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Objective To screen an ssDNA aptamer for rabbit mesenchymal stem cells (MSCs) and to identify the ability of the aptamer to recognize MSCs of a variety of species origin.Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively.Aptamers were screened by cell SELEX (systematic evolution of ligands by exponential enrichment) technique targeting to isolated MSCs.Enrichment of the 5th pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool was then cloned into pGE-T vector and the cloned sequences were determined randomly.The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit, rat, and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions.Aptamer 5-1-12 from 5th enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit, rat and human origin.Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.
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@#Objective To investigate the effects of heat preconditioning (HP) on the expression of protein kinase C (PKC) δ and PKCε in the myocardium of overload exercise rats. Methods 25 male three-month-old Sprague-Dauley rats were divided into control group (n=5), exercise group (n=10) and HP group (n=10). The expression of PKCδ and PKCε in the myocardium was detected with immunohistochemistry, real-time PCR and Western blotting 8 weeks after overload exercise. Results The expression of PKCδ significantly increased in the exercise group compared with the control group and the HP group (P<0.05). In another hand, PCR and immunohistochemistry showed the expression of PKCε in the exercise group decreased compared with the control group and the HP group (P<0.05), but it was not significantly different among the groups using Western blotting (P>0.05). Conclusion PKCs may play an important role in the HP-induced cardio-protection during overload exercise.
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Objective To investigate the effects of heat preconditioning (HP) on the expression of protein kinase C (PKC)δand PKCεin the myocardium of overload exercise rats. Methods 25 male three-month-old Sprague-Dauley rats were divided into control group (n=5), exercise group (n=10) and HP group (n=10). The expression of PKCδand PKCεin the myocardium was detected with immunohistochemis-try, real-time PCR and Western blotting 8 weeks after overload exercise. Results The expression of PKCδsignificantly increased in the exer-cise group compared with the control group and the HP group (P0.05). Conclusion PKCs may play an important role in the HP-induced cardio-protec-tion during overload exercise.
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Objective To evaluate the surgical therapy on dislocated fracture of talus. Methods Retrospective analysis was mode in 21 patients with dislocated fracture of talus collected from Jan. 2004 to Jan.2010, which were treated with open reduction, cannulated screw fixation, and kept neutral position plaster fixation with no weight loading, to do functional exercise depending on the Ⅹ film demonstrations. Results All the patients were followed up from 6 months to 3.8 years post-operation, and according to the evaluation standard by American Foot-Ankle Surgery Society, good rate was 61.91%. Conclusion Treating dislocated fracture of talus with emergency operation, anatomical reduction, valid internal fixation and no weight loading plaster fixation post-operation, shows good effect with low rate of complication.
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BACKGROUND: The deficiency of perfect animal femoral head necrosis model limited its further investigation. OBJECTIVE: To verify the feasibility of establishing rabbit femoral head necrosis models using liquid nitrogen rsfdgeration method, and to provide a foundation for subsequent research. METHODS: A total of 20 adult, New Zealand, white rabbits were selected in the study. The round ligament of femur was not cut off and femoral head was not dislocated, and the exposed femoral head were quick frozen using cotton bud carrying liquid nitrogen for successive 25 times, with 10 s per time. The specimens were examined by gross anatomy, X-ray film, MRI and histological observation at day 3, 7 and weeks 2, 4, 6, and 8 after operation. RESULTS AND CONCLUSION: The histolOgical section showed that chondrocyte, osteccyts, and myelold tissues presented necrosis in freezing and periphery at 3days after model preparation, and the repair process appeared at 2weeks after operation. The articular surface of femoral heads appeared collapse at 4 weeks after operation, and these changes became obvious at 6 weeks. The femoral head presented ostecarthdtis-like disorder, with seriously collapsed articular surface at8 weeks, and the contour of femoral head changed in 2 animals. The results demonstrated that without hip dislocation, rabbit femoral head necrosis models can be established successfully using liquid nitrogen refrigeration method. This method is simple, feesible, with high succeed rate, which can be used in subsequent research.
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@# Maximal oxygen uptake (VO2max) is an important indicator for cardiac function. This paper reviewed the VO2max and related index applied in the physical training.
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@# The apoptosis of cardiac muscle cells refers to an initiative cell death process that is controlled by a series of genes, and in fact it is a kind of physiological cell death. The authors of this article reviewed and analyzed the biology characteristics and development mechanisms of the apoptosis of cardiac muscle cells, summarized the research progress of the apoptosis of cardiac muscle cells and exercise, so as to offer theoretical reference for exercise training and heart protecting.
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[Objective]To investigate the osteogenic ability in vivo of mesenchymal stem cells(MSCs).[Method]Ten-fifteen ml bone marrow aspirates were harvested from the iliac crest of sheep and enriched for MSCs by density gradient centrifugation over a Percoll cushion(1.073 g/ml).After cultured and proliferated,the tissue-engineered bones were constructed with these cells and seeded onto porous 13-tricalcium phosphate ceramics(13-TCP).Then,the constructs were implanted into left metatarsus defect(21 mm in length)of 8 sheep as an experimental group.Porous ?-TCP composed with bone marrow was implanted into defects of same size and position in 8 sheep as a control group.Sheep were sacrificed in the 6th,12th,and 24th week postoperatively,and the implanted samples were examined by radiograph,histology,and biomechanical analysis.[Result]New bone tissue was observed either radiographically or histologically at the defect area of experimental group as early as the 6th week postoperatively;but was not observed in the control group.Because of the new bone formed in a direct manner without progression through a cartilaginous process intermediately,osteoid tissue,woven bone and lamellar bone,occurred earlier in the experimental group than in the control group,in which the bone defects were repaired in a "creep substitution" manner.At the 24th week,radiographs and biomechanical tests revealed an almost complete repair of the defect in experimental group,but only a partial repair in the control group.[Conclusion]MSCs have good reproductive activity not only in vitro,but also in vivo.The new bone formed in a direct manner without progression through a cartilaginous process intermediately when MSCs were transplanted in vivo.The results demonstrated that MSCs was an excellent seed cells for bone tissue engineering.
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Tissue-engineering bone with porous ,betatricalcium phosphate (3-TCP) ceramic and autologous bone marrow mesenchymal stem cells (MSC) was constructed and the effect of this composite on healing of segmental bone defects was investigated. 10-15 ml bone marrow aspirates were harvested from the iliac crest of sheep, and enriched for MSC by density gradient centrifugation over a Percoll cushion (1. 073 g/ml). After cultured and proliferated, tissue-engineering bones were constructed with these,cellS seeded onto porous f-TCP, and then the constructs were implanted in 8 sheep left metatarsus defect (25 mm in length) as experimental group. Porous ,-TCP only were implanted to bridge same size and position defects in 8 sheep as control group, and 25 mm segmental bone defects of left metatarsus were left empty in 4 sheep as blank group. Sheep were sacrificed on the 6th, 12th, and 24th week postoperatively and the implants samples were examined by radiograph, histology, and biomechanical test. The 4 sheep in blank group were sacrificed on the 24th week postoperatively. The results showed that new bone tissues were observed either radiographic or histologically at the defects of experimental group as early as 6th week postoperatively, but not in control group, and osteoid tissue, woven bone and lamellar bone occurred earlier than in control group in which the bone defects were repaired in "creep substitution" way, because of the new bone formed in direct manner without progression through a cartilaginous intermediate. At the 24th week, radiographs and biomechanical test revealed an almost complete repair of the defect of experimental group, only partly in control group. The bone defects in blank group were non-healing at the 24th week. It was concluded that engineering bones constructed with porous -TCP and autologous MSC were capable of repairing segmental bone defects in sheep metatarsus beyond "creep substitution" way and making it healed earlier. Porous ,-TCP being constituted with autologous MSC may be a good option in healing critical segmental bone defects in clinical practice and provide insight for future clinical repair of segmental defect.
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Bone Marrow Cells/cytology , Calcium Phosphates/pharmacology , Cells, Cultured , Fractures, Bone/therapy , Implants, Experimental , Mesenchymal Stem Cells/cytology , Metatarsus/injuries , Porosity , Sheep , Tissue EngineeringABSTRACT
Tissue-engineering bone with porous β-tricalcium phosphate (β-TCP) ceramic and autologous bone marrow mesenchymal stem cells (MSC) was constructed and the effect of this composite on healing of segmental bone defects was investigated. 10-15 ml bone marrow aspirates were harvested from the iliac crestof sheep, and enriched for MSC by density gradient centrifugation over a Percoll cushion (1. 073 g/ml). After cultured and proliferated, tissue-engineering bones were constructed with these cells seeded onto porous β-TCP, and then the constructs were implanted in 8 sheep left metatarsus defect (25 mm in length) as experimental group. Porous β-TCP only were implanted to bridge same size and position defects in 8 sheep as control group, and 25 mm segmental bone defects of left metatarsus were left empty in 4 sheep as blank group. Sheep were sacrificed on the 6th, 12th, and 24th week postoperatively and the implants samples were examined by radiograph, histology, and biomechanical test. The 4 sheep in blank group were sacrificed on the 24th week postoperatively. The results showed that new bone tissues were observed either radiographic or histologically at the defects of experimental group as early as 6th week postoperatively, but not in control group, and osteoid tissue, woven bone and lamellar bone occurred earlier than in control group in which the bone defects were repaired in "creep substitution" way, because of the new bone formed in direct manner without progression through a cartilaginous intermediate. At the 24th week, radiographs and biomechanical test revealed an almost complete repair of the defect of experimental group, only partly in control group. The bone defects in blank group were non-healing at the 24th week. It was concluded that engineering bones constructed with porous β-TCP and autologous MSC were capable of repairing segmental bone defects in sheep metatarsus beyond "creep substitution" way and making it healed earlier. Porous β-TCP being constituted with autologous MSC may be a good option in healing critical segmental bonedefects in clinical practice and provide insight for future clinical repair of segmental defect.
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BACKGROUND:Sirius red is a strong acid anionic dye. Being not-easyto-fade and specific, sirius red becomes the best dye for collagen staining.Collagen is a major component of extracellular matrix and has some specific physiological functions. Through synthesis and reconstruction of collagen, bone fracture repair will be accomplished.OBJECTIVE: Picric acid-Sirius red stained slides were observed under a polarized light microscopy for evaluation the dynamic changes in the ratio of different collagen types and their distributions in bone fracture healing.DESIGN: It was a controlled observation.SETTING: It was conducted in the Department of Orthopedics, Renmin Hospital, Wuhan University; Department of Traumatic Orthopaedics, Tianjin Hospital; Department of Traumatic Orthopaedics, Jishuitan Hospital,Medical Department, Peking University; Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLAMATERIALS: It was conducted at Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA from March 2002 to September 2003. Three healthy adult Chinese sheep, male and in weight from 25 to 35 g, were selected.METHODS: All the animals were anesthesized and sterilized; a transverse osteotomy of the trunk of metatarsus was performed; and the end of fracture was fixed with a six-hole Medoff sliding plate. At the post-operative month 1, 3 and 6, samples were taken from bone fractures. After decalcification with EDTA, they were stained with Picric acid-sirius red, and the types and distribution of collagens were observed under a polarized light microscopy.MAIN OUTCOME MEASURES: Types and distributions of collagens in bone lesion in different period of bone healing were investigated.RESULTS: Three sheep used in this study entered the statistical analysis.①Morphological features of various collagens under a polarized light microscopy postoperatively: Type Ⅰ collagen packed tightly, with a strong refraction and yellow, orange or red thick fibres. Type Ⅱ collagen formed a loose reticulation with fibres exhibiting different colour and a weak refraction. Thin fibres of type Ⅲ collagen with weak refraction and green colour formed a loose reticulation. ②Quantitative studies on various collagens under a polarized light microscopy postoperatively: At postoperative month 1,red or orange fibres (type Ⅰ collagen) were rarely seen in bone fracture,while green fibres (typical of type Ⅲ collagen) were dominant with a disorder pack. At postoperative month 3, red or orange fibres increased significantly and the ratio of type Ⅲ collagen reduced. The collagen fibres assembled regularly. At postoperative month 6, thick yellow-red collagen became dominant and thin green type Ⅲ collagen decreased dramatically and arranged in an obvious oblique, spiral and crossed orientations.CONCLUSION: Picric acid-sirius red stain combined with polarized light microscopy technique is not only capable of identifing type Ⅰ and type Ⅲ collagens in bone fraction, but also can reflect the morphological features,distribution and the ratio of these two type collagens. This approach has the virtues of easiness in operation, strong specificity and high sensitivity.
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BACKGROUND:Bone marrow derived mesenchymal stem cells (BMMSCs) have multi-differentiation potentials, possessing a repairing capability for the sectional bone defects if combined with degradable porous β-tricalcium phosphate china. This provides a new idea in clinical repair of various bone defects.OBJECTIVE: To explore in radiology the curative effect of implanting porous β-tricalcium phosphate and autogenous BMMSCs compound to treat bone defects.DESIGN: A randomized controlled study with callus growth at various healing periods as subjects for observation. SETTING: Department of Orthopaedics, Renmin Hospital, Wuhan University; Tissue Engineering Center, Research Institute of Basic Medicine,Academy of Military Medical Sciences of Chinese PLAMATERIALS: This experiment was carried out at the Tissue Engineering Center, Research Institute of Basic Medicine, Academy of Military Medical Sciences of Chinese PLA between March and September 2002. Twenty China healthy adult sheep were randomized into the groups of blank control group (4 sheep), simple implantation group (8 sheep) and complex implantation group (8 sheep).METHODS: Under general anesthesia and aseptic condition, 10-15 mL of sheep marrow was extracted; MSCs were separated and cultured before combined with porous β-tricalcium phosphate china for tissue engineering bone construction. Rats in each group were cut off 21mm long metatarsus in the middle section of metatarsus bonestem. Β-tricalcium phosphate china and autogenous MSC compound was implanted into the sheep of the complex implantation group; β-tricalcium phosphate china was implanted into the simple implantation group; and the bone defects in the blank control group remained untouched. Then the incision was sutured.X-ray filming was carried out right after the operation, as well as 1, 3,and 6 months after the operation for radiological appraisal (scored for1 if bone union formed in one surface of bone defect, but scored 0 if no boneunion formed in any surface of bone defect, and scored 4 if bone union formed in front, back, lateral surfaces and the center of bone defect), Xray radiation-resisting density was analyzed to compare the results of bone defect repair.MAIN OUTCOME MEASURES: The postoperative general condition and general observation, as well as the results of the radiological analysis of the bone defects of all sheep.RESULTS: Totally 20 sheep were brought into this xperiment and all entered the stage of result analysis. ① The postoperative general condition:Sheep regained consciousness 2-6 hours after the operation without incision infection and loosing of internal fixation. Their spirit gradually was back to normal 1 week after the operation, at which time the injured legs could touch ground but were incapable of bearing load, and the affected legs could bear load 2 weeks after the operation, walked slightly lamely 3 weeks after the operation, and even moved freely without limp 4 weeks after the operation. ②The general condition 6 months after the operation: In pure implantation group, the surface white hyaline cartilage-like tissues were gradually calcified, with both ends connected with the host bone by bone bridge, but china granules could still be easily observed; while no implantation substance could be observed in compound implantation group,with the boundary between implantation substance and host bone vanished,and bone defect became basically the same as host bone. However there was no bone tissue formed in bone defect at various postoperative time points in the blank control group. ③ Radiological analysis of the bone defects at various postoperative time points: The radiological rating score was obviously higher in complex implantation group at the time poin ts of 3, 6 months after the operation compared with the pure implantation group [(2.3±0.3), (1.8±0.5); (3.3±0.5), (2.6±0.6), P ＜ 0.05]. ④ Radiological analysis of bone callus thickness and the relative value of radiation-resisting density at various postoperative time points: The bone callus thickness in the complex implantation group was obviously lower than that of the pure implantation group at the postoperative time point of 6 months (4.62 vs 7.64, P ＜ 0.05), with relative value of radiation-resisting density obviously higher than that of the pure implantation group (70.4±1.5 vs 61.18±1.2, P ＜ 0.05).CONCLUSION: Radiological appraisal and bone defect density measurement can well reflect the dynamical repairing process of bone defects; the implantation of porous β-tricalcium phosphate china and autogenous BMMSCs compound into sheep can enhance the repair of large sectional bone defect.
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BACKGROUND: To eliminate effectively and promptly sports fatigue and prevent excessive fatigue that are associate with the effects of training, improvement of sports results and health of athletes directly.OBJECTIVE:To explore the effects of acupoint iontophoresis on eliminating sports fatigue.DESIGN: Randomized controlled experiment was designed, in which, the athletes were taken as the objects.SETTING: Research and Teaching Room of Sport Medicine of Wuhan In stitute of Physical Education and College of Acupuncture and Massage of Shanghai University of Traditional Chinese Medicine.PARTICIPANTS:The experiment was performed in Wuhan Institute of Physical Education from June 2002 to June 2003. Totally 32 male athletes were selected as the objects,with similar level in training of kayak in Wuhan Institute of Physical Education. They were randomized into 5groups, named training group, training+ acupoint-dermal electric stimulation (dermal electric group), training + oral herbal administration (Chinese herb group), training + acupoint iontophoresis group (iontophoresis group)and training + Chinese herb + iontophoresis group(Chinese herb + iontophoresis group).There were 4 persons in the training control and 7 persons in the rest groups.METHODS:The experiment was performed in the training stage of large sports load in summer. The training methods were same in each group, totally for 8 weeks. ① Training group was taken as the control. ② In dermal electric group,after training,dermal electric stimulation was applied on shanzhong (CV 17), xinshu (BL 15), baihui (GV 20) and mingmen (GV 4).③ In Chinese herb group, after training, self-prepared shenwensan was applied for oral administration as decoction. ④ In iontophoresis group, after training, iontophoresis was used. ⑤ In Chinese herb + iontophoresis group,both self-prepared shenwensan and iontophoresis were applied.Trace observation of heart and lung functions were performed with subjective physical sensation scoring (before, after and on the 4th week of experiment), test of sports results,test of heart and lung functions and radioimmunoassay(RIA) (before and after experiment) in athletes.MAIN OUTCOME MEASURES: Subjective physical sensation scoring,test of sports results,test of heart and lung functions and determination of levels of endothelin (ET) and calcitoningene-related peptide (CGRP).RESULTS:The athletes in all of groups accomplished the training and tests of every index and the results had all entered final analysis.① Subjective sensation of athletes was all improved obviously and the effect of iontophoresis was improved significantly compared with dermal electric group and Chinese herb group (P ＜ 0.05). ② The results of rowing projects were improved to different extents in each group.The improved amplitude of 12 km training in every treating group was different significantly compared with training group (P ＜ 0.05). The result of 2 km training was improved most significantly in Chinese herb + iontophoresis group (P ＜ 0.05).③ VO2max was increased in Chinese herb group,iontophoresis group and Chinese herb + iontophoresis group to different extents. Maximal respiratory quotient (Rqmax)was in tendency of increasing in every group after experiment, HR-AT was all in tendency of decreasing and it was decreased in large amplitude in Chinese herb + iontophoresis group (P ＜ 0.05). The time of exhaustive sports was prolonged in all of groups,but the effect was the most obvious in iontophoresis group. ④ Endothelin (ET) was in tendency of increasing after experiment in each group, but the increasing amplitude was the largest in training group.The calcitoningene-relatedpeptide (CGRP) was in tendency of increasing in every group, but CGRP was increased with the best result in Chinese herb + iontophoresis group and that was with the least result in training group. The difference was no significant in comparisons among groups and before and after experiment(P ＞ 0.05).It was indicated that the training load of athletes had not reach the fatigue limitation.CONCLUSION:Cardiac sports fatigue is relevant to multiple factors.Acupoint iontophoresis can protect myocardium in multiple links and layers, improve cardiac function, reduce HR-AT, increase oxygenic endurance and promote the secretion of nervous peptide ET and CGRP; retard fatigue and promote fatigue elimination so as to delay cardiac sports fatigue.
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In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Phenotype , Sheep , Tissue EngineeringABSTRACT
In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering. MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-beta3, IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.
Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cell Separation , Cells, Cultured , Chondrocytes , Cell Biology , Chondrogenesis , Mesenchymal Stem Cells , Cell Biology , Phenotype , Sheep , Tissue EngineeringABSTRACT
@#Objective To approach the mechanism of foot reflective therapeutics to sub-health state. Methods 35 patients with sub-health state were treated with foot reflective therapeutics, to press the foot reflective section by using many manipulations (such as press, rotation, push, etc) or apparatuses, once every other day, ten times constituting one therapeutic course. Results In 35 patients, 27 (77.1%) had excellent results, 7 good and one fair, the good rate was 97.1%. Conclusions Foot reflective therapeutics can regulate functions of viscera and dredge meridians on the basis of its convenient and effective characters.