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.
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 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 effects of titanium modified by ultrasonic acid etching/anodic oxidation (UAT) loaded with endothelial progenitor cells-exosome (EPCs-exo) on proliferation and osteogenic and angiogenic differentiations of adipose-derived stem cells (ADSCs). Methods The adipose tissue and bone marrow of 10 Sprague Dawley rats were harvested. Then the ADSCs and EPCs were isolated and cultured by collagenase digestion method and density gradient centrifugation method, respectively, and identified by flow cytometry. Exo was extracted from the 3rd to 5th generation EPCs using extraction kit, and CD9 and CD81 were detected by Western blot for identification. The three-dimensional printed titanium was modified by ultrasonic acid etching and anodic oxidation to prepare the UAT. The surface characteristics of UAT before and after modification was observed by scanning electron microscopy; UAT was placed in EPCs-exo solutions of different concentrations (100, 200 ng/mL), and the in vitro absorption and release capacity of EPCs-exo was detected by BCA method. Then, UAT was placed in DMEM medium containing different concentrations of EPCs-exo (0, 100, 200 ng/mL), and co-cultured with the 3rd generation ADSCs to construct UAT-ADSCs-exo. Cell morphology by laser confocal microscopy, live/dead cell staining, and cell proliferation were observed to evaluate biocompatibility; alkaline phosphatase (ALP) staining and alizarin red staining, RT-PCR detection of osteogenesis-related genes [osteocalcin (OCN), RUNT-related transcription factor 2 (Runx2), ALP, collagen type 1 (COL-1)] and angiogenesis-related gene [vascular endothelial growth factor (VEGF)], immunofluorescence staining for osteogenesis (OCN)- and angiogenesis (VEGF)-related protein expression were detected to evaluate the effect on the osteogenic and angiogenic differentiation ability of ADSCs. Results Scanning electron microscopy showed that micro-nano multilevel composite structures were formed on the surface of UAT. About 77% EPCs-exo was absorbed by UAT within 48 hours, while EPCs-exo absorbed on the surface of UAT showed continuous and stable release within 8 days. The absorption and release amount of 200 ng/mL group were significantly higher than those of 100 ng/mL group (P<0.05). Biocompatibility test showed that the cells in all concentration groups grew well after culture, and the 200 ng/mL group was better than the other groups, with fully spread cells and abundant pseudopodia, and the cell count and cell activity were significantly higher than those in the other groups (P<0.05). Compared with the other groups, 200 ng/mL group showed enhanced ALP activity and mineralization ability, increased expressions of osteogenic and angiogenic genes (OCN, Runx2, COL-1, ALP, and VEGF), as well as increased expressions of OCN and VEGF proteins, with significant differences (P<0.05). Conclusion EPCs-exo can effectively promote the adhesion, proliferation, and osteogenic and angiogenic differentiation of ADSCs on UAT surface, the effect is the most significant when the concentration is 200 ng/mL.
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.
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.
ObjectiveTo investigate the effect of human subcutaneous adipose-derived stem cells (hADSCs) local transplantation on orthodontically induced root resorption (OIRR) and provide theoretical and experimental basis for the clinical application of hADSCs to inhibit OIRR. MethodsForty 8-week-old male Sprague Dawley rats were randomly divided into experimental group and control group, with 20 rats in each group, to establish the first molar mesial orthodontic tooth movement (OTM) model of rat right maxillary. The rats in the experimental group were injected with 25 μL of cell suspension containing 2.5×105 hADSCs on the 1st, 4th, 8th, and 12th day of modeling, while the rats in the control group were injected with 25 μL of PBS. The rat maxillary models were obtained before and after 7 and 14 days of force application, and 10 rats in each group were killed and sampled after 7 and 14 days of force application. The OTM distance was measured by stereomicroscope, the root morphology of the pressure side was observed by scanning electron microscope and the root resorption area ratio was measured. The root resorption and periodontal tissue remodeling of the pressure side were observed by HE staining and the root resorption index was calculated. The number of cementoclast and osteoclast in the periodontal tissue on the pressure side was counted by tartrate resistant acid phosphatase staining. Results The TOM distance of both groups increased with the extension of the force application time, and there was no significant difference (P<0.05). There was no significant difference in OTM distance between the experimental group and the control group after 7 and 14 days of force application (P>0.05). Scanning electron microscope observation showed that small and shallow scattered resorption lacunae were observed on the root surface of the experimental group and the control group after 7 days of force application, and there was no significant difference in the root resorption area ratio between the two groups (P>0.05); after 14 days of application, the root resorption lacunae deepened and became larger in both groups, and the root resorption area ratio in the experimental group was significantly lower than that in the control group (P<0.05). The range and depth of root absorption in the experimental group were smaller and shallower than those in the control group, and the root absorption index in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). The number of cementoclast in the experimental group was significantly lower than that in the control group after 7 and 14 days of force application (P<0.05); the number of osteoclasts in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). Conclusion Local transplantation of hADSCs may reduce the area and depth of root resorption by reducing the number of cementoclasts and osteoclasts during OTM in rats, thereby inhibiting orthodontic-derived root resorption.
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.
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.