The Effects Of Transplantation Of Adipose-derived Stem Cells In Senile Osteoporosis Mice

Background:Osteoporosis (OP) is one of common metabolic bone disease. Primary osteoporosis is not accompanied by other disease or dysfunction, including postmenopausal osteoporosis (PMOP) and senile osteoporosis. With the development of aging society, osteoporosis patients increased rapidly, how to effectively prevent and treat osteoporosis has become a hot topic at home and abroad. The concept of osteoporosis has been proposed for over one hundred years. After continuous in-depth research, and with the development of science and technology, the scholars of the pathogenesis of osteoporosis update a cellular level and molecular level understanding, understanding of its fulfillment, inspired people from cellular and molecular level and gene level to the treatment of osteoporosis.Osteoporosis is a kind of metabolic disease that characteristic by osteopenia and bone microstructural destroy, which could cause fragility increasing and be able to fractured. Current clinical treatment of osteoporosis drugs, its mechanism is mainly inhibit bone resorption and prevent the loss of bone and promoting bone formation, thereby correcting the abnormal bone turnover, and improve the bone quality, reduce fracture and other complications occurred. Although the long-term medication can improve symptoms in a large extent, but the treatment cycle is long, can not achieve the purpose of cure, the cost is not low and there are some adverse reactions. Thus, new therapeutic methods of research has been in the ascendant, and the pathogenesis of osteoporosis is bone absorption and undifferentiated stem cells failed to fully to osteoblast differentiation, resulting in insufficient bone formation and the emergence of a disease. Therefore, how to intervene the emergence of negative balance of bone metabolism, effectively complement the osteoblasts, promote bone formation, reconstruct the positive balance of bone metabolism is the key to the treatment of osteoporosis.Adipose-derived stem cells (ADSCs) are a kind of mesenchymal stem cells. With the development of regenerative medicine, ADSCs research has become one of the focus of stem cell research. Compared with bone marrow mesenchymal stem cells, they have more sources, easier to be isolated, brings less trauma and pain to the patient. Adipose-derived stem cells are isolated more easily and culture, and have differentiation of multipotent. They also have the capacity of cross-mesodermal differentiation. In vitro and in vivo assay have shown that ADSCs can be differentiated into several cell lineages, including adipocytes, osteoblasts, chondrocytes (cartilage cells), myocytes, and neuronal cells dependent upon the culture conditions employed. They have low immunity, immune suppression and immune regulation function, which provid advantageous condition for allograft of ADSCs. Thus, increasing the undifferentiation ADSCs, inducing osteogenic differentiation and increasing bone formation may have preventing and treating effects with osteoporosis.In order to investigate the prevention and treatment effect of adipose tissue derived stem cell transplantation on senile osteoporosis mice, we use micro computed tomography imaging (micro CT) detection and serological detection et al to observe the impact of adipose-derived stem cells transplantation on the spontaneous senile osteoporosis model mice SAMP6, from free radical, serum testosterone, osteocalcin, calcium, phosphorus, alkaline phosphatase metabolism, bone density, bone biomechanics properties and bone histomorphometry. Then to carry out comprehensive evaluation and explore a new treatment for osteoporosis, for the prevention and treatment of osteoporosis clinical provide new research ideas.Objective1.To isolate and culture the ADSCs from the adipose tissue of sprague-dawley (SD) rats, and to test their multiple differentiation multipotent and surface markers.2. To observe the effect of transplanting the adipose-derived stem cells (ADSCs) on serological detection, such as serum testosterone (T), osteocalcin (BGP), serum superoxide dismutase (SOD) and malondialdehyde (MDA), nitric oxide (NO) of osteoporosis SAMP6 mice; to explore the mechanism for anti-aging.3. To observe the effect of transplanting the adipose-derived stem cells (ADSCs) on bone mineral metabolism and bone formation of osteoporosis SAMP6 model by detecting the levels of serum calcium (Ca), serum phosphorus (P) and serum alkaline phosphatase (ALP).4. To observe the effect of transplanting the adipose-derived stem cells (ADSCs) on bone quality and bone microenvironment of osteoporosis SAMP6 mice by micro CT detection.5. To observe the effect of transplanting the adipose-derived stem cells (ADSCs) on bone strength and bone stiffness of SAMP6 mouse by three-point bending test.6. To observe the effect of transplanting the adipose-derived stem cells (ADSCs) on microenvironment in bone tissue of SAMP6 mouse by histomorphometry.7. To provide the evidence for the theory of undifferentiated stem cell could treat degenerative diseases and aging diseases.Methods1. To isolate the ADSCs from the SD rats’adipose tissue, and then cultured the cells in vitro. The 5th passage ADSCs were observed by their morphology, differentiatial potent by induced them into adipogenesis, osteogenesis and germ cell. Flow cytometry instrument were used to observe their biological markers on the cell membrane, such as CD29, CD90, CD11b, CD49d, CD106 and CD45. Using quantitative PCR to observe 5th passage ADSCs under retinoic acid induced can express germ cell associated genes Oct4, Dazl, Nobox, Piwil, which determine whether ADSCs have the potential to differentiate into germ cell; And respectively after the osteogenetic differentiation and into adipose differentiation, accordingly alizarin red and oil red O staining, and then determine whether the cells cultured by adipose source of stem cells with multidirectional differentiation potential.2.20 male 6-month spontaneous senile osteoporosis SAMP6 mice and 10 male 6-month SAMR1 were randomly divided into 3 groups (A, B, and C),10 in each group. A were SAMR1 control group, B were SAMP6 aging model group, C were ADSCs treating group.3. Harvest and culture purified ADSCs from SD rats. All rats in group C were injected with ADSCs. In brief, the purified ADSCs of the 5th passage were harvested and resuspended in the physiological saline at 3×106cells/mL. Each rat in group C was injected with 3*106 of ADSCs into its tail vein. The rats in groups B were similarly administered with same volume of physiological saline. Sacrificed and drew the materials after 4 weeks.4. After micro CT detection, the rats were anesthetized by chloral hydrate and the blood was got from the hearts of mice into the Pro-Coagulation Tubes, and stored them at room temperate for 8h. Then centrifugated 15min by the speed of 3000r/min to prepare serum, stored at -70℃ as backup. Radioimmunoassay method for the determination of serum testosterone (T) and Osteocalcin (BGP) level, superoxide dismutase (SOD) activity was determined by the method of xanthine oxidase, the content of malondialdehyde (MDA) measured by thiobarbituric acid (TBA) method, nitric oxide (NO) measured by nitrate reductase method. The levels of serum serum calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) were measured respectively by Arsenazo III method, phosphomolybdic acid colorimetric method and velocity method with automatic biochemistry analyzer.5. After the rats were anesthetized by chloral hydrate, bone densities at third to tifth lumbar vertebras and right femurs were measured by micro CT instrument with small animal measure software. When micro CT scanning is completed, selected the region remote distance growth plate 1.0 mm, layer thickness 3.0 mm bone tissue in cancellous bone as interested area (region of interest, ROI) for 3D reconstruction. Quantitative analysis the reconstruction images with software. Then measure the histomorphometric parameters, included percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), structure model index (SMI), degree of anisotropy (DOA).6. After micro CT scanning, sacrificed the mice by heart bloodletting, took out of two sides of femur, and remove muscular tissue around. Three-point bending tests were performed at the left femur using BOSE biomaterial dynamic test machine at a span of 18mm and a displacement rate of 0.05mm/s. Bones were positioned in the test apparatus with the dorsal side in compression and ventral side in tension. Mechanical properties measured included strength (ultimate force that the specimen sustained) and stiffness (the slope of the initial, linear portion of the load-deformation curve).7. Sacrificed the mice by heart bloodletting, took out of the up 1/3 parts of right tibias, and remove muscular tissue around. Then split them across sagittal plane. The undecalcified slices were produced by used the methyl methacrylate, and then stained by Giemsa and Von kossa, respectively. Used the light microscope to observe the histology and measure the histomorphometric parameters, osteoclast number (OC.N).8. Statistical analysis was carried out using SPSS13.0 statistical software, ANOVA followed by Fisher’s post hoc analysis, variance, the mean were compared with single factor analysis of variance, two-two compared with LSD method; variance not neat, multi-average number were compared by Welch method, two-two were compared with Dunnett’s T3, P<0.05 were significant differences.Results1. ADSCs can be extracted from the rats’fat tissue. Observation by light microscope, the primary cultured ADSCs exhibited a fibroblast-like morphology, long spindle, and polygon, and can form cell colony. The ADSCs were passaged at ratio 1:3 after 6 days, and then passaged every 3 days. The 5th passage cells were adherent and had a spindle-shaped morphology in the third days; Flow cytometry demonstrated that ADSCs were positively labeled by APC-CD29, FITC-CD90, and low phycoerythrin-CD106, and were negative for FITC-CD 49d, APC-CDllb and phycoerythrin-CD45 surface antigens; Culturing of undifferentiated cells for 14 days under adipogenic conditions induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were characteristics of adipocytes. Induction of osteogenic differentiation of the cells for 21 days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were characteristics of osteoblasts. QPCR showed a comprehensive profile of germ cell-associated genes including Oct4, Dazl, Nobox and Piwil expression during differentiation of rat ADSCs in the presence of 10-5 RA and 10-6 RA. Our observations demonstrate the capacity of rat ADSCs to express genes characteristic of male germ cells in vitro. ADSCs can differentiate in vitro into adipogenic, osteogenic and germ cells in the presence of lineage-specific induction factors.2. In this experiment, the spontaneous senile osteoporosis SAMP6 mice has been successfully established. Compared with the SAMR1 control group mice, the SAMP6 mice showed decreased testosterone content and increased bone glaprotein (BGP). After transplanting ADSCs, the SAMP6 mice in ADSCs treatment group showed a significant decrease in BGP level and a significant increase in testosterone content.3. Compared with the SAMR1 control group, the osteoporosis SAMP6 aging mice model showed increased MDA activities, decreased SOD levels and NO content. After transplanting ADSCs, the SAMP6 mice in ADSCs treatment group showed a significant decrease in MDA activities and a significant increase in SOD levels and NO content.4. After transplanting ADSCs, the spontaneous senile osteoporosis SAMP6 mice could significantly increase the level of serum Ca and ALP, bone mineral density, bone strength and bone stiffness. The levels of serum P obviously decreased. Bone trabecular became thicken and harden with an increasing quantity. The quantity, volume and thickness of bone trabecular were all increased significantly but Trabecular separation and osteoclast number decreased. Structure model index increased and degree of anisotropy decreased.Conclusion1. ADSCs have the morphological features of long spindle, and polygon, and can form cell colony. ADSCs can differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. The 5th passage of ADSCs in adipogenic culture 14d, oil red O staining showed that the specificity of lipid droplets. After osteogenic induction medium for 21 days, alizarin red staining showed specific pink mineralized nodules; quantitative PCR results showed that the retinoic acid induced ADSCs can express germ cell associated dazl, oct4, nobox, piwil genes. ADSCs were uniformly expressed CD29, CD90, and low CD 106, and negative for CD 49d, CD11b and CD45 surface antigens, in line with mesenchymal stem cell characteristics.2. Through transplanting ADSCs into the spontaneous senile osteoporosis SAMP6 mice can effectively increase testosterone content, decrease BGP level, improve Ca and P metabolism, which provid a new method of preventing and treating senile OP in clinical.3. Through transplanting ADSCs to the spontaneous senile osteoporosis SAMP6 mice, SOD level, NO level have a significant increase in ADSCs treatment group rats while contrast with the senile osteoporosis SAMP6 model rats. And MDA activities showed a significant decreased.4. Through transplanting ADSCs to the osteoporosis SAMP6 mice, analysis of micro CT detection combined with bone histomorphometry showed bone mineral density, bone strength and bone stiffness was significantly increased; an increase in the number of trabecular bone, trabecular thickening thicker, inter trabecular appear to be connected, reduce lacuna; a significant increase in trabecular number and considerable volume and thickness, and trabecular separation degree decreased significantly, unit bone trabecular osteoclast number reduced. Structure model index increased and degree of anisotropy decreased.In summary, transplantation of adipose tissue derived stem cells on the spontaneous senile osteoporosis mice can effectively improve the bone microenvironment and improve trabecular bone structure, promote bone mineralization and bone formation, reduce bone resorption, increasing bone mass, improve bone quality, and improve the biomechanical properties of bone and enhance the bone strength, so as to reduce the incidence of fractures. The experiments further suggest that ADSCs transplantation could fundamentally improve the osteoporosis disease, directly or indirectly promote bone formation. Therefore, this method has great potential to preventing and treating senile OP in clinical.