CN112972495A

CN112972495A - Therapeutic agent for atrophic facial stem cells and application thereof

Info

Publication number: CN112972495A
Application number: CN202110192652.XA
Authority: CN
Inventors: 龙笑; 王晨羽; 王晓军
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2021-02-20
Filing date: 2021-02-20
Publication date: 2021-06-18

Abstract

The invention discloses a therapeutic agent for a facial atrophy stem cell, which comprises a fat-derived stem cell, wherein the concentration of the fat-derived stem cell in the therapeutic agent for the facial atrophy is 5 multiplied by 10⁵ADSCs/mL. The invention also provides an application of the therapeutic agent for the face atrophy. The treatment agent for the atrophic facial stem cells has strong clinical application and production value, can be repeatedly reproduced, has small influence on a medical person, and has the advantages of strong operability, safety, effectiveness, low cost, good rejection response effect and obvious treatment effect.

Description

Therapeutic agent for atrophic facial stem cells and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a therapeutic agent for a facial atrophy stem cell and application thereof.

Background

Facial atrophy refers to progressive unilateral facial atrophy or Parry-Romberg syndrome, manifested by progressive atrophy of unilateral facial skin, subcutaneous tissue, muscles, bones. The disease is frequently seen in young years, the sex difference is not obvious, the skin damage is more than one side of forehead, mandible, cheek and other parts begin to appear irregular pigmentation or pigmentation reduction spots, local hair is occasionally whitened, muscle spasm or neuralgia is occasionally seen, and further the progressive atrophy of skin, subcutaneous tissues, muscles and bones is locally generated within months or years. Lesions generally progress rapidly within 6 months to 2 years after onset, more slowly later, and even quiescence. The affected part has clear skin damage area boundary, and the skin is sunken and deformed like scar adhered to the lower tissue. The skin becomes thin, dry, shiny, ivory or dark gray, sebum and sweat gland secretion is reduced, hair is thin and thin, and scalp involvement can cause alopecia. It is usually confined to a branch of the trigeminal nerve and affects the face of one side of the face. Typical clinical manifestations can be diagnosed in conjunction with histopathological examination. This disease is common in young adults, with typical unilateral facial skin, subcutaneous connective tissue, and up to skeletal atrophy. Histopathology was characterized by dermal thickening, hardening of collagen bundles, loss of appendages, replacement of subcutaneous tissue by fibrous tissue, muscle atrophy, edema, vacuolization, focal inflammation, and loss of striated muscle.

Localized fibrotic diseases comprise a series of rare diseases that usually begin to appear in childhood. Localized fibrotic diseases are characterized by localized skin fibrosis or sclerosis, eventually atrophy. Local fibrotic diseases can be divided into several subtypes, including scleroderma, systemic scleroderma and linear scleroderma, wherein linear scleroderma is a disease in which subcutaneous fat gradually decreases with changes in skin pigment, and the affected skin area is banded.

Localized scleroderma (LoS) is a rare autoimmune connective tissue disease characterized by inflammation and fibrosis of the skin and subcutaneous fat in the affected area and 0.3 to 3 cases per hundred thousand residents per year. The disease is mainly manifested by atrophy and pigmentation of subcutaneous tissues, and seriously affects the quality of life and mental health of patients. Autologous fat transplantation (AFG) has the advantages of minimal invasion, rapid recovery and natural appearance, replaces a microvascular free flap transfer method, and becomes a main surgical treatment method for improving the facial atrophy of LoS patients. Due to the local inflammatory microenvironment, and the reduction of adipose-derived stem cells (ADSCs) in adipose tissue induced by corticosteroid hormone therapy, most fat grafts are difficult to survive in LoS patients, and therefore multiple transplants are necessary to maintain the appearance outcome and to burden financial and mental stress procedures. Several studies have shown that Stromal Vascular Fraction (SVF) assisted fat transplantation can improve the retention of fat grafts by about 35% in healthy people, but this increase in LoS patients has not been demonstrated. In recent years, it has been clinically reported that ADSCs have various advantages of an immunomodulatory effect, an ability to promote angiogenesis and an ability to differentiate into mature adipocytes, showing potential effects in treating many autoimmune diseases and improving fat retention.

The early research result of the invention shows that fat transplantation has obvious effect on improving wound fibrosis and promoting angiogenesis, and how to maintain the fat transplantation effect obviously is the problem to be mainly solved by the invention.

Disclosure of Invention

The invention aims to focus on the matching relation between fat and adipose-derived stem cells, so as to focus on improving the retention mechanism of the adipose-derived graft, prolong and improve the treatment effect of a patient to the greatest extent.

The invention compares the feasibility and efficacy of ADSCs-assisted, Stromal Vascular Fraction (SVF) -assisted and traditional AFG methods for improving fat retention in LoS patients. Follow-up studies for 6 months were performed in 18 LoS patients. Participants were randomly divided into three groups: AFG conventional group, SVF helper group and ADSCs helper group. SVF auxiliary group SVF: fat ratio 1: 1 received SVF assisted AFG. ADSCs adjuvant group reception mixed with 5X 10⁵ADSCs/mL fat enriched fat graft mixture of ADSCs. The volume preservation imaging effect is measured by magnetic resonance imaging for evaluation of the results.

First, the present invention provides a therapeutic agent for facial atrophy comprising adipose-derived stem cells at a concentration of 5 x 10 in the therapeutic agent for facial atrophy⁵ADSCs/mL。

The therapeutic agent of the present invention is preferably 5X 10⁵The concentration of ADSCs/mL is convenient for operation, and can provide guidance standard for clinical application, namely that the cultured stem cells in vitro are not less than 5 × 10⁵Under ADSCs/mL conditions, the concentration of stem cells cultured in vitro in the preferred embodiment of the present invention is 1X 10⁷ADSCs/mL, the concentration of the ADSCs after the stem cells are mixed with the filler is5×10⁵ADSCs/mL, can reach the clinical application value that the fat retention rate is stably improved by 1.5 times at least in the clinical application process; the second aspect provides a uniform production standard for industrial applications, namely 20mL fresh fat: according to the proportion, in the actual production and application process, only the filling volume to be shaped needs to be accurately measured, so that standard unified operation can be realized, repeated reproduction can be realized, and simplicity and convenience are realized; in a third aspect, most of the patients with facial atrophy to be reshaped and filled in the plastic department are accompanied by other diseases of the immune system, especially for scleroderma patients, the number of ADSCs is reduced by 50% compared with healthy people, and for new clinical technical application, the number of ADSCs is 5 multiplied by 10⁵The ADSCs/mL can be easily obtained under the condition of meeting the minimum effect, and for people who are difficult to obtain autologous fat, the ADSCs/mL can be easily obtained regardless of the concentration of the stem cells or the volume of the filler, so that the difficulty in application is reduced; finally, the therapeutic agent provided by the invention is safe and effective, has low cost and good rejection response effect, is effective and safe in the aspect of improving the graft retention of facial atrophy of LoS patients, is superior to simple fat transplantation or SVF assisted fat transplantation, and has obvious effect.

In a preferred embodiment, the filler can also be a commonly used in vivo filling material, such as silica gel and physiological saline, and the more commonly used filling material in the department of cosmetology is autologous fat, hyaluronic acid and other materials.

The therapeutic agent for the facial atrophy stem cells is mixed with fresh fat and then injected, wherein the fresh fat is derived from fresh fat obtained by performing autologous abdominal liposuction under low and constant negative pressure.

Preferably, the immunophenotype of the adipose derived stem cells is: positive, CD13, CD29, CD44, CD73, CD90 and CD105(> 80%); negative, CD31, CD45 and CD235a (< 2%).

In a preferred embodiment, the facial atrophy stem cell therapeutic further comprises one or more compounds that facilitate implantation. Such as anti-T cell antibodies, immunosuppressive agents, glucose, and the like.

Preferably, the formulation of the facial atrophy stem cell therapeutic agent is an injection, preferably subcutaneous injection, and more preferably, subcutaneous injection is performed using a fan pattern technique of a 1.4mm blunt cannula.

Preferably, the method for preparing the adipose-derived stem cells comprises:

(1) performing abdominal liposuction under low and constant negative pressure to obtain fresh fat, digesting with collagenase I, centrifuging, collecting bottom cells, preparing a matrix blood vessel component SVF solution with sterile saline, and culturing the SVF cells for two weeks;

(2) suspending SVF in a serum-free culture medium to obtain separation of ADSCs to obtain an ADSCs separation solution, wherein the separation method comprises centrifuging at 600 Xg for 5 minutes;

(3) the ADSCs separating solution of step (2) was again centrifuged at 600 Xg for 5 minutes, resuspended in the medium, inoculated into culture flasks, and cultured in 5% CO₂Culturing at 37 deg.C; cells were cultured to 3 passages, medium was changed every 2 days, 1X 10⁷ADSCs/mL suspension solution.

On the other hand, the invention also provides application of the facial atrophy stem cell therapeutic agent in preparing a medicine for treating facial atrophy.

Facial atrophy in the present invention refers in particular to progressive unilateral facial atrophy or Parry-Romberg syndrome, which is manifested as progressive atrophy of unilateral facial skin, subcutaneous tissue, fat, muscle, mostly originating from trauma, vascular injury, manifested as ulcers and/or dermal fibrosis.

In still another aspect, the invention also provides the application of the facial atrophy stem cell therapeutic agent in preparing a local fibrosis disease medicament.

In still another aspect, the invention also provides application of the facial atrophy stem cell therapeutic agent in preparation of a medicine for treating scleroderma.

Preferably, the scleroderma is localized scleroderma, in particular linear localized scleroderma, more preferably facial atrophy in linear localized scleroderma.

Preferably, the ADSCs can secrete various cytokines to promote angiogenesis, inhibit apoptosis of adipocytes, and increase survival and regeneration of fat; the ADSCs also have the advantage of immunomodulation to limit inflammation and reduce fibrosis in scleroderma models.

More preferably, the cytokines include: aFGF, bFGF, angiogenin (angiogenin), PD-ECGF (platelet-derived endothelial growth factor), TGF, TNF, and Vascular Endothelial Growth Factor (VEGF).

Advantageous effects

The present invention measures fat retention volume by magnetic resonance imaging and takes clinical pictures to evaluate the results. At the follow-up visit of the sixth month, the fat retention of the ADSCs helper group was 49.83 + -3.61%, significantly higher than 31.75 + -1.73% (p ═ 0.0004) of the SVF helper group and 21.86 + -1.68% (p <0.0001) of the conventional group. No serious adverse events occurred during surgery and follow-up. This preliminary study shows that ADSCs-assisted AFG is safer and a more viable alternative to traditional AFG and SVF-assisted methods in correcting facial atrophy in LoS patients.

The treatment agent for the atrophic facial stem cells has strong clinical application and production value, can be repeatedly reproduced, has small influence on a medical person, and has the advantages of strong operability, safety, effectiveness, low cost, good rejection response effect and obvious treatment effect.

Drawings

FIG. 1 is an overview of the intervention of the present invention; ADSC, adipose stem cells, MRI, magnetic resonance imaging, SVF: stromal vascular fraction, AFG: and (4) autologous fat transplantation.

FIG. 2 is a statistical chart of the fat retention results of the present invention; a, injection amount of three groups of fat grafts; b, fat retention results in a three month follow-up; c, fat retention results in six month follow-up; d, trend of change in fat retention over six months.

FIG. 3 MRI scan results of the present invention show changes in subcutaneous adipose tissue of a patient in buccal lesions (in boxes) of ADSCs assisted AFG; a, preoperative; b, immediately performing postoperation; c, after three months; and D, six months later.

FIG. 4 is a graph of the results of exploratory data analysis of the present invention, with the AFG method being the only variable affecting the resulting variable (fat retention) among all the exposure variables (box).

FIG. 5 clinical outcome satisfaction evaluation by the expert of the invention for three groups of patients.

FIG. 6 shows a specific example of the clinical evaluation result of the present invention. A, forehead of a 24 year old female LoS patient received AFG assisted by ADSCs. (A1 preoperatively; A2 six months later). B, the forehead of a female LoS patient 25 years old received AFG assisted by ADSCs. (B1 preoperatively; B2 six months later). C, the cheek of a 22 year old female LoS patient received AFG assisted by ADSCs. (C1, preoperative; C2, six months later).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The reagents used in the present invention are as follows:

fat preservation solution: preparing raw materials of the preservation solution according to the proportion: liquid cell culture medium DMEM/F12, every 100mL fat preservation solution contains 4000 units of heparin sodium as an anticoagulant, 50mg ascorbic acid as an antioxidant and 2000 units of gentamicin as an antibiotic.

(1) Adding anticoagulant, antioxidant, antibiotic and/or other nutritional factors into 100mL of liquid cell culture medium in sequence according to a proportion, and fully and uniformly mixing;

(2) filtering and sterilizing with a filter membrane to obtain the fat preserving fluid, subpackaging, and freezing and preserving; when in use, the mixture is placed at 2-6 ℃ in advance for unfreezing.

Stem cell preservation solution: TC199, physiological saline containing 2% human albumin and compound amino acid.

The Exploratory Data Analysis (EDA) in the invention refers to a Data Analysis method for exploring the structure and rule of Data by means of drawing, tabulating, equation fitting, calculating characteristic quantity and the like under the condition of least prior hypothesis on the existing Data, and the method is proposed by american statistician j.k.tukey in the last 70 th century. In the conventional statistical analysis method, data is usually assumed to conform to a statistical model, and then parameters and statistics of the model are estimated according to data samples, so as to know the characteristics of the data. EDA is a more realistic analysis method, emphasizes that data "speak" itself, and we can most truly and directly observe the structure and features of data through EDA.

After the appearance of EDA, the process of data analysis was divided into two stages, the exploration stage and the verification stage. The exploration phase focuses on discovering patterns or models contained in the data, and the validation phase focuses on evaluating the discovered patterns or models. When we get a piece of data, if the purpose of data analysis is not very clear and targeted, we may feel somewhat at a loss, and we are more necessary to do EDA, which can help us to preliminarily understand the structure and characteristics of the data, even find some patterns or models, and combine with the background knowledge of the industry, perhaps to get some useful conclusions directly.

Example 1 sample Collection and protocol

In this study, 18 cases (female: 11; male: 7) of patients with facial atrophy and deformity caused by localized scleroderma were selected, 6 cases of patients received ADSCs-assisted fat transplantation (experimental group), 6 cases of vascular stromal fraction (SVF) -assisted fat transplantation (standard control group), and 6 cases of simple fat transplantation (control group).

Inclusion criteria for samples:

(1) linear localized scleroderma patients diagnosed with dermatology;

(2) localized scleroderma is in the stationary phase;

(3) age 18-40 years old;

(4)BMI18.5-24.5kg/m²；

(5) desire to correct facial atrophy;

(6) signing the informed consent.

Exclusion criteria for samples:

(1) a soft tissue transplant or fat transplant has been previously performed in scleroderma patients;

(2) no lower abdominal fat donor sites;

(3) patients undergoing hormone, anti-fibrotic and anti-scarring drug therapy;

(4) localized scleroderma patients in unstable or progressive stages;

(5) smoking;

(6) chronic diseases requiring long-term medication;

(7) weak or no immune function;

(8) patients with a BMI greater than 2.

The present example refers to localized scleroderma, preferably linear localized scleroderma, and more preferably to patients with localized fibrosis due to facial atrophy as in linear localized scleroderma.

The invention was performed in LoS patients with facial atrophy (forehead and cheek). All participants received AFG to correct facial atrophy according to a randomized sequence. The conventional group received the conventional AFG group and the SVF-assisted group received the SVF: fat ratio of 1: 1, receiving SVF assisted AFG, and the ADSCs helper assembly receiving the ADSCs enriched in vitro for fat transplantation. Adding 5 × 10 into fat graft mixture rich in ADSCs⁵ADSCs/mL fat. The clinical effect at this concentration was selected to be superior to other concentrations according to previous protocols of ADSC transplantation clinical studies. Magnetic Resonance Imaging (MRI) analysis was used to preoperatively measure the amount of facial lipoatrophy in each participant. The fat retention volume of each participant was also measured by MRI after surgery and in two visits of six months.

EXAMPLE 2 liposuction, graft preparation

Adipose tissues are derived from healthy volunteers, the age and sex of the donor are recorded in detail before collection, and the health condition is known by routine physical examination, and the serological detection of HBV, HCV, HIV and syphilis is required to be negative. Fasting and water are collected in the morning of the day, and biochemical whole items and blood routine are checked before entering an operating room.

At study entry, the amount of facial atrophy was measured by MRI analysis for each participant. Evaluation of The severity of Facial atrophy and graft volume was recorded for each participant by comparing The volume difference of subcutaneous adipose tissue between The preoperative target lesion site and The unaffected symmetric site (evaluation criteria, ref. Hayson Chenyu Wang, etc., The Development and initiative dissolution of The PUMC Localized diagnosis Facial analytical Index: A Pilot Study, analytical plastics Surgery, 15January 2021). All participants underwent outpatient liposuction and subsequent facial fat transplantation surgery under local anesthesia, and autologous SVF and ADSC were prepared in a good manufacturing practice facility (New drug development and Stem cell therapy clinical trials, Beijing City Key laboratory, BZ 0381). Abdominal liposuction was performed using a 50 ml syringe at low and constant negative pressure to limit damage to the adipocytes.

Example 3 cell culture, identification and transplantation

Performing abdominal liposuction under low and constant negative pressure to obtain fresh fat, digesting with collagenase I, centrifuging, collecting bottom cells, preparing SVF solution with sterile saline, and culturing SVF cells for two weeks.

Isolation of ADSCs was achieved by suspending SVF in serum-free medium (Lonza Group AG, Switzerland). The solution was centrifuged again at 600 Xg for 5 minutes, resuspended in medium, inoculated into culture flasks and incubated at 5% CO₂The cells were cultured at 37 ℃. The following day the cultures were examined and cell adhesion was confirmed under a microscope. Cells were cultured to passage 2 to 3, with medium changed every 2 days.

Cells were confirmed by flow cytometry as adipose-derived stromal cells according to the international society for cell therapy standards, positive: CD13, CD29, CD44, CD73, CD90 and CD105(> 80%); negative: CD31, CD45 and CD235a (< 2%).

The evaluation of ADSCs prior to clinical use includes: 1) no visible clumps in the cell suspension; 2) absence of pathogen contamination by sterility testing of the medium; 3) ADSC (all-dielectric self-supporting ceramic) raw materialCapacity measurement, should exceed 90%; 4) characteristic morphology of ADSC. Each batch is 1 × 10⁷The ADSCs were suspended in 1mL of ADSCs solution in sterile saline.

Solutions of either SVF or ADSC are stored in sterile containers labeled according to current regulations and transferred to the operating room in a sealed sterile box. The target transplant amount is determined by overcorrection of 20% for the measured amount of facial atrophy. The conventional group received fat transplantation directly. Will vary depending on the ratio of fat to SVF 1: 1(20mL fresh fat: 1mL fresh SVF solution collected from 20mL adipose tissue) of the transplantation mixture was injected into SVF helper patients. Mixture of fresh fat and cultured ADSC, corresponding to 5X 10 per ml fat⁵ADSCs (20mL fresh fat: 1mL non-cryopreserved ADSCs solution) were transplanted into the ADSCs helper group (Table 1). The homogeneity of the adipocyte and ADSCs solution or SVF solution is ensured by rotating and gently stirring the mixed transplant product until the color and consistency are uniform. For all groups, AFG or AFG mixture with SVF/ADSC was transferred into the subcutaneous tissue of the target lesion by fan pattern technique using a 1.4mm blunt cannula.

TABLE 1 fat transplantation amount and total number of ADSCs injected in ADSCs-assisted AFG group

Example 4 clinical evaluation

Preoperative variables include age, gender, LoS severity index (LoSSI) and BMI. After surgery, participants were asked to return for follow-up assessments three and six months later. At each follow-up visit, surgical complications were recorded and participants were photographed. Five orthopedic specialists evaluated the clinical outcome separately. Photographs of each participant pre-and follow-up were presented blindly and as was the case with each expert, and the expert's satisfaction with the problem was assessed: "how do you have a satisfactory level of retention and aesthetic improvement for this participant? "expert satisfaction includes five levels: below average, above average, good and excellent.

1. Magnetic resonance imaging analysis (MRI analysis)

MRI a 3.0 tesla MR scanner (Discovery MR 7503T, GE Healthcare, milwaukee, wisconsin, usa) was used before, after and at each follow-up visit using 32-channel head and neck coils. MRI examinations were performed from frontal to cervical, including all six cervical lymph node levels, excluding only the supraclavicular region. Axial TSE T1-FLAIR weighting was performed (acquisition parameters: TR 1725ms, TE 24ms, flip angle 111 °, TA 3min6 s; 50 slices; FOV 22 × 20mm) with a slice thickness of 4 mm. Volume measurements were performed using Horos V3.3.1 (Horos project, usa). The imaging examination before and after surgery and at each follow-up visit included the location, appearance and volume of the target diseased adipose tissue (fig. 1). Percent change in adipose tissue volume for LoS lesions was measured as follows: retention ═ volume of remaining fat at follow-up-volume of starting adipose tissue/volume of fat grafts. In addition, the presence of complications is noted. All imaging was performed by a radiologist with a 6-year experience.

2. Statistical analysis

The calculated sample size was large, with an average distribution of 18 participants in total (1: 1: 1), showing a significant difference of 20% in retention, 5% on both sides, and 80% efficacy. Statistical analysis was performed using GraphPad Prism 8.0.2(GraphPad Software, usa). Significance of difference was assessed by one-way anova with Tukey's multiple comparison test. Significance was set at p < 0.05. All data are expressed as mean ± SEM. Exploratory data analysis was further performed using Python 3.8.3 (Python (tm) usa) to visualize potential relationships between exposed and result variables.

3. Results

18 participants (11 females and 7 males) participated in the study and remained throughout 6 months, with a mean age of 26.68 + -1.28 years and a mean BMI of 21.22 + -0.34 kg/m²The average LoSSI was 3.46. + -. 0.11 minutes. There were no significant differences in age, BMI, scleroderma severity (LoSSI) or facial atrophy severity (fat graft volume) between the three groups (table 2, fig. 2A). No experience of the participantsInfection, hematoma, seroma or oil cyst. There was no perioperative use of antibiotics. No serious adverse or unexpected events occurred.

TABLE 2 participant characteristic statistics

In table 2, P value1 is the difference between the ADSCs adjuvant group and the conventional AFG group, P value2 is the difference between the ADSCs adjuvant group and the SVF adjuvant group, P value3 is the difference between the SVF adjuvant group of the conventional AFG group, BMI is body mass index, and initial volume of subcutaneous fat of ISFV.

In the follow-up three months, the fat retention rate of the ADSCs adjuvant group was 61.25 ± 2.03%, which was significantly higher than 45.69 ± 4.11% (p ═ 0.0089) of the SVF adjuvant group and 41.26 ± 3.00% (p ═ 0.0012) of the conventional group. However, there was no significant difference between the conventional group and the SVF-assisted group (p-0.5929). In the follow-up visit at the sixth month, the fat retention rate of the ADSCs adjuvant group was 49.83 ± 3.61%, which was significantly higher than 31.75 ± 1.73% (p ═ 0.0004) of the SVF adjuvant group and 21.86 ± 1.68% (p <0.0001) of the conventional group. A significant difference in fat retention was also observed between the SVF-assisted group and the conventional group (p-0.0346) (table 3, fig. 2B-D).

TABLE 3 participant fat retention and Retention Rate statistics

In table 3, P1 is the difference between the ADSCs adjuvant group and the conventional AFG group, P2 is the difference between the ADSCs adjuvant group and the SVF adjuvant group, and P3 is the difference between the SVF adjuvant group of the conventional AFG group.

As shown in fig. 3, MRI scan results show changes in the patient's subcutaneous adipose tissue in buccal lesions (in boxes) of ADSCs-assisted AFG. A, preoperative; b, immediately performing postoperation; c, after three months; and D, six months later.

As shown in fig. 4, the present invention confirmed by exploratory data analysis that the AFG method is the only exposure variable affecting the outcome variable (fat retention), as shown by the data in the box.

As shown in fig. 5 and 6, the expert satisfaction of the ADSCs adjuvant group was 3.98 ± 0.11, which was significantly higher than 3.07 ± 0.18(p ═ 0.0092) of the SVF adjuvant group and 2.20 ± 0.24(p <0.0001) of the conventional fat transplantation.

SVF obtained from collagenase digested adipose tissue comprises a plurality of heterogeneous cell populations including endothelial cells, hematopoietic cells, pericyte origin cells and a small amount of ADSCs (less than 3%). The use of SVF in healthy human samples is reported: the fat ratio is 1: 1, up to about 1X 10 per ml⁴The concentration of ADSCs, which is equal to a doubling of the physiological number of ADSCs in healthy human adipose tissue. As a method of cell-assisted lipid transfer, SVF assisted lipofection is considered to be a time-saving technique, and supplement of ADSCs can be obtained by collagenase isolation without additional culture. However, to date, there is no existing data that convincingly demonstrates that SVF-assisted methods can consistently produce comparable or increased fat graft retention compared to two conventional fat transplants. In our study, fat retention in the SVF helper group increased 45% over the conventional group after 6 months. However, a method which can stably increase the retention rate by 1.5 times or more is not considered to have clinical application value. Therefore, the ADSCs of the invention have clinical application value in terms of clinical application.

In addition, the number and activity of ADSCs vary widely among patients due to the composition of SVF, resulting in unstable results of SVF-assisted methods. Although our previous studies compared the biological activity of ADSCs between LoS patients and healthy people, and found that the isolated ADSC lines did not differ in morphology or adipogenic differentiation potential, the number of ADSCs in scleroderma patients was reduced by 50% compared to healthy people, which means that the number of ADSCs in scleroderma SVF was also lower than in healthy people SVF samples. These factors also limit the potential clinical use of SVF-enriched methods for treating scleroderma patients.

The ADSC auxiliary method for in-vitro amplification not only can obtain enough stem cells, but also can identify the biological activity of the cells before transplantation. This advantage makes it a more unique application advantage in the field of correcting facial atrophy in patients with LoS.

Our in-clinical in-precursor studies show that mesenchymal stem cell transplantation has low tumorigenic risk, and we also performed MRI scans 6 months after surgery, and no complications or hazards were found in LoS patients associated with the use of ADSC. As shown in a number of clinical and basic studies on ADSCs, there are no reports on cell mutation or tumor formation, and expansion of passaged ADSCs does not affect the differentiation capacity of stem cells, nor does it confer a cancerous state or capacity in vitro. Furthermore, by tracking labeled stem cell products using novel monitoring methods (e.g., imaging), determining the distribution and tendency of injected cells to migrate and migrate in vivo, it would be beneficial to understand the potential benefits and complications of cell therapy. Homing of transplanted cells also warrants the use of such techniques in future clinical trials.

The therapeutic agent of the present invention is preferably 5X 10⁵The concentration of ADSCs/mL is convenient for operation, and can provide guidance standard for clinical application, namely that the cultured stem cells in vitro are not less than 5 × 10⁵Mixing ADSCs/mL with filler to obtain ADSCs concentration of 5 × 10⁵ADSCs/mL, can reach the clinical application value that the fat retention rate is stably improved by 1.5 times at least in the clinical application process; the second aspect provides a uniform production standard for industrial applications, namely 20mL fresh fat: according to the proportion, in the actual production and application process, only the filling volume required for accurately measuring the facial atrophy is needed, so that standard unified operation can be realized, repeated reproduction can be realized, and simplicity and convenience are realized; in the third aspect, most of the patients suffering from facial atrophy faced by plastic surgery at present are accompanied by other diseases such as immune system, particularly for scleroderma patients, the number of ADSCs is reduced by 50% compared with that of healthy people, and for the application of new clinical technology, the number of ADSCs is 5 multiplied by 10⁵The ADSCs/mL can be easily obtained under the condition of meeting the minimum effect, and for people who are difficult to obtain autologous fat, the ADSCs/mL can be easily obtained regardless of the concentration of the stem cells or the volume of the filler, so that the difficulty in application is reduced; finally, the invention providesThe provided therapeutic agent is safe and effective, has low cost and good rejection response effect, is effective and safe in the aspect of improving the retention of the facial atrophy transplant of LoS patients, is superior to simple fat transplantation or SVF assisted fat transplantation, and has obvious effect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1.A therapeutic agent for facial atrophy stem cells, comprising adipose-derived stem cells at a concentration of 5 x 10 in the therapeutic agent for facial atrophy⁵ADSCs/mL。

2. The facial atrophy stem cell therapeutic agent of claim 1, further comprising a filler material.

3. The therapeutic agent for the stem cell of facial atrophy according to claim 1, wherein the immunophenotype of the adipose-derived stem cell is: positive, CD13, CD29, CD44, CD73, CD90 and CD105(> 80%); negative, CD31, CD45 and CD235a (< 2%).

4. The facial atrophy stem cell therapeutic agent of claim 1, further comprising one or more compounds that facilitate implantation.

5. The therapeutic agent for the stem cells for facial atrophy according to any one of claims 1 to 4, wherein the dosage form of the therapeutic agent is an injection.

6. The method for preparing the adipose-derived stem cells of claim 1, comprising the steps of:

7. Use of the therapeutic agent for the stem cells for facial atrophy according to any one of claims 1 to 5 in the preparation of a medicament for the treatment of facial atrophy.

8. Use of the therapeutic agent for the face atrophy stem cells according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of a localized fibrotic disease.

9. Use of the therapeutic agent for facial atrophy stem cells according to any one of claims 1 to 5, in the preparation of a medicament for treating scleroderma, the therapeutic agent for facial atrophy comprising adipose-derived stem cells, the concentration of the adipose-derived stem cells in the therapeutic agent for facial atrophy being 5 x 10⁵ADSCs/mL。

10. Use according to claim 9, wherein the scleroderma is localized scleroderma, in particular linear localized scleroderma.

11. The use of any one of claims 7 to 9, wherein the ADSCs secrete cytokines to promote angiogenesis, inhibit apoptosis of adipocytes, and increase survival and regeneration of fat; the ADSCs also have the advantage of immunomodulation to limit inflammation and reduce fibrosis in scleroderma models.