significantly (P<0.01). Immunofluorescence stain showed that the positive rate of CK14 in group B was higher than that in group A [(19.5±7.0)%vs. (10.8±5.7)%, P<0.01], and the expression of CK14 was negative in group C. Conclusion HaCaT condition medium can enhance the ability of ASCs differentiation into epidermal cells in the culture medium containing ATRA.
ObjectiveTo explore the feasibility of three-dimensional (3D) bioprinted adipose-derived stem cells (ADSCs) combined with gelatin methacryloyl (GelMA) to construct tissue engineered cartilage.MethodsAdipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs (hADSCs). The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink. The hADSCs-GelMA composite scaffold was prepared by 3D bioprinting technology, and it was observed in general, and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days. After cultured for 1, 4, and 7 days, the composite scaffolds were taken for live/dead cell staining to observe cell survival rate; and cell counting kit 8 (CCK-8) method was used to detect cell proliferation. The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group, and the composite scaffolds cultured in complete medium for 14 days were used as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect cartilage formation. The relative expression levels of the mRNA of cartilage matrix gene [(aggrecan, ACAN)], chondrogenic regulatory factor (SOX9), cartilage-specific gene [collagen type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D bioprinted hADSCs-GelMA composite scaffold (experimental group) and the blank GelMA hydrogel scaffold without cells (control group) cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively, and the materials were taken after 4 weeks, and gross observation, Safranin O staining, Alcian blue staining, and collagen type Ⅱ immunohistochemical staining were performed to observe the cartilage formation in the composite scaffold.ResultsMacroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure. The cell viability could be maintained at 80%-90% at 1, 4, and 7 days after printing, and the differences between different time points were significant (P<0.05). The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing. After 14 days of chondrogenic induction and culture on the composite scaffold, the expressions of ACAN, SOX9, and COLⅡA1 were significantly up-regulated (P<0.05), the expression of COLⅩA1 was significantly down-regulated (P<0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue.ConclusionThe 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D structure and high cell viability, and can be induced differentiation into cartilage tissue, which can be used to construct tissue engineered cartilage in vivo and in vitro.
The biological pacemaker has become a new strategy in the treatment of severe bradycardias, in which a kind of ideal pacemaker cells is a pivotal factor. Here we reviewed the progress in the differentiation of bone-marrow mesenchymal stem cells and adipose-derived stem cells into pacemaker-like cells by means of gene transfer, chemical molecules, co-culture with other cells and specific culture media, and we also analyzed the potential issues to be solved when they are used as seeding cells of biological pacemaker.
ObjectiveTo investigate the effect of silk fibroin-poly-L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs).MethodsADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells.ResultsADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar (P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences (P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs.ConclusionThe ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.
Objective To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.
Objective To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. Methods Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups (n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×107cells/mL), group B received 0.1 mL of rat ADSCs (1×107cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×107cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×107cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. ResultsAll groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences (P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences (P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups (P<0.05). Conclusion ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
ObjectiveTo explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.
ObjectiveTo prepare adipose-derived stem cells (ADSCs) and chitosan chloride (CSCl) gel complex to study the biocompatibility and the feasibility of repairing the wounds of deep partial thickness scald in rats. MethodsADSCs were prepared by enzymogen digestion and differential adherence method from the subcutaneous adipose tissue of SPF grade 6-week-old male Sprague Dawley (SD) rats. Temperature sensitive CSCl gel was prepared by mixing CSCl, β glycerol phosphate, and hydroxyethyl cellulose in 8∶2∶2.5 ratio. The proliferation of ADSCs was measured by cell counting kit 8 (CCK-8) assay and the survival of ADSCs was detected by the Live/Dead flurescent staining in vitro. A deep partial thickness burn animal model was made on the back of 72 SPF grade 6-week-old male SD rats by boiled water contact method and randomly divided into 3 groups (n=24). Group A was blank control group, group B was CSCl hydrogel group, group C was ADSCs/CSCl gel group. The wound closure rate at 3, 7, 14, 21 days was observed after operation. The number of inflammatory cells at 7 days and epidermal thickness at 21 days were observed by HE staining after operation. The angiogenesis at 7 days was evaluated by immunohistochemistry staining with CD31 expression. ResultsCSCl had a temperature sensitivity, at 4℃, the temperature-responsive hydrogel was liquid and became solid at 37℃. The CCK-8 assay and Live/Dead flurescent staining confirmed that ADSCs could grow and proliferate in the ADSCs/CSCl hydrogel complex. General observation showed the wound closure ratio in group C was superior to groups A and B after operation (P<0.05). HE staining showed that at 7 days after operation, the wound healing of the three groups entered fibrous proliferation stage. Collagen deposition and inflammatory cell infiltration were observed in the dermis of each group. The proportion of inflammatory cells in group C was significantly lower than that in groups A and B, and in group B than in group A (P<0.01). At 21 days after operation, the fibrous connective tissues of neoepithelium and dermis in groups B and C were arranged neatly, and fibroblasts and neocapillaries could be seen. In group A, neoepidermis could also be seen, but the fibrous connective tissues in dermis were arranged disorderly and sporadic capillaries could be seen. The thickness of neonatal epidermis in group C was significantly larger than that in groups A and B, and in group B than in group A (P<0.01). CD31 immunohistochemistry staining showed that the neovascularization could be seen in all groups. The number of neovascularization in group C was significantly higher than that in groups A and B, and in group B than in group A (P<0.05). ConclusionThe ADSCs/CSCl hydrogel complex has a good biocompatibility and possessed positive effects on promoting the deep partial thickness scald wound repairing in rats.
ObjectiveTo discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects.MethodsHuman adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts.ResultsThe cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] (t=3.929, P=0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] (t=30.700, P=0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] (t=8.678, P=0.000).ConclusionThe hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo, which may stimulate angiogenesis, and improve retention rate of new-born tissue.
Objective To investigate the effects of adipose-derived stem cells (ADSCs) and endothelial cells (ECs) on the survival and neovascularization of fat tissue transplants. Methods The ADSCs were isolated by collagenase digestion from the adipose tissues voluntarily donated by the patients undergoing mastectomy, and subcultured. The passage 3 ADSCs were used for subsequent experiments. The residual fat tissues were used to prepare fat particles (FPs). The human umbilical vein endothelial cells (HUVECs) were used as ECs for subsequent experiments. Eighty healthy male nude mice, aged 4-6 weeks, were randomly divided into 4 groups (n=20). The mice were received subcutaneous injection at the dorsum of 1 mL FPs+0.3 mL normal saline (NS) in control group, 1 mL FPs+2×106 ECs+0.3 mL NS in ECs group, 1 mL FPs+2×106 ADSCs+0.3 mL NS in ADSCs group, and 1 mL FPs+1×106 ECs+1×106 ADSCs+0.3 NS in ADSCs+ECs group. General observations of the injection sites were performed, and the survival of the mice was recorded. At 2, 4, 8, and 12 weeks after injection, grafted fat tissues were firstly assessed by ultrasonography, then they were collected for volume measurement (water displacement method) and histology observation (HE staining and immunofluorescence staining). Results All mice survived until the end of experiment. At each time point, no significant difference was noted between groups in ultrasonography assay. There was no significant blood flow signal in the grafted fat tissues, or cysts, calcification, solid occupying in recipient area. Generally, the volume of grafted fat tissues decreased with time in all groups. Specifically, the volumes of grafted fat tissues were larger in ADSCs group and ADSCs+ECs group than that in control group and ECs group (P<0.05) at each time point, and in ADSCs group than in ADSCs+ECs group (P<0.05) at 8 and 12 weeks. HE staining showed that all groups had similar tendencies in general histology changes, and remodeling in ADSCs group was the fastest than in the other groups. By immunofluorescence staining for neovascularization, the new vessels in all groups were increasing with time. The vessel densities were higher in ECs group, ADSCs group, and ADSCs+ECs group than in control group (P<0.05) at each time point, in ADSCs group than in ECs group and ADSCs+ECs group (P<0.05) at 4 weeks, in ADSCs group and ADSCs+ECs group than in ECs group (P<0.05) at 8 and 12 weeks. Conclusion ADSCs can significantly increase the survival of transplanted fat tissue, which may be related to promoting the neovascularization.