CN109876012B - Medicinal composition and application thereof in promoting skin wound healing - Google Patents
Medicinal composition and application thereof in promoting skin wound healing Download PDFInfo
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Abstract
The invention relates to a medicinal composition and application thereof in promoting skin wound healing. The pharmaceutical composition comprises mesenchymal stem cells and a mixture of traditional Chinese medicines, wherein the mixture of traditional Chinese medicines comprises aloe and mint. The medicinal composition utilizes the strong proliferation and differentiation capacity of the mesenchymal stem cells, is applied to skin wound healing by being supplemented with traditional Chinese medicines such as aloe, mint and the like, can obviously and effectively shorten the period of wound healing, reduces the formation of scars and meets the treatment requirement.
Description
Technical Field
The invention relates to the technical field of wound treatment, in particular to a medicinal composition and application thereof in promoting skin wound healing.
Background
In daily life, the skin of people is inevitably scratched, burned or scalded. Skin healing is a rather complex and synergistic process involving multiple factors, requiring stages of inflammatory response, granulation tissue proliferation and epithelial tissue formation, with a healing cycle typically around 21 days. For minor wound conditions, patients are often treated with anti-inflammatory, anti-pain, and other medications, and then the wound will heal naturally. However, when the wound is severe, such as scald and burn, the depth and area of the skin wound are large, and the wound is difficult to heal quickly, so that the patient suffers more continuous pain, the treatment period is long, the wound is easy to be infected repeatedly, the treatment cost is increased, a large scar is generated after the wound is healed, the luster and flexibility of the skin are lost, the overall appearance of the skin is affected, and a large psychological shadow is caused to people. Although there are many kinds of conventionally used drugs such as external application drugs and ointments, there is no specific drug for promoting rapid healing of wounds and reducing scars on the market at present, and thus the need for treating wounds cannot be satisfied.
Disclosure of Invention
In view of the above problems, the present invention provides a pharmaceutical composition and its use in promoting skin wound healing, wherein the pharmaceutical composition comprises mesenchymal stem cells and a mixture of traditional Chinese medicines. The mesenchymal stem cell is a pluripotent stem cell, has strong proliferation capacity and multidirectional differentiation capacity, and can be differentiated into dermal tissues to improve the healing speed of a wound after being externally applied to the wound of skin. Meanwhile, the traditional Chinese medicine mixture contains substances such as aloe, mint and the like, the aloe contains various functional components beneficial to the skin, such as aloin, wound hormone, polypeptide mannitol (Ke-2) and the like, and the aloe has the effects of resisting bacteria, diminishing inflammation, stopping bleeding, reducing swelling and promoting wound healing and recovery, can disintegrate dead skin cell tissues and can naturally drop dead skin to reduce scars generated after wound healing. In addition, another main ingredient, namely the menthol, contains the functional ingredient, and when the menthol is applied to a skin wound, the menthol can stimulate the nerve endings of a human body to generate the effects of cooling, relieving pain and relieving itching. In order to accelerate wound healing and reduce scars, the invention provides a pharmaceutical composition and application thereof in promoting skin wound healing. The purpose of the invention is realized by the following technical scheme:
a pharmaceutical composition comprising the following components: mesenchymal stem cells, a mixture of traditional Chinese medicines; wherein the Chinese medicinal mixture comprises Aloe and herba Menthae; preferably, the mesenchymal stem cells account for 5 to 10 parts and the traditional Chinese medicine mixture accounts for 15 to 20 parts based on the total weight of the pharmaceutical composition; preferably, the aloe is 30-60 parts by weight and the mint is 10-15 parts by weight based on the total weight of the traditional Chinese medicine mixture.
Further, the medicinal composition also comprises one or more of human serum albumin, low molecular weight heparin calcium and vitamin C.
Further, the pharmaceutical composition also comprises human serum albumin, low molecular weight heparin calcium and vitamin C. The human serum albumin in the medicinal composition can supply nutrition for the stem cells so as to maintain the number and the survival rate of the stem cells, the low molecular heparin calcium can prevent the cells from aggregating so as to ensure the uniform distribution of the cells in a medicinal matrix, thus ensuring the dosage and the medicinal effect of the medicinal composition, and the two components are common clinical medicines so as not to influence the use safety of a human body. In addition, vitamin C can maintain the activity of peroxidase and preserve the activity of cells.
Further, the medicinal composition also comprises one or more of ointment, a preservative, a humectant and water; preferably, the pharmaceutical composition further comprises an ointment, a preservative, a humectant and water.
Further, the Chinese medicinal mixture also comprises one or more of fructus Kochiae, Polygoni Multiflori radix, Cera flava, herba plantaginis and herba Taraxaci.
Further, the traditional Chinese medicine mixture also comprises belvedere fruit, tuber fleeceflower root, beewax, plantain herb and dandelion. The other components in the traditional Chinese medicine mixture, such as the belvedere fruit and the tuber fleeceflower root, have the effects of relieving itching of skin, the beewax has the effects of detoxifying, healing sore, promoting granulation and relieving pain, the dandelion and the plantain herb have the effects of clearing away heat and toxic materials, and reducing swelling and dissipating stagnation, and the comprehensive use of the five traditional Chinese medicine components can further assist the wound to heal quickly and relieve the pain of a patient.
Preferably, the mesenchymal stem cell is a human umbilical cord mesenchymal stem cell.
Further, the pharmaceutical composition comprises the following components: human umbilical cord mesenchymal stem cells, human serum albumin, low molecular weight heparin calcium, vitamin C, a traditional Chinese medicine mixture, ointment, a preservative, a humectant and water; the Chinese medicinal mixture comprises Aloe, herba Menthae, Kochiae fructus, Polygoni Multiflori radix, Cera flava, herba plantaginis, and herba Taraxaci; preferably, based on the total weight of the pharmaceutical composition, the human umbilical cord mesenchymal stem cells are 5-10 parts, the human serum albumin is 1-3 parts, the low molecular heparin calcium is 0.1-0.2 part, the vitamin C is 0.3-0.5 part, the traditional Chinese medicine mixture is 15-20 parts, the ointment is 20-30 parts, the preservative is 0.2-0.6 part, the humectant is 1-1.5 parts, and the water is 86-93 parts; wherein the total weight of the traditional Chinese medicine mixture is taken as a reference, the aloe accounts for 30-60 parts by weight, the mint accounts for 10-15 parts by weight, the fructus kochiae accounts for 8-15 parts by weight, the polygonum multiflorum accounts for 10-18 parts by weight, the beeswax accounts for 5-10 parts by weight, the plantain accounts for 3-8 parts by weight, and the dandelion accounts for 8-15 parts by weight.
Preferably, the ointment is glycerin; the preservative is selected from one or more of sorbic acid, butyl hydroxybenzoate and nisin; the humectant is selected from one or more of butanediol, sorbitol and sodium hyaluronate.
All the traditional Chinese medicines of the medicinal composition are in powder form, and the finer the powder is, the better the powder is.
The invention also provides a method for preparing the medicinal composition, which comprises uniformly mixing all the components of the medicinal composition. For a non-aqueous pharmaceutical composition, a suitable amount of water may be added to formulate the pharmaceutical composition into a paste at the time of use.
Preferably, the pharmaceutical composition is prepared according to the following steps: (1) mixing the components except for the human umbilical cord mesenchymal stem cells uniformly; (2) adding human umbilical cord mesenchymal stem cells into the substance obtained in the step (1).
The invention has the advantages that the main component of the mesenchymal stem cell in the medicinal composition, such as the human umbilical cord mesenchymal stem cell, is used as a stem cell with multidirectional differentiation potential and strong paracrine effect, not only can directly provide repair cells to promote the healing of a damaged wound surface and accelerate the proliferation rate of the repair cells, but also can secrete various anti-inflammatory factors to inhibit inflammatory reaction and play an anti-inflammatory role, and can also induce the expression of endogenous endothelial growth factors, promote the proliferation of microvessels, repair damaged blood vessels, regulate the healing of the wound surface and reduce the formation of scars by inhibiting the generation amount of wound surface fibroblasts and the amount of extracellular matrix generated by the wound surface fibroblasts. Meanwhile, the human umbilical cord mesenchymal stem cells are easily obtained, do not violate ethics, have low immunogenicity, have no obvious rejection reaction after being transplanted into the body, and have higher biological safety. In addition, the traditional Chinese medicine in the medicinal composition contains aloe, mint, fructus kochiae, polygonum multiflorum, beeswax, plantain and dandelion, and the components have the effects of sterilizing, resisting inflammation, reducing swelling, relieving pain and the like. Therefore, the synergistic effect of the components in the medicinal composition can obviously and effectively reduce the time for wound healing, effectively accelerate the treatment speed, reduce the pain of patients, reduce the formation of scars and meet the treatment requirement.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the invention, all parts and percentages are weight units, and all equipment, raw materials and the like can be purchased from the market or are commonly used in the industry, if not specified. Unless otherwise indicated, the methods employed in the examples are those commonly used in the art.
Example 1
1. Preparation of human umbilical cord mesenchymal stem cells
Cutting umbilical cord into segments of about 3cm under aseptic condition, removing blood vessel, cutting substances around blood vessel, digesting with 1 ‰ type IV collagenase for 2 hr, centrifuging at 2000r/min for 10min, collecting the lower layer precipitate, further digesting with 0.25% trypsin-PBS at 37 deg.C for 25min, centrifuging at 2000r/min for 10min, collecting the cell precipitate, digesting with 0.25% trypsin solution, centrifuging at 800r/min for 10min, collecting the cell precipitate at 1 × 106The cells/ml are suspended in a culture medium, and the components of the culture medium are commercial mesenchymal stem cell serum-free culture medium. Placing at 37 deg.C, 100% humidity and 5% CO2Culturing in dark environment, changing the culture medium 1 time every 3 days, growing fibroblast on the bottom of the bottle after 2 days, digesting with 0.25% trypsin-PBS when 80% confluence can be reached after about 10 days, and then culturing at cell density of 5 × 104Per cm2And (5) subculturing.
2. Detection of biological characteristics of human umbilical cord mesenchymal stem cells
a. Identifying the molecular surface markers of the cells by a flow cytometer:
and (3) taking the 3 rd generation human umbilical cord mesenchymal stem cells with good growth state to perform flow cytometry analysis to see whether the 3 rd generation human umbilical cord mesenchymal stem cells express surface molecules such as CD34, CD44, CD45, CD73, CD90, CD105, HLA-DR and the like.
The results show that mesenchymal cell surface markers CD44, CD73, CD90 and CD105 are expressed, hematopoietic cell markers CD34 and CD45 are not expressed, and the surface marker HLA-DR closely related to the occurrence of transplant immune rejection is not expressed.
b. Cell immunofluorescence staining experiment:
taking 3 rd generation human umbilical cord mesenchymal stem cells with good growth state, adjusting cell concentration, making cell slide, fixing the cells with 4% paraformaldehyde after the cells are overgrown, making cell immunofluorescence staining, and observing and detecting whether the cells contain Vimentin under a fluorescence microscope.
The results show that the cells express the cytoskeletal protein Vimentin.
c. In vitro multi-directional differentiation experiments:
after subculture, culturing the human umbilical cord mesenchymal stem cells in an osteogenic induction system such as dexamethasone, beta-phospholipid glycerol, ascorbate and the like to differentiate the human umbilical cord mesenchymal stem cells into osteoblasts, and performing staining identification by using alizarin red and alkaline phosphatase; after subculture, the human umbilical cord mesenchymal stem cells are cultured in a adipogenic induction system containing 3-isobutyl-1-methylxanthine and indonesemetic magnesium zinc to differentiate into adipogenic cells, and are identified by oil red O staining.
The results show that human umbilical cord mesenchymal stem cells can be differentiated into osteoblasts and adipoblasts.
3. Preparation of pharmaceutical compositions
a. Based on the total weight of the medicinal composition: 3 parts of human serum albumin, 0.2 part of low molecular weight heparin calcium, 0.5 part of vitamin C, 20 parts of traditional Chinese medicine mixture, 30 parts of ointment, 0.6 part of preservative, 1.5 parts of humectant and 93.9 parts of water are placed in a stirrer to be stirred; wherein, the traditional Chinese medicine mixture comprises 30 parts of aloe powder, 10 parts of mint powder, 8 parts of belvedere fruit powder, 18 parts of polygonum multiflorum powder, 10 parts of beeswax powder, 8 parts of plantain powder and 15 parts of dandelion powder by taking the total weight of the traditional Chinese medicine mixture as a reference. Further, the ointment is glycerin; the preservative is a mixture of sorbic acid, butyl hydroxybenzoate and nisin, and the weight ratio of the sorbic acid to the butyl hydroxybenzoate to the nisin is 2:2: 3; the humectant is a mixture of butanediol, sorbitol and sodium hyaluronate, and the total amount ratio of the butanediol, the sorbitol and the sodium hyaluronate is 1:1: 2.
The stirring time was 2 hours, the stirring direction was changed every 4 minutes, and a paste-like object was obtained after the stirring was completed.
b. And (b) injecting 10 parts of human umbilical cord mesenchymal stem cells based on the total weight of the pharmaceutical composition into the pasty object prepared in the step (a), stirring at the temperature of 37 ℃ for 5min, and then completing stirring.
Example 2:
a pharmaceutical composition was prepared in the same manner as in example 1. The difference is that the traditional Chinese medicine mixture comprises 13 parts of fructus kochiae powder, 12 parts of polygonum multiflorum powder, 8 parts of beeswax powder, 4 parts of plantain herb powder and 9 parts of dandelion powder by taking the total weight of the traditional Chinese medicine mixture as a reference. In addition, the ointment is glycerin, the preservative is a mixture of butyl hydroxybenzoate and nisin, the weight ratio of butyl hydroxybenzoate to nisin is 2:3, the humectant is a mixture of sorbitol and sodium hyaluronate, and the weight ratio of sorbitol and sodium hyaluronate is 1: 2.
In addition, the stirring time was 4 hours, the stirring direction was changed every 6 minutes, and a paste-like object was obtained after completion of the stirring.
Example 3:
a pharmaceutical composition was prepared in the same manner as in example 1. The difference is that the medicinal composition comprises the following components by taking the total weight of the medicinal composition as a reference: 5 parts of human umbilical cord mesenchymal stem cells, 1 part of human serum albumin, 0.1 part of low molecular heparin calcium, 0.3 part of vitamin C, 15 parts of traditional Chinese medicine, 20 parts of ointment, 0.2 part of preservative, 1 part of humectant and 86.8 parts of water, wherein the traditional Chinese medicine mixture comprises 37 parts of aloe powder, 12 parts of mint powder, 11 parts of belvedere fruit powder, 15 parts of polygonum multiflorum powder, 8 parts of beeswax powder, 5 parts of plantain herb powder and 12 parts of dandelion powder by taking the total weight as a reference. In addition, the ointment is glycerin, the antiseptic is sorbic acid, and the humectant is butylene glycol.
Example 4:
a pharmaceutical composition was prepared in the same manner as in example 1. The difference is that the medicinal composition comprises the following components by taking the total weight of the medicinal composition as a reference: 8 parts of human umbilical cord mesenchymal stem cells, 2 parts of human serum albumin, 0.15 part of low molecular heparin calcium, 0.4 part of vitamin C, 18 parts of traditional Chinese medicine, 25 parts of ointment, 0.4 part of preservative, 1.2 parts of humectant and 90 parts of water. The traditional Chinese medicine mixture comprises 60 parts of aloe powder, 15 parts of mint powder, 15 parts of broom cypress fruit powder, 10 parts of polygonum multiflorum powder, 5 parts of beeswax powder, 3 parts of plantain powder and 8 parts of dandelion powder by taking the total weight of the traditional Chinese medicine mixture as a reference. In addition, the ointment is glycerin, the preservative is a mixture of sorbic acid and nisin, the weight ratio of sorbic acid to nisin is 2:3, the humectant is a mixture of butanediol and sodium hyaluronate, and the weight ratio of butanediol to sodium hyaluronate is 1: 2.
Example 5
A pharmaceutical composition was prepared in the same manner as in example 1. The difference is that the medicinal composition comprises the following components by taking the total weight of the medicinal composition as a reference: 5 parts of human umbilical cord mesenchymal stem cells, 20 parts of traditional Chinese medicine and 90 parts of water. Wherein the traditional Chinese medicine mixture comprises 60 parts of aloe powder and 10 parts of mint powder by taking the total weight of the traditional Chinese medicine mixture as a reference.
Example 6
A pharmaceutical composition was prepared in the same manner as in example 1. The difference is that the medicinal composition comprises the following components by taking the total weight of the medicinal composition as a reference: 10 parts of human umbilical cord mesenchymal stem cells, 15 parts of traditional Chinese medicines and 90 parts of water. Wherein the traditional Chinese medicine mixture comprises 32 parts of aloe powder and 15 parts of mint powder by taking the total weight of the traditional Chinese medicine mixture as a reference. Verifying the therapeutic efficacy of a pharmaceutical composition
Surgical excision was performed on the back of 42 nude mice to form 1.5cm x1.5cm full-thickness skin wounds, and the nude mice were randomly divided into 7 groups of 6 mice each: the first 6 groups are respectively and independently coated with the medicinal compositions of the examples 1 to 6 of the invention; the last group was coated with physiological saline only. On days 0, 7, 14, 21, 28, 1 was sacrificed. Taking a wound tissue, fixing the wound tissue by 4% paraformaldehyde, carrying out eosin hematoxylin staining, immunohistochemistry and immunofluorescence identification on a paraffin-embedded section, and carrying out histological examination on the wound tissue, wherein examination indexes relate to wound healing time, scar formation and skin flexibility.
Detection of donor cells in recipient wound tissue:
a. identification of green fluorescent protein: in order to research whether the human umbilical cord mesenchymal stem cells participate in wound repair, the human umbilical cord mesenchymal stem cells are transfected by green fluorescent protein plasmids, the new wound tissues of the recipient mice are fixed by 4 percent paraformaldehyde after 2 weeks, the tissues are sliced by a freezing section embedding medium and a constant-cold box slicer at minus 20 ℃, and the expression of the green fluorescent protein of the human umbilical cord mesenchymal stem cells is detected by a fluorescence microscope.
b. Detection of cytokeratin and collagen: in order to study whether the human umbilical cord mesenchymal stem cells differentiate epithelial cells and form collagen in vivo, newborn sections of recipient mice are incubated with rabbit anti-human keratin (pan-CK) and rabbit anti-human collagen type 1 polyclonal antibodies respectively, and then incubated with fluorescent phycoerythrin-labeled goat anti-rabbit immunoglobulin G (IgG), and detected by a fluorescent microscope at 0, 7, 14, 21 and 28 days.
As shown by the results listed in table 1 below, the human umbilical cord mesenchymal stem cells in the pharmaceutical compositions of examples 1 to 6 of the present invention can be well attached to the wound surface tissue and participate in the repair of epidermis and dermis.
TABLE 1 comparison of the wound treatment effects of the experimental groups 1-6 with the control group
The foregoing description is intended to be illustrative rather than limiting, and it will be appreciated by those skilled in the art that many modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. The application of an external medicinal composition in preparing a medicament for promoting skin wound healing and reducing scars is characterized in that the external medicinal composition comprises the following components: mesenchymal stem cells, a traditional Chinese medicine mixture, blood albumin, low molecular weight heparin calcium and vitamin C; wherein, based on the total weight of the external pharmaceutical composition, the mesenchymal stem cells account for 5-10 parts by weight, the traditional Chinese medicine mixture accounts for 15-20 parts by weight, the human serum albumin accounts for 1-3 parts by weight, the low molecular heparin calcium accounts for 0.1-0.2 part by weight, and the vitamin C accounts for 0.3-0.5 part by weight; wherein the Chinese medicinal mixture comprises Aloe, herba Menthae, Kochiae fructus, Polygoni Multiflori radix, Cera flava, herba plantaginis and herba Taraxaci; wherein the total weight of the traditional Chinese medicine mixture is taken as a reference, the aloe accounts for 30-60 parts by weight, the mint accounts for 10-15 parts by weight, the fructus kochiae accounts for 8-15 parts by weight, the polygonum multiflorum accounts for 10-18 parts by weight, the beeswax accounts for 5-10 parts by weight, the plantain accounts for 3-8 parts by weight, and the dandelion accounts for 8-15 parts by weight.
2. The use according to claim 1, wherein the pharmaceutical composition for external use further comprises one or more of an ointment, a preservative, a moisturizer and water.
3. The use according to claim 2, wherein the pharmaceutical composition for external use further comprises an ointment, a preservative, a humectant and water.
4. The use according to claim 3, wherein the ointment is 20 to 30 parts, the preservative is 0.2 to 0.6 part, the humectant is 1 to 1.5 parts, and the water is 86 to 93 parts, based on the total weight of the pharmaceutical composition.
5. The use according to claim 3, wherein the ointment is glycerol; the preservative is selected from one or more of sorbic acid, butyl hydroxybenzoate and nisin; the humectant is selected from one or more of butanediol, sorbitol and sodium hyaluronate.
6. Use according to claim 1, wherein the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.
7. The use according to any one of claims 1 to 6, wherein the pharmaceutical composition for external use is prepared by the following steps: (1) mixing the components except for the human umbilical cord mesenchymal stem cells uniformly; (2) adding human umbilical cord mesenchymal stem cells into the substance obtained in the step (1).
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| CN102120031B (en) * | 2003-08-07 | 2012-12-05 | 希尔洛有限公司 | Pharmaceutical compositions and methods for accelerating wound healing |
| KR20100105138A (en) * | 2009-03-20 | 2010-09-29 | (주)아모레퍼시픽 | Composition for promoting the volume of skin |
| CN101919380B (en) * | 2010-08-06 | 2014-10-22 | 青岛奥克生物开发有限公司 | Improved mesenchyme stem cell protection solution and application thereof |
| CN102228485B (en) * | 2011-06-02 | 2013-06-12 | 叶松 | Composition for burn rehabilitation and preparation method thereof |
| CN102920735A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, preparation method and application thereof in preparing medicine for treating diabetes |
| CN103494723B (en) * | 2013-06-26 | 2014-11-12 | 马玉贤 | Stem cell skin care composition and manufacturing method and application thereof |
| CN103550117A (en) * | 2013-10-18 | 2014-02-05 | 上海润生生物技术有限公司 | Beauty and skin care product capable of promoting skin repairation and regeneration as well as preparation method and application thereof |
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| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application |
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