Decellularized Extracellular Matrix Hydrogel From Human Placenta To Recover Hair Inductive Characteristic Of Dermal Papilla Cells For Hair Follicle Regeneration

BackgroundHair follicle(HF)is appendage with self-regeneration ability of skin,undergoing cyclical bouts of the stage of growth(anagen),destruction(catagen)and rest(telogen),which controls the hair growth.Besides the basic functions of hair such as regulating body temperature,secretion and excretion,the hair also has cosmetic emphasis on physical appearance.Nowadays,hair loss caused by aging,diseases,trauma and medications has distressed impact on people’s appearance and social activity.At present,there are two options to treat hair loss.One is medicine including finasteride and minoxidil,however these two drugs have some adverse effects.Another is hair transplantation surgery,which transplant hair follicles from unbald area to bald area through surgical treatment.Nevertheless,these two methods all fail to generate new hair follicles in bald scalp.In recent years,with the progress of tissue engineering and regenerative medicine,reconstructing the hair follicle by tissue-engineering technology becomes the effective way to solve this problem.Seeding cells,growth factors and scaffolds are three main factors of the tissue engineering.The hair follicle is composed of epidermal(epithelial)and dermal(mesenchymal)compartments and the interaction between these two parts is essential for hair follicle regeneration.The epidermal components include hair follicle stem cells,hair germinal matrix cells,the inner and outer root sheath,while the dermal components mainly contain dermal papilla(dermal papilla cell,DPC)and connective tissue sheath.When hair follicle formation first occurs during embryogenesis,the epithelial part requires the guidance of the DPC to be organized into complicated hair structure.In other words,the DPC is capable in the induction of new hair follicles,the regulation of morphogenesis,and the control of the hair growth cycle,which is considered to be the optimal seeding cell in hair follicle regeneration.Studies have shown that the hair-inducing ability of DPCs is closely related with their aggregative growth pattern.However,it is a pity that DPCs tend to lose their inductive ability during subculture when cells were cultured on traditional 2D surfaces.Thus,the important issue to the regeneration of human hair follicle by tissue engineering is the maintenance of the hair-inducing ability of DPCs when expanding them in vitro.Physiologically,nearly all tissue cells reside in an extracellular matrix(ECM)composed of collagens,proteoglycans,glycoproteins,and glycosaminoglycans.,ECM provide natural cellular environment that regulates cell behaviors such as cell survival,proliferation,morphogenesis,migration,and differentiation.ECM interact with distinctive cells through its structural features and other cues such as its bound growth factors and intracellular signaling,each of which result in cell fate decision.In vivo,DPCs are packed with ECM,showing an aggregative pattern of cell growth.Therefor ECM extracts were introduced as cell culture matrices,also known as scaffolds in which cultured DP cells can grow into 3D spheres to maintain their hair inductive potential.According to these cognition,we think it is feasible that ECM extracts were introduced as cell culture scaffold on which DPCs can grow into spheres to maintain their hair inductive potential.However,it is difficult to simulate the ECM microenvironment in vitro because of the complex three-dimensional structural organization and functional molecules of the native ECM.With the development of the decellularized technology,preparing the decellularized ECM(DECM)scaffold becomes an alternative to solve this problem and is gaining a lot of attention.DECM material is typically fabricated through physical,chemical,and enzymatic treatments or their combinations to remove cellular material while preserving the native ECM ultrastructure and composition.When processed appropriately,harvested DECM can retain the biochemical complexity,nanostructure,and bio-inductive properties of the native ECM.At present,DECM material have been successfully used in the field of regeneration of skin and adipose tissue,but in the field of HF regeneration not been reported.ObjectiveThis study was divided into three parts.The first part includes fabrication of decellularized extracellular hydrogel derived from human placenta(HPECM hydrogel).The second part includes observation of growth pattern of human dermal papilla cells(hDPCs)cultured on the HPECM hydrogel.The third part is to evaluate efficacy of the HPECM hydrogel to recover the hair inductive property of DPCs for hair follicle regeneration.Methods1.Establishment of 3D decellularized human placenta hydrogel culturing model of human dermal papilla cellsHuman placenta was obtained from donors undergoing normal or caesarean deliveries at Nanfang Hospital of Nanfang Medical University after ethic approval.Whole frozen human placenta stored at-80℃ was allowed to thaw at 4℃.The placenta was washed in one liter of 4℃ cold distilled water supplemented with 1%penicillin-streptomycin(Gibco,NY,USA)for 30 mins at 1200 RPM on an orbital shaker.This process was repeated until excess blood was significantly reduced.To decellularize the placenta,the washed tissue was shaken at 200 RPM in one liter of devitalization buffer supplemented with 2.5 g N-Ethylmaleimide(Sigma Aldrich,St.Louis,MO,USA),20 g N-laurylsarcosine(Sigma Aldrich),and 1%penicillinstreptomycin(Gibco)overnight at room temperature.Next,10.2 g of wet tissue was digested in 250ml 1 mg/ml pepsin(Sigma Aldrich)/0.1 M Hydrochloric Acid(HCL)solution for 64-72h.Following the about 60 hours,the pepsin HCI was brought to pH 8.0 briefly then to pH 7.0-7.2 using sodium hydroxide and HCL.The resulting liquid product of hpECM appeared viscous and near translucent in appearance and it was induced to form a gel at 37℃.To evaluate the effectiveness of the decellularization process,hematoxylin-eosin(HE)staining technique was conducted.The concentration of fibroblast growth factor(FGF)and platelet-derived growth factor(PDGF)in the solubilized hpECM were quantitatively measured with a Quantikine enzyme-linked immunosorbent assay kit(Sigma)following the manufacturer’s instructions.The hpECM solution was stored at-80℃ for long-term storage,or at 4℃ for use within 2 weeks.Human scalp samples were collected from patients who underwent face lifting surgery after obtaining informed consent.DP was isolated by microinjection combined with enzymatic digestion,then isolated dermal papillae were transferred onto plastic dishes and cultured in the Dulbecco’s modified Eagle’s medium(DMEM)supplemented with 1%(v/v)penicillin-streptomycin and 20%(v/v)fetal bovine serum(FBS)in a humidified 5%CO2 incubator at 37℃.Once the outgrowth reached to 80%confluence,hDPCs were harvested with 0.25%(w/v)trypsin/EDTA and transferred to new culture dishes with a split ratio of 1:2.Afterward,hDPCs were maintained in the DMEM supplemented with 10%FBS.HDPCs at passage 2 were used in positive control group,and hDPCs at passage 8 were used in experimental group and negative control group.For 3D cell culture,HPECM hydrogel was formed by placing 50μL/well of liquid HPECM solution in a 96-well plate and incubating the solution for 15-30min in a humidified incubator with 5%CO2.Following gelation,1 ×104 hDPCs/well,a cell number commonly used for Matrigel morphogenesis assay,were cultured on the HPECM hydrogel.The cells were incubated at 37℃ and imaged under a reverse phase-contrast microscope.HDPCs cultured on HPECM hydrogel were stained using a commercial Live/Dead Viability/Cytotoxicity kit according to the manufacturer’s instruction.After 20min incubation at 37℃,the stained samples were visualized using a fluorescence microscope.2.Evaluation of the HPECM hydrogel to recover the hair inductive property of DPCs for hair follicle regeneration.HDPCs at Passage 2 and Passage 8 were prepared for HPECM hydrogel culture or conventional 2D culture according to experimental design.All HPECM-cultured DP spheres or 2D-cultured DPCs were characterized with the hair inductive property as the followings:qRT-PCR was introduced to evaluate the gene expression of versican,ALP and β-catenin;immunofluorescence staining was introduced to qualitatively evaluate the protein expression of versican,ALP and β-catenin;western blotting was introduced to quantitatively evaluate the protein expression of versican,ALP and β-catenin.3.The application of 3D decellularized human placenta hydrogel culturing model of human dermal papilla cells for in vivo hair-follicle regenerationFor in vivo HF induction assay,freshly isolated epidermal cells from newborn mouse with DPCs(2D-P8-DPCs as negative control group;2D-P2-DP cells as positive control group;HPECM-P8-DP spheres as experimental group)were injected subcutaneously into the dorsal side with 29-gauge needles.The grafted specimens were evaluated under macroscopic observation and histological examination by H&E staining.Results1.The establishment of 3D HPECM hydrogel culturing model of human dermal papilla cellsMicroinjection combined with enzymatic digestion isolated DP exhibited an completely pyriform or oval shape.Migrated DPCs were observed to exhibit a longspindle growth pattern.Self-aggregation behavior of cultured DPCs were gradually lost during passaging.When DPCs were cultured on HPECM hydrogel with the seeding density as 1×104/well,DPC exhibited spherical growth pattern on day 3.As time goes on,there was no obvious change of DP sphere appearance.The result of Live/dead staining verified good viability of DP sphere,which indicated the HPECM hydrogel has favorable compatibility.2.Evaluation of the HPECM hydrogel to recover the hair inductive property of DPCs for hair follicle regenerationCompared with 2D-P2-DPCs which acted as positive control,qRT-PCR showed that mRNA expression of genes associated with hair-inducing ability—ALP,βcatenin,and versican—was lost in 2D-P8-DPCs,whereas expression of these characteristic markers were restored when cells formed spheres on HPECM hydrogels.The protein expression of ALP,β-catenin,and versican were verified by immunofluorescence staining,and results were similar those of qRT-PCR,confirming that expression of these three proteins was higher in HPECM-P8-DP spheres compared with 2D-P8-DPCs.Further quantitation by immunoblotting showed that the HPECM-P8-DP spheres expressed all these signature markers,whereas in 2D-P8DPCs they were barely expressed.3.The application of 3D decellularized human placenta hydrogel culturing model of human dermal papilla cells for in vivo hair-follicle regenerationThree weeks after implantation,large numbers of newly-formed HFs were observed within the hypodermis of nude mice in the positive control group(2D-P2DPCs),and a few HFs were visible in the HPECM-P8-DP spheres group;whereas nothing could be seen in the negative control group(2D-P8-DPCs).Corresponding haematoxylin and eosin-stained sections showed that the organization of these newlyformed HFs induced by 2D-P2-DPCs group and HPECM-P8-DP spheres group resembled that of a mature follicle,while 2D-P8-DPCs group did not present any follicle structure.Conclusion1.HPECM hydrogel was designed as biological scaffold successfully.Compare to 2D cultured DPCs,HPECM hydrogel did not significantly influence the viability of DPC.2.DP spheres formed on HPECM hydrogel can restore and maintain the hair-inducing ability of high-passaged DPC,suggests that HPECM hydrogel supply DPC with a favorable micro-environment.3.HPECM hydrogel culturing model not only can restore and maintain the biological characteristics and functions of high passaged DPC,but also an induce hair follicle reconstruction in vivo.