The Establishment Of Nano-scale Microenvironment For Single Dermal Papilla Cells And Its Application For Hair Induction

Background and objectiveWith the continuous development of regenerative medicine,the hair follicle regeneration through construction of tissue engineering hair follicle is the best option for hair loss treatment.The dermal papilla cell(DPC)is the most important cell in construction of tissue engineering hair follicle,due to its hair-inducing capacity.However,the inductive ability of DPCs tends to gradually lose during passaging once they are cultured in traditional 2D culture in vitro.Therefore,the establishment of in vitro DPC ecological cultivation model to maintain its biological characteristics and functions after passaging,is key to the reconstruction of tissue engineering hair follicles.Layer-by-Layer self-assembly(LbL)is an ultrathin film nano-fabrication technique that functions by depositing multilayer coatings of oppositely charged material-polycation and polyanion.This functional assembly of biocompatible polymers on the cell surface to fabricate cell-extracellular matrix(ECM)offers a biomimetic microenvironment for cell.Thus,in this study,we employed LbL technology to encapsulate single DPC for suitable biomimetic microenvironment and microtissue construction by "spheroid formation chemically".This study not only provide a large number of biologically functional seed cells for tissue engineering hair follicles,but also a new model for cell three-dimensional ecological culture in vitro and a new method for the treatment of hair loss.Methods1.The establishment and evaluation of single DPC encapsulation culturing model using LbL technologyIsolation and cultivation of mice DPCs.Gelatin and alginate were selected for encapsulation by ultrathin matrices of gelatin/alginate/gelatin around the membrane to LbL-DPCs.Further,in order to explore if LbL nano-encapsulation was available,we carried out SEM,TEM,zeta-potential assessment and fluorescent reagents labeling.Live/dead staining,CCK-8,immunofluorescent staining and PCR were performed to examined whether single DPC-based LBL encapsulation would impact the intrinsic properties of DPC.2.The effect of hair-inducing capacity when FGF-2 was loaded into the LBL-DPC nano-structureFGF-2 was loaded with alginate into the LBL-DPC nano-structure.To test the release of FGF-2 and its effect on the viability,proliferation,and intrinsic properties of DPCs.LBL(FGF-2)-DPC were co-transplanted with mice newborn epidermal cells,to investigate hair-inducing capacity by macroscopic appearance and histologic assay of the grafts after 2 weeks of subcutaneous implantation.3.The establishment of chemical crosslinking LbL-DPCs for 3D culturing model and its hair-inducing capacityWe product cell spheroids by aggregation of LbL-DPCs through crosslinking alginate and Ca2+.To determine the optimal conditions for DP spheroids formation,we tested the size of DP spheroids under seeding various cell densities in a low-adhesive plate.SEM,TEM,fluorescent reagents labeling,Live/dead staining and H&E staining was carried out for morphological and histological evaluation.Construction of DP cell spheres by high-passaged DPC to detect of DPC cell marker gene and protein expression.Being co-transplanted with mice newborn epidermal cells,to investigate hair-inducing capacity.Results1.The establishment and evaluation of single DPC encapsulation culturing model using LbL technologyIsolation of mice DP by microinjection combined with enzymatic digestion did not significantly affect the cell morphology,growth pattern and the expression of ALP and a-SMA of the relative specific markers of DPC.SEM,TEM,zeta-potential assessment and fluorescent reagents labeling test results shown that LbL nano ultra-thin ECM was applied on a single DPC surface successfully.Live/dead staining,CCCK-8 and Ki67 immunofluorescence indicated that LbL nano-coating did not significantly influence the viability,proliferation of DPC.At last,the expression levels of a-SMA in LbL-DPC were significantly lower than DPC,and there was no significant difference in expression level of ALP and ?-Catenin in DPC and DPC.2.The effect of hair-inducing capacity when FGF-2 was loaded into the LBL-DPC nano-structureFGF-2 was loaded with alginate into the LBL-DPC nano-structure.FGF-2 immunofluorescence indicated that it was loaded into the LbL-DPC nano-structure successfully.ELISA assay shown that the LbL-loaded FGF-2 exhibited a sustained and time-dependent release profile.Cells were individed into four groups,DPC,DPC+FGF-2,LbL-DPC and LbL(FGF-2)-DPC.The proliferation activity and expression of cell markers of LbL(FGF-2)-DPC were higher than the other groups.Similarly,the statistical results demonstrated a significantly higher HF-induced density arising from LbL(FGF-2)-DPCs than that of other groups.Through the cell fluorescence tracer,it was confirmed that the regeneration of hair follicles derived from the transplanted cells.3.The establishment of chemical crosslinking LbL-DPCs for 3D culturing model and its hair-inducing capacityWe product cell spheroids by aggregation of LbL-DPCs through crosslinking alginate and Ca2+.We tested the size of DP spheroids under seeding various cell densities in a low-adhesive plate.The percentage of DP spheroids with diameters ranging from 80 to 150?m generated at 4×104 cells/well was significantly higher than other groups.Live/dead staining was performed to verify the viability of DP spheroids shown green fluorescence with good viability.As time proceeding,the diameter of DP spheroids did not significantly increase.After reseeding,DP spheroid adherence and cell migration were similar to those of mice primary DP.We constructed DP cell spheres with P2 and P8 DPCs,respectively.There was no significant difference in the number of DP spheroids with diameter between 80-150?m.Detection of gene and protein expression of DPC specific markers ALP,NCAM and Versican shown that,compared to P8 DPCs under 2D culture,the expression of these signature genes and proteins were well preserved in P8 DP spheroids,which was similar to P2 DPC in 2D culture.High-passaged DPC combined with epidermal cells after 3D culture can induce hair follicle regeneration in vivo,but failed in the high-passaged DPC in 2D culture.Conclusions1.LbL nano ultra-thin ECM was applied on a single DPC surface successfully.Compare to untreated DPCs,LbL nano-coating did not significantly influence biological characteristics of DPC(include the viability,proliferation and hair-inducing expressional profile).2.On the basis of LbL-coated single DPC culture model,FGF-2 was loaded in LbL-DPC nano-structure,which further enhance the hair-inducing capacity of a single DPC with a feasible micro-environment.3.The establishment of chemical crosslinking LbL-DPCs for 3D culturing model(DP spheroid)mimic primary DP in morphology and structure.It restored the biological characteristics and functions of high passaged DPC and displayed an excellent hair follicle inductive capacity in vivo.