Hedgehog Signalling Contributes To Trauma-Induced Tendon Heterotopic Ossification

Background and aimsTendon heterotopic ossification(HO)is a common manifestation of chronic functional and structural disorders caused by tendon injury.The molecular mechanisms of tendon HO are not fully revealed.Osteogenic differentiation of tendon-derived stem cells(TDSCs)is pivotal in tendon HO,but the mechanism of erroneous differentiation of TDSCs in the occurrence of HO is not fully clear.Hedgehog(Hh)signalling is reportedly critical in hereditary HO,but how Hh signalling impacts the maintenance and function of TDSCs in HO has not been reported.The effects of Hh signalling on such differentiation of TDSCs may be critically involved in the physiology and pathobiology of tendon HO.Herein,with the Achilles tenotomy model and cell model of TDSCs,we sought to determine the effect of Hh signalling in the progression of trauma-induced HO in tendons.In summary,we aimed to clarify the impact of Hh signalling on the pathogenesis of trauma-induced tendon HO and provide a new therapeutic target for trauma-induced tendon HO.Methods1.The injured tendon specimens from mice at 1,4,7 and 10 weeks after Achilles tenotomy were collected.The gene and protein expression of Hh signalling-related molecules was detected by qRT-PCR and immunohistochemical(IHC)staining.2.GANT58,a Hh signalling antagonist,was injected intraperitoneally(i.p.)into mice from day 1 to week 6 after Achilles tenotomy.Injured tendon were sampled at 1,4,7,and 10 weeks.Micro-CT and H&E staining were used to detect the formation of mineralized bone.Alcian blue staining was used to detect the formation of cartilage nodules.The expression of osteogenic and chondrogenic specific genes and proteins was detected by qRT-PCR and IHC.3.SAG,a Hh signalling agonist,was injected intraperitoneally(i.p.)into mice from day 1 to week 6 after Achilles tenotomy,and tendon specimens were collected at 1,4,7,and 10 weeks.Ectopic bone formation was detected by micro-CT and H&E staining.Alcian blue staining was used to detect the formation of cartilage nodules.The expression of osteogenic and chondrogenic specific genes and proteins was detected by qRT-PCR and IHC.4.Under osteogenic culture conditions,TDSCs were stimulated by GANT58 and SAG respectively.After 7 and 14 days,the expression of osteogenic specific genes was detected by qRT-PCR.After 7 days,the osteogenic differentiation ability of TDSCs was evaluated by ALP staining.Under chondrogenic culture conditions,TDSCs were stimulated by GANT58 and SAG respectively.After 7 days,gene expression of chondrogenic markers was detected by qRT-PCR.Meanwhile,the chondrogenic differentiation ability of TDSCs were evaluated by Alcian blue staining.Results1.The specific gene and protein expressions of Hh signalling-related molecules significantly increased in the injured tendons from mice after Achilles tenotomy.2.GANT58 significantly reduced the formation of cartilage nodules and ectopic mineralized bones in tendons.GANT58 significantly downregulated the expressions of bone-related and cartilage-related genes and proteins in tendon.3.SAG significantly accelerated the formation of cartilage nodules and ectopic bone tissues in the injured tendons.SAG significantly upregulated the expressions of bone-related and cartilage-related genes and proteins in tendon.4.Under osteogenic induction,the gene expressions of osteogenic markers in TDSCs were suppressed after treatment with GANT58,but promoted after treatment with SAG.GANT58 reduced ALP staining,while SAG increased ALP staining.After chondrogenic induction,the gene expressions of chondrogenic markers were suppressed in TDSCs in response to GANT58 treatment,but were promoted in response to SAG treatment.At the same time,the GANT58 group exhibited less intense Alcian blue staining,while the SAG group displayed more intense staining.ConclusionIn conclusion,Hh signalling contributes to trauma-induced tendon HO and the regulation of Hh signalling affects the formation of tendon ossification by modulating the differentiation direction of TDSCs.GANT58,a Hh signalling antagonist,inhibits trauma-induced tendon ossification by restricting chondrogenesis and osteogenesis of tendons.Therefore,targeting Hh signalling by GANT58 may be a potential therapeutic strategy for useful prevention of trauma-induced tendon HO.