Therapeutic Effects Of Bone Marrow Mesenchymal Stem Cells Trasplantation On The Experimental Avascular Necrosis Of Femoral Head In Rabbits

The avascular or ischemic necrosis of femoral head (ANFH) is a devastating disease usually leading to hip joint destruction in the third through fifth decades of life. Epidemiologic studies have revealed associations between osteonecrosis and a multitude of factors. For traumatic cases such as a hip dislocation, in which the arteries to the femoral head have been injured (lateral epiphyseal artery), the pathogenetic mechanism is clear. However, in other situations (osteonecrosis associated with alcohol or steroids), the exact mechanism is uncertain. Recently, cytoreduction of bone marrow mesenchamal stem cells (BMSCs) have been identified as pathogenetic in many cases. In the current series, grafting was done with autologous bone marrow obtained from the iliac crest of patients operated on for osteonecrosis of the hip. Patients who had the greater number of progenitor cells transplanted in their hips had better outcomes. Adult mesenchymal stem cells are a population of multipotent cells found primarily in the bone marrow. They have long been known to be capable of osteogenic, adipogenic and chondrogenic, myogenic, neural differentiation and are currently the subject of a number of trials to assess their potential use in the clinic. We investigate here the cellular characteristics and differentiating potential of BMSCs, and the promising applications for future in treatment of ANFH.1. BMSCs induced differentiating into osteoplasts and their morphological characteristicsObjective: Bone marrow mesenchymal stem cells (BMSCs) are a population of multipotent cells which are the most popular seeds in bone tissue engineering. This experiment is to investigate their culture, proliferation, osteoblastic differentiation in vitro and to observe their morphological characteristics.Methods: The BMSCs were isolated, cultured and proliferated by cultivating whole bone marrow. BMSCs were induced differetiating into osteoplast by dexamethasone, ascorbate and ?-glycerol phosphate. BMSCs and induced MSCs were identified by alkaline phosphatase (ALP), calcificatalized node staining and immunohistochemistry. The ultrastructural features of them were observed by transmission and scanning electron microscope.Results: The results showed that BMSCs were positive for ALP staining and negtive for bone morphgenetic protein (BMP) by immunohistochemical staining and calcificatalized node staining, but induced BMSCs were positive for ALP staining, BMP immunohistochemistry and calcificatalized node stainings. BMSCs showed long fusiform shape mostly and a few of them showed astero-form by light microscope and scanning electron microscope. After induced, cell volum increased, the abundant organellas in BMSCs were observed by transmission electron microscope. After induced, mitochondrion increased and a lot of matrix vesicles,layers-like structure and vacuolus-like structure appeared in cytoplasm.Conclusion: Whole bone marrow-cultivating method is a best way for BMSCs amplification. BMSCs can be induced to osteoplast and either of them possess different morphological characteristics.2. Therapeutic effects of BMSCs trasplantation on the experimental ANFH in rabbitsObjective: Epidemiologic studies have revealed associations between osteonecrosis and a multitude of factors. Recently, cytoreduction of bone marrow mesenchamal stem cells (BMSCs) have been identified as pathogenetic in many cases. This experiment is to investigate their therapeutic effects and repair mechanism by treating ANFH in rabbits with BMSCs transplantation. Methods: The 48 Newzland rabbits were divided into 4 groups randomly, as liquid nitrogen-freezing group (A), decompression group (B), BMSCs autografting group (C) and allografting group (D). The animal model was made using of freezing method with liquid nitrogen. The specimens from every group were observed with radioscopy and histology respectively at 2, 4, 6, 8 weeks after operation.Results: Freezing method with liquid nitrogen is a reliable way to made animal model in rabbit ANFH. We observed necrosis and repair phenomenon in all of the specimens. (1)Radioscopic observation: After freezing by liquid nitrogen, the density of femoral head in group A increased gradually at 2, 4, 6, 8 weeks. In the meantime, trabecula became disorder and appeared cystic degeneration. These phenomenon were more magnification at 8 weeks.Several speciments became flat and presented osteoarthritis-like change.The density around the drilled hole area (DHA) in group B, C, D increased at 2 weeks. At 8 weeks, the surrounding DHA presented low density and formed sclerotic line in group B, while more trabecula appeared in the DHA in group C, D. (2)Histological observation: The liquid nitrogen-freezing model in group A appeared the pathologic proceeding of ANFH. The repair effect was unsatisfaction in gronp B at 8 weeks. In group C, D, there were a large amount of BMSCs in the DHA at 2 weeks. And then DHA were filled of trabeculae at 4weeks. At 8 weeks, the matured trabeculae and marrow appeared in the intertrabecular space. There is no statistical difference between group C and D, by analyzing trabeculae area image.Conclusion: Freezing method with liquid nitrogen is a reliable way to made animal model in rabbit ANFH. Because of cytoreduction of BMSCs, decompression is limited to treat ANFH. There have resemble repair effect by treating ANFH in rabbits with BMSCs autografting and allografting, although some lymphocytes and plasmacytes appeared in the early stage. It is feasible to establish BMSCs bank and present clinical apply.3. Therapeutic effects of induced BMSCs trasplantation on the experimental ANFH in rabbitsObjective: There is lack of evidence to convince that it is better to treat ANFH with induced BMSCs than to treat ANFH with BMSCs. This experiment is to compare the therapeutic effects of the two methods and to supply reasonable way for BMSCs clinical transplantation.Methods: The 24 Newzland rabbits were divided into 2 groups randomly, as BMSCs autografting group (A), induced BMSCs autografting group (B), The animal model was made using freezing method with liquid nitrogen. The specimens from every group were observed with radioscopy, histology and immunohistochemical staining respectively at 2, 4, 6, 8 weeks after operation. Induced BMSCs were examined by BMP immunohistochemical staining respectively at 1, 2, 3 weeks after inducing.Results: The induced BMSCs in partial area were positive for BMP immunohistochemical staining after 2 weeks of inducing. Implanted BMSCs in all area were positive for BMP immunohistochemical staining in specimens of the two group at 2 weeks. (1)Radioscopic observation is similar in the two groups. The bone density in the drilled hole area (DHA) increased at 4 weeks. Bone trabecula appeared in the DHA at 6weeks. At 8 weeks, more normal bone trabecula formed in the DHA. (2)Histological observation is also similar in the two groups. There were a large amount of BMSCs in the DHA at 2 weeks. And then DHA were filled of bone trabeculae at 4 weeks. At 8 weeks, the matured bone trabeculae and marrow appeared in the intertrabecular space. There is no statistical difference between group C and D, by analyzing trabeculae area image.Conclusion: There have resemble repair effect by treating ANFH in rabbits with induced BMSCs autografting and BMSCs autografting. As to reduce BMSCs differentiating into osteoblasts, inducte effect in internal enviroment of femoral head is more effective than induction in vitro.4. Therapeutic effects of different concentrational BMSCs trasplantation on ANFH in rabbits Objective: To treat bone defect with BMSCs trasplantation need to consider two of the points. The first point is the cell number to repair the defect; the second is the implanted cell concentration. This experiment is to investigate a optimal cell concentration by comparing the therapeutic effects of the different concentrational BMSCs trasplantation on the experimental ANFH in rabbits.Methods: The 40 Newzland rabbits were divided into 5 groups randomly, as 1×104/cm2 BMSCs autografting group (A), 1×105/cm2 BMSCs autografting group (B), 1×106/cm2 BMSCs autografting group (C), 1×107/cm2 BMSCs autografting group (D), 1×108/cm2 BMSCs autografting group (E). The animal model was made using freezing method with liquid nitrogen. The specimens from every group were observed with radioscopy, histology and image analysis respectively at 2, 4, 6, 8 weeks after operation.Results: (1)Radioscopic observation: The bone density in part area of drilled hole area (DHA) increased at 4 weeks in group A. Part area of DHA present clearly bone trabeculae, part area of DHA present low density at 8 weeks. Radioscopic observation is similar in B, C, D, E groups. The bone density in DHA increased at 4 weeks. Bone trabecula appeared in the DHA at 6weeks. At 8 weeks, more normal trabecula formed in the DHA. (2)Histological observation: A sandwich-like structure was found in DHA at 2 weeks, in which new-formed trabeculae were located lateral layer, osteoblasts were located in intermedial layer, and inflammatory cells were located in interior layer. Part area of DHA formed new-formed bone trabeculae at 4 weeks. DHA in group A presented incomplete repair at 8 weeks. Histological observation is also similar in the B, C, D, E groups. There were a large amount of BMSCs in the DHA at 2 weeks. And then DHA were filled of bone trabeculae at 4 weeks. At 8 weeks, the matured bone trabeculae and marrow appeared in the intertrabecular space. There is no statistical difference among group B, C, D, E, by analyzing bone trabeculae area image.Conclusion: There have resemble repair effect by treating ANFH in rabbits with 1×105-108/cm2 BMSCs autografting. Treating ANFH in rabbits with 1×104/cm2 BMSCs autografting may lead to incomplete repair.5. Therapeutic effects of spongy bone autografting combined with BMSCs trasplantation on ANFH in rabbitsObjective: The spongy bone autografting is a common procedure to treat clinically ANFH, and unsatisfactory results maybe related to cytoreduction of BMSCs. This experiment is to investigate their therapeutic effects of spongy bone grafting combined BMSCs transplantation on the experimental ANFH in rabbits.Methods: The 24 Newzland rabbits were divided into 2 groups randomly, as spongy bone grafting combined BMSCs transplantation group (A), alone spongy bone grafting group (B). The specimens from every group were observed with radioscopy, histology, scanning electron microscope and biomechanics respectively at 2, 4, 6, 8 weeks after operation. P and Ca content were subjected to electromagnetic spectrum analysis at 8 weeks.Results: (1) Radioscopic observation: The bone density in the DHA in group A increased at 2 weeks. With time going, bone trabeculae in the DHA and good conjunction at junctional zone appeared. At 8 weeks, there were normal tension trabeculae and pressure trabeculae formed in the DHA. while some specimens in group B which were unsatisfaction in result presented bone resorption and collapse. (2) Histological observation: In group A, spongy bone grafting combined BMSCs transplantation were revascularized early, a large amount of new-formed bone tissue encircled grafted spongy bone and seamed them together at 2 weeks. Remodeling was active at 6 weeks, there were remodeling bone trabeculae and matured bone marrow formed at 8 weeks. At 2 weeks, there were limited revascularization and sparse osteoblasts in group B in some specimens which were unsatisfaction in results. Severe resorption appeared at 4-6 weeks. Dead bone trabeculae still existed in the DHA at 8 weeks. (3) Scanning electron microscope observation: At 8 weeks, the bone trabeculae in satisfactory specimens were different from normal biomechanics structure, which arranged closely and had bone narrow within intertrabecular space. Collagen fibers were irregular. There was no statistical difference between group A and B, by analyzing P and Ca content at 8 weeks. Specimens of the two groups which were satisfaction in results presented increasing biomechanics from 2 weeks to 8 weeks. There was statistical difference between group A and B, by analyzing their biomechanics.Conclusion: Because of limited revascularization and limited recruiting BMSCs, treating ANFH with alone spongy bone autografting could lead to failure. The treatment of ANFH by spongy bone autografting combined with BMSCs can raise achievement rate.