Rabbit Bone Marrow Mesenchymal Stem Cells Research Articles

The IFT80/Hedgehog Pathway Regulates the Osteogenic-adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Steroid-induced avascular necrosis of the femoral head (SANFH) is a typical refractory disease that often progresses irreversibly and has a high disability rate. Numerous studies have confirmed that abnormal osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) is one of the major factors of SANFH. However, the mechanism remains to be elucidated. This study aimed to investigate the mechanism and effect of the IFT80/Hedgehog-mediated osteogenic-adipogenic differentiation of BM-MSCs in SANFH. Femoral head specimens of SANFH patients and femoral neck fractures (FNF) patients were collected to detect the expression of IFT80, Shh and osteogenic-adipogenic differentiation-related genes by immunohistochemistry (IHC), western blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Based on the rabbit SANFH model, the mRNA expression and protein level of IFT80 and Shh were detected by RT-qPCR and WB. After the osteogenic/adipogenic differentiation based on rabbit BM-MSCs, the IFT80, Gli1, PPAR-γ, and Runx2 expression were detected. Differences in alkaline phosphodiesterase activity, calcium nodule, quantification/distribution of lipid droplets, expression of IFT80/Hedgehog axis, and the level of osteogenic- adipogenic associated factors were determined after IFT80 overexpression. RT-qPCR, WB and IHC revealed that IFT80 and Shh lowly expressed in the osteoblasts and intra-trabecular osteocytes at the edge of trabeculae and in the intercellular matrix of the bone marrow lumen in the SANFH specimens. The Runx2 expression was low, while the PPAR-γ expression was high in both human specimens and animal models of SANFH, suggesting that the balance of osteogenic-adipogenic differentiation was dysregulated. Rabbit BM-MSCs with stable overexpression of IFT80 showed increased alkaline phosphatase activity after induction of osteogenic differentiation, increased calcium nodule production, and decreased adipogenesis after induction of adipogenic differentiation. There is a dysregulation of the balance of osteogenic-adipogenic differentiation in SANFH. IFT80 may inhibit adipogenic differentiation while promoting osteogenic differentiation in rabbit BM-MSCs by activating the Hedgehog pathway.

Experimental Studies on the Effects of Glycitinregulated TGF-β Signaling Pathway on the Proliferation, Osteogenic, and Lipogenic Differentiation of BMSCs

Background Glycitin, generated from the seeds of the legume soybean, has an essential function in antioxidant, obesity inhibition, and wound healing promotion. Furthermore, recent research has shown that it has anti-inflammatory and cartilage-protective properties. Objectives This study was designed to investigate the osteogenic effects of glycitin and the transforming growth factor-β (TGF-β) signaling pathway on bone marrow mesenchymal stem cells (BMSCs), as well as their interaction. Materials and Methods We isolated rabbit BMSCs by whole bone marrow adherent method and cultured, identified, and induced differentiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the proliferation ability of BMSCs with different concentrations of glycitin at different times; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of osteogenesis-related genes runt-related transcription factor 2 (Runx2), collagen type I (Col-1), and osteocalcin (OC) mRNA; and a kit was used to determine the activities of alkaline phosphatase and triglycerides (TG) in BMSCs. A Western blot was used to detect the expression of TGF-β protein in each group. Results On the fifth day of BMSC primary culture, the morphology of long spindle-shaped cells might be plentiful, dominated by a daisy-like or whirling arrangement. They proliferated rapidly and fused to 70%–80% on the eighth day of culture; the third generation of BMSCs was taken for phenotyping, and the detection results of flow cytometry analysis showed that the surface antigenic markers of BMSCs, CD44, and CD90 were positive, whereas CD34 and CD45 were negative; after osteogenic induction of BMSCs, visible calcified nodules were evident. There was cartilage matrix formation after osteogenic induction; glycitin promoted BMSC proliferation in a time-dependent and dose-dependent manner, and BMSC proliferation increased significantly at 20 µM glycitin for 3–5 days; glycitin increased the expression of Runx2, Col-1, and OC mRNA in BMSCs and increased the activity of alkaline phosphatase (ALP) while decreasing the activity of TG. Meanwhile, 20 µM glycitin dramatically increased the expression of TGF-β. Conclusion These findings imply that glycitin increases BMSCs proliferation and osteogenic differentiation. By regulating the TGF-β signaling pathway, glycitin modulates the differentiation of BMSCs into osteoblasts.

A composite membrane with microtopographical morphology to regulate cellular behavior for improved tissue regeneration.

Tissue engineering scaffolds with specific surface topographical morphologies can regulate cellular behaviors and promote tissue repair. In this study, poly lactic(co-glycolic acid) (PLGA)/wool keratin composite guided tissue regeneration (GTR) membranes with three types of microtopographies (three groups each of pits, grooves and columns, thus nine groups in total) were prepared. Then, the effects of the nine groups of membranes on cell adhesion, proliferation and osteogenic differentiation were examined. The nine different membranes had clear, regular and uniform surface topographical morphologies. The 2 µm pit-structured membrane had the best effect on promoting the proliferation of bone marrow mesenchymal stem cells(BMSCs) and periodontal ligament stem cells (PDLSCs), while the 10 µm groove-structured membrane was the best for inducing osteogenic differentiation of BMSCs and PDLSCs. Then, we investigated the ectopic osteogenic, guided bone tissue regeneration and guided periodontal tissue regeneration effects of the 10 µm groove-structured membrane combined with cells or cell sheets. The 10 µm groove-structured membrane/cell complex had good compatibility and certain ectopic osteogenic effects, and the 10 µm groove-structured membrane/cell sheet complex promoted better bone repair and regeneration and periodontal tissue regeneration. Thus, the 10 µm groove-structured membrane shows potential to treat bone defects and periodontal disease. STATEMENT OF SIGNIFICANCE: PLGA/wool keratin composite GTR membranes with microcolumn, micropit and microgroove topographical morphologies were prepared by dry etching technology and the solvent casting method. The composite GTR membranes had different effects on cell behavior. The 2 µm pit-structured membrane had the best effect on promoting the proliferation of rabbit BMSCs and PDLSCs and the 10 µm groove-structured membrane was the best for inducing the osteogenic differentiation of BMSCs and PDLSCs. The combined application ofa 10 µm groove-structured membrane and PDLSC sheet can promote better bone repair and regeneration as well as periodontal tissue regeneration. Our findings may have significant potential for guiding the design of future GTR membranes with topographical morphologies and clinical applications of the groove-structured membrane/cell sheet complex.

The Antibacterial Activity Comparison between Novel Carbon-Based Nanofilm Coated Titanium Alloy and Co-Cr-Mo Alloy.

Objective The purpose of this study was to reveal the antibacterial activity of carbon-based nanofilm titanium alloy. Methods The experiments were performed both in vitro and in vivo of animals using two circular-piece test specimens of the same specification, which were made from carbon-based nanofilm coated titanium alloy and commonly used in artificial joints Co-Cr-Mo alloy, respectively. In the in vitro experiments, the two test specimens were cocultured with standard strains of Staphylococcus aureus and Escherichia coli, and the antibacterial properties of the two test specimens were evaluated via inhibition zone size, scanning electron microscopy, fluorescence staining, colony forming unit count, and others; the cytotoxicities of the two test specimens were evaluated by coculturing and them with rabbit bone marrow mesenchymal stem cells (BMSCs). In the in vivo experiments, the two test specimens were implanted in the muscle tissue of experimental rabbits to evaluate their histocompatibilities. Results Both in vitro cocultures of the carbon-based nanofilm titanium alloy and the Co-Cr-Mo alloy with Staphylococcus aureus and Escherichia coli failed to form inhibition zone. However, some biofilms were observed on the surface of the Co-Cr-Mo alloy. Fewer bacteria adhered to the carbon-based nanofilm titanium alloy can be observed via scanning electron microscopy and fluorescence staining techniques. Meanwhile, the colony forming counts showed that, compared with the Co-Cr-Mo alloy, the carbon-based nanofilm titanium alloy had fewer bacteria adhered (P < 0.05). After coculture of the two test specimens with rabbit BMSCs, there was no significant difference in cell count, and both cell counts showed no cytotoxicity. In the in vivo experiment of animals, there are relatively fewer giant cells and better histocompatibility in tissues near the carbon-based nanofilm titanium alloy. Conclusion Compared with Co-Cr-Mo alloy, the novel carbon-based nanofilm titanium alloy enjoys stronger in vitro antibacterial activity and better in vivo histocompatibility.

Hyaluronan size alters chondrogenesis of mesenchymal stem cells cultured on tricalcium phosphate-collagen-hyaluronan scaffolds.

Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.

Repopulation of multipotent stem cells derived from adult male rabbits on a polycaprolactone scaffold: an in vitro study

ABSTRACT Background: MSCs (mesenchymal stem cells) are multipotent, spindle-shaped cells that give them the ability to differentiate into a broad lineage of tissues. Polycaprolactone (PCL) is a synthetic material that has been used several times for the fabrication of scaffolds to be utilized in the field of tissue engineering. Aim of the work: The main goal of this research is to develop a protocol for isolating, cultivating, and characterizing rabbit bone marrow mesenchymal stem cells (BMSCs), as well as to investigate their capacity to repopulate PCL scaffolds. Results: In Dulbecco's Modified Eagle's Medium (DMEM) supplied with 10% fetal bovine serum (FBS), BMSCs were successfully isolated, cultivated, and grown. The cells were characterized morphologically by their spindle-shape, processes of cytoplasmic, plastic adhesion tendencies, and formation of colonies. They were characterized by flowcytometric analysis as having a positive CD73 expression and a negative CD45 expression. Before cell seeding, the PCL scaffold was inspected under a scanning electron microscope to assess the PCL material characteristics and pour size, and after cell seeding to detect BMSC attachments to the scaffold. Conclusion: The protocol used in this investigation was effective for isolation, culture, and characterization of rabbit BMSCs and the cells that were obtained successfully repopulated the PCL scaffold.

Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels

To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD +) levels. The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H 2O 2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H 2O 2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H 2O 2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD + and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment. The rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased ( P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H 2O 2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD + and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant ( P<0.05). NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD + levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.

A study of Drynaria fortunei in modulation of BMP–2 signalling by bone tissue engineering

Background/aim Drynaria fortunei (Gusuibu; GSB) is a popular traditional Chinese medicine used for bone repair. An increasing number of studies have reported that GSB induces osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). These results provide insight into the application of GSB for bone tissue engineering techniques used to repair large bone defects. However, few studies have described the molecular mechanisms of GSB.Materials and methodsIn the present study, the effects of GSB and naringin, a marker compound, on the binding of BMP-2 to BMPR and BMP-2-derived signal transduction were investigated using surface plasmon resonance (SPR) and coculturing with BMPR-expressed cell line, C2C12, respectively. Furthermore, naringin was also used to prepare naringin contained scaffolds for bone tissue engineering. The physical and chemical properties of these scaffolds were analysed using scanning electron microscopy (SEM) and highperformance liquid chromatography (HPLC). These scaffolds were cocultured with rabbit BMSCs in vitro and implanted into rabbit calvarial defects for bone repair assessment. ResultsThe results showed that GSB and naringin affect the binding of BMP and BMPR in SPR experiments. GSB is a subtle BMP modulator that simultaneously inhibits the binding of BMP-2 to BMPR-1A and enhances its binding to BMPR-1B. In contrast, naringin inhibited BMP-2 binding to BMPR-1A. In vitro studies involving the phosphorylation of signals downstream of BMPR and Smad showed that GSB and naringin affected stem cell differentiation by inhibiting BMPR-1A signalling. When using GSB for bone tissue engineering, naringin exhibited a higher capacity for slow and gradual release from the scaffold, which promotes bone formation via osteoinduction. Moreover, control and naringin scaffolds were implanted into rabbit calvarial defects for 4 weeks, and naringin enhanced bone regeneration in vivo significantly.ConclusionGSB and its marker compound (naringin) could inhibit the binding of BMP-2 and BMPR-1A to control cell differentiation by blocked BMPR-1A signalling and enhanced BMPR-1B signalling. GSB and naringin could be good natural BMP regulators for bone tissue engineering.

Overexpression of Bone Morphogenetic Protein-1 Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells In Vitro.

BackgroundOsteogenesis of bone marrow mesenchymal stem cells (BMSCs) is an important research topic in the application of bone tissue engineering. Bone morphogenetic protein-1 (BMP-1) is important in bone formation and stability, but its effects on the osteogenesis of BMSCs are unclear. This study aimed to investigate the association of BMP-1 with the osteogenic capacity of BMSCs.Material/MethodsPrimary rabbit BMSCs were cultured and divided into a BMP-1-overexpressing group, a Green Fluorescent Protein-expressing (GFP) group, and a Control group. The transfection efficiency of BMP-1 was tested by Western blotting. Cell viabilities, alkaline phosphatase (ALP) activities, Ca2+ concentrations, and gross examinations of BMSC sheets were examined at different times. The osteogenic marker collagen I was assessed by immunohistochemical analysis.ResultsThe cell viability, ALP activity, and Ca2+ content of the BMP1-overexpressed group were significantly enhanced compared with the GFP group and Control group. Immunohistochemistry staining results showed that BMP-1 promoted the expression of type I collagen in BMSCs sheets.ConclusionsOur results suggest that the overexpression of BMP-1 can promote the osteogenesis of BMSCs and provides an improved method of cell-based tissue engineering.

Study of the timing of tooth movement after repair of alveolar bone defects by rabbit BMSCs combined with beta-TCP

To investigate the effect of tooth movement at different time after repair of alveolar bone defects with tissue engineering bone constructed by rabbit bone marrow mesenchymal stem cells (BMSCs) and beta-tricalcium phosphate (β-TCP). Alveolar bone defect (6 mm×4 mm×8 mm) was made on the right side of 40 New Zealand rabbits, which was filled with tissue engineering bone constructed by BMSCs and β-TCP as experimental sides. Tooth extraction on the other side was performed as control. The mandibular second molars in both sides were moved mesially 2, 4, 8 and 12 weeks after surgery. The specimens were taken 4 weeks after exertion. The distance of mandibular second molar moved was measured. The periodontal tissues were observed after H-E staining. TRAP staining was performed and osteoclasts were counted in the periodontal tissues on the pressure side. BMP-2 immunohistochemical staining was used to observe the average optical density of periodontal tissue on tension side. The results were analyzed with SAS 8.0 software package. The distance of tooth movement, the amount of TRAP positive cells and the optical density of BMP-2 in the experimental side of the 2-week and 4-week groups were all significantly lower than those in the control side, while there was no significant difference between the 8-week and 12-week groups. Eight weeks after repair of alveolar bone defect with rabbit BMSCs and β-TCP is an appropriate time for orthodontic tooth movement.

Glucocorticoid Enhanced the Expression of Ski in Osteonecrosis of Femoral Head: The Effect on Adipogenesis of Rabbit BMSCs.

Glucocorticoid (GC)-induced osteonecrosis has been considered as the most serious side effect in long-term or over-dose steroid therapy. The decreased bone mass and increased marrow fat tissue demonstrated that GC can destroy the normal differentiation of bone marrow mesenchymal stem cells (BMSCs), which accelerates adipogenesis but not osteogenesis. However, the underlying mechanisms are still unclear. Ski, an evolutionary conserved protein, is a multifunctional transcriptional regulator that involved in regulating signaling pathways associated with adipogenesis differentiation, but the concrete function remains unclear. In this work, we first established a methylprednisolone (MPS)-induced osteonecrosis of femoral head (ONFH) rabbit model, in which the expression of Ski, PPAR-γ, and FABP4 was up-regulated compared with control group, and then we induced the isolated BMSCs from rabbit with dexamethasone (Dex) in vitro and the results showed that the Ski expression was up-regulated by Dex in a dose- and time-dependent manner. Therefore, we demonstrated that the expression of Ski was up-regulated in glucocorticoid-related osteonecrosis disease in vivo and in vitro. Moreover, the adipogenesis differentiation capacity of BMSCs was enhanced after induced by Dex, which was identified by Oil Red O staining, and the up-regulated PPAR-γ and FABP4 expression. To further study the function of Ski in BMSC after induced by Dex, Ski specific small interfering RNA (Ski-siRNA) was used. Results showed that knockdown of Ski obviously decreased adipogenesis differentiation evident by Oil Red O staining, and the expression of PPAR-γ and FABP4 was down-regulated simultaneously. Collectively, our findings suggest that Ski increased significantly during glucocorticoid-induced adipogenic differentiation of BMSCs, and the expression level was consistent with adipogenic-related proteins including PPAR-γ and FABP4. Based on the above data, we believe that Ski might become a new molecule in the treatment of GC-induced ONFH and our study could provide a basis for further study on the detailed function of Ski in ONFH.

Dynamic mechanical loading facilitated chondrogenic differentiation of rabbit BMSCs in collagen scaffolds.

Mechanical signals have been played close attention to regulate chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In this study, dynamic mechanical loading simulation with natural frequencies and intensities were applied to the 3D cultured BMSCs–collagen scaffold constructs. We investigated the effects of dynamic mechanical loading on cell adhesion, uniform distribution, proliferation, secretion of extracellular matrix (ECM) and chondrogenic differentiation of BMSCs–collagen scaffold constructs. The results indicated that dynamic mechanical loading facilitated the BMSCs adhesion, uniform distribution, proliferation and secretion of ECM with a slight contraction, which significantly improved the mechanical strength of the BMSCs–collagen scaffold constructs for better mimicking the structure and function of a native cartilage. Gene expression results indicated that dynamic mechanical loading contributed to the chondrogenic differentiation of BMSCs with higher levels of AGG, COL2A1 and SOX9 genes, and prevented of hypertrophic process with lower levels of COL10A1, and reduced the possibility of fibrocartilage formation due to down-regulated COL1A2. In conclusion, this study emphasized the important role of dynamic mechanical loading on promoting BMSCs chondrogenic differentiation and maintaining the cartilage phenotype for in vitro reconstruction of tissue-engineered cartilage, which provided an attractive prospect and a feasibility strategy for cartilage repair.

Effects of extracorporeal fucosylation of CD44 on the homing ability of rabbit bone marrow mesenchymal stem cells

BackgroundThe aim of the study was to investigate the effects of extracorporeal fucosylation of CD44 on the homing ability of rabbit bone marrow mesenchymal stem cells (BMSCs). MethodsThe rabbit BMSCs were extracorporeal fucosylated using alpha-(1,3)-fucosyltransferase VI (FTVI), then the positive rate of sialyl-LewisX (sLeX) and the binding rate of E-selectin were detected by flow cytometry, as well as the fluid adhesion of rabbit BMSCs were detected by the parallel flow chamber adhesion test. Then BMSCs were constructed to stably express enhanced green fluorescent protein (EGFP) and were injected intravenously into the model rabbits with tibial fractures. After 6 weeks of injection, the levels of stromal cell-derived factor (SDF-1) and monocyte chemoattractant protein-1 (MCP-1) in rabbit serum and damaged bone tissues were detected. The positive rate of EGFP expressions was detected by immunohistochemistry staining. ResultsAfter fucosylation, the positive rate of sLeX and the binding rate of E-selectin were significantly higher than those in the no fucosylated group. The results of fluorescence microscopy showed that BMSCs with stable expression of EGFP were successfully constructed. The results of ELISA and Western Blot showed that the secretion of SDF-1 and MCP-1 and the expression of SDF-1 and MCP-1 protein in BMSCs treatment group processed by fucosylated were significantly higher than those in BMSCs treatment group processed by no fucosylated. The results of immunohistochemical staining showed that the positive rate of EGFP expression was also significantly increased, which indicated that the BMSCs at the injured bone tissues were significantly increased and helpful in the repair of bone injury. ConclusionsExtracorporeal fucosylation of CD44 molecules can significantly enhance the homing ability of rabbit BMSCs, which may be achieved by SDF-1 and MCP-1 regulation.

Enhancement of osteogenesis of rabbit bone marrow derived mesenchymal stem cells by transfection of human BMP-2 and EGFP recombinant adenovirus via Wnt signaling pathway.

Bone marrow mesenchymal stem cells (BMSCs) are considered the most important seed cells in bone tissue engineering. The present study aimed to investigate the potential of rabbit BMSCs in osteogenesis after the transfection of human BMP-2 and EGFP recombinant adenovirus. Rabbit BMSCs were isolated and the surface stem cell makers, including CD29, CD44 and CD45 were detected by flow cytometry. The expression of BMP-2 mRNA and protein in BMSCs were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. After an induction with osteogenic medium, the alkaline phosphatase (ALK) activity at 7 days, the type I collagen at 14 days, and the calcium nodules at 21 days were performed using an ALK activity kit, immunohistochemical staining and alizarin red S staining, respectively. The expression levels of proteins related to the Wnt signaling pathway were detected by western blot analysis. The positive rates of CD29, CD44 and CD45 were 62.92±1.99, 93.55±0.99 and 0.21±0.12%. The expression of BMP-2 mRNA and protein was significantly upregulated in Ad-BMP-2/EGFP transfected BMSCs. Furthermore, Ad-BMP-2/EGFP induced ALP activity, promoted the production of type I collagen and calcium nodule formation in rabbit BMSCs. The levels of β-catenin, cyclin D1, Runx2 and c-myc were upregulated in Ad-hBMP-2/EGFP transfected BMSCs, while the level of GSK3β was significantly decreased. Results also indicated that the overexpression of BMP-2 by Ad-hBMP-2/EGFP enhanced the osteogenic differentiation ability of cultured rabbit BMSCs via stimulating the Wnt signaling pathway with the accumulation of β-catenin and suppression of GSK3β. The Ad-hBMP-2/EGFP transfected rabbit BMSCs are expected to be a good seed cell in bone tissue engineering.

Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165

The effects of CD44 fucosylation on fluid adhesion force of rabbit bone marrow mesenchymal stem cells

To investigate the effect of CD44 fucosylation on fluid adhesion force of rabbit bone marrow mesenchymal stem cells (BMSCs). The rabbit BMSCs were isolated and purified by density gradient centrifugation combined with adherent culture method. The morphology of cells were observed by inverted microscope, and the cell surface markers of CD44, CD34, CD29, and CD105 were assessed by flow cytometry. BMSCs fucosylated by alpha-(1, 3)-fucosyltransferase Ⅵ (FTⅥ) were as the experimental group, and the non-fucosylated BMSCs were as the control group, and then the positive rate of sialyl-LewisX (sLe X) and the binding rate of E-selectin were detected by flow cytometry. The fucosylated BMSCs resuspended in Hank balanced salt solution (HBSS) were assigned as the experimental group (group A), at same time, the non-fucosylated BMSCs resuspended in HBSS solution as the study control group (group B), and the fucosylated BMSCs resuspended in HBSS solution which was added EDTA as negative control group (group C). The fluid adhesion force of rabbit BMSCs were detected by the parallel flow chamber adhesion test. Primary BMSCs mainly shaped as spindle and kept strong growth. The third generation BMSCs were negative for CD34, but positive for CD44, CD29, and CD105. After fucosylation, the positive rate of sLe X in the experimental group was 32.52%±1.76%, which was significantly higher than that in the control group (1.48%±0.51%) ( t=29.277, P= 0.000). The binding rate of E-selectin in the experimental group was 41.05%±1.84%, which was also significantly higher than that in the control group (4.33%±0.92%) ( t=35.674, P=0.000). With the increase of fluid shear force, the number of BMSCs adhering to the surface of human umbilical vascular endothelial cells (HUVEC) in group A was increased at first and then decreased, while there was few BMSCs adhering to the surface of HUVEC in groups B and C. Under the different fluid shear stress, the number of BMSCs adhered to the surface of HUVEC in group A was significantly higher than that in groups B and C ( P<0.05), and there was no significant difference between groups B and C ( P>0.05). CD44 fucosylation on BMSCs can enhance the fluid adhesion force of rabbit BMSCs.

Osteogenesis Differentiation of Rabbit Bone Marrow-mesenchymal Stem Cells in Silk Scaffold Loaded with Various Ratios of Hydroxyapatite

Osteogenesis Differentiation of Rabbit Bone Marrow-mesenchymal Stem Cells in Silk Scaffold Loaded with Various Ratios of Hydroxyapatite

ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGULATING OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS

To investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. The recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. WST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. The over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.

Inhibition of bone marrow mesenchymal stem cell autologous transplantation on inflammation following rabbit corneal alkali burn

Background Ocular alkali burns leads to corneal ulcer and angiogenesis and even corneal opacity. There is still no ideal treatment method. Studies showed that mesenchymal stem cells (MSCs) can repair corneal wound in vivo, but the specific mechanism is still not clear. Objective This study aimed to observe the histopathological change after the early transplatation of bone marrow MSCs (BMSCs) for corneal alkali burn model in rabbits and explore the anti-inflammatory effects of MSCs after corneal alkali burn. Methods Bone marrow of 4 ml was collected from 2-3 month-old Japanese rabbit. BMSCs were isolated and cultured from the bone marrow of rabbits, and the third generation of cells were used in this study. Cultured cells were identified by morphology and the expressions of surface markers. Corneal alkali burn models were extablished in the right eyes of 24 rabbits by attaching the filter paper with 0. 1% NaOH at the central cornea for 30 seconds, and then the models were randomized into 2 groups. BMSCs suspension of 300 μl (concentration 5×106/μl) was subconjunctivally injected 1 hour after modeling in the BMSCs group, and equal volume of PBS was used in the same way in the PBS group. Corneal opacification was scored under the slim lamp microscope in 3, 14 and 28 days after injection. The polymorphonuclear neutrophils (PMNs) were counted by histopathological examination, and the expression of matrix metalloproteinase-2 (MMP-2) in the corneal tissue was evaluated by immunochemistry in various time points. The use and care of the rabbits followed the statement of ARVO. Results The rabbit BMSCs were plastic-adherent cells that exhibited a fibroblast-like shape. Cultrued cells highly expressed surface adhesion molecular markers CD29 and CD90 (99. 18% and 97. 94%) and lowly expressed hematopoietic cell markers CD34 and CD31 (0. 74% and 0. 15%). Opacification of cornea, defect of corneal epithelium, stromal edema and neovascularization appeared after modeling. In 14 days and 28 days after modeling, the opacification scores in the BMSCs group were 2. 37±0. 52 and 2. 25±0. 50, which were significantly lower than 3. 00±0. 53 and 3. 25±0. 50 in the PBS group (t=2. 376, 2. 828, both at P<0. 05). After subconjunctival injection, the number of PMNs was (34. 17±1. 85) /12 fields and (25. 64±3. 86)/12 fields in the BMSCs group, showing significant decrease in comparison with (42. 70±1. 54) /12 fields and (32. 67±1. 42)/12 fields in the PBS group (t=10. 021, 4. 832, both at P=0. 000). The expression levels of MMP-2 (A value) in cornea were 0. 388±0. 016 and 0. 384±0. 006 in the BMSCs group, with considerable decreases in comparison with 0. 438±0. 006 and 0. 412±0. 005 in the PBS group (t=10. 205, 13. 514, both at P=0. 000). Conclusions Early transplantation of BMSCs can arrest the occurrance of corneal ulcer by suppressing the infiltration of PMNs, alleviateing the inflammation reaction, downregulating the expression of MMP-2 in cornea and inhibiting the degradation of stromal collagen fibers. Key words: Bone marrow mesenchymal stem cell transplantation/methods; Biological markers; Burns, chemical/therapy; Corneal diseases/therapy; Neovascularization, pathologic/chemically induced; Inflammation/prevention & control; Matrix metalloproteinase; Disease models, animal

A tissue-engineered conduit for urinary diversion using bone marrow mesenchymal stem cells and bladder acellular matrix

Ileal conduits are commonly used in bladder cancer treatment and in pediatric patients who require urinary diversion surgeries. We constructed a tissue-engineered conduit using bone marrow mesenchymal stem cells (BMSCs) and a bladder acellular matrix (BAM) and transplanted it into rabbits for urinary diversion to evaluate the feasibility of its clinical application. Rabbit BMSCs were isolated, expanded in vitro, and were then treated with a conditional medium for 3 weeks, allowing the BMSCs to transform into urothelium-like cells. These cells were seeded onto BAM and cultured for another week. The cell-matrix grafts were then sewn into a conduit approximately 4 cm in length and 1 cm in diameter and were implanted as conduits for urinary diversion in 12 male rabbits. Rabbits were sacrificed at 1, 2, and 4 week after operation. Histologic examinations were performed using hematoxylin and eosin staining and tissue structures were evaluated by using immunohistochemistry. BMSCs can transform into urothelium-like cells that express the urothelium-specific proteins uroplakin 1A (UPK1A), cytokeratin 7 (CK7), and cytokeratin 13 (CK13). These cells are able to generate epithelial coverage in the conduit lumen based on BAM. Immunohistochemistry showed positive staining with AE1/AE3, UPK1A, and tight junction protein 1 (ZO-1), indicating the presence of mature and functional epithelial cells on the lumen of the conduit. BMSCs may represent a new source cell for urinary tissue engineering, and it is feasible to construct a conduit with transformed BMSCs and BAM for urinary diversion in rabbits.