| Background:Osteoporosis is a metabolic disease characterized by osteopenia and bone microstructural destroy, which could cause the brittleness increasing and become easy to fracture. Due to the increasing osteoporosis patients, researched how to prevent and treat osteoporosis have an important clinical significance. Recently, Prof. L. Yuan had put forward a new hypothesis, fasciology hypothesis. In fasciology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells from the network of unspecialized connective tissues (fascia network). The other one is the functional system, which is consisted of differentiated functional cells and enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. Undifferentiated stem cells in the supporting-storing system incessantly migrate to target areas, differentiate into committed-stem cells, and further differentiate into functional cells. The supporting-storing system provides energy and cell reserve for the functional system. The structures and functions of an organism are maintained by the incessant supplement and refreshment from the supporting-storing system to the functional system. Meanwhile, under the regulation of the nervous system and immune system, the fascia network throughout the body regulates the functional and living status of cells and provides a stable environment for the survival of functional cells. According to fasciology, the process of a life is just that of continuous consumption of the supporting-storing system. The supplementary of functional cells to repair the functions of damaged tissues ensures normal activities of the functional system. Once one of the functional cells could not be updated by the supporting-storing system in metabolism, the body will be disorder. Osteoporosis is the disease that the osteoblasts, supplemented from the undifferentiated stem cells in the supporting-storing system, is insufficient after bone resorption caused by many factors, e.g. menopause, age and glucocorticoid et al. Therefore, when negative balance exists in bone metabolism, how to increase the undifferentiated stem cells in the supporting-storing system to differentiate into osteoblasts may be an effective treatment for osteoporosis.Adipose-derived stem cells (ADSCs), which isolated from adipose tissue, are main undifferentiated cells in the supporting-storing system. Currently, ADSCs have become one of the focuses in stem cell research. They are easy to be cultured, rich in source and have differentiation of multipotent, even low in immunity. They also have the capacity of cross-mesodermal differentiation, such as osteogenic, chondrogenic, adipogenic, and myogenic et al. Furthermore, ADSCs are isolated more easily, have more sources and less pain in patients than BMSCs, 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.Glucocorticoid has been found to induce osteoporosis rats. Long-term and high-dose glucocorticoid could lead to abnormality of calcium and phosphorus metabolism, increase of parathormone, decrease osteoprotegerin. Proliferation and activity of osteoblasts could be inhibited while activity of osteoclast upgraded, which could induce bone resorption increasing and bone formation decreasing, lead to osteoporosis finally.The aim of this experiment is to investigate the effect of bone mineral density, biomechanical properties and histomorphometry in glucocorticoid-induced osteoporosis (GIOP) by systemic transplantation of ADSCs, to explore a new method for prevention and treatment of osteoporosis in clinical. Objective1. To detach and culture the ADSCs from the adipose tissue of rats, and to test their multiple differentiation multipotent and surface markers. To judge that whether the ADSCs exist in the supporting-storing system;2. To judge whether glucocorticoid-induced osteoporosis model succeed by detecting bone mineral density.3. To observe the effect of transplanting the ADSCs on bone mineral metabolism and bone formation of GIOP rats by detecting the levels of serum Ca,P and ALP.4. To observe the effect of transplanting the ADSCs on bone weight of GIOP rats by detecting bone mineral density.5. To observe the effect of transplanting the ADSCs on bone strength and bone stiffness of GIOP rats by three-point bending test.6. To observe the effect of transplanting the ADSCs on microenvironment in bone tissue of GIOP rats by histomorphometry.7. To provide the evidence for "fasciology hypothesis", which consider that mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative diseases and aging diseases.Methods1. To detach the ADSCs from the rats'adipose tissue, and then cultured the cells in vitro. The 6th passage cells were observed by their morphology, differentiatial potent by induced them into adipogenesis and osteogenesis, and their biological markers on the cell membrane, such as CD11b,CD29,CD45,CD49d,CD90 and D106.2.40 female Wistar rats were randomly divided into 4 groups:blank control group(A), model group(B), treatment group(C) and treatment control group (D),10 in each group. Rats in B, C and D groups were injected with prednisolone into the neck subcutaneously at a dose of 8 mg/kg of body weight, three times per week for 12 weeks. The rats in group A were injected with physiological saline at the same volume for 12 weeks. Group A and B were sacrificed and specimens were procured to judge whether model succeed after 12 weeks. 3. Harvest and culture purified ADSCs from Wistar rats. After the GIOP models were succeed, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the 6th passage were harvested and resuspended in the physiological saline at 3×106 cells/mL. Each rat in group C was injected with 3×106 of ADSCs into its tail vein. The rats in groups D were similarly administered with same volume of physiological saline. Sacrificed and drew the materials after 4 weeks.4. After the rats were anesthetized by chloralization, the blood was got from the hearts of rats 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, and stored at-70℃as backup. The levels of serum Ca,P and ALP were measured respectively by azoⅢ,PMA chromatometry and velocity method with automatic biochemistry analyzer. Then, sacrificed the rats by heart bloodletting, took out of the third to fifth lumbar vertebras and two sides of femurs, and remove muscular tissue around. Bone densities at lumbar and right femur were measured by X-ray bone mineral density instrument with small animal measure software. 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).5. The up 1/3 parts of right tibias were got out after sacrificed, then split them across sagittal plane. The undecalcified slices were produced by used the methyl methacrylate, then stained by Giemsa and Von kossa, respectively. Used the light microscope to observe the histology and measure the histomorphometric parameters, included percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and osteoclast number (OC.N).Results1. Observation by light microscope, primary cultured ADSCs have the morphological features of long spindle, and polygon, and can form cell colony. The ADSCs were passaged at ratio 1:3 after 7 days, and then passaged every 3 days. After passaged the cells to the 6th passage, the ADSCs had the single cell morphology, all of them were spindle or polygon. The cells could be induced to differentiate into fat cells and osteoblasts. Flow cytometry demonstrated that CD29 and CD90 were high positive, CD106 low positive, while CD11b,CD45 and CD49d were negative.2. Long-term and high-dose glucocorticoid can successfully establish osteoporosis rats model, which shows progressive decreasing of the levels of serum Ca and ALP, as well as bone mineral density, bone strength and bone stiffness, but the levels of serum P increased obviously. Trabecular bone became thinner, disarranged, and destroyed with a wide intertrabecular distance, even had lacunae on the surface. The quantity, volume and thickness of trabecular bone were all decreased significantly, along with a significant increasing of trabecular separation and osteoclast number.3. Transplanted allograft ADSCs could significantly increase the level of serum Ca and ALP, bone mineral density, bone strength and bone stiffness. The levels of serum P decreased obviously at the same time. Trabecular bone became thicken and harden with a increasing quantity. The quantity, volume and thickness of trabecular bone were all increased significantly but Trabecular separation and osteoclast number decreased.Conclusion1. There were ADSCs, which was one kind of undifferentiated mesenchymal stem cells, in supporting-storing system,2. Long-term and high-dose glucocorticoid could successfully establish osteoporosis rats model.3. Transplanted ADSCs into the osteoporosis rats could effectively improve Ca and P metabolism, increase bone formation, effectively prevent and treat osteoporosis, which provid a new method of preventing and treating GIOP in clinical.4. This experiment provided the evidence for one hypothesis of "fasciology", mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative disease.In summary, transplanting ADSCs to glucocorticoid-induced osteoporosis rats have effects on bone formation, increasing bone density, improving biomechanics property, enhancing bone strength, thereby decrease incidence of bone fracture. The study further tell us that we should pay more attention to increase quantity of stem cells and induce them osteogenesis in research of GIOP, to provid a new research pathway of preventing and treating GIOP in clinical. |
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