Role Of Collagen Type X During Endochondral Ossification Using Antler Model

Collagen type X(Col X)is a member of collagen family whose function is not well understood.Col Xis specifically expressed by terminallydifferentiating chondrocytes,i.e.hypertrophic chondrocytesandrestricted in the matrix of hypertrophicchondrocytes duringendochondral ossification,suggesting that it is of major importance in endochondral bone growth and development.However,thus far the detailed molecular mechanism underlying Col X regulation is not fully understood.This situation is at least partially due to the limitation of the currently available models,i.e.embryo long bone formation,as in the model transition between each differentiation stage is very brief and the hypertrophied chondrocytes are only made up of a narrow line(1-2 cells wide).Deer antler is the only annual renewable bone organ which can annually regenerate,and each regeneration undergoes a special endochondral ossification,known as “modified endochondral ossification”,which is similar to classic endochondral ossification,which mainly undergoes an entire process of bone formation from progenitor cells to matureosteocytes.But the process of endochondral ossification in antlers is different from its classic counterpart.As theformer is richly vascularized,whereas the latter is relativelyavascular.On the other hand,in comparison with the narrow layers of the growth-plate,antlers can offer more detailed cellular events than that of prenatal long bone formation,the gold standard for endochondral ossification study,as the thickness of different layers of antler growth center including mesenchymal,pre-cartilage,and transitional and cartilage zone can reach 2.9 mm,6.5mm,3.22 mm,8 mm respectively.So antler can serve as a better model to study the role of Col X during endochondral ossification and the relationship between Col X and angiogenesis.In this study we intended to address these two important questionsfrom both in vitro and vivo aspects respectively.In the in vitro study: Four RNAi target sequence of Col X named as S1,S2,S3 and S4 were designed and inserted into carrier vector Plvthm.The recombinant lentivirus Plvthm-S1,Plvthm-S2,Plvthm-S3 Plvthm-S4 were collected and concentrated from the supernatant of packaging cell line 293 T,and subsequently used to infect AP cells.qPCR and Western Blot showed that the expression of Col X significantly down-regulated compared with negative control.Among these,S3 was the most effective and its inhibition ratio to Col X could reach 96.4%.Histological assay showed that down-regulating Col X could inhibit AP cells to differentiate into chondrocytes during endochondral ossification.On the other hand,qPCR and Western Blot assay showed that the distribution of Col X in antler tip changed significantly,among these different layers: mesenchyme,precartilage,transitional zone and cartilage,transitional zone contained the most Col X which was the growth center of blood vessel in antler.In the cartilage zone,distribution of Col X was also associated with blood vessels which could be found at the vascular wall and the matrix of precartilage cells surrounding the blood vessel.So,Col X could play a signal role in inducing angiogenesis in antlers.In the in vivo study: First,two lentiviral Plvthm and Pll3.7 were used to infect AP cells and AP tissue to find out which one was more effective.The results showed that Plvthm was more powerful at both cell and tissue levels which was suitable for in vivo research model of antler.Second,AP tissue wascollected from three sika deer stag calves named as model-1,model-2 and model-3.Then Plvthm-S3 was used to deliver RNAi target sequence to AP tissue.After infection,the AP tissue was transplanted into the inner skin pockets of the donor deer.But these transplanted tissues failed to develop into full antler,and underwent self-resorption.Then severalexperiments were carried out to investigate the reason of resorption of AP tissue after self-transplantation.The results showed that the damage of mechanical chopping of AP tissue could not interrupt the process of AP development but delayed it.And the stem cells in AP tissue did have the ability to induce angiogenesis both in vitro and in vivo.But during the process of in vitro culture,the stem cells in AP tissue differentiated into chondrocytes and lost the abilityduring which the expression of Col X and VEGF both dereased significantly.So when the cultured AP tissue has transplanted into the inner skin of donor deer,the AP tissue failed to connect to blood vessel system of donor deer and died soon,then was absorbed by the immune system of the donor deer.Conclusion: 1.Col X was an essential part of the matrix of cartilage and played an important role in endochondral ossification of deer antlers.Down-regulation of Col X could distort the matrix and cause the death of chondrocytes.2.The distribution of Col X in antler tip changed significantly,among these different layers: mesenchyme,precartilage,transitional zone and cartilage,transitional zone contained the most Col X which was the formation center of blood vessels in antlers.In the cartilage zone,distribution of Col X was also associated with blood vessels which could be found at the vascular wall and the matrix of precartilage cells surrounding the blood vessels.So Col X could play as a signal to induce angiogenesis in antler.3.The damage of mechanical chopping of AP tissue could not interrupt the process of AP development but delay it.And the stem cells in AP tissue did have the ability to induce angiogenesis both in vitro and in vivo.But during the process of in vitro culture,the stem cells in AP tissue differentiated into chondrocytes and lost the ability during which the expression of Col X and VEGF both dereased significantly.So when the cultured AP tissue has transplanted inner skin of donor deer,the AP tissue failed to connect to blood vessel system of donor deer and died soon,then was absorbed by the immune system of donor deer.