Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
Objective To investigate the effect of human placental-derived mesenchymal stem cells (PMSCs) on immunological rejection in mouse allogeneic skin transplantation. Methods The placenta fetal tissues from voluntary donors were used to isolate and culture the PMSCs, and the 3rd passage PMSCs were used in the experiment. Thirty Vr ∶ CD1 (ICR) mice at age of 1-2 days were used as skin donors for allogeneic skin transplantation. Thirty C57BL/6 mice at age of 6-8 weeks as recipients were made back skin defect of 12 mm in diameter and were randomly divided into 3 groups (n=10): group A, autograft; group B, allogeneic graft + PBS tail vein injection; and group C, allogeneic graft + human PMSCs (1 × 105 cells/mouse) tail vein injection. The flap survival was observed. At 7 days after skin transplantation, blood leukocyte counting, abdominal fluid macrophage activation, and the expression levels of interleukin 4 (IL-4), interleukin 17 (IL-17), and interferon γ (INF-γ) in blood and spleen were detected by ELISA and RT-PCR, respectively. Results The flap survival time was significantly longer in group A [(58.33 ± 4.04) days] than in groups B and C [(3.80 ± 0.92) days and (6.80 ± 0.82) days] (P lt; 0.05), and in group C than in group B (P lt; 0.05). At 7 days after transplantation, the blood leukocyte number was (6.32 ± 0.45) × 109/L in group A, (7.45 ± 0.52) × 109/L in group B, and (6.35 ± 0.39)× 109/ L in group C, and it was significantly more in group B than in groups A and C (P lt; 0.05). The macrophage activation rate of the abdominal fluid was 6.87% ± 2.40% in group A, 7.84% ± 0.44% in group B, and 15.98% ± 2.87% in group C; group C was significantly higher than groups A and B (P lt; 0.01). ELISA results showed that there was no significant difference in the concentrations of IL-4 among 3 groups (P gt; 0.05). Compared with group B, the concentrations of IL-17 and IFN-γ were significantly reduced in group C (P lt; 0.05), while the concentration of IFN-γ was significantly increased in group B when compared with group A (P lt; 0.05). RT-PCR results showed that there were significant differences in the expressions of IL-4, IL-17, and IFN-γ mRNA between groups B, C and group A (P lt; 0.05); the expressions of IL-4 and IFN-γ mRNA were significantly lower in group C than in group B (P lt; 0.05). Conclusion Human PMSCs transplantation can suppress the acute immunological rejection in allogeneic skin transplantation. The possible mechanism may be partially related to the inhibitory effect on the secretion of IL-17 and IFN-γ.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
Objective Tri ptol ide can suppress immunological rejection reaction. To investigate the effect of tri ptol ide on allogenic tendon transplantation in repairing tendon defect in chicken. Methods The defect model of the third toes tendon was establ ished in 64 healthy-cleaning male Leghorn chickens (4-month-old, weighing 1.9-2.3 kg), which underwent allogenic tendon transplantation for repairing and were divided into 2 groups randomly (n=32). Tri ptol ide feeding[100 μg/(kg·d)] was given for 3 weeks in the experimental group and normal feeding in the control group. General condition of the chickens was observed after operation. The transplanted tendons were harvested from 4 chickens in each group for gross observation at 1, 2, 3, and 4 weeks after operation; the histological observation was performed at 1 and 3 weeks, and transmission electron microscope observation at 2 and 4 weeks. The blood and tendon were harvested from another 8 chickens in each group for flow cytometry and biomechanical tests respectively at 3 and 6 weeks. Results All chickens survived to the experiment end. Gross observation: with time extending, hyperemia and edema around transplanted tendon were rel ieved. Rarefaction adhering zone was seen in experimental group, and pyknotic adhering zone in control group. Histological observation: inflammatory reaction in experimental group was sl ighter than that in control group at 1 and 3 weeks. Transmission electron microscope observation: at 2 and 4 weeks, fibroblasts had big cell nucleus, more euchromatin, and l ittle heterochromatin in experimental group; however, there were small amount of rough endocytoplasmic reticulums with gentle expanded capsular space in control group, which contained sparse content. Flow cytometry test: at 3 and 6 weeks, peri pheral blood contained less CD4+ and CD8+ T lymphocytes in experimental group than in control group, and the ratio of CD4+ to CD8+ T lymphocyte significantly decreased in experimental group when compared with control group (P lt; 0.05). Biomechanical examination: at 3and 6 weeks, the maximum tensile strength in experimental group was bigger than that in control group, and tensile adhesion power in experimental group was smaller than that in control group. There were significant differences in the indexes between 2 groups (P lt; 0.05). Conclusion Tri ptol ide can suppress immunological rejection reaction, strengthen tendon healing strength, and reduce tendon adhesion in allogenic tendon transplantation.
Objective To evaluate the cl inical results of allogeneic bone graft for interbody fusion in cervical tuberculosis. Methods Between January 2000 and January 2008, 30 cases of cervical tuberculosis were treated with allogeneic (group A, n=15) or autologous (group B, n=15) il iac crest bone graft combined with anterior fixation after radical debridement. In group A, there were 8 males and 7 females with an average age of 38 years; the disease duration was 6 to 14 months; the preoperative kyphosis Cobb angle was (8.6 ± 11.3)°; the preoperative Japanese Orthopaedic Association (JOA) score was 13.0 ± 3.1 for neurological function; and the length of bone graft was 32 mm on average. In group B, there were 9males and 6 females with an average age of 42 years; the disease duration was 4 to 17 months; the preoperative kyphosis Cobb angle was (4.9 ± 7.4)°; the preoperative JOA score 12.3 ± 4.2; and the length of bone graft was 34 mm on average. There was no significant difference in general data between 2 groups (P gt; 0.05). Results The operation time and bleeding volume in group A were significantly less than those in group B (P lt; 0.05). Wound effusion were found in 2 cases of group A, and the other incisions healed by first intention. No infection occurred in group B. In group A, 13 cases were followed up 12-48 months; in group B, 14 cases were followed up 13-46 months. The time of bone graft heal ing in group A [(7.6 ± 2.1) months] was significantly longer than that in group B [(4.2 ± 1.1) months] (t=2.773, P=0.005). The kyphosis Cobb angles were significantly improved at 6 months and last follow-up after operation in 2 groups when compared with that before operation (P lt; 0.05), but no significant difference was found between 2 groups at different time after operation (P gt; 0.05). There was no significant difference in JOA score at 6 months after operation between group A (14.1 ± 2.6) and group B (14.3 ± 2.4) (t=1.655, P=0.162). The improvement rate for neural function were 83.7% in group A and 87.8% in group B, showing no significant difference (χ2=3.150, P=0.071). There was no loosening of internal fixation and recurrence of tuberculosis in 2 groups during follow-up. Five cases had chronic pain at il iac donor sites in group B. According to Bridwell et al. evaluation standard, the bone fusion was satisfactory in 11 cases (84.6%) and unsatisfactory in 2 cases (15.4%) in group A, and was satisfactory for all in 14 cases (100%) in group B. The satisfactory rate of bone fusion showed no significant difference between 2 groups (χ2=2.680, P=0.115).Conclusion Allogeneic bone grafting has a good cl inical result for spinal fusion in cervical tuberculosis surgery, which can treat tuberculosis bone defect effectively.
Objective To investigate the value of computer-aided design (CAD) in defining the resection boundary, reconstructing the pelvis and hip in patients with pelvis tumors. Methods Between November 2006 and April 2009, 5 cases of pelvis tumors were treated surgically using CAD technology. There were 3 males and 2 females with an average age of 36.4 years (range, 24-62 years). The cause was osteosarcoma, giant cell tumor of bone, and angiosarcoma in 1 case, respectively,and chondrosarcoma in 2 cases. According to the Enneking system for staging benign and mal ignant musculoskeletal tumors, regions I, I + II, III, IV, and I + IV is in 1 case, respectively. According to the principle of reverse engineering, 5 patients with pelvis tumors were checked with lamellar CT/MRI scanning, whose two-dimensional data were obtained in disease area. The three-dimensional reconstruction of pelvic anatomical model, precise resection boundary of tumor, individual surgical template, individual prosthesis, and surgical simulation were precisely made by computer with CAD software. Based on the proposal of CAD, the bone tumor was resected accurately, and allograft il ium with internal fixation instrument or allogeneic il ium with personal ized prosthetic replacement were used to reconstruct the bone defect after tumor was resected. Results The operation was successfully performed in 5 cases. The average operation time was 7.9 hours, and the average blood loss was 3 125 mL. Hemorrhage and cerebrospinal fluid leakage occurred in 1 case, respectively, and were cured after debridement. Five patients were followed up from 24 to 50 months (mean, 34.5 months). All patients began non-weight bearing walk with double crutches at 4-6 weeks after operation, and began walk at 3-6 months after operation. Local recurrence developed in 2 patients at 18 months after operation, and resection and radiotherapy were performed. According to International Society of Limb Salvage criteria for curative effectiveness of bone tumor l imb salvage, the results were excellent in 2 and good in 3. Conclusion The individual surgical template, individual prosthesis, and surgical simulation by CAD ensure the precision and rel iabil ity of pelvis tumors resection. The CAD technology promotes pelvis tumor resection and the reconstruction of pelvis to individual treatment stage, and good curative effectiveness can be obtained.
Objective To investigate the method and short-term effectiveness of arthroscopic reconstruction of anterior cruciate l igament (ACL) using RetroButton-allogeneic tendon-interference screw. Methods Between June 2009 and October 2009, 23 patients with ACL rupture were treated by arthroscopic reconstruction with RetroButton-allogeneic tendon-interference screw. There were 15 males and 8 females with an average age of 32.5 years (range, 19-46 years), including 17 left knees and 6 right knees. The injury causes were sport trauma (13 cases), traffic accident (8 cases), and fall ing injury(2 cases). There were 7 acute cases (lt; 6 weeks) and 16 chronic cases (gt; 6 weeks). Among the cases, 11 cases compl icated by medial meniscus injury, 6 by lateral meniscus injury, 3 by the both injuries, and 5 by articular cartilage injury. All cases had no injuries of posterior cruciate l igament, medial or lateral collateral l igament, or posterolateral structure. The time from injury to operation ranged from 3 weeks to 32 months. Lysholm and International Knee Documentation Committee (IKDC) scores were used for subjective evaluation, while Lachman test and KT-1000 measurement for objective evaluation. Results All wounds healed by first intention. The symptoms of unstable knee were improved obviously. No high fever, infection, or immunologic rejection were observed. Refractoriness synovitis and joint effusion occurred in 1 case after operation, and was improved after articular cavity flushing for 7 times within 3 weeks. All cases were followed up 10-17 months (mean, 14.7 months). There were significant differences in Lysholm score, IKDC score, Lachman test, and KT-1000 measurement between pre-operation and last follow-up (P lt; 0.05). Conclusion Arthroscopic reconstruction of ACL with RetroButton-allogeneic tendon-interferencescrew is simple and safe, and its short-term effectiveness is satisfactory.
Objective To investigate the effectiveness of meniscal allograft in treatment of discoid meniscus tear. Methods Between May 2005 and September 2009, 7 cases of discoid meniscus tear were treated with meniscal allograft. There were 2 males and 5 females with an average age of 22.6 years (range, 18-33 years). The locations were the left knee in 5cases and the right knee in 2 cases. The causes were sport trauma in 4 cases, impact injury in 2 cases, and no apparent reason in 1 case. Of them, 7 cases had joint tenderness gap, 2 cases had positive results in floating patella test, 7 cases had positive results in McMurray test, and 6 cases had positive results in squatting test. The range of knee motion was 0-60°. The disease duration ranged from 3 months to 17 years (median, 26 months). Results The number of the pins was 9-13 (mean, 11). The operative time ranged from 1.5 to 2.7 hours (mean, 1.7 hours). The postoperative fever time ranged from 31 to 57 hours (mean, 46.4 hours). Incisions healed by first intention in 6 cases; infection occurred in 1 case after operation and was cured after 18 days of local irrigation with antibiotic sal ine. Seven patients were followed up 21.6 months on average (range, 12-53 months). The range of knee motion was 0-125°. All patients had the negative results of floating patella test, McMurray test, and squatting test. MRI showed that there was a good heal ing between the transplanted meniscus and the surrounding synovium. The Japanese Orthopaedic Association (JOA) score was 80.71 ± 4.82 at 6 months after operation, showing significant difference (t=10.11, P=0.00) when compared with the score (41.71 ± 8.07) before operation. Conclusion Meniscal allograft in treatment of discoid meniscus tear is a rel iable and effective method, which can ease knee pain.
Objective To compare the effectiveness between arthroscopic double-bundle and single-bundle anterior cruciate l igament (ACL) reconstruction with deep-frozen allografts. Methods Between January 2008 and January 2009, 105 patients undergoing arthroscopic ACL reconstruction with deep-frozen allografts were selected and randomly divided intosingle-bundle (n=59) or double-bundle (n=46) groups. Patients were evaluated preoperatively and postoperatively 6, 12, and 24 months. Of the patients, 93 (51 in the single-bundle group and 42 in the double-bundle group) were available for full evaluation. There was no significant difference in gender, age, height, weight, disease duration, compl ication, the International Knee Documentation Committee (IKDC) score, and Lysholm score between 2 groups (P gt; 0.05). The anterior drawer test and the Lachman test for all were rated as + in 2 groups, and the pivot shift test were rated as ++ before operation. Results All wounds healed by first intention, and no related compl ication such as fracture, infection, or deep venous thrombosis was observed. There was no significant difference in the IKDC score, Lysholm score, anterior drawer test, pivot shift test, and Lachman test between 2 groups at 6-month and 12-month follow-up (P gt; 0.05). The IKDC score, Lysholm score, pivot shift test, Lachman test, and the side-to-side difference measured by KT-1000 arthrometer in the double-bundle group were significantly better than those in single-bundle group at 24-month follow-up (P lt; 0.05). Conclusion The double-bundle ACL reconstruction with deep-frozen allografts has better effectiveness than the single-bundle ACL reconstruction.
Objective To compare the recovery of proprioception between autograft and allograft for anterior cruciate l igament (ACL) reconstruction. Methods Between January 2008 and January 2010, 40 patients underwent ACL reconstruction with autologous tendon (autograft group, n=20) and allogeneic tendon (allograft group, n=20). No significant difference was found in gender, age, disease duration, and function scores between 2 groups (P gt; 0.05). All the patients underwent the ACL reconstruction with single-bundle technique. The knee range of motion (ROM), International Knee Documentation Committee (IKDC) score, and Lysholm score were measured after operation. The proprioception was assessedby the joint position sense (JPS) at 3 and 12 months postoperatively. The normal knee was used as control. Results Thepatients of 2 groups achieved heal ing of incision by first intention without compl ication of infection or haemarthrosis. Allpatients were followed up 12-18 months (mean, 13.5 months). There were significant differences in knee ROM, IKDC score, and Lysholm score between preoperation and 12 months postoperatively in 2 groups (P lt; 0.05). There was no significant difference in JPS 30°, JPS 60°, and JPS 90° between affected knees and normal knees in autograft group at 3 months postoperatively (P gt; 0.05). No significant difference was found in JPS 30° between affected knees and normal knees in allograft group at 3 months postoperatively (P gt; 0.05); but significant differences were found in JPS 60° and JPS 90° between affected knees and normal knees in allograft group at 3 months postoperatively (P lt; 0.05). There was no significant difference in JPS 30°, JPS 60°, and JPS 90° between affected knees and normal knees in 2 groups at 12 months postoperatively (P gt; 0.05). Significant differences were also found in JPS 60° and JPS 90° between affected knees of 2 groups (P lt; 0.05) at 3 months postoperatively, whereas no significant difference was found in JPS 30° between affected knees of 2 groups (P gt; 0.05). No significant difference was found in JPS 30°, JPS 60°, and JPS 90° between affected knees of 2 groups at 12 months postoperatively (P gt; 0.05). Conclusion Autologous andACL reconstruction is better than allogeneic ACL reconstruction in the recovery of proprioception at early time after surgery.