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.
ObjectiveTo investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits.MethodsThe bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm (n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope.ResultsADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides (P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week (P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C.ConclusionADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.
ObjectiveTo investigate the effect of human adipose-derived stem cells (hADSCs) on pressure ulcers in mouse.MethodsThe subcutaneous adipose tissue from voluntary donation was harvested. Then the hADSCs were isolated and cultured by mechanical isolation combined with typeⅠcollagenase digestion. The 3rd generation cells were identified by osteogenic, adipogenic, chondrogenic differentiations and flow cytometry. The platelet rich plasma (PRP) from peripheral blood donated by healthy volunteers was prepared by centrifugation. The pressure ulcer model was established in 45 C57BL/6 mice by two magnets pressurized the back skin, and randomly divided into 3 groups (n=15). The wounds were injected with 100 μL of hADSCs (1×106 cells) transfected with a green fluorescent protein (GFP)-carrying virus, 100 μL human PRP, and 100 μL PBS in hADSCs group, PRP group, and control group, respectively. The wound healing was observed after injection. The wound healing rate was calculated on the 5th, 9th, and 13th days. On the 5th, 11th, and 21st day, the specimens were stained with HE staing, Masson staining, and CD31 and S100 immunohistochemical staining to observe the vascular and nerve regeneration of the wound. In hADSCs group, fluorescence tracer method was used to observe the colonization and survival of the cells on the 11th day.ResultsThe cultured cells were identified as hADSCs by induced differentiation and flow cytometry. The platelet counting was significantly higher in PRP group than in normal peripheral blood group (t=5.781, P=0.029). General observation showed that the wound healing in hADSCs group was superior to those in PRP group and control group after injection. On the 5th, 9th, and 13th days, the wound healing rate in hADSCs group was significantly higher than those in PRP group and control group (P<0.05). Histological observation showed that compared with PRP group and control group, inflammatory cell infiltration and inflammatory reaction were significantly reduced in hADSCs group, collagen deposition was significantly increased, and skin appendage regeneration was seen on the 21st day; at each time point, the expression of collagen was significantly higher in hADSCs group than in PRP group and control group (P<0.05). Immunohistochemical staining showed that the number of neovascularization and the percentage of S100-positive cells in hADSCs group were significantly better than those in PRP group and control group on the 5th, 9th, and 13th days (P<0.05). Fluorescent tracer method showed that the hADSCs could colonize the wound and survive during 11 days after injection.ConclusionLocal transplantation of hADSCs can accelerate healing of pressure ulcer wounds in mice and improve healing quality by promoting revascularization and nerve regeneration.
Objective To review the research progress of miRNA regulation in the differentiation of adipose-derived stem cells (ADSCs). Methods The recent literature associated with miRNAs and differentiation of ADSCs was reviewed. The regulatory mechanism was analyzed in detail and summarized. Results The results indicate that the expression of miRNAs changes during differentiation of ADSCs. In addition, miRNAs regulate the differentiation of ADSCs into adipocytes, osteoblasts, chondrocytes, neurons, and hepatocytes by regulating the signaling pathways involved in cell differentiation. Conclusion Through controlling the differentiation of ADSCs by miRNAs, the suitable seed cell for tissue engineering can be established. The review will provide a theoretical basis for molecular targeted therapy and stem cell therapy in clinic.
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 investigate the effects of exosomes from adipose-derived stem cells (ADSCs) on peripheral nerve regeneration, and to find a new treatment for peripheral nerve injury. MethodsThirty-six adult Sprague Dawley (SD) rats (male or female, weighing 220-240 g) were randomly divided into 3 groups (n=12). Group A was the control group; group B was sciatic nerve injury group; group C was sciatic nerve injury combined with exosomes from ADSCs treatment group. The sciatic nerve was only exposed without injury in group A, and the sciatic nerve crush injury model was prepared in groups B and C. The SD rats in groups A and B were injected with PBS solution of 200 μL via tail veins; the SD rats in group C were injected with pure PBS solution of 200 μL containing 100 μg exosomes from ADSCs, once a week and injected for 12 weeks. At 1 week after the end of the injection, the rats were killed and the sciatic nerves were taken at the part of injury. The sciatic nerve fiber bundles were observed by HE staining; the SCs apoptosis of the sciatic nerve tissue were detected by TUNEL staining; the ultrastructure and SCs autophagy of the sciatic nerve were observed by transmission electron microscope. ResultsGross observation showed that there was no obvious abnormality in the injured limbs of group A, but there were the injured limbs paralysis and muscle atrophy in groups B and C, and the degree of paralysis and muscle atrophy in group C were lighter than those in group B. HE staining showed that the perineurium of group A was regular; the perineurium of group B was irregular, and there were a lot of cell-free structures and tissue fragments in group B; the perineurium of group C was more complete, and significantly well than that of group B. TUNEL staining showed that the SCs apoptosis was significantly increased in groups B and C than in group A, in group B than in group C (P<0.01). Transmission electron microscope observation showed that the SCs autophagosomes in groups B and C were significantly increased than those in group A, but the autophagosomes in group C were significantly lower than those in group B. ConclusionThe exosomes from ADSCs can promote the peripheral nerve regeneration. The mechanism may be related to reducing SCs apoptosis, inhibiting SCs autophagy, and reducing nerve Wallerian degeneration.
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.
Objective To assess the effect of pregnant rat adipose-derived stem cells (ADSCs) on repair of acute liver injury. Methods ADSCs were isolated from 18-week pregnant Sprague Dawley rats and were identified by flow cytometry. Twenty Sprague Dawley rats were randomly divided into groups A, B, C, and D (n=5); rats in group A were not treated as normal controls; rats in groups B, C, and D were injected intraperitoneally with CCl4 to establish the acute liver injury model. At 2 hours after modeling, DPBS, 0.1 mL normal rat ADSCs (2×106cells/mL), and pregnant rat ADSCs (2×106cells/mL) were injected into the spleen in groups A, C, and D respectively; rats in group B was not treated. After 7 days, total bilirubin (TBIL), alanine aminotransferase (ALT), aspartic acid transaminase (AST), albumin (ALB), and total protein (TP) in serum were measured. The liver tissue sections were stained with HE. The expressions of Ki67, alpha-fetoprotein (AFP), and ALB were measured by immunohistochemistry. Results The serum levels of TBIL, ALT, and AST in group B were significantly higher than those in groups A, C, and D (P<0.05), but ALB and TP were significantly lower than those in groups A, C, and D (P<0.05). The levels of TBIL, ALT, and AST were significantly higher in groups C and D than group A, and in group C than group D (P<0.05). There was no significant difference in serum levels of ALB among groups A, C, and D (P>0.05). The serum level of TP in groups C and D was significantly lower than that in group A (P<0.05), but no significant difference was found between group C and group D (P>0.05). HE staining showed that the liver tissue of group A had clear structure; the cells arranged neatly with uniform size. The hepatocytes in group B showed obvious edema, disorderly arrangement, dot necrosis in liver lobules, and diffuse infiltration of inflammatory cells. In groups C and D, the inflammation and hepatocellular necrosis were obviously reduced when compared with group B, and the number of vacuoles caused by dilation of mitochondria and rough endoplasmic reticulum was decreased; especially in group D, improvement of liver injury was more effective. The Ki67 positive cell rate was significantly higher in groups C and D than groups A and B (P<0.05), in group B than group A (P<0.05), and in group D than group C (P<0.05). There was no expression of AFP in groups A and B, but positive expression was observed in groups C and D, and AFP positive cell rate of group D was significantly higher than that of group C (t=3.006,P=0.017). ALB expression was significantly higher in groups C and D than groups A and B (P<0.05), and in group D than group C (P<0.05). Conclusion Pregnant rat ADSCs could promote repair of liver injury induced by CCl4.
ObjectiveTo summarize the research progress of the effects of high glucose microenvironment on the biological activity of adipose-derived stem cells (ADSCs).MethodsThe literature on the high glucose microenvironment and ADSCs at home and abroad in recent years was reviewed, and the effects of high glucose microenvironment on the general characteristics, differentiation potential, angiogenesis, and nerve regeneration of ADSCs were summarized.ResultsThe accumulation of advanced glycosylation end products (AGEs) in the high glucose microenvironment led to changes in the biological activities of ADSCs through various pathways, including cell surface markers, proliferation, migration, multi-lineage differentiation, secretory function, and tissue repair ability. The ability of ADSCs to promote angiogenesis and nerve regeneration in high glucose microenvironment is still controversial.ConclusionHigh glucose microenvironment can affect the biological activity of ADSCs, and the effect and mechanism of ADSCs on angiogenesis and nerve regeneration in high glucose microenvironment need to be further studied.
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.