Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5-azacytidine enhances therapeutic efficacy for myocardial infarction.

BACKGROUND AND PURPOSE: The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (BMSCs) after myocardial infarction (MI) is controversial. Our previous results suggested that hypergravity promoted the cardiomyogenic differentiation of BMSCs, and thus we postulated that ex vivo pretreatment of BMSCs using hypergravity and 5-azacytidine (5-Aza) would lead to cardiomyogenic differentiation and result in superior biological and functional effects on cardiac regeneration of infarcted myocardium. METHODS: We used a rat MI model generated by ligation of the coronary artery. Homogeneous rat BMSCs were isolated, culture expanded, and differentiated into a cardiac lineage by adding hypergravity (2G) for 3 days and 5-Aza (50 lmol/L, 24 h). Rats underwent BMSCs (labeled with DAPI) injection after the infarction and were randomized into five groups. Group A rats received the control medium, Group B rats received unmodified BMSCs, Group C rats received BMSCs treated with hypergravity, Group D rats received BMSCs treated with 5-Aza, and Group E rats received BMSCs treated with 5-Aza and hypergravity (n = 6). RESULTS: After hypergravity and 5-Aza treatment, BMSCs showed positive for the early muscle and cardiac markers GATA-4, MEF-2, and Nkx2-5 with RT-PCR. We also found that hypergravity could enhance the activities of MEF-2 via promoting the nuclear export of HDAC5. The frozen section showed that the implanted BMSCs labeled with DAPI survived and angiogenesis was identified at the implantation site. In Groups B, C, D, and E rats, pre-treated BMSCs colocalized with α-actinin, and Group E rats showed a significantly larger increase in left ventricular function. CONCLUSIONS: The biological ex vivo cardiomyogenic differentiation of adult BMSCs with hypergravity and 5-Aza prior to their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium.

The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension.

BACKGROUND: The ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. It has been shown that chronic hindlimb unloading downregulates the satellite cell activity. This study investigated the role of low-frequency electrical stimulation on satellite cell activity during a 28 d hindlimb suspension in rats. RESULTS: Mechanical unloading resulted in a 44% reduction in the myofiber cross-sectional area as well as a 29% and 34% reduction in the number of myonuclei and myonuclear domains, respectively, in the soleus muscles (P < 0.001 vs the weight-bearing control). The number of quiescent (M-cadherin(+)), proliferating (BrdU(+) and myoD(+)), and differentiated (myogenin(+)) satellite cells was also reduced by 48-57% compared to the weight-bearing animals (P < 0.01 for all). Daily application of electrical stimulation (2 × 3 h at a 20 Hz frequency) partially attenuated the reduction of the fiber cross-sectional area, satellite cell activity, and myonuclear domain (P < 0.05 for all). Extensor digitorum longus muscles were not significantly altered by hindlimb unloading. CONCLUSION: This study shows that electrical stimulation partially attenuated the decrease in muscle size and satellite cells during hindlimb unloading. The causal relationship between satellite cell activation and electrical stimulation remain to be established.

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells. METHODS AND RESULTS: Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and beta-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes). CONCLUSION: Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.

[Effects of rotation on osteonectin and osteopontin mRNA level of cultured osteoblasts in rats].

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood except the suppression of osteoblast function. To investigate the effect of simulated weightlessness on mRNA level of extracellular matrix proteins, osteoblasts were rotated in horizontal plane as a model of simulated microgravity. Primordial osteoblasts of rats were grown for 2 d and then rotated for 24, 48 and 72 h, respectively. After isolating total RNA in cells, reverse transcription polymerase chain reaction (PT-PCR) was made to examine the mRNA level of osteopontin (OPN) and osteonectin (ON). Meanwhile, the levels of alkaline phosphatase (ALP) and osteocalcin (BGP) in the cultured medium were measured to evaluate the calcific function of cell. The expression of OPN and ON mRNA fell significantly after rotating for 24, 48 and 72 h, respectively. The contents of BGP descended significantly, meanwhile, the activity of ALP also showed a degressive tendency. Horizontal rotation decreased the expression of ON and OPN as well as diminished the secretion of BGP and ALP, which affected the calcific function of osteoblast. The results obtained suggest that depression of extracellular matrix proteins expression plays a key role in bone loss during weightlessness.

[Simulation of the rat tibial bone density changes with the finite element method].

OBJECTIVE: To calculate changes of rat tibial density induced by mechanical load with the finite element method. METHOD: A three-dimensional computer model was formed by processing the industrial CT image of rat tibia; bone remodeling results were predicted by using the internal bone remodeling theory presented by Beaupre in conjunction with the finite element method. RESULT: The increments of the elements density and its distribution plot were obtained. CONCLUSION: The simulation results agree well with the results of the biological experiment, so the simulation results were proved to be accurate.

[Effects of Naoyaojia on structural mechanical properties of femur in rats].

OBJECTIVE: To study the effect of long term Naoyaojia administration on the structural mechanical properties of rat femurs. METHOD: Young wistar rats were treated orally with Naoyaojia at doses of 3, 6, and 12 mg/kg body weight every 2 weeks for 1 year. Beary's bending test was used for determining physical properties of the femur. RESULT: The thickness of the cortex and cross-sectional area of femur increased significantly in animals treated with 3 mg/kg dose of Naoyaojia (P<0.01), significant increase in maximum bending load was also found in this group compared with the control group (P<0.01). But an opposite change was found in animals treated with 12 mg/kg dose of Naoyaojia. CONCLUSION: Stimulation on the thickness of the cortex along the axis as well as the maximum bending load of femurs are found in animals treated with 3 mg/kg dose of Naoyaojia, but negative effects were observed at higher dose level.

[Effects of simulated weightlessness and mechanical loading on bone interstitial fluid flow in rats].

OBJECTIVE: To investigate the effects of simulated weightlessness and mechanical loading on bone interstitial fluid flow. METHOD: Thirty-six male Sprague-Dawley [correction of Spargue-Danley] rats were divided into 3 groups: the control group, the tail-suspension group, and the tail-suspension plus mechanical loading group, with four rats in each group. All the rats were injected via a lateral tail vein with horseradish peroxidase on the 21st day of the experiment. Tibial tissue specimens were explanted, fixed, decalcified and cut into 30 micrometers frozen sections 3 h after the intravenous injection. RESULT: There were less peroxidase reaction product in the bony matrix and bone lacunae in tail-suspended rats than the control and tail-suspended plus mechanical loading rats. CONCLUSION: Tail-suspension decreased fluid movement through the bone, while mechanical loading increased it.

[Changes of endocrine hormones during -6 degrees head down bed rest in human].

OBJECTIVE: To investigate changes of endocrine hormones during 7 d head down bed rest (HDBR). METHOD: Six healthy male volunteers served as the subjects and experienced 7 d -6 degrees HDBR. Urine was collected from 6:00-22:00 and from 22:00-6:00. Serum was collected 48 h before HDBR, 48 h and 120 h during HDBR. Then the endocrine indices in urine and serum were determined. RESULT: 1) The levels of serum CORT and ALD increased at 48 h during HDBR, while serum T3, T4, TP, UN decreased but they all recovered to normal at 120 h during HDBR. 2) The level of urine CORT, ALD and NE reached its peak in 24-48 h, and then gradually decreased to normal level. CONCLUSION: The endocrine indices in serum and urine changed in the early period but returned to normal level gradually with the proceeding of HDBR.

[Effects of hindlimb unloading on bone histomorphometry and bone mass in rats].

OBJECTIVE: To study the effects of hindlimb unloading on bone histomorphometry, bone local growth factor, bone biomechanical properties and bone contents in rats. METHOD: Male SD rats were arranged into free active control group (CON) and tail-suspended group (TS) with 9 rats in each group. The experiment lasted for 3 weeks. Bone histomorphometry, bone local growth factor, biomechanical properties and bone contents were measured before and after tail suspension. RESULT: Structure of the trabecular bone was disorganized. Compared with CON, trabecular bone volume (% Tb. Ar), mean trabecular plate thickness (Tb. Th), osteoblast surface (Ob. S) were significantly reduced in TS. The eroded surface (Oc. N/BS) tended to be higher, though not significant at this stage of tail suspension; alkaline phosphatase (ALP) activity in the tibia were significantly reduced, but NO content in the femoral trunk was significantly decreased; bone biomechanical properties, bone mineral content (BMC), bone density (BD) and bone collagen density (BCD) in the femur were significantly reduced. CONCLUSION: Hindlimb unloading may lead to regression of bone microstructure, change of bone local growth factor content, reduction of bone biomechanical properties and bone content.

[Effects of simulated weightlessness on reproduction in male rats].

OBJECTIVE: To study the effects of tail-suspension on gonad and spermatic hormone in male rats. METHOD: After tail-suspension and reambulation some times in male rats, weight of testis was measured, sperm of epididymis was inspected, morphology of testicular cell was compared, and antent of testosterone assay by radioimmunoassay ratio of LH-beta positive cell in pituitary was measured by immunohistochemic. RESULT: After tail-suspension, weight of testis in male rats decreased significantly. Shape of the convoluted tubules and number of all levels of the spermatocyte in testis atrophied markedly. No sperm was found in the epididymis. Content of testosterone decreased markedly. Number of positive cell of beta-subunit of LH in the pituitary increased markedly. CONCLUSION: Tail-suspension has negative effect on sex gland and reproduction function of male rats.