Objective To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days (P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture (P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group (P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points (P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days (P>0.05). Conclusion The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after inductionin vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.
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.
Objective To investigate the effects of human acellularamnion membrane on SD rat tendon adhesion and to obtain the experimental data for clinical application in preventing postoperative tendon adhesion. Methods The tendons of 28 adult SD rats hindlimb were cut and sutured. The tendons of left hindlimb were encapsulated by human accellular amnion membraneas the experimental group and the ones of the other side were not encapsulatedas control group. The rats were killed 1, 2, 4, 6, 8 and 12 weeks after operation. The results were evaluated grossly and histologically. Results There were no differences in healing of injury tendon and inflammatory response between the two groups. The anatomical and histological results showed the experimental group had less adhesion than the control group(Plt;0.05). Conclusion Human acellular amnion membrane can prevent adhesion of tendonwithout affecting tendon healing and is an optimal biological material to prevent tendon adhesion.
Objective To compare the effects of the denudedfreeze-dried-amniotic-membrane and the denuded freeze-dried bovine corneal stroma when they were explanted to repair the corneal defect of rabbits. Methods The amnia from healthy human placentae were prepared with the method reported by LUO Jingcong, which were freeze-dried and sterilized. The bovine cornea was also denuded by typsin, rinsed, freeze-dried, and sterilized. Twenty Japan rabbits weredivided into group A(the amniontic group) and group B(the bovine-corneal-stroma group) at random. The defect was made, which was 7.5 mm in diameter and 1/3 ofthe thickness of the cornea, and the two kinds of materials were explanted to repair the defect. The vascularization and the changes of the operated eye were observed. The samples were taken at 2, 4 and 8 weeks for histologicalexamination. Results The explanted materials were not melted or excluded. There were visible neovessels in both groups, yet there was no significant difference between them. According to the histological observation, there was severe inflammation in both groups 2 weeks after operation, the fibroblasts were proliferated, and the collagen fibers were disorganized; however,the reactions became milder from 4 weeks after operations,andthe neovessles could be seen in groups A and B; at 8 weeks, the collagen fibers were more organized in groups A and B; however,there was still a small area of disorganized fibers left. Conclusion The two materials can lead to rejection to some extent, and so they need to be improved.
To investigate the immunoreaction, histological reaction and turnover by comparing the xenotransplantation of fresh human amniotic membrane (HAM) with that of preserved HAM, and to analyze the cl inical appl ication value of different kinds of HAM preparations. Methods Subcutaneous implant models were establ ished in 150 BALB/C mice, which were randomized into 5 groups of 30 mice each, based on different implants: fresh amniotic membrane (FAM), double fresh amniotic membrane (DFAM), glycerin preserved amniotic membrane (GPAM), chorion (positive control) or merely operation (negative control). The tissue samples from grafted area were observed with SABC and HE staining, and the inflammatory cells were calculated with l ight microscopy. 1, 2, 4, 8 and 12 weeks after surgery. Results The mice in all of groups were normal in eating and moving, and the wound surface healed well. In all of AM groups, the expression of MHC Ⅱ and the calculation of inflammatory cells were much less than those in chorion groups, showing significant differences (P lt; 0.01). At 1, 8 and 12 weeks after surgery, there were no significant differences in the expression of MHC Ⅱ and the calculation of inflammatory cells in all of AM groups, compared with other groups (P gt; 0.05). From 2 weeks to 4 weeks after surgery, there were no significant differences in the expression of MHC Ⅱ and the calculation of inflammatory cells between FAM and DFAM groups (P gt; 0.05), but they were both more than those in GPAM groups, showing significant differences (P lt; 0.05). At the 4th week after surgery, in all of AM groups, the expression of MHC Ⅱ and the calculation of inflammatory cells were less than those at the 2nd week, showing significant difference (P lt; 0.01).The amniotic epithel ium was still al ive in fresh AM groups until 4 weeks after transplantation. Early after surgery, fibroblasts infiltrated AM from the substantia basilaris layer while made fibrous capsule around the epithel ium. After 12 weeks, the amnion absorbed. Conclusion As a kind of heterologous biomaterial, whether fresh or preserved, HAM can be seemedof ideal immunocompatibil ity and histocompatibil ity. Fresh HAM with al ive epithel ium may be more successful in area ofrepair and reconstruction.
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.
Objective To observe clinical effects of burn wounds treatment with bovine amnion and to screen the best method of preparing and storing of bovine amnion. Methods From January 2004 to January 2005,We selected randomly 58 patients with superficial Ⅱ° wound, deepⅡ° wound, autografting area for removal of eschars and tangential excision, fetching skin area or residual burn wound . Using auto-control, every burn wound was divided into 3 parts and was treated with 3 dressings: bovine amnion dealt with by 0.1% chlorhexidine（group A）, bovine amnion dealt with by 0.4% glutaraldehyde（group B） and vaseline gauze dressing（group C as control）. The clinical effects were compared between different groupsand the method of preparing and storing bovine amnion was evaluated. Results The dressing texture of group A was softer than that of group B, and its flexibility was fine. The pretreatment was not necessary for dressing in group A. When the dressing was used on burn wounds in groups A and B, painwas slight, but pain was obvious in group C; healing time in groups A and B was much less than that in group C, showing statistically significant difference(P＜0.01). There was no statistically significant difference in healing time between groups A and B (P＞0.05). The infection ratio of burn wound in deepⅡ° wound and residual burn wound of groups A and B is much lower than that of group C, showing statistically significant difference (P＜0.05); in theother burn wounds there was no significant difference (P＞0.05). There was no statistically significant difference between groups A and B (P＞0.05). Conclusion Bovine amnion could make benefit on burn wounds healing, reduce infection ratio of burn wounds, could be used on different kinds of burn wounds. The clinical effect between bovine amnion dealt with by glutaraldehyde and by chlorhexidine is similar. Whereas the latter is more easy to be popularized.
ObjectiveTo observe the survival, migration, and effect of human amniotic epithelial cells (hAECs) on hepatic fibrosis in immune rats so as to provide the experimental theory for the clinical treatment with hAECs.
MethodsSixty-four 10-week-old male Sprague Dawley rats (weighing, 220-280 g) were randomly divided into 4 groups, sixteen rats in each group. Rat hepatic fibrosis model was induced in groups A, B, and C; hepatic fibrosis rats were injected with 4×106 hAECs in group A, and with normal saline in group B, and no treatment was given in group C; group D served as control group. After 2 weeks of transplantation, the expression of human Alu gene repeat sequence was detected by DNA-PCR method and human leucocyte antigen G (HLA-G) by immunohistochemical staining in heart, liver, spleen, kidney, lung, and brain in group A, and then the percentage of positive expression was compared between organs except spleen. Semi-quantitative analysis was done for liver fibrosis with HE staining according to Chevallier semi-quantitative histological liver fibrosis scoring system, and immunohistochemical staining for TGF-β1 was used to record immunohistochemical score (ISH), the concentrations of aspartate transaminase (AST), alanine aminotransferase (ALT), and albumin (ALB) were determined to analyze hepatic fibrosis.
ResultsAlu gene repeat sequence and HLA-G could be detected in liver, heart, brain, lung, and kidney in group A, the percentage of positive expression in the liver was significantly higher than that in the other organs (P<0.05). The histological semi-quantitative score of group A (10.47±3.20) was significantly lower than that of groups B and C[(13.84±3.46) and (13.85±3.16)](P<0.05), but no significant difference was found between groups B and C (P>0.05). The ISH scores in groups A, B, C, and D were 3.60±1.50, 5.38±2.60, 5.50±2.40, and 1.87±1.36, respectively; groups A, B, and C were significantly higher than group D, and group A was significantly lower than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The concentrations of ALT and AST in groups A, B, and C were significantly higher than those in group D, and group A was significantly lower than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The concentration of ALB in groups A, B, and C was significantly lower than that in group D, and group A was significantly higher than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05).
ConclusionhAECs can survive in immune rats by intrasplenic transplantation and migrate to liver, heart, brain, lung, and kidney, and the liver shows the largest migration. The transplantation of hAECs in immune rat with cirrhosis can alleviate hepatic fibrosis and improve the serum indexes of liver function.
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.
