The Role And Mechanism Of Indian Hedgehog In Cartilage Development And Repair Of Growth Plate Injury

Objective:Growth plate injury（GPI）,also known as epiphyseal injury,is an injury to the growth plate of long bones caused by trauma,infection,etc.Limb shortening and angulation deformities in patients with growth plate injury have always been a major challenge for pediatric orthopaedic surgeons,and there is no reliable treatment option to date.Therefore,it is of great significance to identify the specific mechanism of homeostasis maintenance of growth plate chondrocyte in the process of bone bridge formation after growth plate injury,which is of great significance to find drug targets for effective treatment or prevention of growth plate injury.Indian Hedgehog（Ihh）belongs to a subtype of the Hedgehog family,which is mainly expressed in the pre-hypertrophic and in hypertrophic layer of the growth plate chondrocytes,and it plays an important role in cartilage development and cartilage homeostasis.At present,there are many studies have demonstrated that the Ihh play a crucial role in osteoarthritis（OA）,but its role in postnatal cartilage development remains unclear.In this study,we firstly found that the knockout of Ihh accelerates the mineralization of growth plate chondrocytes and promotes endogenous bone formation in the postnatal growth plate.Therefore,we speculate that overexpression of Ihh can repair growth plate injury by inhibiting the bone formation after growth plate injury.This study intends to further clarify the effect of overexpression of Ihh on growth plate and its specific molecular mechanism,and it could provide a new target for the prevention and treatment of growth plate injury.Methods:1.To establish the conditional knockout mice of chondrocyte-specific deletion of Ihh,the heterozygous Ihh conditional knockout mice(Ihh ^Flox/+)was hybridized with chondrocyte-specific Col2a1-Cre ER^T2mice to obtain Ihh ^Flox/+;Col2a1-Cre ER^T2mice,then the Ihh ^Flox/+;Col2a1-Cre ER^T2mice were mated with heterozygous Ihh ^Flox/+mice to obtain Ihh ^Flox/flox;Col2a1-Cre ER^T2（Ihhc KO）and control mice(Ihh ^Flox/flox,Control).Intraperitoneal injection of 1mg/10g/day Tamoxifen（TM）on the 1st day after birth,continuous injection for 2 days.The control and Ihhc KO mice at 3 days,7 days,10 days and 14 days after birth were observed and photographed,and the body length changes of the mice were dynamically measured.The X-ray scanning was performed on 14-day-old mice using the Faxitron X-ray scanner,and the development of the hindlimb growth plate was observed.Computed Tomography Scans were performed on mouse hindlimb specimens using Micro-CT,the region of interest（ROI）was selected for 3D reconstruction in the region of the proximal tibia growth plate.After the hindlimb specimens were fixed in 4%paraformaldehyde,and the dehydrated with graded ethanol,cleared with chloroform,embedded in paraffin,and finally sliced with a thickness of 5μm.Routine dewaxing,rehydration and Safranin O/Fast Green staining were used to observe the growth and development of mouse growth plate chondrocytes after Ihh deletion.2.To established a growth plate injury model of adolescent wild-type C57BL/6 mice,the Ihh-hydrogel mixed drug(25μg recombinant Ihh protein was dissolved in 313μl Vitro Gel^TM3D hydrogel)was injected into the injury site after injury immediately.And the proximal tibia of mice was taken for the histopathological observation at 8 weeks after surgery.Furthermore,the Ihh-hydrogel mixed drug(25μg recombinant Ihh protein was dissolved in 313μl Vitro Gel^TM3D hydrogel)was injected into the injury site after injury after the establishment of the model of growth plate injury in adolescent SD rat.Small animal tomographic fluorescence scanning（FMT）and Osteo Sence680EX osteogenic probe were used to monitor new bone formation after growth plate injury at 2 weeks after surgery.Safranin O/Fast Green staining was used to evaluate the repair effect of growth plate injury site,and the immunohistochemistry and Laser Microdissection were performed to assess the repair tissue properties in growth plate injury site.3.Extraction of rib growth plate chondrocytes of C57BL/6 mice of P3,the green fluorescent protein（GFP）-labeled adenovirus vectors overexpressing Ihh（GFP-Ad-Ihh）and silencing Ihh（GFP-Ad-Ihh si RNA）were expanded and transfected into the growth plate chondrocytes in vitro.The RNA was extracted by TRIzol method after the successful transfection was confirmed.The RNA-Seq was used to observe the differential gene expression of Ihh overexpressed and Ihh silenced in growth plate chondrocytes.Furthermore,the GO analysis and KEGG pathway analysis were performed to analysis the biological function after the variation of Ihh.The signal pathways enriched by differential genes by RNA sequencing were further analyzed to verify the effects of Hedgehog signal pathway and Wnt/β-catenin signal pathway"Crosstalk"on the proliferation,differentiation and metabolism of growth plate chondrocytes.Results:1.Ihh chondrocyte-specific knockout mice were obtained by hybridization and identification.Compared with Control mice,the body weight of Ihhc KO mice on P14 was significantly reduced,and the body length and limbs of the mice were significantly shortened;The result of Faxitron X-ray demonstrated that the length of the whole skeleton system of the Ihhc KO mice on P14 was reduced,the limbs were significantly shortened.And the proximal tibial epiphysis,secondary ossification center and growth plate structure were disappeared.The three-dimensional reconstruction of Micro-CT showed that the closure and abnormalities of growth plate,the deficiency of the secondary ossification center and the abnormal ossification of growth plate.Safranin O/Fast Green staining showed that the cartilage structure of the tibial growth plate of the Ihhc KO mice on P3 day was normal,and the chondrocytes in the rest zone,proliferative zone and hypertrophic zone nonnally grow up;At P5,the proliferation zone chondrocytes arranged in a flat column of growth plates were replaced by a few hypertrophic chondrocytes.At P10,the maturation and hypertrophy of chondrocytes were observed in the growth plate,the chondrocytes of hypertrophic layer was mineralized,and the structure of the growth plate was obviously lost.At P14,the mineralization of the growth plate cartilage extended to most areas of the epiphysis and metaphysis,and the structure of the growth plate was disappeared completely.The cartilage layers of the tibial growth plate were evenly arranged at each time point in the Control group,and the tibial growth plate developed normally.In general,the deletion of Ihh accelerates the mineralization of growth plate chondrocytes and promotes endogenous bone formation in the growth plate.This suggests that overexpression of Ihh may prevent bone formation after growth plate injury,thereby promoting the repair of growth plate injury.2.The formation of bone bridge and bone trabeculae were observed in situ and a small amount of new cartilage was occasionally distributed around the defect site at 8 weeks after growth plate injury in Hydrogel group.Clusters of new cartilage filled the defect site in Ihh-hydrogel group.Compared with the Hydrogel group alone,the cartilage regeneration area increased and the Pineda score decreased in Ihh-hydrogel treatment group and the difference was statistically significant（P<0.05）.Furthermore,the rat model of growth plate injury was established and the Scanning electron microscopy showed that the continuity of the growth plate was interrupted and a cylindrical defect was formed after injury.Micro-CT scan showed that the high-intensity contrast agent filled the defect after the injection of Ioversol-Hydrogel gel into the defect site,which was confirmed that the Hydrogel drugs could successfully stay in the defect.A rat model of bilateral growth plate injury was established.After two weeks of treatment with Ihh-hydrogel,normal saline or osteogenic fluorescent probe Osteo Sence 680EX was injected into the tail vein.FMT showed that no fluorescence signal was observed in both knees in the bilateral sham-operated+normal saline group;the fluorescence intensity of left knee stays in step with right knee in the bilateral sham operation+Osteo Sence 680EX group.After treatment of Ihh-hydrogel,the fluorescence signal was significantly weakened,and the difference was statistically significant（P<0.05）;Compared with the Hydrogel group alone,the fluorescence signal was significantly weakened in treatment with Ihh-hydrogel and the difference was statistically significant（P<0.05）.Faxitron X-ray showed that the proximal tibia epiphyseal line was continuous in the sham group and no epiphyseal calcification was found at 4weeks after surgery.The injury spanned the epiphyseal line of proximal medial epiphysis in Hydrogel group alone and the pathologicallycalcification in injury site was observed,and the epiphysis in the injured area was significantly closed.Compared with Hydrogel group,the continuity of the epiphyseal line was better and a small amount of calcification was observed at the injury site after treatment of Ihh-hydrogel.And the epiphysis in the injured area was incompletly closed.The result of coronal Micro-CT of the injured area showed that the bone bridge at the defect site of the growth plate of the rats treated with Hydrogel alone was almost completely replaced by the bone bridge,and conversely the formation of bone bridge was significantly inhibited after Ihh-hydrogel treatment.Faxitron X-ray and Micro-CT results showed that the formation of bone bridge was less in Ihh-Hydrogel group when compared with Hydrogel group（P<0.05）.Safranin O/Fast Green staining showed that the cluster regeneration of growth plate cartilage in situ of growth plate injury site and the good continuity of growth plate was observed in Ihh-hydrogel group.Compared with the hydrogel group alone,the cartilage regeneration area increased and the Pineda score decreased in Ihh-hydrogel group（P<0.05）.The immunohistochemical analysis showed that the positive area of Col2a1 were observed in Ihh-hydrogel group,while a few positive areas of Col2a1 were occasionally observed in Hydrogel group alone.The results of Laser Microdissection of paraffin tissue combined with RT-q PCR demonstrated that a higher expression levels of Col2a1 and Aggrecan m RNA and a lower expression of Runx2,Col10a1 and Osterix m RNA of repaired tissue were detected in Ihh-hydrogel group when compared with Hydrogel group.3.Mouse growth plate chondrocytes were successfully isolated and cultured in vitro.The adenovirus vector containing GFP-Ihh was successfully amplified in vitro and transfected into mouse chondrocytes.Western Blot confirmed that both overexpressed Ihh and silenced Ihh adenovirus vectors were highly transfected.The results of RNA-Seq analysis showed that overexpression of Ihh has359 up-regulated genes and 370 down-regulated genes;And the silencing of Ihh has335 up-regulated genes and 394 down-regulated genes.GO analysis showed that the differential genes were mainly enriched in biological processes such as cartilage development and differentiation.Furthermore,KEGG enrichment found that the"Crosstalk"phenomenon occur between the Hedgehog pathway and the Wnt/β-catenin signaling pathway,which jointly regulate the metabolic function of growth plate chondrocytes.The result found that overexpression of Ihh increased the expressions of GLI1,PTHr P,Col2a1,Aggrecan,and Sox9.Conversely,the deletion of Ihh activated the Wnt/β-catenin signaling pathway,and then promoted the increased expressions of Col10a1,Runx2,and ADAMTS5 in vitro.Conclusions:1.The functional loss of Ihh accelerates the mineralization of growth plate chondrocytes and promotes endogenous bone formation,leading to growth plate developmental disorders;2.Exogenous overexpression of Ihh inhibits the formation of bone bridge after growth plate injury,and promotes regeneration of growth plate cartilage in situ to repair the injuried growth plate;3.Ihh mediates the"Crosstalk"between Hedgehog signaling pathway and Wnt/β-catenin signaling pathway,thereby regulating the metabolic function of growth plate chondrocytes.