Mechanisms Of Hypertrophic Scarring Through TGF-?1 Mediating TGF-?1/Smad And ERK/MAPK Signaling Pathway In Rat Osteomyelitis Model

CHAPTER I Establishment and assessment of rat experimental model of osteomyelitisBACKGROUND:Osteomyelitis is an acute or chronic inflammatory process of the bone and its related structures caused by pathogenic bacteria.It can be classified into hematogenous osteomyelitis,traumatic osteomyelitis and contiguous osteomyelitis.Traumatic osteomyelitis is a complication resulting from trauma or operation.Traumatic osteomyelitis often progresses to chronic osteomyelitis,which is charactered by existence of bacteria,perseverative inflammation and the soft tissue scar surrounding the infected bone.The treatment of traumatic osteomyelitis is very difficult due to its prolonged therapy and recurrence.And traumatic osteomyelitis becomes a significant public health problem.Because of the various pathophysiology of osteomyelitis,it is difficult to evaluate in clinical studies.Therefore,an ideal animal model is necessary to evaluate the pathogenic mechanism,prevention and treatment of osteomyelitis.ObjectiveThe goal of this study was to eatablish an ideal and repeatable osteomyelitis animal model,representing a process of traumatic osteomyelitis clinically.Method30 Wistar rats were randomly divided into control group(group A)and osteomyelitis model group(group B).Animals were given an intraperitoneal anesthesia.The leg of the animals was disinfected,and soft tissue was dissected from the lateral of the femur.In group A,the femur was exposed and then the wound was sutured.In group B,the osteomyelitis model was established.Followed by dissection of sofe tissue.the femur was fractured by vascular forceps.Then the fracture was fixed with Kirschner wire through the bone cavity.The medullary cavity received an intramedullary contamination of 106 CFU of Staphylococcus aureus in 50ul.Then the skin was closed.Status of insicion and radiographs of the femur in both groups were observed at day 7,14 and 28 to evaluate wound healing and changes in femur.Muscle and bone tissues in group A and group B,were harvested at 7,14 and 28 day.Tissue samples were fixed in 4%formaldehyde for hematoxylin-eosin staining to observe changes in muscle and femur tissue.Bacterial culture of bone samples were underwent in the both groups.ResultsAll incision healed well in group A.While in group B,tissue around the incision were swollen at 7 day.There were pus surround the incision at 14 day,and sequestrum was observed in the incision at 28 day.Radiographs of the femur were normal in group A.In group B,X-ray showed minimal periosteal bone response at 7 day.Signs of infected tissue cortical bone response were observed at 14 day.Evident bone destruction and calcification were present at 28 day.The hematoxylin-eosin staining of mussle tissue indicated the muscle fibers were well-arranged in group A.In group B,muscle tissue was necrotic with neutrophil infiltration at 7 day.A high degree of inflammation,an increased number of blood vessels and fibroustissue hyperplasia were observed at 14 day.Disarranged collagen tissue with a vortex-like distribution was present at 28 day.According to the hematoxylin-eosin staining of bone tissue,bone cortex in group A showed intact.In group B,inflammatory cell infiltration was observed in cortical bone at 7 day.Cortex absorbed cavities were formed at 14 day and obvious osteonecrosis present at 28 day.Staphylococcus aureus cultures were negative in group A.The diagnosis of osteomyelitis was confirmed by a positive culture Staphylococcus aureus in group B.ConclusionOur study demonstrated a successful result in establishing osteomyelitis model in rat mocked traumatic osteomyelitis.CHAPTER ? Role of TGF-?1/Smad and ERK/MAPK signaling pathway in scaring formation in rat osteomyelitis modelBACKGROUND:Osteomyelitis is charactered by recurrence years after cure,and it often progresses to affect the surrounding soft tissue forming hypertrophic scar.The scarring surrounding soft tissues result in lack of blood supply and poor antimicrobial treatment to the focus of infection.On the other hand,the scaring can result in deformities and restriction of the mobility of extremities.Although most of osteomyelitis should be managed by anti-inflammatory combined with debridement to establish blood supplied microenvironment,reducing the formation of scaring is very important for the treatment of osteomyelitis.TGF-?1 is an important cytokine in fibrosis disease,and TGF-?1 mediating TGF-?1/Smad and ERK/MAPK signaling pathway is wildly studied in fibrosis disease.However,the precise mechanism of hypertrophic scar formation in osteomyelitis still remains unknown.Investigating the mechanism of scar formation in osteomyelitis is necessary for its prevention and treatment.ObjectiveThe goal of this study was to1.Investigate the time-point of hypertrophic scarring formation in a rat osteomyelitis model.2.Investigate the role of TGF-?1 mediated TGF-?1/Smad singnal pathway and ERK/MAPK singnal pathway for formation of hypertrophic scarring in a rat osteomyelitis model.Method30 Wistar rats were randomly divided into control group(group A)and osteomyelitis model group(group B).Each group was divided into three subgroups(Al-3,B1-3)according to sampling time.The osteomyelitis model was established as described in chapter ?.Muscle tissues were harvested at 7,14 and 28 day for sampling.The expression of TGF-?1 was analyzed by immunohistochemistry.Collagen deposition in muscle was observed by MASSON stain.The activation levels of Smad2/3 singnal pathway,ERX1/2 and collagen ? was assessed by immunoblotting.ResultsThe immunohistochemistry of mussle tissue showed the expression intensity of TGF-?1 in group A1,A2,A3,B1,B2 and B3 were 11.08%,13.86%,13.62%,19.07%.56.35%and 83.12%.The expression intensity of TGF-?1 was significantly difference in group B2 and B3 compared with group A2 and group A3.The MASSON stain showed collagen fraction volume(CFV)in group Al,A2,A3,B1,B2 and B3 were 0.19%,0.11%,0.85%,1.37%,43.72%and 83.59%.CFV%was significantly difference in group B2 and B3 compared with compared with group A2 and group A3.The immunoblotting analysis showed the expression of TGF-?1 in group A1,A2,A3,B1,B2 and B3 were 35.80%,34.86%,33.94%,34.47%,80.52%and 225.95%.The expression of pSmad2 in group Al,A2,A3,B1,B2 and B3 were 8.10%,8.61%,8.93%,8.34%,17.61%and 33.71%.The expression of pSmad3 in group Al,A2,A3,B1,B2 and B3 were 7.45%,6.35%,7.69%,7.39%,15.54%and 51.95%.The expression of pERK1/2 in group Al,A2,A3,B1,B2 and B3 were 15.33%,13.86%,9.51%,16.37%,51.29%and 49.30%.The expression of collagen I in group A1,A2,A3,B1,B2 and B3 were 39.48%,33.51%,31.69%,43.90%,15.54%and 19.55%.The expression TGF-?1,pSmad2,pSmad3,pERK1/2 and collagen I was significantly higher in group B2 and B3 compared with group A2 and group A3.Conclusion(1)The hypertrophic scarring formed at 14 day in the rat osteomyelitis model,and the degree of hypertrophic scarring increased at 28 day.(2)TGF-?1/Smad singnal pathway was activated at 14 and 28 day in the rat osteomyelitis model.(3)ERK/MAPK singnal pathway was activated at 14 and 28 day in the rat osteomyelitis model.(4)The activation of TGF-?1/Smad signaling pathway and ERK/MAPK singnal pathway might contribute to the synthesis of collagen type I which leads to hypertrophic scarring formation in the rat osteomyelitis model.TGF-?1/Smad signaling pathway and ERK/MAPK singnal pathway might as a target to specifically inhibit hypertrophic scarring formation in the rat osteomyelitis.CHAPTER III Role of blocking TGF-?1/Smad and ERK/MAPK signaling pathways in inhibiting scaring formation in rat osteomyelitis modelBACKGROUND:TGF-?1 regulates a variety of cellular processes such as differentiation and proliferation through TGF-?1/Smad signal pathway.TGF-?1 binding to T?RI and T 3 RII receptor complex causes the recruitment and subsequent activation of smad2 and smad3.Activatied smad2/3 combined with smad4 subsequently translocate into the nucleus to regulate gene expression.TGF-?1 also induced smad-independent signaling cascades,such as ERK/MAPK signal pathway to regulate gene expression.In this non-Smad singnal pathway,TGF-?1 activates of Ras results in activated Raf,which activates MEK,followed by ERK activation.Activation of ERK was necessary for TGF-?1 induced fibroblast replication.In addition,there existed cross-talk between ERK and Smad signaling pathway by phosphorylating the linker region of Smads.The phosphorylated linker region,has been shown to either inhibit or enhance smad mediated transcriptional activity depended on different cells.However,the role of blocking ERK and Smad signaling pathways and cross-talk between these two signaling pathways in inhibiting scaring formation in osteomyelitis is still unknown.ObjectiveThe aim of this part was to evaluate1.The role of blocking TGF-?1,which mediating TGF-?1/Smad and ERK/MAPK signaling pathways,in inhibiting scaring formation in rat osteomyelitis model.2.The role blocking ERK1/2,and the cross-talk between ERK1/2 and Smad2/3,in inhibiting scaring formation in rat osteomyelitis model.Method1.20 Wistar rats were randomly divided into Decorin-treated group(group A)and osteomyelitis model group(group B).Each group was divided into two subgroups(A 1-2,B1-2)according to sampling time.The osteomyelitis model was established as described in chapter I.A dose of 50?g Decorin was injected in group A and 0.5ml PBS was injected in group B in the muscle tissue surrounding the fracture on post-wounding days 1,3.and 5.Muscle tissues were harvested at 14 and 28 day for sampling.Collagen deposition in muscle was observed by MASSON stain.The expression of TGF-?1,Smad2/3,ERK1/2 and collagen I was analyzed by by immunoblotting.2.20 Wistar rats were randomly divided into PD98059-treated group(group A)and osteomyelitis model group(group B).Each group was divided into two subgroups(A 1-2,B1-2)according to sampling time.The osteomyelitis model was established as described in chapter I.A dose of 10 mg/kg PD98059 was injected in group A and 0.5ml PBS was injected in group B in the muscle tissue surrounding the fracture on post-wounding days 1,3,and 5.Muscle tissues were harvested at 14 and 28 day for sampling.Collagen deposition in muscle was observed by MASSON stain.The expression of ERK1/2,Smad2/3 and collagen I was analyzed by by immunoblotting.Results1.The MASSON stain showed collagen fraction volume(CFV)in group Al,A2,B1 and B2 were 15.55%,39.27%,48.57%and 68.22%.CFV%was significantly difference in group A1 and A2 compared with group B1 and group B2.The immunoblotting analysis showed the expression of TGF-?1 in group A1,A2,B1 and B2 were 1.16%,3.93%,8.57%and 15.22%.The expression of pSmad2 in group Al,A2,B1 and B2 were 5.26%,4.01%,8.60%and 11.68%.The expression of pSmad3 in group A1,A2,B1 and B2 were 1.04%,2.44%,9.02%and 13.65%.The expression of pERK1/2 in group A1,A2,B1 and B2 were 17.94%.14.75%,48.99%and 53.75%.The expression of collagen I in group A1,A2,B1 and B2 were 5.94%,4.75%,15.99%and 26.75%.The expression TGF-?1,pSmad2,pSmad3,pERK1/2 and collagen I was significantly higher in group A1 and A2 compared with group B1 and group B2.2.The MASSON stain showed collagen fraction volume(CFV%)in group A1,A2,B1 and B2 were 21.13%.39.79%,41.34%and 63.61%..CFV%was significantly difference in group A1 and A2 compared with group B1 and group B2.The immunoblotting analysis showed the expression of pERK1/2 in group Al,A2,B1 and B2 were 9.18%,12.95%,39.99%and 46.54%.The expression of pSmad2 in group A1,A2,B1 and B2 were 3.24%,9.20%,15.31%and 21.44%.The expression of pSmad3 in group A1,A2,B1 and B2 were 0.96%,2.14%,3.02%and 12.35%.The expression of collagen I in group A1,A2,B1 and B2 were 4.40%,5.15%,11.69%and 18.65%.The expression pERK1/2,pSmad2,pSmad3 and collagen I was significantly higher in group A1 and A2 compared with group B1 and group B2.Conclusion1.Decorin reduces scar formation in rat osteomyelitis model through blocking of the TGF-?1 mediating TGF-?1/Smad signaling pathway and ERK/MAPK signaling pathway.2.Blocking ERK1/2 signaling pathway and cross-talk between ERK1/2 and Smad2/3 signaling can reduce scar formation in rat osteomyelitis model.