Noninvasive optical system and method for stimulating hair regeneration

Abstract

A non-invasive optical system for stimulating hair regeneration is disclosed. The non-invasive optical system comprises a femtosecond laser unit used for generating femtosecond laser; and the optical focusing unit is used for focusing the femtosecond laser to a basal layer of skin tissue so as to stimulate basal cells in the basal layer to form hair follicles and further stimulate hair regeneration. According to the non-invasive optical system, femtosecond laser directly acts on basal cells through cuticles, no wound is caused, hair follicles can be formed within one day after single treatment, and the non-invasive optical system has good transformation application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a non-invasive optical system and method for stimulating hair regeneration. Background Technology

[0002] Skin cells are organized into a multi-layered structure. Skin cell populations exhibit high plasticity. Skin stem cells (SCs), comprising several distinct populations, are responsible for maintaining skin homeostasis and repair. These stem cell pools are located in different regions of skin units, such as hair follicles, interfollicular epidermis, and sebaceous glands. Although they originate from a common embryonic progenitor cell, the heterogeneity of the microenvironment determines their different fates and functional specializations. Among them, hair follicle stem cells (HFSCs) located in the bulge region of the hair follicle are the engine of cyclical hair regeneration. Epidermal stem cells (EpSCs, also known as basal cells (BCs)) are located in the basal layer of the epidermis and coordinate epidermal homeostasis. Under the induction of wound stimulation, physiological signals, or microenvironmental remodeling, these stem cells enter a lineage plasticity state and differentiate to their final cell fate.

[0003] Existing research has made significant progress in understanding the mechanisms and techniques of hair regeneration. Hair follicle stem cells (HFSCs), located in different parts of the hair follicle such as the dermal papilla, hair bulb, and follicular ridge, finely coordinate the activation states of different follicle sites to promote the hair cycle, hair growth, and regeneration. This process involves the Wnt, BMP, and Hedgehog signaling pathways. Three hair regeneration mechanisms have been revealed: First, wound healing is highly correlated with hair regeneration. Wound-induced hair follicle regeneration and regrowth is a well-established model. The epidermal Wnt signaling pathway is crucial for this process and influences the skin's mechanosensitivity at both the cellular and tissue levels. Recent studies have shown that in skin wound models, local retinoic acid levels in the HFSC microenvironment determine the plasticity of HFSCs to address their lineage issues, thereby enabling hair regeneration and damaged epidermal repair. The neuropeptide calcitonin and TRPV1 neurons also play key roles in epidermal abrasion-induced hair growth, which is associated with macrophages. Secondly, innate skin immune cells, including mast cells, γδT cells, natural killer cells, and macrophages, also contribute to the hair cycle and the activation of hair follicle stem cells. Macrophages participate in hair cycle-related angiogenesis under both physiological and pathological conditions, while γδT cells promote hair follicle regeneration after trauma. Regulatory T cells promote the proliferation and differentiation of hair follicle stem cells, initiating the hair cycle and hair follicle regeneration. Thirdly, metabolic and oxidative stress regulate the state of hair follicle stem cells and hair growth. Obesity, by producing excessive reactive oxygen species (ROS), promotes the development of activated hair follicle stem cells toward epidermal keratinization, leading to hair follicle miniaturization and eventual hair loss. Conversely, local hypoxia induced by resident skin bacteria enhances glutamine metabolism in keratinocytes, promoting skin and hair follicle regeneration. These findings gradually elucidate how stem cells regulate the hair growth cycle, opening prospects for the clinical development of small molecule drugs and biotechnologies for hair regeneration. However, these fundamental findings are relatively complex and difficult to translate into feasible hair follicle technologies. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the present invention provides a non-invasive optical system and method for stimulating hair regeneration.

[0005] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0006] A first aspect of the present invention protects a non-invasive optical system for stimulating hair regeneration, the non-invasive optical system comprising:

[0007] Femtosecond laser unit, used to generate femtosecond lasers;

[0008] An optical focusing unit is used to focus the femtosecond laser onto the basal layer of skin tissue to stimulate basal cells in the basal layer to form hair follicles and thus stimulate hair regeneration.

[0009] In some embodiments, the femtosecond laser unit includes a femtosecond laser 100 for generating femtosecond lasers;

[0010] The optical focusing unit includes a galvanometer assembly and an objective lens 500;

[0011] The galvanometer assembly is used to guide the femtosecond laser to the objective lens 500;

[0012] The objective lens 500 is used to focus the femtosecond laser, which has passed through the galvanometer assembly, onto the basal layer of the skin tissue;

[0013] The galvanometer assembly and the femtosecond laser 100 are coupled via a first dichroic mirror 105.

[0014] In some embodiments, a beam expander 400 is provided between the galvanometer assembly and the objective lens 500, the beam expander 400 being used to increase the beam diameter of the femtosecond laser.

[0015] In some embodiments, the femtosecond laser unit further includes a half-wave plate 101, a polarizing beam splitter 102, and a control switch 103. The femtosecond laser generated by the femtosecond laser 100 is transmitted to the basal layer of the skin tissue by passing sequentially through the half-wave plate 101, the polarizing beam splitter 102, the first dichroic mirror 105, the galvanometer assembly, the beam expander 400, and the objective lens 500.

[0016] In some embodiments, the femtosecond laser has a wavelength of 700-1000 nm.

[0017] In some embodiments, the femtosecond laser has a power of 60-100mW.

[0018] In some embodiments, the pulse width of the femtosecond laser is 30-300 fs.

[0019] In some implementations, the scanning time of the femtosecond laser is ≤5s.

[0020] In some embodiments, the non-invasive optical system further includes an analysis unit for determining the location of the basal layer based on an image formed after the excitation light or excitation wave passes through the skin tissue.

[0021] A second aspect of the present invention protects a method of using the non-invasive optical system as described above, the method comprising:

[0022] A femtosecond laser is generated and the femtosecond laser is focused onto the basal layer of the skin tissue.

[0023] In some embodiments, the femtosecond laser has a wavelength of 700-1000 nm.

[0024] In some embodiments, the femtosecond laser has a power of 60-100mW.

[0025] In some embodiments, the pulse width of the femtosecond laser is 30-300 fs.

[0026] In some implementations, the scanning time of the femtosecond laser is no more than 5 seconds.

[0027] A third aspect of the present invention protects a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the usage method as described above.

[0028] A fourth aspect of the present invention protects a computer processing apparatus, including a processor and a computer-readable storage medium as described above, wherein the processor executes a computer program on the computer-readable storage medium to implement the steps of the usage method as described above.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The system and method of this invention stimulate the basal layer with a femtosecond laser, thereby specifically stimulating endogenous retinol in the skin (especially basal cells). Under the influence of retinol, basal cells transform into hyperproliferating cells and Sox9. + The system and method of this invention provide an effective, non-invasive technique for hair follicle regeneration, and thus hair regeneration. This technique has promising prospects for translational applications. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the non-invasive optical system for stimulating hair regeneration according to the present invention.

[0032] Figure 2 This is a schematic diagram of the non-invasive optical system for stimulating hair regeneration according to the present invention.

[0033] Figure 3 This is a flowchart illustrating the method of using the non-invasive optical system for stimulating hair regeneration according to the present invention.

[0034] Figure 4This diagram illustrates the effect of photostimulation of basal cells (BCs) on hair regeneration. In the diagram, A represents the experimental procedure for photostimulation of basal cells (red line). Hair removal (blue) and observation (yellow) were performed at designated time points. The upper inset shows the photostimulation optical apparatus and basal cell microscopic imaging. Mice were placed back-down on a microscopic platform for back skin microscopic observation and photostimulation. A second laser was coupled to the first laser and focused through an objective lens (30x, oil immersion, numerical aperture = 1.05). The first laser excited basal cells to produce autofluorescence, which guided the second laser to precisely locate the basal layer. The second laser stimulation was controlled by a switch and a galvanometer, performing a single 3-second laser scan of a preset area. Abbreviations: CW for first laser; fs for second laser; λ / 2 for half-wave plate; PBS for polarizing beam splitter; DM for dichroic mirror; FOV for field of view; BC for basal cells. The scale bar is 20 micrometers. The lower inset shows a photograph (left) of the back skin of mice on day 0 before laser treatment and β-catenin immunofluorescence (right). Scale bar: 50 micrometers. B shows representative images of skin autofluorescence on days 0, 2, and 4 after photostimulation. Arrows indicate hair autofluorescence. Scale bar: 50 micrometers. C and D are quantitative analyses of the average hair length (C) and hair density (D) over time in Figure B (n = 16-18 fields of view for 4 mice). Statistical comparisons were made against the control group. E shows representative images of mouse skin on days 0 and 21 after photostimulation (n = 4 mice). Red boxes indicate the photostimulated areas. F shows representative images of the mouse backs on days 0, 31, and 34 after re-hairing on day 31 (n = 4 mice, 3 of which showed this phenomenon).

[0035] Figure 5 This diagram illustrates hair regeneration in other skin layers after photostimulation. A shows a schematic diagram of photostimulation in different skin layers. B shows typical autofluorescence images of the skin on days 0, 2, and 4 after photostimulation of the stratum corneum, basal layer, and dermis, respectively. Scale bar: 50 μm. C and D represent quantitative analyses of the average hair length (C) and hair density (D) in image (B), respectively (control group: 24 fields of view from 4 mice; laser group: 16-18 fields of view from 4 mice). Statistical comparisons were based on day 0.

[0036] Figure 6This diagram shows the morphogenesis of hair follicles after photostimulation. In Figure A, the morphology of hair follicles at different stages of regeneration after photostimulation (displayed by β-catenin immunofluorescence) is shown. The epidermal-dermal junction is marked with a dashed line. Scale bar: 50 μm. Inset: Enlarged image of a typical hair follicle (HF). Epi represents the epidermis; Bg represents the bulge region; JZ represents the junction region; SG represents the sebaceous gland; DP represents the dermal papilla. Arrows indicate laser-induced hair follicles. Scale bar: 20 μm. Figure B shows the quantitative analysis of the average hair follicle length in Figure A. At 0 h, 28 fields of view were obtained from 4 mice; at 3 h, 23 fields of view were obtained from 4 mice in the control group and 29 fields of view were obtained from 4 mice in the laser group; at 6 h, 23 fields of view were obtained from 4 mice; at 12 h, 28 fields of view were obtained from 4 mice in the control group and 30 fields of view were obtained from 4 mice in the laser group; at 24 h and 6 d, 28 fields of view were obtained from 4 mice in the control group and 33 fields of view were obtained from 4 mice in the laser group. Statistical comparisons were based on the control group. C shows typical CD34 immunofluorescence images of skin 12 hours and 6 days after light stimulation. The epidermal-dermal junction is marked with a dashed line. Scale bar: 30 μm. Inset: Enlarged image of the hair follicle bulge area. Arrows indicate typical CD34-positive cells. Scale bar: 10 μm.

[0037] Figure 7 Two-photon excitation of retinol in basal cells promotes hair follicle formation. A shows representative immunofluorescence images of β-catenin in the skin 24 hours after laser stimulation at different wavelengths (700-1000 nm). Scale bar: 30 μm. B and C are quantitative analyses of the mean hair follicle length (B) and normalized hair follicle density (C) in image A, respectively. Control group: 8 mice, 56 fields of view; Laser group: 4 mice, 28-33 fields of view. Statistical comparisons were based on the control group.

[0038] Figure 8 This diagram illustrates laser-induced hair follicle formation in a high-fat diet mouse model. A shows a schematic diagram of the photostimulation (red-marked) experimental procedure in the high-fat diet mouse model. Mice (6-8 weeks old) were fed a high-fat diet (green-marked) starting from day 70, as shown above. Hair removal (blue-marked) and skin harvesting (purple-marked) were performed at designated time points. B shows representative images of mice in the high-fat diet group (n=4) and the normal diet group (n=3). C shows β-catenin immunofluorescence images of the skin on days 0, 1, and 3 after photostimulation. Red arrows indicate epidermal thickening caused by the high-fat diet, white arrows indicate laser-induced hair follicles, and yellow arrows indicate hair. Scale bar: 50 micrometers. D and E show the quantitative analysis of the mean hair follicle length (D) and normalized hair follicle density (E) in image (C) (23-38 fields of view for 4 mice). Statistical comparisons were made against the control group. BC: basal cells; TAC: transiently expanded cells.

[0039] Figure 9This section presents the results of a study on the mechanism by which retinol two-photon excitation in basal cells promotes hair follicle formation. D is a schematic diagram of the retinoic acid signaling pathway steps and related inhibitors, including transcriptional activity. RAR:RXR: Retinyl acid receptor-retinoid X receptor heterodimer. E shows the metabolism of retinol-FITC (1 μM) in Hepa 1-6 cells, with or without ALDH1A inhibitor (10 μM). Scale bar: 10 μm. Right panel: Quantitative retinol-FITC levels (18 fields of view in 3 independent experiments). Statistical comparisons were made against the control group. F shows the fluorescence changes of retinol-FITC (1 μM) before and after light stimulation in Hepa 1-6 cells. Pink box: Light-stimulated area; Blue box: Control area. Scale bar: 5 μm. Right panel: Retinol-FITC ratio before and after stimulation (18 fields of view in 3 independent experiments). Statistical comparisons were made against the control group. G represents representative immunofluorescence images of skin β-catenin 24 hours after photostimulation with ALDH1A inhibitor 673A (20 μM) or AGN193109 (200 nM), or 24 hours after treatment with atRA (200 nM) alone. Scale bar: 30 μm. H and I represent quantitative analyses of mean hair follicle length (H) and normalized hair follicle density (I) in (G) (28-33 fields of view from 4 mice). Statistical comparisons in the atRA group were based on the untreated control group, and other groups were based on the Base group.

[0040] Figure 10 This section describes the transformation of basal cells into transiently proliferating cells induced by photostimulation. A, C, E, and G represent the immunofluorescence staining of K14 (A), Ki67 (C), CD71 / α6 (E), and Sox9 (G) in the skin 3-24 hours after photostimulation. The epidermal-dermal junction is marked with a dashed line. Arrows indicate representative K14. - Cell (A), Ki67 + Cells (C), CD71 + / α6 + Cells (E) and Sox9 + Cells (G). Scale bar: 30 micrometers. B shows the quantitative analysis of K14 immunofluorescence in the epidermis (27-31 fields from 4 mice). D shows the Ki67 in the epidermis. + Cell percentage (25-27 fields of view, 4 mice). F represents the fluorescence ratio of CD71 to α6 in the epidermis (29-32 fields of view, 4 mice). H represents the fluorescence ratio of Sox9 in the epidermis. + Cell percentage (26-27 fields of view from 4 mice) and Sox9 in all hair follicles + Accumulated percentage of cells (26-28 fields of view, 4 mice). Statistical comparisons were made with the control group as the baseline. I represents CD71 at 3 hours and 6 hours. + Cells and Sox9 +Cells. Scale bar: 20 micrometers.

[0041] Figure 11 This is a schematic diagram illustrating the mechanism by which the present invention stimulates hair regeneration.

[0042] in, Figure 1 The accompanying diagram is labeled as follows: 11, femtosecond laser unit; 12, optical focusing unit.

[0043] in, Figure 2 The attached figures are labeled as follows:

[0044] 100 femtosecond laser

[0045] 101 Half-wave film

[0046] 102 Polarization beam splitter

[0047] 103 Control Switch

[0048] 104 Reflectors

[0049] 105 First Dichroic Mirror

[0050] 106 Second dichroic mirror

[0051] 107 First Galvanometer

[0052] 108 Second Galactic Mirror

[0053] 200 Excitation Laser

[0054] 300 detectors

[0055] 400 beam expander

[0056] 500 objective lens. Detailed Implementation

[0057] This application proposes a non-invasive optical system and method for stimulating hair regeneration. It utilizes femtosecond laser stimulation of the basal layer of skin tissue to efficiently activate endogenous retinol in basal cells. Endogenous retinol is converted into all-trans retinoic acid, prompting basal cells to transform into transiently proliferating cells and activating Sox9. + Within 24 hours, hair follicles are formed from the cells, leading to hair regeneration. The non-invasive optical system and method of this application directly initiate the formation of new hair follicles by instantaneously stimulating skin tissue with a single femtosecond laser, providing a simple, non-invasive, and efficient strategy for hair regeneration with significant application potential.

[0058] like Figure 1 and 2 As shown, this is a non-invasive optical system for stimulating hair regeneration according to this application. The non-invasive optical system includes:

[0059] Femtosecond laser unit, used to generate femtosecond lasers;

[0060] An optical focusing unit is used to focus the femtosecond laser onto the basal layer of skin tissue to stimulate basal cells in the basal layer to form hair follicles and thus stimulate hair regeneration.

[0061] The skin consists of three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis is further divided into five layers: the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum is the outermost layer, composed of several to dozens of layers of flattened keratinized cells filled with keratin, forming an important protective layer of the skin. The stratum lucidum is composed of several layers of flattened cells with indistinct cell boundaries, homogeneous and transparent cytoplasm, and absent cell nuclei. The stratum basale is the innermost layer of the epidermis, composed of a single layer of low columnar basal cells with a strong capacity for division and proliferation, serving as the source of epidermal cell renewal.

[0062] The non-invasive optical system of this application stimulates the basal layer of skin tissue with a femtosecond laser, promoting the conversion of endogenous retinol in basal cells into all-trans retinoic acid (atRA). AtRA binds to retinoic acid receptors (RAR) and retinol X receptors (RXR), activating the expression of downstream genes such as Sox9, causing basal cells to transform into transiently proliferating cells (TA cells). Ultimately, TA cells differentiate into cells expressing Sox9 (Sox9...). + The process involves inducing epidermal cell thickening to form a basal plate, which then grows downwards to form a hair bulb. The hair bulb continues to grow downwards to develop into a hair follicle, leading to hair regeneration. The femtosecond laser in this application's non-invasive optical system has an extremely short pulse width and extremely high peak pulse power, enabling two-photon excitation of retinol in the basal layer. Furthermore, the focusing unit confines the energy of the femtosecond laser within a focusing ellipsoid, spatially enabling specific action on a very small area and increasing the peak pulse power. This achieves multi-photon excitation of endogenous retinol in the basal layer of skin tissue with high targeting, while having no effect on the stratum corneum or dermis.

[0063] In some embodiments, the femtosecond laser unit includes a femtosecond laser 100 for generating femtosecond lasers;

[0064] The optical focusing unit includes a galvanometer assembly and an objective lens 500;

[0065] The galvanometer assembly is used to guide the femtosecond laser to the objective lens 500;

[0066] The objective lens 500 is used to focus the femtosecond laser, which has passed through the galvanometer assembly, onto the basal layer of the skin tissue;

[0067] The galvanometer assembly and the femtosecond laser 100 are coupled via a first dichroic mirror 105. A dichroic mirror is a special optical element that selectively reflects and transmits light of a specific wavelength using the principle of thin-film interference. The femtosecond laser emitted by the femtosecond laser 100 is projected through the first dichroic mirror 105, its beam direction is adjusted by the galvanometer assembly, and it is focused onto the basal layer of the skin tissue by the objective lens 500.

[0068] In some embodiments, the femtosecond laser 100 is a fiber femtosecond laser, a Ti:sapphire femtosecond laser, or a femtosecond laser-pumped optical parametric oscillator. Preferably, it is a Ti:sapphire femtosecond laser (wavelength 680-1080nm; 140±20fs, 80MHz; Chameleon Ultra II, Coherent).

[0069] In some specific embodiments, the galvanometer assembly includes a first galvanometer 107 and a second galvanometer 108. A galvanometer is a rapidly rotating mirror that changes the direction of the reflected light beam by altering its angle. The first galvanometer 107 is used to adjust the scanning of the light beam along the X-axis; it scans the beam along the X-axis of the sample by rapid rotation. The second galvanometer 108 is used to adjust the scanning of the light beam along the Y-axis; it scans the beam along the Y-axis of the sample by rapid rotation. The first and second galvanometers need to operate synchronously to achieve scanning of skin tissue.

[0070] In some specific embodiments, the femtosecond laser unit further includes a half-wave plate (referred to as 1 / 2λ) 101, a polarizing beam splitter 102, and a control switch 103. The femtosecond laser generated by the femtosecond laser (100) is focused onto the basal layer of the skin tissue by passing sequentially through the half-wave plate (101), the polarizing beam splitter (102), the first dichroic mirror (105), the galvanometer assembly, the beam expander (400), and the objective lens (500). The femtosecond laser unit also includes a reflecting mirror 104; the reflecting mirror 104 is used to reflect the femtosecond laser emitted after passing sequentially through the half-wave plate 101 and the polarizing beam splitter 102, and transmit it to the first dichroic mirror 105. Control switch 103 controls the exposure stimulation time of the femtosecond laser on the basal layer. Control switch 103 has a time resolution of 10 ms. The switching of control switch 103 is synchronized with the galvanometer assembly (first galvanometer 107 and second galvanometer 108). In scanning stimulation mode, control switch 103 turns on when scanning reaches the basal layer. In scanning stimulation mode, the exposure time is defined as the time required to scan one frame of the basal layer; in point stimulation mode, an exposure time of 0.1–0.2 s is typically selected.

[0071] In some embodiments, a beam expander 400 is also provided between the galvanometer assembly and the objective lens 500. The beam expander 400 is used to expand the beam diameter of the femtosecond laser and to match the back pupil of the objective lens 500.

[0072] In some embodiments, the objective lens is a 10×-60× lens with a numerical aperture of 0.9-1.5. Preferably, it is a 30× lens with a numerical aperture of 1.05.

[0073] In some embodiments, the wavelength of the femtosecond laser can be 700-1000 nm, 700-850 nm, 800-1000 nm, or 700 nm, 800 nm, 900 nm, or 1000 nm. This application conducted experiments on the stimulation of hair follicle regeneration with femtosecond lasers of different wavelengths, finding that femtosecond lasers of 700-1000 nm can stimulate hair follicle formation, with the longest hair follicles formed at wavelengths of 800 nm and 900 nm. The hair follicle density produced by stimulation at 800 nm was significantly higher than that at 900 nm and 700 nm. Furthermore, studies using obese mice as experimental subjects also showed that femtosecond lasers with wavelengths of 700-1000 nm can still stimulate hair follicle regeneration.

[0074] In some embodiments, the power of the femtosecond laser can be 60-100mW, 60-85mW, 80-100mW, or 60mW, 70mW, 80mW, 90mW, or 100mW.

[0075] In some embodiments, the pulse width of the femtosecond laser is 30-300 fs, specifically 30-135 fs, 130-250 fs, 240-300 fs, or even 30 fs, 50 fs, 80 fs, 100 fs, 120 fs, 130 fs, 140 fs, 150 fs, 160 fs, 210 fs, 240 fs, 260 fs, or 300 fs. In this application, the femtosecond laser pulse interval is relatively large compared to the pulse width, thus avoiding strong thermal effects and causing no damage to skin tissue.

[0076] In some embodiments, the scanning time of the femtosecond laser is ≤5s. In this application, the scanning time for femtosecond laser stimulation of hair follicle regeneration cannot be too long, as excessive time can lead to excessive photodamage and photobleaching of the skin.

[0077] In some embodiments, the non-invasive optical system further includes an analysis unit for determining the location of the basal layer based on an image formed after the excitation light or excitation wave passes through the skin tissue. The location of the basal layer can be determined using various non-invasive techniques. These techniques primarily utilize the interaction between excitation light or excitation waves of different wavelengths and skin tissue to present subcutaneous structures non-invasively, including but not limited to ultrasound imaging, excitation laser imaging (such as confocal microscopy), and near-infrared imaging (such as optical coherence tomography, OCT); the location of the basal layer can also be determined empirically. The excitation light can be an excitation laser or near-infrared light, and the excitation wave can be ultrasound. Excitation laser imaging utilizes laser excitation of fluorescence signals in the skin tissue, generating an image of the skin tissue by receiving the fluorescence signals, thereby determining the location of the basal layer; near-infrared imaging utilizes the high penetration and low scattering characteristics of near-infrared light in skin tissue, generating an image by receiving reflected or transmitted near-infrared light, thereby determining the location of the basal layer; ultrasound imaging utilizes the propagation and reflection characteristics of high-frequency sound waves in skin tissue, generating an image of the skin tissue by receiving reflected signals, thereby determining the location of the basal layer.

[0078] In some embodiments, the location of the basal layer is determined by excitation laser imaging, wherein the analysis unit is used to determine the location of the basal layer based on the fluorescence image of the skin tissue after the excitation laser passes through it.

[0079] In some specific embodiments, the analysis unit includes: an excitation light module for generating an excitation laser and focusing it onto skin tissue via the optical focusing unit to excite the skin tissue to emit a fluorescence signal;

[0080] An imaging module generates a fluorescence image based on the fluorescence signal and determines the basal layer of the skin tissue based on the fluorescence image.

[0081] In some more specific embodiments, the excitation light module includes an excitation light source 200. Preferably, the excitation light source 200 is an excitation laser with a wavelength of 400-600nm, or 400-550nm, or 500-600nm, or 400nm, 500nm, or 600nm, and in a specific embodiment, 473nm.

[0082] In some more specific embodiments, the imaging module includes a detector 300. The detector 300 is a photodetector (PMT) or a charge-coupled device (CCD). Preferably, it is a PMT. The PMT detects fluorescence signals, generates single-point light intensity data, and can generate a two-dimensional image through rapid scanning. The detector 300 and the excitation light source 200 are connected via an optical signal.

[0083] In some specific embodiments, the analysis unit can form an independent optical path, or the excitation light source 200 can be coupled to the galvanometer assembly in the optical focusing unit through the first dichroic mirror 105, and the detector 300 can be coupled to the galvanometer assembly in the optical focusing unit through the second dichroic mirror 106. One branch optical signal of the second dichroic mirror 106 is connected to the galvanometer assembly, one branch optical signal is connected to the first dichroic mirror 105, and one branch optical signal is connected to the detector 300. The excitation laser emitted by the exciter 200 is reflected by the first dichroic mirror 105, transmitted by the second dichroic mirror 106, the beam is adjusted by the galvanometer assembly, and then focused on the skin tissue after passing through the beam expander 400 and the objective lens 500 to excite the basal layer of the skin tissue to emit fluorescence signals; then, the fluorescence signals pass through the objective lens 500, the beam expander 400, the galvanometer assembly in sequence, and are reflected by the second dichroic mirror 106 and transmitted to the detector 300.

[0084] In a specific embodiment, the optical focusing unit and the analysis unit are provided by a confocal microscope, specifically, it can be an Olympus FV1200 / IX83 laser scanning confocal microscope. The excitation laser wavelength is adjusted and imaging analysis are performed by using the Olympus FV1200 system control software. The excitation laser excites the fluorescence signals in the skin basal cells, and the basal layer is determined through the fluorescence images in confocal imaging. The confocal microscope uses a water immersion objective lens with a magnification of 60X and a numerical aperture of 1.2 or an oil immersion objective lens with a magnification of 30X, and uses the 405nm, 473nm and 543nm semiconductor lasers supporting the FV1200 as the excitation light sources for fluorescence and bright field imaging. The femtosecond laser optical path is coupled to the confocal microscope through the first dichroic mirror 105.

[0085] The basal cells usually appear columnar and have a compact structure under the confocal microscope. When stimulating the skin with the excitation light, generally half of the imaging area (the number of pixels is 640×329) or the entire area (640×640) is selected. When scanning to determine the basal layer, the control switch 103 of the femtosecond laser optical path is synchronously turned on, and after one-frame scanning stimulation of the basal layer, the control switch 103 is turned off to complete the optical stimulation of the basal layer. The size of the basal layer in the target area is about 4mm×4mm.

[0086] Specifically, the excitation laser 200 emits an excitation laser beam, which is reflected by the first dichroic mirror 105, transmitted through the second dichroic mirror 106, and then adjusted by the first galvanometer mirror 107 and the second galvanometer mirror 108 before entering the microscope 500 through the beam expander 400. This allows the excitation laser to be focused on the skin, and the skin emits a fluorescent signal after being stimulated by the excitation laser. The emitted fluorescent signal passes sequentially through the microscope 500, the beam expander 400, the second galvanometer mirror 108, and the first galvanometer mirror 107, and is reflected by the second dichroic mirror 106 before being transmitted to the detector. The detector 300 determines the location of the basal layer based on the fluorescence image in the detector 300; the control switch 103 is turned on, and the femtosecond laser generated by the laser generator 100 is adjusted by the half glass plate 101 and the polarizing beam splitter 102 in sequence, reflected by the mirror 104 and transmitted by the first dichroic mirror 105 and the second dichroic mirror 106, and after the beam is adjusted by the first galvanometer 107 and the second galvanometer 108, it enters the objective lens 500 through the beam expander 400, so that the femtosecond laser stimulates the selected basal layer, thereby realizing hair follicle regeneration and hair regeneration.

[0087] In this invention, promoting hair growth refers to increasing the average hair follicle length and / or hair follicle density.

[0088] like Figure 2 The diagram illustrates a method of using the non-invasive optical system described in this application, comprising:

[0089] S100, generate a femtosecond laser and focus the femtosecond laser onto the basal layer of the skin tissue.

[0090] In some embodiments, the wavelength of the femtosecond laser is 700-1000nm, or it can be 700-850nm, or it can be 800-1000nm, or it can be 700nm, 800nm, 900nm, or 1000nm.

[0091] In some embodiments, the power of the femtosecond laser is 60-100mW, or it can be 60-85mW, or it can be 80-100mW, or it can be 60mW, 70mW, 80mW, 90mW, or 100mW.

[0092] In some embodiments, the pulse width of the femtosecond laser is 30-300 fs, which can be 30-135 fs, 130-250 fs, 240-300 fs, or 30 fs, 50 fs, 80 fs, 100 fs, 120 fs, 130 fs, 140 fs, 150 fs, 160 fs, 210 fs, 240 fs, 260 fs, or 300 fs.

[0093] In some implementations, the scanning time of the femtosecond laser is no more than 5 seconds.

[0094] The present invention also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the method described above.

[0095] The present invention also provides a computer processing device, including a processor and the aforementioned computer-readable storage medium, wherein the processor executes a computer program on the computer-readable storage medium to implement the steps of the aforementioned method.

[0096] Computer-readable storage medium: As will be understood by those skilled in the art, all or part of the steps of the above-described method embodiments can be implemented by hardware related to a computer program. The aforementioned computer program can be stored in a computer-readable storage medium. When the program is executed, it performs the steps of the above-described method embodiments; and the aforementioned storage medium includes various media capable of storing program code, such as ROM, RAM, magnetic disks, or optical disks.

[0097] In this application, the skin refers to both real skin samples isolated from organisms and model skin prepared through non-biological methods. Preparation of real skin samples: First, the desired skin portion (epidermis, whole skin, etc.) is separated from human or animal tissue, pressed into a suitable shape (generally sheet-like), and stored in a storage solution for later use. Preparation of model skin: Suitable skin cells are cultured in a cell culture chamber, and different substructures of the skin (stratum corneum, granular layer, spinous layer, basal layer, etc.) are obtained through cell division, ultimately yielding artificial skin which is then stored in a storage solution for later use; alternatively, various phospholipids, proteins, and other biomolecules are mixed according to the actual proportions in organisms, and a multi-layered model skin is prepared using a self-assembly membrane method or a vesicle rupture method, and stored in a refrigerator for later use.

[0098] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0099] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0100] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0101] Example 1: Optical System and Stimulation of Hair Follicle Regeneration

[0102] In this embodiment, mice were used as experimental subjects, and femtosecond laser stimulation was applied to them to stimulate hair follicle regeneration. The non-invasive optical system in this embodiment is based on a confocal microscope and a femtosecond laser optical path. It includes the following:

[0103] 1.1 Mice and Experimental Procedure

[0104] Animal husbandry and experimental protocols followed the guidelines of the School of Biomedical Engineering, Shanghai Jiao Tong University, and the Animal Ethics Committee of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval Nos.: 202101332, A2024148). Mice were housed in a temperature- and humidity-controlled environment, with a maximum of 5 mice per cage, maintaining a 12-hour light-dark cycle. Six- to eight-week-old C57BL / 6 male and female mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. All experiments were conducted using anesthesia with a mixture of isoflurane (1-2%) and oxygen (0.5-1.0 L / min).

[0105] Hair removal was performed two days before the experiment, and a second hair removal was performed 31 days later for follow-up observation. First, the hair on the back was shaved, then hair removal cream (Veet, Reckitt Benckiser) was applied for 3 minutes, and then rinsed off with warm water.

[0106] Following laser or drug treatment, in vivo observation (photography or microscopic imaging) or tissue sampling and analysis were performed on the dorsal skin of mice at predetermined time points. Skin samples were fixed in 4% paraformaldehyde (Beyotime) for 24 hours at 4°C, dehydrated in 30% sucrose solution for 24 hours, rapidly frozen, and then embedded in OCT (Sakura Medical, Japan) and stored at -80°C. The samples were then sectioned into 15μm thick sections using a cryostat (Leica).

[0107] 1.2. Photostimulation

[0108] like Figure 4A. Mice were fixed in a custom-made support, with coverslips pressed flat against their backs. They were placed dorsally down on the stage of a confocal microscope, and anesthesia was maintained with isoflurane (Anesthesia mask). The basal layer to be stimulated by subsequent femtosecond laser stimulation was located using epidermal basal layer autofluorescence microscopy (excitation 473nm, detection 490-525nm). A confocal microscope (Olympus, FV1200) acquired fluorescence images at 640×640 pixels / frame and 2μs / pixel. Only when basal cell autofluorescence was clearly visible (approximately 20μm depth in mouse skin) was a femtosecond laser focused on the same objective lens scanned the basal layer of the pre-defined target area in the same plane. The basal layer position needed to be recalibrated using autofluorescence before changing the field of view.

[0109] A Ti:sapphire femtosecond laser (tunable wavelength 680-1080 nm, pulse width 140±20 fs, repetition rate 80 MHz; Coherent, Chameleon Ultra II) is coupled to a confocal microscope (for autofluorescence imaging). The femtosecond laser shares the optical path with the confocal system, achieving synchronization between femtosecond laser stimulation and microscopy. The femtosecond laser beam is jointly controlled by a control switch and a galvanometer assembly, and focused through a 30x oil immersion lens (objective) (NA=1.05). The focused femtosecond laser scans the basal layer of the preset target area (approximately 4×4 mm) in a continuous field of view. 2 Vertically adjust the scanning plane to target the basal layer (approximately 20 μm below the skin surface, localized based on basal cell autofluorescence). Each field of view (FOV) is 420 × 420 μm. 2 It accepts a single 3-second continuous point scan (640×640 pixels, 2μs / pixel, 2 frames) using an 800nm, 80mW femtosecond laser.

[0110] 1.3 Multiphoton excitation of the basal layer promotes hair regeneration

[0111] Figure 4 A illustrates the experimental setup. A femtosecond laser (Chameleon Ti: sapphire laser, Coherent, 690-1060nm, 80MHz, 75fs) was coupled to a confocal microscope via a dichroic mirror DM1. The beam was then expanded and aligned to the objective lens (30×, numerical aperture (NA) = 1.05, oil immersion). This enabled simultaneous confocal microscopy imaging and femtosecond laser scanning on the same plane.

[0112] C57BL / 6 mice (6-8 weeks old) underwent hair removal two days prior to femtosecond laser scanning. After shaving the back, the skin was treated with a hair removal cream (Veet, Reckitt Benckiser) for 3 minutes, followed by rinsing with warm water. This treatment completely removed the hair and significantly damaged the hair follicles in the skin. Figure 4 (Illustration A)

[0113] Laser scanning (Day 0) is achieved by using a focused femtosecond laser to perform a single-frame continuous field of view (FOV) scan of the predetermined photostimulation region (i.e., the target stimulation region) on the skin. The focal point localization of the femtosecond laser (depth approximately 20 μm) is guided by real-time autofluorescence imaging of basal cells (BCs). Figure 4 (Illustration A)

[0114] After acquiring clear images of basal cells, a femtosecond laser focused by the same objective lens begins scanning in the same plane. Each field of view (420 μm × 420 μm) is scanned in a single 3-second burst using an 800 nm femtosecond laser (80 mW).

[0115] Therefore, a single full-coverage scan of a 4mm×4mm area takes 300 seconds. Skin stimulated by light was examined immediately after treatment (day 0) and on days 2, 4, 21, 31 (with a second hair removal treatment on day 31), and 34.

[0116] from Figure 4 As shown in B, autofluorescence images from days 2 to 4 revealed significant hair regeneration and growth in the light-stimulated areas, while no such effect was observed in the control areas.

[0117] from Figure 4 As shown in C and 1D, compared with the control group, the average hair length and hair number of the light-stimulated area in the laser group were significantly increased.

[0118] from Figure 4 E shows that by day 21, the regenerated hair became unusually thick.

[0119] from Figure 4 As shown in F, after hair removal again on day 31, the area of ​​skin stimulated by light turned black, indicating that melanocytes in this area proliferated and melanin was upregulated, which is consistent with the spontaneous hair regeneration phenomenon on day 34.

[0120] In summary, scanning the skin with an 800nm ​​femtosecond laser to stimulate basal cells can significantly promote hair regeneration in mice.

[0121] Example 2: Screening of different skin layers

[0122] In this embodiment, the target stimulation area and stimulation wavelength under the skin were screened using femtosecond laser stimulation. This included the following:

[0123] 2.1 Screening of different skin layers

[0124] Mice were divided into four groups of four mice each: a corneum group, a stratum corneum group, a dermis group, and a control group. Each group of mice underwent femtosecond laser stimulation using the non-invasive optical system described in Example 1. The control group did not receive femtosecond laser stimulation. The average hair length and hair follicle density (hair number / mm²) were recorded immediately after femtosecond laser stimulation (day 0), day 2, and day 4. 2 ).

[0125] The wavelengths and frequencies of femtosecond laser treatment on the stratum corneum (0 μm depth), basal layer (approximately 20 μm depth), and dermis (>60 μm depth) are shown in [reference needed]. Figure 5 A. Laser power for different skin layers is compensated for by tissue attenuation: 60mW for stratum corneum (0μm depth), 80mW for basal layer (approximately 20μm depth), and 100mW for dermis (>60μm depth). When adjusting the wavelength within the range of 700-1000nm, the power at the sample location remains constant at 80mW.

[0126] from Figure 5 According to BD, on the 2nd and 4th days, only the basal layer group was able to induce hair regeneration and growth after femtosecond laser stimulation, and the average hair follicle length and hair follicle density of the basal layer group were the highest; the stratum corneum and dermis did not produce regeneration effect after laser stimulation.

[0127] 2.2 Immunofluorescence labeling with β-catenin was used to examine changes in epidermal and hair follicle morphology.

[0128] The basal layer group from Example 2 was used as the subject of investigation. β-catenin immunofluorescence staining was employed to examine changes in epidermal and hair follicle morphology.

[0129] Immunofluorescence staining included: fixation of frozen sections of skin tissue with 4% PFA (Beyotime), permeabilization with 0.3% Triton X-100 (Beyotime), and then staining with QuickBlock. TM Blocked with Beyotime buffer. Sections were then incubated overnight at 4°C with primary antibody, followed by incubation with secondary antibody at room temperature for 2 hours. Finally, they were blocked with Fluoroshield containing DAPI. TM Mounting solution (Sigma, F6057) for mounting.

[0130] The primary antibodies used are as follows: anti-β-catenin rabbit antibody (Abcam, ab32572, 1:250), anti-CD34 rabbit antibody (Abcam, ab81289, 1:250), and anti-K14 rabbit antibody (Huabio, ET1610-42, 1:1000). 488 anti-Ki67 rabbit antibody (Abcam, ab281847, 1:200), anti-CD71 mouse antibody (Invitrogen, 13-6800, 1:250), anti-α6 rabbit antibody (Proteintech, 27189-1-AP, 1:500), anti-Sox9 rabbit antibody (Huabio, ET1611-56, 1:500). Secondary antibody as follows: goat anti-rabbit IgG H&L ( 488,Abcam,ab150077,1:500; 555, Abcam, ab150078, 1:500), goat anti-mouse IgG H&L ( 488, Abcam, ab150113, 1:500).

[0131] Images were acquired using a confocal microscope (FV1200, Olympus) at 1024×1024 pixels / frame and 2 microseconds / pixel.

[0132] Figure 6 A shows the timeline of hair follicle morphogenesis in the epidermis (labeled Epi) and dermis after laser stimulation: Placode formation is observed 3 hours after scanning; typical morphological evolution from hair germ to hair peg is observed between 6 and 12 hours; mature hair follicle (labeled HF) structures form within 24 hours; and by day 6, the bulge (Bg), sebaceous gland (SG), and dermal papilla (DP) structures are clearly visible. Figure 6 A).

[0133] from Figure 6 As shown in B, the average HF length of laser-induced hair follicles increases significantly over time, which is consistent with the hair follicle morphogenesis process.

[0134] from Figure 6 As shown in C, CD34 (a marker of hair follicle stem cells in adult ridges) was not detected at 12 hours, but was clearly located in the ridges at day 6.

[0135] In summary, sections 2.1 and 2.2 demonstrate that femtosecond laser scanning of the basal layer induced hair follicle regeneration.

[0136] 2.3 Selection of Laser Wavelength

[0137] Furthermore, the basal layer of the skin was stimulated using femtosecond lasers of different wavelengths (700, 800, 900, and 1000 nm). The frequency, wavelength, and pulse width of the femtosecond lasers were the same as in Example 1. The average hair follicle length and density were measured 24 hours after the femtosecond laser treatment. The results are shown in […]. Figure 7 .

[0138] from Figure 7 As shown in A and 7B, hair follicles can be formed within the wavelength range of 700-1000nm 24 hours after light stimulation, with the longest hair follicles formed at wavelengths of 800nm ​​and 900nm.

[0139] from Figure 7 As shown in C, the hair follicle density generated by stimulation at 800nm ​​is significantly higher than that at 900nm. This indicates that hair follicle regeneration depends on the wavelength of the femtosecond laser.

[0140] Example 3: Femtosecond laser scanning to rescue obesity-induced hair loss

[0141] In Example 3, the hair follicle regeneration effect of this method was further verified in an obese mouse model established by feeding the mouse with a high-fat diet (HFD) for more than two months.

[0142] Six- to eight-week-old male C57BL / 6 mice (males are easier to use to establish a high-fat diet model) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. A 60% high-fat diet (D12492) was purchased from Research Diets. Male C57BL / 6 mice in the high-fat diet model group were fed a high-fat diet for 70 days prior to photostimulation and maintained this diet throughout the procedure. All experiments were performed under anesthesia using a mixture of isoflurane (1-2%) and oxygen (0.5-1.0 L / min). Hair removal was performed two days before photostimulation. The experimental flowchart is shown below. Figure 8 A.

[0143] Simultaneously, an ND group (normal diet group) was established, differing from the high-fat diet group (HFD group) in that the mice were fed a normal diet, otherwise the same. After the experiments in both the HFD and ND groups, the mice were euthanized, and skin samples were collected and cut into 15μm thick sections (same as in Example 1). β-catenin immunization was performed as described in step 2.2, and the average hair follicle length and density were calculated. Results are shown below. Figure 8 In B, the left column represents -70 days and the right column represents -3 days.

[0144] from Figure 8 B indicates that mice in the HFD group fed a high-fat diet for 67 days (from day -70 to day -3) developed seborrheic alopecia.

[0145] After determining the location of the basal layer by irradiating the skin with autofluorescence and imaging using a confocal microscope in a non-invasive optical system, femtosecond laser scanning was performed to stimulate basal cells using the same protocol as in Example 1. Results are shown below. Figure 8 C, where Control indicates no laser stimulation and Laser indicates laser stimulation.

[0146] from Figure 8 As shown in C, the β-catenin immunofluorescence images of the skin on days 0, 1, and 3 after light stimulation showed that the mouse epidermis was significantly thickened; only the hair germ structure could be detected on day 1 after light stimulation, and it was not until day 3 that a mature hair follicle containing growing hair was formed.

[0147] from Figure 8 According to DE, statistical data on average hair follicle length and density show that the average hair follicle length and density on the 3rd day after femtosecond laser stimulation were significantly higher than those in the control group.

[0148] Example 4: Mechanism Study

[0149] In this embodiment 4, the mechanism of femtosecond laser stimulation for hair follicle regeneration using a non-invasive optical system was studied.

[0150] 4.1 Validation of retinol target

[0151] A FITC-labeled retinol (retinol-FITC) was synthesized, with FITC covalently bound to the hydroxyl group of retinol. When retinol is oxidized, the OH bond breaks, releasing free FITC and causing fluorescence decay. Based on this property, Hepa 1-6 cells, which have high retinol metabolic activity, were selected, and the metabolic process was visualized by treating Hepa 1-6 cells with retinol-FITC.

[0152] 1) Hepa 1-6 cells were cultured in high-glucose DMEM medium (Gibco, C11995500) containing 10% fetal bovine serum (SERANA, FBS-AUS500) and 1% penicillin-streptomycin (Gibco, 15140122) at 37°C and 5% CO2. Then, cells were seeded at a density of 2-3 × 10⁶ cells per dish. 5 Cells were incubated with 1 μM FITC-labeled retinol for 5 hours, washed, and then replaced with fresh culture medium containing or without ALDH1A inhibitor (10 μM). The cells were then observed. The group without ALDH1A inhibitor served as the control group.

[0153] from Figure 9 E indicates that retinol-FITC entering Hepa 1-6 cells is consumed within 27 hours after entering fresh culture medium, and this process can be inhibited by the ALDH1A inhibitor (ALDH1Ai).

[0154] 2) Hepa 1-6 cells were cultured in high-glucose DMEM medium (Gibco, C11995500) containing 10% fetal bovine serum (SERANA, FBS-AUS500) and 1% penicillin-streptomycin (Gibco, 15140122) at 37°C and 5% CO2. Then, cells were seeded at a density of 2-3 × 10⁶ cells per dish. 5 (1 cell) was incubated with 1 μM FITC-labeled retinol for 5 hours, washed, and then replaced with fresh culture medium without ALDH1A inhibitors. Then, a light stimulation experiment was performed.

[0155] from Figure 9 F indicates that after light stimulation of microregions in Hepa 1-6 cells, the retinol-FITC metabolic rate in the stimulated regions was significantly accelerated. Figure 9 F).

[0156] To further validate this, the basal layer of mice was pretreated with an ALDH1A inhibitor or a RAR antagonist (AGN193109) one hour before and one hour after photostimulation (epidermal thickness approximately 20 μm). Mouse skin was then acutely exposed to 200 nM atRA for 2 hours as a positive control.

[0157] from Figure 9 GI analysis showed that both treatments significantly inhibited femtosecond laser-induced hair follicle regeneration, reducing average hair follicle length and density. Figure 9 (GI). The positive control showed direct induction of some hair follicle formation within 24 hours. These data strongly suggest that endogenous retinol is the target of photostimulation-induced hair follicle formation.

[0158] Based on this, the mechanism by which femtosecond lasers stimulate hair regeneration is as follows: by stimulating endogenous retinol in basal cells (where electrons gain higher energy), retinol is more easily oxidized by aldehyde dehydrogenase 1 (ALDH1) to produce all-trans retinoic acid (atRA)—the main active metabolite of retinol in the skin. The generated atRA then activates the retinoic acid receptor (RAR) in the cell nucleus, which, in conjunction with Wnt signaling, reprograms basal cells into transiently expanded cells (TA cells), such as... Figure 9 As shown in D.

[0159] 4.2 Transformation process of basal cells after light stimulation

[0160] Immunofluorescence staining was used to detect the expression of K14, Ki67, CD71, α6, and Sox9 in mice at 3h, 6h, 12h, and 24h after laser stimulation, and quantitative statistics were performed. The results are shown below. Figure 10AI. Immunofluorescence staining is performed as described in step 2.2.

[0161] from Figure 10 As shown in A and 10B, 3 hours after light stimulation, the level of keratin 14 (K14) in some basal cells of the epidermis was significantly lower than that in the control group, suggesting an acute transition from the resting state to the differentiated state. 6 hours after stimulation, the epidermal K14 level partially recovered, but remained relatively low for 6-24 hours.

[0162] from Figure 10 C and 10D showed that Ki67 was significantly upregulated in some stimulated basal cells at 3 and 6 hours and remained highly expressed up to 24 hours, which may provide proliferation momentum for substrate formation.

[0163] from Figure 10 From E and 10F, we know that CD71 + / α6 + TA cells appeared 3 hours after stimulation, but disappeared within 12 hours.

[0164] from Figure 10 According to GI, Sox9 + These cells may indicate the destination of TA cells. These Sox9 cells... + Cells appeared in the epidermis 3 hours after stimulation, possibly due to CD71. + / α6 + Derived from TA cells—because some Sox9 can be observed after 3 hours. + CD71 + Double-positive cells. Subsequently, these Sox9... + Cells migrate downwards to the dermis within 6-24 hours, forming hair germs, hair plugs, and eventually settling into the hair follicle sheath. Figure 10 GI). By 6 hours, Sox9 + The cells are no longer associated with CD71 + Cell colocalization occurs because the former has migrated to the dermis while the latter remains in the epidermis. Figure 10 I). Comprehensive evidence: Light stimulation triggers K14 + Basal cells first transform into TA cells, and then into Sox9 cells. + Cells form hair follicles.

[0165] Figure 11 The mechanism of action of the non-invasive optical system and method of this application in stimulating hair regeneration is as follows: femtosecond laser excites endogenous retinol in basal cells (BC), prompting it to be released into TA cells and Sox9 cells. + Cells transform and form hair follicles.

[0166] 1) Laser stimulation of retinol in basal cells (BC) promotes its oxidation to all-trans retinoic acid (atRA). All-trans retinoic acid (atRA) binds to retinoic acid receptor (RAR) and retinol X receptor (RXR) to promote the transcription of downstream genes such as Sox9, thereby promoting basal cell transformation.

[0167] 2) Basal cell (BC) transformation process: The expression of K14 (a cytokeratin) decreases, while the expression of Ki67 (a cell proliferation marker) increases, indicating that cells begin to transition from a quiescent state to a proliferative state. The expression of α6 and CD71 (TA cell markers) and Sox9 (an early marker of hair follicle stem cells) increases, indicating that basal cells (BC) transform into TA cells, and ultimately, TA cells differentiate into cells expressing Sox9. + cell);

[0168] 3) Hair follicle formation process: 3 hours after laser stimulation, the placode gradually forms; 6 hours after laser stimulation, the hair germ begins to form; 24 hours after laser stimulation, the hair germ develops into a complete hair follicle; after the hair follicle continues to mature, CD34 (a marker of hair follicle stem cells in the bulge area) is expressed in the bulge area.

[0169] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A non-invasive optical system for stimulating hair regeneration, characterized in that, The non-invasive optical system includes: Femtosecond laser unit, used to generate femtosecond lasers; An optical focusing unit is used to focus the femtosecond laser onto the basal layer of skin tissue to stimulate basal cells in the basal layer to form hair follicles and thus stimulate hair regeneration.

2. The non-invasive optical system as described in claim 1, characterized in that, The femtosecond laser unit includes a femtosecond laser (100) for generating femtosecond lasers; The optical focusing unit includes a galvanometer assembly and an objective lens (500); The galvanometer assembly is used to guide the femtosecond laser to the objective lens (500); The objective lens (500) is used to focus the femtosecond laser passing through the galvanometer assembly onto the basal layer of the skin tissue; The galvanometer assembly and the femtosecond laser (100) are coupled via a first dichroic mirror (105).

3. The non-invasive optical system as described in claim 2, characterized in that, A beam expander (400) is also provided between the galvanometer assembly and the objective lens (500), and the beam expander (400) is used to expand the beam diameter of the femtosecond laser.

4. The non-invasive optical system as described in claim 3, characterized in that, The femtosecond laser unit also includes a half-wave plate (101), a polarization beam splitter (102), and a control switch (103); The femtosecond laser generated by the femtosecond laser (100) is focused onto the basal layer of the skin tissue by passing sequentially through a half-wave plate (101), a polarizing beam splitter (102), a first dichroic mirror (105), the galvanometer assembly, a beam expander (400), and an objective lens (500).

5. The non-invasive optical system as described in claim 1, characterized in that, The wavelength of the femtosecond laser is 700-1000nm; And / or, the power of the femtosecond laser is 60-100mW; And / or, the pulse width of the femtosecond laser is 30-300 fs; And / or, the scanning time of the femtosecond laser is ≤5s.

6. The non-invasive optical system according to any one of claims 1-5, characterized in that, The non-invasive optical system also includes an analysis unit for determining the location of the basal layer based on an image formed after the excitation light or excitation wave passes through the skin tissue.

7. The method of using the non-invasive optical system as described in any one of claims 1-6, characterized in that, The method includes: A femtosecond laser is generated and the femtosecond laser is focused onto the basal layer of the skin tissue.

8. The method of use as described in claim 7, characterized in that, The wavelength of the femtosecond laser is 700-1000nm; And / or, the power of the femtosecond laser is 60-100mW; And / or, the pulse width of the femtosecond laser is 30-300 fs; And / or, the scanning time of the femtosecond laser is ≤5s.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the program is executed by the processor, it implements the usage method as described in any one of claims 7 to 8.

10. A computer processing apparatus, comprising a processor and a computer-readable storage medium as described in claim 9, characterized in that, The processor executes a computer program on the computer-readable storage medium to implement the steps of the usage method as described in any one of claims 7 to 8.

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