The Effect Of PRP On Hair Follicle Stem Cells And Hair Follicle Regeneration And Their Mechanisms

[Background and Objectives]Androgenic alopecia(AGA),also known as male pattern baldness,is a common disorder that affects men and women.Recent studies have shown that platelet-rich plasma(PRP)had different effects on hair regrowth.However,the underlying cellular and molecular mechanisms along with its effects on hair follicle stem cells are poorly understood.In this study,the effects of PRP on the hair regrowth and their cellular and molecular mechanisms are explored,in order to develop novel PRP-based therapies for AGA.[Methods]1.The platelets in PRP were induced to release factors by calcium chloride(PC)or by sonication where platelet lysates(PS)or the supernatants of platelet lysate(PSS),which were injected subcutaneously into the back of C57 male mice(6 weeks old),to evaluate their effects on the hair follicle activation.2.The supernatants of platelet lysate by sonication(PSS)were injected subcutaneously into the back of K14-H2B-GFP transgenic male mice,with normal saline(NS)as control,to evaluate their effects on the hair follicle stem cell activation.3.The supernatants of sonicated platelet lysate(PSS)and platelet lysates by calcium chloride(PC)were incubated with human immortal keratinocytes(HaCaT cells)and skin-derived precursors(SKPs)isolated from the dorsal skin of neonatal C57BL mice 1-3 days after birth respectively,in order to observe their biological effects on hair follicle cycling.4.The epidermal cells and SKPs derived from neonatal K14-H2B-GFP mice in 20%PSS or equal volume of saline were implanted into 2.5 mm excisional wounds of BALB/c nu/nu mice(5 weeks old),to observe their effects on hair follicle reconstitution.5.PRP samples(PC,PS and PSS)were subjected to antibody-based protein array analysis to detect 41 human growth factors.[Results]1.Quantitation of the hair regrowth areas showed that PSS induced enhanced hair regrowth compared to NS and PC.2.There was a dramatic increase in the number of K14-GFP-positive cells in the hair follicle at the injection site of PSS,compared to the saline injection site,which indicates that factors released from platelets activate quiescent HFSCs,and promotes their proliferation,resulting in hair follicle TAT.3.Both PC and PSS significantly promoted the proliferation of keratinocytes.In the absence of regular supplement,SKPs formed small cell aggregates in basal medium compared to SKPs in regular growth medium.The supplementation of PC to basal medium resulted in the formation of large cell aggregates.However,when PSS was added to the culture,SKPs formed large aggregates with healthy morphology compared to PC,which indicates that PSS exhibited enhanced effect in promoting SKP survival.4.The sites of grafts containing PSS generated more hairs than sites of grafts containing control saline,with the PSS-supplemented grafts generating larger hair follicles than saline-supplemented grafts by two-photon microscopy,which indicates that PRP sonicates enhance de novo hair regeneration and hair growth.5.Out of 41 growth factors analysed,16 factors showed higher levels(?2 folds)in PSS than in PC,which included VEGF-D,PDGF-AA,PDGF-BB,PDGF-AB,HGF,SCF,GDNF,FGF-4,FGF-7,bFGF and IGF-1.The result suggests that sonication better releases growth factors from platelets in PRP compared to conventional calcium-induced platelet contraction.[Conclusions]This study has provided convincing data to show that PRP preparations activate HFSCs and promote hair follicle regeneration,and platelet sonicates contain high levels of numerous growth factors and exhibit superior effects in promoting SKP survival,suggesting a better preparation for PRP therapy.