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 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.
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.
Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
Icariin（ICA） is one of the main active ingredients in the Berberidaceae family Epimedium. It makes a variety of biological activities, such as promoting bone formation, antibacterial and anti-inflammatory, and regulating immunity. Periodontitis is a chronic inflammatory disease that is present in the soft and hard tissues of the periodontium. The ultimate goals of its treatment are the reconstruction of periodontal tissues and bone defect repairing. At present, conventional treatment of periodontitis fails to achieve the ideal periodontal tissue regeneration. In recent years, the rapid development of tissue engineering technology has brought new ideas for the treatment of periodontal disease and bone defect repairing. Because of its anti-inflammatory and osteogenic effects, ICA has great potential for the treatments of periodontitis and bone defect repairing. This paper summarizes the effect and the molecular mechanism of ICA in the treatment of periodontitis and bone defect repairing, and discusses its application prospect as a drug for periodontal adjuvant therapy. This paper aims to provide a theoretical basis for the research and application of ICA in periodontitis treatment and bone defect repairing.
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.
Objective To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. Methods Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups (n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×107cells/mL), group B received 0.1 mL of rat ADSCs (1×107cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×107cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×107cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. ResultsAll groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences (P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences (P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups (P<0.05). Conclusion ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Objective To establish an effective model of myocardial infarction in black goat so as to provide a safe, convenient and credible model of myocardial infarction for treatment and research. Methods Sixteen black goats were made chronic myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess. Electrocardiogram(ECG) and serum myocardial enzymes were investigated before and after occlusion. Echocardiographic measurements were performed, and left coronary artery angiography was performed with digital subtraction angiography (DSA) before infarction and 6 weeks after infarction. The myocardial ultrastructure were observed. Results All goats survived more than 6 weeks. ECG showed ambulatory change, ST-segment elevated half an hour after occlusion and pathologic Q waves 6 weeks after infarction, CK-MB significantly increased. Echocardiographic indexes showed significant decrease of maximal peak A, percent wall thickening(WHT) and ejecting fraction (EF), increase ofend-systolic volume (ESV), end-diastolic volume (EDV), and dilation of left ventricle. DSA showed block or decrease of perfusion of far end of left anterior descending coronary artery. Conclusion It is safe, convenient and credible to establish model of myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess in black goat.
Objective To evaluate the clinical efficiency of fresh amniotic membrane transplantation in treatment of stenosis of conjunctival sac. MethodsThirty cases (30 eyes) of stenosis of conjunctival sac were treated with fresh amniotic membrane transplantation. Amniotic membrane was obtained under sterile conditions after elective cesarean delivery. The woman’s serum was negative for HBsAg, syphilis, and human immunodeficiency virus. The placenta was first washedfree of blood clots with sterile saline. Under sterile conditions, the inner amniotic membrane was separated from the chorion by blunt dissection, and was cleaned of blood with the sterile saline again. The membrane was then flattened onto a surgidrape adhesive paper with the epithelium surface up. The paper with the adherent amniotic membrane was then cut into 5 cm×8 cm pieces, and then rinsed in solution containing 4×106 U/L of gentamycin and stored at 4℃. It could bestored for 12 hours after preparation. The-adhesiotomy was performed firstly. The separation between the conjunctiva and scar tissue should be complete and wide enough to reach to the orbital margin. The adhesiectomy was taken secondly. The scar tissues were removed completely. The fresh amniotic membrane was flattened onto the conjunctival defect with epithelium side up. The fresh amniotic membranewas 10 mm more than the conjunctival defect by trimming off the excess portion.This fashioned membrane was then secured to surrounding conjunctival edge with continuous 7-0 nylon sutures. The necessary mattress suture of inferior conjunctival fornix via skin next to the inferior orbital margin was performed simultaneously. The retrobular implantation of the an artificial globe made of hydroxyapatite was performed on some patients with sunken eye. Correction of traumatic ptosis was performed on a few patients.Results The operation ofreconstruction of partial conjunctival sac for 30 cases was successful. All amniotic membrane grafts were alive. The cosmetic result was complete favorable. The infection and contracture of the graft, immunologic rejection and amniotic lysis were not observedin all cases during the follow-up period of 13-18 months.Conclusion Fresh amniotic membrane transplantation can be used in reconstruction of the partial conjunctival sac effectively and can be popularized in thelocal hospital in China because the amniotic membrane can be obtained easily.