Objective To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. ResultsFlow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection (P<0.05), Fibronectin significantly increased at 3 days (P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days (P<0.05). CCK-8 detection showed that there was no significant difference (P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Objective
To review the latest development of amniotic fluid-derived stem cells (AFSCs) in regenerative medicine, and to discuss issues related to the studies in the field of AFSCs.
Methods
The recent articles about AFSCs were extensively reviewed. The important knowledge of AFSCs was introduced in the field of regenerative medicine, and the basic and clinical researches of AFSCs were summarized and discussed.
Results
Currently, it is confirmed that AFSCs have a multi-directional differentiation capacity, therefore, they have a wide application prospect in regenerative medicine, anti-tumor, and other fields.
Conclusion
AFSCs will become one of the ideal seed cells in the field of regenerative medicine with extensive research value because of the advantages of easy amniotic fluid sampling, little maternal and child trauma, no tumorigenesis, and no ethical restrictions.
Objective To investigate the effectiveness of mosaicplasty in repair of large-sized osteochondral compound defects and the integrity of transplanted tissue with recipient sites so as to lay a foundation for clinical application. Methods Twenty-four adult goats were divided into 3 groups randomly. The diameters of defect were 6 mm for the medium-sized defects and 9 mm for the large-sized defects, which were created by a trepan. All of the defects were repaired with osteochondral plugs in diameters of 2 mm(the mediumsized defects) or 3 mm(the large-sized defects). The osteochondral plugs were harvested around the intercondylar fossa or intertrochlea groove, and pressed into the recipient sites by specialized instruments in a mosaic mode. No internal fixation was needed and the animal wereallowed to move freely after operation. From 4 to 24 weeks postoperatively, thespecimens were observed in gross and under electromicroscopy. X-ray detection and glycosaminoglycan(GAG) analysis were also performed to testify the healing processand the integrity of the cartilage and subchondral bone. Results The transplanted subchondral bone was integrated firmly with each other or with recipient sites in both mosaicplasty groups. But 24 weeks postoperatively, transplanted cartilage was not integrate with each other apparently. Obvious cleavage between cartilage plugs could be seen. But in the largesized defect groups, some of the osteochondral plugs were relapsed into the defects leaving the recipient sites some steps, leading to some degree of abrasion in the opposing articular cartilage. There was no significant difference in the GAG content between the transplanted cartilage and normal cartilage. X-ray analysis also demonstrated the healing process between the subchondral bone. Conclusion Mosaicplasty can repair the medium or small-sized osteochondral defects efficiently.
Human amnion (hAM), as a biomaterial, has made significant progress in the field of ophthalmology, particularly in the treatment of retinal diseases. hAM possesses biological properties such as promoting tissue repair, inhibiting inflammation and neovascularization, and reducing fibrosis, which have led to its promising clinical outcomes in treating macular holes, retinal detachment, proliferative vitreoretinopathy, optic disc depression-related macular detachment, and age-related macular degeneration. The application of hAM can improve surgical success rates and promote vision recovery, with no significant rejection reactions observed due to its low immunogenicity. Nevertheless, the use of hAM still faces challenges in optimizing preparation and storage techniques, enhancing therapeutic efficacy, and reducing the risk of infectious disease transmission. Future research should focus on addressing these issues to further promote the application of hAM in retinal disease treatment and enhance its effectiveness.
Objective To observe the effect of amniotic homogenate on closing holes in experimental rhegmatogenous retinal detachment and investigate its mechanism. Methods Forty rabbits were randomly divided into group A, B, C and D with 10 rabbits in each group. Group A and C were the treatment groups, and group B and D were the control groups. All eyes of rabbits underwent pars plana vitrectomy, retinectomy, and fluidair exchange. The surface of the breaks was treated with 01 ml amniotic homogenate in experimental groups and 0.1 ml PBS in control groups. At the end of operation, 20% SF6 was tamponaded and the retina reattaced. The animals were executed 14 (group A and B) and 28 days (group C and D) after the surgery. The tissue sections were observed by light microscope, electron microscope and immunocytochemistry method. Results Fourteen days after the surgery, the retina reattached in 6 eyes in group A (60%) and 2 eyes in group B (20%) （P=0.021）. Twenty-eight days after the surgery, the retina reattached in 8 eyes in group C (80%) and 3 eyes in group D (30%) (P=0.046). The difference of the rate of retinal reattachment among the 4 groups were statistical significant (Plt;0.05). Light postoperative inflammation of ocular anterior segment was observed, which was controlled 3-5 days after treated with topical steroids. The result of light microscopy showed that the eyes in treatment groups had multilayer of fibroblastlike cells around the retinal breaks, adhering to the choroid and retinal pigment epithelial cells. The proliferative cells around the retinal breaks obvious less in control groups than that in the treatment groups, and the retina could not adhere to the choroid. The results of electron microscopy were the same as that of light microscopy. Immunohistochemistry staining of the fibroblastlike cells revealed positve glial fibrillary acidic protein, which suggested that the proliferative cells around the retinal breaks were retinal glial cells. Conclusions Amniotic homogenate helps to seal retinal breaks and promote retinal reattachment by stimulating the proliferation of retinal glial cells around the breaks.
ObjectiveTo evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).MethodsBMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10?5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.ResultsWith the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=?10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).ConclusionIcariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.
ObjectiveTo investigate the effect of microRNA-135a (miR-135a) in human amnion mesenchymal stem cell exosome (hAMSC-Exo) on the migration of fibroblasts.MethodsThe hAMSC-Exo was extracted with exosomes separation kit and identified, the effect of hAMSC-Exo on fibroblasts migration was detected by scratch test. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-135a gene in hAMSC-Exo after overexpression of miR-135a. Scratch test was used to detect the effect of hAMSC-Exo on the migration of fibroblasts after overexpression and knockdown of miR-135a. Western blot was used to detect the migration related proteins of fibroblasts [large tumor suppressor 2 (LATS2), E-cadherin, N-cadherin, and α smooth muscle actin (α-SMA)] after overexpression and knockdown of miR-135a. The 293T cell exosomes and hAMSC-Exo were used as control.ResultshAMSC-Exos were extracted successfully. Scratch test results showed that hAMSC group had the strongest ability to promote fibroblasts migration, and GW4869 (exosome inhibitor) treatment group had reduced ability to promote fibroblasts migration. qRT-PCR test showed that the relative expression of miR-135a gene in hAMSC-Exo increased significantly after over expression of miR-135a. Scratch test results showed that after over expression of miR-135a, hAMSC-Exo enhanced the migration ability of fibroblasts, while after knockdown of miR-135a, hAMSC-Exo weakened the migration ability of fibroblasts. Western blot results showed that the expressions of E-cadherin, N-cadherin, LATS2 were down regulated and α-SMA was up regulated in each hAMSC-Exo treatment group when compared with 293T cell exosomes group; after over expression of miR-135a, hAMSC-Exo decreased the expressions of E-cadherin, N-cadherin, LATS2 and increased the expression of α-SMA; while after knockdown of miR-135a, the ability of hAMSC-Exo was weakened.ConclusionmiR-135a in hAMSC-Exo can promote fibroblasts’ migration, inhibit the expressions of E-cadherin, N-cadherin, LATS2, and promote the expression of α-SMA.
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction.
MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining.
ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001).
ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.
ObjectiveUnder hypoxic conditions, the survival and apoptosis of human amniotic mesenchymal stem cells (hAMSCs) were observed by transient transfection of hypoxia-inducible factor 1α (HIF-1α) gene, to investigate the effect of HIF-1α on hypoxic tolerance of hAMSCs.MethodsThe hAMSCs were isolated and cultured from amniotic membrane tissue from voluntary donors who were treated with cesarean section. And the morphological observation by inverted phase contrast microscope and immunofluorescence detection of the expressions of stem cell markers OCT-4 and NANOG were performed to identify the cultured cells. The third generation hAMSCs were treated with 200 μmol/L CoCl2, and transient transfection of plasmids were added according to the following grouping: group A was hAMSCs blank group; group B was pcDNA3.1 negative control group; group C was short hairpin RNA (shRNA) negative control group; group D was shRNA-HIF-1α interference group; group E was pcDNA3.1-HIF-1α over expression group. Cell survival rate of each group was measured by cell counting kit 8 (CCK-8) at 12, 24, 48 hours after hypoxia treatment. Flow cytometry was used to detect apoptosis rate of each group at 24 hours after hypoxia treatment. The expression levels of HIF-1α, vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bax, and cleaved Caspase-3 (C-Caspase-3) proteins were detected by Western blot at 24 hours after hypoxia treatment.ResultsCCK-8 assay showed that the cell survival rate of group D was significantly lower than those of groups A and C at all time points after hypoxia treatment; while the cell survival rate in group E was significantly increased than those in groups A and B, and the diffrences at 24 hours were significant (P<0.05). In group E, the cell survival rate at 24 hours was significantly higher than those at 12 and 48 hours (P<0.05). The results of flow cytometry showed that the apoptosis rate in group D was significantly higher than those in groups A and C (P<0.05), and the apoptosis rate in group E was significantly lower than those in groups A and B (P<0.05). Western blot showed that the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group D were significantly decreased when compared with those in groups A and C, and the expressions of Bax and C-Caspase-3 proteins were significantly increased (P<0.05). On the contrary, the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group E were significantly higher than those in groups A and B, and the expressions of Bax and C-Caspase-3 proteins were significantly decreased (P<0.05).ConclusionOverexpression of HIF-1α gene can significantly improve hAMSCs tolerance to hypoxia, the mechanism may be related to up-regulation of VEGF and Bcl-2 expressions, and down-regulation of Bax and C-Caspase-3 expressions.
