A cell-extracellular matrix complex for hair follicle regeneration and a preparation method and application thereof

Abstract

The application relates to the field of tissue engineering and regenerative medicine, and particularly discloses a cell-extracellular matrix complex for hair follicle regeneration and a preparation method thereof. The complex comprises autologous hair follicle stem cells, a human scalp-derived acellular ECM scaffold, time-sequentially added growth factors and an anti-scarring component. In preparation, the autologous hair follicle stem cells are first induced by BMP4 and TGF-beta 1 in cooperation, then inoculated into the human scalp-derived acellular ECM scaffold, and the complex is constructed under dynamic culture conditions, and VEGF is added to promote vascularization. The complex can efficiently form a structure-intact hair papilla-like tissue, has good biocompatibility, low immunogenicity and anti-scarring characteristics, and is significantly superior to a traditional collagen scaffold system. The application provides a new regeneration treatment strategy with clear clinical transformation prospects for scaleable preparation for androgenetic alopecia and other hair follicle deficiency diseases.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and regenerative medicine technology, specifically relating to a cell-extracellular matrix (ECM) complex for hair follicle regeneration, its preparation method, and its application. Background Technology

[0002] Hair loss, especially androgenetic alopecia (AGA), is one of the most common hair disorders worldwide, affecting more than 50% of adult men and a significant proportion of women. Current mainstream clinical treatments include topical minoxidil and oral finasteride, but these methods can only slow the hair loss process or slightly promote hair thickening; they cannot rebuild functional hair follicle structures, and relapse is common after discontinuation of these medications.

[0003] In recent years, cell therapy and tissue engineering technologies have provided new approaches to hair follicle regeneration. For example, Chinese patent CN113350484A discloses a hair loss treatment based on bone marrow mesenchymal stem cells (BMSCs). However, BMSCs lack hair follicle lineage specificity, have low in vivo differentiation efficiency, and preclinical data show that their hair follicle-related proliferation rate is only 45%, and they are difficult to form complete hair follicle units with dermal papilla structures. Another type of technology, such as Chinese patent CN116688100A, uses synthetic surfactants or cell-free formulations, which can promote epidermal thickening but completely lack hair follicle regeneration capabilities, failing to solve the fundamental problem.

[0004] In tissue engineering strategies, scaffold materials are the key "soil" determining regeneration effectiveness. While traditional collagen scaffolds possess some biocompatibility, they exhibit high immunogenicity (e.g., IL-6 secretion levels often exceed 50 pg / mL), and their physical properties, such as porosity and elastic modulus, do not match those of the natural scalp dermis, leading to poor cell adhesion and limited nutrient exchange. Furthermore, existing scaffolds are mostly derived from xenogeneic tissues or chemically synthesized, lacking the specific extracellular matrix signals required for hair follicle development, such as fibronectin and laminin subtypes.

[0005] Although some studies have attempted to combine stem cells with scaffolds, the following technical bottlenecks are generally encountered: (1) Cell-scaffold origin mismatch: Cells not derived from hair follicles are less likely to respond to the induction signals of scalp ECM; (2) Lack of dynamic microenvironment regulation: Static culture cannot simulate the blood supply and mechanical stimulation required for hair follicle development in vivo; (3) Low regeneration efficiency: The formation rate of hair follicle structure in most systems is less than 50%, and the newly grown hair is fine, soft and easy to fall out; (4) Accompanied by obvious scar formation: The regeneration process activates the TGF-β1 pathway, leading to fibrosis, which affects appearance and function.

[0006] Therefore, there is an urgent need for a hair follicle regeneration technology that can efficiently reconstruct functional hair follicle units, has good biocompatibility, can be scalably prepared, and is clinically feasible. This application is proposed against this backdrop, by integrating tissue-specific seed cells (autologous hair follicle stem cells) to construct a biomimetic "seed-soil" synergistic regeneration system, significantly improving hair follicle regeneration efficiency and clinical applicability. Summary of the Invention

[0007] The purpose of this application is to provide a hair follicle regeneration complex based on the synergistic effect of human scalp acellular ECM scaffold and autologous hair follicle stem cells, in order to solve the technical problems of existing hair loss treatment technologies, such as the inability to functionally reconstruct hair follicle structure, low regeneration efficiency, high immunogenicity, and easy formation of scars.

[0008] To achieve the above-mentioned objectives, this application adopts the following technical solution: A cell-extracellular matrix (ECM) complex for hair follicle regeneration, the complex comprising: (1) An ECM scaffold obtained from human scalp tissue through decellularization, wherein the ECM scaffold has an average pore size of 50-80 μm, a porosity of 85-92%, and an elastic modulus of 0.5-1.2 kPa; and (2) Autologous hair follicle stem cells induced by bone morphogenetic protein 4 (BMP4) were seeded onto the ECM scaffold at an initial seeding density of 1×10⁻⁶. 5 Up to 5×10 5 cells / cm 2 .

[0009] Furthermore, the complex comes into contact with vascular endothelial growth factor (VEGF) during the preparation process.

[0010] Furthermore, the ECM scaffold is prepared by decellularization using 0.1% sodium dodecyl sulfate (SDS) at 4°C for 12 hours, resulting in a nucleic acid residue of less than 50 ng / mg, and then cross-linked with transglutaminase to form a hydrogel.

[0011] Furthermore, the complex also contains asiaticoside at a concentration of 50-200 μg / mL.

[0012] This application also discloses a method for preparing a cell-extracellular matrix (ECM) complex for hair follicle regeneration, comprising the following steps: (i) Pretreatment of autologous hair follicle stem cells in induction medium containing BMP4; (ii) The pretreated cells were mixed with ECM gel and injected into a pre-molded mold to form a cylinder with a diameter of 3 mm and a height of 2 mm; (iii) The shaped composite was placed in a bioreactor and dynamically cultured at a 5% oxygen concentration and a perfusion rate of 0.5 mL / min; (iv) Add VEGF to the culture system on day 7 of culture.

[0013] Furthermore, the induction medium containing BMP4 mentioned in step (i) is DMEM / F12 basal medium supplemented with 10 ng / mL bone morphogenetic protein 4 (BMP4) and 5 ng / mL transforming growth factor-β1 (TGF-β1).

[0014] Furthermore, in step (ii), the cell seeding density is 1×10⁻⁶. 5 Up to 5×10 5 cells / cm 2 .

[0015] Use of the cell-ECM complex in the preparation of a medicament for the treatment of androgenetic alopecia.

[0016] The drug is administered via local injection, with a single dose of 0.5 mL, once a month, for at least 3 consecutive months.

[0017] Furthermore, on day 14 of in vitro culture, the K15 / K17 immunofluorescence double positivity rate reached over 80%, and dermal papilla-like structures with a diameter of 50-80 μm were formed.

[0018] Furthermore, the human scalp tissue is derived from a healthy donor and has a thickness of 2-4 mm.

[0019] The hair follicle regeneration complex and its preparation method provided in this application achieve a specific match between hair follicle-derived stem cells and human scalp abscess cells (ECM) at the tissue development level, constructing a highly biomimetic "seed-soil" regeneration microenvironment that significantly promotes the formation and maturation of functional hair follicle units. Combined with dynamic perfusion culture and a time-sequential growth factor delivery strategy, it effectively simulates the mechanical and biochemical signals required for in vivo hair follicle development, further enhancing the integrity and long-term survival of the regenerated structure. Simultaneously, the anti-scarring component introduced into the complex, synergistically with the low immunogenicity of ECM, significantly inhibits fibrosis and improves the quality of tissue repair in the regenerated area. This technical solution not only boasts high regeneration efficiency and good biosafety but also features stable processes and is adaptable to large-scale biomanufacturing systems, providing a novel treatment pathway for refractory hair loss diseases such as androgenetic alopecia that combines scientific advancement with clinical translational feasibility. Attached Figure Description

[0020] Figure 1 Example 2: HE staining results of cell-ECM complex; Figure 2Example 4: HE staining results of control group A; Figure 3 Example 4: HE staining results of control group B; Figure 4 Example 4: HE staining results of control group C; Figure 5 In Example 5, the AGA-DHT model was administered the drug on day 0. Figure 6 Example 5: Week 4 of drug administration in the AGA-DHT model; Figure 7 Example 5: Week 12 of AGA-DHT model administration. Detailed Implementation

[0021] The present application will be further illustrated by the following embodiments, but the scope of protection of the present application is not limited to the embodiments.

[0022] In this embodiment, all human scalp tissue used is derived from healthy donors or patients' autologous tissue approved by an ethics committee. All donors have signed written informed consent forms, explicitly agreeing to the use of their tissue for scientific research and regenerative medicine product development. The entire process of tissue acquisition, transportation, processing, and use strictly complies with the Declaration of Helsinki and relevant Chinese laws and regulations, ensuring legal sourcing, compliant processes, and protected privacy. The scalp tissue is excess healthy tissue obtained during surgery or biopsy.

[0023] Example 1: Preparation of human scalp decellularized ECM scaffold Healthy human scalp tissue (2-4 mm thick) obtained after ethical approval and signed informed consent forms were processed as follows: (1) Wash three times with PBS buffer containing 1% penicillin-streptomycin, each time for 30 minutes; (2) Digest with 0.25% trypsin at 37°C for 2 hours, then peel off the epidermal layer; (3) Transfer to 0.1% sodium dodecyl sulfate (SDS) solution and decellularize at 4°C for 12 hours; (4) Dialyze with distilled water for 48 hours, changing the water 6 times during the period; (5) After sterilization by γ-ray, 0.5 U / mg transglutaminase ECM is added and crosslinked at 4°C for 6 hours to form a hydrogel ECM scaffold.

[0024] The obtained ECM scaffold has an average pore size of 65 μm, a porosity of 89%, an elastic modulus of 0.8 kPa, and a nucleic acid residue of less than 50 ng / mg, while retaining natural collagen, fibronectin, laminin, and other hair follicle development-related signaling molecules.

[0025] Example 2: Preparation of cell-ECM complex Hair follicle units were extracted from the healthy scalp area at the back of the subject's head. After digestion with collagenase IV, the follicular ridge tissue was separated and primary cultured in DMEM / F12 basal medium (Gibco, Cat. No. 11330032) containing 10% fetal bovine serum (v / v). After cell adhesion, the medium was replaced with induction medium: DMEM / F12 basal medium supplemented with 10 ng / mL bone morphogenetic protein 4 (BMP4) and 5 ng / mL transforming growth factor-β1 (TGF-β1), and cultured for another 48 hours to obtain autologous hair follicle stem cells synergistically induced by BMP4 and TGF-β1.

[0026] The stem cells were resuspended in the human scalp decellularization ECM gel prepared as in Example 1, and the cell concentration was adjusted to an inoculation density of 3 × 10⁻⁶ cells / mL. 5 cells / cm². Take 50 μL of cell-ECM suspension and inject it into a polydimethylsiloxane (PDMS) pre-molding mold (3 mm in diameter and 2 mm in height), and let it stand for 30 minutes to form a gel.

[0027] The matured complex was transferred to a perfusion bioreactor and dynamically cultured under conditions of 5% oxygen concentration and a perfusion flow rate of 0.5 mL / min. On day 7 of culture, vascular endothelial growth factor (VEGF) was added to the culture system to a final concentration of 20 ng / mL, and culture was continued until day 14 to obtain mature cell-ECM complexes.

[0028] Further analysis of cell viability in the cell-ECM composite formulation: After mixing human hair follicle stem cells (HFSC) with different proportions of compound preparations, the mixtures were placed at 4°C, and cell viability was measured at 0h, 12h, 24h, and 48h. The method was to add 20μL trypan blue to an equal volume of cell mixture, mix well, and then add the mixture to a Countstar counting chamber (Countstar, CO010101). Cell viability was then measured directly using a Countstar cell counter (Countstar, GDZ002097). The results are shown in Table 1.

[0029] Table 1 0h - Cell viability 12h - Cell viability 24h - Cell viability 48h - Cell viability HFSC-ECM (1 / 1) 99.15% 90.32% 69.15% 49.15% HFSC-ECM (2 / 1) 99.77% 95.62% 92.51% 81.17% HFSC-ECM (5 / 1) 99.43% 92.87% 60.73% 32.13% HFSC-ECM (10 / 1) 99.73% 75.81% 48.73% 22.13% HFSC-ECM (1 / 2) 99.95% 85.02% 70.67% 51.48% HFSC-ECM (1 / 5) 99.84% 80.14% 62.51% 44.91% HFSC-ECM (1 / 10) 99.72% 75.14% 52.41% 30.91% Viability analysis revealed that the composite parameter ratio of HFSC-ECM (2 / 1) best maintained cell viability, achieving a viability of over 81% after 48 hours. The table shows that excessively high ratios of HFSC-ECM, such as HFSC-ECM (1 / 5), HFSC-ECM (1 / 10), HFSC-ECM (5 / 1), and HFSC-ECM (10 / 1), failed to maintain good cell viability after 48 hours. This is because the cell-ECM ratio is crucial for microenvironmental homeostasis; excessively high ratios lead to physical blockage and signal overload, while excessively low ratios result in lack of support and signal dissipation, ultimately leading to low cell viability. Other ratios, such as HFSC-ECM (1 / 1) and HFSC-ECM (1 / 2), maintained cell viability to around 50% after 48 hours, but this was still relatively lower than the viability of HFSC-ECM (2 / 1) after 48 hours. Therefore, it has been verified that the composite parameter ratio of HFSC-ECM (2 / 1) is optimal.

[0030] Example 3: Functional Verification The complex prepared in Example 2 was tested in vitro. Immunofluorescence staining showed a significant increase in the proportion of K15 / K17 double-positive cells, and histological observation revealed the formation of typical dermal papilla-like structures.

[0031] The complex was transplanted subcutaneously into the back of nude mice, and new hair was observed penetrating the skin surface after 8 weeks; see [link / reference needed]. Figure 1 HE staining showed that the hair follicle structure was intact, including the hair bulb, inner root sheath and other basic components; CD31 immunohistochemistry confirmed that the vascular network in the regeneration area was well formed; ELISA showed that the IL-6 level in the culture supernatant was extremely low, indicating that the complex has excellent biocompatibility and low immunogenicity.

[0032] Example 4: Comparative Experiment To verify the synergistic effect of the various technical features of this invention, the following control group was set up: Control group A: Commercially available type I collagen (Corning RatTail Collagen I, catalog number 354236) was prepared into a 3 mg / mL hydrogel according to the instructions, replacing the human decellularized scalp ECM scaffold prepared in Example 1. The remaining steps were the same as in Example 2. Control group B: Autologous hair follicle stem cells that were not induced by BMP4 and TGF-β1 were used, and the remaining steps were the same as in Example 2; Control group C: No VEGF was added during the dynamic culture process, and the remaining steps were the same as in Example 2.

[0033] The results show that: Figure 2 The results showed that the hair follicle structure formed in control group A was loose and sparse, and the hair papilla-like tissue was not obvious. Figure 3 The results showed that the B cell differentiation efficiency in the control group was significantly reduced, and the number of hair follicles was small and degenerated. Figure 4 The control group C showed that although it had rudimentary hair follicles, it lacked vascular support, had a small number of hair follicles, and was prone to degeneration.

[0034] In contrast, only the present invention's solution (Example 2, see...) Figure 1 It can efficiently construct functional hair follicle units with complete structure, good vascularization, and long-term stability, with a large number of hair follicles and complete structure, which fully demonstrates the necessity and innovation of the synergistic effect of human scalp ECM, BMP4 / TGF-β1 synergistic induction, and VEGF time-sequential addition.

[0035] Example 5: Validation of the use of cell-ECM complex in the treatment of androgenetic alopecia To verify the feasibility of using the cell-ECM complex of this invention for the treatment of androgenetic alopecia, a dihydrotestosterone (DHT)-induced hair follicle miniaturization mouse model was established. The specific methods are as follows: Eight-week-old male C57BL / 6 mice were selected. After hair removal on their backs, they were subcutaneously injected with DHT (1 mg / kg / day) for 21 consecutive days to induce hair follicles to enter the resting phase and exhibit miniaturization. Subsequently, the cell-ECM complex prepared in Example 2 was resuspended in physiological saline to a total volume of 0.5 mL and injected into the bald areas on the backs of the mice (n=10) via local multi-point injection. The control group was injected with an equal volume of PBS or uninduced cell suspension.

[0036] The dosing regimen simulates clinical practice: once every 4 weeks for a total of 3 times (i.e., continuous treatment for 3 months).

[0037] The results show that... Figures 5-7 ,in Figure 5 Photographs of the AGA model constructed on day 21 of DHT administration, and the AGA-DHT model on day 0 of administration; Figure 6 Week 4 of administration to the AGA-DHT model; Figure 7 Week 12 of drug administration in the AGA-DHT model: The mice in the treatment group began to show hair regrowth in the 4th week after the first injection, and the hair had already turned black in some areas with the growth of micro-hairs; by the 12th week, the hair gradually increased and basically completely covered the previously bald area. The hair density, hair shaft diameter and hair follicle depth were significantly better than those in the control group. Histological analysis showed that the number of hair follicles in the treatment group recovered to more than 85% of the normal level, and the collagen in the dermis was arranged in an orderly manner with no obvious fibrosis. Immunohistochemical analysis showed that the expression of dermal papilla markers (Versican, Sox2) was significantly upregulated, while the activity of the androgen receptor (AR) signaling pathway was inhibited.

[0038] Furthermore, with ethical approval and informed consent from the subjects, a preliminary exploratory application was conducted on three patients with mild to moderate androgenetic alopecia (Norwood grade II–III): 0.5 mL of the complex suspension was injected intradermally at multiple points into the balding area once a month for three consecutive months. Follow-up observation showed that all subjects experienced the conversion of vellus hair to terminal hair after three months of treatment, subjective assessment of scalp hair density improved, and no significant adverse reactions occurred.

[0039] The above results indicate that the cell-ECM complex described in this invention can be used to prepare a drug for treating androgenetic alopecia, and that the dosing regimen of a single 0.5 mL dose, once a month for local injection, and continuous treatment for at least 3 months is feasible and has preliminary effectiveness.

[0040] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A cell-extracellular matrix complex for hair follicle regeneration, characterized in that, The composite includes: (1) An ECM scaffold obtained from human scalp tissue through decellularization, wherein the ECM scaffold has an average pore size of 50-80 μm, a porosity of 85-92%, and an elastic modulus of 0.5-1.2 kPa; and (2) Autologous hair follicle stem cells induced by bone morphogenetic protein 4 were seeded onto the ECM scaffold at an initial seeding density of 1×10⁻⁶. 5 Up to 5×10 5 cells / cm 2 .

2. The cell-extracellular matrix complex for hair follicle regeneration according to claim 1, characterized in that, The ECM scaffold was prepared by decellularization using 0.1% sodium dodecyl sulfate at 4°C for 12 hours, resulting in a nucleic acid residue of less than 50 ng / mg, and then cross-linked with transglutaminase to form a hydrogel.

3. The cell-extracellular matrix complex for hair follicle regeneration according to claim 1, characterized in that, The complex also contains asiaticoside at a concentration of 50-200 μg / mL.

4. A method for preparing a cell-extracellular matrix complex for hair follicle regeneration as described in any one of claims 1-3, characterized in that, Includes the following steps: (i) Pretreatment of autologous hair follicle stem cells in induction medium containing BMP4; (ii) The pretreated cells were mixed with ECM gel and injected into a pre-molded mold to form a cylinder with a diameter of 3 mm and a height of 2 mm; (iii) The shaped composite was placed in a bioreactor and dynamically cultured at a 5% oxygen concentration and a perfusion rate of 0.5 mL / min; (iv) Add VEGF to the culture system on day 7 of culture.

5. The method for preparing a cell-extracellular matrix complex for hair follicle regeneration according to claim 4, characterized in that... The induction medium containing BMP4 mentioned in step (i) is DMEM / F12 basal medium supplemented with 10 ng / mL bone morphogenetic protein 4 (BMP4) and 5 ng / mL transforming growth factor-β1 (TGF-β1).

6. The method for preparing a cell-extracellular matrix complex for hair follicle regeneration according to claim 4, characterized in that, In step (ii), the cell seeding density is 1×10⁻⁶. 5 Up to 5×10 5 cells / cm 2 .

7. The method for preparing a cell-extracellular matrix complex for hair follicle regeneration according to claim 4, characterized in that, The final concentration of VEGF described in step (iv) is 20 ng / mL.

8. Use of the cell-extracellular matrix complex as described in any one of claims 1-3 in the preparation of a medicament for treating androgenetic alopecia.

9. The use according to claim 8, characterized in that, The drug is administered via local injection, with a single dose of 0.5 mL, once a month, for at least 3 consecutive months.

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