| Background:Osteoporosis is a metabolic disease that characteristic by osteopenia and bone microstructural destroy, which could cause fragility increasing and able to fractured. Due to the increasing osteoporosis patients, research that 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 is constituted 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 is 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.The repair of bone defects is one of the common difficult problem in clinical department of orthopedics, the bone transplantation in the treatment of bone defect has obtained the certain effect, but this method has limitation. Autogenous bone transplantation has been considered the gold standard for the repair of bone defect, but the bone graft is limited, can cause donor deformity, bone operation also additional trauma and anguish; allogeneic bone transplantation can reduce the times of operation, convenient, but because of its antigenicity, can cause immune rejection at the same time, there is the potential spread of disease risk, and allograft bone complications are more, such as infection, fracture and nonunion. Therefore, the demand for the new substitute transplantation increasing. Bone cement has favored. With the increasing of aging population, osteoporosis has become a major disease that threatens the health of middle-aged and elderly people, osteoporosis is very easy to cause fracture, the fractures are often accompanied with cancellous bone compression and bone defects, in addition with the increase of osteoporosis absolute population, due to various causes of patients with bone defect with osteoporosis caused by more and more. Under circumstance of osteoporosis, enhance the activity of osteoclasts, bone resorption and bone formation, decrease new bone formation ability, bone defect healing was delayed, the new callus biomechanical strength reduction, bone healing after the poor fracture easily, therefore, compared with the conventional bone defect, bone defect costly and traditional methods poor efficacy. Auxiliary treatment of bone cement is gradually applied in osteoporotic fracture bone defect, it can enhance the fixing screw pull-out strength, prevent osteoporosis fracture internal fixation loosening, filling the bone defect, make better fracture reduction and healing, reduce the occurrence of sequelae of fracture, in recent years, calcium phosphate cement was used in vertebroplasty surgery for the treatment of osteoporotic vertebral compression fractures, and achieved good effect. Treatment of sustained-release bone length factor has gradually been applied in osteoporotic bone defects, which not only significantly accelerated the healing of bone defects, but also can improve the local osteoporosis in a certain extent, enhancing local bone biomechanics, to reduce the incidence of fracture.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 adipose-deived stem cells (ADSCs) on bone mineral metabolism and bone formation of GIOP rats by detecting the levels of serum Caã€P and ALP.4. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone weight of GIOP rats by detecting bone mineral density.5. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone strength and bone stiffness of GIOP rats by three-point bending test.6. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on microenvironment in bone tissue of GIOP rats by histomorphometry.7. To provide the evidence for the theory of "fasciology hypothesis", which consider that mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative diseases and aging diseases.8. Different bone filling materials were observed the morphology of bone in ovariectomized rats were filled, and provides some basis for the clinical application of new materials.Methods1. To detach the ADSCs from the rats’adipose tissue, and then cultured the cells in vitro. The6th 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 CD11bã€CD29ã€CD45ã€CD49dã€CD90and CD106. Then to judge whether the cultured cells were ADSCs. 2.40female Wistar rats were randomly divided into4groups (A, B, C and D),10in each group. Rats in B, C and D groups were injected with prednisolone into the neck subcutaneously at a dose of8mg/kg of body weight, three times per week for12weeks. The rats in group A were injected with physiological saline at the same volume for12weeks. Group A and B were sacrificed and specimens were procured to judge whether model succeed after12weeks.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 the6th passage were harvested and resuspended in the physiological saline at3×106cells/mL. Each rat in group C was injected with3×106of ADSCs into its tail vein. The rats in groups D were similarly administered with same volume of physiological saline. Sacrificed and drew the materials after4weeks.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 for8h. Then centrifugated15min by the speed of3000r/min to prepare serum, and stored at-70℃as backup. The levels of serum Caã€P and ALP were measured respectively by azo â…¢ PMA chromatometry and velocity method with automatic biochemistry analyzer. Then, Sacrificed the rats by heart bloodletting, took out of Third to Fifth lumbar vertebra and two sides of femur, 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 of18mm and a displacement rate of0.05mm/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 up1/3parts 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).6. Osteoporosis model operation:normal female rats after successful anesthesia, abdominal shaving, iodophor disinfection. A ventral midline incision into the abdominal cavity, layer by layer. Find one ovary ligation and resection. Act with the removal of the other side. Suture of peritoneum and abdominal muscles, and skin suture. A week stitches.3months after the osteoporosis model.7. The model is established successfully, make drilling wood vertical bone surface5mm deep a bone tunnel. In the tunnel inner implantation material and lightly compacted to the bone surface, then with bone wax sealing material implant operation:Zhou anteromedial shaving anesthesia after the success of double knee. Iodophor disinfection. From the knee anterior tibial spine incision along the length of about2cm. Step by step up into the periosteum, subperiosteal dissection revealed proximal tibia front inner side, with a diameter of3mm drill bit. Hemostatic, rinse, selected four kinds of biological materials of β-TCP,β-phosphate calcium:three comparison of CS calcium silicate:after degradation, the release of the beneficial element silicon,Sr-CS strontium-doped calcium silicate:after degradation, release of the beneficial elements of silicon, strontium,45S5biochemical ceramic glass filled bone defect. Suture the wound (exposure). Animals were killed at9weeks after.8. Application of HE staining, Giemsa, T-blue, Van Gieson, safranin O fast green staining. To observe the changes of bone slice morphology with different filling materials.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 ratio1:3after7days, and then passaged every3days. Results In passage6th, the ADSCs have the homogenicity with a spindle or polygon shaped. The cells can be induced to differentiate into fat cells and osteoblasts. Flow cytometry demonstrated that CD29and CD90were high positive, CD106low positive, while CD11bã€CD45and CD49d were negative.2. Long-term and high-dose glucocorticoid can successfully establish osteoporosis rats model, which shows progressive decreasing of the level of serum Ca and ALP, as well as bone mineral density, bone strength and bone stiffness, but the levels of serum P increased obviously. Bone trabecular became thinner and disarranged, and destroyed with a wide intertrabecular distance, even had lacunae on the surface. The quantity, volume and thickness of bone trabecular 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. Bone trabecular became thicken and harden with a increasing quantity. The quantity, volume and thickness of bone trabecular were all increased significantly but Trabecular separation and osteoclast number decreased.Conclusion1. There are ADSCs, one kind of undifferentiated mesenchymal stem cells, in supporting-storing system,2. Rats injected with long-term and high-dose glucocorticoid can successfully establish osteoporosis model.3. Through transplanting ADSCs into the osteoporosis rats can 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 theory of "fasciology hypothesis", mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative disease.5. Different bone filling materials for bone trauma, surface effects. New materials of CS and Sr can promote bone formation.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. |
