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With the development of the 3rd-generation high-throughput sequencing technology and tissue engineering, recent studies show that many long-chain non-coding RNAs (LncRNAs) have played an important role in osteogenic differentiation of mesenchymal stem cells (MSCs). LncRNAs, which are involved in the regulation of mechanical regulation, further regulate bone-related cell functions and play a regulatory role at multiple levels, including transcription, post-transcriptional and epigenetic. LncRNAs may be involved in the osteogenic differentiation and bone remodeling of MSCs, the regulation of bone-related cell functions as a mechanical response molecule, as well as the pathological process of skeletal diseases.
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To investigate the effect of icaritin (ICT) combined with GDF-5 on chondrogenic differentiation of bone marrow stromal cells (BMSCs), and discuss the action of Wnt signaling pathway, full bone marrow adherent method was used to isolate and culture SD rats BMSCs, and the cells at P3 generation were taken and divided into 6 groups： BMSCs group, ICT group, GDF-5 group, GDF-5+ICT group, GDF-5+ICT+SB216763 group, and GDF-5+ICT+ XAV-939 group. The cells were induced and cultured for 14 days. The morphology change was observed by inverted microscope. Alcian blue staining method was used to detect the changes of proteoglycans. RT-PCR was used to detect the mRNA expressions of aggrecan, Col2, Sox9, Dvl1, Gsk3β, and β-catenin. The protein expressions of collagen 2 (COL2) and β-catenin were detected by Western blot. The results indicated that, compared with the BMSCs group, gradual increase was present in proteoglycan Alcian blue staining; mRNA expressions of cartilage differentiation marker genes aggrecan, COL2, Sox9 and the protein expression of COL2, as well as mRNA and protein expressions of Wnt signaling pathway-related gene β-catenin, but with gradual decrease in Gsk3β mRNA expressions in GDF-5 group, GDF-5+ICT group and GDF-5+ICT+SB216763 group. On the contrary, compared with GDF-5+ICT group, there was a decrease in expressions of Dvl1, and β-catenin related to chondrogenic differentiation and Wnt signaling pathway, a increase in Gsk3β mRNA expression, and also a decrease in protein expressions of COL2 and β-catenin in GDF-5+ICT+XAV-939 group, with statistically significant difference between two groups. GDF-5 in combination with icaritin can induce chondrogenic differentiation of BMSCs in rats, and icaritin (ICT) can promote the chondrogenic differentiation. ICT can promote the chondrogenic differentiation of BMSCs in vitro probably by activating the Wnt/β-catenin signaling pathway.
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Objective To evaluate differences in genes expression of rat bone marrow stromal cells (rBMSCs) under continuous mechanical strain by gene microarray technology．Methods rBMSCs were isolated and cultured in vitro. Continuous stresses with amplitude of 10% and frequency of 1 Hz were applied on rBMSCs for 6 hours by Flexercell mechanical loading system to investigate rBMSC gene expression profiles, and quantitative PCR was used to verify gene expression changes related to osteoblastic differentiation. Results Compared with the control group, 1 244 differentially expressed genes were found in mechanical loading group, among which 793 genes were up-regulated, while 451 genes were down-regulated．GO (gene ontology) analysis suggested that differentially expressed genes were mainly involved in multicellular organismal development, cell differentiation, chemotaxis, cell adhesion and so on. Four signaling pathways as Notch, Wnt, FGF and IGF might participate in the regulation of stress-induced osteoblastic differentiation. PCR validation results were consistent with the gene chip results. Conclusions Mechanical stress could induce osteoblastic differentiation of the BMSCs, while several differentially expressed genes screened by gene microarray may attribute to this process.
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Objective To study the effect of continuous strain on the proliferation and osteogenic differentiation of rat bone marrow stromal cells(BMSCs) in vitro. Methods Rat BMSCs were obtained from adult female Sprague-Dawley rats (3-month old), and purified by full-blood attachment culture. BMSCs between passage 3—5 were seeded on Flexercell mechanical loading system(10%, 1 Hz), and divided into 1 h group, 6 h group, 12 h group, 24 h group, 48 h group, respectively, according to the time subjected to strain. Effects of continuous strain on the morphology, proliferation and osteogenic differentiation of BMSCs were observed and analyzed. Results (1) Compared with the control group, cells subjected to 10% strain showed the particular orientation. Their alignment elongated mostly in the direction perpendicular to the strain axis in a time-dependent manner. (2)10% continuous strain could significantly decrease the proliferation of BMSCs. (3) Continuous strain could increase mRNA expression of ALP, COLⅠand Runx2 in a time-dependent manner. Compared with the control group, mRNA expression of ALP was increased significantly at 24 h, COLⅠat 24 h and 48 h, and Runx2 at 6 h. mRNA expression of osteocalcin (OC) ascended greatly in the beginning, but went down gradually and was significantly lower than that of control at 48 h（P＜0.05）. (4) Continuous strain could induce an increase in Runx2 protein level. A sharp increase in Runx2 protein was observed at 6 h(P＜0.05) , then Runx2 protein level decreased slowly with its mRNA expression being significantly lower than that of control at 24 h（P＜0.05） Conclusions Continuous strain could induce rat BMSCs to orient in an orderly manner, suppress its proliferation activity, but stimulate the osteoblastic differentiation at the early stage.
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Objective To explore the feasibility of building tissue engineered cartilage by bone marrow stromal cells and pbotografting modified copolymers of 3-hydroxybutymte and 3-hydroxyvalerate.Methods Sheep BMSCs were seeded in three-dimensional photografting modified PHBV scaffoids.Twenty-four hours later.composites were cultured with ehondrogenically inductive medium(DMEM)containing TGF-B(10 ng/m1),IGF-1(150 ng/m1)and 20% fetal bovine serum.Three weeks later,the constructs were evaluated by scanning electron microscopy(SEM)and light microscopy with alcian blue,safrine 0 and type Ⅱ collage immunohistochemical staining.GAG contents of constructs were determined by DMB(1,9-dimethylmethylene blue)binding assay at weekly intervals up to 3 weeks.The composites were implanted subcutaneously in sheep abedoml and were evaluated macroscopically and bistologically at 4 weeks postoperatively.Results SEM photograph showed.after one week culture,cell morphology changed from fibroblast-like elongated spindle to the flat rounded like chondrocytes,and the extra cellular matrix also increased obviousl~.Furthmore,with the culture time extension,this change were more evident.HE staining showed that cells filled all the inter-connected pores in the constructs.And more cells were observed in the outer layer of the constructs.ECM(extraeellular matrix)Was strongly positive by Aleian blue,Safrine O staining and type Ⅱ collage immunohistechemical staining.DMB binding assay revealed that the induced BMSCs GAG secretion(1306.7±192.3)wag significantly higher than BMSCs(205.0±26.2)(P<0.001),but it was significantly lower than passage 2 ehondrocytes(1969.2±235.3)(P<0.001).Saltine O and type Ⅱ collage immunohistochemical staining were positive in constructs implanted subcutaneously.Conclusion Tissue engineered cartilage could be obtained using BMSCs and photografting modified PHBV,but there are still gaps physiologically between the constructs and the nature cartilage.
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Objective To evaluate the effects of adeno-associated virus-enhanced green fluorescent protein (AAV-EGFP)on the biologic behavior of rabbit's bone marrow stromal cells(BMSCs)by means of a simple method of culturing and osteogenic induction in vitro so as to find an ideal viral vector and cell tracing mark for tissue en- gineering.Methods Total bone marrow culture was conducted to obtain rabbit BMSCs which were then induced in the osteogenic direction.The morphology of the cells was observed continuously,and their surface antigen and ossification were detected by alkali phosphatase stain and Von Kossa stain.On the basis of the above results, AAV-EGFP was transfected into the induced cells.The morphologic changes of the cells,the expression time and intensity of fluorescent light were observed.The transfection efficiency was detected to find the best multiplicity of infection(MOI)value.The cell growth curves were drawn to evaluate the biologic effects of AAV-EGFP on the cyto-activity.Results The morphology and purity of the rabbit BMSCs obtained were good.The ossification of the cells was significant after osteogenic induction.The best MOI value was found to be 1?10~5.The expression intensity of fluorescent light was strong with the expression time more than eight weeks so that the fluorescent light could be observed after cell generations.The transfection efficiency of AAV was high without significant biologic effects on the cyto-activity.Conclusions The total bone marrow culture and in vitro cell induction can satisfy the requirements for seeding cells in tissue engineering.AAV is an ideal viral vector for tissue engineering.Transfection of AAV-EGFP to cells could be an ideal method for cell tracing mark.
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Objective To study the osteogenic effects of induced endothelial cell(EC)on bone marrow stromal cells(BMSCs)of rabbits in co-culture condition.Methods BMSCs were obtained from rabbits by density gradient centrifugation.The adhesive ceils were preserved to passage in culture.The cultured cells were divided into four groups:group A(BMSCs),group B(BMSCs osteogenic induction),group C(EC induction)and group D (co-culture of induced BMSCs and EC).The cell morphology,immunofluorescence,cell proliferation,alkaline pbosphatase(ALP)activity,osteocalcin synthesis were observed to determine the effects of induced autologous EC on the osteogenic potential and cellular compatibility of BMSCs.Results The immunochemical staining showed that the BMSCs were induced into EC in group C.The cellular compatibility of BMSCs and EC was good.The ALP activity and osteocalcin content were obviously higher in group D than in any other groups(P＜0.05).The cell proliferation difference was not obvious between groups(P＞0.05).Conclusions The cellular compatibility of induced osteoblasts and induced EC is perfect.The ECs can significantly increase the viability and ALP activity of induced osteoblasts.
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Objective To investigate the effects of cryopreservation on the growth and osteogenesis capa- bility of human bone marrow stromal cells(BMSCs)on demineralized bene matrix(DBM).Methods Bone marrow aspirates were obtained from the lilac crests of three donors.The BMSCs were isolated from the bone marrow by density gradient centrifugation.Cells of passage 3 were cryopreserved in liquid nitrogen for 24 hours,and then re- covered.The non-cryopreserved BMSCs were used as the control,The cryopreserved and control BMSCs were cul- tured in osteogenic media,collected and labeled with Dil to be seeded onto the DBM when cells were confluent.The percentage of BMSCs adhered to the DMB was detected.The cell morphology and matrices secreted by BMSCs on the DBM were observed by the inverted phase-contrasted microscope,fluorescence microscope and scanning electron microscope(SEM).The growth and viability of BMSCs on the DBM were determined using the modified MTT ashy. The osteogenesis ability of BMSCs on the DBM was determined by assessment of the alkaline phosphatase(ALP) activity and osteocalcin(OCN)content.Results The percentages of the cryopreserved and control cells adhered to DBM were(97.25?1.17)% and(97.00?1.09)% respectively.The cells adhered well to the DBM and grew rapidly.Large amounts of matrices on the DBM were observed by the light microscope and SEM.The cells embedded in the matrices could be observed by fluorescence microscope.There were no significant differences in the assay values of MTT,ALP and OCN between the cryopreserved and control BMSCs on the DBM.Conclusion Since cryopreservation does not affect the growth and osteogenesis capability of BMSCs on DBM,the cryopreserved BMSCs can be used as a cell source in bone tissue engineering.
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Objective To explore the effects of surface modification of PLGA-[ASP-PEG] scaffold with typeⅠcollagen on the adhesion,proliferation and osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs).Methods After PLGA-[ASP-PEG] materials were modified with typeⅠcollagen chemically,the collagen was coated onto the materials physically.The BMSCs obtained from rabbits were cultured on the modified PLGA-[ ASP-PEG] and on the unmodified PLGA-[ ASP-PEG] as control.The adhesion and proliferation behavior of the cells was analyzed and the expressions of osteogenie marker alkaline phosphatase,osteocalcin,osteopontin,typeⅠcollagen and core binding factor al were also detected.Results X-ray photoelectron spectrometry(XPS) confirmed that TypeⅠcollagen was grafted onto the surface of PLGA-[ASP-PEG] successfully and the collagen content on the materials modified chemically and physically was significantly increased.The abilities of adhesion and proliferation and the expressions of osteogenie makers of the BMSCs were significantly greater than those in the control group(P＜0.05).Conclusion Since Type collagen I can improve the biocompatibility of PLGA- [ASP-PEG] scaffold materials,it can be used as a new way to optimize scaffolds in tissue engineering.
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Objective To investigate effects of co-grafts of bone marrow stromal cells (BMSCs) combined with nerve growth factor (NGF) suspension on repair of spinal cord injury (SCI) in adult rats so as to determine whether the co-grafts are more effective than a single protocol.Methods Spinal cords of adult rats (n=32) were injured by the modified Allen's method.One week after injury,the injured cords were injected with Dubecco-modified Eagles Medium,BMSCs,NGF,and BMSCs plus NGF respectively.One month and two months after injury,rats were respectively sacrificed-and their injured cord tissues were sectioned to identify the transplanted cells under fluorescent microscopy.We observed axonal regeneration and differentiation of BMSCs through immunocytochemical staining conducted with neurofilament (NF) and Neuron-Specific Nuclear Protein (NeuN) and glial fibrillary acidic protein (GFAP).We measured the cavity volume by staining with hematoxylin-eosin (HE).At the same time,rats were subjected to behavioral tests using the open-field BBB (blood brain battier) scoring system.Results One month and two months after transplantation,immunocytochemical staining showed that transplanted cells partially expressed NeuN and GFAP.At the same time,significant reduction in lesion area (P＜0.05) and improvement in BBB lo- comotor rating scale (P＜0.05) were observed in the cases that received implantation,as compared with those that received vehicle injection.More importantly,further reduction in lesion area and improvement in function were ob- served in the combined treatment group.Conclusions BMSCs can differentiate into neurons and astroeytes. BMSCs and NGF can reduce lesion size,promote axonal regeneration and improve functional recovery,and may have a synergistic effect.
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Objective To investigate the stimulative effects of CollagenⅠon the increased adhesion of rabbit bone marrow stromal cells (BMSCs), cytoskeleton actin organization and intracellular free Ca~(2+) concentration. Methods The third generation BMSCs isolated from mature rabbits were cultured at different initial concentrations on cover-slice coated by collagenⅠin RPMI1640 containing 10% fetal calf serum, and cultured on the same kind of cover-slice untreated with collagenⅠas control. The cells adhesive behavior at different times was assessed. Cellular actin organization was described as either typeⅠor typeⅡcells. In general, typeⅠcells are round and represent a preliminary stage of actin assembly, while typeⅡcells are elongated with organized actin fiber network. At the same time intracellular free Ca~(2+) concentration was measured by using calcium fluorescent probe Fluo-3/AM and laser confocal microscope. Results We found more typeⅡcells in BMSCs cultured with collagen typeⅠsix hours after culture than in the control group. At 12 hours 89% of the BMSCs were typeⅡcells, while only 55% were typeⅡcells in the control group. This indicated active cellular actin organization after being modified by collagen typeⅠ. We also found that the BMSCs cultured with collagen typeⅠincreased intracellular free Ca~(2+) concentration in monolayer culture. Conclusions CollagenⅠis effective in promoting the cellular adhesion, which suggests that a kind of internal relationship or cross-talk may exist between cellular actin organization, intracellular free Ca~(2+) concentration and cell adhesion. Further study, however, is needed.
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Objective To qualitatively analyze the expressions of key marker genes involved in osteogenic differentiation of bone marrow stromal cells (BMSCs) of mice at defined stages in vitro using real-time RT-PCR. Methods The BMSCs of mice were cultured in vitro and induced to osteoblasts for 7, 14, and 21 days. The total RNA was extracted from the cultured cells and the gene expression levels of collagenⅠ, bone alkaline phosphatase (APL), osteopontin (OPN) and bone sialoprotein (BSP) in the osteoblasts at the defined stages were determined by real-time RT-PCR. Results The ColⅠmRNA levels in 14 and 21 days were (0. 75?0. 04) and (0. 34?0. 03) times respectively of that in 7 days. ALP, OPN and BSP mRNA levels in 21 days were (19. 70?2. 36), (150. 12?9. 31) and (7. 73?0. 58) times of those in 7 days respectively. Conclusions ColⅠexpression levels tend to gradually decline while ALP, OPN and BSP levels gradually increase along with the culture time. Real-time RT-PCR is a reliable method for investigation of gene expressions in BMSCs cultured in vitro.
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Objective To develop a new method, the bone marrow stromal cells (BMSCs)-mediated tissue engineering technique combined with mosaicplasty, for repair of osteochondral defects and integration of gaps. Methods BMSCs from 12 Chinese goats were cultured and proliferated in vitro. Prior to the BMSCs harvest, osteochondral defects, 5 mm in diameter and 3 mm in depth, were created in the femoral medial condyles of both the goat's hind limbs. When the mosaicplasty (osteochondral autograft transplantation) was performed, the BMSCs, which had been harvested and compounded with hyaluronic acid, were injected into the gaps between the osteochondral autografts in the left hind limb. The right hind limb which only received osteochondral autograft transplantation without BMSCs served as a control. At four, eight and 16 weeks post-operatively, samples of the repaired defects were harvested and assessed by histological evaluation, immunohistochemical analysis and glycosaminoglycan (GAG) quantification. In both groups 16 weeks post-operatively, the GAG quantification was analyzed by one-way ANOVA and least significant difference (LSD) method. Results At all the time points, the cartilage autografts in both groups survived as hyaline cartilage and presented no significant difference from the surrounding native cartilage. In the group filled with BMSCs compound, the gaps were replaced by regenerated hyaline cartilage and disappeared; however, in the control group, the osteochondral autografts were still distinct from the surrounding normal cartilage, though the gaps were replaced by fibrous tissue or fibrous cartilage. Immunohistochemical analysis of typeⅡcollagen showed positive staining in the matrix of transplanted and regenerated cartilage. The Alcian blue method also confirmed a significantly less GAG content in the regenerated tissue in gaps in the control group than in the treatment group and in the normal cartilage. Conclusion Since tissue engineering combined with mosaicplasty can promote gap integration and cartilage healing, the method can be an ideal way for osteochondral defect repair.
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Objective To observe whether the full-thickness defects of articular cartilage at the knee joint of rabbits could be repaired by implantation of polyglycolic-acid(PGA) composites adhered with bone marrow stromal cells (BMSCs). Methods Culture-expanded rabbits’BMSCs were seeded onto porous PGA scaffolds. After a 72-hour co-culturing, the cell-adhered PGA was implanted into the articular cartilage defect at the intercondylar fossa of the femur. The rabbits were sacrificed 12 weeks later after the operation and the specimens were examined histologically for morphologic features, and stained immunohistochemically for type Ⅱcollagen. Results The specimens harvested from BMSCs-PGA composite demonstrated a hyaline cartilage formation. No obvious progressive degeneration sign was found in the newly formed tissue. The control groups showed no hyaline-like cartilage formation. Conclusion PGA composites adhered with in vitro culture-expanded autologous BMSCs can facilitate formation of hyaline-like cartilage in rabbits.
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Objective To use the new technique of seeding cells labeled with fluorescent protein for monitoring the construction process of tissue-engineering bone in vitro.Methods Human bone marrow stromal cells(BMSCs)were isolated and culture-expanded from femoral marrow.BMSCs were transfected with the recombinant retrovirus pLEGFP-Nl containing enhanced green fluorescent protein(EGFP).Those expressing EGFP were cultured in the experiment groups,and the unmarked BMSCs were in the control groups.The cell-scaffold complexes were constructed with BMSCs and hydroxyapatite in vitro.Some complexes were used to assess the adhesion efficiency and the others were cultured and observed under fluorescence microscope for four weeks.Results EGFP was successfully expressed after BMSCs were transfected with the retroviral vector pLEGFP-N1.The labeled cells were easy to observe while the cell-scaffold complexes were constructed and cultured.The adhesion efficiency was(90.3?2.1)%for the group of labeled BMSCs and(92.0?1.5)%for the control group,so there was no significant difference between the two groups(P=0.236).Conclusions After BMSCs are transfected with the recombinant retrovirus containing EGFP,the adhesion of cells will hardly be affected so that seeding cells labeled with fluorescent protein can well be used for monitoring the construction and culture of tissue-engineering bone in vitro.
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Objective To discuss a new method of BMSCs culture which can reduce the lead-in of extraneous antigenic substances in the process of cell culture, and can thus better meet the demand on the quantity and biological characteristics of BMSCs in the clinical use of tissue engineering bone. Methods BMSCs from a human adult in vitro were induced by using autologous platelet-rich plasma instead of animal serum in high-carbohydrate DMEM (Dulbecco's minimum essential medium). After the cultured BMSCs were amplified rapidly by Rotary Cell Culture System, their growth status was observed and their biological characteristics were detected. Results The cells were in good growth status and presented fine biological characteristics. Their growth speed in Rotary Cell Culture System was observed to be evidently faster than that by conventional methods. Conclusions Rotary Cell Culture System by autologous platelet-rich plasma instead of animal serum is a good culture method to induce human BMSCs in vitro. The quantity and biological characteristics of BMSCs cultured in this way can meet the demand in clinical use.
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To investigate the feasibility of in vitro inducing differentiation of bone marrow stromal cells (BMSCs) into neural stem cells and mature neural cells, and to offer reference for the application of BMSCs in the field of neuroscience, BMSCs were acquired from the marrow of dogs. Basic fibroblast growth factor (bFGF), all trans retinoic acid (RA) and glial cell line derived neurotrophic factor (GDNF) etc were used as proliferation or differentiation factors. Immunocytochemistry was employed to identify the cells at every culture stage. When BMSCs were proliferatively cultured for 24～72h, cleavage phase and cluster like clones appeared. On the 3rd day, some of the BMSCs derived cells started to express neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP). The same stage cells could be cloned, that is one of the characters of stem cells, when they were cultured in the proliferation medium. The neural cell modality like cells appeared on the 10th day after adding inducing factors into culture medium, which were proved by testing the express of NSE and GFAP. BMSCs can differentiate into neural stem cells and mature neural cells in vitro, and can be used as "seed cells" in the field of neuroscience.
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Objective To investigate the affect of body positions on biochemical indexes. Methods By autogenous contrast and cross matched survey, 107 volunteers divided into 3 season patches of winter, spring and summer, blood samples were drawn from the same part in both standing and lying positions。From19 persons, blood samples were collected respectively after standing and sitting for 15 min, lying for 15 min and 30 min and then sitting for another 15 min。 The blood samples were analyzed for 32 biochemical indexes on analyzer。Results 25 biochemical indexes in sitting position were significantly different from those in lying position (P0。05)。Conclusions Changing body position can result in obvious physiological variation of 28 biochemical indexes, particularly of those related to protein. Such result may lead to abnormality in some marginal values. It suggests body position should not be neglected in analyzing laboratory data.