The Study On Hair Follicle Reconstruction With The Organoid-loaded Stratified GelMA-cryomicroneedles

Background:Human mature organs and even tissues have obvious limitations in their regenerative ability.Once they experience atrophy and necrosis of tissues or organs,it is almost difficult to repair.Hair loss caused by atrophy and necrosis of hair follicles is also difficult to repair.Therefore,the research of hair follicle reconstruction in regenerative medicine and tissue engineering is a promising method to solve the hair follicle defect.However,the current classical hair follicle reconstruction methods still have some limitations.In addition to the need to use surgery to construct a certain microenvironment required by the wound to provide cells,there is also the difficulty of directly rebuilding hair that can control the number and location and break through the growth of the skin.The application of hair follicle organs in the reconstruction and regeneration of hair follicles has been paid more and more attention.By simulating the cell state of hair follicles during the embryonic stage,hair follicles with normal physiological structure can be realized through surgical implantation in the form of 3D culture.However,in addition to in vitro culture and model construction,this method still requires the use of surgical instruments for wound implantation,which requires a certain threshold of operation.Microneedles are an emerging minimally invasive delivery method.They form short water-based micro-channels through the cuticle through micron-level needle tips to painlessly deliver carrying contents to the dermis where hair follicles are located.It seems to be a good choice to deliver organoids to achieve hair follicle reconstruction.However,in the preparation of traditional microneedles,in order to have a certain mechanical strength,the use of excessively high concentration of materials will affect the migration,aggregation and differentiation ability of cells.The drying and drying steps in the preparation process will severely damage living cells,making it difficult for cells to maintain vitality.Therefore,it is necessary to apply a new way to achieve the improvement of mechanical strength.Liquid water forms solid ice through the physical change of cryogenic freezing,which has obvious mechanical strength improvement and can be well shaped.However,the formation of ice crystals will cause damage to cells,and the addition of cell cryopreservation is necessary.In this study,a minimally invasive new hair follicle reconstruction method was achieved by coordinating the balance among hydrogels carrying organoids,freezing to improve the mechanical strength of micronedles,and maintaining the vitality and dryness of cells in organoids.Method:1.Screening and optimization of three-dimensional culture hydrogels carrying cells:The suitable hydrogel materials for the construction of frozen microneedles were selected based on the morphology maintenance ability of methacrylate gelatin,hyaluronic acid and matrix glue in freezing and melting.To further verify the degradation of methacrylic anhydride gelatin in vitro and optimize the appropriate concentration of hydrogels for organoid culture.2.Preparation and characterization of stratified frozen microneedles carrying organoids:By adjusting the ratio between cell cryopreservation and methacrylate gelatine,and by screening the ratio of broken needles,1×106mL-1 neonatal mouse dermal cells and 0.5×106mL-1 epidermal cells were used to construct microneedle needle tips carrying hair follicular organs.The microneedle base was filled with 2%DMSO frozen solution.Then it was programmed to refrigerate at-20℃ for 4h,80℃ for 4h and-196℃ for 1h.Frozen microneedles were prepared by mixing Rhodamine B with tip solution and fluorescein isothiocyanate with base solution.Cell distribution consisted of CM-DiO labeled dermal cells and DAPI labeled epidermal cells,and Rhodamine B labeled cells were mixed into the needle body.Morphological observation was performed using fluorescent stereopicroscope and L M980 laser confocal microscope.The frozen GelMA-cryo microneedles were dried in a freeze dryer and prepared by drying method.After being sprayed with gold powder and fixed with conductive tape,the microneedles were observed and photographed by scanning electron microscope.A material mechanics measurement system was used to detect microneedles prepared by four different methods and materials:dried GelMA,frozen pure water microneedles,frozen Gelma-Cryo microneedles,and melted Gelma-Cryo microneedles.The frozen microneedles were precooled using liquid nitrogen on the loaded base.The cells attached to the stratified frozen microneedles were labeled with DAPI and applied to pig skin and nude mouse skin.The transdermal delivery results were observed with a positive fluorescence microscope.3.Evaluation of frozen methacrylic anhydride gelatin hydrogel as cell carrier:The cell viability of neonatal rat dermal cells,epidermal cells and 2:1 ratio of dermal cell suspension hydrogel loaded with GelMA-cryo hydrogel after photocrosslinking and programmed cooling was detected using cell viability dye.As a control,the preparation process of microneedles by drying method was simulated,and the cells of the same group were photocrosslinked and dried for 24-48h to detect cell viability.The results were observed with an inverted fluorescence microscope.GelMA-cryo hydrogel with 2:1 ratio of dermal cells was used to prepare the frozen micrhenedles.After the programmed cooling was completed,the cells were quickly resuspended at 37℃,and cell viability was tested with cell viability dye.Similarly,microneedles equipped with 2:1 ratio of dermal cells were prepared by drying method,and cell viability was detected by cell viability dye after demoulding.The results were observed by L M980 laser confocal microscope and 3D reconstruction.Frozen microneedles were used to transfer organoids to confocal dishes containing PDMS models for in vitro culture,and 10%FBS/DMEM medium was added for culture.The results were observed under stereopicroscope and inverted microscope.Dermal cells were labeled with CM-DiO and epidermal cells with CM-Dil.The ice needle was prepared and delivered to a confocal dish.Cell changes were observed by LSM980 laser confocal microscope and three-dimensional reconstruction.4.Evaluation of freezing methacrylate gelatine with organoids for hair follicle reconstruction:Frozen microneedles(containing 1×106mL-1 dermal cells and 0.5×106mL-1 epidermal cells)equipped with organoids prepared by programmed cooling were applied to the back of nude mice,and the organoids were implanted into the skin by microneedles.The CM-Dil labeled cells were photographed and the results were analyzed using the in vivo imager.At day 0,5,7 and 14,the stereopicroscope was used to observe and shoot.The material was fixed on the 14th day.HE staining and double standard immunofluorescence staining of K1,K14,K15,VCAN,α-SMA,TH and other proteins were performed.The same concentration of dermal cells was injected into the back subcutaneous area of nude mice using injection,a traditional method of hair follicle reconstruction.At day 0,5,7 and 14,the stereopicroscope was used to observe and shoot.The material was fixed on the 14th day.HE staining was performed.The results of HE staining were observed and recorded by an upright optical microscope,and the results of immunofluorescence double-label staining were observed and recorded by an LSM980 laser confocal microscope.Result:1.Screening and optimization of three-dimensional cell culture with scaffolds for cryopreservation conditions:Compared with non-crosslinked GelMA,hyaluronic acid and Matrigel,crosslinked GelMA hydrogels have better morphological maintenance ability after freezing and melting.GelMA hydrogel has good degradability and can be completely degraded in vitro in 12 days.Neonatal mice dermal cells were cultured with 10%GelMA hydrogel,and the cells were difficult to migrate and aggregate to form cell aggregates.The cells were cultured with 5%GelMA hydrogel and Matrigel,and the cells could aggregate into several cell aggregates.2.Preparation and characterization of stratified GelMA-cryoMNs equipped with organoids:The stratified frozen GelMA-cryoMNs were prepared by 5%GelMA-cryo hydrogel and 2%cryopreservation solution,and the 5×5 array conical microneedles with the distance from base to tip～1200 μm and the base width～800 μm were obtained.The needle structure composed of GelMA-cryo hydrogel is～700 μm high and～470μm wide.The epidermal cells gathered at the vertical distance of 0-600 μm from the tip of the needle.Scanning electron microscopy(SEM)showed that the GelMA-cryoMNs had dense,uniform and interconnected pores on the surface of the needle tip,which contained organoid constituent cells.Drying MNs are compact,smooth and flat without obvious pores.According to the compression modulus test,both drying and freezing MNs have high mechanical strength,while melting MNs do not have transdermal mechanical strength.The physical change of freezing increases Young’s modulus by 110 times.The results of skin puncture test showed that the frozen MNs had the ability to penetrate the skin stratum corneum to deliver organoids to the dermis.3.Evaluation of frozen methacrylate gelatin hydrogels as cell carriers:DCs or ECs and their combinations maintained good survival rates after freezing resuscitated cells using GelMA-cryo hydrogels.The survival rates of DCs and ECs were 58.31 ± 1.48%and 63.08±0.97%,respectively.The cell viability was 68.65±0.90%after cryopreserved in 2:1 ratio of DCs and ECs.As a control,the effect of drying method on cell viability was observed.After drying by drying method,all the cells of DCs and ECs embedded alone or mixed in GelMA hydrogel completely lost vitality after ultraviolet light crosslinking and oven drying,and the cell viability rate was 0%.The results of in vitro delivery culture showed that organoids delivered and resuscated by frozen microneedles still had the ability of cell migration,aggregation and self-assembly,and hair bud differentiation structure could be seen after 14 days of in vitro culture.The cell color trace showed that the cells were further clustered into cell clusters by discrete cells.4.Evaluation of the freezing of methacrylate gelatine with organoids for hair follicle reconstruction:Among the 25 sites reconstructed with frozen microneedles,hair follicle reconstruction was achieved at 22 sites(88%),and among the 22 sites(50%),1-3 hairs broke through the skin growth.Only the disordered growth of reconstructed black hair was seen under the skin by injection.In vivo imaging showed that the implanted organoids remained active in the mouse skin for 14 days.HE staining showed that hair follicles reconstructed with frozen microneedles showed black hair shafts extending vertically from the dermis through the stratum corneum.The structures of the outer and inner root sheaths are seen.The outer root sheath is attached to the skin of the host,and the cytoplasmic sac-like sebaceous glands are attached to the outer root sheath.The muscle fibers are seen to extend,namely,the arector pili muscle.As a control,the hair follicles reconstructed by injection,HE staining sections showed that the hair follicles reconstructed by injection were clumps wrapped under the skin,with disordered growth directions between hairs,and no reconstructed hair passed through the skin surface.Reconstruction of hair follicles by immunofluorescence staining.Cytokeratin 1 is expressed in the upper basal layer of the epidermis and the inner root sheath of the reconstructed hair follicle,and cytokeratin 15 is expressed in the eminence region at the end of the isthmus of the hair follicle.Dermal papilla cells specifically express multifunctional proteoglycans.Cytokeratin 14 is expressed in the outer root sheath of hair follicles and sebaceous duct epithelial cells).The eminence is connected to the erector pili muscle,which expresses alpha-smooth muscle actin.You can see that the sympathetic nerve accompanying it expresses tyrosine hydroxylase in the pili erector muscle.Conclusion:1.The crosslinked GelMA hydrogel has good morphological maintenance ability,which conforms to the degradation time required for hair follicle reconstruction.GelMA hydrogel with 5%concentration is suitable for 3D cell culture,which can realize cell migration and aggregation to form cell aggregates.2.5%GelMA-cryo hydrogel is used to make the tip of the needle and 2%freeze-storage solution is used to make the base.Frozen microneedles with complete form can be obtained,and the cells on board can gather in the tip of the needle and be wrapped in the porous hydrogel.The freezing process greatly improves the mechanical force of low-concentration hydrogels,and the frozen microneedles can achieve transdermal delivery of GelMA organs.3.Drying method is not suitable for preparation of microneedles loaded with living cells;5%GelMA-cryo hydrogel can effectively protect cell viability,and in vitro culture proved that the resuscated organoids had the ability to further aggregate and differentiate,forming hair follicle-like hair bud structure.4.Frozen layered GelMA-cryo microneedles successfully reconstructed hair follicles that broke through the skin surface in the back of nude mice.The reconstructed sites contained 1-3 hair shafts,which was consistent with the physiological growth of hair follicles.The complete structure of the normal hair follicle that forms interconnections with the host.