[Objective]To investigate if allogeneic human serum(alloHS)could replace fetal bovine serum(FBS)for the expansion of human bone marrow mesenchymal stem cells(hBMSCs)in vitro.[Method]Human BM-MSCs were isolated either from spongy bone residues obtained during hip surgery or from washed filters used during bone marrow graft processing for allogeneic bone marrow transplantation.We culture human bone marrow mesenchymal stem cells were cultivated using alloHS and FBS,then observe its growth character,antigen presentation and differentiation potency through condition medium directional induction were observed.[Result]Isolated MSCs were positive for the markers CD105,CD29 and CD44 and negative for typical hematopoietic and endothelial markers CD34,CD45,CD106 and HLA-DR.The choice of serum affected hMSCs at several different levels.Firstly,hMSCs in alloHS proliferated markedly faster than hMSCs in FBS.Secondly,hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in alloHS.[Conclusion]hMSCs may be expanded rapidly in alloHS in the absence of growth factors,this may be a safely culture method suitable for clinical demand.

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: Spinal cord injury occurs frequently and its consequence is very severe. There is no effective method to rebuild the function of demylinated nerves. Transplantation of a kind of special glial cells in olfactory system of mammal attracts more attention.OBJECTIVE: To observe whether combined transplantation of human fetal olfactory ensheathing cells (human OECs) and rat embryonic spinal cord tissues (rat ECS) possesses synergistic effect in promoting axonal regeneration in the rats following spinal cord transection.transection.DESIGN: Open experiment.SETTING: Cell Room, Third People's Hospital of Wuxi City; Department of Neurology, First Hospital Affiliated to Soochow University; Department of Neurosurgery, Zhongda Hospital Affiliated to Central South University MATERIALS: This experiment was carried out in the cell laboratory of the Third People's Hospital of Wuxi from September 2002 to October 2004. ① Totally 36 adult female SD rats, of clean grade, were selected and randomly divided into 4 groups: human OECs group (n=10), rat ECS group (n=10), combined transplantation group (n=10) and sham-operation group (n=6). ② Fresh 12-week aborted human embryo was used for culture and purification of human OECs (Informed consent was obtained from the parturient). ③One SD rat at embryonic 14 days underwent caesarean operation, and fetal rat and fetal membrane were taken out together and used for preparing new embryonic spinal cord.METHODS: ①Rats of 4 groups were all created into hemisection cavity models. Gelatin sponge and complete culture medium of 8 μL were packed into the injured cavity of rats in the model group, and the same culture medium of 2 μL was injected at 1 mm above or below injure; Human OECs suspension of 8 μL was added to gelatin sponge in human fetal Human OECs group, and human OECs suspension of 2 μL was injected at 1 mm above and below injure; rat embryonic spinal cord tissue of rat ECS group was chipped into pieces, which were packed into the cavity,and gelatin sponge was spread on the injury part. Embryonic spinal cord with the same size was packed into the cavity of combined transplantation group, then 8 μL human OECs suspension was injected into cavity with micro sample injector, and gelatin sponge was spread on the injury part, and then cellular suspension of 2 μL was injected at 1 mm above and below the cavity, and muscular layer skin was sutured layer by layer. ②The rats of each group were performed ethological evaluation periodically. Combined with pathological observation, effect of human OECS and rat ECS on neuronal survival and regeneration was evaluated by performing horseradish peroxidase-tetramethyl benzidine tracer technique.MAIN OUTCOME MEASURES: ①In vitro culture and purification of human fetal human OECs. ② In vitro immunocytochemistrical analysis. ③BBB scoring of motor function of hindlimb of rats. ④ Immunohistochemical detection of implants and injured spinal cord repair⑤ Quantitative analysis on labeled neurons at the cortex and mesencephalic red nucleus ineach group with horseradish peroxidase-tetramethyl benzidine tracer technique.RESULTS: ① Most of human fetal OSCs presented double-polar spindle.Five to seven days after culture, OSCs weaved into net and a lot of mitosis phases were found. The cellular purity was 85%. ② The rate of P75 positive cells was (83±7)%. Glial fibrillary acidic protein was found in about (81±6)% of cells and Vimentin in (91±9)% of cells and the rate of Nestin positive was (77±5)%. ③Three to five days after operation, affected limb of rats of sham-operation group began to contract, the activity of hindlimb of intact side was limited a little. Fewer obvious contraction symptoms were found in the other 3 groups. From 2 weeks after operation,behavioral function recovered significantly fast in each group. BBB scores of combined transplanted group were significantly high than those of human OECs group, rat ECS group and sham-operation group [(6.2±1.13) vs.(5.0±1.15)vs.(3.9±0.88)vs.(3.3±1.03)scores,P ＜ 0.05]. ④In bipolar or multipolar cells, in which basic protein(+)granules were found, P75 and glial fibrillary acidic protein positive were found at the implanted part in the range of 2.0 to 5.0 mm of transplanted region in the human OECs group and combined transplantation group. A great many of small MAP2 positive neurons were found in the spinal defected focus in the rat ECS group and combined transplantation group. Nerve plexus positive fibers were observed in spinal defected region of human OECs group, rat ECS group and combined transplantation group to different extents, especially significantly in the combined transplantation group, but they were not found in the model group. ⑤ Horse radish peroxidase labeling was hardly found in neurons at the injured side of sham-operation group, while the number of labeled neurons at the cortex and nesencephalic red nucleus was significantly higher in the combined transplantation group than in the human OECs group and rat ECS group (P ＜ 0.05).CONCLUSION: Combined transplantation of OECs and ESC can obviously protect injured spinal cord, promote host spinal axonal regeneration and play s a complementary and synergetic effect in speeding up the functional recovery of rats.

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: Umbilical cord blood mesenchymal stem cells (UCBMSCs) have been shown to differentiate into nerve-like cells under the condition of in vitro culture. Brain-derived neurotrophic factors (BDNF) combined with ciliary neurotrophic factors (CNTF) at certain concentration can in vitro induce the differentiation of UCBMSCs into high proportion of neuronal-like cells. OBJECTIVE: To investigate the feasibility of BDNF, CNTF and their combination for in vitrodifferentiation of UCBMSCs into nerve-like cells.METHODS: UCBMSCs of passage 5 were induced to differentiate into nerve-like cells using 5,10, 20 ug/L BDNF, 5,10, 20 ug/L CNTF or their combination. A blank control group was set, with no any interventions. Cell morphology was observed under inverted phase contrast microscope. At 1, 3, and 6 days of experiment, immunocytochemical staining for neuron specific enolase and glial fibrillary acidic protein was performed. The proportion of differentiated neuronal-like cells and glial cell-like cells was calculated.RESULTS ANDCONCLUSION:After induced differentiation into nerve-like cells, human UCBMSCs exhibited changeable morphology with contracted cell body, enhanced refraction of nucleus, and dendrite- and axon-like structure. Compared with blank control group, BDNF and CNTF couldsignificantly enhance the differentiation proportion of UCBMSCs into nerve-like cells. 20 ug/LBDNF combined with 20 ug/L CNTF yielded highest differentiation proportion of human UCBMSCs into nerve-like cells. These findings indicate that human UCBMSCs can differentiate into nerve-like cells after in vitro induction of BDNF, CNTF or their combination.

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

BACKGROUND: The repair of periodontal tissue is dependent on the number and proliferation and differentiation abilities of periodontal ligament (PDL) cells. PDL cells have the potentiality of multi-directional differentiation such as cementoblast,osteoblast and fibroblast to fonn cement, alveolar bone and periodontal ligament and finally achieve periodontal tissue regeneration.OBJECTIVE:To observe the effects of Shuanghuangbu extract on the proliferation and differentiation of PDL cells.DESIGN:Observation trail.SETTING:Central Laboratory of Wuxi Third People's Hospital. MATERIALS: Periodontal tissue was provided voluntarily by the healthy young patients with deformity correction, and golden thread,skullcap,and rhizoma drynariae by the National Institute for the Control of Pharmaceutical and Biological Products.METHODS: The experiment was conducted in the Central Laboratory of Wuxi Third People's Hospital from July to October 2003.The crushed golden thread.skullcap,and rhizoma drynariae were mixed with distilled water at ratio of 1:10 (m:V),and refluxed in boiling water for 5 hours.The extract was collected,and after colation,the residue was refluxed in boiling water for another 3 hours. Both extract was blended, rotary evaporated and condensed, finally the liquid extract of 3 kg/L was obtained.There were 8 groups in the study including golden thread group, skullcap group,rhizoma drynariae group,golden thread plus skullcap group,golden thread plus rhizoma drynariae group, skullcap plus rhizoma drynariae group, Shuanghuangbu group and control group. PDL cells were cultured/n vitro assisted with Shuanghuangbu.The proliferation of cells was detected with MTT method and the ratio of collagen content in total protein was evaluated with hydroxyproline (HP).MAIN OUTCOME MEASURES:A value of proliferated PDL cells and the proportion of collagen protein in total protein.RESULTS:①Proliferation of PDL cells:Except golden thread group,all Chinese medicine promoted the proliferation of PDL cells significantly compared with control group (P＜0.05). The A value of Shuanghuangbu group was significantly increased.A value was increased with time and reached the peak on day 5.There were significant differences among each group at different time (P＜0.05).②Ratio of collagen content in total protein:Except golden thread group,the percentage was significantly increased by other Chinese medicines compared with control group (P＜0.05), especially Shuanghuangbu. The percentage was increased with time and reached the peak on day 5. There were significant differences among each group at different time (P＜0.05). CONCLUSION:The findings suggest that as a traditional Chinese herb,Shuanghuangbu can significantly stimulate the proliferation and differentiation of PDL cells.and increase the proportion of collagen content in total protein.It may act as an ideal Chinese medicine helper factor for the regeneration of PDL cells.

BACKGROUND: Some researches suggest that placenta is regarded as a new source of mesemchymal stem cells (MSCs). Adherent cells derived from placenta tissue have similar morphological characteristics and surface markers to MSCs; meanwhile, they can differentiate into osteoblasts and nerve cells.OBJECTIVE: To find a new source and way for MSCs separation.DESIGN: Observational study.SETTING: The Third People's Hospital of Wuxi.MATERIALS: Placenta was sourced from cesarean in our department of obstetrics and gynecology and provided confirmed consent from the relatives and ethics committee. The main reagents contained culture medium, sABC kit, DAB staining kit, caprine-anti-rat FITC, recombinant human basic fibroblast growth factor (rh-bFGF), rabbit-anti-human glial fibriliary acidic protein (GFAP).METHODS: The experiment was carried out in the Laboratory of Cells & Molecular Biology, the Third People's Hospital of Wuxi from May 2005 to August 2006. Placenta entity tissue was digested, adherent cells were cultured, morphological characteristics were observed, and growth curves of placenta-derived multipotent cells (PDMCs) in various generations were drawn. On the 1st and 7th days, supernatant was derived from cells in primary culture to measure content of β-glycerophosphate disodium (β-HCG) with chemiluminescence technique. In addition, expression of surface antigen and differentiating potency were detected at the same time. At 24 hours (at phase of nerve cells) and 2 weeks (at phase of osteoblasts) after inducible differentiation, cultured cells were dealt with routine immunocytochemical stain, and then they were observed under routine microscope or fluorescence microscope.MAIN OUTCOME MEASURES: ① Morphological indexes and growth curves of PDMCs; ② measurement of β-HCG in supernatant of PDMCs and expression of antigen of PDMCs with flow cytometer; ③ immunohistochemical analysis of PDMCs during and after inducible differentiation.RESULTS: ① Primary culture of PDMCs: After digestion of placenta tissue, a few of adherent cells were obtained and gradually formed thin and flat monolayer cells two weeks later. The monolayer cells grew like whirlpool or cluster. With the increase of cell density, soma was slenderer and slenderer and like fibroblast. ② Growth curves of PDMCs: Growth latency ranged from 2 to 8 days after cell inoculation. During this period, adherence was observed gradually but not obviously amplified. Eight days later, cells entered log growth phase. During this period, proliferation was active and cell process surrounding extended under phase contrast microscope. A lot of MSCs were observed in division phase between two nuclei. In addition, density was increased and cells connected to each other. Within 11-14 days, growth curve gradually entered platform phase. MSCs covered the bottom of bottle, cells slowly expanded, and primary culture stopped. ③ Measurement of β-HCG: Expression of β-HCG was not detected in supernatant at two time points. ④ Characteristics of surface antigen of PDMCs: PDMCs could express CD29, CD44 and CD105, but not CD34, CD45,CD19 and CD106. ⑤ Inducible differentiation of PDMCs: At 24 hours after inducing to nerve cells, form of PDMCs obviously changed, soma rebounded, refraction of nucleus was partially reinforced, structure was similar to dendrite and axis-cylinder, and positive neuro-specific enolase and GFAP were observed after staining.CONCLUSION:Placenta tissue contains PDMCs whose morphological function is similar to MSCs; in addition, placenta is regarded as an effective source of MSCs.