BACKGROUND: Researches show that erythropoietin (EPO) can stimulate the proliferation and protraction of endothelial progenitor cells to form new vessels, so EPO may play an important role in proliferation and differentiation of bone marrow-derived mesenchymal stem cells (MSCs). OBJECTIVE: To explore the effects of EPO on the proliferation and cell cycle of MSCs. DESIGN, TIME AND SETTING: From July to November 2007, the observation of comparative cell trial was performed at the Hematologic Diseases Institute of General Hospital Lanzhou Military Area Command of Chinese PLA. MATERIALS: Bone marrow liquid specimens were provided by voluntary donors. The informed consents were obtained from all patients, and the experiment was approved by Hospital Ethics Committee. METHODS: Using Percoll solution, MSCs were isolated from bone marrow by the method of density centrifugation. Flow cytometry was applied to detect the cell cycles of the second and third passages. MSCs were incubated with different doses of EPO (0.25, 0.50, 1.00, 2.00, 5.00 U/mL) in serum free culture media, and cells cultured with no EPO were regarded as control. All cells were cultured for 24, 48, and 72 hours. MAIN OUTCOME MEASURES: ①Proliferation of MSCs was measured by MTT assays after 24, 48, and 72 hours; ②Cell cycle was measured by flow cytometry assays after incubated with 10 U/mL EPO for 72 hours. RESULTS: After MSCs was incubated with EPO, the cell proliferation index was significantly increased in a dose and time dependent manner. The effects on the proliferation of MSCs were highest in 5 U/mL group. Compared with the control group, EPO could significantly decrease G0 /G1 ratio, and increase S and G2/M stage ratio (P

BACKGROUND: No particular marker molecule of bone marrow mesenchymal stem cells (BMSCs) is presently found, so its determination is difficult. BrdU marker has no radioactive pollution. Some studies have confirmed that BrdU marker has no damage to cell function without ultraviolet radiation. OBJECTIVE: To investigate in vitro identification and labeling methods and biological characteristics of adult BMSCs. DESIGN, TIME AND SETTING: The cell experiment was conducted at the General Hospital of Lanzhou Military Area Command of Chinese PLA between June and December 2007. MATERIALS: Bone marrow was collected from 5 healthy adult volunteers. BrdU was purchased from Sigma, USA. METHODS: 10 mL adult bone marrow was harvested to isolate mononuclear cells by density gradient. Cells were cultured in DMEM containing 10% fetal bovine serum and proliferated at a density of 2?108 L-1. At the third passage, BMSCs was inoculated in medium supplemented with osteoblast inductor and stained with alkaline phosphatase. Subsequently, BMSCs were inoculated in medium supplemented with lipoblast inductor and stained with oil red O. Cells were incubated with BrdU at different concentrations of 5, 10 and 15 ?mol/L for 12, 24, 48, 72 and 96 hours, and then detected by immunocytochemistry. MAIN OUTCOME MEASURES: Cell growth and proliferation were observed under an inverted light microscope. Cell phenotype, osteoblast and lipoblast differentiation were identified by flow cytometry. BrdU-positive labeling rate and cell growth after labeling were investigated. RESULTS: In vitro cultured BMSCs were homogenous population and exhibited a spindle-shaped fibroblastic morphology. BMSCs expressed CD44 and CD71, but did not express CD34 and HLA-DR. BMSCs can differentiate into osteoblasts and lipoblasts. Most BMSCs were labeled by BrdU. With the increase in labeling concentration and over time, BrdU positive rate gradually increased and exceeded 90% after labeling with BrdU (10 ?mol/L) for 72 hours, with the labeling identifiable in nine consecutive passages. At the third passage, 90% BMSCs were in G0/G1 phase, and 88.62% in G0/G1 phase after labeling. BrdU has little effect on morphous and proliferation of labeled cells. CONCLUSION: Adult BMSCs can be identified through morphological character, specific surface antigens and multipotential differentiation in vitro. BrdU labeling provides a feasible means for a dynamic observation of the survival, growth and differentiation of the implanted BMSCs. The optimal dosage and timing of BrdU labeling are respectively 10 ?mol/L and 72 hours.

BACKGROUND: At present the strategy of nerve regeneration and repairng are main promoting nerve intrinsic regeneration capacity and improving the micro-environment. Studies have shown a number of combined treatment which could promote the regeneration and growth of nerve axon.OBJECTIVE: To explore the feasibility and effect of rat spinal cord injury repaired by peripheral nerve combined growth factor. METHODS: Sixty healthy adult female SD rats were randomly divided into 4 groups: nerve graft group, nerve graft combined growth factor group, spinal cord transaction group and laminectomy group. Taking T_9 as the center, a longitudinal incision was conducted in rat skin, revealing dural sac, spinal cord was transected and removed 3 mm, 2-cm segment of the eighth to tenth intercostal nerve was obtained from nerve graft group and nerve graft combined with growth factor group, autologous intercostal nerve was cross-transplanted into spinal defect (proximal white matter and distal gray matter, distal white matter and proximal gray matter) after pruning appropriately. The transplanted intercostal nerves were fixed with fibrin glue in nerve graft group, while those in nerve graft combined growth factor group were fixed with fibrin glue containing 2.1 mg/L acidic fibroblast growth factor, followed by dural suture～ Stump of broken ends was done in spinal cord transection group, while laminectomy was performed in laminectomy group. RESULTS AND CONCLUSION: At 90 days post-surgery, somatosensory evoked potential (SEP) and motor evoked potential (MEP) were determined, the motor function of hind limbs was evaluated by the Basso. Beattie.Bresnahan (BBB) test at 70 days. Both SEP and MEP were led in the laminectomy group, but not lead in spinal cord transection group; in nerve graft group, 3 rats showed bilateral SEP, 4 led unilateral SEP, 4 led bilateral MEP, 3 led unilateral MEP; in nerve graft combined with growth factor group, 5 led bilateral SEP and 2 led unilateral SEP, 5 led bilateral MEP and 2 led unilateral MEP. The SEP and MEP latency and amplitude in the nerve graft group and nerve graft combined growth factor group were significantly superior to the spinal cord transection group (P < 0.01), autologous rib nerve graft group was better than nerve graft combined growth factor group (P <0.01). In the laminectomy group, awake rats following anesthesia returned to normal exercise, rats in spinal cord transection group continued to extend limbs and rotated within 3 months, rats in other two groups recovered functions obviously 3 weeks post-surgery and gradually restored throughout the entire observation period. Nerve graft group and nerve graft combined growth factor group showed significantly increased BBB score compared with spinal cord transection were (P < 0.01), and the nerve graft combined growth factor group was superior to nerve graft group (P < 0.01). The peripheral nerve graft can promote the spinal function following spinal cord injury, while the nerve combined growth factor can better restore the function.

BACKGROUND: Transforming growth factor (TGF)-β_1, a potent cell growth and proliferation regulatory proteins, plays an important role in development of anti-graft rejection and graft vascular disease. OBJECTIVE: To observe local injection of TGF-β_1 effects on transplant immune rejection following freezing disposal and nerve allograft. DESIGN, TIME AND SETTING: The randomized controlled animal study was performed at the Animal Experimental Center, Harbin Medical University from June 2007 to June 2008. MATERIALS: A total of 60 clean SD rats (recipients) were divided into 3 groups: autogenous nerve graft group, nerve allograft group, TGF-β_1 plasmid + nerve allograft group, 20 in each group. A total of 40 Wistar male rats served as donors. pAdTrack-CMV-TGF-β_1 plasmid, pAdEasy-1-Bj51833 cells were presented by the Orthopedic Laboratory of Fourth Hospital of Harbin Medical University. METHODS: Longitudinal posterolateral incision was made in 40 donor rats to expose sciatic nerve. The whole bilateral sciatic nerve was cut and placed in sterile frozen tubes for 1 week for use. Under the microscope, connective tissue was cut in the biceps muscle and semi-tendon and semi-membrane gap of recipient rats to expose the sciatic nerve. 1-cm sciatic nerve was cut 0.5 cm below the muscle from the plow-shaped hole. Transplantation of frozen autogenous nerve graft and nerve allograft (nerve at equal size) was separately performed in the autogenous nerve graft and nerve allograft groups. In the TGF-β_1 plasmid + nerve allograft group, pAdTrack-CMV-TGF-β_1 plasmid (40 μg) was injected into the local muscle and two sides of transected sciatic nerve of each rat following nerve allograft transplantation. MAIN OUTCOME MEASURES: Motor nerve conduction velocity, pathology and axonal counting were examined 3, 6, 9 weeks after surgery. RESULTS: Motor nerve conduction velocity was higher in the TGF-β_1 plasmid + nerve allograft group than in the nerve allograft group (P < 0.01), which did not show significant difference compared with the autogenous nerve graft group. Axonal counting was greater in the autogenous nerve graft and TGF-β_1 plasmid + nerve allograft groups compared with the nerve allograft group 9 weeks following surgery (P < 0.01). Using optical microscope and electron microscope, nerve fibers were normal and well arranged in the TGF-β_1 plasmid + nerve allograft group. Nerve fibers presented vascular proliferation, good myelin sheath. Abundant regenerated myelin sheath was found in nerve fiber. The number of Schwann cells was obviously increased, and there were prosperous cytoplasm, a large amount of rough endoplasmic reticulum, clear mitochondria. In regenerated axons, microfilament closely arranged, which was similar to the autogenous nerve graft group. In the nerve allograft group, the optical microscope and electron microscope showed a few nerve fibers, disorderly arranged, significant demyelination, axon degeneration and disappearance, without regenerated fibers. CONCLUSION: Local injection of TGF-β_1 plasmid could reduce immune rejection after cold sciatic nerve allograft transplantation.

BACKGROUND:It is proved by a number of experiments that such a structure as Bungner band-Schwann cell-basilar membrane,which is formed at 2 or 3 weeks after nerve injury,is the ideal microenvironment for neural regeneration. However,the sprouting of nerve fiber close to broken ends takes place at several hours after nerve injury,which shows that the regeneration of nerve fiber and the formation of required microenvironment don't occurred at the same time.OBJECTIVE:To investigate the best repairing time for peripheral nerve injury.DESIGN,TIME AND SETTING:A randomized control animal experiment was performed in the Animal Experiment Centre,Harbin Medical University from June 2007 to June 2008.MATERIALS:A total of 20 New Zealand rabbits were randomly divided into four groups,namely,an immediate repairing group and the other three groups that were repaired respectively at week 2,week 4 and month 3 after injury.METHODS:Peripheral nerve injury models of New Zealand rabbits were established. The immediate repairing group received suture immediately after injury;For the other three groups,the two broken ends of their nerves were fixed on sarcoiemmas temporarily and their wounds were sutured layer by layer. Then they were opened respectively at week 2,week 4 and month 3 after injury,to receive epineural suture with non traumatic 10-0 nylon suture under operating microscope,after which wounds were sutured again.MAIN OUTCOME MEASURES:Nerve electrophysiological observation,axon number,light microscope and electron microscope observation of sutured nerve segments in each group.RESULTS:Nerves repaired at week 2 after injury had a slower nerve conduction velocity than those at week 4 and month 3 after injury (P＜0.01);There was no difference of significance between the immediate repairing group and the group repaired at week 2 after injury (P＞0.05). According to the comparison among the four groups:it had the best repairing effect to repair nerve at week 2 after injury,with normal course and neat arrangement of nerve fibers,vascular proliferation in nerve fibers,myelin sheaths with better structure,Schwann ceils with active function,as well as regenerated axons with intensively arranged microfilaments;Repairing at week 4 after injury had the worst effect,with rare nerve fivers disorderly arranged,myelin sheath and axons significantly degenerated,most nerve fibers demyelinated with axons disappeared,and no regenerated nerve fibers seen;Repairing at month 3 saw the worse repairing effect,with more nerve fiber damaged and disorderly arranged,myelin sheath and axons significantly degenerated,nerve fibers rarely regenerated,less Schwann cells,as well as cytoplasm did not well develope;The effect of immediate repairing after injury was better,with nerve fibers unobviously damaged and well arranged,myelin sheath and axons lightly degenerated,large amounts of myelin sheaths regenerated in nerve fibers,Schwann cells increased obviously,as well as cytoplasms better-developed. Axon counting result was better in the group repaired at week 2 after injury than the otherthree groups,with the minimum in the group repaired at week 4 after injury.CONCLUSION:Repairing at week 2 after injury can get a better result than at any other time points,accordingly two weeks after nerve injury is the best time for repairing peripheral nerve injury.

Objective To analyze and evaluate the effects of the tissue engineering bladder reconstruction on the upper urinary tract structure and function.Methods The 8 male beagles were randomly divided into the two groups:sham-operation group (group A,n=4)and the collagen scaffold repair group (group B,n=4).The bladder defect animal model was established in the group B by using the collagen scaffold materials to repair the bladder.The renal function related biochemical indicators were detec-ted and the renal Doppler ultrasonic examination was performed in each group before repair and in 23 weeks after repair.The speci-mens from the two groups were performed the gross morphology observation and the histology examination on postoperative 24 weeks.Results The renal Doppler ultrasound examination showed the normal kidney morphology and normal blood flow signal.In the general observation,no calculi and neoplasm were found in the kidney and ureter of the experimental dogs.The renal function related biochemical indicators had no statistically significant differences between the two groups(P>0.05).The histological exami-nation indicated that the organization structure was integrity,the nephrons in each group had no obvious pathological changes.Con-clusion Using the collagen scaffold materials to reconstruct the canine bladder has no adverse influence on the upper urinary tract structure and function,this tissue engineering approach has good feasibility.

Objective:To evaluate the effects of Sander Ⅱ appliance in the orthodontic treatment of adolescent ClassⅡmalocclusion. Methods:15 cases (6 male and 9 female)of adolescent ClassⅡ malocclusion with mandibular retrution were treated with Sander Ⅱappliance.Pre-treatment and post-treatment Lateral cephalogram measurements were traced and analyzed in terms of 28 indicators. SPSS 19.0 software package was used for paired t test.Results:After treatment,15 patients achieved remarkable improvement in the maxillofacial profile and normal overjet of teeth.The cephalometric analysis showed that ANB,OJ,H°and U1-E decreased(P <0. 05),SNB,B-OLP,Pg-OLP(mm),Ii-OLP,Ms-OLP(mm),Mi-OLP(mm),N-Me,ANS-Me,S-Go,Go-Gn,N'-Pg'/FH,Cm-Sn-Ls and Pg-Pg'increased(P <0.05).There was no significant change in SN-MP,SN-OL and Y-axis before and after treatment. Conclusion:Sander Ⅱ appliance is effective in the treatment of adolescent early Class Ⅱmalocclusion.