Study Of Heterotopic Ossification Related Cytokines In Mouse Muscle Traumatic Injury Model

PURPOSE:To investigate the early changes of cytokines in the standardized traumatic model.Identify the surface markers of cells isolated from the traumatic muscle tissue and thenormal muscle tissue, and compare their differentiation ability.MATERIALS AND METHODS:Untreated CD-1murine carcasses obtained as waste from other studies wereobtained shortly after euthanasia. The left leg was exposed to a pressure waveproduced2.5cm from the leg following rupture of a1250psi blast disc under1500psiof pressure. The right leg served as control (n=4). The injured muscle was separatedfrom the leg immediately after the blast and all particulate debris, fibrous tissue andfat were removed. The muscle-tissue was minced (100-150mm3) and cultured inbasal medium for3days. Live/Dead assays (Invitrogen) were used to observe thecell viability. Twenty-five cell and inflammatory factors were tested by Q-PCR toassess the changes in cytokine profile within muscle. Immunohistochemistry was usedto confirm these changes. FACS was employed to characterize surface epitopes ofputative MPCs isolated from normal and blast-traumatized muscle. Compare with thepotential of the differentiation about the traumatic and normal MPCs.RESULTS:Both control and blast-traumatized murine muscle tissue cultured for up to3days, retained high cell viability, suggesting that this tissue may possibly be used tostudy early cell and tissue changes that induce HO. Q-PCR revealed increases in,BMP4, BMP6, BMP7, TGFβ-1, IL-6, IL-15and NODAL cytokine inblast-traumatized muscle tissue compared with normal muscle tissue. Immunohistochemistry confirmed these Q-PCR results revealing that the traumaticmuscle tissue can produce more cell factors associate with bone formation thannon-traumatic muscle. Putative MPCs from blast-traumatized muscle tissue that thesurface epitopes CD34（±）、CD45（-）、CD105（+）、CD90（+） are similaras the MPCs isolated from human muscle. To compare of the differentiation of theputative MPCs from the traumatic and normal muscle, both of the MPCs have thepotential of osteogenesis and adipogenesis.CONCLUSION:The study demonstrate that blast-traumatized murine muscle tissue producesincreased osteogenic cytokines including BMP4、BMP6、BMP7、TGF-β、IL-6、IL-15and NODAL than normal tissue after3days in organ culture similar to humanblast-traumatized tissue. BMPs and TGFβ-1are important osteogenic factors in boneformation that may induce HO. The MPCs isolated from the muscle tissue have thepotential of osteogenesis and adipogenesis. So there is more uniform in the model ofblast-traumatized murine muscle. It is the foundation to research the formationmechanism of HO.