BACKGROUND: Spinal cord can regenerate after injury in certain microenvironment. Olfactory ensheathing cells (OECs)have the characteristics of astrocytes and Schwann cells and can accelerate the spinal cord axonal regeneration.OBJECTIVE: To make injured thoracic cord rat models and observe the effect of OECs on injured spinal cord axonal regeneration.DESIGN: Observational experiment.SETTING: Second Affiliated Hospital of Sun Yat-Sen University.MATERIALS: The experiment was performed at the Second Affiliated Hospital of Sun Yat-Sen University from January 2001 to November 2002.Totally 20 adult SD male rats with the body mass of (380±20) g were provided by Experimental Animal Center of Sun Yat-Sen University (number of institution license SYXK2004-0020). There were DMEM culture solution with low glucose (L-DMEM, GibcoBRL), fetal calf serum (FCS) (Hyclone), myelin basic protein (MBP) (Sigma) and nerve growth factor receptor antibody (Sigma). They were divided into cell transplantation group and control group by the method of random digits table with 10 in each group.METHODS: The adult SD rats were anaesthetized and decapitated to obtain the whole olfactory bulb and then isolate olfactory nerve with a sterile operation. Thoracic cord injury models were established by modified Allen method. 10μL OECs suspension (2.5×1010 L-1) was injected into injured spinal cord of the cell transplantation group, whereas DMEM/F12 (1:1) culture solution of the same dose was injected in the control group. The influence of OECs on spinal cord axonal regeneration was observed by histological and immunohistochemical method 6 weeks after transplantation.MAIN OUTCOME MEASURES: ①OECs were identified by nerve growth factor receptor antibody staining. ②Repair of myelin sheath was observed by MBP staining. ③Nerve axonal regeneration was observed by argentaffin staining. RESULTS: Two rats in the cell transplantation group and 3 rats in the control group died, so totally 15 rats were involved in the result analysis. ①In the cell transplantation group,injured spinal meninges were integrated,but spinal cord became thin as compared with the normal spinal cord. By hematoxylin-eosin (HE) staining, multipolar cells appeared in injured region and the cells were fused excessively with myeloid tissues. It proved that survival OECs were integrated well within the host. New axons were clustered in bundles and infiltrated by small round lymphocytes. By argentaffin staining, regenerated axons grew in tissues of injured region, which mostly accompanied with fascicular-arranged multipolar cells. In the control group, spinal cord became thin markedly and spinal meninges were integrated. No new axon appeared in the injured spinal cord by HE staining. No regenerated axon appeared by argentaffin staining, either. ②In the cell transplantation group, most multipolar cells were clustered in bundles. A mass of positive granules of nerve growth factor receptor antibody appeared in cytoplasm, which further verified that OECs still survived and integrated well within the host 6 weeks after transplantation. Linear MBP positive fibers appeared in the injured region by MBP staining,which indicated that myelin-like substance appeared and drew closely in both ends of injured region. At the same time,MBP positive substance also appeared in the multipolar cells, which illustrated that transplanted OECs could induce the occurrence of myelin-like substance. No regenerated axon was found in the control group.CONCLUSION: Delayed transplantation of OECs can survive and induce the occurrence of myelin-like substance in injured spinal cord of adult rats.

AIM: human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, and hTERT mRNA expression contributes to telomerase activity. We therefore assessed the significance of hTERT expression in the biological behavior of giant cell tumor(GCT) of bone. METHODS: In situ hybridization was used to identify the expression of hTERT mRNA in 27 formalin-fixed paraffin-embedded clinical specimens from human GCT. RESULTS: 8 cases (8/27)displayed positive hTERT mRNA signal, positive hTERT mRNA signal located in the cytoplasms of mononuclear stromal cells and some multinuclear giant cells. CONCLUSION: The expression of telomerase in paraffin-embedded tissue can be evaluated efficiently and effectively by in situ hybridization, and the results suggest that there may be a neoplastic multinuclear giant cell type in human GCT.

Objective To observe the effects of cryopreserved olfactory ensheathing cells(OECs )transplantation on spinal cord axonal regeneration and spinal func tional improvement.Methods Twenty-four rats were divided into experimental a nd control groups,with each group of 12rats.The spinal cord in-jury was es tablished by transecting the spinal cord with microsurgery scissors.OECs were p urified from SD rat olfactory bulb and cultured in DMEM and cryopreserved(-1 20℃)for 2weeks.OECs suspension[(1～1.4)?10 5 /ml ]was transplanted into transected spinal cord at T 10 level,while the DMEM solution was in ject-ed instead in the control group .At 6weeks and12weeks after transplantation,the rats were evaluated with cl imbing test and MEP monitoring.The samples of spinal cord were procured and st udied with histological and immunohistochemical measurement.Results At 6weeks after transplantation,all of the rats in both transplanted and control group were paraplegic,and MEPs could not be recorded.Morphol ogy of trans-plante d OECs was normal,and OECs were interfused with host well.Axons could regrow i nto gap tis sue be-tween the spinal cords.Both OECs and regrowed axons were im munoreactive for MBP.No re grown axons were found in the control group.At 12weeks after transplantation,2rats(2/7)had lower ex tremities mus-cle contraction,2rats(2/7)had hip and /or knee active movement,and MEP of 5rats(5/7)could be recorded in the calf in the transplantation group.Non e of the rates(7/7)in the control group had functional improvement,and n one of them had MEPs recorded.In the transplanted group,histological and imm unohis-tochemical methods showed the number of transplanted OECs reduced and s ome regrown axons had reached the end of transected spinal cord.However,no r egrown axons could be seen except scar forma tion in the control group.Concl usion Cryopreserved OECs could integrate with the host and promote re growing a x-ons across the transected spinal cord ends.

BACKGROUND:Olfactory ensheathing cells (OECs) have been shown to possess the potential of repairing injured spinal cord, but their biological characteristics after transplantation in vivo are not well understood.OBJECTIVE: To investigate the migration of OECs after transplantation into the injured spinal cord of adult rats.DESIGN: Randomized and controlled experiment.SETTING: Department of Orthopedics, Guangdong Provincial People's Hospital; Experimental Animal Center of the North Campus of Sun Yat-sen UniversityMATERIALS: Totally 38 2-month-old male SD rats with body mass of (350 ±20) g were used in this study.METHODS: This experiment was conducted in the Experimental Animal Center, North Campus of Sun Yat-sen University between February 2004and May 2004. Two SD rats were used to extract the OECs, which were stained with Hoechst 33342. Totally 36 SD rats were subsequently randomized into 3 groups, namely rostral transplantation group, caudal transplantation group and control group with 12 rats in each group. The rats in the rostral and caudal transplantation groups subjected to operations to establish thoracic spinal cord injury model and OEC suspension was injected; in the control group, the rats were spared of thoracic spinal cord injury with only OEC suspension injection.MAIN OUTCOME MEASURES: Distribution of OECs in the spinal cord was observed under fluorescence microscope 1, 2, 4 and 6 weeks after operation, respectively.RESULTS: Of the rats in the 3 groups, 1 died in the rostral group, and 2in each of the caudal transplantation and control groups, leaving 29 rats for result analysis. The OECs in the rostral and caudal transplantation groups migrated longitudinally along the long axis of the spinal cord to a farthest distance of 8 mm and penetrating the scar tissues, but very few cells could reach the contralateral side. The OECs in the control group diffused locally without migration.CONCLUSION: OECs mainly migrate along the axons in white matter of the injured spinal cord, and their rostral and caudal migration does not differ in speed or amount. Only a small amout of OECs can across the transected gap of the spinal cord.

[Objective]This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene.[Methods]Total RNA was extracted from mt osteoblast and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone throush the LR recombination reaction.The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer.Ad-LMP-1 was infected into the 3rd passage MSC,the expression of LMP-1 was detected by Western blot.The osteogenic activity of MSC was evaluated by the expression of collagen Ⅰ,ALP,osteocalcin and the formation of bone nodule.[Result]The LMP-1 gene was successfully acquired and confirmed,the entry clone and the expression clone were both verified by enzymes digestion,and the expression clone was further confirmed by sequenced.The expression of LMP-1 was detected successfully in MSC.The increasing expression of collagen Ⅰ,osteocalcin.ALP and bone nodule were observed by comparing to the control group.[Conclusion]Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast,but also get a high transfection efficacy in MSC.LMP-1 gene can induce the osteoblast differention of MSCs,and improve its osteogenic activity.The adenovirus vector is reliable to be used in further gene therapy research.

Objective To investigate the operation key points, instrument improvement and shortterm effects in total en bloc spondylectomy (TES) via a single posterior approach for thoracic and lumbar tumors. Methods A series of modified instruments have been designed for the TES, including threadwire saw (T-saw) with a diameter of 0.81 mm, director and clamping for the saw, L shape and furcation osteotomes.The corpectomy of original TES which was defined as "one step dissection" from anteriorly to posteriorly, was modified into "two step dissection" which means that corpectomy was performed with saw cutting anteriorposteriorly and the L shape cutting posterior-anteriorly. In the cases with difficulty in pediculotomy using a T-saw, furcation osteotome was used for pediculotomy. Ten patients with thoracic or lumbar tumors were treated with the modified TES. There were 1 case of bone giant cell tumor, 1 case of bone neurilemmoma and 8 cases of metastatic tumors. All patients suffered moderate-severe pain and neurological deficit. Results The average follow-up period was 8.1(3.3-18.1) months. The average operating time was 7.8 h(6.0-10.3 h),and average blood loss was 2100 ml (1200-3500 ml). No disruption of dural mater, the leakage of cerebrospinal fluid, iatrogenic spinal cord injury and major vessel damage occurred. Two patients who underwent pleura disruption happened during the operation were treated with intrathoracic drain remedy. Among 7 cases with thoracic tumors, significant improvement in neurological function were achieved in 5 patients with the improvement of one grade in ASIA classification, while no change was found in 2 cases. In 3 cases with lumbar tumor, lumbar nerve root pain relieved and the muscle strength had recovered to grade 4 at least postoperatively. Conclusion Significant improvement has been achieved in the maneuverability and safety of the modified surgical techniques in TES with a single posterior approach for thoracic and lumbar tumors.

Objective To study the incidence and prognosis of avascular necrosis after talus fracture. Methods A retrospective survey was performed in 12 patients ( 13 feet) with talus fractures admitted into hospital from July 2004 to November 2009 to analyze necrosis rate, ankle function recovery and disability rate. According to Hawkin' s classification system, there were two patients with type Ⅰ feet, four with type Ⅱ feet, five with type Ⅲ feet and two with type Ⅳ feet. Results All patients were followed up for average period of 19.6 months (range 11-52 months). Avascular necrosis was detected in eight feet, of which one foot was treated with ankle fusion, one with subtalar arthrodesis and one with bone implantation. The other five feet had good ankle and subtalar function, with no collapse or osteoarthritis. According to Maryland foot score, the result was excellent in eight patients, good in two, fair in one and failure in two, with excellence rate of 77%. Conclusion The incidence of avascular necrosis after talus fracture is related to the location and energy of trauma. However, the function prognosis of the talus shows no correlation with necrosis.

BACKGROUND:How to obtain sufficient autogeneic chondrocytes is a problem which must be answered as soon as possible in both the transplantation of chondrocytes and the development of cartilage engineering.Bone marrow-derived mesenchymal stem cells have the potential of multidirectional differentiation. Under different induced conditions, they can differentiate into multiple tissue cells, such as chondrocyte, osteoblasts , sarcoblast, nerve cells and so on. Insulin-like growth factor-I plays an important role in regulating the formation and development of limb and cartilage.OBJECTIVE:To observe the effect of the insulin-like growth factor-I and culture solution of chondrocyte on inducing bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes in vitro.DESIGN:Open experiment.MATERIALS:This experiment was carried out at the Medical Study Center, Second Hospital Affiliated to Sun Yat-sen University from March to November 2003.Human bone marrow-derived mesenchymal stem cells were harvested from 4 to 6-month old embryos, all of which were from pregnant women who needed to terminate of pregnancy by induction delivery with water bag for health.METHODS:Human embryonic mesenchymal stem cells were isolated with Percoll separating medium. Subsequently, the cells were amplified in vitro,and the expression of surface makers of bone marrow-derived mesenchymal stem cells, such as CD44, CD71, CD34 and CD45 were measured with flow cytometer to identify the cells in our experiment. 100 μg/L insulin-like growth factors and culture solution of chondrocytes were added in the culture medium of bone marrow-derived mesenchymal stem cells of the fourth generation. The morphological changes of induced cells were observed with an inverted microscope. The expression of type Ⅱ cartilage matrix was observed by collogen immunohistochemistry. The proteoglycan level in the cells was detected, too.MAIN OUTCOME MEASURES:Phenotype of bone marrow-derived mesenchymal stem cells was identified through detecting the expressions of CD34, CD44 and CD4.Type Ⅱ collagen immunohistochemistry and the change of cellular ability to secrete proteoglycan after induction were observed to determine whether bone marrow-derived mesenchymal stem cells can differentiate into chondrocytes.RESULTS: ① Being observed under an inverted microscope,the bone marrow-derived mesenchymal stem cells presented a morphology like fibroblasts when they were cultured in vitro. ②Identification of surface antigen of bone marrow-derived mesenchymal stem cells: These cells were detected to have good homogenicity with flow cytometer. The fourth generation of bone marrow-derived stem cells positively expressed CD44, negatively expressed CD34 and CD45, suggesting these cells had the characteristics of bone marrow-derived mesenchymal stem cells. ③Observation of the morphological change of chondrocytes induced by bone marrow-derived mesenchymal stem cells under optical microscope: chondrocyte condition culture solution and insulin-like growth factors-Ⅰ were added to co-culture.During the process of culture, bone marrow-derived mesenchymal stem cells were seen to have a shape of round gradually. Fifteen days later, some cells presented a shape of short-shuttle or polygon with short mutations,which were the shape characteristics of chondrocytes. ④Immunohistochemical staining of Type Ⅱ collagen: In the insulin-like growth factor group,72.5% cells had many brown granules in cytoplasm, which were weakly positive or strongly positive expression of Type Ⅱ collagen. In the control group, bone marrow-derived mesenchymal stem cells was negative expression of type Ⅱ collagen on the 15th day. ⑤ Measurement of proteoglycan level: After co-culture with insulin-like growth factor-Ⅰ and chondrocyte culture solution for 15 days, proteoglycan was higher in the cells of co-culture group [ (8.92±0.91) μg/L ] than in culture group [(2.56±0.26) μg/L,P ＜ 0.05], but lower than in the chondrocyte group[(13.69±1.51) μg/L, P＜ 0.05].CONCLUSION:Insulin-like growth factor-Ⅰ and chondrocyte culture solution can induce bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes.

Lumbar spine fusion surgeries have adverse effects on adjacent segments and lead to adjacent segment disease and degeneration which increase the possibility of revision surgery and affect the final outcome.If new degeneration or aggravation of the primary degeneration in the adjacent segment only occurs in the images but without corresponding clinical symptoms,it would be called adjacent segment degeneration;if the corresponding clinical symptoms co-exist with degeneration in images,it would be called adjacent segment disease.Generally,the stress concentration on adjacent intervertebral discs and facet joints caused by spine fusion is the main pathogeny of adjacent segment degeneration and disease.The damage of normal spinal anatomy structure in surgery and the natural spine degeneration process are also important pathogenic factors.The occurrence of adjacent segment degeneration and disease after lumbar surgery is related to age,body weight,postoperative spinal-pelvic sagittal balance and the preoperative degeneration of adjacent segment.Surgical programs including numbers of the fusion segments,surgical approach and whether to adopt non-fusion technology have great effect on the occurrence of adjacent segment degeneration and disease.In the planning of surgeries,necessary measures and methods should be taken to prevent adjacent segment disease and degeneration according to the different patients.Non-fusion technology,minimally invasive spine surgery technique and Topping-off technique can help reduce the occurrence of adjacent segment disease and degenerative.If the patients are combined with high risk factors of adjacent segment degeneration and disease,more attention should be paid and appropriate and individualized therapies should be chosen.

AIM: To study the expression of mic2/CD99 protein and their correlation with Eber-1/LMP-1 in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma. METHODS: Immunohistochemical staining, in situ hybrization and tissue microarry technique were used to detect the expressions of mic2/CD99 and Eber-1/LMP-1 of H/RS cells in 43 cases of cHL and 16 cases of NHL. RESULTS: The positive rate of CD99 protein expression in 43 cases of cHL was 2.3% (1/43) , mic2 was 55.8% (24/43), LMP1 was 58.1% (25/43) and Eber-1 was 53.5% (23/43). The expressions of CD99 and mic2 in the NHL group were higher than those in cHL group (P0.05). There was a negative correlation between the expression of CD99 protein and LMP1 in H/RS cells (P0.05). There was a significant correlation between the high expression of LMP1 and a low expression of CD99 in the young patients (P0.05). CONCLUSION: There is a negative correlation between the expression of LMP1 and CD99 in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma.