BACKGROUND: Longterm therapeutic effects of routine drug treatment, intervention or vascular bypass transplantation on lower extremity arterial occlusion are not ideal. During recent years, angiogenesis of stem cells possibly becones a new method to repair or rebuild an effective collateral circulation at infarct regions.OBJECTIVE: To verify the effect of autologous bone marrow mesenchymal stem cell transplantation on promoting angiogenesis in rabbit ischemic hind limbs.DESIGN, TIME AND SETTING: A randomized controlled animal study was performed in Jiangsu Institute of Schistosomiasis Control between August 2005 and November 2006. MATERIALS: Eight rabbits were used to prepare ischemic models of hind limbs, and then they were randomly divided into experimental group (n=4) and control group (n=4).METHODS: Bone marrow mesenchymal stem cells were isolated and cultured from New Zealand rabbits in the experimental group, and they were then marked 5-bromo-2-deoxyuridine (Brdu). Suspension of bone marrow mesenchymal stem cells was injected into the ischemic hind limbs in the experimental group, while the same volume of saline was injected into the controls. MAIN OUTCOME MEASURES: After two weeks, two-dimensional and color Doppler ultrasound detection was used on rabbit femoral artery to measure inner diameter of blood vessel, peak velocity and acceleration time of blood flow before and after transplantation. Muscle tissues were obtained from ischemic regions to observe distribution of transplant cells and state of angiogenesis using immunofluorescence staining and hematoxylin-eosin (HE) staining. RESULTS: Two weeks after transplantation, the inner diameter of femoral artery and the peak velocity of blood flow in the experimental group were higher than those in the control group (P < 0.01), but the acceleration time of blood flow was shorter than that in the control group (P < 0.01). Lmmunofluorescence staining showed that anti-Brdu-staining positive cells were found out in transplant part in the experimental group; while HE staining indicated that vessel density of ischemic region in the experimental group was higher than that in the control group. CONCLUSION: Bone marrow mesenchymal stem cells can promote angiogenesis, while autologous bone marrow mesenchymai stem cell transplantation will become a simple and effective method to treat lower limb ischemia.

BACKGROUND: Bone bone bone marrow derived mesenchymal stem cells (MSCs) has the capacities of self-renewal and multi-directional differentiation, which can differentiate into osteoblasts after induction. Combined with basal hydroxyapatite materials, MSCs can repair the bone defect with satisfactory shape and function.OBJECTIVE: To observe the bone formation performance of segmental bone defect repaired by autologous transplantation after combination of MSCs and basal hydroxyapatite materials.DESIGN: Open experiment.SETTING: Department of Cells, Third People' s Hospital of Wuxi.MATERIALS: The experiment was conducted in the Department of Cells,Third People's Hospital of Wuxi between May 2004 and March 2005.Eighteen New Zealand rabbits of clean grade, aged 6 months were selected and randomly divided into cytoskeleton group, simple scaffold group and blank control group with 6 rabbits in each group. Nano-collagen basal hydroxyapatite bone were provided by the Department of Material, Tsinghua University, which is characteristic of natural-bone microstructure. Composition: about 57% were hydroxyapatite,and 30% were collagen and 13%were polylactic acid.METHODS: ①bone bone marrow derived MSCs of rabbits were isolated and cultured. Well-grown cells of the second generation were inspected of the expression of cell surface antigen (CSA) with EPICS-ALTRA flow cytometry. ②Scaffold materials were cut into blocks with the size of 6 mm ×6 mm×4 mm, sterilized by 60Co irradiance and infiltrated with DMEM-LG before using. Cells of the 2nd and 3rd generations were collected, cultured,the concentration of which was regulated to 109 L-1. Cells were co-cultured with nano-collagen basal hydroxyapatite in vitro. ③Model of segmental defects in radial of rabbits were established in all groups. The prepared compounds in the cytoskeleton compound group were embedded in the bone defect according to autograft principle, and blank scaffold materials were embedded in the bone defects of simple scaffold group. Nothing was transplanted in the blank control group. Rabbits in all groups received no internal or external fixation, whose soft tissues and skins were closely sutured.④The surface structure of materials and cell adherence in all groups were observed under scanning electron microscope (SEM).⑤Bone formation was studied by general observation, histological analysis and X-rays at 4, 8 and 16 weeks after operation respectively.MAIN OUTCOME MEASURES: ①The isolated culture and identification of MSCs.②Observation of all groups under SEM.③Results of radiological inspection of all groups. ④General morphous of cells in each group after operation.⑤Results of histological inspection of all groups after operation.RESULTS: A total of 18 enrolled New Zealand rabbits were involved in the analysis of results.①There were adherent cells at 4 hours after primary culture, which were like cloned at 48 hours after static culture. Cells were in irregular scale-shape with large cell body and the nucleus was in middle. Inspection with flow cytometer showed that CD29,CD44, CD-105 were positive, and the ratios of three positive cells were 97.4% ,98.1% and 86.2% respectively. ②The surface of simple scaffold group was irregular with many ventages. There were cells adherent to the surface of materials as well as the inside of ventage in the cytoskeleton compound group. ③The absorbance of X-rays at 4, 8 and 16 weeks after operation were higher in the cytoskeleton compound group than blank control group and simple scaffold group, moreover, bone union could be seen at the 16th week in the cytoskeleton compound group. ④The implants was embedded in the osteotylus at 16 weeks after operation in the cytoskeleton compound group,while parts of the middle segment of implants in the simple scaffold group was not covered by bone tissues. No porosis was found in the blank control group.⑤The ossifying capacity at each time point after operation was better in the cytoskeleton compound group than simple scaffold group, moreover,there was bone trabecula formed at the 16th week, and the osteoblasts were arranged in order. The bone tissues in the simple scaffold group mainly concentrated on the surface of materials, which sucked little. There were some bony tissues formed in the proximal end of defects in the blank control group, while none was seen in the middle parts.CONCLUSION: Rabbit MSCs can greatly proliferate in vitro, which has strong osteogenesis by being co-cultured with nano-collagen basal hydroxyapatite. It is a good kind of seed cells in tissue engineering.

BACKGROUND: There have been no effective means for clinical treatment of large regions of bone defects.Nano-hydroxyapatite/collagen(nHAC)composite would provide a new pathway for repair of bone defects owing to its similar structure to natural skeleton and better biocompatibility.OBJECTIVE: To investigate the role of nHAC composite co-cultured with bone marrow-derived mesenchymal stem cells(BMSCs)in repair of bone defects.METHODS: Following isolation and culture,human BMSCs were co-cultured with nHAC composite.Gross observation,histological analysis,and electron microscope observation were performed to analyze osteogenesis for repair of bone defects in the clinic.RESULTS AND CONCLUSION: Human nHAC could greatly proliferate in vitro.X-ray photography revealed that bone defects well healed after implantation of nHAC/BMSCs composite.These findings indicate that BMSCs exhibit osteogenic potential and nHAC is a satisfactory scaffold material for construction of tissue-engineered bone.

BACKGROUND:Several studies have demonstrated that stem cells can differentiate into vascular endothelial cells, and then further differentiate and form blood capillary. Based on this principle, autologous bone marrow mesenchyrnal stem cell (BMSC) transplantation promotes angiogenesis to treat ischemia in lower limb.OBJECTIVE: To evaluate the angiogenesis in rabbit ischemic limbs following autologous BMSC transplantation using high-frequency two-dimensional ultrasound detection in conjunction with Doppler color-flow imaging examination. DESIGN, TIME AND SEI-FING: A randomized, controlled, animal experiment was performed in the Third People's Hospital of Wuxi between March 2007 and April 2008.MATERIALS: Twenty-four New Zealand rabbits were randomized to a control group and a cell transplantation group, with 12 rabbits in each.METHODS: Ischemia in lower limbs was induced in all rabbits. One week following ischemia induction, the cell transplantation group was injected with 0.5 mL cell suspension, comprising 2×106 BrdU-labeled autologous BMSCs cultured in vitro, through multiple sites in the region of gastrocnemius muscle. Simultaneously, the control group received the same amount of physical saline in the same region.MAIN OUTCOME MEASURES: The initial segment of rabbit femoral artery and superficial femoral artery was subjected to high-frequency two-dimensional ultrasound and Doppler color-flow imaging examinations to measure femoral artery vascular internal diameter, blood flow peak velocity, and blood flow acceleration time. Ischemic muscular tissue was taken for immunohistochemical staining to detect transplanted cell distribution and for pathological examination of angiogenesis. RESULTS: Two weeks following autologous BMSC transplantation, high-frequency ultrasound results revealed that femoral artery internal diameter and blood flow peak velocity were greater, but blood flow acceleration time was shorter, in the cell transplantation group than in the control group (P<0.01). Immunohistochemical staining results demonstrated the presence of BrdU-positive cells. Pathological sections displayed that vascular density was significantly higher in the cell transplantation group than ih the control group. CONCLUSION: Autologous BMSC transplantation is a promising, simple, and effective method of treating ischemia in lower limbs owing to its promotion of angiogenesis. Meanwhile, high-frequency ultrasound detection of femoral artery is an effective, practical method to evaluate the clinical outcomes of autologous BMSC transplantation.

10.3969/j.issn.2095-4344.2013.23.006

Objective:To summarize the clinical characteristics, common images, pathogens, and gene mutation types of chronic granulomatosis disease (CGD) in 19 children.Methods:The clinical manifestations, laboratory findings, treatment, and prognosis of 19 patients diagnosed with CGD in Hong Kong University-Shenzhen Hospital from December 2012 to December 2018 were analyzed.Results:The 19 patients were all males and confirmed as CGD by the dihydrorhodamine test and gene sequencing.The age of the first infection was mostly 1 month after birth(13 cases), and the age of clinical diagnosis ranged from 2 months to 10 years.Sixteen mothers were carriers.The patients presented with pulmonary fungal infection (19/19 cases), Bacillus Calmette Guerin (BCG)-osis (14/19 cases), lymphadenitis (14/19 cases), perianal abscess (9/19 cases), skin abscess (5/19 cases) and ulcerative colitis (2/19 cases). There were 59 positive cultures.Pathogens included fungi (9 cases), Klebsiella pneumonia (8 cases), mycobacteria (7 cases), Streptococcus Viridans (5 cases), Escherichia coli (3 cases), gram-positive bacteria (3 cases), Staphylococcus aureus (3 cases), and Burkholderia cenocepacia (2 cases). Gene mutations were found in all 19 patients, including 17 cases of CYBB, 1 case of CYBA and 1 case of NCF2.The type of mutations included nonsense mutations (6 cases), deletion mutations (5 cases, including 2 large fragment deletions), splice mutations (3 cases) and missense mutations (5 cases). Five mutations were novel.Splice mutations in 3 cases often led to skin abscess, perianal abscess and lymphadenitis.Two patients with large deletion mutations had more serious infection than other patients. Conclusions:In China, CGD is characterized with pulmonary infection and disseminated BCG-osis.Mycobacteria are common pathogens of CGD, and fungi are dominant pathogens of CGD.The most common infection is respiratory infection. Klebsiella pneumonia and Escherichia coli often lead to perianal abscess.The relationship between gene mutation types and clinical phenotypes requires further verification by big data.