| Tendon injury is the most common sport injury,include acute and chronic injury.The process of tendon healing is slow,often healed with scar tissue and fibrosis.It is difficult to make the injured tendon restored with normal function and structure.Achilles tendon,as the indispensable tendon for the actions of walking,running and jumping,is vulnerable.With the development of stem cells biology and technology,mesenchymal stem cells(MSCs)are more and more attractive.Bone marrow,fat,muscle,embryonic tissues and other sources of stem cells have be used to promote the repair of tendon injury in preclinical and clinical studies,but many of them have their own disadvantages,such as heterotopic ossification,pimelosis,risk of tumor formation,invasive sampling,and so on.Human umbilical cord mesenchymal stem cells(hUC-MSCs)come mainly from Wharton’s jelly of umbilical cord tissue.They have many advantages,include more primitive,stronger ability of proliferation and differentiation,low immunogenicity,immunomodulatory effects,anti-tumor,suitable for xenograft,convenient sampling,sufficient quantity,no ethical issues and so on.An animal experiment showed that allogeneic h UC-MSCs achieved good results in treatment of tendinopathy of the racing horse.Platelet-Rich plasma(PRP)is a portion of the plasma fraction that has a platelet concentration of more than 1000000/u L by centrifuge extraction from blood plasma.PRP contains a variety of growth factors,which makes it become another hotspot of tendon healing.Relevant preclinical and clinical studies have showed that PRP was effective for promoting tendon healing.One animal study suggested that PRP combinated with rats’ tendon stem cells(TSCs)could get better outcomes than PRP or TSCs been used alone in promoting the repair of injured tendons in rats.In most studies about PRP promotion healing of injured tendon,PRP concentration was different.An in vitro study found that concentrations of PRP could affect proliferation,differentiation,migration and secretion of tenocytes,and high concentrations of PRP did harm to the cells.Based on the above basis,we speculate that PRP concentration can affect hUC-MSCs’ proliferation and differentiation,application of hUC-MSCs combinated with PRP may be effective on repair of tendon injury.Because there are no related reports about PRP combinated with hUC-MSCs for repair of injured tendon so far,therefore this research tries to clarify effects of PRP of different concentrations on the proliferation of h UC-MSCs,on the tenogenic differentiation of h UC-MSCs and to explore the effect of PRP combinated with h UC-MSCs on repair of tendon injury.Objective:⑴ To verify the effect of different concentrations PRP on the proliferation of h UC-MSCs in vitro.⑵ To observe the effect of different concentrations PRP on the tenogenic differentiation of h UC-MSCs in vitro.⑶ To assessment the effect of h UC-MSCs combinated with PRP on healing of Achilles tendon injury in rats.Methods:⑴ The effect of different concentrations PRP on proliferation of hUC-MSCs: PRP was activated and became PRP-clot releasate(PRCR);Passage 3 hUC-MSCs were cultuerd in medium for 24 hours and then added DMEM/F12 medium respectively containing 0%(control group),10%,20%,and 40% volume PRCR.Tested the optimal density(OD)values in each group using Cell counting kits-8(CCK-8)to reflect the cells’ proliferation rate at 1,2,3,and 4 days.⑵ The effect of different concentrations PRP on the tenogenic differentiation of h UC-MSCs in vitro: Passage 3 h UC-MSCs were cultuerd in medium for 24 hours and then added DMEM/F12 medium respectively contain 0%(control group),10%,20%,and 40% volume PRCR.Collected cells in each group and extracted total protein of them,expression of tendon markers(TNC,and SCX,Col I)of h UC-MSCs at protein level were detected and calculated through Western blot at 1,3,and 5 days.⑶ To assess the effect of hUC-MSCs combinated with PRP on healing of Achilles tendon injury in rats: 48 male SD rats 8 weeks aged were prepared,2 and 4 weeks after received h UC-MSCs,PRP,h UC-MSCs with PRP or Saline(control)injection into the site of injuried rat Achilles tendon 3 day after type I collagenase injection,tendons were harvested and were evaluated by HE staining histology and Western blot of tendon markers.Results:⑴ CCK-8 detects OD value of each group in 450 nm at day 1,2,3,and 4.Proliferation rate of hUC-MSCs in experimental groups were significantly faster than control group(P<0.05),except for that of 40%PRCR group at day 2(P>0.05).Proliferation rate of h UC-MSCs was fastest in 40%PRCR group at day 1,and it was fastest in 20%PRCR group at day 2,3 and 4.Different concentrations of PRCR could promote the proliferation of h UC-MSCs,and 20%PRCR group had the most obvious effect of promotion for proliferation.⑵ Collected cells in each group and extracted total protein of them,expression of tendon markers(TNC,SCX and Col I)of h UC-MSCs at protein level were detected and calculated through Western blot at day 1,3 and 5.Expression of tendon markers of h UC-MSCs in experimental groups were obviously higher than control group(P<0.05)at protein level,except for the expression of Col I in 40%PRCR group at day 1(P>0.05)and the expression of TNC,SCX in 40%PRCR group at day 3 and 5(P>0.05).In all time,the expression of tendon markers in 20%PRCR group were significantly higher than other groups(P<0.05).⑶ 2 and 4 weeks after received hUC-MSCs,PRP,hUC-MSCs+PRP or Saline(control)injection therapy into the site of injured rat Achilles tendon,expression of tendon markers in rat Achilles tendon at protein level were detected through Western blot.Expression of tendon markers in experimental groups were obviously higher than control group(P<0.05)at protein level,except for the expression of TNC in PRP group and h UC-MSCs group at week 4 after operation(P>0.05).The expression of tendon markers in PRP+h UC-MSCs group are the highest at all experimental time.⑷ 2 and 4 weeks after operation,slice of rat Achilles tendons were stained with hematoxylin eosin(HE).Fiber structure,fiber arrangement and inflammatory reaction of rat Achilles tendon were assessed by the means of inverted microscope.2 weeks after operation,disordered arrangement of collagenous fiber and more serious infiltration of inflammatory cells were observed in control group and PRP group,while neater fiber structure,more ordered arrangement and less infiltration were observed in hUC-MSCs group and PRP+hUC-MSCs group.PRP+h UC-MSCs group showed superiority in histological assessment.At 4 weeks,histological assessment in all groups was better than that of 2 weeks after operation,histological assessment of the healing rat Achilles tendons in h UC-MSCs group and PRP+h UC-MSCs group were better than control group,but PRP therapy showed little improvement to the healing of injured Achilles tendons.PRP+hUC-MSCs group also showed superiority in histological assessment at 4 weeks.Conclusion:⑴ PRP concentration can affect the proliferation rate of h UC-MSCs,suitable concentration of PRP can significantly promote the proliferation of h UC-MSCs in vitro.⑵ PRP concentration can affect the differentiation of h UC-MSCs,suitable concentration of PRP can obviously enhance tenogenic differentiation of h UC-MSCs in vitro.⑶ PRP combinated with h UC-MSCs can preferably promote the expression of tendon markers at protein level in rat Achilles tendon and improve the histological assessment in the process of tendon repair than PRP or h UC-MSCs used alone.This study makes us to realize the synergistic effect of PRP and hUC-MSCs in promoting tendon healing,and to provide animai experimental foundation which makes the therapy of PRP combinated with h UC-MSCs be potentially used in treatment of tendon injuries clinically. |
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