| PART ONE An experimental study of the expression of osteolytic cytokines stimulated by micrometer-diameter wear particlesObjective: To investigate the biological reaction of micrometer-diameter wear particles ( Ti-6Al-4V and UHMWPE ) in vivo in a rat subcutaneous air pouch model of aseptic loosening, and evaluate the influence of the expression of osteolytic cytokines induced by the two wear particles. Methods: Filtration air was injected subcutaneously into rats'back 6 times ( 3ml qod ). Air pouch tissue treated with wear debris was obtained from rats killed after day 14. Tissue was also taken from physiological saline treated pouches ( control group ) at day 14. Pouch tissues were weighted, wax embedded and stained with hematoxylin and eosin, observed under microscope. AKP of serium, IL-6 and TNF-a expression with immunohistochemical method, and mRNA expression of EMMPRIN with real time PCR method were measured. Results: Air pouch tissues situated in subcutaneous tissues, with distinct border, horsebean-size, small proliferous blood vessels in the surface, and fibroid tissues in some fields, similar to limiting membrane of periprothesis tissue in the cases of aseptic loosening. As to pouch tissue weight, there was a significant increase in group B than in group C (p<0.05). Microscopy illustrated a large amount of phagocytes in group A and B, compared with no tissular and cellular reaction in group C. Evaluating AKP of serium, there was a significant increase in group A,B than in group C (p<0.05). With immunohistochemical method, there was a significant increase with IL-6 expression in group A,B than in group C. With real time PCR method, there was a significant increase with mRNA expression of EMMPRIN in group A,B than in group C. Conclusions: The rat air pouch model was an optical animal model studying aseptic loosening , which reflected the tissular and biological properties of aseptic loosening.The two wear particles both can cause tissular reactions, modulate the production and expression of osteolytic cytokines, induce osteolytic reactions, lead to aseptic loosening.PART TWO An experimental study of the biological reactions induced by rhBMP-2 slow release formulation in the rat air pouch tissueObjective: To investigate the subcutaneous biological reaction of rhBMP-2 slow release formulation in the rat air pouch model, analysis the possible mechanism of new bone ingrowth and the inhibition of osteolysis indueced by rhBMP-2. Methods: Filtration air was injected subcutaneously into rats'back 6 times(3ml qod).Air pouch tissue treated with rhBMP-2 slow release formulation was obtained from rats killed after day 7,day 14. Tissue was also taken from physiological saline treated pouches (control group) at day 7,day 14. Pouch tissues were weighted,wax embedded and stained with hematoxylin and eosin,observed under microscope. AKP of serium,IL-6 and TNF-a expression with immunohistochemical method, mRNA expression of EMMPRIN with real time PCR method and protein expression of EMMPRIN with westrern blotting method were measured. Results: Air pouch tissues situated in subcutaneous tissues, with distinct border, horsebean-size, small proliferous blood vessels in the surface, and fibroid tissues in some fields, similar to limiting membrane of periprothesis tissue in the cases of aseptic loosening. Air pouch tissues were incrassate in group A, it contained a small flexible nodule, which indicated the new bone ingrowth. As to pouch tissue weight,there was a significant difference between group A(day 14) and group A(day 7),group B(p<0.05). Microscopy illustrated no tissular and cellular reaction in both group A and group B.Evaluating of AKP of serium, there was a significant difference between group A(day 7) and group A(day 14),group B(p<0.05). With immunohistochemical method, there was a significant decrease with IL-6 expression in group A than in group B. With real time PCR method,there was a significant decrease with mRNA expression of EMMPRIN in group A than in group B,and in group A(day 14) than in group A(day 7). With western blotting method,there was a significant decrease with protein expression of EMMPRIN in group A than in group B,and in group A(day 14) than in group A(day 7). Conclusions: In one hand, rhBMP-2 slow release formulation stimulated the chemotaxis of osteoblasts/osteoblastlike cells, modulated expression of integrin, increased the speed of cell proliferation, so as to integrate osteoblasts into extracellular matrix, lead to new bone ingrowth;In the other hand, rhBMP-2 slow release formulation inhibited the production and activities of osteolytic cytokines, so as to inhibit osteolytic reaction.It afforded the possibility of using rhBMP-2 in the early prevetion of aseptic loosening.PART THREE The experimental study of the feasibility of using rhBMP-2 slow release formulation to prevent prothetic aseptic looseningObjective: To investigate the subcutaneous biological reaction interfered with rhBMP-2 slow release formulation in vivo in the rat air pouch model of aseptic loosening, analysis the biological activities of using rhBMP-2 to improve new bone ingrowth and inhibit the production and activities of osteolytic cytokines, so as to inhibit osteolytic reaction, evaluate the feasibility of early using rhBMP-2 in prevetion of aseptic loosening. Methods: Filtration air was injected subcutaneously into rats'back 6 times(3ml qod).Air pouch tissue treated with wear debris and interfered with rhBMP-2 slow release formulation was obtained from rats killed after day 7,day 14. Tissue was also taken from wear debris and physiological saline treated pouches (control group) at day 7,day 14. Pouch tissues were weighted,wax embedded and stained with hematoxylin and eosin,observed under microscope. AKP of serium,IL-6 and TNF-a expression with immunohistochemical method, mRNA expression of EMMPRIN with real time PCR method and protein expression of EMMPRIN with western blotting method were measured. Results: Air pouch tissues situated in subcutaneous tissues, with distinct border, horsebean-size. Air pouch tissues were incrassate in group A and group C, it contained a small flexible nodule, which indicated the new bone ingrowth.As to pouch tissue weight,there was a significant decrease in day 14 than in day 7 in each group(p<0.05). Microscopy illustrated a large amount of phagocytes in group B and D, compared with no tissular and cellular reaction in group A and group C.Evaluating AKP of serium, there was a significant difference in group A than in group B,in group C than in group D(p<0.05) in different stage of time. With immunohistochemical method, there was a significant decrease with IL-6 expression in group A than in group B,in group C than in group D. With real time PCR method,there was a significant decrease with mRNA expression of EMMPRIN in group A than in group B, in group C than in group D,and decrease in day 14 than in day 7. With western blotting method,there was a significant decrease with protein expression of EMMPRIN in group A than in group B, in group C than in group D,and decrease in day 14 than in day 7.Conclusions: RhBMP-2 slow release formulation improved new bone ingrowth, inhibited the expression of osteolytic cytokines mediated by wear particles. It indicated that early using rhBMP-2 to prevent prothetic aseptic loosening was feasible.
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