Use of multipotent stem cell-derived mesenchymal stem cells in preparation of a drug for promoting sebaceous gland regeneration

Abstract

The application belongs to the field of regenerative medicine and biotechnology, and particularly relates to application of mesenchymal stem cells derived from pluripotent stem cells in preparation of a drug for promoting sebaceous gland regeneration. ‑ / CD105 + High-purity mesenchymal stem cells are obtained by sorting and purification as active ingredients, and are embedded in PG hydrogel to form a local delivery system. The system can activate the hypoxic adaptation and antioxidant pathways of stem cells, reduce the level of reactive oxygen species and inhibit cell apoptosis, and maintain the survival and paracrine function of cells in the damaged microenvironment. The application is mainly used in the wound and scar areas, can significantly promote the de novo regeneration and functional reconstruction of sebaceous glands, and restore the skin barrier and moisturizing functions.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine and biotechnology, specifically relating to the application of mesenchymal stem cells derived from pluripotent stem cells in the preparation of drugs that promote sebaceous gland regeneration. Background Technology

[0002] Sebaceous glands are important appendages of the skin, secreting through holoserine secretion (…). holocrine secretion Sebaceous glands produce sebum, which participates in the formation of the lipid membrane on the skin surface, maintaining the skin's moisturizing, barrier, antibacterial, and thermoregulatory functions. Scar areas resulting from trauma, burns, surgery, and skin grafts share the common pathological feature of irreversible loss of skin appendages, including sebaceous glands. Due to the lack of sebum secretion, scar areas commonly experience dry skin, impaired barrier function, increased transepidermal water loss, itching, and cracking, severely impacting patients' quality of life. Currently, there are no effective methods for reconstructing sebaceous glands in scar areas; only symptomatic relief with emollients is possible.

[0003] Skin wound healing ( wound healing Sebaceous gland regeneration and epidermal regeneration are two processes at different biological levels. The former aims to restore the epidermal barrier and dermal continuity, and the final product is scarred skin without appendages; the latter involves the de novo reconstruction of secretory cells in the healing tissue. sebocyte The sebaceous gland structure, with its differentiation and lipid synthesis capabilities, is closer to the development of appendages during development in terms of cell origin and morphogenesis than to simple epithelial coverage. Therefore, any technical solution that "accelerates wound healing" or "repairs skin damage" cannot be automatically deduced to have the effect of "promoting sebaceous gland regeneration."

[0004] During development, sebaceous glands and hair follicles originate from the same stem / progenitor cells in the basal layer of the epidermis. The fate determination of sebaceous cells involves the regulation of the Wnt / β-catenin, c-Myc, Hedgehog, and PPAR pathways, and depends on the support of the dermal microenvironment (nutrient supply and paracrine signaling). In adult mammals, de novo regeneration of sebaceous glands usually does not occur after extensive full-thickness skin injury; however, existing literature reports that hair follicles (WIHNs) can regenerate de novo under specific conditions in the center of the wound, accompanied by the appearance of sebaceous gland structures, suggesting that modification of the healing microenvironment may reactivate the appendage generation program.

[0005] Mesenchymal stem cells (MSCs) have low immunogenicity and strong paracrine function, and can improve the local microenvironment and regulate immunity by secreting growth factors such as VEGF, HGF, and IGF. MSCs derived from pluripotent stem cells (human embryonic stem cells hESCs or human induced pluripotent stem cells hiPSCs) have advantages over MSCs derived from adult tissues, including stable source, scalability, high batch consistency, low heterogeneity, and small age-related variability, making them more suitable as standardized cell therapy products.

[0006] Existing methods for promoting skin appendage regeneration using MSCs (adult-derived) or their exosomes / conditioned media all target hair growth, not sebaceous gland regeneration. Adult MSCs suffer from significant donor variability, batch instability, and difficulty in large-scale standardization, while exosomes / proteins lack the sustained paracrine capacity of live cells. Existing de novo sebaceous gland regeneration protocols rely on composite transplantation of epidermal stem cells and skin-derived progenitor cells (Wang et al., Stem Cells Transl. Med. 2016), which differs from this invention in cell type and preparation complexity, and has not achieved standardization.

[0007] In summary, although existing patents involve tissue-engineered skin containing sebaceous gland-like structures, the stem cells used are mainly placental mesenchymal stem cells or amniotic epithelial cells. There are no reports of using iPSC-derived MSCs combined with hydrogel systems to promote sebaceous gland regeneration by regulating the activity state of stem cells. Summary of the Invention

[0008] The purpose of this invention is to provide the application of pluripotent stem cell-derived mesenchymal stem cells in the preparation of drugs that promote sebaceous gland regeneration, for promoting the regeneration and de novo reconstruction of sebaceous glands (especially sebaceous glands in trauma / scar areas), thereby overcoming the problems of existing technologies such as scar repair not containing sebaceous glands, long-term defects in the skin barrier and moisturizing function of scar areas, and lack of effective reconstruction methods.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of mesenchymal stem cells in the preparation of drugs for promoting sebaceous gland regeneration, wherein the mesenchymal stem cells are derived from human embryonic stem cells or human induced pluripotent stem cells.

[0010] Preferably, the promotion of sebaceous gland regeneration is to induce de novo reconstruction of sebaceous glands in areas of skin trauma or scarring.

[0011] Preferably, the drug further comprises a biocompatible carrier in which the mesenchymal stem cells are embedded, enabling the mesenchymal stem cells to upregulate the transcriptional levels of HIF-α, CXCR4, PHD3 and SOD1 and reduce intracellular reactive oxygen species levels.

[0012] More preferably, the biocompatible carrier is a hydrogel; The hydrogel is selected from collagen, gelatin, hyaluronic acid, alginate, chitosan, methacrylated hydrogel, PEG-gelatin crosslinked hydrogel, self-assembled peptide hydrogel, or a combination thereof.

[0013] The present invention also provides a sebaceous gland regrowth-promoting PG hydrogel, wherein the sebaceous gland regrowth-promoting PG hydrogel comprises the above-mentioned mesenchymal stem cells and PG hydrogel as a carrier; The mesenchymal stem cells are embedded in the PG hydrogel.

[0014] Preferably, the mesenchymal stem cells can highly express CD44, CD90, and CD105, but do not express CD24 and CD45.

[0015] This invention also provides a method for preparing the above-mentioned sebaceous gland-promoting hair growth PG hydrogel, comprising the following steps: (1) Mix the polyethylene glycol derivative and the gelatin derivative evenly in a sterile buffer solution to prepare a PG hydrogel precursor solution; (2) The above-mentioned mesenchymal stem cells were resuspended in the PG hydrogel precursor solution prepared in step (1) to obtain a cell-gel mixture; (3) The cell-gel mixture is incubated at 37°C and physiological pH until it is completely cross-linked into a gel to form a hydrogel scaffold in which the mesenchymal stem cells are embedded.

[0016] Preferably, in step (1), the polyethylene glycol derivative is 4-arm polyethylene glycol maleimide, and the gelatin derivative is thiolized gelatin; the 4-arm polyethylene glycol maleimide and the thiolized gelatin are mixed in a 1:1 molar ratio.

[0017] Preferably, in step (1), the PG hydrogel precursor solution is cross-linked by a Michael addition reaction under physiological conditions to construct a solid hydrogel scaffold with a three-dimensional network structure.

[0018] Preferably, in step (2), after resuspending the mesenchymal stem cells in the PG hydrogel precursor solution, the final cell concentration is adjusted to 1×10⁻⁶. 7 cells / mL.

[0019] The beneficial effects of this invention are: This invention is the first to use MSCs derived from pluripotent stem cells to promote sebaceous gland regeneration. The invention utilizes live MSCs derived from pluripotent stem cells (hESC / hiPSC), which, compared to adult MSCs or exosome / protein protocols, offers advantages such as stable source, scalability, high batch-to-batch consistency, and sustainable paracrine function.

[0020] The mesenchymal stem cells described in this invention can upregulate the transcriptional levels of HIF-α, CXCR4, PHD3 and SOD1, and reduce the level of intracellular reactive oxygen species. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A is a representative crystal violet-stained colony-forming unit culture plate after 14 days, scale bar = 10 mm; B is Oil Red O staining after adipogenic induction, scale bar = 200 μm; C is Alizarin Red S staining after osteogenic induction. Figure 2 This is a diagram of the MSC surface markers for material identification. Figure 3 Figure 1 shows the results of HIF-α / CXCR4 / PHD3 / SOD1 transcriptional level detection after in vitro conditionation. Figure 4 ROS / PI flow cytometry quantification plot; Figure 5 This is a graph showing the results of flow cytometry analysis. Figure 6 H&E staining images of skin tissue from different groups in the in vivo trauma model; Figure 7 Quantitative sebaceous gland counts for each group in an in vivo trauma model; Figure 8 Image showing the results of Masson staining. Detailed Implementation

[0023] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0025] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0026] Example 1: Preparation of Stem Cell Active Components (iPSC-derived MSCs) This embodiment provides a method for preparing and identifying mesenchymal stem cells derived from induced pluripotent stem cells—the core stem cell active component in the pharmaceutical composition of the present invention. The specific steps are as follows: 1.1 iPSC culture and feederless mesenchymal differentiation induction Undifferentiated iPSCs were inoculated onto Matrigel-coated culture plates and cultured without a feeder layer in Essential 8 (E8) medium (supplemented with 50 U / mL penicillin and 50 μg / mL streptomycin) at 37°C, 5% CO2, and 95% humidity. The medium was changed daily, and cultures awaiting confluence were passaged every 3–4 days.

[0027] Once the iPSCs reached the predetermined confluence, mesenchymal lineage differentiation was induced. The culture medium was replaced with mesenchymal induction medium based on Knockout DMEM, supplemented with 10% Knockout serum substitute, 5 ng / mL bFGF, 10 ng / mL EGF, and 5 ng / mL PDGF-AB, along with 50 U / mL penicillin and 50 μg / mL streptomycin. This induction medium was changed every 3 days and induction continued for one week to obtain a mixed cell population containing mesenchymal-like cells.

[0028] 1.2 Enrichment, purification, and clonal amplification of MSCs derived from iPSCs One week after induction, the mixed cell population was digested and stained with fluorescent antibodies. Cells were labeled with CD24-AF488 and CD105-AF555 antibodies and then sorted using a flow cytometer (such as CytoFLEX). High-purity mesenchymal-restricted cell populations (iPSC-MSCs) were precisely recovered by combining negative selection with positive selection with CD105 markers.

[0029] The sorted and purified iPSC-MSCs were seeded in 0.1% gelatin-coated culture dishes and expanded using the aforementioned mesenchymal induction medium. By the third generation (P3), microscopic observation showed that most cells exhibited a uniform fibroblast-like mesenchymal morphology.

[0030] 1.3 Quality control and phenotypic identification of active ingredients To ensure the uniformity of the prepared active ingredients and their stem cell characteristics, the above iPSC-MSCs were subjected to the following quality control identification.

[0031] 1.3.1 Phenotypic Immunophenotyping Cell surface markers were analyzed using flow cytometry. Cells were stained with direct-conjugated antibodies against CD24-AF488, CD105-AF555, CD44-AF555, CD90-AF555, and CD45-AF555, and then resuspended in 1×PBS containing 2% FBS. 10,000 events were collected per sample by flow cytometry.

[0032] The results showed that the prepared iPSC-MSCs highly expressed CD44, CD90, and CD105, but did not express CD24 and CD45, which met the definition criteria of MSCs of the International Society for Cell Therapy (ISCT).

[0033] 1.3.2 Colony Forming Unit (CFU) Experiment iPSC-MSCs were digested into single-cell suspensions and seeded at clonal density in 6-well plates, then cultured in MSC growth medium for 14 days. Colonies were subsequently stained with crystal violet and counted under a bright-field microscope. The results showed that the cells possessed excellent colony-forming ability, confirming their strong self-renewal and proliferative stemness.

[0034] 1.3.3 Verification of Multidirectional Differentiation Potential iPSC-MSCs were directionally induced for 21 days using adipogenic differentiation kits and osteogenic differentiation kits. Results are as follows: Figure 1 As shown in Figures AC, the adipogenic culture exhibited obvious intracellular lipid droplets after Oil Red O staining; the osteogenic culture showed obvious mineralized calcium deposition after Alizarin Red S staining. These results confirm that the prepared active ingredient possesses typical mesenchymal multi-lineage differentiation potential.

[0035] Example 2: Preparation of PG hydrogel for promoting sebaceous gland hair growth 2.1 Preparation of PG hydrogel (1) Preparation of hydrogel framework: Polyethylene glycol (PEG) derivatives and gelatin derivatives are mixed evenly in sterile buffer at a predetermined ratio (e.g., 1:1 molar ratio) to prepare PG hydrogel precursor solution. For example, 4-arm polyethylene glycol maleimide (PEG-4MAL) is mixed with thiolized gelatin. This system can rapidly undergo Michael addition reaction crosslinking under physiological conditions, making it suitable for constructing solid hydrogel scaffolds with a three-dimensional network structure.

[0036] (2) Cell complexation and embedding: Cells differentiated without feeder layer and CD24 were collected. - / CD105 + The third-generation (P3) iPSC-derived MSCs obtained through cell enrichment and purification were used as the core active ingredient. The iPSC-MSCs were resuspended in the PG hydrogel precursor solution prepared in step (1), and the final cell concentration was adjusted to 1 × 10⁻⁶. 7cells / mL. The cell-gel mixture was incubated at 37°C and physiological pH until completely cross-linked to form a hydrogel scaffold encapsulating iPSC-MSCs. The hydrogel scaffold was then washed three times with sterile PBS to remove unreacted precursors and impurities, yielding the PG hydrogel for topical application.

[0037] 2.2 Verification of the purity and dryness maintenance of active ingredients Results identified by flow cytometry are as follows Figure 2 As shown, CD24 is differentiated and bound through the feederless layer. - / CD105 + The iPSC-MSCs obtained through the sorting strategy exhibited extremely high mesenchymal purity: CD44 expression rate was 98.0%, CD90 was 98.4%, CD105 was 97.7%, while the expression of the hematopoietic marker CD45 was extremely low (0.02%). Simultaneously, colony formation assays (CFU) and adipogenic / osteogenic multi-lineage differentiation potential tests were both positive, confirming that this high-purity active ingredient maintained good stem cell stemness and pluripotency both before and after embedding in the PG hydrogel scaffold.

[0038] Example 3: Validation Experiment of Hypoxia Adaptation and Anti-apoptosis Mechanism of iPSC-MSCs under PG Hydrogel Embedding Conditions 3.1 Experimental grouping and cell treatment The P3 generation iPSC-derived MSCs prepared in Example 1 were divided into two groups: Control group (ctrl): Cells were seeded in 0.1% gelatin-coated culture dishes and cultured in conventional mesenchymal induction medium; Experimental group (PG gel mixed): Cells were resuspended in PG hydrogel precursor solution (hydrogel framework is a mixture of 4-arm polyethylene glycol maleimide (PEG-4MAL) and thiolized gelatin), and the final cell concentration was adjusted to 1×10⁻⁶. 7 The cells / mL were incubated at 37°C until completely gelled, forming a PG hydrogel embedding system.

[0039] The two groups of cells were cultured for 24 hours and 48 hours, respectively, and samples were collected for subsequent testing.

[0040] 3.2 Detection of transcriptional levels of hypoxia and antioxidant-related genes (qPCR) RNA extraction: Total RNA was extracted from the two groups of cells using TRIzol reagent, and the RNA concentration and purity were detected by NanoDrop (A260 / A280≈2.0).

[0041] Reverse transcription: 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0042] qPCR detection: Using cDNA as a template, specific primers (primer sequences designed by Primer-BLAST and synthesized by Sangon Biotech (Shanghai) Co., Ltd.) were designed for HIF-α, CXCR4, PHD3, and SOD1 genes using the SYBR Green dye method. GAPDH was used as an internal reference gene for real-time quantitative PCR.

[0043] Data analysis: The relative expression levels of each gene were calculated using the 2^-ΔΔCt method. Using the control group as a baseline (set as 1), the upregulation folds of gene transcription levels in the experimental groups were compared. Results are as follows: Figure 3 As shown, after PG hydrogel embedding, compared with the gelatin-encapsulated control group, the transcriptional levels of HIF-α, CXCR4, PHD3, and SOD1 increased by approximately 6-fold, 4-fold, 4-fold, and 18-fold, respectively. This transcriptional pattern indicates that PG hydrogel embedding induces a hypoxia response and antioxidant-related state in cells.

[0044] 3.3 Horizontal flow cytometry detection of reactive oxygen species (ROS) Staining: Collect two groups of cells, wash twice with PBS, resuspend in serum-free medium containing 10 μM CM-H2DCFDA (reactive oxygen fluorescent probe), and incubate at 37°C in the dark for 30 minutes.

[0045] Flow cytometry analysis was performed to detect intracellular ROS-positive signals (FITC channel) using a flow cytometer, with 10,000 events collected per sample.

[0046] The results are as follows Figure 4 As shown, the proportion of ROS-positive cells in the control group (coated with 0.1% gelatin) was 34.2–36.4%; after 24 hours of PG hydrogel embedding, the proportion of ROS-positive cells decreased to 4.95%, and remained at 8.9% after 48 hours.

[0047] 3.4 Flow cytometry detection of apoptosis levels (Annexin V-FITC / PI double staining method) Cell collection and processing: Cells from the control group and experimental group (PG gel mixed) were collected at 12 h, 24 h, and 48 h. For the PG hydrogel-embedded group, an equal volume of 0.25% trypsin-EDTA solution was added, and the cells were incubated at 37°C for 10–15 min to allow the hydrogel to gently degrade and digest the adherent cells. Digestion was then terminated by adding PBS containing 2% FBS. The cell pellet was collected by centrifugation at 300 g for 5 min and washed twice with pre-cooled PBS.

[0048] Staining procedure: Double staining was performed using the Annexin V-FITC / PI apoptosis detection kit. Washed cells were resuspended in 1×Binding Buffer, and the cell density was adjusted to 1×10⁶ cells / year. 6 cells / mL. Transfer 100 μL of cell suspension to a flow cytometry tube, add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, vortex gently to mix, and incubate at room temperature (20-25℃) in the dark for 15 minutes. Analyze the cells within 1 hour, adding 400 μL of 1×Binding Buffer before analysis.

[0049] Flow cytometry analysis: Annexin V's green fluorescence signal was detected using the FITC channel (Ex=488 nm, Em=525 nm) and PI's red fluorescence signal was detected using the PE channel (Ex=535 nm, Em=615 nm) on a flow cytometer. 10,000 events were collected per sample. Analysis was performed using a gating strategy: lower left quadrant (Q3, Annexin V...) - / PI - ) are live cells; lower right quadrant (Q4, Annexin V) + / PI - ) are early apoptotic cells; the upper right quadrant (Q2, Annexin V) + / PI + These are cells in late apoptosis or secondary necrosis; the upper left quadrant (Q1, Annexin V) represents cells in late apoptosis or secondary necrosis. - / PI + Cells with mechanical damage or simple necrosis are considered to be affected. The total apoptosis rate (AP) is the sum of the proportions of early apoptosis and late apoptosis (Q4 + Q2).

[0050] Results Analysis: The results are as follows Figure 5 As shown, the total apoptosis rate (AP) of the control group at 12 h, 24 h, and 48 h was 1.62%, 1.85%, and 1.71%, respectively, indicating stable cell status. In the PG hydrogel-embedded group, the AP was 4.85% at 12 h, decreasing to 3.46% at 24 h, and remaining at 4.05% at 48 h. This data indicates that the initial microenvironmental changes during hydrogel embedding (12 h) triggered mild cellular stress, but did not lead to extensive cell necrosis. Over time (24 h–48 h), cells gradually adapted to the three-dimensional microenvironment, and the apoptosis rate stabilized at a low level. Combined with the aforementioned significant reduction in ROS, this confirms that the PG hydrogel embedding system not only did not cause irreversible cell damage, but also effectively maintained the survival of iPSC-MSCs in the scar microenvironment by activating antioxidant mechanisms, providing a mechanistic guarantee for stem cells to continuously exert paracrine functions and promote sebaceous gland regeneration.

[0051] The above experiments demonstrate that the PG hydrogel embedding system can significantly reduce ROS accumulation in the scar microenvironment by activating the hypoxia adaptation pathway (HIF-α / CXCR4 / PHD3) and antioxidant pathway (SOD1) of iPSC-MSCs, while inhibiting cell apoptosis, providing key mechanistic support for the survival and function of stem cells in the trauma / scar area (promoting sebaceous gland regeneration).

[0052] Example 4: Application in a full-thickness skin excision scar model 4.1 Constructing an animal model Eight-week-old male BALB / c nude mice (immunodeficient, to avoid xenogeneic cellular rejection) were selected and housed in an SPF-grade animal facility (temperature 22±2℃, humidity 50±10%, 12-hour light / dark cycle). A full-thickness skin trauma model was established. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and the skin was prepared and disinfected. Full-thickness skin defects of 1.5 cm in diameter were created on both sides of the spine on the back (removing the entire skin thickness down to the subcutaneous fat layer), and the wounds were exposed after hemostasis. Wound healing was observed daily, and infection was prevented (e.g., by topical disinfection with povidone-iodine).

[0053] The study included a group design (n=6 / group): Untreated group: no treatment was given to the wound; PG gel group: cell-free PG hydrogel scaffolds were placed directly into the wound; Injected MSC group: iPSC-MSCs were suspended in culture medium and subcutaneously injected around the wound edges (control group); PG gel MSC group: PG hydrogel scaffolds were washed three times with sterile PBS and placed directly into each wound (local delivery), and covered with a dressing.

[0054] Within 24 hours of wound formation (or after partial wound healing, adjusted according to the scar formation stage; this experiment selected immediate administration after wound formation to simulate early intervention), the prepared hydrogel scaffold was directly applied to the wound and fixed with sterile dressings for the PG gel and PG gel MSC groups, according to the group design. For the Injected MSC group, a local subcutaneous injection (100 μL / wound area) was performed at the wound edge to ensure uniform distribution of the preparation in the wound and surrounding subcutaneous tissue.

[0055] On day 14 after drug administration (scar maturation period), mice were sacrificed, and skin tissue from the wound and surrounding areas (including the normal skin margins) was collected. Tissue samples were fixed in 4% paraformaldehyde for 24 hours, then routinely dehydrated and embedded in paraffin.

[0056] 4.2 Histological analysis (H&E staining) 5 μm thick serial sections were prepared, dewaxed, and hydrated using standard procedures. Hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin. Skin tissue structure was observed under an optical microscope, with a focus on recording the morphology, integrity of glandular structure, number of acini, and distribution of sebaceous glands in the dermis.

[0057] Five high-power fields (×200) were randomly selected from each slice. The number of sebaceous glands in each field was counted, and the average value was calculated as the sebaceous gland density of the sample.

[0058] Data analysis: Statistical analysis (such as One-way ANOVA) was performed using GraphPad Prism software to compare the differences in the number of sebaceous glands among the groups.

[0059] The results are as follows Figure 6 As shown, the Untreated group and PG gel group had almost no sebaceous gland structures in the dermis; the Injected MSC group had a few scattered sebaceous gland-like structures; and the PG gel MSC group had a large number of well-formed sebaceous glands in the dermis with regular acini, indicating a significant effect of hair regrowth from the root.

[0060] like Figure 7 As shown, quantitative statistics showed that the number of sebaceous glands in the PG gel MSC group (approximately 6 per field of view) was significantly higher than that in the Injected MSC group (approximately 3 per field of view), the PG gel group (approximately 0.5 per field of view), and the Untreated group (approximately 0.3 per field of view), demonstrating that the compound preparation described in this invention has the strongest ability to promote sebaceous gland regeneration in vivo.

[0061] 4.3 Masson staining The results are as follows Figure 8 As shown, the PG gel MSC group exhibited a denser collagen pattern that was closer to the woven pattern of a basketball hoop, which, compared to the intermediate state of the injection group and the sparse linear arrangement of the untreated group, indicated a higher quality of repair.

[0062] The results in summary indicate that, compared with simple cell suspension injection, local delivery of iPSC-MSCs using PG hydrogel can more significantly promote wound closure, greatly promote the regeneration of skin appendages such as hair follicles and sebaceous glands, and improve collagen arrangement, demonstrating the strongest ability to promote skin structure and function regeneration.

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of mesenchymal stem cells in the preparation of drugs for promoting sebaceous gland regeneration, characterized in that, The mesenchymal stem cells are derived from human embryonic stem cells or human induced pluripotent stem cells.

2. The application according to claim 1, characterized in that, The promotion of sebaceous gland regeneration involves inducing de novo reconstruction of sebaceous glands in areas of skin trauma or scarring.

3. The application according to claim 1, characterized in that, The drug also includes a biocompatible carrier in which the mesenchymal stem cells are embedded, enabling the mesenchymal stem cells to upregulate the transcriptional levels of HIF-α, CXCR4, PHD3 and SOD1 and reduce intracellular reactive oxygen species levels.

4. The application according to claim 3, characterized in that, The biocompatible carrier is a hydrogel; The hydrogel is selected from collagen, gelatin, hyaluronic acid, alginate, chitosan, methacrylated hydrogel, PEG-gelatin crosslinked hydrogel, self-assembled peptide hydrogel, or a combination thereof.

5. A PG hydrogel that promotes sebaceous gland hair growth, characterized in that, The sebaceous gland-promoting PG hydrogel includes the mesenchymal stem cells as described in claim 1, and the PG hydrogel as a carrier; The mesenchymal stem cells are embedded in the PG hydrogel.

6. The PG hydrogel for promoting sebaceous gland growth according to claim 5, characterized in that, The mesenchymal stem cells expressed high levels of CD44, CD90, and CD105, but not CD24 and CD45.

7. The method for preparing the PG hydrogel for promoting sebaceous gland growth as described in claim 5, characterized in that, Includes the following steps: (1) Mix the polyethylene glycol derivative and the gelatin derivative evenly in a sterile buffer solution to prepare a PG hydrogel precursor solution; (2) The mesenchymal stem cells described in claim 1 are resuspended in the PG hydrogel precursor solution prepared in step (1) to obtain a cell-gel mixture; (3) The cell-gel mixture is incubated at 37°C and physiological pH until it is completely cross-linked into a gel to form a hydrogel scaffold in which the mesenchymal stem cells are embedded.

8. The preparation method according to claim 7, characterized in that, In step (1), the polyethylene glycol derivative is 4-arm polyethylene glycol maleimide, and the gelatin derivative is thiolized gelatin; the 4-arm polyethylene glycol maleimide and the thiolized gelatin are mixed in a 1:1 molar ratio.

9. The preparation method according to claim 7, characterized in that, In step (1), the PG hydrogel precursor solution is cross-linked by a Michael addition reaction under physiological conditions to construct a solid hydrogel scaffold with a three-dimensional network structure.

10. The preparation method according to claim 7, characterized in that, In step (2), after resuspending the mesenchymal stem cells in PG hydrogel precursor solution, the final cell concentration is adjusted to 1×10⁻⁶. 7 cells / mL.

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