| Part Ⅰ Induction of Osteoarthritis in the mice by surgical tear of anterior cruciate ligamentObjective:Build a model of osteoarthritis(OA)in the mice by surgically transecting anterior cruciate ligament(ACLT).Materials and Methods:The anterior cruciate ligament of the knee of the 8-week,male C57BL/6J mice was transected.A paraffin section of the affected knee joint was made 8 weeks after the surgery.The sections were stained with Alcian blue/hematoxylin&orange G(AB/H&OG)and studied microscopically by histology.The severity of gross cartilage and subchondral bone damage was assessed with OARSI scoring system.Results:Surgical transection of anterior cruciate ligament resulted in severe damage of the cartilage both in depth and area as measured histologically.OARSI score of the ACLT group was significantly higher than the control group.Conclusion:These results demonstrate that ACLT is effective in induction of OA model in the mice.The pathological changes as measured histologically are similar to that of human knee OA in terminal stage.Part Ⅱ Histological analysis of tibial cartilage and subchondral bone of OA in the mice treated with ICAObjective:OA in the mice was treated with early-or late-stage administration of ICA.Histological analysis of the knee joint was made to investigate the protective effect of ICA on tibial cartilage and subchondral bone of OA in the mice.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.ICA was given by gavage(10mg/kg/day)upon surgery and 4 weeks after ACLT,respectively.A paraffin section of the knee joint was made 8 weeks after the surgery.The sections were stained with Alcian blue/hematoxylin&orange G(AB/H&OG)and studied microscopically by histology.The area of tibial cartilage was calculated with software Image J.The depths of tibial hyaline cartilage and calcified cartilage were observed microscopically.The average depth of tibial subchondral bone plate(which was marked out by green line)was measured with bone histomorphometric software Bioquant.Results:1.A severe tibial cartilage loss of late-stage OA in the mice was noted as compared to the sham operation control group.The extent of tibial cartilage loss in the early-stage ICA administration group was much better than in the non-administered control group.The extent of tibial cartilage loss in the late-stage ICA administration group was close to the non-administered control group(no significance,NS).2.The OARSI score of late-stage OA in the mice was significantly higher than the sham operation group(P<0.01).The OARSI score of the early-stage ICA administration group is significantly lower than the non-administered control group(P<0.05).The OARSI score of the late-stage ICA administration group was close to the non-administered control group.(P>0.05).3.The remaining area of tibial cartilage of late-stage OA in the mice was much smaller than the sham operation group(P<0.05).The proportion of remaining tibial cartilage area in the early-stage ICA administration group to that in the shamgroup is much lower than the non-administered control(P<0.05).The proportion of remaining tibial cartilage area in the late-stage ICA administration group to that in the sham operation group is close to the non-administered control(NS).4.Both the hyaline and calcified cartilage were severely damaged,and subchondral bone exposed in late-stage OA in the mice.The tibial hyaline and calcified cartilage layer were still clearly identified in the early-stage ICA administration group 8 weeks after the ACLT surgery.The tibial hyaline cartilage layer in the late-stage ICA administration group vanished 8 weeks after the ACLT surgery.Only a little calcified cartilage remained in the late-stage ICA administration group.5.The average subchondral bone plate thickness of late-stage OA in the mice wassignificantly higher than that of the sham operation group.The average subchondral bone plate thickness of the early-stage ICA administration group was significantly thinner than the non-administered control group.The average subchondral bone plate thickness of the late-stage ICA administration group was close to the non-administered control(NS).Conclusion:In a mouse model of OA,(1)The tibial cartilage is severely damaged,the subchondral bone exposed and the subchondral bone plate thickness increased in late-stage OA.(2)ICA exerts protective effect on both hyaline and calcified cartilage.ICA can prevent the subchondral bone plate from thickening effectively.(3)The therapeutic effect of ICA is associated with drug administration time.Early-stage administration yields better effect than late-stage administration.Part Ⅲ The Level of Serum Bone Turnover Markers of OA in the Mice Treated with ICAObjective:OA in the mice was treated with early-or late-stage administration of ICA.The effects ofICA on serum bone turnover markers were explored.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.ICA was given by gavage(10mg/kg/day)upon surgery and 4 weeks after ACLT,respectively.The serum of the mouse was prepared from the blood collected 8-weeks after the surgery.The generation of serum C-telopeptide of type Ⅰ collagen(CTX),Osteocalcin(OC),IL-6,TNF-α and IL-1β were measured by ELISA(enzyme linked immunosorbent assay,ELISA).Results:(1)The level of serum CTX of late-stage OA in the mice was significantly lower than that of the sham operation group(P<0.05).The level of serum CTX both in ICA early-and late-stage administation group was significantly increased(P<0.05).(2)The level of serum OC of late-stage OA in the mice was significantly lower than that of the sham operation group(P<0.05).The level of serum OC in ICA early-stage administration group was significantly increased(P<0.05).The level of serum OC in ICA late-stage administration group was close to that in the non-administered control group(NS).(3)The level of serum IL-6 of late-stage OA in the mice was significantly higher than that of the sham operation group(P<0.05).The level of serum IL-6 in ICA early-stage administration group was significantly reduced(P<0.05).The level of serum IL-6 in ICA late-stage administration group was close to that in the non-administered control group(NS).(4)The level of serum TNF-α of late-stage OA in the mice was significantly higher than that of the sham operation group(P<0.05).The level of serum TNF-α was close to that in the control both in ICA early-and late-stage administration group(NS).(5)The level of serum IL-1β of late-stage OA in the mice was significantly higher than that of the sham operation group(P<0.05).The level of serum IL-1β was close to that in the control both in ICA early-and late-stage administration group(NS).Conclusion:In a mouse model of OA,(1)Bone remodeling and bone turnover in subchondral bone decrease in late-stage OA.The level of serum CTX and OC decrease in late-stage OA,too.(2)The levels of serum IL-6,TNF-α and IL-1β increase in late-stage OA.(3)Bone turnover in subchondral bone increases after ICA treatment.Early treatment yields better therapeutic effect than late treatment.(4)The level of serum IL-6 may be associated with the severity of cartilage damage and the level of serum TNF-α and IL-1β may have no relevance with the severity of cartilage damage.Part Ⅳ Expression of Cartilage-related Marker Genes of OA in the Mice Treated with ICAObjective:OA in the mice was treated with early-or late-stage administration of ICA.The effects of ICA on the expression of cartilage-related marker genes were explored.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.ICA was given by gavage(10mg/kg/day)upon surgery and 4 weeks after ACLT,respectively.The proximal tibial cartilage was collected and total RNA extracted.The expression of mRNA of Mmp family(including 1,2,3,8,13)and Adamts5,IL-1β,TNF-αwas observed by RT-PCR.Results:(1)The expression of Mmp 1,3,8,13 significantly increased in late-stage OA as compared to the sham operation group(P<0.05).The expression of Mmp1,3,8,13 significantly decreased in ICA early-stage administration group as compared to non-administrated control group(P<0.05).(2)The expression of mRNA of Adamts5,IL-1β and TNF-α significantly increased in late-stage OA as compared to the sham operation group(P<0.05).The expression of mRNA of Adamts5,IL-1β and TNF-α significantly decreased in ICA early-stage administration group as compared to non-administrated control group(P<0.05).(3)As compared to non-administrated control group,the expression of Mmpl,3,8,13 was not significant different in ICA late-stage administration group(NS);the expression of Mmp 13 significantly decreased(P<0.05).(4)The expression of mRNA of Adamts5,IL-1β and TNF-α was not significant different in ICA late-stage administration group as compared to non-administrated control group(NS).Conclusion:In a mouse model of OA,(1)The expression of mRNA of Mmp family(including 1,3,8,13)and Adamts5,IL-1β,TNF-α increases in late-stage OA.(2)The expression of mRNA of Mmp family(including 1,2,3,8,13)and Adamts5,IL-1β,TNF-α decreases after ICA treatment.(3)The therapeutic effect of ICA is associated with drug administration time.Early-stage administration yields a better result than late-stage administration.Part Ⅴ Effect on Bone Marrow Stromal Cells in tibial subchondral bone of OA in the miceObjective:OA in the mice was treated with early administration of ICA.Bone marrow stromal cells(BMSCs)in tibial subchondral bone of the mice were isolated and cultured in vitro.The osteogenetic differential potential,capacity of mineralization and collagen synthesis of BMSCs in tibial subchondral bone of the mice were measured after ICA treatment.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.ICA was given by gavage(10mg/kg/day)upon surgery.The proximal tibia was collected and BMSCs were isolated from subchondral bone.BMSCs were:(1)inoculated in 10-cm dishes and osteoblastic induced in vitro for 25 days.Von kossa’s staining was used numbers of osteoblast colony were calculated under microscope;(2)The proportion of Nestin and Osterix positive cells was measured by flow cytomertry;(3)induced directly to osteoblast with BMP-2.The cells were stained with Alizarin red 4 weeks later and pictured under microscope.The staining was eluted and quantitatively analyzed with the spectrophotometry;(4)the total RNA and protein were extracted.The expression of mRNA and protein of CollA1 CollA2 were measured by quantitative RT-PCR and western-blot,respectively;the ratio of CollAl/CollA2 was calculated.(5)The knees of the mice were collected and paraffin sections were made.Immunohistochemical staining for Nestin and Osterix was performed and pictured under the microscope.Results:(1)The number of osteoblast colony induced from BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).The numbers of osteoblast colony in BMSCs deceased significantly after the ICA treatment as compared to non-administrated control group(P<0.05).(2)The expression of Nestin and Osterix in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).The expression of Nestin and Osterix deceased significantly after the ICA treatment as compared to the non-administrated control group(P<0.05).(3)The Nestin and Osterix positive cells in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).The Nestin and Osterix positive cells in BMSCs deceased significantly after the ICA treatment as compared to the non-administrated control group(P<0.05).(4)The number of mineral node stained with alizarin red in tibial subchondral bone of late-stage OA in the mice significantly decreased as compared to the sham operation group(P<0.05).The number of mineral node stained with alizarin red in tibial subchondral bone of late-stage OA in the mice significantly increased after the ICA treatment as compared to the non-administrated control group(P<0.05).(5)The expression of mRNA and proteins of Coll Al,Col1A2 significantly increased as compared to the sham operation group(P<0.05).The ratio of Col1Al1Col1A2 significantly increased as well.The expression of mRNA and proteins of CollAl,CollA2 significantly decreased after the ICA treatment as compared to the non-administrated control group(P<0.05).The ratio of CollAl/Coll A2 decreased as well(P<0.05).Conclusion:In a mouse model of late-stage OA,(1)The capacity of osteoblast differentiation of BMSCs in tibial subchondral bone increases.However,this capacity can be inhibited by ICA.(2)The expression of Nestin and Osterix in BMSCs in tibial subchondral bone increases.However,the expression can be inhibited by ICA.(3)The capacity of type I collagen synthesis of BMSCs in tibial subchondral bone increases.However,the ratio of Col1A1/Col1A2 increases as well,which leads to defects in mineralization of tibial subchondral bone.The capacity of type I collagen synthesis of BMSCs can be inhibited effectively by ICA.The ratio of CollAl/CollA2 declines,thus,the mineralization dysfunction of subchondral bone can be corrected.Part Ⅵ The Molecular Mechanism of ICA on Bone Marrow Stromal Cells in tibial subchondral bone of OA in the mice Objective:OA in the mice was treated with early adminstration(?)A.The(?)mechanism of ICA on bone marrow stromal cells in tibial subchondral bone of OA in the mice was explored.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.ICA was given by gavage(1mg/kg/day)upon surgery.The proximal tibia was collected and BMSCs were isolated from subchondral bone.Then,(1)the total protein was extracted and the expression of 36 common bone metabolism biochemical markers was measured by Antibody array.The protein expression level was represented by fluorescence intensity.(2)the total RNA and proteins were extracted.The expression of mRNA and protein of TGF-β1 were measured by quantitative RT-PCR and ELISA,respectively.(3)the knees of the mice were collected and paraffin sections were made.Immunohistochemical staining for TGF-β1 was performed and pictured under the microscope.(4)BMSCs were cultured in vitro.The carriers of Topflash and Renilla luciferase were co-transfected into these cells.The activity of the luciferase carriers was detected.(5)BMSCs were cultured in vitro.Cells mineralization was induced by BMP-2.ICA,TGF-β1 and/or DKK2 were added to the medium.The cells were stained with alizarin red 4 weeks later and pictured under micro scope.The staining was eluted and quantitatively analyzed with the spectrophotometry.(6)BMSCs were cultured in vitro.ICA,TGF-β1 and/or DKK2 were added to the medium.The expression of proteins of CollAl,Col1A2 was measured with western blot.The ratio of CollAl/CollA2 was calculated.Results:(1)The expression of TGF-β1 of BMSCs in tibial subchondral bone of late-stage OA in the mice significantly decreased after ICA early-administration as compared to non-administrated control group(P<0.05).(2)The expression of mRNA and proteins of TGF-β1 in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).The expression of mRNA and proteins of TGF-β1 in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly decreased after ICA early-administration as compared to non-administrated control group(P<0.05).(3)The expression of mRNA and proteins of DKK2 in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).The expression of mRNA and proteins of DKK2 in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly decreased after ICA early-administration as compared to non-administrated control group(P<0.05).(4)The activity of the luciferase carriers(Topflash and Renilla luciferase)of p-catenin in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly decreased as compared to the sham operation group(P<0.05).The activity of the luciferase carriers(Topflash and Renilla luciferase)of β-catenin in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased after ICA early-administration as compared to non-administrated control group(P<0.05).(5)The number of mineral node stained with alizarin red in tibial subchondral bone of late-stage OA in the mice significantly increased after ICA early-administration as compared to non-administrated control group(P<0.05);the number of mineral node significantly decreased after adding TGF-β1 or DKK2(P<0.05).(6)The expression of proteins of Col1A1 and Col1A2 in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly decreased after ICA early-administration as compared to non-administrated control group(P<0.05);the ratio of Col1A1/CollA2 decreased as well.The expression of proteins of Col1A1 and Co1lA2 significantly increased after adding TGF-β1 or DKK2(P<0.05);the ratio of CollAl/CollA2 increased as well.Conclusion:In a mouse model of late-stage OA,(1)The expression of TGF-β1 in BMSCs in tibial subchondral bone increases.However,the expression can be inhibited by ICA.(2)The expression of DKK2 in BMSCs in tibial subchondral bone increases.However,the expression can be inhibited by ICA.(3)The activity of β-catenin in tibial subchondral bone decreases.The activity can be enhanced by ICA.(4)The synthesis of type I collagen in BMSCs in tibial subchondral bone can be regulated by ICA.However,the regulation can be inhibited by TGF-β1 or DKK2.(5)Mineralization of tibial subchondral bone can be improved by ICA.However,the function of ICA can be inhibited by TGF-β1 or DKK2.The molecular mechanism of ICA on OA can be hypothesized as below according our experimental results:Mineralization of subchondral bone dysfunctions in OA.The hyper-secretion of TGF-β1 in BMSCs in OA is inhibited by ICA.DKK2,the inhibitor of Wnt/β-catenin signaling pathway is then down-regulated.Therefore,the activity of Wnt/β-catenin signaling pathway is unregulated.As a result,defects in mineralization of tibial subchondral bone are corrected to some extent.In conclusion,ICA yields protective effects on subchondral bone in OA. |
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