Methods for preparing dermal papilla cell spheroids and their applications

Abstract

This invention provides a method for preparing dermal papilla cell spheres, comprising mixing hydrophobic hyaluronic acid grafted with hydrophobic groups and cyclodextrin modified with crosslinking groups to obtain a hydrogel prepolymer, adding a dermal papilla cell suspension to the hydrogel prepolymer to initiate a crosslinking reaction of the crosslinking groups to form a dynamically crosslinked hydrogel loaded with cells, and then adding a cell culture medium. The hydrophobic groups are selected from tert-butylbenzene, ibuprofen, menthol, etc. This method can rapidly obtain dermal papilla cell spheres and maintain the stemness of dermal papilla cells and the induction ability of hair follicle tissue, which can be used to achieve hair follicle tissue repair and regeneration, and promote hair growth. This invention also relates to the application of the method for preparing dermal papilla cell spheres in the preparation of compositions that promote hair follicle tissue repair, regeneration, or hair growth, wherein the composition includes a dynamically crosslinked hydrogel loaded with dermal papilla cells, which can be used to prevent and treat hair loss.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing dermal papilla cell spheroids and its application. Background Technology

[0002] Dermal papilla cells (DPCs) are specialized mesenchymal cells located at the base of the hair follicle, within the dermal papilla structure. DPCs secrete various growth factors, cytokines, and signaling molecules (such as FGF, BMP, WNT, and Shh) to regulate the behavior of the entire hair follicle. They can induce epidermal stem cells to initiate and form a new hair follicle. DPCs also regulate the hair follicle cycle, directing the hair follicle from the anagen (growth) phase to the catagen (regression) phase, then to the telogen (resting) phase, and then initiating a new anagen phase. Furthermore, DPCs can transmit signals to control hair thickness, length, color (by regulating melanocytes), and type (such as scalp hair and body hair). The dermal papilla (DP) is a multicellular microstructure composed of DPCs located at the base of the hair follicle and is a key inducing factor in regulating hair follicle morphogenesis, the hair growth cycle, and hair morphology. DPs are surrounded by an abundant extracellular matrix (ECM) and are accompanied by rich blood vessels and neural networks. The signaling molecules secreted by DPC determine the degree of activation of hair follicle stem cells, which in turn regulate the volume of the hair bulb, the thickness of the hair, the growth rate, and the cycle length.

[0003] In vivo, the "cellular microenvironment" upon which dermal papilla cells (DPCs) maintain their phenotype and function is composed of surrounding cells, cytokines, and the extracellular matrix (ECM). Nanoscale interactions between cells and the ECM play a crucial role in maintaining cellular function. Studies have shown that DPCs form spherical aggregates, which helps maintain their hair follicle-inducing ability and can induce new hair follicle growth in human skin; conversely, once transferred from a three-dimensional environment to a traditional two-dimensional culture system, their hair-inducing ability is rapidly lost. Therefore, the formation of dermal papilla cell clusters is a core issue. The size of dermal papilla cell clusters varies significantly with the hair follicle growth cycle, follicle type, and body location. In hair follicle biology and regeneration research, the number of dermal papilla cells and the size of their clusters directly determine the strength of their growth signals. Sufficiently large, compact three-dimensional clusters are key to maintaining their "instruction" function. In hair transplantation, the transplanted follicular unit must contain an intact, undamaged dermal papilla to survive and grow. Studies have shown that only when in vitro expanded and cultured dermal papilla cells spontaneously aggregate into sufficiently large clusters (typically requiring a diameter > 100 micrometers) can they be transplanted into the skin to induce the formation of new hair follicles. Traditional two-dimensional culture methods are not ideal for obtaining dermal papilla cell clusters, and reported three-dimensional culture methods are cumbersome and cannot rapidly obtain cell spheroids. Furthermore, due to the limited in vitro DPC expansion capacity, it remains difficult to stably obtain a sufficient number of bioactive DPC cells for tissue engineering reconstruction of hair follicles. Existing three-dimensional culture systems face the following main problems:

[0004] (1) Cell recovery is difficult and the operation is complicated: Some hydrogel systems are difficult to recover cells gently and quickly after gel formation. The preparation and operation process is complicated, which affects experimental efficiency and clinical translation feasibility.

[0005] (2) Mismatch of mechanical properties and limited cell behavior: Traditional irreversible cross-linked hydrogels, due to their stable cross-linking network and high rigidity, limit the three-dimensional migration, aggregation and mechanical signal transmission between DPC and cell-ECM, making it difficult to simulate the dynamic microenvironment of native DP, thus leading to the decline of DPC function.

[0006] (3) Poor stability of the culture system and uneven structure formation: The existing hydrogels have uneven distribution of physicochemical properties in three-dimensional space, resulting in significant differences in the growth state of DPC on the surface and inside of the hydrogel, making it difficult to achieve a uniform and high-quality multi-cell aggregate structure (i.e., the formation of artificial DP).

[0007] In summary, the current three-dimensional culture system for DPC still has significant shortcomings in terms of cell function maintenance, dynamic signal transduction, biosafety, and operability, making it difficult to meet the large-scale culture requirements of functional dermal papilla cell spheres for hair follicle tissue engineering regeneration. New methods for obtaining bioactive dermal papilla cell spheres need to be developed. Summary of the Invention

[0008] To address the problems urgently needing to be solved in the prior art, one objective of this invention is to provide a method for preparing dermal papilla cell spheres. These dermal papilla cell spheres possess the ability to maintain the stemness of dermal papilla cells and induce hair follicle tissue, and can be used to achieve hair follicle tissue repair and regeneration, as well as promote hair growth. The method includes the following steps:

[0009] 1) A hydrogel prepolymer is obtained by mixing hydrophobic hyaluronic acid grafted with hydrophobic groups and cyclodextrin modified with crosslinking groups, wherein the hydrophobic groups grafted onto the hyaluronic acid can act as guest molecules and interact with cyclodextrin as host molecules.

[0010] 2) Take the hair papilla cell suspension and add it to the hydrogel prepolymer to initiate the cross-linking reaction of the cross-linking groups to form a dynamic cross-linked hydrogel loaded with cells, and add cell culture medium.

[0011] In some embodiments, the hydrophobic group is selected from hydrophobic structural groups such as tert-butylbenzene, ibuprofen, and menthol.

[0012] In some embodiments, the crosslinked group-modified cyclodextrin is acrylated cyclodextrin. The hydrophobic group-grafted hyaluronic acid in the hydrogel prepolymer has a mass-volume percentage of 1%-10%, and the crosslinked group-modified cyclodextrin has a mass-volume percentage of 1%-10%.

[0013] In some embodiments, the hydrogel prepolymer further comprises gelatin. Optionally, tert-butylbenzene-grafted hyaluronic acid and gelatin are mixed as guest molecules to form a composite dynamically cross-linked hydrogel, wherein the mass-volume percentage of the hydrophobic group-grafted hyaluronic acid is 1%-5% and the mass-volume percentage of the gelatin is 1%-5%.

[0014] In some embodiments, the cell culture medium is DMEM, DMEM / F12, etc.

[0015] Another objective of this invention application is to provide an application of a dynamically cross-linked hydrogel in the preparation of dermal papilla cell spheroids. The hydrogel is a hydrogel prepolymer composed of hydrophobic groups grafted onto hyaluronic acid and cross-linked groups modified cyclodextrin, formed by initiating a cross-linking reaction of the cross-linked groups; the hydrophobic groups grafted onto the hyaluronic acid are selected from tert-butylbenzene, ibuprofen, menthol, etc.

[0016] Another object of this invention application is to provide a composition for promoting hair follicle tissue repair and regeneration. This composition comprises a dynamically cross-linked hydrogel loaded with dermal papilla cells, wherein the dynamically cross-linked hydrogel is formed by cross-linking initiated after mixing the dermal papilla cells and a hydrogel prepolymer solution. The hydrogel prepolymer comprises hyaluronic acid grafted with hydrophobic groups and cyclodextrin modified with cross-linking groups, wherein the hydrophobic groups grafted onto the hyaluronic acid interact with the cyclodextrin via a host-guest interaction. Further, the hydrogel prepolymer also includes gelatin. Preferably, the hydrophobic groups are selected from at least one of tert-butylbenzene, menthol, or ibuprofen.

[0017] In some embodiments, the dynamically cross-linked hydrogel is further loaded with at least one of hair matrix cells, hair follicle stem cells, outer root sheath cells (ORS cells), fibroblasts (FB), endothelial progenitor cells (EPC), or human umbilical vein endothelial cells (HUVEC).

[0018] Another object of this application is to provide the use of the composition in the following aspects: i) preventing and treating hair loss; ii) promoting hair growth; iii) hair follicle repair and regeneration.

[0019] The method for preparing dermal papilla cell spheres in this application can mimic the ECM microenvironment within dermal papilla cells, thereby rapidly forming dermal papilla cell spheres. It also significantly enhances the expression of relevant markers (such as ALP and β-catenin) and hair-inducing ability within the dermal papilla cell spheres, allowing for in vitro proliferation to form dermal papilla tissue and maintaining its hair follicle regeneration and repair function in vivo. Furthermore, the dynamically cross-linked hydrogel loaded with dermal papilla cells used in this method can reconstruct a hair follicle-like microenvironment, maintaining its function long-term after implantation and promoting hair follicle repair and regeneration. Attached Figure Description

[0020] Figure 1 Materials characteristics of hydrogels HA-CA and HA-MT: a) DMT modulus; b) Normalized stress, stress test and relaxation time.

[0021] Figure 2 Microscopic images of dermal papilla cells in a dynamic hydrogel.

[0022] Figure 3 The expression levels of ALP, β-catenin, and α-SMA in DPC cells obtained by hydrogel in vitro expansion (p < 0.01).

[0023] Figure 4 Microscopic observation of pore size and statistical chart of pore diameter of HA-MT dynamic cross-linked hydrogels with different ratios.

[0024] Figure 5a) Schematic diagram of the hair induction ability of artificial hair follicles constructed with ultra-dynamic hydrogel; b) Gross image of the transplantation site 3 weeks after transplantation; and c) HE section of the transplantation site (*p<0.05, **p<0.01). Detailed Implementation

[0025] Unless otherwise stated, the reagents used in the following examples are commercially available products or obtained according to existing methods in the art.

[0026] This invention focuses on the design requirements of functional biomaterials in the treatment of hair loss through tissue engineering. It adopts a strategy centered on adapting the mechanical microenvironment to maintain cell function and constructs a dermal papilla cell adaptive ultradynamic hydrogel system to achieve highly biomimetic mechanical properties and controllable release of cell derivatives. This system promotes the formation and function maintenance of dermal papilla cell spheres while restoring hair follicle regeneration capabilities.

[0027] Example 1 (GelCD)

[0028] This embodiment uses dynamically cross-linked hydrogels to simulate the cytoplasmic environment of dermal papilla cells, and is used to expand dermal papilla cells in vitro and form dermal papilla cell spheres with hair follicle induction function. The specific experimental method is as follows:

[0029] 1. Preparation of dermal papilla cell (DPC) suspension

[0030] 1.1) Cell source and processing:

[0031] Fresh hair follicle units were selected from patients undergoing autologous hair follicle transplantation (FUE), rinsed with PBS, and separated under a stereomicroscope to harvest the hair bulb. The hair bulb was then digested with 0.1% neutral protease, and the dermis and epidermis of the hair follicle were separated.

[0032] 1.2) Cell digestion and culture:

[0033] Hair follicle dermal tissue was digested with a mixture of 0.2% collagenase and 0.1% neutral protease (37°C, 1–2 hours, mixing every 15 minutes). After terminating the reaction, the tissue was centrifuged, resuspended in PBS, and cultured in DMEM medium containing 10% FBS.

[0034] 2. Construction of dynamically cross-linked hydrogels

[0035] 2.1 Preparation steps of dynamic hydrogel prepolymer:

[0036] a. Synthesis of acrylamide cyclodextrin (Ac-β-CD):

[0037] At 0°C, β-cyclodextrin was dissolved in DMF, and 5–10 molar amounts of acryloyl chloride and triethylamine were added dropwise to react. After filtering off the byproducts, the mixture was dried by rotary evaporation to obtain Ac-β-CD solid.

[0038] c. Preparation of hydrogel prepolymer:

[0039] Ac-β-CD and gelatin were dissolved separately in PBS buffer, and a photoinitiator was added to prepare a prepolymer hydrogel solution; the viscoelastic properties of the hydrogel could be adjusted by controlling the ratio of the two. Preferably, the gelatin content in the hydrogel prepolymer solution was 3%-5% (w / v), and the content of acryloyl β-cyclodextrin was 3%-5% (w / v).

[0040] 3. Construction of a three-dimensional cell culture and expansion system

[0041] Take a suspension of P3–P5 generation dermal papilla cells and add a hydrogel prepolymer solution (containing I2959 photoinitiator). The cell density is approximately 2*102 4 / μL. Cross-linking was initiated using UV light (365nm, 2–5 min) to form a hydrogel encapsulating DPCs. Complete culture medium (45ml DMEM solution + 5ml FBS + 500ul penicillin-streptomycin) was added, and the mixture was incubated statically at 37°C and 5% CO2.

[0042] 4. Cell recovery methods

[0043] To recover cell clusters, host-guest molecules with higher affinity (such as adamantane hydrochloride, 0.2–1 mM) can be added to the culture system to achieve hydrogel degradation and release of cell aggregates. This method is mild, biocompatible, and does not damage cell structure.

[0044] Example 2 (HA-TP)

[0045] The difference between this embodiment and Example 1 is that the dynamically cross-linked hydrogel prepolymer used includes tert-butylbenzene-grafted hyaluronic acid and acrylated cyclodextrin. The synthesis method of tert-butylbenzene-grafted hyaluronic acid is as follows: HA-TBA is dissolved in DMSO, and with the number of HA-TBA structural units as 1 equivalent, 2-3 equivalents of p-tert-butylphenylacetic acid, 0.75 equivalents of DMAP, and 1.2-2.0 equivalents of di-tert-butyldicarbonate are added sequentially to the above solution system; after reacting at 45°C, the product is freeze-dried; the product is then dialyzed and freeze-dried to obtain the final product.

[0046] Preparation of hydrogel prepolymer:

[0047] Ac-β-CD grafted with tert-butylbenzene hyaluronic acid (HA-TP) was dissolved in PBS buffer, and a photoinitiator was added to prepare a prepolymer hydrogel solution; the tert-butylbenzene grafted hyaluronic acid content in the hydrogel prepolymer solution was 1%-10% (w / v), and the content of acryloyl β-cyclodextrin was 1%-10% (w / v).

[0048] Example 3 (HA-TP-GEL)

[0049] To further regulate the dynamic properties of hydrogels, hydrophobic groups grafted onto hyaluronic acid and gelatin can be mixed as guest molecules to form a composite dynamic cross-linked hydrogel.

[0050] This embodiment uses the method of Example 2 to prepare a dynamic cross-linked hydrogel, the difference being that the hydrogel prepolymer further includes gelatin, wherein the content of tert-butylbenzene-grafted hyaluronic acid is 1%-5%, and the content of gelatin is 1%-5%.

[0051] Example 4 (HA-MT)

[0052] This embodiment uses the method of Example 2 to prepare a dynamically cross-linked hydrogel. The difference is that menthol-grafted hyaluronic acid is used in the hydrogel prepolymer. The synthesis method of menthol-grafted hyaluronic acid is as follows: HA-TBA is dissolved in DMSO. With the number of HA-TBA structural units as 1 equivalent, 2-3 equivalents of menthol succinate monoester, 0.75 equivalents of DMAP, and 1.2-2.0 equivalents of di-tert-butyl dicarbonate are added sequentially to the above solution system. After 24 hours of reaction, the product is obtained by dialyzing and lyophilization.

[0053] Example 5 (HA-CA)

[0054] This embodiment uses the method of Example 2 to prepare a dynamically cross-linked hydrogel. The difference is that hyaluronic acid grafted with cholic acid is used in the hydrogel prepolymer. The synthesis method of cholic acid grafted hyaluronic acid is as follows: HA-TBA is dissolved in dimethyl sulfoxide to prepare a 1% solution. Using HA-TBA molar equivalent as 1, 3 equivalents of the corresponding type of hydrophobic cholic acid, 0.75 equivalents of 4-dimethylaminopyridine, and 1.2 equivalents of di-tert-butyl dicarbonate are added. The reaction is carried out for 24 hours, dialyzed, and lyophilized to obtain the product.

[0055] Example 1

[0056] This example examines the dynamic performance of different dynamically cross-linked hydrogels in three-dimensional in vitro expansion simulating a dermal papilla cell environment. The hydrogels were prepared using the same methods as in the previous examples, and the following dynamically cross-linked hydrogels were specifically examined:

[0057] Table 1 Composition of dynamically cross-linked hydrogels

[0058]

[0059] The proliferation of dermal papilla cells in dynamically cross-linked hydrogels was observed under a microscope. The experiment revealed that the proliferation and morphology of dermal papilla cells differed in different dynamically cross-linked hydrogels. They generally exhibited the following states: minimal proliferation, rapid spheroidization and expansion; or expansion but without spheroidization. The observation results are shown in Table 2 below.

[0060] Table 2. Proliferation of dermal papilla cells in hydrogel

[0061]

[0062] Through the above experiments, it was found that dermal papilla cells in Gel-CD did not form cell spheroids, and their proliferation was also poor. In HA-TP hydrogel, a small number of cells could be observed to form spheroids, but the spheroidization time was relatively long. The composite hydrogel formed by HA-TP and gelatin showed improved cell spheroidization compared to HA-TP hydrogel. Adding gelatin also improved the shear-thinning properties of the hydrogel, enhancing its injectability. Further experiments showed that HA-MT hydrogel had better cell culture results, producing cell spheroids within one day. In the composite hydrogel of HA-MT and gelatin, cell spheroids could also be rapidly obtained in various proportions. HA-IB could also form dermal papilla cell spheroids, but the time was longer, and the spheroid size was smaller.

[0063] In the embodiments of this application, the guest small molecules of grafted hyaluronic acid are mostly derived from natural drugs, thus the prepared hydrogel has excellent biocompatibility and biochemical activity; the introduction of some functional guest molecules can bring more functions to the hydrogel. For example, menthol has the effect of promoting hair growth, and ibuprofen hyaluronic acid hydrogel has the effect of anti-inflammatory, which can significantly improve the condition of hair follicle atrophy, inflammation or mild damage.

[0064] Example 2

[0065] This example further investigates the mechanical properties of the HA-MT hydrogel. The HA-CA hydrogel was also tested as a control.

[0066] Atomic force microscopy (AFM) is used to test mechanical properties (DMT modulus).

[0067] Mechanical properties (DMT modulus) were tested using atomic force microscopy (AFM). Figure 1 The results show that the HA-MT hydrogel has significantly higher elastic strength. The stress and relaxation time of the hydrogel were tested using a rotational rheometer, and the results are as follows: Figure 1 As shown in b. Figure 1The stress comparison shown in b indicates that the HA-MT dynamically crosslinked hydrogel obtained in Example 4 has high viscoelasticity. Figure 1 b shows that the relaxation time of the hydrogel in the HA-MT group was significantly lower than that in the HA-CA group, indicating that it has superior dynamic adaptive properties.

[0068] We further investigated HA-MT hydrogels with different grafting rates (20%-40%), and experimented with grafting rates of 20%, 25%, 30%, 35% and 40%, all of which showed good gelation and in vitro expansion to form cell spheroids.

[0069] SEM and AFM analysis revealed that, by adjusting the grafting rate and hydrogel monomer concentration, the dynamically cross-linked hydrogel exhibited advantages such as rapid stress relaxation and tunable pore size structure, which are helpful in regulating the adaptive spheroidization and secretion of different tissue cells. Microscopic observation of the hydrogel's microstructure showed adjustable pore sizes (e.g., ...). Figure 4 As shown in the figure, the hydrogel formulations are as follows: 35% grafting rate, 5% grafted hyaluronic acid and 5% acrylated cyclodextrin (35-5-5); 35% grafting rate, 3% grafted hyaluronic acid and 3% acrylated cyclodextrin (35-3-3); 25% grafting rate, 5% grafted hyaluronic acid and 5% acrylated cyclodextrin (25-5-5); 25% grafting rate, 3% grafted hyaluronic acid and 3% acrylated cyclodextrin (25-3-3).

[0070] The above test results show that the high viscoelasticity of HA-MT dynamic hydrogel provides a good ECM-like environment for the in vitro expansion of dermal papilla cells. Compared with low viscoelastic hydrogels (such as HA-CA), dermal papilla cells proliferate faster and form larger spheres.

[0071] Example 6: Experiment on the efficacy of dermal papilla cells in dynamically cross-linked hydrogels

[0072] Based on a host-guest supramolecular assembly strategy, the aforementioned embodiments prepared a hyperdynamic hydrogel system with self-regulating microenvironment mechanical properties. Acrylated cyclodextrin (AC-β-CD) served as the host molecule, and hyaluronic acid modified with hydrophobic groups served as the guest molecule. The hydrophobicity and host-guest recognition between the two molecules formed a reversible cross-linked network. AC-β-CD provided structural stability to the hydrogel, while the hydrophobic-grafted hyaluronic acid achieved dynamic response and adaptive regulation through controllable hydrophobic interactions. Further experiments in this embodiment revealed that the highly viscoelastic hydrogel system can accurately simulate the ECM mechanical properties of the dermal hair follicle, thereby providing a more suitable biomechanical environment for DPCs and enhancing their dryness maintenance and secretory functions.

[0073] (1) Enhanced ability of dermal papilla cells to form spheres

[0074] In this embodiment, dermal papilla cells were loaded onto highly viscoelastic HA-TP gelatin (HA-TP-GEL) composite dynamic hydrogels (1%-3% grafted hyaluronic acid hydrogel, 3% gelatin, 5% acryloyl hyaluronic acid, w / v) and HA-MT dynamic hydrogels (1%, 3%, or 5% grafted hyaluronic acid, 3% or 5% acryloyl cyclodextrin, w / v) for three-dimensional culture to construct spherical dermal papilla cell tissue structures. The formation of dermal papilla cell spheroids observed in HA-TP gelatin composite dynamic hydrogels and HA-MT is shown below. Figure 2 As shown in the diagram, in the HA-TP-GEL composite hydrogel, dermal papilla tissue with a diameter greater than 120 μm was obtained after 4 days, after which the expansion stopped. In the HA-MT hydrogel, spherical cell clusters were observed within 24 hours, and the cell spheres gradually increased in size over time. After 7 days, the diameter of the cell clusters formed by DPC was approximately 120–200 μm, with an average diameter greater than 150 μm and a maximum diameter of up to 250 μm, similar to the in vivo DP structure. With continued culture, the maximum cell sphere diameter reached 300 μm. Live / dead staining results showed that the cells cultured in the dynamic hydrogel had good viability. The HA-MT hydrogel has high viscoelasticity, and its mechanical properties precisely match the mechanical properties of the natural dermal papilla ECM; while the HA-CA hydrogel (low viscoelasticity), as a control, had insufficient dynamic performance.

[0075] (2) Enhanced expression of key biomarkers

[0076] Compared with the conventional 2D culture method (DMEM medium containing 10% FBS and bFGF), the expression of ALP and β-catenin in DP cell spheres obtained in the HA-MT culture group was significantly increased. Furthermore, qPCR results showed that the mRNA levels of VEGF, FGF-7, and ALP in in vitro expanded dermal papilla cells were all higher than those in the control group (e.g., ...). Figure 3 As shown (p < 0.01). Compared with two-dimensionally cultured dermal papilla cells, the expression of SMA in the dermal papilla cell tissue cultured in this application was significantly reduced, and almost not expressed, indicating that it is closer to the state of normal in vivo natural dermal papilla tissue. Therefore, the dermal papilla cell cluster tissue obtained by the method of this application has the functional characteristics of normal in vivo dermal papilla cells and has outstanding effects on hair follicle reconstruction and functional recovery.

[0077] The viscoelasticity, pore structure, and surface topology of the hydrogel were characterized using rheological analysis, scanning electron microscopy (SEM), and atomic force microscopy (AFM) to ensure its physical consistency with the natural dermal papilla (DP) microenvironment. In contrast, the control group, using a low-viscoelastic hydrogel (with HA-CA as the guest molecule), exhibited poorer cell aggregation efficiency and secreted protein expression levels, further validating the importance of material mechanical properties in regulating the function of dermal papilla cells (DPCs). Microstructural regulation promotes the three-dimensional aggregation and functional expression of dermal papilla cells.

[0078] (3) Constructing a three-dimensional culture system with drug delivery function to improve treatment efficiency and durability. The menthol structure contained in the HA-MT side chain of this invention can not only form hydrophobic microregions of hydrogel, but also has a wide range of functions in the structural modification of hair growth drugs, such as improving drug stability, cell membrane permeability and receptor binding ability. Based on the hydrophobic self-assembly of this structure, lipid vesicle structures can be formed, which significantly improves the loading efficiency and sustained release ability of hydrophobic drugs in the hydrogel system, solves the problem that traditional hydrogels are difficult to load hydrophobic drugs and have unstable release, thus endowing the material with dual functions: it can be used as a three-dimensional cell culture substrate and as a local drug delivery system, providing effective drug efficacy support for tissue-engineered hair follicle regeneration.

[0079] Furthermore, the cyclodextrin-based main structure in the hydrogel can further enhance the encapsulation and sustained release of hydrophobic small molecules, thereby improving the material's ability to regulate the secretion behavior and morphological changes of DPCs.

[0080] The hydrogel system of this invention is formed based on non-covalent weak interactions, exhibiting reversibility and responsiveness. The hydrogel of this invention is degradable; upon the addition of guest molecules with stronger binding affinity to cyclodextrin, the hydrogel can achieve rapid degradation. Preferably, adamantane hydrochloride, terpineol and its structural isomers, tert-butylbenzoate, Triton 100X, etc., can be added. By introducing guest molecules with stronger competitive binding affinity, the hydrogel structure can rapidly disintegrate, achieving a gentle release of cell clusters. This avoids the damage to cellular bioactivity caused by traditional irreversible cross-linking materials, greatly enhancing the feasibility and biofriendliness of cell recovery, and meeting the continuous application requirements of subsequent in vitro expansion, functional evaluation, and in vivo implantation.

[0081] Example 7: In vivo experiment on hair follicle regeneration

[0082] This embodiment uses a chamber assay model to verify the hair follicle regeneration and hair growth efficacy of DPC cells loaded with dynamically cross-linked hydrogels, i.e., the in vivo hair regeneration and growth capacity of the three-dimensional culture system of hair follicle organoids. A schematic diagram of the transplantation is shown below. Figure 5 As shown in a, the operation process is as follows:

[0083] I. Experimental Materials and Equipment

[0084] Laboratory animals: 6–8 weeks old nude mice (BALB / c-nu / nu), male or female, weighing 18–22 g

[0085] Chamber material: 1.5 mL polyethylene EP tube;

[0086] Surgical instruments: ophthalmic scissors, ophthalmic forceps, suture needles, 5-0 sutures;

[0087] Sterile dressing: 75% ethanol, medical petroleum jelly gauze;

[0088] Cell materials: HA-MENTHOL hydrogel-encapsulated dermal papilla cells (DPCs) + epidermal cell clusters (primary extract from newborn mouse epidermis); HA-CA control hydrogel-loaded cells; control group: cell mixture resuspended in PBS.

[0089] Auxiliary reagents: Anesthetics (such as sodium pentobarbital or isoflurane)

[0090] II. Experimental Procedure

[0091] 1. Chamber preparation:

[0092] Take a 1.5 mL EP tube and cut off about 1 / 5 of the top length to form an "open ring"; the outer diameter of the chamber is about 8–10 mm and the inner diameter is about 6 mm; immerse the chamber in 75% alcohol for 30 min for disinfection and then irradiate it with ultraviolet light for 15 min before use.

[0093] 2. Animal anesthesia and preparation:

[0094] Nude mice were deeply anesthetized by intraperitoneal injection of anesthetic or inhalation of isoflurane; the hair on their backs was shaved off using an electric shaver, and the skin surface was disinfected with 75% ethanol.

[0095] 3. Wound preparation:

[0096] Use sterile scissors to cut a full-thickness skin of about 1 cm in diameter from the back of a nude mouse; the wound should penetrate to the muscle layer without damaging the muscle or underlying tissue; remove surface bleeding and rinse with PBS to keep it moist.

[0097] 4. Small chamber implantation:

[0098] Place the sterilized EP tube chamber in the center of the wound, with the tube body adhering to the muscle layer; use 4-0 sutures to suture the wound edge and the outer wall of the chamber in a circular manner to fix the chamber; ensure the sutures are tightly sealed to prevent material leakage or detachment.

[0099] 5. Material Implantation: According to the method in Example 1 (2.2), a precursor was prepared by mixing 3% menthol-grafted hyaluronic acid (HA-Menthol) and 3% acryloyl cyclodextrin (Ac-CD), and then 2×10 6 dermal papilla cells and 2×10 6 Newborn mouse epidermal cells (mixed at a 1:1, 2:1, or 3:1 cell ratio) and a photoinitiator were prepared. After 24 hours in culture medium, the mixture was ready for inoculation. Each inoculation group consisted of 150 μL of material, placed in the center of the lesion within a small chamber, ensuring close contact with the skin base. Dynamic cross-linked hydrogels loaded with hair follicle cell primordia were obtained under 405 nm blue light.

[0100] Experimental group 1 (HA-MT): HA-MENTHOL hydrogel encapsulating clusters of dermal papilla cells and neonatal mouse epidermal cells; 2×10 6 dermal papilla cells and 2×10 6 Newborn mouse epidermal cells (mixed in a 1:1, 2:1, or 3:1 cell ratio).

[0101] Experimental group 2 (HA-CA): The same cells were loaded onto HA-CA hydrogel;

[0102] Plate group: A mixture of dermal papilla cells and epidermal cells resuspended in PBS.

[0103] 6. Postoperative care:

[0104] Cover the cell with a layer of Vaseline gauze to keep it moist and prevent contamination; keep nude mice in individual cages to observe their postoperative condition and prevent them from biting or colliding with each other; check the wound and cell stability daily.

[0105] 7. Removal and post-processing of the small chamber

[0106] On the 7th day post-surgery, mice were anesthetized; the sutures were removed, and the chamber was carefully dismantled; the central tissue of the wound was observed for the formation of granulation tissue, cell aggregation, or neovascularization. Hair growth was continuously observed for 10–20 days post-surgery. During the observation period, photos were taken every 3–5 days to record the area, quantity, density, and polarity of hair growth; histological sections or immunostaining analysis were performed 20 days post-surgery.

[0107] III. Post-experiment evaluation of hair regeneration in mice; results are as follows: Figure 5 As described in Table 3.

[0108] Table 3

[0109]

[0110] Sixteen to twenty days after the surgery, hair protrusions were visible on the transplanted area of ​​the mice, with experimental group 1 showing the best hair regeneration effect. Figure 5Figures b and 5c demonstrate the in vivo hair induction ability of the hyperdynamic hydrogel in constructing artificial hair follicles. Three weeks after transplantation, gross images of the transplantation site and HE sections showed that the PBS group induced a small amount of hair growth, HA-CA produced a large number of follicle-like structures, and HA-MT induced the most hair growth. HA-MT produced clear follicle structures and the largest number of growing hairs. HA-CA also produced a relatively large number of follicle-like structures, but their hair growth ability was poor, suggesting a defect in hair follicle function (*p<0.05, **p<0.01). The key to hair follicle repair is to promptly prevent further damage to the hair follicles and promote their functional recovery through scientific methods. This example illustrates that the hair follicle structure constructed from dermal papilla cells encapsulated in HA-MT hydrogel in nude mice has superior (complete) function, with its hair growth ability far exceeding that of the low-viscoelastic HA-CA group. Therefore, the highly viscoelastic host-guest dynamic cross-linked hydrogel has a better effect in mimicking the dermal papilla cell microenvironment and inducing the generation of normal hair follicle structures. The method of reconstructing hair follicles in nude mice (without hair follicles) in this embodiment demonstrates that HA-TP and HA-MT hydrogels loaded with dermal papilla cells can be used for hair follicle tissue engineering reconstruction, and have good application prospects in the field of hair regeneration such as hair loss and hair transplantation.

Claims

1. A method for preparing dermal papilla cell spheroids, characterized in that, Includes the following steps: 1) A hydrogel prepolymer is obtained by mixing hydrophobic hyaluronic acid grafted with hydrophobic groups and cyclodextrin modified with crosslinking groups, wherein the hydrophobic groups grafted onto the hyaluronic acid and the cyclodextrin interact as host and guest. 2) Take the hair papilla cell suspension and add it to the hydrogel prepolymer to initiate the cross-linking reaction of the cross-linking groups to form a dynamic cross-linked hydrogel loaded with cells, and add cell culture medium; The hydrophobic group is at least one of tert-butylbenzene, ibuprofen, or menthol.

2. The method as described in claim 1, characterized in that, The cyclodextrin modified with the crosslinking group is an acryloyl cyclodextrin, and the hydrogel prepolymer further includes a photoinitiator.

3. The method as described in claim 1 or 2, characterized in that, The hydrogel prepolymer contains 1%-10% by mass and volume of hyaluronic acid grafted with hydrophobic groups and 1%-10% by mass and volume of cyclodextrin modified with crosslinking groups.

4. The method as described in claim 1, characterized in that, The hydrogel prepolymer further includes gelatin, and the mass-volume percentage of hyaluronic acid grafted with the hydrophobic group is 1%-5%, and the mass-volume percentage of gelatin is 1%-5%.

5. The method according to any one of claims 1-4, characterized in that, The grafting rate of the hydrophobic group-grafted hyaluronic acid is 10%-60%.

6. A composition for hair follicle regeneration or hair growth, comprising a dynamically cross-linked hydrogel loaded with dermal papilla cells, characterized in that, The dynamic crosslinked hydrogel is formed by mixing hydrophobic hyaluronic acid grafted with hydrophobic groups and cyclodextrin modified with crosslinking groups to obtain a hydrogel prepolymer and initiating crosslinking. The hydrophobic groups are at least one of tert-butylbenzene, ibuprofen, or menthol.

7. The composition according to claim 6, characterized in that, The hydrogel prepolymer contains hyaluronic acid grafted with hydrophobic groups at a mass-volume percentage of 1%-10%, and the cyclodextrin modified with crosslinking groups is acrylated cyclodextrin with a mass-volume percentage of 1%-10%.

8. The composition according to claim 6, characterized in that The hydrogel prepolymer further contains gelatin, and the mass-volume percentage of hyaluronic acid grafted with the hydrophobic group is 1%-5%, and the mass-volume percentage of gelatin is 1%-5%.

9. The composition according to claim 6, characterized in that, The dynamically cross-linked hydrogel is further loaded with at least one of the following: hair matrix cells, hair follicle stem cells, hair follicle outer root sheath cells, fibroblasts, endothelial progenitor cells, or human umbilical vein endothelial cells.

10. The use of the method for preparing dermal papilla cell spheroids as described in claims 1-5 or the composition as described in claims 6-9 in the following i)-iii): i) Prevent and treat hair loss; ii) Promotes hair growth; iii) Hair follicle repair and regeneration.

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