Culture Of Human Melanocytes Based On Carboxymethyl Chitosan Hydrogel

Backgr ound and obj ectiveVitiligo is a common depigmented dermatological disease,and yet the pathogenesis is not well understood.In clinical implications,vitiligo is divided into stable phase and advanced phase.Surgical procedures are often associated with the treatment of stable or resistant vitiligo,including autologous grafting,melanocyte suspension transplantation,needling and dermabrasion.Specifically,the principle of melanocytes suspension transplantation is to transplant melanocytes into the depigmented site to achieve repigmentation directly.However,the contribution of fluidity and instability of melanocyte suspension cause the loss and uneven distribution of melanocytes,potentially limited the optimization of repigmentation.Further,with the implement of tissue engineering,transplantation can be completed with biomaterials with melanocyte fixed ontoThe purpose of our experiment is to prepare genipin-crosslinked carboxymethyl-chitosan hydrogel(CMCS-GEL)to culture the human primary melanocytes.physicochemical properties of CMCS-GELs with different proportions of carboxymethyl-chitosan and genipin are tested,in order to study the activity and proliferation of melanocytes cultured on CMCS-GEL.We investigated the optimal preparation conditions of CMCS-GEL,as wellas assessed the biocompatibility of CMCS-GEL.We aim to provide the experimental results to shed some lights on the future clinical treatment of vitiligo Methods3%carboxymethyl-chitosan solution and 0.1%genipin aqueous solution were mixed with the proportion of 1:0.5,1:1,1:1.5,and stored in a 37? incubator to crosslink for 24h,indexed as GEL-1,2,3.The prepared CMCS-GELs appeared pale gray-blue hydrogels with the thickness of 0.2cmTesting protocol of physicochemical properties of CMCS-GELs:1)carboxymethyl-chitosan,genipin and CMCS-GEL were tested by Fourier Transform Infrared Spectrometer(FTIR);2)the characterizations of CMCS-GEL were observed by scanning electron microscope(SEM);3)elastic modulus G'and viscous modulus G"of CMCS-GELs were tested by Rotational Rheometer;4)swelling ratios were calculated after putting freeze-dry CMCS-GELs into thermostatic water and interrupted weighed.Assess of biocompatibility of CMCS-GEL:1)Melanocytes were cultured on CMCS-GELs and observed by optical microscope and environmental scanning electron microscope(ESEM);2)cell counting kit-8(CCK-8)was used to test the cell activity of melanocytes cultured on CMCS-GELs;3)S-100 antibody was tested via immunofluorescence.Results1.FTIR showed the change and interactions between chemical groups and chemical bonds in CMCS-GEL compared to genipin and carboxymethyl-chitosan,indicating the finish of crosslinking.2.Under SEM,the structure of CMCS-GEL appeared to be cellular spongy three-dimensional,the pore size was about 50?m.3.Rotational Rheometer showed elastic modulus G' and viscous modulus G" of all groups of CMCS-GELs remained stable with the changed frequency.G'>G".all groups of CMCS-GELs conform the characteristics of hydrogel.The yield value of stress of GEL-1,2 was higher than GEL-3,and GEL-2 showed better stress resistant than other groups.4.Swelling ratios of GEL-1,2,3 presented gradual step-down trend,with the number of350%,275%,248%.This result indicated that all CMCS-GELs had strong ability of water absorption.5.The activity of melanocytes cultured on CMCS-GELs was tested by CCK-8 for three consecutive days.The cell viabilities GEL-1,2,3 were 0.975,0.98,0.892 at 24h,0.94,0.976,0.847 at 48h;0.848,0.969,0.748 at 72h.the results had no statistically significant difference(P>0.05).We concluded that cell viabilities of all the groups decreased,and GEL-2 had slightest change.6.The morphology of melanocytes cultured on CMCS-GELs and culture plates was observed under optical microscope and ESEM.Melanocytes have already attached after culture for 8h via optical microscope.After incubation for 48h,we observed the large nucleus of melanocytes and short dendrites,the nucleo-cytoplasmic ratio is big.While with the gradual growth of melanocytes,dendrites are thinner and longer,nucleo-cytoplasmic ratio is smaller under ESEM.7.S-100 antibody was tested via immunofluorescence.It showed that S-100 was expressed in melanocytes on culture plates and CMCS-GELs.The melanocytes on culture plates present the long dendrites,and small soma.The adjacent cells form connections like a network.The melanocytes on CMCS-GELs had edge blurring,bigger soma,less dendrites.ConclusionsCarboxymethyl chitosan hydrogel(CMCS-GEL)has stress resistance and good absorbency.The excellent biocompatibility of CMCS-GELis capable of keeping the growth and morphology of melanocytes,and maintaining the normal functions of melanocytes.