The Effect Of Platelet-rich Plasma On Cell Biological Function Of Dermal Papilla Cells And Its Application For Hair Follicle Reconstruction

Background and objectiveHair follicle regeneration through bioengineering is a promising alternative for treatment of alopecia.Engineered hair follicle reconstitution methods require a combination of trichogenic cells,three-dimensional scaffolds,and biomolecular signals.Dermal papilla cells(DPCs)are important seed cells for hair follicle reconstruction.However,DPCs gradually lose hair-inductive capacity during subculture and it is difficult to obtain large numbers of DPCs with inductive capacity under current culture conditions in a short time.There is a need of improved culture conditions for DPCs.In addition,it is a necessary to select a suitable scaffold for hair follicle regeneration.Platelet-rich plasma(PRP)can release a large number of growth factors when activated,these growth factors can promote cell proliferation and differentiation.Clinical studies show that PRP can be used for the treatment of alopecia and promote hair growth.PRP-gel is a suitable scaffold material for tissue engineering,which had a three-dimensional mesh-like structure,and cells can survive and proliferate in it.Therefore,in the study we improve the culture condition of DPCs in vitro by adding activated PRP in the culture medium,and investigate the feasibility of PRP gel as a cell scaffold for hair follicle reconstruction and as a three-dimensional scaffold for DPCs culture.Methods1.The effects of PRP on cell biological function of mouse vibrissa DPCs and human scalp DPCsPRP was extracted by two-step centrifugation.After activation,different concentrations of activated PRP supernatant were added to the culture medium to culture mouse and human DPCs,and the optimal concentration was selected by CCK8 proliferation assay.The effects of PRP on the biological activity and properities of DPCs were detected by immunofluorescence,Western-blot and PCR.Hair follicle reconstruction model in vivo were used to detect the hair-inductive capacity of DPCs.2.The mechanism of PRP on the biological effect of human DPCsExpression of phosphorylated growth factor receptors(GFRs)in cultured DPCs were ananlyzed by immunofluorescence and western blot.Signal pathways mediated by GFRs were ananlyzed by human phospho-kinase array.3.PRR gel as a cell delivery vehicle for hair follicle reconstructionDPCs,epithelial cells and appropriate amount of PRP were mixed,and PRP gels comprising trichogenic cells were formed after activated by thrombin.PRP gels containing trichogenic cells were transplanted onto the back wound of nude mice,and the hair follicle reconstruction was observed.4.PRP gel as a three-dimensional scaffold for human DPCs cultureDPCs of different numbers and different passages were seeded onto the PRP gel,appearance of cell growth and aggregation were observed.Results1.The effect of PRP on cell biological function of mouse vibrissa DPCs and human scalp DPCsLow concentration of activated PRP can promote the proliferation of human and mouse DPCs,5%activated PRP enhance mouse and human DPC proliferation maximally.The biological function markers of DPCs were detected through protein and gene level.The results show that the expression of ALP,?-catenin and Versican which were associated with hair-inductive capacity of DPCs were up-regulated in medium with 5%PRP than in control medium.Animal experiments conformed that the hair-inductive capacity of mouse DPCs treated with 5%activated PRP was stronger than that DPCs in control cultures.However,when cultured human DPCs and neonatal mouse epidermal cells were transplanted in the mini-chamber assay,no hair follicle induction was observed.2.The mechanism of PRP on the biological effect of human DPCsPhosphorylated FGFR1,PDGFR a and PDGFR ? of human DPCs were up-regulated after treated with 5%activated PRP,Human phospho-kinase membrane arrays show that phosphorylation levels of ERK,JNK,p38 and Akt were elevated and GSK-3 was lower in the PRP group compared to controls.3.PRR gel as a cell delivery vehicle for hair follicle reconstructionPRP gels containing mouse DPCs and neonatal epidermal cells were transplanted into nude mice can reconstruct hair follicle.However,PRP gels containing human DPCs and foreskin epidermal cells were transplanted into nude mice hair follicles were not induced.4.PRP gel as a three-dimensional scaffold for human DPCs cultureWhen human DPCs were seeded at the density of 0.5×104,1×104 and 2×104 per well on the PRP gel,the cells neither grow adherently nor aggregate into a sphere.Increasing the cell density to 5×104,only a few cell aggregates were observed per well,the shape of aggregates was irregular and the connection between cells was loose.The majority of other cells show a round shape at the time of seeding.The growth of low and high passages of DPCs were similar.Conclusion1.The addition of 5%activated PRP supernatant in the culture medium significantly promoted the proliferation of mouse and human DPCs and enhanced the hair-inductive capacity of DPCs.2.PRP promotes the proliferation and enhances the hair-inductive capacity of human DPCs by up-regulating the expression of phosphorylated receptors FGFR1,PDGFR a and PDGFR ?,then activating MAPK,Akt and Wnt signaling pathways.3.PRP gel could be employed as a cell delivery vehicle for hair follicle tissue engineering.4.PRP gel are not suitable for three-dimensional culture of human DPCs.