Research On The Kartogenin And P-PRP Promots Tendon-Bone Healing And Fibrocartilage Zone Regeneration

Part 1.Test the effect of KGN to induce tendon stem cells into chondrocytesObjective: In vitro experiments to test the ability of KGN to promote TSCS differentiate into chondrocytes to see the feasibility of using KGN for in vivo research of tendon-bone interface healing.Methods: We used collagenase and dispase to digest tendon to get tendon stem cells and purified the tendon stem cells using single cell culture method.Then identify these cells by using immunofluorescence and flow cytometry to test its surface markers.TSCs were treated with different concentration of KGN(1n M,10 n M,100 n M,1?M,10?M)and CCK-8 was used for the proliferation assay;RT-PCR was used for differentiation assay to measure the expression of chondrocyte related markers including Col-2,Sox-9 and Aggrecan.Results: Almost pure TSCs were obtained after using methods above.Proliferation results by CCK-8 was shown that no significant difference were detected in KGN with different concentrations(1n M,10 n M,100 n M,1?M,10?M)which were also similar with control group.RT-PCR results showed that KGN could increase the expression of chondrocyte related markers including Col-2,Sox-9 and Aggrecan.The effect is positive correlation with the concentration of KGN.Conclusion: Our method to obtain TSCs is quite mature to get pure tendon stem cells.KGN does not has an obvious effect to promote TSCs proliferation but does have a strong effect to induce TSCs differentiate into chondrocyte,which indicate KGN is available for the next in vivo study.Part 2.The differential effects of leukocytes-containing and pure platelet-rich-plasma(PRP)on tendon stem/progenitor cellsIntroduction: Platelet-rich plasma(PRP)is widely used to treat tendon injuries in clinics.These PRP preparations often contain white blood cells(WBCs)or leukocytes and the precise cellular effects of L-PRP(leukocyte-rich PRP)on tendons are not well defined.Therefore,in this study we determined the effects of L-PRP on tendon stem/progenitor cells(TSCs),which play a key role in tendon homeostasis and repair.Methods: TSCs isolated from the patellar tendons of rabbits were treated with autologous L-PRP or P-PRP(pure PRP without leukocytes)in vitro followed by cellular analyses including cell proliferation,stem cell marker expression by immunostaining,and tenocyte specific,catabolic and inflammatory gene expression by q RT-PCR and protein expression by western blot analysis and ELISA.Results: Cell proliferation was induced by both L-PRP and P-PRP in a dose-dependent manner with maximum proliferation at 10% PRP dose.Both PRP treatments also induced differentiation of TSCs into tenocytes.Nevertheless,the two types of PRP largely differed in several effects exerted on TSCs.L-PRP induced predominantly catabolic and inflammatory changes in differentiated tenocytes;its treatment increased the expression of catabolic marker genes,MMP-1,MMP-13,IL-1?,IL-6 and TNF-?,and their respective protein expression and PGE2 production.In contrast,P-PRP mainly induced anabolic changes;that is,P-PRP increased the gene expression of anabolic genes,?-SMA,collagen types I and III.Conclusion: These findings indicate that while both L-PRP and P-PRP appear to be "safe" in inducing TSC differentiation into active tenocytes,L-PRP may be detrimental to the healing of injured tendons because it induces catabolic and inflammatory effects on tendon cells and may prolong the effects in healing tendons.We would like to choose P-PRP for the in vivo study.Part 3.Kartogenin and P-PRP Promotes the Formation of Fibrocartilage Zone Between the Tendon Graft and BoneObjective: Treatment of tendon graft-bone tunnel with KGN in conjunction with platelet-rich plasma(PRP)results in fibrocartilage transition zone formation in a time dependent manner.Methods: The rat tendon graft-bone tunnel model was created by surgery.Immediately after surgery,rats received KGN+PRP or PRP injections in the tendon graft-bone tunnel interface.The control group was untreated.After 4,8 and 12 weeks,tendon graft-bone tunnels were harvested for histological,immunohistochemical and biomechanical analyses.Results: We monitored the release of KGN from the PRP gel in vitro for 7 days and found that the release of KGN from PRP gel lasted for 4 days and decreased over time.KGN release was quick in the initial 4 hrs,slow from 4-48 hrs and plateaued thereafter.Safranin O staining detected the abundance of proteoglycans in the KGN+PRP group indicating the formation of cartilage-like transition zone.Furthermore,immunohistochemical and immuno-fluorescent staining revealed collagen types I(Col-I)and II(Col-II)in the newly formed cartilage zone.The extent of this fibrocartilage zone formation and maturation was healing time dependent.In contrast,at all healing time points(4,8,and 12 weeks),PRP and control groups had no cartilage-like tissues and minimal Col-I and Col-II staining,and some gaps were present between the tendon graft and bone tunnel in the control group.Finally,pull-out strength of the fibrocartilage transition zone in the KGN+PRP treated group at 8 weeks was 1.4-fold higher than the PRP treated group and 1.6-fold higher than the control group.Conclusion: KGN,with PRP as a carrier,promotes the formation of fibrocartilage transition zone between the tendon graft and bone tunnel.PRP alone does not promote fibrocartilage formation,but functions as an effective "carrier" that supplies a fibrin gel scaffold and growth factors necessary to enhance the tendon graft-bone tunnel interface healing.