Objective To compare the reparative effects between the acellular small intestinal submucosa andthe acellular amnion as dressings for traumatic skin defects. Methods Three full-thickness skin defects, which wereclose to the vertebral column of the pig, were created on both sides of the dorsum. The skin defects were randomlydivided into three groups. In each group, the following different materials were used to cover the skin defects: theacellular amnion in Group A, the acellular small intestinal submucosa (SIS) in Group B, and the physiological saline aguze in Group C (the control group). The specimens from the skin defects were harvested for a histological evaluation and for determination of the hydroxyproline content at 10 (2 pigs), 20 (2 pigs), and 30 days (3 pigs). We observed the healing process of the wound and its healing rate, counted the inflammatory cells, vasecular endothelial cells, and proliferating cells, and determined the hydroxyproline content. Results The acellular amnion in Group A and acellular SIS in Group B adhered to the wound tightly, but they did not adhere to the dressing; when the dressing was changed, the wound did not bleed. The saline gauze in Group C adhred to the wound tightly, but when the dressing was changed, the wound bled until 22 days after operation. Under the microscope, the collagen in the tissue below the epithelium was arranged more regularly and there were fewer cells concerned with inflammation in Groups A and B than in Group C at 10, 20, and 30 days after operation. At 10, 20, and 30 days after operation, the wound healing rate was greater in Groups A and B than in Group C, The number of the inflammatory cells and the proliferating cells were greater in Groupo C than in Groups A and B. There was a statistically significant difference (P lt; 0.05),At 20 and 30 days after operatin, the content of hydroxyproline was greater in Group c than in Group A and B. There was a statistically significant difference (P lt; 0.05). However, there was no statistically significant difference between Group A and Group B in the wound healing rate, the numbers of the inflammatory cells, vascular endothelial cells and prokiferating cells, and the content of hydroxyproline(P gt; 0.050). There was no statistically significant difference among the three groups in the number of the vascular endothelial cells. Conclusion Compared with Group C........
Objective To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. Methods The primary porcine keratinocytes were cultivated with DKSFM(Defined keratinocyteSFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 005% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. Results The proliferation of the primry porcine keratinocytes cultured with the medium ofDKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFMcontaining 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 005% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygong and arranged like slabstone. After 14 and 16 days,the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. Conclusion To culture primary porcine keratinocytes with the medium that DKSFMcontaining 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscafold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
Objective To investigate the clinical therapeutic effects of two types of vaginoplasty. Methods From January 1996 to March 2005, 63 patients wih the congenital absence of the vagina were treated by two types of vaginoplasty. Of the 63 patients, 37 underwent vaginoplasty using the amnion and 26 underwent an improved laparoscopic Vecchitti operation. The durations ofthe operation and hospitalization, as well as the blood loss were compared between the two types of vaginoplasty. The vaginal moulds were improved during the operations. Results According to the follow-up for 2 months to 4 years in the 35 patients. Compared with vaginoplasty using the amnion, vaginoplasty by an improved laparoscopic Vecchitti operation had advantages of significantly shorter surgical duration, shorter hospitalization, and less blood loss (Plt;0.05). After the operations, the artificial vagina of all the 63 patients could hold a speculum and the mucosa appeared so soft and smooth with normal lubrication. The married patients were satisfied with the intercourse. However, after vaginoplasty using the amnion, an infection of the amnion occurred in 3 patients, scar contracture in 2 patients, one of whom underwent scar incision 13 months after operation with a success; but the other refuse to accept another operation. But the improved laparoscopic Vecchitti operation achieved a success in the patients without any infectionor scar contracture, according to the 2 month-2.5 years follow-up. Conclusion The improved laparoscopic Vecchitti operation is a preferred procedure of constructing a vagina for the patients suffering from the congenital absence of the vagina.
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 prepare human acellular amniotic membrane(HAAM) and to measure its cytocompatibility and biocompatibility. Methods HAAM were preparedby chemical detergent-enzymatic extraction. Fresh human amnion was crosslinkedwith glutaradehyde, shaken in 0.5% SDS for 24 hours, and then treated with 0.25%trypsin for 4 hours. The production were freeze-drying and sterilized using ethylene oxide. Human fibroblasts were isolated from embryo and expanded in vitro. The fibroblasts were seeded in HAAM. HAAM and specimen were stained with HE and Mallory, and observed grossly, under light microscopy and scanning electron microscopy. The HAAM were implanted in the back of SD rats. Results There wereno residues of cells in the HAAM (HE, Mallory staining). One side of HAAM had reticular and porous structure, the other side had compact fibrous structure.Pore size was from 10 to 80 nm. The HAAM could be seeded with expanded fibroblasts in vitro,and fibroblasts had the potential of spread and proliferation. The SD rat in the implant test had no death, convulsions and other abnormal response. Conclusion The detergent-enzymatic extraction process can remove cellsand solvable components effectively and preserve the tissue matrix well and keep the reticular structure. The HAAM can be used as an ideal scaffold of biological membrane for tissue engineering.
Objective To observe the differentiation effect of rabbit amnion-derived stem cells (ADSC) induced into neural cells.Methods ADSC of New Zealand female rabbits were isolated and cultured. Its mRNA level of Fibronectin, Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction. The selfreplication ability of ADSC was confirmed by monoclonal formation experiments. These ADSC were further induced into neural cells in vitro. Five days after induced differentiation, the expression of -tubulin and glial fibrillary acidic protein (GFAP) were detected by immunofluorescent staining. Results ADSC were separated from amnion tissue gradually after 24 hours. There were polygonal cells gathered around the amnion tissue at 72 hours, and were distributed compactly around the amnion at 120 hours. The morphology of cleavage daughter cells was basically the same as parent cells. ADSC has the ability of self-replication. The Nestin, Vimentin, Fibronectin mRNA expressions in ADSC were 15.79, 1.91, 7.65 times those in spleen cells. The differences were statistically significant(Z=-5.243, -3.972, -2.524; P＜0.05). The beta;-tubulin expression was found in cytoplasm of most cells. The GFAP expression was found in cytoplasm in some cells. Conclusions ADSC has self-replication ability. It can be induced into neurons and neuroglial cells under the right conditions.
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.
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.
Objective To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind l imbs. Methods The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n=4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under themicroscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological sal ine were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindl imbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biopinylated dextan amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 ± 1.26) in group A was significantly higher than those of other groups (P lt; 0.05), and the recovery in latency (3.52 ± 2.45) ms and ampl itude (480.68 ± 18.41) μV of MEP was also obviously improved in group A (P lt; 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was higher than those of other groups (P lt; 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. Conclusion The BMSCs-DHAM composite matrix can improve hindl imb motor function to some extent after spinal cord injury. It will be widely appl ied as the matrix material in the future.